Bioactive Natural Products (Part G), Volume 26 (Studies in Natural Products Chemistry)

Studies in Natural Products Chemistry Volume 26 Bioactive Natural Products (Part G)

studies in Natural Products Chemistry edited by Atta-ur-Rahman

Vol. 1 Vol. 2 Vol. 3 Vol. 4 Vol. 5 Vol. 6 Vol. 7 Vol. 8 Vol. 9 Vol. 10 Vol. 11 Vol. 12 Vol. 13 Vol. 14 Vol. 15 Vol. 16 Vol. 17 Vol. 18 Vol. 19 Vol. 20 Vol. 21 Vol. 22 Vol. 23 Vol. 24 Vol. 25 Vol. 26

Stereoselective Synthesis (Part A) Structure Elucidation (Part A) Stereoselective Synthesis (Part B) Stereoselective Synthesis (Part C) Structure Elucidaton (Part B) Stereoselective Synthesis (Part D) Structure and Chemistry (Part A) Stereoselective Synthesis (Part E) Structure Chemistry (Part B) Stereoselective Synthesis (Part F) Stereoselective Synthesis (Part G) Stereoselective Synthesis (Part H) Bioactive Natural Products (Part A) Stereoselective Synthesis (Part I) Structure and Chemistry (Part C) Stereoselective Synthesis (Part J) Structure and Chemistry (Part D) Stereoselective Synthesis (Part K) Structure and Chemistry (Part E) Structure and Chemistry (Part F) Bioactive Natural Products (Part B) Bioactive Natural Products (Part C) Bioactive Natural Products (Part D) Bioactive Natural Products (Part E) Bioactive Natural Products (Part F) Bioactive Natural Products (Part G)

Studies in Natural Products Chemistry Volume 26 Bioactlve Natural Products (Part G)

Edited by

Atta-ur-Rahman H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

2002

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FOREWORD The present volume of "Studies in Natural Product Chemistry" presents the chemistry of a large number of exciting natural products. The triterpenoid saponins from Caryophyllaceae family have been reviewed by Jia et al. Marine fatty acids present an unusual array of structural types. Recent developments in the total synthesis of bioactive marine fatty acids are reviewed by Carballeira. The chemistry of secologanin, a monoterpenoid glucoside which is a precursor of about 3000 indole, isoquinoline and related alkaloids, is presented by Szabo. Lignans represent an important and widely distributed class of dimeric phenylpropanold derivatives, many of which have shown antimicrobial, anti-viral or anti-feedant activities, some of which play an important role In plant defense. The chemistry of the lignans of Podophyllum has been reviewed by Moraes et al, while the bioactivity of lignans has been reviewed by Rios et al. Saffron, the yellow orange stigmas from the Crocus sativus flower, is the world's most expensive spice which contains some 34 volatile components responsible for the powerful odor and a number of non-volatile components responsible for its color. The chemistry and biological activities of the constituents of saffron are reviewed by Liakopoulou-Kyriakides et al. The bark of Fraxunus emus has been employed In the traditional systems of medicine for treatment of wounds, inflammation, arthritis and dysentry. The chemistry and biological activity of the constituents of the plant have been reviewed by Kostova et al. The management of undesirable fresh water phytoplankton blooms, which can cause losses in aquaculture, farm livestock and waterfowl, using natural campounds is discussed by Schrader etal. Aromatic plants have been used in folk medicine, cosmetics and food industry, largely because of the essential oils that they contain. The pharmacological activities and applications of essential oils of Salvia sclarea and Salvia desoleana are presented by Peana et al. Plant- and animal-parasitic nematodes are responsible for considerable crop damage each year and they also contribute to malnutrition and disease in humans and domestic livestock. The chemistry and bioactivity of secondary metabolites is reviewed by Ghisalberti. Resveratrol is one of the most widely known stilbenes which undergoes polymerization in plants and fungi to afford a host of complex oligomers. The structure, chemistry and biological activity of resveratrol oligomers is reviewed by Cichewicz et al. Phytopathogenic bacteria and plants produce a number of bioactive metabolites, many of which are plant growth regulators and phytotoxins. The chemistry and pharmacological activity of such compounds is reviewed by Evidente et al. The chemistry and biological activities of the constituents of Rubia tlnctomm L are presented by Derksen et al. The Pteridium species are In a group referred to as the "bracken fern". Recent developments in the chemical behaviour, toxicology and chemical ecology of compounds found in bracken fern are reviewed by Alonso-Amelot. Flavonoids can serve as important neutraceutlcals as they exhibit interesting anti-oxidant properties and play a role on ascorbic acid preservation. This field is discussed by Marin et al. The occurrence, structure and bioactivity of 1,7-diarylheptanoids is reviewed by Claeson et al. The structures and biological activities of natural insecticides from Lauraceae, Compositae, Ranunculaceae and Boraginaceae species are presented by Gonzalez-Coloma et al, while potential anti-parasitic substance from natural sources are presented by Kayser et al. The chemistry and bioactivity of nitric oxide (NO) in plant and animal cells is reviewed by Wendehenne et al. while the role of 0-aminophenol-type tryptophan metabolites in living organisms is discussed by Rescigno et al. Recent developments on structural studies and pharmacology of strychnos alkaloids is reviewed by Rasoanaivo et al. while the chemistry and biological activity of certain compounds present in the pungent principles of radish is presented by Uda et al. The occurrence, structure, properties, metabolism, biological activities and uses of carbohydrates having the hexo-D-manno configuration are reviewed by Matheson. Sponges belonging to the llthistid order have

VI

proved to be spectacular sources of several interesting classes of biologically active compounds. This is discussed in a review by D'Auria et al. The chemistry and biological properties of copper/topa quinone-containing amine oxidases is reviewed by Sebela. The present volume contains articles written by eminent experts in their respective fields. It should prove to be of wide interest to medicinal chemists, phamriacologists and organic chemists working in academia and industry. This 26^^ volume of this encyclopaedic series represents a landmark. The 25 volumes published previously during the last 12 years contain a huge amount of exciting chemistry. It is hoped that it will be received with the same order of enthusiasm as its predecessors. I would like to express my thanks to Dr. Shakeel Ahmad for his assistance in the preparation of the index. I am also grateful to Mr. Muhammad Asif and Mr. Waseem Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.

Atta-ur-Rahman Ph.D. (Cantab), Sc.D. (Cantab)

July, 2001

Vll

PREFACE It is testimony to the healthy state of Natural Products chemistry over the last decade that the present volume is the 26**^ in a series which is only 12 years old. Professor Atta-urRahman is once again to be congratulated for his great skill and perseverance needed to sustain such a valuable project and for his ability to attract first-rate investigators to write chapters covering the full range of the field. Volume 26 Is of particular Interest since all of the chapters reflect the importance of the biological activity of nature's molecules activity which continues to Inspire the evolution of new drugs against disease. It is significant that a high percentage of clinically approved drugs have structures based on natural product leads. Although the biological receptors for the majority of the structures described in this volume are still unknown, progress at the chemistry- biology interface is now accelerating rapidly and we look forward to future volumes devoted to the molecular description of natural product bioactivity. Also, thanks to modern, rapid spectroscopic methodology, there appears to be an exponential rate of discovery and description of novel structures from natural sources and we can confidently expect this trend to continue. Finally, I would like to express the hope that in spite of his heavy duties as Federal Minister for Science and Technology in Pakistan, Professor Atta-ur-Rahman will continue to find time to produce this very valuable series of reviews on Natural Product Chemistry, which taken as a whole serve as essenfial reading for active researchers in the field.

A. Ian Scott, F.R.S.

Davidson Professor of Science Director of Center for Biological NMR August. 2001

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IX

CONTENTS Foreword Preface Contributors Triterpenoid saponins from caryophyllaceae family ZHONGHUA JIA, KAZUO KOIKE, NIRANJAN P. SAHU, AND TAMOTSU NIKAIDO

v vii xiii 3

Recent developments in the total synthesis of bioactive marine fatty acids NESTOR M. CARBALLEIRA

63

Some aspects of the chemistry of secologanin LASZLO F. SZABO

95

The lignans of Podophyllum RITA M. MORAES

149

New findings on the bioactivity of lignans JOSE LUIS RIOS, ROSA M. GINER, JOSE M. PRIETO

183

Crocus sativus-biological active constituents M. LIAKOPOULOU-KYRIAKIDES, D.A. KYRIAKIDIS

293

Chemical components of Fraxinus ornus bark-structure and biological activity IVANKA N. KOSTOVA, TANYA lOSSIFOVA

313

Natural compounds for the management of undesirable freshwater phytoplankton blooms KEVIN K. SCHRADER, AGNES M. RIMANDO, STEPHEN O. DUKE

351

Pharmacological activities and applications of Salvia sclarea and Salvia desoleana essential oils ALESSANDRA T. PEANA, MARIO D.L. MORETTI

391

Secondary metabolites with antinematodal activity EMILIO L. GHISALBERTI

425

Resveratrol oligomers: structure, chemistry, and biological activity ROBERT H. CICHEWICZ, SAMIR A. KOUZI

507

Bioactive metabolites from phytopathogenic bacteria and plants ANTONIO EVIDENTE AND ANDREA MOTTA

581

Rubia tinctorum L. GOVERDINA C.H. DERKSEN AND TERIS A. VAN BEEK

629

The chemistry an toxicology of bioactive compounds in bracken fern {Pteridium Sp.), with special reference to chemical ecology and carcinogenesis MIGUEL. E. ALONSO-AMELOT

685

Flavonoids as nutraceuticals: structural related antioxidant properties and their role on ascorbic acid preservation F.R. MARIN, M.J. FRUTOS, J.A. PEREZ-ALVAREZ,

741

F. M A R T I N E Z - S A N C H E Z , J.A. DEL

RIO

Natural products as potential antiparasitic drugs OLIVER KAYSER, ALBRECHT F. KIDERLEN, SIMON L. CROFT

779

Natural insecticides: structure diversity, effects and structure-activity relationships. A case study A. G O N Z A L E Z - C O L O M A , M . REINA, C. GUTIERREZ, B.M. FRAGA

849

Occurrence, structure, and bioactivity of 1,7-diarylheptanoids PER CLAESON, UBONWAN P. CLAESON, PATOOMRATANA TUCHINDA AND VICHAI REUTRAKUL

881

Nitric oxide: chemistry and bioactivity in animal and plant cells DAVID WENDEHENNE, LAURE DUSSABLY, JEAN-FRANCOIS JEANNIN AND ALAIN PUGIN

909

o-Aminophenol-type tryptophan metabolites: 3-hydroxykynurenine, 3Hydroxyanthranilic acid, and theu* role in living organisms ANTONIO RESCIGNO AND ENRICO SANJUST

965

New contributions to the structure elucidation and pharmacology of Strychnos alkaloids P. RASOANAIVO, M- T. MARTIN, E. GUITTET AND F. FRAPPIER

1029

Occurrence of biologically active 2-thioxopyrrolidines and 3,5-disubstituted 2-thiohydantoins from the pungent principle of radish (Raphanus sativus L.) YASUSHI UDA, YOSHIO OZAWA AND KOICHI YONEY AMA

1073

Structure, occurrence and roles of carbohydrates with the hexo-D-manno configuration NORMAN K. MATHESON

1113

The chemistry of lithistid sponge: a spectacular source of new metabolites M. VALERIA D'AUPUA, ANGELA ZAMPELLA AND FRANCO ZOLLO

1175

XI

Copper/topa quinone-containing amine oxidases - recent research developments MAREK SEBELA, IVO FREBORT, MAREK PETRIVALSKY AND PAVEL PEC

1259

Subject Index

1301

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Xlll

CONTRIBUTORS

Miguel. E. AlonsoAmelot

Grupo de Quimica Ecologica, Departamento de Quimica, Universidad de Los Andes, Merida, Venezuela

Koichi Yoney Ama

Center for Research on Wild Plants, Universit)^ Utsunomiya, 321-8505, Japan

Teris A Van Beek

Laboratory of Organic Chemistry, Phytocheniical Section, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Ntherlands

Nestor M. Carballeira

Department of Chemistry, University of Puerto Rico, P.O. Box 23346, San Juan, Puerto Rico 00931-3346, Division of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, Louisiana 71209, USA

Robert H. Cichewicz

Utsunomiya

Per Claeson

Division of Pharmacognosy, Department of Pharmacy, Upssala University, Biomedical Centre, Box 579, SE-751 23 Uppsala, Sweden

Ubonvvan P. Claeson

Institute for Bioactive Natural Products, Uppsala Science Park, SE-751 83 Uppsala, Sweden

Simon L. Croft

London School of Hygiene and Tropical Medicine Department of Infectious and Tropical Diseases Keppel Street London, WCIE 7HT, United Kingdom

M. Valeria D'Auria

Dipartimento di Chimica Delle Sostanze Naturali, Via D. Montesano 49 80131 Napoli, Italy

Goverdina C.H. Derksen

Laboratory of Organic Chemistry, Phytochemical Section, Wageningen University, dreijenplein 8, 6703 HB Wageningen, The Netherlands

Stephen O. Duke

United States Department of Agriculture, Agricultural Research Service, Natural products Utilization Research Unit. P.O. Box 8048, University MS 38677-8048, USA

Laure Dussably

EPHE/INSERM, Laboratory of Cancer Immunotherapy and Unit 517, Faculty of Medicine, 7 Bd Jeanne d'Arc, 21033 Dijon Cedex, France

Antonio Evidente

Dipartimento di Scienze Chimico-Agrarie, Universita di Napoli "Fderico \V\ Via Universita 100, 80055 Portici, Italy

XIV

B.M. Fra",a

Institute de Productos Naturales y Agrobiologia, CSIC, Avda. Asti'ofisico F. Sanchez, 38206 La Laguna, Tenerife, Spain

F. Frapp ier

Laboratoire de Chimie des Substances Maturelles, ESA 8041 CNRS, Museum National d'Histoire Naturelle, 63 rue Buffon, 75231 Paris Cedex 05, France Department of Biochemistry, Faculty of Science, Palaci% Slechtitelu 11, 783 71 Olomouc, Czech Republic

Jose M. Prieto

Departament de FaiTnacologia, Facultat de Famiacia, Universitat de Valencia, Vicent Andres Estelles, 46100 Burjassot, Valencia, Spain

Alain Puein

Unite Mixte INRA/Universite de Bourgogne, BBCE-IPM Laboratory, INRA BV 1540, 17 rue Sully, 21034 Dijon Cedex, France

P. Rasoanaivo

Laboratoire de Phytochimie et de Pharmacologic Cellulaire et Parasitaire, Institut Malgache de Recherches Appliquees, B.P. 3833, 101-Antananarivo, Madagascar

M. Reina

institute de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico F. Sanchez, 38206 La Laguna, Tenerife, Spain

Antonio Rescigno

Cattedra di Chimica Biologica, Dipartimento di Scienze Mediche, Universita di Cagliari, Cittadella Universitaria, 09042 MonseiTato, Cagliari, Italy

Vichai Reutrakul

Department of Chemistry, Faculty of Science, Mahidol Universit)/, Bangkok 10400, Thailand

Agnes M. Rimando

United States Department of Agriculture, Agricultural Research Service, Natural products Utilization Research

XVll

Unit. P.O. Box 8048, University MS 38677-8048, USA J.A. Del Rio

Departamento de biolgia Vegetal (Unidad de Fisiologia Vegetal), Facultad de Biologia, Univesidad de Murcia, 30100, Espinardo (Murcia), Spain

Jose Luis Rios

Departanient de Farmacologia. Facultat de Farmacia, Universitat de Valencia, Vicent Andres Estelles, 46100 Burjassot, Valencia, Spain

Miranjan P. Sahu

Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Raod, Calcutta-700 032, India

Kevin K. Schrader

United States Department of Agriculture, Agricultural Research Service, Natural products Utilization Research Unit. P.O. Box 8048, University MS 38677-8048, USA

Marek Sebela

Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc. Czech Republic

Enrico Sunjust

Cattedra di Chimica Biologica, Dipartimento di Scienze Mediche, Universita di Cagliari, Cittadella Universitaria, 09042 Monserrato, Cagliari, Italy

Laszlo F. Szabo

Institute of Organic Chemistry, Semmelweis University, Hogyes u. 7. H-1092 Budapest, Hungary

Patoomratana Tuchinda

Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

Yasushi Uda

Department of Bioproductive Sciences, and Center for Research on Wild Plants, Utsunomiya University, Utsunomiya, 321-8505, Japan

David Wendehenne

Unite Mixte I>JRA/Universite de Bourgogne, BBCE-IPM Laboratory, INRA BV L540, 17 rue Sully, 21034 Dijon Cedex, France

Angela Zampella

Dipartimento di Chimica Delle Sostanze Naturali, Via D. Montesano 49 80131 Napoli, Italy

Franco Zollo

Dipartimento di Chimica Delle Sostanze Naturali, Via D. Montesano 49 80131 Napoli, Italy

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Bioactive Natural Products

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 26 © 2002 Elsevier Science B.V. All rights reserved.

TRITERPENOID SAPONINS FROM CARYOPHYLLACEAE FAMILY ZHONGHUA JIA"'^, KAZUO KOIKE", NIRANJAN P. SAHU^ AND TAMOTSU NIKAIDO' ^School of Pharmaceutical Sciences, Toho University, Miyama l-l-l, Funabashi, Chiba 275-8510, Japan. ^Indian Institute of Chemical Biology, 4 Raja S, C. Mullick Road, Calcutta-700 032, India ABSTRACT: Triterpenoid saponins from the Caryophyllaceae family are reviewed with special emphasis on recent developments in purification and structural elucidation aspects. Structural characterization using high field NMR techniques is examplified using saponarioside A, a triterpenoid saponin isolated from Saponaria officinalis and possessing nine sugar units in complex chains. A brief report on the biological activities of the saponins of Caryophyllaceae family is also included.

INTRODUCTION The family Caryophyllaceae is a group of mostly herbaceous flowering plants occurring mainly in temperate and warm-temperate regions of the Northern Hemisphere with its center of diversity in the Mediterranean region. The family consists of more than 2000 species with about 90 genera and is fairly easy to recognize because of its uniformity, most species having swollen nodes and opposite leaves. A salient feature of the plants of this family is their capacity to produce voluminous stable froth when shaken with water, indicating the presence of saponins. In fact, the name saponin was derived from the Caryophyllaceae plant Saponaria officinalis (soapwort), the root of which was used as soap since time immemorial {sapo—hdiXm for soap). Other well-known saponinbearing plants in the family are Gypsophila paniculata/muralis (baby's breath) and Acanthophyllum gypsophiloides (Turkestan soap root). Many plants of the family viz. Dianthus chinensis, Vaccaria segetalis, Silene species are well documented as herbal drugs in Asia, especially in traditional Chinese medicine. Saponaria officinalis and Gypsophila species have also been used as folk medicine in Europe. Besides, the family is the source of many ornamental plants, the most widely known species being Dianthus caryophyllus (carnation), which is cultivated around the world. Others in^Present address: Complex Carbohydrate Research Center, The University of Georgia, 220 Riverbend Road, Athens, Georgia 30602-4712, USA

elude Gypsophila, Saponaria, and Silene genera. The roots of several Gypsophila species (commercially known as Saponariae alba radix) are used for the production of a number of industrial products viz detergents and chemicals, film emulsion, and as ingredients of fire extinguishers. Saponins of this family are based on the P-amyrin type of triterpenoid aglycones (Fig. 1) with one (monodesmoside) or two (bisdesmoside) sugar chains. The oligosaccharide chains are mostly branched rather than linear, and possess as many as 9 to 10 monosaccharide units. Most of the saponins have sugars attached to the hydroxy group at C-3 and the carboxy group at C-17. The linkage of a glucuronic acid to C-3 and of a fucose unit (often acetylated) to C-28 of the aglycones is a common feature of the Caryophyllaceae family. Unlike most of the triterpenoid saponins found in other families, Caryophyllaceous saponins have the tendency to have more sugar constituents at C-17 than at C-3. The presence of only an acyloside sugar chain, i.e. one attached to the carboxy group at C-28 of the aglycone, is not uncommon. The last review on the Caryophyllaceae family was published almost two decades ago [1]. Since then, purification and structural elucidation techniques have changed enormously. Many of the earlier chemical studies have been reinvestigated and new findings published. The present review deals with the chemistry of the triterpenoid saponins of Caryophyllaceae family with special emphasis on recent developments in purification techniques and structural study aspects. GENERAL METHOD OF ISOLATION The defatted, air-dried plant material is extracted with methanol, either cold or hot, or with 50% aqueous methanol at ambient temperature. Fresh plant materials have to be processed immediately after collection to prevent fungal growth and enzymatic hydrolysis of the saponins. During extraction, care should be taken as saponins may undergo transformation, e.g. esterification of acidic saponins, hydrolysis of labile ester groups or transacylation, etc. Often the crude extract is suspended in water and partitioned using ethyl acetate and «-butanol successively. Most of the saponin constituents are found in the w-butanol solublefi-action.However, the low-polar saponin constituents may be present in the ethyl acetate part while the most highly polar saponins may be found in the aqueous layer, as in the case oi Saponaria officinalis [2]. Saponins are usually highly polar compounds occurring as complex mixtures, and their separation into individual components is a formidable and time-consuming task. The traditional purification method consists of repetitive chromatography on silica gel columns using chloroform-methanol-water

as eluent, and the method is being widely used even today to get rid of pigments and other non-saponin constituents. Such process may yield pure products in few cases; but usually it separates the crude saponin mixture into different fractions according to their polarity, final purification being achieved by high performance liquid chromatography (HPLC). Following is the method of purification for saponins which is usually adopted by various workers including us. The crude saponin mixture is passed over a column of Diaion HP-20 using water-methanol as eluent in various ratios (0, 10, 30, 50, 70 and 100% in methanol). Often the saponin fraction is obtained from the 70-100% methanol eluates. The saponin fractions thus obtained sometimes afforded pure compounds on repeated silica gel and ODS chromatographic purification. However, in most of the cases, fractions found to be homogeneous on TLC turned out to be mixtures on HPLC analysis. Final purification is usually done by HPLC over a reversedphase column (Cig or so-called ODS) with methanol-water or acetonitrilewater as eluent. Saponins of Caryophyllaceae family usually contain a free carboxyl group either in the aglycone part or in the monosaccharide chain (the so-called acidic saponins), and suppression of ion formation is necessary otherwise peak broadening may occur and separation becomes impossible. This can be achieved by adding a small amount of trifluoroacetic acid (TFA) or acetic acid to the solvent system. We usually use 60-75%) methanol containing 0.06%) TFA and the system works alright in most of the cases. However, care has to be taken while recovering the saponins from the acidic medium. Removal of the acid under reduced pressure and at a relatively lower temperature (2 Glc-p fragment could be explained by the changes in the corresponding torsion angles. For simple oligosaccharides, use of ^^C NMR and MS techniques in combination with some chemical reactions is good enough to establish the structure. However for branching, complex oligosaccharide chains, these methods cannot provide exact structure at the branching point, for the glycosylation shifts can only indicate that a substitution occurred but provides no information about the nature of the substituents. This problem can be overcome by examining the products from mild acidic (partial) hydrolysis or enzymatic degradation. Under such condition, the oligosaccharide chain will usually be cleaved at either of the branching points and lead to the formation of corresponding linear products. Comparison of the ^"^C NMR data of the degraded products with the original one immediately establishes the oligosaccharide structure. The anomeric configurations can also be deduced from Klyne's rule of molecular rotation [10] and ^H, ^^C NMR data.

Modern NMR approaches The advent of modern multidimensional NMR techniques based on high field magnet and versatile computer programs has greatly advanced the structural study of saponins. The structural characterization of saponins involves determining: (i) the structure of the sapogenin; (ii) the nature and number of the constitutent sugar units including the ring size and anomeric configuration; (iii) the interglycosidic linkages and the sequence including the location of the sugar chain attachment to the sapogenin, and (iv) the 3D structure. The following discussion mainly focuses on the structural study of the sugar moiety. The first step in the structural analysis of a saponin is to obtain the ID ^Hand ^^C-NMR spectra. Saponins are usually investigated as deuterium exchanged samples and the most commonly used solvent is pyridine-^is although use of DMS0-(i6 or methanol-(i4 is reported in the literature in few cases. The hydroxyl protons can be exchanged by adding several drops of D2O or deuteriated methanol. The ID ^H NMR spectrum displays only some recognizable signals, especially those for the anomeric protons at 4.5-6.4 ppm, methyl doublets of 6-deoxy sugar units at 1.3-1.5 (e.g. H-6 of rhamnose), and the methyl groups of the aglycones. The ^^C NMR spectra give a better dispersion over a 200 ppm range; additionally the protonation levels are deducible fi-om a DEPT experiment [11]. The sugar anomeric carbons can be located at 6 90-110 and the non-anomeric carbons at 8 60-83 ppm. Most resonances of the aglycone part appear below 6 55 in the higher field region, the oxo-substituted carbon signals appearing in the same range as the non-anomeric sugar carbons. The signal for C-3 is usually found at about 5 84 ppm and that for C-28 at about 5 176 for the acylosides. For the (3amyrin type sapogenins, the signals for the A^^ double bonds are typically observed at ca 5 122 (C-12) and 144 (C-13). ID ^H and ^^C NMR spectra usually give partial structural information only, such as the nature of the sapogenin and the number (by counting the anomeric protons and carbons) of sugars in the molecule. The identification of constituent sugars The identity of sugars and the sugar sequence of the oligosaccharide can be determined by a combination of COSY [12], HOHAHA [13, 14] or TOCSY [15], HETCOR [16] or HMQC [17], HMBC [18] and NOESY [19] techniques. The prerequisite for the identification of the component sugars is the unambiguous assignment of the ^H signals. The through-bond connectivties of the ring protons within each sugar residue can be established by COSY

10

and HOHAHA experiments. Since the anomeric protons of each residue resonate in a characteristic region well isolated from that of the nonanomeric protons, they are usually the starting points for analyzing the spectra. In favorable cases (i.e. less than four or five sugar units), a COSY, preferably DQF-COSY [20] spectrum, which reveals the connectivities for each pair of vicinal and geminal protons, may map out all the spin systems. For saponins with more complex sugar moiety, a HOHAHA experiment can be of further help in the assignment. The individual spin-system can be discerned from the sub-spectra corresponding to the anomeric or the methyl groups (for the deoxy sugars) in the HOHAHA spectrum. Fig. (2) is the HOHAHA spectrum of saponarioside C isolated from Saponaria officinalis [21]. As seen from the example, HOHAHA has the potential of reducing the composite oligosaccharide ^H-NMR spectrum into a subset of spectra. Sometimes, several HOHAHA experiments with different mixing times may be necessary to trace the spin systems from the anomeric to the terminal proton step by step. However, the distribution of magnetization within the spin system can be impeded by small coupling, such as typically found between H-4 and H-5 in a galactosyl residue (system G'" in Fig. 2), though it has been reported that ID-HOHAHA has the potential to obtain correlation past this threshold [22].

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-i-

^»

Fig. (2). The HOHAHA spectrum (!„, = 150 ms) for the sugar moiety of saponarioside C from Saponaria officinalis showing the sub-spectra corresponding to each anomeric proton. Note that for the glucose (G, G', G") and xylose (X) units, the correlations were observed from the anomeric protons till terminals. However, the correlation stopped at H-4 of the galactose (G"') moiety due to the small coupling between H-4 and H-5. Spectrum measured at 500 MHz in pyridine-tis.

11

Additionally, the intra-residue NOEs from 2D-N0ESY or ROESY experiments could furnish further information concerning the proton assignment. In general, 1,3-diaxial and 1,2-eq- ax proton pairs in pyranosyl rings produce intra NOE cross peaks, viz. for P-galactopyranosyl residue NOEs are observed between H-1 and H-3 (and H-5), while only the H-1 to H-2 intraresidue NOE is noticed in a-galacto- pyranosides; these greatly simplify the mapping of the spin systems. After all the spin systems are mapped out and the number of different spin systems established by the above methods, one can go ahead to identify the sugars as well as their anomeric stereochemistry. In pyranosides, the six-membered ring generally forms a stable conformation providing a classification of protons as axial or equatorial. Therefore, the coupling patterns are characteristic of the stereochemistry for this type of sugars. The VHH coupling constants are large (> 6 Hz) for the anti-periplanar orientation of the vicinal ring protons (Z H-C-C-H « 180°), and small (< 5 Hz) for the gauche orientation (Z H-C-C-H « 60°). Several methods have been proposed to measure the proton coupling constants [23, 24]. For conformational study, the coupling constant should have a high accuracy. Such accuracy is not required for the identification of the sugar residues. But it is necessary to distinguish between small and large J values. In order to prevent misassignment, it is recommended that the sugar composition be confirmed by chemical analysis, and the molecular weight confirmed by FAB or MALDI mass spectroscopy. Sugar identity and stereochemistry can also be inferred from ^"^C NMR data. If the *H spectrum be completely assigned, a HETCOR (^^C detected) or HMQC/HSQC (^H detected) experiment would be enough to establish the ^•^C signal identity. Sugars can be identified by comparing their ^^C data with those of the standard methyl glycosides or the published data of saponins. As glycosidation tends to shift the a-carbon to lower field and P-carbon to somewhat higher field positions, careful attention should be paid to the determination of the ring size and anomeric configuration on the basis of chemical shifts as these may lead to ambiguity. In addition, a reliable criterion for determining the anomeric configuration of D-sugars in the "^Ci pyranose form is from one- bond ^^C-^H couplings (^JCH) [25]. Determination of the inter-glycosidic linkage and sugar sequences Once each sugar residue has been identified and its anomeric configuration determined using a combination of the above-mentioned techniques, all that is required is to identify the sugar sequence and the inter-glycosidic linkage. Proton-proton scalar couplings that are four bonds apart (VHH) are usually too small to observe. Therefore, it is necessary to make use of either homo-

12

nuclear dipolar coupling (NOE measurements), or the long-range heteronuclear coupling constant VCH across the glycosidic linkages. Once the ^H assignment is complete within each individual sugar spin system, measurement of NOE enhancement provides a very powerful means for determining linkage and sequence. The presence of an inter-glycosidic NOE from the anomeric proton of a particular sugar residue to a proton of the other sugar residue or non-sugar residue (sapogenin) defines the glycosidic linkage between the two residues. NOE connectivities are most often observed between the anomeric proton and the proton connected to the carbon atom of the linkage. 1

II glc ^^^^--^^^

r 1

B.2

93'

^--

6.0

5.8

5.6

H.12

S'c

xyl

glc

^—U__AJI_

5.4

5.2

5-0

H-3Q G'"-2

/f"^r'S^%o

G-3

" ^ ^

G.5

0 G"-6

G-3 Q G'-3 0 x-3 G'.S

y

^-

^ ^G".S

X.5 ©

1

if

1

HOr^" p-D-Glc'(G') OH

H GOGH

H ^ .H ^ SC i ( >L^ S HO P-D-Xyl (X)

H^;. Q^Y^Qj/^OH's^Q

P-D-GIc (G)

' OH a-D-Gal (G'")

P-D-GIc" (G")

Fig. (3). Phase-sensitive NOESY spectrum of saponarioside C from Saponaria officinalis (the mixing time was 600 msec). Note that both the inter-residue (bold labeled) and intra-residue NOEs were observed. For the a-D-galactose moiety, the only intra-residue NOE noted is between Hi and H2. For all other sugar units, the intra-residue NOEs between H1/H3 and H1/H5 are observed.

13 This has been found to be of wide appHcabihty for the structure determination of the naturally occurring glycosides. An example of a NOESY spectrum is given in the section on the structural study of saponarioside C isolated from Saponaria officinalis (Fig. 3) [21]. Both the inter-residue NOEs and the intra-residue NOEs were observed. The conventional NOEs in the laboratory frame can be positive or negative and pass through zero when cooXc, the product of spectrometer angular frequency and molecular rotational correlation time (which depends on the size and shape of the molecules and on the viscosity of the rotating medium) is approximately equal to unity. Such a problem, which is typical of middlesized molecules like saponins, can be solved by performing the experiment in the rotating frame, the so-called ROESY [26, 27]. In NOE-based linkage and sequence analysis, it is necessary to be certain that all resonances are unequivocally assigned. Even then, the observed NOEs may be inconclusive, if the chemical shift for the aglyconic proton located at the glycosylated carbon coincides with the chemical shifts for protons of other sugar residues. This situation is encountered very often with complex glycosides. Another serious drawback is that the interglycosidic NOE between the anomeric proton (H-1) and the proton on the attachment site of the sugar or the aglycone is not necessarily the only and the largest inter-residue NOE effect. Therefore, NOEs should not be used as the sole criterion for establishing the position of a glycosidic linkage, especially in the branching center. An example is the saponin mimusopin isolated from the seeds of Mimusops elengi [28]. The structure was established by a combination of COSY, HOHAHA, HETCOR, HMBC and phase-sensitive NOESY. The preferred conformation calculated from molecular modeling study for the sugar chain at C-28 of the sapogenin is shown in Fig. (4). The NOE relationship from NMR is indicated by arrows. Besides the NOEs between protons across the glycosidic bonds (R1/A2, RVX3, X1/R4, R'VRs), NOEs between R1/A3, R"i/R2, Xi/R"2 as well as between A1/R5 were observed. Before looking at the molecular modeling result, we were not able to make the inter-glycosidic assignment by using NOE data only. By careftilly examining the preferred conformation, we found that the arabinose adopts a ^C^ conformation placing H-3 in the equatorial position (vicinal to the linkage site). NOEs from such environment (another example is H-4 in 3-glycosylated galactose residue) have been reported and well discussed before [29, 30]. Such protons may exhibit NOEs of approximately the same magnitude as the rest and determination of the linkage might appear equivocal. The NOEs from the pair R"i/R2 is of the same origin. R2 is in an equatorial position vicinal to the linkage site at C-3. However, the observed NOEs between Xi/R"2 and A1/R5 appear somewhat unusual. Molecular modeling results showed that the distance between these two pairs are 2.64 and 2.41 A, re-

14 spectively, within the effective NOE distance. This means that substitutions at both C-3 and C-4 cause the branching center to be very crowded and distort the torsion angles. In fact, anomaly is also found in the ^"^C NMR data of the disubstituted rhamnose (R). Molecular modeling results showed that the corresponding dihedral angles were distorted compared to another saponin, isolated from the same source but lacking the substitution at C-3 of rhamnose (R) [28]. From the above discussion, we can see that using only the observed inter-residue NOEs may lead to wrong conclusions about the primary structure of the sugar chain.

The Optimized Conformation of Mimusopin from Molecular Modeling Study

2.29 A

'R"ha NOEs observed between the fragments Rhal~2Ara and R"hal-3 Rha of Mimusopin Fig. (4). The calculated preferred conformation (part) of mimusopin and NOEs observed from phasesensitive NOESY.

15 Another, more effective way to determine the sugar Hnkage and sequence is to detect the long-range VCH coupling across the glycosidic bond. The most practical technique is HMBC (heteronuclear multibond correlation). An HMBC experiment can furnish inter-glycosidic multi-bond correlation between the anomeric proton and the aglycone carbon, and thus serve to identify the linkage. Besides, HMBC also furnishes intra-residue multi-bond correlations, which are very valuable for confirming the H and C assignments. As with proton-proton vicinal coupling constants, the three-bond carbon-proton couplings as observed in HMBC also conform to the Karplus relationship, the maximum of VCH being usually observed at a dihedral angle of 180° and the minimum near 90°. Therefore, HMBC also furnishes information concerning anomeric configurations and the overall conformations. For example, for a-L-rhamnose possessing an equatorial anomeric proton, intra-residue multi-bond correlations between the anomeric proton and C-3, C-5 are observed (the dihedral angles between H-1 and C-3, H-1 and C-5 are about 180°). The same is true for the a-D-galactose unit of saponarioside C found in the plant Saponaria officinalis [21]. 3D-Structure by NMR and Molecular Modeling studies It is generally believed that biological activity is determined by the 3D structure (conformation) of the compounds. For saponins, the most flexible and biologically important part is the sugar unit. Thus, conformational analysis of the sugar part is crucial for the elucidation of the biological action of these kinds of compounds. The conformational properties of sugars in solution have been largely determined by ^H NMR NOE techniques. It originates in dipolar cross relaxation between protons, which is a function of distances and molecular motions. Because of the inverse sixth power dependence of the NOEs on the interproton distances (r), a highly sensitive conformational "ruler" is available when r is small (< 5A). The most important NOE contacts in sugars are those between protons across the glycosidic bonds. Sometimes other through-space contacts, which are very important in probing local conformations, are also observed. The latter situation is not uncommon in a branching center with attachment of two or more sugars to the same aglycone. Another probe for the three-dimensional structure is the ^JCH value across the glycosidic bonds [31, 32]. This value is an indication of the dihedral angle between the sugars. Thus the local conformation can be determined from the VcH magnitudes. Additionally, ^"^C chemical shifts of the carbons involved in the interglycosidic linkages can also be used as an indication con-

16 ceming the local conformation. But the specific relationship between conformation and glycosidation shifts has not been fully understood yet [33]. In recent years, with the development in computational methods, molecular mechanics (MM) and molecular dynamics (MD) calculations have provided a powerful tool for evaluating intermolecular distances and dihedral angles for comparing with the results obtained from NOE data and VHH measurements respectively. Analyzing 2D NMR data using the energyminimized structures usually leads to more reliable assignments [28]. Fig. (5) is a summary of the general guideline of the modem NMR techniques employed in the structural study of triterpenoid saponins in our laboratory.

'HNMR

13

CNMR

COSY, HOHAHA: Mapping out all of the proton J-coupled spin system of each sugar units

DEPT: assign ^^C multiplicity HETCOR/HMQC; assign the C signals

NOE/ROESY (through space interactions)

HMBC (long range heteronuclear ^H,^-decouplings)

Assign the inter-residue connectivites using NOE and HMBC

Establish the stereochemistry from NOEs and molecular modeling study

Fig. (5). Structure elucidation strategy of saponins from 2D-NMR approaches.

17 TRITERPENOID SAPONINS FROM SAPONARIA OFFICINALIS Saponaria officinalis L., popularly known as fuller's herb or soapwort, is native to Europe and western to central Asia, and is presently cultivated in different parts of the world for its medicinal properties. This plant was well known for its detergent property and was used as soap in ancient times. In folk medicine it is used for various skin diseases, rheumatic disorders and as an expectorant for bronchitis [35]. In Europe, different parts of the plant, viz. the roots, rhizomes and shoots are used for the preparation of herbal medicines [36]. It has long been known that the bioactivity of the plant is due to the triterpenoid saponin constituents. Earlier chemical investigation during 70s led to the isolation of four saponins; the structures of two major ones, saponasides A and D, were established partially, with the latter containing up to ten sugar units (Fig. 6) [37, 38]. The structures were elucidated by various chemical methods such as permethylation, hydrolysis, periodate oxidation, partial hydrolysis and LiAlH4 reduction. During the same period, one more saponin designated saponaroside was isolated from the same source and its structure was established too (Fig. 6) [39]. Since then, no further reports are available on the chemical investigation on the saponin constituents of the plant.

'COOR2 R,0' CHO

Saponaside A Saponaside D

D-GluA

D-Glc-(l-6) I D-Glc-( 1-3)1 D-Glc

D-Gal-(l-2) I L-Ara-(l-4) I D.Xyl-(l-3) I ^ , ^ L-Rha-( 1 -4) I D-GluA

Saponaroside

D-Glc-(1-2)| D-Gal-(1-4)| D-XyKl-S) I L-Rha-( 1 -4) | D-Fuc

COOH

COOH O H ^ H

Fig. (6). Saponasides A, D and saponaroside from Saponaria officinalis.

18 Chemical investigations on the sapogenin component have been carried out as early as 1910s and gypsogenin was proposed as the sapogenin [40]. Some confusion concerning the structures of the sapogenins lasted until the 1980s, when Kubota et al. could isolate only quillaic acid and gypsogenic acid as the sapogenin constituents from S. officinalis [41]; very recently Henry et al. confirmed the presence of quillaic acid in the rhizomes using ^^C NMR techniques [42]. The medicinal and commercial importance of the plant coupled with our continuing interest in the chemistry of triterpenoid saponins prompted us to reinvestigate the saponin constituents of S. officinalis [2, 6, 21]. The MeOH extract of the freshly collected whole plant of S. officinalis was suspended in water and partitioned successively with EtOAc and nBuOH. The aqueous part, on chromatography over Diaion HP-20 followed by repeated MPLC and HPLC purification afforded two major triterpenoid saponins, saponariosides A and B. Similarly, the «-BuOH soluble fraction afforded six triterpenoid saponins designated as saponariosides C-H. Investigation of the plant material collected from different geographical locations led to the isolation of previously reported saponariosides C, E, F and G along with five more new saponins, saponariosides I-M (Fig. 7), from which two new sapogenins have been characterized as VIII and XII (Fig. 1). However, saponarioside D, the major constituent of the «-BuOH soluble fraction as reported previously by us [21], could not be detected. Saponariosides A and B, the major constituents isolated from the aqueous part, are highly soluble in water and water-MeOH mixture but have poor solubility both in MeOH and EtOH. They are bisdesmosides of quillaic acid, glucuronic acid being linked to C-3 of the genin. The other sugar constituents are galactose, xylose, and three 6-deoxy hexoses (rhamnose, fucose, and quinovose). It is of interest to note that both the saponins possessed one acetyl function appended to the quinovose residue at C-4. The saponins isolated from the «-BuOH soluble fraction have aglycones other than quillaic acid, and displayed a distinct and characteristic color (sky blue) on silica gel TLC plates when sprayed with 5% H2SO4 and heated. The aglycones are either gypsogenic acid or 16a-hydroxy gypsogenic acid, with a free carboxyl group at the a-face of C-4. Saponarioside C has a somewhat different substitution pattern and possesses an unprecedented a-D-galactose moiety linked to C-6 of the inner glucose. The minor compound saponarioside H, with only one glucose unit at C-28, is presumed to be the biosynthetic intermediate for the other saponins. Due to the acidic nature of these saponins, a small amount of TFA (0.05% v/v) had to be added to the mobile phase for HPLC purification of the saponins.

19

Saponariosides

GIcA (I)

HO' " ^ ^ ^ ^ " HO Xyl(D) HO '

o OH

HO

Rha (A)

Gal (C) A R = Xyl(H) B R=H HO HO Xyl (F) HOfOH

HO OH

,X:^°'

HO HO

p.Gic(G)

^^^^^^s:;^z:oH/0

OH

COOH

OH

P-Glc (G")

a-Gal (G"')

P-Xyl C

R=H

I

R = OH HCK-OH ^ ^ n ^ O H

"N/

f^^^^^^Y^^

^^

/

/

G' "^OH

^ R2O'

G tOOH R2

\

RaO-T--^ ^ O H O ^ ^ O ^ ^ O H

R3

D

H

Xyl

Glc (G'")

E

H

Glc

Glc

F

OH

Xyl

Glc

G

OH

Xyl

H

20

Minor saponin

OH OH OH

COOH

COOR

' " H ^ ^ : ^

COOH

^^^^^

HO/fOH _ OH O

COOR

HCV^li^r^ HO^^'^OH

COOH

R=

Ho:

^

G

OH .OH HOT^---^^^'' \2-»0,^^^^/^0H G"

WO'^ST^^ir^

' COOH

OH

OH OH

Fig. (7). Triterpenoid saponins from Saponaria officinalis.

Saponarioside A is a white, bitter-tasting powder that induces sneezing. It produces a stable froth when shaken with water. The molecular formula C82H128O45 was determined from its negative ion HRFAB-MS ([M-H]" peak at m/z 1831.7649) in combination with ^^C DEPT NMR data. Of the 82 carbons, 30 were assigned to the aglycone part, 50 to the oligosaccharide moiety and the remaining two to an acetoxy group. The IR spectrum showed absorptions at 3406 cm"^ (-0H) and 1728 cm"^ (ester carbonyl). The six methyl

21

carbon signals at 5 11.1, 15.8, 17.4, 24.5, 27.0 and 33.2 ppm, and the two sp^ carbon signals at 5 122.2 (d) and 144.4 (s), coupled with the information from ^H NMR (six methyl proton singlets and a broad triplet for a vinyl proton at 5 5.54), indicated that the aglycone had an olean-12-ene skeleton. After an extensive 2D-NMR analysis, the aglycone was identified as quillaic acid [43, 44]. The downfield shift of C-3 signal (6 84.4) and upfield shift of C-28 signal (5 175.9) compared to those of the aglycone indicated that it was a bisdesmoside. The ^H and ^"^C NMR spectra displayed signals for nine anomeric sugar protons [8 4.89 d (J= 7.3 Hz), 4.99 d (2H, J = 7.6 Hz), 5.13 d (2H, y = 7.1 Hz), 5.32 d (J= 7.7 Hz), 5.55 d (J= 7.3 Hz), 5.93 d (J= 8.2 Hz), 6.29 s] and carbons (5 94.4, 100.9, 103.8, 104.2, 104.9, 105.5, 105.8, 106.2, 106.9), Fig. (8). Alkaline hydrolysis furnished a prosapogenin, identified as quillaic acid 3-0-P-D-galactopyranosyI-(l->2)[P-D-xylopyranosyl(l->3)]-P-D-glucurono-pyranoside from its spectral data. Acid hydrolysis afforded quillaic acid, and the monosaccharide components were identified as fucose, galactose, xylose, quinovose and rhamnose in the ratio 1:1:4:1:1 from GLC analysis of their TMS derivatives. The other monosaccharide was identified as glucuronic acid by co-TLC analysis (both saponarioside A and the authentic sugar were applied to the TLC plate and then hydrolyzed under HCl vapor at 65^C for 1 h; developing solvent: CHCb-MeOH-HsO, 10:5:1). From the above evidences, it was concluded that saponarioside A was a bisdesmosidic triterpenoid glycoside with glucuronic acid, galactose and xylose linked to the C-3 position of the aglycone, the other six monosaccharides being linked to C-28 of the aglycone through an ester bond. The sequence of the oligosaccharide chains was established using the NMR protocol as discussed in the previous section. In order to facilitate proton assignments, the nine monosaccharides were labeled by the letters A to I. The resonance position for anomeric protons corresponding to E and F as well as G and H appeared overlapped, thus making the proton assignment difficult. The individual spin-systems were discerned from the subspectra corresponding to the anomeric protons or the methyl groups (for the deoxy sugars) in the HOHAHA experiment (Xm = 150 ms) (Fig. 9). Interpretation of the COSY and 2D-H0HAHA spectra revealed the presence of nine monosaccharide units. As shown in the HOHAHA spectrum, three monosaccharide units (A, B and G) were 6-deoxy sugars. Spin-system of A displayed two broad singlets (H1: 5, 6.29 s and H-2: 4.68 s), a double doublet (H-3: 5 4.60, dd, 8.5, 3.2 Hz), two overlapped signals for H-4 and H-5 protons and a terminal doublet for a methyl group (6 1.53, d, 5.8 Hz) (Fig 9). Furthermore, the large ^CH (171 Hz) and strong three-bond HMBC correlations from the anomeric proton to C-3 and C-5 indicated the equatorial orientation of the anomeric proton, thus suggesting a a configuration.

22

-T—I—I—i—r

V W V * l i n - ^ V V'rf^i'Ww^^i^vn^'N ' \V^)^^»^»»>M[g.v^lV»^««»^WWrt^M»>•^^>V^^

""'|""""'l

210

200

"'I'

I'"

I'"

190

180

170

|"M.iii.|

160

150

|.

140

|M.mM.|....nM.|

130

120

110

'^/•f

|.nmni|mM.iM|immM|rimiiM|Mmim|mmm|immM|MiMim|iiiiiiiM|.M

100

Fig. (8). The ^H and ^^C spectra of saponarioside A.

90

BO

70

50

50

40

30

20

10

23 E(Xyl') G(Qui) F(Xyl")-H(Xyl"')"••*^'»\7G4(0«I)/ I (GJcA)

I A (Rham) B(FllC)

6.0

IM2(agl)

CU(Gan (GJ

5.8

^ 4

%

%2

F5

t$

1^3

3&4"

8

«"^i3

A

* ¥.

/ ^ R R2O

^^^^^7^

5^^^^^

HO R5'

R4O

HO

Rha *Note that the absolute configuration of the arabinoses was given as D by the authors.

Fig. (24). Triterpenoid saponins from Gypsophila paniculata and G. arrostii.

In early 1990s, Frechet et al. reported the isolation and structure elucidation of four new triterpenoid saponins from the roots of G. paniculata and G. arrostii (Fig. 24) [76], the roots being imported from Hebei, P. R. China. The

40

saponins were purified by HPLC on an RP-18 column and the authors used modern spectroscopic techniques (a combination of homo- and heteronuclear 2D NMR plus FAB-MS) to elucidate the structures without going through chemical degradation. This seems to be a pioneering work in the application of modern NMR techniques for establishing the structures of complex saponin molecules. As can be seen from Fig. (24), none of the compounds was identified as gypsoside and it is presumed that gypsoside is absent in the roots of G. paniculata and G. arrostii. In 1992, Kim et al. reported the isolation and structure elucidation of two major saponins, MS-1 and MS-2 from commercial Merck saponin, a crude saponin fraction from G. paniculata. Fig. (25) [77]. The separation of the saponins was achieved by a combination of normal silica gel column, reversed-phase (C-8) column, and Sephadex LH-20 column chromatography. The structures were established by the hydrothermal degradation method (hydrothermolysis) in combination with methylation studies and ID ^H and ^"^C NMR techniques. For hydrothermolysis, the compound was heated at lOO^C or at a higher temperature for 10-30 h, when the complex oligosaccharide chain was dissected into smaller fragments (Fig. 25). The technique is helpful for 3,28-0-bidesmosides as the ester glycosidic bond is selectively cleaved under such condition. The method is somewhat similar to traditional methods described earlier and needs a large amount of sample.

HO o^^^^^^^^ HO HO^-^'^^H

^ O"^

MS-1: R=H MS-2: R=OH

ii-x:^

HO' HO

OH Qui

Xyl

Xyl

O) CHy^^llN^U CH

HjO 100°C, 30h

H3cz^: ^^-ST;;^

HHO ^ O - '^ X ^ / OH

^^

0 CH3/O. \ f < ^ O W HO

" ^ HO' „"^^^^^-^^ OH

HO^ ^ ^ - ^ ur>^ OH HO

HO

HO

0rH3/O

"?.o 24 h 140*^0, H3C HO HO

HO ^ HO

Fig. (25). The hydrothermolysis of MS-1 and 2 from Merck saponin.

^^^ OH

41 In 1995, Liu et al. reported the isolation of three new saponins from the roots of G. oldhamiana [78], which has been used as a substitute for the wellknown traditional Chinese medicinal herb Sterllaria dichotoma var. lanceolata in the treatment of fever, consumptive disease, and infantile malnutrition. The most polar saponin was a bisdesmoside and its structure was established as shown in Fig. (26). The other two saponins were monodesmosides derived from C-28 methyl esters of quillaic acid and gypsogenin and having identical sugar moieties at C-3.

Rha HO'

HOCH3 /0>

HOHO' X > ^

HO

"OH

Glc

Fig. (26). Saponin from Gypsophila oldhamiana.

Very recently. Delay et al. reported the isolation and characterization of two highly hemolytic saponins from the commercial Merck saponin (roots of G. paniculatd) (Fig. 27) [24]. The two compounds (SAPO 50 and SAPO 30), which possessed 25% and 1% of the total hemolytic activity of the crude Merck saponin extract, were purified by dialysis, reverse phase C-18 column chromatography, and HPLC with monitoring of hemolytic activity. The structures were determined by high-field gradient-enhanced NMR methods (600 MHz for proton and 125 MHz for carbon) including DQF-COSY, ID and 2D z-TOCSY, ID-NOE, ge-PEP-HSQC, 2D-HSQC-T0CSY, and geHMBC experiments. The proton and carbon chemical shifts of each sugar residue were assigned by a combination of DQF-COSY, z-TOCSY and 2DHSQC-TOCSY. The authors made extensive use of the HSQC-TOCSY technique, which has the potential to locate all the proton and carbon resonances within each sugar residue. When recorded with a small mixing time (16 ms) the HSQC-TOCSY displayed only direct and vicinal correlations as in the TOCSY/HOHAHA experiments; the connectivities disappeared when the VHH value was small (e.g. between H4 and H5 of galactose). But complete proton

42

and carbon assignments could only be made by using a longer mixing time (80 ms). Both compounds had the same aglycone, gypsogenin, with eight and seven monosaccharide units in SAPO 50 and SAPO 30, respectively. The latter had the same structure as G4 described by Frechet et ai. Fig. (24) [76]. It may be mentioned that SAPO 50 had similar oligosaccharide moiety as found in saponarioside A isolated from the whole plant of Saponaria officinalis [2]. The only difference is that the quinovose unit has one more xylose unit at C-3 and an acetoxy group at C-4 in saponarioside A.

Gal HO

H OQ.:^ -X/^H OH

•X/^

HO HO

HO

Rha

Xyl

Xyl

Fig. (27). Triterpenoid saponin from commercial Merck saponin (roots of Gypsophila paniculata).

Apart from the above-mentioned complex bisdesmosides, saponins with simple sugar structures were also reported from this genus. Elgamal et al reported the isolation of seven new saponins (Fig. 28) with quillaic and gypsogenic acids as aglycones from the whole plant of G. capillaris grown in the eastern part of Egyptian desert [79, 80]. The sugar moieties contained 2 to 5 units of glucoses and/or galactoses. The structures were determined by ID and 2D-NMR study. The ^H and ^"^C data for both the glucoses and galactoses presented in the paper were almost identical. The authors had mentioned that it is usually not possible to distinguish glucose and galactose from the ^H and ^^C chemical shifts. They had identified the monosaccharides from the change in shape and multiplicity of the H-3 signals observed on irradiation of the respective H-1 signals in the NOB difference experiment.

43

COOR,

HO' CHO

COOR2

Aglycone

Glc-(l-6)-Glc

OH

Glc-(l-2) I Gal-(1-6) Glc

OH

Glc-(l-3)-Glc-(l-2)| OH

Gal-(l-6) |2)-[P-D-G1C-(1-^6)]-P-D-G1C at C-28 of the aglycones.

HO;r%H D-Glc

D-Glc Ri

R2

1

H

CHO

2

SOs"

CHO

^

H

CH20S03-

Fig. (29). Triterpenoid saponins from Gypsophila bermejoi.

Gypsophila saponins have been shown to possess hypocholesterolemic effects in rat [84]. The serum cholesterol levels were significantly lower in saponin-fed rats than in control. In another study, Gypsophila saponins were tested in young rats and the results showed that Gypsophila saponins may reduce Fe absorption and have an adverse effect on the Fe status in human and monogastric animals. However, Gypsophila saponins had no effect on Zn absorption [85, 86]. Another report showed that the purified saponins from G. capitata were responsible for the hypocholesterolemic effects in rabbits [87].

45 TRITERPENOID SAPONINS FROM SILENE GENUS The Silene genus consists of about 400 species and shows an Eurasic distribution. Plants of this genus are annual, biennial or perennial herbs or small shrubs of varied habitats. While some plants are omamental, utility of several species in folk medicines have been well documented. For example, the roots of 5. jenisseensis found its name in traditional Chinese medicine as one of the substitutes for the Chinese herbal medicine Yin-Chai-Hu (the roots of Stellaria dichotoma var. lanceolata Bge.). Chemical investigations on this species have been limited. In 1965, Tegisbaev et al reported a saponin named silenoside from the roots of S. latifolia [88], but only the aglycone (gypsogenin) and the sugar components (D-GluA, D-Glc, D-Fuc, L-Rha, D-Xyl, L-Ara) could be identified. Later, Gan et al identified quillaic acid from the acid hydrolyzate of the saponins from S. jenisseensis using NMR and MS techniques [89]. In early 1990s, Karawya et al reported the identification of the sapogenins quillaic acid and oleanolic acid from S. succulenta [90]. In another report from the same group, two new 28-nortriterpenoids, villosagenins I and II, were isolated from S. villosa after acid hydrolysis of the saponin fraction (Fig. 30) [91] and their structures were established by ID and 2D-NMR techniques.

I Villosagenin I J

CHO

CHO

Fig. (30). Triterpenoid sapogenins from the acid hydrolyzate of Silene villosa.

Several reports on full structure determination of saponins from Silene species have appeared since 1990s. Tan et al. reported the isolation and characterization of rubicunosides A-D from S. rubicunda (Fig. 31) [92-94]. Quillaic acid was identified as the sapogenin. The structure elucidations of the saponins were based on partial acidic hydrolysis, enzymatic cleavage (using glycyrrhizinic acid hydrolase), FAB-MS and ID NMR analysis. All the four saponins contained either one or two acetoxy groups in the fucose or in the quinovose moieties of the oligosaccharide chain attached to C-28 of the aglycone. The structures of the oligosaccharide moieties were determined by comparing

46 13/

the C NMR data of the partially hydrolyzed or enzymatically cleaved products with the original saponins. However, chemical and enzymatic degradation did not afford any linear products from cleavage at either C-2 or C-4 of the fucose, i.e. the branching centers. Thus there was lack of direct evidence in supporting the substitution pattern since the C glycosylation shifts can only indicate the presence of substitution but provide no information about the nature of the substituents.

Ara

HOJ ^

1

HO

HO

Rha

"K,o^^ Xyl OH Ri

R2

R3

Rubicunoside A

CH3CO

AcO-2Qui-

Xyl

Rubicunoside B

CH3C0

AcO-2Qui-

Xyl

Rubicunoside C

CH3C0

GlcMQui-

Xyl

Rubicunoside D

H

AcO-2Qui-

H

Fig. (31). Rubicunosides A-D from Silene ruhicunda.

Lacaille-Dubois et al, [95, 96] reported a series of saponins from two Silene species, S. jenisseensis and S. fortunei collected from Hebei province, P. R. China. The roots of both plants find their place in traditional Chinese medicine as a substitute for the well-known Chinese herbal medicine YinChai-Hu (roots oiStellaria dichotoma var. lanceolata), used for the treatment of various types of fevers. From the roots S. jenisseensis, four stereoisomeric acylated triterpenoid saponins, jenisseensosides A-D (Fig. 32), were isolated and their structures were established both by chemical strategies and spectroscopic methods (COSY, HMQC and HMBC). The saponins were obtained as two inseparable pairs with the acyl groups attached to C-4 of the fucose moiety [existing as geometrical isomers {trans- or c/i'-p-methoxycinnamoyl)]. Jenisseensosides A and B showed weak inhibitory effect in in vitro cyclooxygenase inhibition assay and jenisseensosides C and D exhibited a significant enhancement of the granulocyte phagocytosis in vitro.

47

Jenisseensosides

RiI Glc

Glc

H

R-

-C-C=C-\ >-OMe H ^—^ O H H /=^ - C - C = C - ^ j>-OMe OH /=x -C-C=C-\ >-OMe H — O H H

HO

^=\

H

Gal

Fig. (32). Jenisseensosides A-D from Silene jenisseensis.

From the roots of S. fortunei din octasaccharide glycoside of quillaic acid (Fig. 33) has been isolated along with two other reported earlier, jenisseensosides C and D [97]. The structure was established by chemical degradation, FAB-MS, and 2D-NMR analysis including DQF-COSY, HMQC and HMBC experiments. It has an acetoxy group attached to C-4 of the fucose moiety rather than a cinnamoyl group as in case of other jenisseensosides. In in vitro bioassay, this saponin showed a significant enhancement of granulocyte phagocytosis.

> ; ^ : : ^ ^ ! ^ « ^ ^ > ^ r Fuc

Oal

HO

O

HO'X/'WI ^'"

HO Rha

HO Ara HO-

Fig. (33). Triterpenoid saponin from Silene fortunei

Quite recently, Glensk et al. reported the isolation of three new saponins, silenosides A-C (Fig. 34) from the roots of iS. vulgaris (syn. S. inflata) [98], a perennial herb of Europe used in folk medicine for the treatment of anemia. The structures of the silenosides were established both by chemical and spec-

48 troscopic evidences. The molar ratios of the monosaccharides were determined by GC-MS analysis of TMS derivatives of the methanolysis products. The absolute configurations w^ere determined by GC analysis of the respective trimethylsilylated methyl 2-(polyhydroxyalkyl)-thiazolidine-4(i?)-carboxylate derivatives. The glycosidic and inter-glycosidic linkages were established from ESI-MS, 2D NMR (COSY, HMQC and HMBC) as well as GC-MS analysis of the permethylated products.

Silenosides A

R, H

R,

R3

R.

H

H

Xyl

B

OH

H

Glc

H

C

OH

Gal

Glc

H

Fig. (34). Silenosides A-C from the roots of Silene vulgaris.

TRITERPENOID SAPONINS FROM OTHER CARYOPHYLLACEAE PLANTS Besides the above-discussed saponin-containing genera, triterpenoid saponins were also reported from other Caryophyllaceae plants. Agrostemma githago (commonly called com cockle), an annual plant native to Eurasia, is now naturalized throughout the world. The seeds are said to be poisonous for both animals and human beings due to its saponin contents. Siepmann et al. reported the isolation and structure elucidation of two acylated saponins from the seeds of A. githago var. githago (Fig. 35) [99]. The structures were determined by ESI-MS, MS-MS, methylation analysis, and 2D homo- and heteronuclear correlated NMR (COSY, HMQC, HMBC) experiments. The absolute configuration of the sugars were determined by conversion of the enzymatically cleaved sugars into (-)-i?-but-2-yl-glycosides followed by GC-MS analysis. Spergularia ramosa is a herbaceous plant used in Peru as a remedy for respiratory ailments, tuberculosis, and rickets. Quite recently, De Tommasi et al. reported the results of chemical investigation on the aerial parts of this plant [100]. Six new saponins (Fig. 36) with gypsogenin or quillaic acid as agly-

49 cones were isolated, and the structures were established on the basis of extensive NMR experiments including DQF-COSY, ID-TOCSY, 2D HOHAHA, ROESY, HSQC, and HMBC. These compounds occur in pairs, which differ in the aglycone part. Saponins 1-4, like most of the saponins reported so far from Caryophyllaceae family, possess a glucuronic acid linked to C-3 of the aglycones. The other two saponins (5, 6) had a P-D-Gal-(l->3)-P-D-Glc moiety at the C-3 position. The pentasaccharide moiety at C-28 consists of arabinose, fucose, glucose (or galactose), rhamnose and xylose with the arabinose unit linked to the carboxyl group.

Xyl

1

R=H

2

R = Xyl

Fig. (35). Triterpenoid saponins from Agrostemma githago var. githago.

1

H

(}-D-Gal-. «__»^

n-C9H j 9 v,^^^.

Br

n-C9Hi9—=—^Br

n-C>9H}9>^

^

H

CHO

n-C9H}9>^

OMe OMe

0 NBS/Me2C0, AgNOs; //) 9-BBN-H, HOAc, HO(CH2)2NH2; ill) 4-bromo-l-butyl-9-borabicyclononane/ NaOH, THF/ Pd(PPh3)4; /v) PCC/CH2CI2; v) LiC(SMe)3/H20/NaH/ Mel; vz) HgCl2/HgO/MeOH/H20 Fig. (11). Soderquist synthesis of methyl (Z)-2-methoxy-6-hexadecenoate

The resulting 5-pentadecen-l-ol was oxidized with PCC to 5pentadecenal. This aldehyde was then reacted with tris(methylthio)methyllithium, affording the corresponding a-hydroxy ortho(trithio)ether, which in turn was methylated in situ using NaH/DMF and Mel to obtain the methoxylated adduct, Fig. (11). Final HgCVHgO catalyzed hydrolysis afforded the desired methyl (Z)-2-methoxy-6hexadecenoate. In this synthesis, the tris(methylthio)methyl group was used as the carboxy synthon. A more recent synthesis by our group utilized Mukaiyama's trimethylsilyl cyanide addition to aldehydes as the key step to introduce the carboxy functionality [33,36]. This approach also required the preparation of (Z)-5-pentadecenal as the key intermediate, Fig. (12). In this case, commercially available decyl aldehyde was coupled with 4carboxybutyltriphenylphosphonium bromide under Wittig conditions, resulting in a 10:1 mixture of the known (Z)- and (F)-5-pentadecenoic acids. The acids were then reduced to the desired (Z)-5-pentadecenal via (Z)-5-pentadecen-l-ol, a known pheromone. Addition of trimethylsilyl cyanide to (Z)-5-pentadecenal, under triethylamine catalysis, yielded

76

OSiMcs

HO

H3CO' OCH3

0 H02C(CH2)3CH2PPh3^Br', n-BuLi, THF/DMSO (1:1), -lO^C; //) IN HCl-MeOH, 3h; //•/) LiAlH4-THF, -78°C; iv) PCC (1.5 eq.) CH2CI2, rt; v) TMS-CN, EtgN, CH2CI2, -lO'^C, 2h; vO HCl (cone.) rt; vii) 50% NaOH, heat; viii) NaH/DMSO, CH3I; ix) KOH-EtOH. Fig. (12). Carballeira's synthesis of (Z)-2-methoxy-6-hexadecenoic acid (9)

2-trimethylsilyloxy-6-hexadecenonitrile. The trimethylsilyl cyanide was then transformed into the intermediate a-hydroxy amide with concentrated HCl, and then hydrolyzed to (Z)-2-hydroxy-6-hexadecenoic acid with 50% NaOH. Double methylation was successfully accomplished with NaH and methyl iodide in DMSO, resulting in the ester that was saponified with KOH in ethanol, affording the desired acid 9. The synthesis of (Z)-2-methoxy-5-hexadecenoic acid (10) was done in a similar fashion, but it first required the preparation of (Z)-4pentadecenal. In this case, the aldehyde was made starting with commercially available 1-dodecyne that was coupled with 2-(2bromoethyl)-l,3-dioxolane and A7-BuLi in tetrahydrofuranhexamethylphosphoramide, resulting in the expected 2-(3-tetradecyne)1,3-dioxolane, Fig. (13). Subsequent catalytic hydrogenation using Lindlar's catalyst afforded the expected 2-(3-tetradecenyl)-l,3-dioxolane. The dioxolane was removed with 5% HCl in acetone-water (1:1), and the equilibriimi favored (Z)-4-pentadecenal. Addition of trimethylsilyl cyanide to (Z)-4-pentadecenal under triethylamine catalysis as described by Mukaiyama ifor other shorter-chain analogues [36] resulted in 2trimethylsilyloxy-5-hexadecenonitrile. Under basic conditions the

77

trimethylsilyloxynitrile easily reverts to the original aldehyde. Therefore, the trimethylsilyloxynitrile had to be first transformed into the corresponding a-hydroxy amide under concentrated acid conditions (HCl) and then hydrolyzed to the a-hydroxy acid with 50% NaOH. Under these conditions, the intermediate (Z)-2-hydroxy-5-hexadecenoic acid was obtained. Double methylation was then successfully accomplished with NaH and methyl iodide in DMSO, resulting in methyl (Z)-2-methoxy-5-hexadecenoate. Final saponification with KOH in ethanol afforded the desired (Z)-2-methoxy-5-hexadecenoic acid (10). We can see that in these two syntheses the nitrile group, delivered using trimethylsilyl cyanide, was used as the carboxy synthon. These two amethoxy fatty acids displayed similar antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus (MIC = 0.35 |Limol/mL) and Streptococcus faecalis (MIC = 0.35 |amol/mL) [33].

/) w-BuLi, THF-HMPA, -70°C; //) Hj, Lindlar; in) 5% HCl, MczCO-HzO, 60''C; /v) TMS-CN, EtjN, CH2CI2, -lO'^C; v) HCl cone, rt; vz) 50% NaOH, heat; vii) NaH/DMSO, CH3I; viii) KOH-EtOH. Fig. (13). Synthesis of(Z)-2-methoxy-5-hexadeeenoicaeid (10)

78

All of the above syntheses resulted in racemic 2-methoxy acids, but the stereospecific synthesis of the naturally occurring (i?)-2methoxyhexadecanoic acid was also recently reported [35]. This synthesis started with commercially available (±)-2-hydroxyhexadecanoic acid for which the S enantiomer was selectively acetylated (47 % yield out of a maximum 50%, > 95% ee) with vinyl acetate in THF using the lipase Pseudomonas fluorescens from Aldrich, Fig. (14). In order to facilitate the separation of the acetylated acid from the non-acetylated acid, both compounds were methylated with diazomethane, thus avoiding cleavage of the acetate functionality. The methyl (i?)-2hydroxyhexadecanoate and the methyl (S)-2-acetoxyhexadecanoate were then separated by silica gel column chromatography. The purified methyl (i?)-2-hydroxyhexadecanoate was further methylated with NaH/Mel in DMSO and finally saponified with KOH in ethanol, which afforded the (i?)-2-methoxyhexadecanoic acid with good enantiomeric purity (>95 % ee). This represented the first synthesis for the (i?)-2methoxyhexadecanoic acid [35].

OH

10

OH

10

OAc

10

O

OCH3 10

/) Pseudomonasfluorescens,CH2=CH0Ac, BHT, THF; //) CH2N2, ether; ///') silica gel column chromatography; /v) NaH, Mel, DMSO; v) KOH/ethanol. Fig. (14). Synthesis of (^)-2-methoxyhexadecanoic acid

Other Methoxylated Fatty Acids Another methoxylated fatty acid of marine origin that has attracted the attention of synthetic chemists has been the (45,7S)-(-)-7-methoxy-4tetradecenoic acid (11), a metaboUte of the marine cyanophyte Lyngbya majuscula [38-39]. This methoxylated compound also displays

79 antimicrobial activity towards the Gram-positive bacteria Staphylococcus aureus and Bacillus sub tills [39]. In a convergent synthetic approach, the synthesis started with (i?)-(2,2-dimethyl-l,3-dioxolan-4-ylmethyl) ptoluenesulfonate that was coupled with 1-hexylmagnesium bromide under LiiCuCU catalysis, Fig. (15). The resulting dioxolane was converted into the desired optically active epoxide by reaction with HBr/ V^o

p-Ts-0^3^0

-h

H3C{H2C)6'i ^ _ J -

of-

° - r ^ -^

H3C(H2C)6^

B.V^^ ^

H3C(H2C)6 OH

H3C(H2C)6

O ' ^

OHH

H

u

H3C(H2C)6^^

C02H

H3C' 11

/) Li2CuCl4, THF, hexylmagnesium bromide; //) HBr/HOAc; ///) KOH/MeOH; /v) CBr4/CH2Cl2/PPh3; v) w-BuLi, 2 eq.; vi) «-BuLi, HMPA, a; vii) YiJ Pd/C; viii) hu, PhSSPh, cyclohexane; ix) A?-BuLi, Mel; jc) MeOH/HCl; xi) KOH/ethanol; xii) H2SO4. Fig. (15). Synthesis of (4£, 75)-(-)-7-methoxy-4-tetradecenoic acid (11)

AcOH followed by ring closure with potassium hydroxide in methanol, which afforded the epoxide. The other coupling unit was made from the reaction of 3-(2,4,10-trioxatricyclo[3.3.1.1"^' ]dec-3-yl)propanal with tetrabromomethane and triphenylphosphine, affording the terminal dibrominated alkene which was subsequently transformed into the terminal alkyne with «-BuLi [40]. Coupling of the lithiated alkyne with the epoxide proceeded stereoselectively to the alcohol with no racemization. The resulting alkyne was then hydrogenated under

80 Lindlar's catalyst into the cis double bond that was subsequently photochemically isomerized to the trans double bond with phenyldisulfide [40]. Final methylation with w-BuLi/Mel and hydrolysis of the orthoester group resulted in the desired (4£,75)-(-)-7-methoxy-4tetradecenoic acid (11) as shown in Fig. (15). Therefore, the key synthetic step for the introduction of the optically active methoxy functionality was the ring opening of an optically active oxirane with a lithioalkyne [40]. Diunsaturated Fatty Acids Straight-Chain A5,9'Fatty Acids The A5,9-diunsaturation is probably the most characteristic diunsaturation in fatty acids from sponge phospholipids [41]. One of the most characteristic sponge fatty acids is the (5Z,9Z)-5,9-hexacosadienoic acid since it is found in the phospholipids of most sponges [41]. More interesting is the published observation that model membranes with symmetrical phosphatidylcholines and phosphatidylethanolamines containing the (5Z,9Z)-5,9-hexacosadienoic acid tend to exclude cholesterol [42]. Other interesting examples of naturally occurring A5,9 fatty acids include the shorter-chain analog (5Z,9Z)-5,9-hexadecadienoic acid (12) that was originally reported from the cellular slime mold Dictyostelium discoideum [43] but later identified in several marine sponges [44]. Another naturally occurring A5,9 fatty acid is the (5Z,9Z)5,9-eicosadienoic acid (13), a major constituent of the phospholipids of the sponge Erylus formosus [45]. Several reports focus on the synthesis of straight-chain A5,9 fatty acids. A recent four-step synthesis was based on acetylide coupling to generate the A9 double bond and the Wittig coupling to generate the A5 double bond [46]. In the synthesis of (5Z,9Z)-5,9-hexadecadienoic acid (12) the 2-(2-bromoethyl)-l,3-dioxolane was coupled with 1-octyne using «-BuLi in tetrahydrofiiran/hexamethyl phosphoric acid triamide, affording the desired 2-(3-decynyl)-l,3-dioxolane, Fig. (16). For the synthesis of (5Z,9Z)-5,9-eicosadienoic acid (13), 1-dodecyne was employed. The advantage of this synthetic scheme is that it is possible to control the chain length of the target fatty acid by simply changing the length of the initial alkyne. Hydrogenation of the alkynyldioxolane in dry

81 hexane under Lindlar's catalysis afforded the desired alkenyldioxolanes, as exemplified by 2-(3-decenyl)-l,3-dioxolane in the synthesis of 12. Removal of the dioxolane was readily accomplished with 5% HCl in acetone/water (1:1) affording the desired aldehydes, namely (Z)-4undecenal as the last synthetic intermediate for 12, or (Z)-4-pentadecenal in the case of 13. Final Wittig olefmation of the aldehydes with 4carboxybutyltriphenylphosphonium bromide and ^7-butyllithium in tetrahydrofuran/dimethylsulfoxide (1:1) afforded the desired (5Z,9Z) diunsaturated acids 12-13, generally in 10:1 ratios favoring the 5Z stereochemistry over the 5E.

C ^ B r * „^

12, n=1 16:2 13, n=5 20:2

0 A?-BuLi, THF-HMPA, -TO'^C; //) H2, Lindlar; ///) 5% HCl, acetone-H20, 60°C; iv) Ph3P^(CH2)4C02H, Br", 2.5 M, w-BuLi, THF/DMSO. Fig. (16). Synthetic sequence for normal-chain A5,9 fatty acids

The first straight-chain A5,9 fatty acid to be synthesized was actually the (5Z,9Z)-5,9-hexacosadienoic acid [47]. The methodology developed by Djerassi et al. is shown in Fig. (17). This six-step synthesis started with the unusual controlled ozonolysis of one of the double bonds of (lZ,5Z)-l,5-cyclooctadiene to secure a 100% Z stereochemistry for what eventually was going to be the A9 double bond of the target molecule. Transformation of the product to the tosylated acetal, followed by Grignard coupling under dilithium tetrachlorocuprate catalysis afforded (Z)-4-heneicosenal, which after Wittig olefmation with 4carboxybutyltriphenylphosphonium bromide resulted in the desired A5,9

82 acid, Fig. (17). This same methodology was used in the synthesis of the corresponding l-^'^C-labeled acid that was used in feeding experiments to elucidate the unusual biosynthesis of A5,9 fatty acids. The C-label was introduced by reaction of K^\4rCN with the mesylate of 4,8-pentacosadienl-ol, followed by basic hydrolysis of the nitrile [48]. Interestingly, these radiolabeling experiments targeting the (5Z,9Z)-5,9-hexacosadienoic acid 0CH3

0CH3

0CH3

H

C13H27

C13H27

^COjH

/) leq. O3, MeOH, -78°C; //) TsOH, Ih; Hi) 1.2 eq. NaBH4, -10°C; iv) w-CnHsTMgBr, THF, Li2CuCl4, 0°C, 2h; v) acetone, cone. HCl (20:1), rt, Ih; v/) Ph3P^CH2CH2CH2CH2C02H Br', KH, Me2S0, rt, 2h.

d;

C13H27

CO2H

^OMs

yf,7/.yx ^Ci3H27

vii) BrTh3P"'CH2CH2CH2C02H; viii) LiAlH4; ix) MsCl, NEt3; ;c) K ' ^ C N , D M F ; xi) KOH, EtOH.

Fig. (17). Synthesis of (5Z,9Z)-5,9-hexacosadienoic acid and its 1- C-labeled analog

in Microciona prolifera suggested that the double-bond introduction follows a random order, i.e., either the A5 double bond or the A9 double bond can be introduced first in the acyl chain, and then either the associated A9 or A5 double bonds are introduced [48]. This contrasts with the order of double bond introduction in fatty acids from animals in which the first double bond tends to be introduced at the A9 position, and the second is inserted between the first bond and the carboxyl group.

83 Monomethylated A5,9'Fatty Acids /yo-branched 5,9-dienoic acids with chain-lengths between 16 and 29 carbons have also been identified in the phospholipids of several marine organisms [49-50]. Recently, the shortest member of the series, namely the (5Z,9Z)-14-methylpentadeca-5,9-dienoic acid (14), was isolated from the Caribbean gorgonian Eunicea succinea and shown to be specifically inhibitory against pathogenic Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus faecalis, but inactive against Gram-negative bacteria [49]. On the other hand, longer-chain isO'/S5,9 analogs, such as the (5Z,9Z)-24-methylhexacosa-5,9-dienoic acid, inhibit DNA topoisomerase I (topo I) with ICso's of 1.1 |aM [51]. The reported synthesis for (5Z,9Z)-14-methylpentadeca-5,9-dienoic acid (14) started with commercially available 4-methylpentan-l-ol, which upon reaction with phosphorous tribromide afforded l-bromo-4methylpentane [52]. Commercially available pent-4-yn-l-ol was also protected as the tetrahydropyranyl ether as shown in Fig. (18). Formation of the lithium acetylide with w-BuLi in THF and subsequent addition of l-bromo-4-methylpentane in hexamethylphosphoric acid triamide resulted in the isolation of the tetrahydropyranyl protected 9-methyldec4-yn-l-ol. Hydrogenation of the alkyne with Lindlar's catalyst and quinoline in dry hexane afforded the cis hydropyranyl-protected 9methyldec-4-en-l-ol. Deprotection of the alcohol with/?-toluenesulfonic acid afforded (Z)-9-methyldec-4-en-l-ol. Pyridinium chlorochromate oxidation of the alcohol resulted in the isolation of the labile (Z)-9methyldec-4-enal. Final Wittig reaction with (4-carboxybutyl) triphenylphosphonium bromide in THF/DMSO resulted in the desired (5Z,9Z)-14-methylpentadeca-5,9-dienoic acid (14). The synthesis of (5Z,9Z)-24-methylpentacosa-5,9-dienoic acid (15) was also reported in the same communication [52], and it started with commercially available 10-bromodecan-l-ol that was oxidized with PCC to 10-bromodecanal, followed by Wittig coupling with 4-methyl-lpentyltriphenylphosphonium bromide resulting in the (Z)- and {E)A' bromo-14-methylpentadec-lO-ene, as shown in Fig. (18). Catalytic

84

OH

»

..^V^^^v^Br a

OH p-^^^«

Hi

p^^CHO

Br

;V

p^/^-^Br.

"'^ ^"^ ^Br

Br

OTHP

.^^J^Xx-'^/OH

Vr CO2H

14 n = 1 15. n = 11

0 PBr3 (70%); //) PPhj/CgHe (95%); ///) PCC/CH2CI2 (83%); iv) 2.5 M /i-BuLi, b, THF/DMSO; v) H2, 5% Pd/C, hexane (92%); vi) 2.5 M w-BuLi, HMPA/THF, a or c; vii) Hz/Lindlar; vm)p-TsOH, MeOH, 45^C; /x) PCC/CH2CI2 (84-89%); x) Ph3P^(CH2)4C02H Bf, 2.5 M w-BuLi, THF/DMSO. Fig. (18). Synthesis of Z50-A5,9-16:2 and /5o-A5,9-26:2 fatty acids

hydrogenation afforded 1 -bromo-14-methylpentadecane. Lithium acetyiide coupling in THF/HMPA resulted in the isolation of the tetrahydropyranyl-protected 19-methyleicos-4-yn-l-ol. In this case, the alcohol was deprotected first with /7-toluenesulfonic acid in methanol, affording 19-methyleicos-4-yn-l-ol that was further hydrogenated with Lindlar's catalyst and quinoline to (Z)-19-methyleicos-4-en-l-ol, Fig. (18). Pyridinium chlorochromate oxidation of the alcohol resulted in (Z)19-methyleicos-4-enal. Final Wittig reaction with (4carboxybutyl)triphenylphosphonium bromide resulted in the desired (5Z,9Z)-24-methylpentacosa-5,9-dienoic acid (15). The synthesis of an iso-anteiso pair of 5,9-21:2 fatty acids, namely the 19-methyleicosa-5,9-dienoic acid (16) and 18-methyleicosa-5,9-dienoic acid (17), prominent constituents of the phospholipids of the sponge

85 Erylus formosus [45], was also reported by Kulkami et al. [53]. As shown in Fig. (19), a common bifunctional nine-carbon aldehyde was used for both syntheses. Both syntheses started with the DHP monoprotection of 1,4-butanediol, followed by subsequent PCC oxidation to the corresponding aldehyde. Reaction of this butanal with (4-carboxybutyl)triphenylphosphonium bromide resulted in the corresponding THP protected olefmic acid that was further deprotected and esterified to the methyl ester. A second oxidization with PCC afforded the desired intermediate methyl 8-formyloct-5-enoate which secured the cis A5 double bond in the final targets, Fig. (19). The

H0(CH2)40H

THPO(CH2)3CHO

O

Me02C(CH2)3

n

A,

(CH2)8C02Me

w7/-x

^'^(CH2)8CH20H

(CH2)2CHO

1^

>

X/-X//

''^^(CH2)9PPh3^Br-

•

^COzH

H ZZ

Jl

(CH2)80H

I ^"'^--'''^(CH2)8PPh3*Br-

^"^(CH2)80H

x/V-xv *•

1^ '^^^^'^(CH2)80H

xvi-xvii ^

^COjH

xviii-xix ^

/) DHP/H"'/CH2Cl2; //) PCC/NaOAc/CHjCIj; ///) Ph3P=CH(CH2)3C027dimsyl/DMSO; /v) MeOH/H"; v) PCC/CH2CI2; v/) CHgPPhjy dimsyl/ DMSO; v/7) LAH/EtjO; viii) H2/ 10%Pd-C/ EtOH; ix) PPh3-Br2; x) PPh3/CH3CN/ heat; xi) KOH/EtOH, H^; xii) a, dimsyl, DMSO; xiii) UgSO^, H^/70% aq. EtOH; xiv) EtPPh3l/dimsyl/DMS0; JCV) H2/10% Pd-C/ EtOH; ;cv/) Ph3P-Br2/CH2Cl2; xvii) Ph3P/CH3CN/heat; xviii) a, dimsyl, DMSO; xix) KOH, EtOH, H"^.

Fig. (19). Synthesis of iso-anteiso 5,9-21:2 fatty acids

86 /50-ramification in 16 was made from methyl 10-oxoundecanoate that was reacted with methylenetriphenylphosphorane followed by LAH reduction to 10-methyleneundecan-l-ol. The further hydrogenation of the methylene group with 10% Pd-C and subsequent bromination with Ph3P*Br2 resulted in 1-bromo-lO-methylundecane, from which the corresponding triphenylphosphonium bromide salt was prepared. Final Wittig reaction of this phosphonium salt with methyl 8-formyloct-5enoate and basic hydrolysis resulted in the synthesis of the 19methyleicosa-5,9-dienoic acid (16). In this particular case, three Wittig reactions were utilized to introduce both double bonds as well as the isoramification. The synthesis of the anteiso isomer, namely the 18-methyleicosa-5,9dienoic acid (17), started from a primary decynol that was converted to the hydroxylated methyl ketone through Hg(II) catalyzed hydration. Condensation of this ketone with ethylidenetriphenylphosphorane and further hydrogenation resulted in the necessary 9-methylundecan-l-ol, with the necessary anteiso functionality. After conversion to the corresponding Wittig salt, coupling with methyl 8-formyloct-5-enoate, and saponification, the expected 18-methyleicosa-5,9-dienoic acid (17) was obtained. Fig. (19). In this case, three Wittig reactions were also used. One of the earlier syntheses of branched A5,9 fatty acids was accomplished by Djerassi's group in the elucidation of the biosynthesis of the unusual sponge fatty acid (i?)-(-)-22-methyl-5,9-octacosadienoic acid (18), a phospholipid fatty acid first identified in the sponge Aplysina fistularis [54]. They established the stereochemistry of the carbon bearing the methyl group in 18 as R, This synthesis started with commercially available (^)-(+)-pulegone that was transformed in ten steps to (J?)-l-bromo-3-methylnonane, Fig. (20). The sequence involved conversion of (i?)-(+)-pulegone to (i?)-(+)-citronellic acid, which was then reduced with lithium aluminum hydride and the resulting alcohol protected with DHP. Ozonolysis of the double bond, NaBH4 reduction, and tosylation was followed by the dilithium tetrachlorocuprate (Li2CuCl4) catalyzed cross coupling with «-propylmagnesium bromide. Final deprotection with acid, tosylation, and reaction with lithium bromide in acetone afforded the stereochemically fixed (i?)-l-bromo-3methylnonane. The A9 cis double bond in (i?)-(-)-22-methy 1-5,9octacosadienoic acid (18) was made, as in previous strategies, from

87

(lZ,5Z)-l,5-cyclooctadiene that was carefully monoepoxidized. This epoxide was opened with H5IO6, and after NaBH4 reduction, the 1,8-oct4-enediol was obtained, monoprotected with DHP, tosylated, and then elongated six carbons in a Li2CuCl4 catalyzed coupling with Me3SiO(CH2)6MgCl. This step was followed by deprotection of the trimethylsilyl group with base, further tosylation, and a third Li2CuCl4

CO2H OTHP

OTHP OH

OTHP

0-^0° IJ-'^^-^'^OTHP

^ CC™ — CC:

OTHP ^OH

xvii-xviii

(i

CO2H

i) HCl; ii) KOH; Hi) LAH, EtjO; iv) DHP, H^; v) O3/ NaBH4; v/) TsCl, Py; v//) LizCuC^, «-PrMgBr; v/7/) TsOH, MeOH; ix) TsCl, py; x) LiBr, acetone; xi) /w-CPBA, CHjClj; xii) HsIG^; xiii) NaBU^; xiv) DHP, H"^, EtzO; xv) TsCl, pyridine; xvi) LijCuCU, Me3SiO(CH2)6MgCl; xvii) K2CO3, MeOH; viii) TsCl, pyridine; xix) LizCuC^, Grignard reagent of a; xx) TsOH, MeOH; xxi) PDC, CH2CI2; xxii) Br'Ph3P''(CH2)4C02H, KH, Me2S0, THF. Fig. (20). Synthesis of (/?)-22-methyl-5,9-octacosadienoic acid

catalyzed coupling with (i?)-l-bromo-3-methylnonane, affording (i?)-17methyltricos-4-en-l-ol after deprotection, Fig (20). Final oxidation of the alcohol to the aldehyde, and Wittig coupling with (4-carboxybutyl)

triphenylphosphonium bromide afforded the desired (i?)-22-methy 1-5,9octacosadienoic acid (18). In this synthesis, the 9Z double bond was obtained from (lZ,5Z)-l,5-cyclooctadiene, the 5Z double bond from a Wittig reaction, and the 22R methyl group from naturally occurring (i?)(+)-pulegone [54]. Cyclopropylidene A5,9-Fatty Acids A recent report describes the isolation of a new class of A5,9 fatty acids from the Australian sponge Amphimedon sp., dubbed amphimic acids. They possess the unusual cyclopropylidene functionality [51]. These compounds were particularly interesting because they displayed DNA topoisomerase I (topo I) inhibition at ICso's of 0.47-3.0 \xM [51]. The HO^^^'"^(CH2)i5CH3

•

HO''^^''^(CH2),5CH3

•

L JL (CH2),5CH3

9"

Ph2(0)P

^^-^(CH2),5CH3

' ^ - \

OH

^^-^(CH2),5CH3

""'^

Ph2(0)P^"^(CH2),5CH3

OH

TBDPSO^^.^v^^A7\ Ph2(0)p'^^^"2^»^^"^

TBDPSO

-

-

-

-

-

-

-

-

-!!^=^

-(CH2).5CH3

HO^''^-^""'^^^^""'^(CH2),5CH3

—

_

^ ._

_

_

_

_

-(CH2),sCH3

'*(CH2),5CH3

19

/) /-BuOOH, (+)-DET, Ti(0-/-Pr)4, CH2CI2, -20°C and then -10°C (76%); //) Red-Al, THF, 0°C (94%); ///) I2, PPhj, HMPA, toluene, rt (72%); /v) PPhj, CaCOj, CH3CN, 80°C; v) NaOH, THF/HjO, SO^'C (72%, 2 steps); v/) MsCl, EtjN, CH2CI2, 0°C (100%); vii) NaN(TMS)2, THF, 0°C (87%); viii) TBDPSO (CH2)3CHO, LDA, THF, -78°C then 0°C; ix) BU4NF, THF, rt (71%); jc) NaH, DMF, 60-70% (80%); xi) DMSO, (C0C1)2, EtjN, CH2CI2, -78°C then 0°C (91%); xii) TBDPS0(CH2)5P'^Ph3r, NaN(TMS)2, toluene, rt (100%); xiii) BU4NF, THF,rt(100%); xiv) DMSO, (C0C1)2, EtjN, -78°C then 0°C (79%); ;cv) NaC102, NaH2P04,2-methyl-2-butene, THF,rt(97%). Fig. (21). Synthesis of amphimic acid A

89 synthesis of one of these acids, namely amphimic acid A (19), started with the Sharpless asymmetric epoxidation of (£r)-2-nonadecen-l-ol, affording the corresponding epoxide, as shown in Fig. (21). Regioselective reduction of the epoxide with Red-Al provided (5)-l,3nonadecanediol. Controlled monoiodination of the diol afforded (iS)-liodononadecan-3-ol, which was subsequently converted into a mesylated phosphine oxide as shown in Fig. (21). Cyclization of the mesylate with NaN(TMS)2 resulted in the optically active cyclopropylphosphine oxide, which after a Wittig-Homer reaction with A'{tert' butyldiphenylsiloxy)butanal resulted in a diastereomeric mixture of cyclopropyl P-hydroxyphosphine oxides. This diastereomeric mixture was separated using silica gel column chromatography. Deprotection of the correct stereoisomer and sodium hydride catalyzed elimination of the P-hydroxyphosphine oxide resulted in a cyclopropylidene with the E stereochemistry, Fig. (21). After Swem oxidation of the alcohol, Wittig reaction, deprotection of the /^rr-butyldiphenylsiloxy group, a second Swem oxidation, and oxidation of the aldehyde to the acid with NaC102, amphimic acid A (19) was obtained. Non-methylene Interrupted Fatty Acids In the family of non-methylene interrupted fatty acids, the total syntheses of the unusual marine acids (IIZ, 15Z)-ll,15-eicosadienoic acid (20) and (lOZ, 15Z)-10, 15-eicosadienoic acid (21), have been reported [55-56]. The (IIZ, 15Z)-11,15-eicosadienoic acid (20) was identified for the first ^ ^

/-//•

lf). The aglucone part of the molecule has three centers of chirahty (possibility of RS isomerism), two of which (C-1 and C-9) are easily epimerized by acid or base after removal of the p-Dglucopyranosyloxy group, however, the third one (C-5) keeps its configuration not only in the aglucones of simple secologanin derivatives, but also in all such alkaloids, in which subsequent reactions did not involve this center [9]. These structural elements give many possibilities not only for transformation of the functional groups, but formation of new rings, as well as cleavage and rearrangement of C-C bonds, too (Ig).

.iiOgIc

H3CO

H3CO

geissoschyzine OCH3

(substructures derived from secologanin are in bold lines)

Fig. (2). Alkaloids derived from secologanin.

Simple chemical transformations of secologanin Already its dissolution in protic solvents initiated changes in the structure of secologanin and resulted multicomponent equilibria, therefore the NMR spectra of secologanin and some of its derivatives should be recorded in aprotic solvents (chloroform, benzene, acetonitril, etc). The

98 equilibria were set in at room temperature in 1-2 hr. Although the compounds of the solvent interactions could not be isolated without shifting the equilibria, the transformations were easily followed in deuterated solvent by ^H NMR spectroscopy as shown in Fig. (3). in deuterium oxide

in deuterated methanol

ilOgIc

ilOgIc

D

(-D3COD)

H NMR chemical shifts in 6. Figures in plain are measured in D2O, those in italics in D3COD

Fig. (3). Acid-base and solvent equilibria of secologanin.

The signals of H-3 and H-7 were sensitive for changes both in the aldehyde and the ester group. The position of the equilibrium of hydrate and hemiacetal (5) depended on the structure of the solvent [10]. The simple chemical transformations of secologanin are summerized in Fig. (4). It is well known from the early literature that secologanin (1) can easily be hydrogenated at position 8,10 to (2) without saturation of the conjugated C=C bond [1]. The presence or absence of the 8(10) double bond (vinyl and ethyl, natural and dihydro derivatives, respectively) often

99 resulted in considerable difference of the chemical properties of the appropriate derivatives.

r~\ Ogle

H;,CO

,,ilOglc(Ac)4

,,lOglc(Ac)4

H""T O

^* X = O o r H N

O

T

9a

MCPBA=3-cliloroperben2oic acid

Fig. (4). Simple chemical transformations of secologanin.

Enzymatic deglucosylation with p-glucosidase gave not the simple primary aglucone, but its bicychc derivative both in the natural (3a) and the dihydro series (3b) [11, 12]. However the acetal (4a), in which the formyl group is protected gave the expected primary aglucone (analogously to Id). Removal of the glucosidic unit either by acid or by base resulted in fragmentation of the aglucone, as will be discussed later.

100

The formyl group could be protected reversibly by acid-catalytic acetalation with ethylene glycol (4a) [13] or methanol (4b) [14]. By temporary protection of the formyl group, methanol could be added to the conjugated C=C bond (6) in aqueous-methanolic sodium hydroxide solution (and with subsequent reesterification) [15]. Acetylation of secologanin with acetic anhydride in the presence of a few drops of pyridine gave the 0',0',(9',0'-tetraacetylsecologanin (7), which could be oxidized in aqueous acidic medium with chromium trioxide to secoxyloganic acid and reesterified with diazomethane to (8). The acetyl groups could be removed by the Zemplen method without changes in the methoxycarbonyl group [16]. After the protection of the formyl group in acetal form (7a), the isolated double bond could be oxidied. 3-chloroperbenzoic acid (MCPBA) epoxidized at first the isolated (9), later also the conjugated (9a) double bond [17]. Osmium tetroxide oxidized only the isolated double bond into a glycol unit (10) [18]. The reactivity of secologanin derivatives against N nucleophiles was influenced mainly by the four potential carbonyl groups. As two of them are protected (C-1) or masked (C-3), in secologanin the most reactive point is C-7. C-11 is rather resistant to external nucleophiles, but sensitive for internal nucleophilic attacks coming from the substituents of C-7. C-3 is sufficiently reactive only in the ester and lactone derivatives, and not reactive in the amides and lactams or in compounds where the cyclic 0-2 is exchanged against N. C-1 is resistant to external nucleophiles in the glucoside form, but reactive in the aglucones. As a further general rule it was observed that the exocyclic alkylamino group had the tendency to take spontaneously the endocyclic position by ring opening and ring closure (ANRORC reaction), according to the higher stability of the new azacycle (tetrahydropyridine ring) over the original oxacycle (dihydropyran ring). The following details support these general remarks. With primary amine, the first product was a labile Schiff base (11) having a signal of H-7 at -7.7 6. However, the excess of the amine (especially in the case of methyl amine) easily transformed the Schiff base into a 7-substituted lactam (12). The substituent of C-7 (methylamino, methoxy or hydroxy) entered first into P equatorial orientation, but then rapidly epimerized to the a axial one, which is stabihzed by a conjugation with the non-bonding orbital of the lactam nitrogen. The methylamino group could easily by exchanged for other groups (methoxy,

101 hydroxy, or even other amino groups) (e. g. 13) [19]. As with secondary amine the formation of the lactam was not possible, it gave 7-substituted lactone (14). Both the lactam and the lactone were formed by intramolecular interaction of the nucleophile of C-7. When the formyl group was protected in form of ethylene acetal or by coupling with the appropriate biogenic amine, the methoxycarbonyl group proved to be rather resistant to nucleophiles, and reactions runned at C-3 (see later). Tertiary amine could not be incorporated into secologanin, but in the presence of a protic solvent (water or methanol), catalyzed the lactonization (15) [20]. Bifunctional amines gave the possibility to annellate a further five- or six-membered ring with a second heteroatom (oxygen, nitrogen, etc.) to the secologanin subunit (16) [20,21]. Chemical transformations of bakankosine derivatives The reactions are summerized in Fig. (5). Bakankosine (15, R=H) , a natural lactam derivative was prepared by Tietze in the reaction of secologanin with benzylamine, followed by reduction with sodium tetrahydridoborate, and after spontaneous lactamization by removal of the benzyl group by catalytic hydrogenation [22]. Further A^-substituted bakankosine derivatives (17a, R is alkyl) were obtained by reduction of the appropriate Schiff bases (11) likewise with sodium tetrahydrido borate, accompanied again by spontaneous lactamization [23]. The vinyl group (in the natural series, e.g. 17a) was easily saturated into an ethyl group (dihydro series, e. g. 17b). The bankosine derivatives proved to be rather stable compounds and could be attacked by nucleophiles neither at C-1, nor C-3. However, after deglucosylation with P-glucosidase or with acid catalysed hydrolysis, the aglucone underwent nucleophilic reactions (see later). The deglucosylation with enzyme resulted in the formation of the primary aglucone (18a, 18b) in both cases in the form of an epimeric pair at C-1. The primary aglucone was easily transformed into an acetal (19a, 19b) in methanol with acidic catalysis. The acidic deglucosylaton gave in the natural series a rearranged epimeric pair (20), in the dihydro series an unrearranged one (23) with inversion of the configuration at C-9. The complicated process was analyzed in detail (see later) [24]. In the presence of a strong base (sodium ethanolate in ethanol, sodium hydride in dimethylsulfoxide) the oxacycle could be cleaved in both (18a) and

102

(23) (strong bathochromic shift in the UV spectrum, see e. g. 21), and alkylated on 0-2 into the enol ether (22) and (24), respectively, having Z geometry in chloroform but E geometry in dimethylsulfoxide. (18a) was isomerized in triethylamine to (20). The dihydro aglycone (18b) gave with ethanolic sodium ethanolate a double lactone (25). Its formation was interpreted as the result of an intramolecular Claisen-Tishchenko reaction.

0

30 X=OorNH

Fig. (5). Chemical tranformations of bakankosine derivatives.

As (20) gave with amines rather unstable products, the reactions with N nucleophiles were investigated in the dihydro series in details. Primary amine attacked the aglucone (23) at C-1, but in the excess of the reagent the oxacycle of the first product (26) was transformed into the azacycle (27) by ring-opening and ring-closure. Like at position C-7, the exocyclic amino group of C-1 could be exchanged for hydroxy or methoxy group (or even another amino group) as well (e. g. 28). The secondary amine

103

piperidine attacked the aglucone in the same position, but the oxacycle remained intact (29). By using bifunctional primary amine, further fiveor six-membered rings containing a second heteroatom could be annellated (30) [results partly not pubHshed yet]. Chemical transformations of sweroside derivatives The reactions are shown in Fig. (6). Reduction of secologanin (1) and its dihydro derivative (2) with sodium tetrahydridoborate in protic solvent gave sweroside (31a) and its dihydro derivative (31b), respectively, [25] which could smoothly be deglucosylized by {i-glucosidase (e. g. to 32), however, the acidic hydrolysis gave no well-defined product. The dihydropyrane ring of (32), Hke that of the bakankosine derivative (23) was cleaved by strong nucleophile in dimethylsulfoxide, and alkylated on 0-2 (33). In ethanol the lactone ring was opened by sodium ethanolate, and after acidification the bridged ester aglucone (34) was formed [unpublished results]. For N nucleophihc attack three sites could be found: C-1, C-3 and C11. Their reactivity is interrelated. Interpretation of the reactions with secondary amine was easy. Unlike to bakankosine, sweroside could be deglucosylized by piperidine, but the amino group was attached to C-3 instead of C-1 (35). It means that the ester group sufficiently activated C3, and the primary adduct was stabilized by deglucosylation and tautomerization of the C=C bond fi-om isolated to conjugated position. Although in the dihydro series the glucoside did not give a well defined product with piperidine, it could be incorporated into the aglucone of dihydrosweroside, but, according to the expectation, at C-1 (36). Propylamine as a primary amine attacked C-3 of the glycosides (31a) and (31b), and afforded the first adduct (37a, 37b), which was stabilized by deglucosylation and spontaneous isomerization into the more stable azacycle (38a) and (38b, respectively) by ring opening and ring closure. In the natural series, even a dehydrated product (39) could be isolated. In the dihydro series, in addition to (38b), a further, bridged bicycUc compound (42) was isolated fi-om the same reaction mixture. (42), in which the ester group was transformed into an amide group, could be obtained from the aglucone of the dihydrosweroside (32), too, but not from (38b). Evidently, N-2 deactivated the C-11 of the ester group.

104

O

35 t^

\

\

...•Ogle p-glucosidase ^ from 31b*

I H, - Pd NaBH4 H2O

0-

f

>^l.C,HsONa +

0-

31b8,10-dihydro H2NCH2CH2CH: from 31a

6v^

J ^

N>^

H,NCH,CH,CH. from 31b

H2NCH2CH2CH3

from 37a

Fig. (6). Chemical transformations of sweroside derivatives.

The rather compHcated situation is not completely clear. From the aglucone (32), the formation of the amide (42) should start with two independent nucleophilic attacks on the same molecule, i. e. at C-1 (to 41) and subsequently at C-11 (to 41a). In the case of glucoside (31b), the two attacks runned on different molecules affording the two intermediates (37b) or (40). This latter should give by a second amino attack the same intermediate (41a), but the mechanism of the deglucosylation is not clear in this case. The common closing steps through (41b) to the final product (42) are usual transformations [26].

105 Acidic deglucosylation of simple bakankosine derivatives The reactivity of secologanin and related compounds is partially blocked by the glucosidic linkage, which effectively regulates the reactivity of secologanin at C-1 and C-3 as well as on the vinyl group. After removal of the P-D-glucopyranosyloxy unit, a cascade of reactions can start. The free aglucone may be represented as an all-carbonyl tautomer, e. g. (le), in which active H atoms are at C-6, C-9 and C-4, i. e. in a position to one (C-7 or C-1, respectively) or two (C-3 and C-11) carbonyl groups. In the aglucone the isolated C=C bond can easily be tautomerized to conjugate position, e. g. (If), which fact has important consequences: also this second C=C bond will be asymmetrically substituted (possibility of E-Z isomerism), a further electrophilic center will be developped at C-9, and the H-10 atoms become activated (H-D exchange). As in acidic hydrolysis, secologanin (1) and 7-substituted bakankosine derivatives (e. g. 13) underwent fragmentation (see later), and sweroside (31a) was destroyed without giving a well defined product, A^-methyl bakankosine (17a) and its dihydro derivative (17b) were selected as model compounds for investigation of the acidic deglucosylation of secologanin derivatives. In the stable educts, C-7 and C-11 had been inactivated by lactamization [24]. As it was shown in Fig. (5), in boiling aqueous diluted hydrochloric acid, (17a) and (17b) gave aglucones (20) (epimeric pair at C-8) and (23) (epimeric pair at C-1), respectively. In (20) the dihydropyran ring was cleaved, the C4 unit rotated around the bond C-5-C-9 and the cycle was reformed to C-8 with simultaneous appearance of a new formyl group. (23) proved to be a simple primary aglycone epimer pair. Treatment of either the educts (17a and 17b) or the products (20 and 23), under the conditions of the deglucosylation in deuterated solvent, resulted in D exchange of H-9 in both the natural and dihydro series, and in addition of H-10 in the natural one. These facts suggested the cleavage and closing of the dihydropyran ring in ring-chain tautomerisms, as well as changes in the configuration of C-9 by oxo-enol and C=C bond tautomerism.

106 possible transformations both in "natural" and dihydro aglucones in "natural" aglucons only

a

/

LrfH*

\

\

Estimated relative free enthalpies of formation in kJ/mol. Energy values in parentheses concern possible aglucones derived from the 8,10-dihydro aglucones * indicates H atoms and C atoms having H atoms which could be exchanged for D under the reaction conditions of deglucosylation both in educts and products Fig. (7). Energy graph of the deglucosylation of bakankosine derivatives.

The possible intermediates were organized into an energy graph shown in Fig. (7), which indicates also the relative enthalpies of formation (in kJ/mol) estimated on simple model reactions. The arrows show the direction of the decrease of the free enthalpy of formation. As each structure has two stereogenic elements (unsymmetrically substituted double bond and/or center of chirality), each formula represents four stereoisomers.

107

However, the configurational and conformational changes could not be considered at the estimation of relative free energies. The structure graph (published in the original paper [24]) involved 48 stereoisomers.

from natural glucoside

Fig. (8). Structure graph of the deglucosylation of bakankosine derivatives.

The simpHfied version of the structure graph presented in this paper as Fig. (8) indicates only those structures, which are on the shortest path from the educts to the products. (In the graph the stereochemical descriptors R, S, Z and E show the configuration of the eventual centers of chirahty at C-1, C-3, C-4, C-8 and C-9, and/or the conformation of the eventual C=C bond at C-3, C-8 and C-9, respectively. Indication of the configuration of a center of chirality precedes that of the conformation of

108 the C=C bond, and a stereogenic element in the "lower" region (C-1, C4) of the structure that in the "upper" region (C-1, C-8, C-9) of it. In both graphs H indicates structures, which are relevant in 8,10-dihydro derivatives, too. Structure numbers in Fig. (7) were italicized. In the interpretations of the changes it was considered that H-D exchange and epimerization at C-9 should run through enol-oxo equilibrium involving C-1 and C-9 at the level of structure 4(H) and/or 5(H), which have the highest energy in the reaction matrix. Equilibrium should go back till this level. As one can see from the energy graph (Fig. (7)), compounds isolated from the reaction mixture have the lowest relative free enthalpy of formation both in the "dihydro" and "natural" series ((20) corresponds to {12), (23) to {IH)), Actually, in the "natural" series, the estimated free enthalpy values of aglucone type 10 and 12 have nearly the same values. Although it could not be isolated or demonstrated, a small amount of (20a) corresponding to {10) can not be excluded to be formed during deglucosylation. However, it should be noted, that R. T. Brown and S. B. Pratt did isolate derivatives according to {10) under slightly different conditions of enzymatic deglucosylation [27]. Moreover, this type of substructure is well known in indole alkaloids of the yohimban type. The results are interpreted as follows (see Fig. (8)): According to experimental data, the enzymatic deglucosylation of (17a) and (17b) is accompanied only by simple epimerization at C-1, which can easily be explained by cleavage of the primary aglucone IRR(H) to 3ER(H) and recyclization either back into IRR(H) {=IRM' 18a-b) or into 1SR(H) (=15',9i?-18a-b) in a ratio of 9:1. The acidic deglucosylation is under thermodynamic control. In the case of the dihydro glucoside (17b), the events could likewise easily be interpreted. After hydrolytic removal of the glucosyloxy unit, the primary aglucone IRRH was spontaneously cleaved to 3ERH and tautomerized to 6RRH, which is already a member of the isomerization cycle, in which, through 5RZH and/or SREH, the configuration of C-9 could be epimerized. Retautomerization of 6RSH to 3ESH and recyclization to IRSH (=li?-23) and ISSH (=15'-23) in a ratio of 8:2 complete the reaction sequence. Trans orientation of C-6 and C-8 in the final product is favoured over the original cis one. In the acidic deglucosylation of the "natural" glucoside 17a, the mechanism is more complicated, because the vinyl group of structure {3) can easily be tautomerized into the more stable ethylidene group of

109 structure (7) (see the appropriate values of free enthalpy of formation in Fig. (7)), which could be recyclized not only into structure (9) but (after rotation around bond C-5-C-9) into structure {11), too. As the energy values of Fig. (7) show, formation of (72), through tautomerization of {11), is most favorable, being the most stable compound. Therefore, the shortest way from the educt to the products was explained as follows: the primary aglucone IRR isomerizes through 3ER to 6RR, and then further into the other members {5RZ, 5RE, 8RZ, 8RE, lORR, lOSR and 6RS) of the isomerization cycle. Finally 7EZ and 7EE could be formed, and recyclization of either of them to IISZ, USE, IIRZ and IIRE gave, after a second tautomerization, the most stable final aglucones 12SS (=85-20)and 12RS (=8i?-20) in a ratio of 6:4. The epimer ratio of the product components can approximately be estimated from the ^H-NMR spectrum. These ratios were interpreted by considering stabilizing anomer-type and destabilizing gauche interactions of the hgands of C-1 and C-9 in {IR'23=1RS) and {IS'23=1SSH) as well as of C-8 and C-9 in {8R'20=12RS) and {SS-20=12SS). Really, the aglucone epimers having the minimum number of gauche and maximum number of anomer-type effects were formed and isolated in higher amount (15'-23 and 85-20). As all steps (equilibria) are reversible (forwards and backwards) and catalized by proton (involving protonations and deprotonations), the complicated reaction matrix involves in the "natural" series 48 aglucones, 92 equilibria and 368 elementary steps, in the "dihydro" series 24 aglucones, 40 equilibria and 160 elementary steps. Although any of these structures and transformations can not be excluded, by graph analysis the shortest rational pathways were found for the interpretation of the events. The results could be used for the investigation of other cases in the bioorganic chemistry of indole and related alkaloids, too (see later). Fragmentation of simple secologanin derivatives As mentioned at the beginning of the previous chapter, in the acidic or basic deglucosylation, secologanin and some of its derivatives underwent deeper structural changes, which will be discussed in this chapter. The structural basis of these fragmentations is the special position of C-5, which has a stable S configuration with three different carbon ligands. The C2 and C4 ligands have one (C-7 and C-1, respectively), the C3 ligand two

no (C-3 and C-11) free or masked carbonyl groups all of them in p position to C-5 (see (If) of Fig. (1)). According to this special arrangement, each of the ligands could be detached from C-5 under different circumstances, depending on the other structural elements and the reaction conditions.

k^x:^ Fig. (9). Deglucosylation and fragmentation of secologanin and its ethylene acetal.

The cleavage of the C-4-C-5 bond is shown in Fig. (9). By acidic deglucosylation, secologanin (1) and its ethylene acetal (4a) gave surprisingly benzaldehyde (49). Probably, in the first phase, all acetalic groups as well as the ester group were hydrolized with formation of the aglucone represented by structure (43) in this case. In the second step, this intermediate underwent afragmentationalong bond C-4-C-5 according to a retro-Michael reaction (indicated by curved arrows in 43) and afforded a Cj (44) and a C3 (45) unit. Unfortunately, the latter fragment could not be detected, because, most probably, it was decomposed into carbon dioxide (46) and acetaldehyde (47). The sum of the oxidation levels of the carbon atoms in (44) already corresponded to that of benzaldehyde (49), which could be formed through (48) in an intramolecular aldol-type reaction (indicated likewise by curved arrows in 44) and aromatization by spontaneous elimination of water. It was mentioned previously that from sweroside no well defined product could be obtained, and from A^-

Ill

methylbakankosine (17a) and its dihydro derivative (17b) epimeric aglucone pairs (20) and (23), respectively, were formed under analogous conditions. As likewise mentioned previously, secologanin with primary amine gave the 7-substituted bakankosine derivative (12), with secondary amine the 7-substituted sweroside derivative (14). In both cases, the first site of the nucleophilic attack was the free formyl group. In secologanin ethylene acetal (6) where the formyl group was protected against nucleophilic attack, the site of the reaction was at C-3. The consequences are shown in Fig. (9). The adduct (50) of primary amine was automatically transformed by deglucosylation and subsequent ring opening and ring closure through (51) into a dihydropyridine derivative (52), which was analogous to (39). With secondary amine the result was again surprising, because from the reaction mixture methyl 3piperidinopropenoate (55) was isolated as the only product. For rationalization of the process, it was taken up that in the primary adduct (53) the p-D-glucopyranosyloxy group was removed in a base-catalyzed elimination, and (54) was formed, in which the structural conditions were appropriate for a re/ro-Diels-Alder reaction. This latter gave (55) and a C7 unit (56) again by the cleavage of bond C-4-C-5. Unfortunately, the hypothetic fragment (56) could not be obtained from the reaction mixture [28]. However, in tryptamine derivatives of secologanin, analogous reactions were observed, and both fragments of the same substrate were isolated (see later). Fig. (10) shows that in acid-catalyzed hydrolysis, 7-substituted bakankosine derivatives underwent an other type of fragmentation, in which bond C-5-C-6 or C-5-C-9, i. e. one of other two-carbon hgands of C-5 were cleaved. The reaction was studied on the oxazolo compound (57a) and its dihydro derivative (57b), but analogous changes were observed in other 7-substituted (hydroxy, methoxy or methylamino) A^alkylbakankosines as well.

112

U

|.,||H

U .iiOglc

Q

I ,,|H

^^X. = ^ ^

.J.OH

R=hydroxyethyl, methyl

3

57a 57b 8,10-dihydro

58b 8,10-dihydro |H30^

Fig. (10). Deglucosylation and fragmentation of 7-substituted bakankosine derivatives.

The reaction products depended on the presence (in the vinyl derivative 57a) or absence (in the ethyl derivative 57b) of the double bond of the side-chain. In both cases a pyridone derivative, (61) or (66, respectively, was formed, and from (57a) a C4 unit (probably crotonic aldehyde (62)), from (57b) a C2 unit (probably acetaldehyde (65)) was eliminated. In addition, from the vinyl compound (57a) afiirtherpyridone derivative (69) was formed as side product, which retained the C4 unit in the form of a C5 side chain. The educts could be deglucosylized by enzyme or aqueous acid to (58a) and (58b), and fiirther acidic treatment cleaved the oxazolidine ring giving the 7-hydroxy-aglucones (59a) and

113 (59b), respectively. These structures represented 12 (in the vinyl derivatives) or 6 (in the ethyl derivatives) structural isomers (and four times as many stereoisomers!), which were analogous to the structures shown in Fig. (7) and probably present in the reaction mixture. Our analysis started from these intermediates. The three most stable isomeric aglucones of them, (60), (63) and (67) corresponded to those of the bakankosine derivatives (20), (23) and (20a), respectively, and each of them were considered as a starting point of the formation of a pyridone aglucone (61), (66) and (69), respectively. The main driving force of the fragmentations is the formation of a stable pyridone system. However, in details the differences were important. The simplest case is the fragmentation of the aglucone (60) (corresponding to aglucone type (12) and formed from (57a)), in which the dihydropyran ring is already preformed for a r^/ro-hetero-Diels-Alder reaction, which could directly give the end product (61) (and probably 62). This fragmentation is impossible in the aglucone (63) (corresponding to aglucone type (IH) and formed from 57b). However, the most basic point of the system is the lactam O atom. It might be supposed that association of a proton to this site could polarize the total ring system (according to the curved arrows in (63)) and facilitate the fragmentation with elimination of a C2 unit (probably (65)). In the intermediate (64), by rotation (involving tautomerization and change in the geometry of the C=C bond), the C-1 atom could obtain a position, which is favourable to cyclization and formation the another pyridone system (66). The formation of the ring-opened side-product (69) might have been interpreted by a subsequent vinylogous intermolecular aldol reaction. However, (69) could not be prepared from the pyridone derivative (61) with, neither its amount increased by addition of crotonic aldehyde under the same conditions. Therefore, an intramolecular mechanism was looked for its formation. Structure (67) (corresponding to the third relatively stable aglucone type (10)) seemed to be a good starting point for the formation of (69). In (67), after elimination of water, the double bonds could be arranged into a position according to (68), which seemed to be favourable for a cycloreversion indicated by the curved arrows. Finally, in the strongly acidic medium the conjugated double bonds took up the trans-trans orientation to give (69) [results to be published].

114 Reinvestigation and completion of the coupling reaction of secologanin with biogenic amines One of the first reactions carried out with secologanin was its coupling reaction with tryptamine and dopamine. The pioneering work of Battersby and his group [29, 30] was later completed mainly by that of R. T. Brown and his co-workers [31-33]. It was demonstrated that the coupling reaction of secologanin (1) with tryptamine (70a) gave strictosidine (3iS'74a) and vincoside (3i?-74a) with low stereoselectivity [29]. Analogously, in the reaction of secologanin and dopamine 2-deacetyl-isoipecoside (IS81) and 2-deacetyl-ipecoside (li?-81) were formed [30]. However, the contemporary experimental techniques were limited in this respect, and some discrepancies and uncertainties were the consequences of this fact. Unfortunately, in both series the configuration of the newly formed center of chirahty (C-3 and C-1, respectively) were given to be opposite. Later, the assignments were corrected on the base of X-ray diffraction analysis of derivatives of vincoside [34] and ipecoside [35], but no direct proof was presented in the S series. The biosynthetic studies gave similar problems. Originally, vincoside (3R) and deacetylipecoside (IR) were considered as the precursors of the indole and ipecac alkaloids, respectively. However, Zenk and his group proved that the exclusive precursor of all indole alkaloids (including the 3R epimers of the la class) is strictosidine (35) [36, 37]. Moreover, it was found that the precursor of the alkaloid emetine (liS) is deacetylisoipecoside (IS), and that of the alkaloid glucoside alangiside (IR) is deacetylipecoside (li?) [38]. The coupling reaction with tryptamine has a single enzyme strictosidine synthase, in that with dopamine the activity of two enzymes was suggested. However, during purification, one of them disappeared [39]. In order to get insight into the stereochemistry of the coupling reactions, it was decided to reinvestigate and extend the reactions. Four type of coupling reactions were carried out, which are shown in Fig. (11) and Fig. (13). (The cyclic skeletons were numbered according to the biogenetic numbering [40]). For the description of the stereogenic elements of the products the following system was used: configuration of the new center of chirality (C-3 in tryptamine and oxotryptamine derivatives, C-1 in dopamine and histamine derivatives): R or S; type of conformation around C-14 (in dopamine derivatives C-11, in histamine derivatives C-10): 11, ... 33, (see Fig. (14)); conformation of the

115 dihydropyran ring (N or P); conformation of the tetrahydropyridine ring: N or P (in the tryptamine, dopamine and histamine derivatives) or configuration of C-7: R and S (in the oxotryptamine derivatives).

-H20. + H20 ,,iOglc(R2)^

I 71aR'=R2=H ^ 71b R'=4-bromobenzyl, R-=acctyl

21 in 3.V-75:S31NP ill 3/ ^ 0 H

3/C-21 0-17-+C-2I N-4-+C-22 0-l7-»C-21

0-17->C-21 N-1->C-21

3/?-90b 18,19-diliydro

0 3/?-90a (=3«-74a)

N-4-+C-22 0-17-»C-l9

3«-94a 19R(e):19 S(a)=3:2

3/?-92b 18.l9-dihydro

3/?-91b 18,19-dihydro

0

0

3;?-91a

3«-92a

0-17-».C-19

3/;-93b=3«-4-dcbciizyl-96b

3/?-93a=3«-4-dcbcn2yl-96a

0

N-J-+C-21

3/?-96a 2 1

3/f-95a 19R(e):l9 S(a)=2:l

+ H3COH

natural series

Fig. (18). Acidic deglucosylation of vincoside derivatives.

0-17^C-19 N-4-»C-21

3/?-97a 1 cpinicr

135

R'=HorMe R*=H or Bn

0-21->C-17toJr//> 0-17->C-21 10 5(H)

0-17^C-19to7 C-18->C-17to9

N( 1 or 4)-^C-17 to type 2(H) N(l or4)^C-21 to type ^ W N(I or 4HC-22 to type 6(H) N(l o r 4 H C - 1 9 t o t y p e »

Fig. (19). Possible interactions in vincoside primary aglucone.

The tetracyclic structures formed by oxa(carba)cyclization (types 3(H), 5(H), 7 and 9), are analogous to structures of the bakankosine derivatives {1(H), 2(H), 12 and 10, respectively in Fig. (7) and Fig. (8)). The tetracyclic structures formed by azacyclization (types 2(H), 4(H), 6(H) and 8) have series A and B according the nucleophiles N-1 and N-4, respectively. Combination of the types of cyclizations by two different ways gave the 16 pentacyclic structures in series A and B. Further explanations can be found in the original paper [52]. Evidently, in most cases the transformations are under thermodynamic control. The structure of secologanin subunit of all isolated aglucones corresponded to the energetically more favoured structures in agreement with the results obtained on the bakankosine derivatives. It means that the favoured direction of the oxacychzation is 0-17-^C-21 in the dihydro derivatives (according to type IH of Fig. (7)), and 0-17->C-19 in the natural ones (according to type 12 of Fig. (7)). The result of azacyclization depended on the atom N-1 and N-4. When both N atoms were protected (3i?-92a) and (3i?-92b), the process stopped at the tetracyclic level (3J?-96a) and (3i?-96b). When both of them were unprotected (in (3i?-90a) and (3J?-90b)), the azacyclization preceded the

136

eglucosylation, and afforded the thermodynamically more stable N-4 lactamized products (3/?-94a) and (3i?-94b). alkaloid types derived from the dihydro glycosides, too

alkaloid types derived from the natural glycosides, only

Fig. (20). Structure graph of the possible aglucones of strictosidine and vincoside derivatives.

When only N-1 was free and N-4 protected (in (3i?-91a) and (3i?-91b)), azacyclization took place in both series with participation of C-21 instead of C-22 and gave the products (3i?-95a) and (3i?-95b). Evidently, the tetrahedric intermediates formed by participation of C-22 could not be stabilezed into the planar lactam group at N-1. It was mentioned above that the compound, in which N-1 was protected and N-4 free could not be prepared. Therefore, only the subsequent azacyclization of their hypothetic aglucones could be studied. It was found that in the natural series N-4 of the aglucone (3i?-93a)=(3/?-4-debenzyl-96a) cyclized to the C=0 in position 21, and in a subsequent reaction the 21-hydroxy group

137 was removed by catalytic hydrogenation to (3i?-97a). In the dihydro series, the cyclic hemiacetal of the aglucone (3i?-93b)=(3i?-4-debenzyl96) was cleaved spontaneously into the hydroxy-oxo compound, and the azacyclization took place again with participation of N-4 and C-21; finally, the subsequent removal of the hydroxy group by hydrogenolysis gave the end product (3i?-97b) [52]. In the vincoside series the reactivity order of the functional groups can be summarized as follows. The preferred nucleophilic site is N-4 over N-1 and 0-17 over 0-21 (C-18 has an intermediate position in the natural series). The preferred electrophile is C-19 over C-21 and C-17 in the natural series, and C-21 over C-17 in the dihydro series. C-22 is the preferred electrophile only in the vincoside series when lactamization preceded the deglucosylation. However, it seems from the work of De Silva and co-workers [53] that in the strictosidine series, in which the lactamization is slow, the preferred electrophile is C-17 over the others. Fragmentations in tryptamine derivatives of secologanin Because in the first product of the coupling reactions of secologanin or its tetraacetyl derivative with biogenic amines, the secologanin subunit remains intact (except the formyl group of C-7), fragmentations analogous to secologanin and its simple derivatives were expected, and really observed. The reactions were investigated in 3S epimer of the oxotryptamine and the 3R epimer of the tryptamine derivative. In order to prevent lactamization, the A^-4 atom was benzylated. In aqueous acid the coupled product (3iS'-78c) of A^-benzyl oxotryptamine (76c) and secologanin (1) gave back the educt A^^-benzyl oxotryptamine (76c), and benzaldehyde (49) (Fig. (21)). As it is known that the coupling reaction with 2-oxotryptamine derivatives is reversible, the fragmentation was interpreted as a retro version of the coupling reaction, followed by the fragmentation of secologanin or its aglucone according to Fig. (9). It is not known, on which level the glucosidic unit was removed. In the tryptamine series, acidic deglucosylation of (3i?-74c) (Fig. (22)) gave the pentacyclic compound (3i?-95a) (see in Fig (18), according to the most stable secologanin aglucone type and cyclized between C-21 and N1 without fragmentation.

138

piperidine

35-78C (R.=benzyl, R2=H)

p^ Q^

76cR'=benzyl(Bn)

^

Fig. (21). Fragmentation of the spirooxo derivative of benzylstrictosidine.

The basic deglucosylation of (35-780) (Fig. (21) and (3i?-74c) Fig. (22) gave methyl 3-piperidinopropenoate (55), and, in addition TV^^^b-dibenzyl2-oxotryptamine (105) or A^b^^b'dibenzyl tryptamine (112), respectively. It was supposed that in both series the first changes in the reaction sequence were analogous to that observed in the case of secologanin ethylene acetal (4a) (Fig. (9)), i. e. the primary adduct (99) and (106) underwent a basecatalyzed deglucosylation followed by a re/ra-Diels-Alder reaction, which afforded (55) and formally the hypothetic fragment (100) and (107), respectively. Evidently, the second benzyl group of (105) and (112) was formed from the C7 subunit of these fragments. The necessary requirement for this transformation was the cleavage of bonds C-3-N-4 and C-3-C-7 in (100) or C-3-C-2 in (107), i. e. the reversal of the coupling (Mannich-type) reaction combined with the formation of the new bond N-4-C-21. In the oxotryptamine series the reaction sequence could easily been interpreted by cooperation of the solvent (and catalytic effect of the base) as indicated by arrows in structures from (100) to

139 (105). However, it is not known, at which point was the reduction step at C-21 inserted. In the triptamine series, the final steps from (107a) to (112) were supposed to be analogous, but no experimental support could be obtained for the existence of the indolenine structure (107a), which should have be formed by reversal of the rearrangement of the spiro into the fused system.

Fig. (22). Fragmentation of benzylvincoside.

A final remark was made concerning the step of the reduction of C-21. It was observed that the yield of the final products (105) and (112), respectively, was less than 50%. However, in the tryptamine series, the formation of a side product with a supposed structure (109) was observed. Unfortunately, it could not be obtained in pure state, but the ^H NMR spectrum of the crude product was close to that of the adduct (106) except the signal of C-17, which appeared at the olefinic region. This fact suggested the dehydrogenation of the bond C-16-C-17 at the cost of the reduction of C-21 in the hypothetic intermediate (108). Thin layer chromatography showed that the main product (112) and side-product

140 (109) were formed in approximately 1:1 ratio. Moreover, if the basic deglucosylation of (3i?-74c) was carried out in the presence of sodium tetrahydridoborate, the total amount of the educt could be transformed into (112), however, in the presence of potassium hexacyanoferrate (112) was not formed at all. The formation of (102) analogous to (109) was expected in the oxindol series, too, but it could not be demonstrated [results to be published].

0

COCH3

stemmadenine derivative (I)

H3CO "

O

114 dehydrosecodine

H3CO

\

115 catharanthine (II)

CO2CH3

H3CO "

O

116 tabersonine (III)

"corynanthean-type derivative"

118 "sarpagan-type derivative"

Fig. (23). Bioorganic significance of the fragmentation of secologanin derivatives.

The bioorganic significance of these fragmentations (together with those observed in simple secologanin derivatives) may be evaluated as follows (Fig. (23)): 1. The cleavage of bond C-15-C-16 is the same, which was supposed to be cleaved in biosynthethic pass from the acetyl derivative (113) of the type I indole alkaloid stemmadenin through a secodine derivative (114) toward the type II (ibogan, e. g. catharanthine (115)) and type III (plumeran, e. g. aspidospermidine (116)) alkaloids, and it may be served as a model in the biogenesis of these alkaloids [54]. 2. Thefragmentationsof Fig. (9), Fig. (10), Fig. (21) and Fig. (22) involved C-5 (in simple secologanin derivatives) or C-15 (in tryptamine derivatives of secologanin), respectively, as only unfixnctionalized carbon atoms. However, the reactivity of C-4 has parallel in the formation of the sarpagan (e. g. (118)) (and ajmalan) derivatives from corynanthean

141

derivatives (117) 55, 56]. 3. A new, possibly biogenetic and really biomimetic mode of aromatization has been detected in the cychzation of a linear subunit of terpenoid origine. Two interesting structures Finally, two special compounds are shown in Fig. (24). They represent the several isolated and many possible structures, which may have interesting physical, chemical and probable biological properties. Compound (119) is a trioxadamantane derivative, which was prepared from the methanol adduct (6) of secologanin dimethylacetal (4a), in which all masked carbonyl groups were in acetal form. Aqueous acidic hydrolysis, under termodynamic control, transformed them groups of (6) into totally cyclized acetal structure. The backbone of the molecule has a high symmetry, which is broken by the functional groups [15]. Compound (120) was prepared from 8,10-dihydrosweroside aglucone (32), which could be alkylated in aprotic solvent in the presence of a strong base to (33). However, under the same conditions, but in the absence of the alkylating agent, a sequence of four base-catalyzed reactions (dimerization by a vinylogous Michael addition, intramolecular aldolization, lactonization and cyclization by hetero Michael addition) gave the final product (120) . Detailed analysis of its formation would exceed the frame of this paper [result to be pubHshed].

Fig. (24). Two interesting structures prepared from secologanin.

The bioactive properties of secologanin and their derivatives Secologanin and their congeners as well as their alkaloid derivatives are bioactive compounds. In the literature many iridoids and secoirdoids were mentioned to have bioactive, pharmacological and sometimes even therapeutical properties.

142 Some of them are shown in Table (3). However, it seems that detailed investigations have not been carried out, the reported activities were not strong or characteristic, and neither of the compounds applied in clinical praxis. Table 3. Biological Activity of some iridoids 1 Compound

Biological activity

lAUamdin

antileukemic

Elenolic acid

antiviral

Geniposide

laxative

Harpagoside

analgetic, antiinflammatory

pieuropeine

hypotensive

Plumericine

antimicrobial

Sweroside

hepatoprotevtive

Swertiamarine

bitter taste

Valepotriates

sedative

The situation is quite opposite in the nitrogen containing derivatives (i. e. alkaloids) of secologanin. Many of them have marked, in most cases strong biological activities, which could even be applied for therapeutical purposes. Table (4) shows some of the representatives of the indole and related alkaloids having more or less expressed biological or pharmacological activity. Although, many of these activities were not well documented neither investigated in details, several of the alkaloids proved to be highly active, and used not only in physiological and pharmacological research but also in clinical praxis. A selection of them is shown in Fig. (25). Strychnine (from Strychnos nux vomica L.), one of the first alkaloids isolated in crystallin form and a notorious poison, has a very strong stimulating effect (LD50: 0.96 mg/kg) on the motorial inhibitory neurons of the spinal cord as a consequence of the competitive antagonism at the glycine receptor. It can not be used as a therapeutic agent, but useful in pharmacological experiments. Ctoxiferine-I (from Strychnos toxifera F. Schomb.), a bis-quaternary dimer of a strychnan type subunit, is a very potent non-depolarizing, curare-like, nicotinic antagonist at the neuromuscular junction (LDjoo* 23 |ag/kg). Its hemisynthetic bisnor-bisallyl derivative Alcuronium was developped as a clinically potent skeletal muscle relaxant, which is used in the therapeutical praxis as well.

143 Table 4. Biological Activity of some alkaloids derived from secologanin Compound

Structure Type

[Source

Ajmalicine

l a (corynanthean)

ICotharantus roseus

Pharmacological Activity Hypotensive

Ajmaline

la (corynanthean)

IRauvolfia sp.

Antiarrythmic

Alcuronium

Ip (strychnan, "dimer")

hemisynthetic

Skeletal muscle relaxant

Camptothecine

special

Camptotheca acuminata

Anticancer

Catharathine

11(3 (ibogan)

Catharanthus roseus

Hypoglycemic

Coronaridine

up (ibogan)

Catharanthus roseus

Diuretic, cytotoxic

Ellipticine

special

Ochrosia elliptica

Antitumor

Emetine

isoquinoline skeleton

Cephaelis ipecacuanha

Antiamoebic, emetic, expectorant

Deserpidine

la (corynanthean)

Rauvolfia canescens

Hypotensive

Ibogain

lip (ibogan)

Tabernanthe iboga

Psychotomimetic

Mitraphylline

Ip (strychnan)

Mitrgyna rotundifolia

Weak depressant, hypotensive

Olivacine

special

Aspidosperma

Antileukemic

Quinidine

ruban

Remijia sp.

Cardiac depressant

Quinine

ruban

Cinchona sp.

Antimalarial

Rescinnamine

la (corynanthean)

olivaceum

Rauvolfia sp.

Hypotensive

Rhynchophyllin Ip (strychnan)

Uncaria rynchophyalla

Neuroprotective

e

la (corynanthean)

Rauvolfia vomitoria

Hypotensive

Reserpine

ip (strychnan)

Strychnos nux vomica

CNS depressant

Strychnine

Ip (strychnan, "dimer")

Strychnos toxifera

Skeletal muscle relaxant

Toxiferine I

IIP+IIIP ("dimer")

Catharanthus roseus

Antineoplastic

Vinblastine

Iip+IIip ("dimer")

Catharanthus roseus

Antineoplastic

Vincristine

Ilia (ebuman)

Vinca major

Hypotensive

Vincamine

Iip+IIIp ("dimer")

hemisynthetic

Antineoplastic

Vindesine

IIip (plumeran)

Catharanthus roseus

Substructure of vinblastine

Vindoline

Iip+IIIp ("dimer")

hemisynthetic

Antineoplastic

[a (corynanthean)

Corynanthe johimbe

a-adrenergic blocker

Vinorelbine Yohimbine

1

Yohimbine (from Corynanthe johimbe K. Schum.) is an a2 adrenoreceptor antagonist and applied in the pharmacological research. Its aphrodisiac activity was not proved, although it is used in the treatment of impotentia of men. Reserpine (from Rauwolfia serpentina L. Benth.) was introduced into the therapy in the middle of the 20*^ century as a hypotensive and neuroleptic agent. It depletes the biogenic amines (among others dopamine and serotonine) in the brain. Because of some unfavoured side effects it is already not used in the therapy, but as a lead compound had an important influence on the treatment of hypertension, strongly catalysed the progress of the physiology of neuronal transmission and (among others) initiated the golden age of the psychotherapy. All these factors contributed to the explosional development of the chemistry of the indole and related alkaloids. Vincamine (from Vinca minor L.) is

144 also an alkaloid, which was first applied in the treatment of hypertension. As a lead compound it stimulated the preparation and investigation of many related natural, hemi- or totally synthetic compounds. Its close derivative vinpocetine is even now used as a cerebral vasodilator. Two "dimer" indole alkaloids, vinblastine and vincristine (from Catharanthus roseus G. Don.), are still in the center of interest of the pharmacologists. They have antineoplastic activity and used in the therapy of certain types of leukemia. In the cells they inhibit the formation of microtubulles and by that way hinder the cell division. Among many newer, hemisynthetic derivatives of them are Vindesine and Vinorelbine.

RO2C' strychnine

vincamine, R=Me vinpocetine, R=Et, 16-dehyclroxy, 16,17-didehydro

H3CO

vinblastine, R'=CH3, R2=C00Me, R^=0C0CH3 vincristine, R'=CHO, R2=C00Me, R3=OCOCH3 vindesine, R'=CH3, R2=CONH2, R^=OH vinorelbine, R'=CH3, R2=C00Me, R3=OCOCH3, 20'-dehydroxy-15',20'-didehydro

R2

C-Toxiferine-I, R=Me Alkuronium, R=allyl

yohimbine, 3a-H, 203-H, lea-MeOiC R^=OH, R^=H rescrpine, 3P-H, 20a-H, 1 l-OMe, lep-MeO^C, R2=OMe, R3=3',4',5'-trimethoxybenzoyloxy

Fig. (25). Some selected therapeutically active derivatives of secologanin.

Two further antineoplastic compounds derived from coupling of secologanine and tryptamine but having a quinoline ring are camptothecine. Fig (2), (fi'om Camptotheca acuminata Decsne.) and olivacine {Aspidosperma olivaceum Miill. Arg.). Both of them are currently being investigated as anticancer drugs, but up to now they were not widely be used in the therapy. One of the oldest medicaments used against malaria was the Cinchona bark whose main active agent is quinine (see in Fig. (2)) (from Cinchona ledgeriana Moens). While it is still used as such, it was the most important lead compound in the search for more potent antimalarial agents during World War II. In a huge international

145 cooperation, several thousands of derivatives and other compounds were prepared and tested, some of which proved to be therapeutically useful. Quinidine, a diastereomer of it, is occasionally used as an antiarrhythmic agent. Among the isoquinoline derivatives derived from secologanin and dopamine, emetine (see in Fig. (2)), (from Cephaelis ipecacuanha (Biot.) A. Rich), one of the first alkaloids isolated from plants, was used as an antiamoebic, emetic and expectorant agent, but it had unfavourable side effects. This brief summary shows convincingly that even now, derivatives of secologanin should be considered as potent and potentially rich source of bioactive and terapeutically useful compounds. CONCLUSIONS The experimental work carried out on secologanin by us and several other groups during the last 30 years as well as the large number of alkaloids with high biological activity synthesized by plants from secologanin proved that this terpenoid glucoside is one of the most versatile synthons of nature. The work with alkaloids derived from secologanin largely contributed to the chemistry of indole and isoquinoline compounds and to the development of important physicalchemical methods as mass spectrometry and nuclear magnetic resonance spectroscopy. It catalyzed the elaboration of new isolation techniques and the biosynthetic research. In the last half century, many of these alkaloids were targets of huge and successful synthetic efforts (although the total synthesis of secologanin still fails). The total syntheses of quinine, strychnine, reserpine, emetine, vincamine, and many others were milestones of organic synthesis and elaboration of new, versatile synthetic methods and conceptions. It may be expected, that the investigation of these topics can give surprising and important results in the future too. Some of the results presented in this paper may contribute to the interpretation of the biogenetic chemistry of secologanin as well. Moreover, the chemistry of secologanin clearly demonstrates one of the important principles of Nature: to construct a large variety of more or less complicated molecular architectures by combination of simple building stones. It is a special type of bioorganic combinatorial chemistry.

146 Acknowledgements The results presented in this paper could not have be realized without the experimental and intellectual work of my co-workers and friends Gy. Beke, L. Karolyhazy, A. Kocsis, A. Patthy-Lukats, A. Schwartz and K. Szabo-Pusztay. I express my special gratitude to all of them. Most of the NMR investigations were carried out by B. Podanyi (Chinoin Pharmaceutical and Chemical Works, Ltd, - a member of the Sanofi-Synthelabo group, Budapest). I am indebted very much for his excellent work. The contributions of K. Bqjthe-Horvath, F. Hetenyi, G. Krajsovszky, Z. Pal and M. Varga-Balazs in different phases of this work are also thankfully appreciated. I express my sincere thanks to Prof. A. I. Scott for the generous supply of the plasmid containing the gene of strictosidine synthase and for the practical instructions concerning the work with it. The financial support of this work by the National Scientific Research Foundation (OTKA) and the Research Institute of Medicinal Plants (Director: P. Tetenyi sen.) is gratefully acknowledged.

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[10] Szabo, L.; Bojthe-Horvath, K.; Hetenyi, F.; Kocsis, A.; Pal, Z.; Varga-Balazs, M.; Tetenyi, P. F. E. C S. Int. Conf. Chem. Biotechnol Biol Act. Nat. Prod., F\ 1981, Short Communications, 1981, 3/1, 87-91. [11] Kinast, G.; Tietze, L-F. Chem. Ber. 1976,109, 3640-3645. [12] Brown, R. T.; Chappie, C. L, Tetrahedron Lett. 1976, 787-790. [13] Tietze, L.-F., Henke, S. Angew. Chem. 1976, 93, 1005. [14] Kawai, H.; Kuroyanagi, M.; Ueno, A. Chem. Pharm. Bull. 1988, 36, 3664-3666. [15] Krajsovszky, G.; Kocsis, A.; Szabo, L. F.; Podanyi, B. Tetrahedron, 1997, 53, 11659-11668. [16] Brown, R. T.; Chappie, C. L.; Duckworth, D. M.; Piatt, R. J. Chem. Soc. Perkin I, 1976, 160-162 [17] Purdy, J. R.; Hamilton, R. G.; Akhter, L.; McLean, S. Can. J. Chem. 1981, 59, 210214. [18] Saunders, G. N.; Purdy, J. R.; McLean, S. Can .J. Chem. 1983, 61, 276-281. [19] Pal, Z.; Varga-Balazs, M.; Szabo, L. F.; Tetenyi, P. In Bio-organic Heterocycles. H. C. van der Plas, L. Otvos, M. Simonyi, Ed.; Akademiai Kiado, Budapest, 1984, pp. 221-224. [20] Kocsis, A.; Pal, Z.; Patthy, A, Mrs.; Szabo, L.; Varga, J. Mrs.; Tetenyi, P. Hung Teljes HU 47304, Febr. 28, 1989; Chem. Abstr. 1990,112, 70033tq. [21] Tietze, L.-F.; Baertels, C; Fennen, J. Liehigs Ann. Chem. 1989, 1241-1246. [22] Tietze, L.-F. Tetrehedron Lett. 1976, 2535-2538. [23] Kocsis, A.; Pal, Z.; Patthy, A. Mrs.; Szab6, L.; Varga, J., Tetenyi, P. Teljes, HU 48,640, June 28, 1989; Chem. Abstr. 1990, 112, 132478c. [24] Schwartz, A.; Szabo, L. F.; Podanyi, B. Tetrahedron, 1997, 53, 10489-10502. [25] Tietze, L. F.; Baertels, C. Tetrahedron, 1989, 45, 681-686. [26] Szabo-Pusztay K.; Szabo L. F.; Podanyi B. Acta Chimica Hungarica. Models in Chemistry, 1994,131, 475-488. [27] R. T. Brown, S. B. Pratt, J. Chem. Soc. Chem. Commun. 1980, 165-167. [28] Karolyhazy, L.; Patthy-Lukats, A.; Szabo, L. F.; Podanyi, B. Tetrahedron Lett. 2000,^7,1575-1578. [29] Battersby, A. R.; Burnett, A. R.; Parsons, P. G. J. Chem. Soc. (C) 1969, 1193-1200. [30] Battersby, A. R., Burnett, A. R., Parsons, P. G. J. Chem. Soc. (C) 1969, 1187-1192. [31] Brown, R. T. In Indole and Biogenetically Related Alkaloids. Phillipson, J. D.; Zenk, M. H. Eds.; Academic Press, 1980, 171-184. [32] R. S. Kapil, R. T. Brown, in: The Alkaloids; R. H. F, Manske, R. Rodrigo, R., Eds.; Academic Press, New York, 1979; Vol. 18, pp. 545-588. [33] Brown, R. T.; Curless, D. Tetrahedron Lett. 1986, 27, 6005-6008. [34] Mattes, K. C; Hutchinson, C. R.; Springer, J, P.; Clardy, J. J. Am. Chem. Soc, 1975,97,6210-6271. [35] Kennard, O., Roberts, P. J., Isaacs, N. W., Allen, F. H., Motherwell, W. D. S., Gibson, K. H., Battersby, A. R. Chem Commun. 1971, 899-900. [36] Stockigt, J; Zenk, M. H. Chem Commun. 1977, 646-648. [37] Rueffer, M.; Nagakura, N.; Zenk, M. H. Tetrahedron Lett. 1978, 1593-1596. [38] Nagakura, N.; Hofle, G.; Coggiola, D.; Zenk, M. H. Planta Med. 34, 381-389. [39] De-Eknamkul, W., Ounaroon, A., Tanahashi, T., Kutchan, T. M., Zenk, M. H. Phytochemistry, 1997, 45, 477-484. [40] Le Men, J.; Taylor, W. L, Experientia, 1965, 21, 508.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

149

THE LIGNANS OF PODOPHYLLUM RITA M. MORAES National Center for Natural Products Research, The Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA FRANCK E. DAY AN and CAMILO CANEL USDA, ARS, NPURU, National Center for Natural Products Research, University, MS 38677, USA ABSTRACT: Lignans are a widely distributed class of dimeric phenylpropanoid derivatives, many of which have strong antimicrobial, antiviral, or antifeedant activity and thus play important roles in plant defense. Of more restricted taxonomic distribution, the aryltetralin lignans have been found in highest abundance in plants of the genus Podophyllum (Berberidaceae). Foremost among these lignans, podophyllotoxin is a particularly cytotoxic inhibitor of microtubule assembly and a strong antiviral agent. Semisynthetic epimeric derivatives of podophyllotoxin having inhibitory activity against DNA-topoisomerase II have been developed as effective antineoplastic drugs. Current work on Podophyllum lignans is focused on two fronts: 1) Structure optimization to generate derivatives with superior pharmacological profiles and broader therapeutic use, and 2) Development of alternative sources of podophyllotoxin. Numerous variations of the basic aryltetralin structure have been created. Some of the new compounds have shown promising activity profiles, but practically little has been achieved besides improvement in solubility. Interest in new derivatives remains strong, which, along with the formulation of existing drugs for new indications, is increasing the demand for podophyllotoxin. While intense collection has severely reduced the natural stocks of Indian Podophyllum, the primary source of podophyllotoxin, a North American species has emerged as a rich and renewable source of this compound.

HISTORICAL BACKGROUND The genus Podophyllum (Berberidaceae) is most prominently represented by P. peltatum L., Fig. (1), in the United States and P, emodi Wall. (syn. P. hexandrum Royle) in northern India and Nepal. Extracts of dried rhizomes of mayapple and bankakri have a long history of use by the

Fig. (2). Crlstal \tructurc and ahwlute conliguration of I‘-broinopodoph) llt#o\in tlIu\trating the mostly planai tetralin hachhone. the envelop confhrination ofthc lactwic ring. and the freely rotating aryl ring. Whitc - carbon. red oxygen: grcen - hrointne; light blue - hydrogen

Fig. (I). f’odoph) Ilutn pcltatuin in natiiral habitat

151 indigenous populations of North America and the Himalayas, respectively, who valued their cathartic and cholagogue properties. European settlers of North America adopted the use of extracts of P. peltatum, which led to its introduction in Western pharmacopoeias. However, podophyllotoxin, Fig. (2), was shown to possess colchicine-like toxicity in 1891 [1], causing a decline in the therapeutic use of the extracts. The demonstration, in 1942, of the resin's efficacy in the treatment of condylomata acuminata [2], rekindled interest in podophyllotoxin and raised hopes for its use in the treatment of malignant tumors. The resin was later shown to possess potent antimitotic properties [3]. A report describing the results of preliminary screening of hundreds of compounds, performed at the National Cancer Institute, highlighted the capacity of a single dose of podophyllotoxin to grossly damage tumors in mice [4]. Subsequent clinical tests showed that podophyllotoxin caused severe abdominal pain, vomiting, and diarrhea, which made the compound unacceptable as a drug. The belief that glycoside derivatives of podophyllotoxin would exhibit superior pharmacological profiles led researchers to extract and test lignan glycosides from Podophyllum tissues. Being more water-soluble, the glycosides indeed proved less toxic, but showed reduced antitumor activity. Although not immediately successful, this approach eventually led to the serendipitous discovery of glycosidic derivatives of podophyllotoxin that proved to be potent anticancer agents. An insightful account of the development of podophyllotoxin into the antineoplastic drug etoposide has been published [5]. DISTRIBUTION AND BIOLOGY OF PODOPHYLLUM By 1970, in the wake of efforts to capitalize on the newly realized medicinal value of podophyllotoxin for Western medicine, more than 130 tons of Podophyllum rhizomes were needed in the US annually to satisfy the demand for the compound [6]. This plant material was wild-harvested from the understory of oak and hickory forests of the eastern and central US. In 1974, mayapple was considered a common plant with a natural habitat extending from South Carolina to Maine along the Atlantic coast in the East, to eastern Texas, Oklahoma, and Kansas beyond the Mississippi basin in the West, and as far North as Lake Michigan [6]. Today, mayapple is commonly seen along roadsides and can still be abundantly found in other readily accessible areas within its natural range. Several

152 colonies have been found near roads in the town of Oxford, Mississippi, where our laboratories are located. Mayapple is a perennial herb that forms highly branched rhizome systems, with new rhizome segments appearing each year. Consequently, colonies of mayapple often consist of hundreds of closely spaced shoots that are interconnected through extensive rhizome systems covering several hundred square meters. Each shoot supports the growth of one or more new rhizome segments, each of which has a terminal bud that remains dormant over the winter. The shoots senesce at the end of the growing season and detach from the rhizome, leaving a dormant bud at the node. The terminal buds develop into annual aerial shoots the following spring. New shoots may also develop from older dormant nodes, especially if apical dominance is affected by damage to the terminal bud. Even though mayapple is self-incompatible, the genetic diversity within mayapple colonies is highly restricted [7]. This is the result of a combination of factors such as high rates of flower and fruit abortion, low rates of germination, and high seedling mortality. Nevertheless, sexual shoots, which can be easily identified by the presence of two or more leaves, are very common in mayapple colonies. Because they carry at least twice the number of leaves, the photosynthetic area of sexual shoots is approximately twice as large as that of asexual shoots [8]. Studies of the relation between rhizome growth and shoot development indicate that photosynthetic productivity, a function of the size of the photosynthetic area and its longevity, strongly affects the development of new rhizome segments. For instance, late senescing shoots produce more numerous, longer, and heavier new rhizome segments than early senescing ones [9]. Similarly, sexual nodes are twice as likely to branch as vegetative nodes [8] and produce more massive rhizomes than asexual, single-leafed shoots [9]. In contrast, successful fruiting results in less rhizome growth; new rhizome segments produced by fruiting nodes are shorter than those produced by vegetative or non-fruiting sexual nodes [7]. Stored photosynthesis-derived resources appear destined to support fruit development, but, upon flower or fruit abortion, are used instead to produce larger and more numerous new rhizome segments. In view of the high cost of fruit production, the low success rate of sexual reproduction, and the added vigor and increased shoot number that result from flower and fruit abortion, the question arises why P. peltatum has not abandoned propagation by seed altogether. The persistence of sexual development in mayapple must have demographic implications that

153

are still not understood. Interestingly, longer and heavier rhizomes are more likely to give rise to sexual, multi-leafed shoots [9]. A model has been proposed, according to which fruit failure increases the chance of success of sexual reproduction in the future and therefore decreases the probability of the colony's extinction [7]. Given the wide geographic distribution of mayapple, seedling establishment, albeit infrequent, appears to be more than just insurance for survival against possible colony destruction by pathogens and severe weather. The colonization of such an extensive area must have resulted from production and dispersal of viable seeds. Interest as a commercial source of raw material turned to Indian Podophyllum when its rhizomes were found to contain more podophyllotoxin than those of the American species [10]. P. emodi is a perennial rhizomatous herb found in the Himalayas at altitudes of 2,000 to 4,000 m. It grows in the understory of subalpine forests, and near boulders and in open meadows above the tree line [11]. The suggestion that P. peltatum is conditioned to a winter rest period induced by coldness and requires a mean January temperature of no less than -5°C and a growth season of at least 150 frost-free days [6] seems to apply to P. emodi as well, and may explain why the latter is not found in the Indian plains or above 4,000 m in the Himalayas. Commercial interest in P. emodi has driven the species to an endangered status. The size of natural populations of P. emodi has declined sharply in recent years due to intensive collection, lack of cultivation, and loss of habitat to grazing. The present distribution of natural populations is relatively restricted. In Garhwal Himalaya, for instance, several populations first studied in 1982 have virtually disappeared [11]. During a recent visit to the Himalayas, specimens of P. emodi were found in the understory of a protected cedar forest with a distribution of approximately one every 100 m^ [Canel, personal observations]. Based on the number of leaves and the presence of reproductive structures, many of these plants were several years old. In all cases, the plants had a rhizome no more than 10 cm long with a profuse root system and no evidence of branching. In the face of declining natural populations of P. emodi, a valiant effort is being made by Indian research institutions to rescue the species. The study of the population biology and genetic diversity of P. emodi is considered essential for the success of these efforts. For instance, under the direction of Prof. S. N. Raina, researchers of the Department of Botany of the University of Delhi are collecting specimens of Indian Podophyllum

154 from natural populations in the Western Himalayas. These populations are being characterized at the cytogenetic level to assess the existing genetic diversity. Representative specimens are being transplanted to experimental plots in the Himalayan foothills. Similarly, a group at the GB Pant Institute of Himalayan Environment and Development in Garhwal has established a demonstration garden for the cultivation of P. emodi and is engaged in replenishing the disappearing natural stocks with rhizome cuttings, viable seeds, and plants regenerated from embryogenic calli. A fairly high level of genetic diversity seems to exist among and within populations of P. emodi. Isozyme analysis indicated that there is considerable inter- as well as intra-population variation [11]. This offers hope for the success of conservation efforts. The seed biology of Indian Podophyllum is also being studied in order to enhance the availability of this plant. At the Institute of Himalayan Bioresource Technology in Palampur, researchers have subjected seeds to various dormancy-breaking treatments such as chilling, application of hormones, and mechanical scarification. Chilling followed by scarification resulted in a germination rate of 80% within 7 days, whereas embryo rescue produced germination rates nearing 100% [12]. These efforts are especially laudable given the remoteness and difficult accessibility of the natural habitat of P. emodi, and the low level of funding with which some of these institutions operate. STRUCTURE AND BIOSYNTHESIS Lignans are a diverse and ubiquitously distributed group of phytochemicals that are biosynthetically derived from the phenylpropanoid pathway. The structural diversity and taxonomic occurrence of lignans and related compounds have been reviewed [13, 14]. Podophyllum tissues contain lignans of the fran^-aryltetralinlactone type and their 4-0-y9-D-glucopyranosides, among which podophyllotoxin is of most interest. The approved nomenclature for compounds having the aryltetralin carbon skeleton is described in Fig. (3). Podophyllum lignans have 5 rings designated A, B, C, D, and E. Ring A consists of a 1,3dioxolane cycle, rings B and C together form the 1,2,3,4tetrahydronaphthalene substructure, ring D is composed of a y- lactone, and ring E, connected at CI, is an aryl side chain. Carbon 4 carries the hydroxyl group involved in the glycosidic linkage, whereas C2 and C3 are involved in the lactone ring D [15].

155

Rj = H, R2 = OH: podophyllotoxin Rj = OH, R2 = H: epipodophyllotoxin Fig. (3). Nomenclature of aryltetralins

Bonds projecting below and above the plane of the tetracyclic backbone are referred to as a and p, respectively. The a diastereomer at C4 is knovm as podophyllotoxin, whereas the fi diastereomer is called epipodophyllotoxin, Fig. (3). The lactone ring always has the a,/?configuration at the C2,C3 bond, except for the unnatural picropodophyllotoxin obtained as an artifact during extraction [16]. The X-ray analysis of crystalized 2'-bromopodophyllotoxin demonstrated that rings A, B, C, and D form a nearly planar structure with the aryl ring connected to CI being pseudo-axial to ring C, Fig. (2) [17]. The dioxolane ring A is coplanar with ring B, and the lactone ring D exhibits the expected envelope conformation with C3 bending down to form a 'flap' and the remaining atoms of the lactone ring being coplanar. The biosynthesis of lignans has received comprehensive coverage in the recent literature [18]. Lignans are formed by the enzymatic stereospecific linkage of monolignol precursors, most commonly through 8-8' bonds. Although the pathway from monolignol to podophyllotoxin has not been unambiguously demonstrated, the experimental evidence

156 accumulated thus far strongly indicates that podophyllotoxin and congeners found in Podophyllum are synthesized from coniferyl alcohol, Fig. (4). For instance, roots of P. hexandrum incorporated isotopically labeled coniferyl alcohol into podophyllotoxin [19]. Similarly, watersoluble forms of coniferyl alcohol, such as its complex with Pcyclodextrin and the glucoside coniferin, greatly increased podophyllotoxin production by cultured cells of P. emodi [20, 21]. Two biosynthetically distinct groups of Podophyllum lignans have been identified, one having a 3,4,5-trimethoxy substituted aryl group, which includes podophyllotoxin and y^peltatin, and the other having a 4hydroxy-3,5-dimethoxy substituted aryl group, which includes a-peltatin, Fig. (4). The efficient incorporation of (~)-[^'^C]matairesinol into podophyllotoxin, /?-peltatin, 4'-demethylpodophyllotoxin, and a-peltatin in Podophyllum demonstrated that matairesinol is a common intermediate, and probably the branch-point compound, leading to the formation of both groups of Podophyllum lignans [22]. Matairesinol, a 4-hydroxy-3methoxy substituted compound is converted to the 3,4,5-trimethoxy substituted yatein or to 4-demethylyatein, which respectively give rise to podophyllotoxin/)5-peltatin or 4-demethylpodophyllotoxin/a-peltatin, via quinonemethide and desoxy intermediates [23]. Carefully designed labeling experiments have conclusively established the biosynthetic origin of (--)-matairesinol in Forsythia spp. Cell-free enzyme preparations of F. suspensa stems catalyze the stereoselective coupling of two molecules of [8-^'^C]coniferyl alcohol to form (+)pinoresinol in enantiomeric excess [24]. This reaction likely involves the coupling of free radicals of coniferyl alcohol and is mediated by two enzymes: an oxidase and a dirigent protein that controls the stereospecificity of the reaction. Variously labeled coniferyl alcohol was also incorporated into optically pure (-)-secoisolariciresinol and (-)matairesinol by tissues of F. intermedia [25]. It is probable that the series of stereospecific and strictly enantioselective reactions that transform two achiral molecules of coniferyl alcohol into (~)-rotatory matairesinol in Forsythia are also responsible for matairesinol production in Podophyllum, Fig. (4). Little is known about the tissue and subcellular compartmentation of lignan biosynthesis in Podophyllum. In vitro cultured cells of P. emodi are capable of synthesizing podophyllotoxin from coniferyl alcohol, implying

V OH O C H ,

'

H3c0m H3CO

OH

HO

"',,//

I

-

H3C0

Yatein

U

R,=H, R,=OH: Podophyllotoxin R,=OH, R2=H: /%Peltatin

OCH3 (+)-Pinoresinol

HO

O O C H , HO (-)-Secoisolariciresinol

HO Demethyl yatein

HO R, =H, R,=OH 4'-Demethylpodophyllotxin R,=OH, R2=H a-Peltatin

Fig. (4). Proposed lignan biosynthetic pathway in Podophyllum (steps 1-3 have been characterized in Forsythia spp.). 1, Oxidase and dirigent protein create an 8-8' bond between two molecules of coniferyl alcohol; 2, NADPH reductase catalyses two sequential reactions; 3, NADP+ dehydrogenase. Dotted arrows indicate multiple uncharacterized steps.

t; 4

158

that the underground parts of this plant have full biosynthetic capacity [20]. The leaves of P. peltatum, in turn, can incorporate matairesinol into podophyllotoxin and the peltatins [22]. To our knowledge, there are no published reports of lignan content in leaves of P. emodi. Analysis of an accession originating from Nepal showed that only minute amounts of J5peltatin were present [Canel, unpublished data]. Whether this is representative of the species as a whole remains to be established. If so, the possibility that transport of water-soluble monolignol precursors, such as coniferin, from the leaves to the roots merits investigation. Lignan accumulation appears to differ in the American and Indian species. Studies involving differential extraction with aqueous and organic solvents indicate that lignans are stored mostly in the form of 4-0-y^Dglucopyranosides in P. peltatum leaves, the efficient extraction of which requires hydrolytic cleavage of the sugar moiety [26]. In contrast, notwithstanding possible conversion during processing and storage of the tissues, most podophyllotoxin present in the rhizomes of P. emodi exists as the aglycone, and can be extracted directly with ethanol [26]. BIOLOGICAL ACTIVITY The wide taxonomic distribution of lignans and their pronounced toxicity and antimicrobial activity suggest that these phytochemicals play important roles in protecting plants from pathogens and herbivores. For instance, in leaves of P. peltatum, the accumulation of large amounts of podophyllotoxin glucoside, which is rapidly hydrolyzed into the more toxic aglycone upon disruption of the structural integrity of the leaves, points to the possible role of podophyllotoxin as a feeding deterrent. Because of the medicinal importance of podophyllotoxin, the biological activity of Podophyllum lignans has been studied extensively. Most attention has been given to the potential use of these compounds and their derivatives as antineoplastic and antiviral agents. The successful derivatization of podophyllotoxin into potent and safe antineoplastic drugs has generated considerable interest and led to numerous studies by research groups throughout the world. Thousands of compounds have been generated in the last several years. The study of the biological activity and medicinal properties of Podophyllum lignans is therefore tightly linked to the manipulation of the basic aryltetralin backbone and is presented accordingly in this section. In general, efforts to

159 optimize the structure of podophyllotoxin to improve its pharmacological properties and expand its application have focused on one of the following aspects: 1) manipulation of the linkage at the C4 carbon, including derivatization of the sugar moiety, 2) modification of the CI aryl group, and 3) modification of the aryltetralin structure. Anticancer Activity Most of the work on the derivatization of podophyllotoxin has targeted the C4 position. These studies can be classified according to the fate of the carbon-oxygen bond, which may be either retained or replaced by a carbon-nitrogen or a carbon-carbon bond. When retaining the C-0 bond, derivatization has involved either the modification of the sugar moiety or its replacement with groups of different chemical nature. Modification of the Sugar Group

The early work on structural optimization of podophyllotoxin was done in the mid 1950's by chemists at Sandoz, in Switzerland, who sought to eliminate the toxic effects of the compound while retaining its antitumor activity. The first attempts to generate pharmacologically useful derivatives of podophyllotoxin involved the use of the glucoside, since the presence of the sugar moiety made the compound more water-soluble and reduced its toxicity [5]. Modification of the sugar group initially involved alkyl derivatization at the 4" position, which did not produce superior compounds, and later acylation at the same position, which reduced the activity. Further work to stabilize the glucoside was done by blocking positions 4" and 6" with various groups. Derivatives having a 4",6"benzylidene group showed much improved oral bioavailability while retaining the cytostatic properties of the unaltered glucoside. Among the benzylidene derivatives, a compound was fortuitously generated that inhibited cellular proliferation via a mechanism other than spindle poisoning and concomitant mitotic arrest. The unexpected activity was traced to a contaminant found in minute amounts in preparations of derivatives of the total glucoside fraction of Podophyllum rhizomes. The new compound differed from other derivatives by having a p configuration at C4, instead of the a configuration of podophyllotoxin.

160 and by inhibiting DNA-topoisomerase II. Extensive studies ensued on the synthesis of C-4/? analogs, referred to as epipodophyllotoxins, resulting in the discovery of teniposide and etoposide, which were launched commercially in 1976 and 1980, respectively. All podophyllotoxin-based anticancer drugs commercially available today are epipodophyllotoxins that have modified sugar moieties. Teniposide has a thionylidene glucoside, whereas etoposide and etoposide phosphate, a water-soluble phosphate ester prodrug of etoposide launched in 1996 under the trade name Etopophos, have ethylidene glucosides, Fig. (5). Etoposide phosphate was developed to overcome the limitations associated with the considerable hydrophobicity of etoposide. The prodrug can be administered in higher doses than etoposide as a short intravenous injection, after which it is rapidly converted to the parent compound by plasma phosphatases, and thus constitutes an improved formulation of etoposide [27].

Ri

•Q

Tenoposide:

R2

OH

Etoposide:

CH3

OH

NK-6n:

CH,

N(CH3)2

Fig. (5). Various glucosides of podophyllotoxin-derived antineoplastic drugs

Numerous other glucosidic substitutions have been tested [28]. One of the most promising compounds with such modification, NK-611, has a dimethylamino side chain instead of a hydroxyl group at the R2 position of the ethylidene-type sugar moiety. This substitution greatly increased water solubility and improved in vitro antitumor activity. Clinical tests of intravenous and oral formulations of NK-611 suggest that the compound

161 has better bioavailability than etoposide [29, 30]. The synthesis and evaluation of a series of 42 new amino- and alkylamino-glycosides of epipodophyllotoxin has recently been reported. Four of these derivatives showed at least as much activity as etoposide on in vivo leukemia models [31]. This study demonstrated the importance of the 4",6"-acetal substitution in the sugar moiety for biological activity. Surprisingly, none of these compounds inhibited DNA-topoisomerase II or bound to tubulin. Replacement ofC4 Sugar Moiety with Other Groups

Studies using compounds having non-sugar groups attached at C4 indicate that the sugar moiety is not essential for anticancer activity, but its replacement affects the mechanism of action of the lignan. Several C4 esters have been generated [32]. These compounds behaved more like inhibitors of microtubule polymerization than like DNA topoisomerase inhibitors. Most compounds of a series of twenty nine C-4/? derivatives possessing linear alkyl, hydorxyalkyl, methylthioalkyl, acetonide, or dihydroxyalkyl side chains were not very active on DNA-topoisomerase II, except for those having dimethylamino and methylpyrrolidino groups [33]. Replacement of the C4 Carbon-Oxygen Linkage with Amino Functionality

The introduction of a C-N bond at the C4 position yields relatively simpler structures and has resulted in compounds having promising therapeutic profiles, Fig. (6).

-ORi HN

I

5'dl Fig. (6). Podophyllotoxin derivatives possessing novel C4 substitution with a carbon-nitrogen bond replacing the carbon-oxygen bond

162

For example, the new etoposide analog GL-311, possessing a C-Apaminoaniline substitution, is currently in phase II clinical trials for various anticancer treatments. This compound is a more potent inhibitor of proliferation of refractory cancer cells than etoposide [34]. Attempts to increase the biological activity of GL-311 by di- and tri- substitution of the aniline ring have produced derivatives up to 10 times as potent as etoposide [35]. Other such compounds have proved at least as active as etoposide as anticancer agents; these include alkylamino-substituted derivatives [36, 37] and synthetic acetyl salicylic acid, 2mercaptobenzothiazole and 2-aminobenzothiazole derivatives [38]. In all cases, it has been necessary to maintain the P stereochemistry at C4 to obtain acceptably high antitumor and anti-topoisomerase activity. A comparison of the activities of several a and /? analogs, including simple alkylamino derivatives, showed that compounds with a configuration were consistently more potent inhibitors of tubulin polymerization than their counterparts with p configurations [39]. An extension of this approach was evaluated, which combined the C-4yS-aminoaryl substitution with the presence of an o-quinone side chain at CI, Fig. (7). The doubly substituted compounds were more potent than etoposide at either inhibiting DNA-topoisomerase II or inducing DNA breaks [40].

,r-^%. H3CO

Y

"O

o Fig. (7). Podophyllotoxin derivatives with 1-o-quinone and 4-aminoaryl substitutions

163

Carbon-Carbon

Linkages

One of the main objectives of structural derivatization of podophyllotoxin has been the stabilization of the C4 linkage. Therefore, it is surprising that relatively few studies have aimed at substituting the C4 carbon-oxygen bond with carbon-carbon bonds, given the likelihood that these molecules would be less sensitive to degradation. One such study involved the synthesis of 17 analogs having alkene, alkyl, alkyl alcohol, and ether groups at C4. Some of these molecules, including a hydroxypropyl derivative, had activities somewhere between etoposide and tenoposide [41]. TOP-53, another highly active podophyllotoxin derivative possessing a C4 carbon to carbon linkage, is in phase-I clinical trials. Fig. (8). This molecule was selected from a series of C-4/?-alkyl derivatives containing hydroxy, amino, and amido substitutions, among which it was the most active [42]. More detailed studies showed that TOP-53 was twice as active as etoposide at inhibiting DNA-topoisomerase II and is as effective as etoposide at 5 to 6 times lower concentration [43].

Fig. (8). Podophyllotoxin derivatives possessing novel C4 substitutions with a carbon-carbon bond replacing the carbon-oxygen bond

Modification of Ring E

Studies of the structure-activity relationship of CI-substituted derivatives of etoposide, albeit few, have shed light on the relative importance of the nature of the substitution. Most compounds generated have either a hydroxy 1 or a methoxy group at C4' of the aromatic E ring. Analysis of the biological activity of these compounds has clearly demonstrated that

164 the hydroxyl group at C4' is instrumental in the ability of the derivative to inhibit DNA topoisomerase [28]. Novel ring-E analogs having o-quinone, ketal, and spyroketal functionalities, Fig. (9) have been synthesized, but their biological activity has not been tested [44].

r-H MeO

\ OMe OMe

Ti^

MeO

Fig. (9). Modified aryl E-ring possessing o-quinone, ketal, and spiroketal functionalities

Modification of the Aryltetralin Backbone

While the greatest emphasis has been placed on the synthesis of derivatives at the C4 position, several research groups have generated sets of analogs with modified tetralin backbones. One recurrent theme has been the opening of the lactone ring. For example, acid hydrazide and C3(2,2,5,5-tetramethylpyrrolinenyloxy) semicarbazide derivatives have been generated from podophyllotoxin, Fig. (10). The latter compound was as active as etoposide and seemed to have weaker immunosuppressive effects [45]. In contrast, several naphthalene lignans either with or without the lactone ring, such as some junaphthoic acid derivatives, were found to be significantly less active than their aryltetralin counterparts [46]. Derivatives of the naturally occurring podophyllotoxin and its C4-deoxy analog lacking the lactone ring were synthesized and tested on neoplastic systems [47]. The set of twenty two compounds included carbonyl, hydrazone, and oxime derivatives, most of which were active on several tumor cell lines, albeit generally less potently than the natural parent compounds. Computer modeling of the new derivatives suggested that the introduction of a double bond in ring C led to more rigid, as well as more active, structures. Fig. (10). It was hypothesized that the less flexible unsaturated ring C caused a better orientation of the ester group and more closely

165

CHO

\^-"i ^r

COOCH3 O

V

Ar /
t ' " / - I ^ N H N M L / ^ ^ ^

Fig. (10). Modifications of podophyllotoxin involving opening of the lactone D-ring and introduction of unsaturation in the C-ring

resembled podophyllotoxin. The preparation of simpler triol and ether derivatives obtained by opening the lactone ring has been reported [16], but the biological activity of these compounds was not tested. Compounds with a catechol-type instead of the dioxolane ring A were poorly active [33]. More radical alterations of the basic natural structure, involving the opening of ring A, have been performed. A recent study reported the 11-step total synthesis of an etoposide analog having modifications in rings A and D, Fig. (11).

D o

Fig. (11). Modifications of podophyllotoxin picropodophyllotoxin lactone configuration

involving

the

A-ring

and

using

the

166 This compound possesses a c/^'-fused-lactone ring D, as in picropodophyllotoxin, and a pyridazine ring as cycle A [48]. Diphyllinlike derivatives where ring A was opened, the tetrahydronaphthalene structure was unsaturated to its naphthalene form (rings B and C), and the aryl ring E of podophyilotoxin was retained, Fig. (12) have also been synthesized [49]. The introduction of the double bonds in rings C caused the entire system to be planar, including the hydroxyl group at C4 and the ;r-lactone ring D. The inhibitory activity of these compounds against the

D

O

Fig. (12). Hybrid backbone possessing characteristics of podophyilotoxin and diphyllin natural products

DNA-topoisomerase II was much lower than commercially available compounds. Some attention is being focused on extending the number of cycles beyond ring A in order to generate derivatives that would have better DNA intercalation [50]. A series of synthetic flavonoid/podophyllotoxin hybrid molecules were synthesized with the aim of combining the well-known antitumor activity of many flavonoids and the structural features of the podophyilotoxin considered to be important for activity, such as the dioxolane ring A and the aromatic ring B, Fig. (13). As a whole, these compounds were only weakly cytotoxic towards cultured tumor cells [51]. Another unsuccessful attempt to improve the anticancer properties of podophyilotoxin involved the replacement of the C2 carbon by a nitrogen atom [52]. The aza derivative of

167

deoxypodophyllotoxin inhibited microtubule assembly but lacked inhibitory activity toward topoisomerase; on the other hand, oxidation of ring E of the aza-analog yielded a quinone having non-intercalative antitopoisomerase activity.

-T-R

Fig. (13). Hybrid backbone possession characteristics of podophyllotoxin and flavonoid natural products

Lessons Learned from Studies of Structure-Activity

Relationships

A study that correlated the cytotoxicity of a set of compounds with their mode of action (inhibition of either tubulin polymerization or DNAtopoisomerase II) demonstrated that the effects of podophyllotoxin derivatives could not be consistently predicted [33]. Compounds that inhibited neither site were still highly cytotoxic, whereas some of the most active compounds inhibited both sites, and some strong topoisomerase inhibitors were not necessarily potent anticancer agents. Similarly, several highly active glucosidic derivatives did not show activity against either site [31]. Yet, DNA-topoisomerase II remains the preferred target of podophyllotoxin-derivatization work. Because cytotoxicity and, particularly, low therapeutic index are equated with non-specific induction of mitotic arrest, compounds that inhibit tubulin polymerization are not desired. In fact, all drugs approved thus far and the most promising new derivatives exert their activity through inhibition of the DNA-modifying enzyme. Some features that differentially affect the inhibitory properties of podophyllotoxin analogs have been identified. A graphical summary of these features is shown in Fig. (14). As a rule, compounds with 4a configuration tend to be quite cytotoxic and inhibit microtubule

168 polymerization, whereas those with the Ap configuration are less cytotoxic and inhibit DNA-topoisomerase II.

4p configuration is essential

frans-lactone with 2a,3p configuration is very important

free rotation of ring E is required

4' hydroxy is essential with esterification tolerated

Fig. (14). Diagram summarizing the key features of the podophyllotoxin backbone obtained from structure-activity relationship studies

Commercial podophyllotoxin-based drugs all possess modified 4y^Dglucosides. However, highly active structures have been synthesized that have either amino or carbon-carbon linkage at position C4, suggesting that the sugar structure of etoposide can be simplified considerably while retaining or even improving biological activity. Although many derivatives lacking the lactone ring have shown high anticancer activity, the ;K-lactone ring is critical for topoisomerase inhibition. With few exceptions, the C-2a, C-3y5 trans configuration has been maintained in most studies; typically, /? cis analogs are much less 7active. The importance of the 4-hydroxyphenyl character of the E ring was recognized early on. Derivatization of the hydroxyl group yields significantly less active compounds. Only minor changes such as esterification at 4'-0(E) can be made without diminishing activitiy. Furthermore, free rotation of the E ring appears to be required to maintain topoisomerase inhibitory

169 activity. The dioxolane ring A is considered optimal for anticancer activity. Studies of ring-A opening yielded relatively inactive compounds. In spite of the large amount of work done on the derivatization of podophyllotoxin, a clear picture of the structural features not already present in etoposide that determine whether the molecules inhibit DNAtopoisomerase or microtubule polymerization has not emerged. Etoposide and teniposide are among a group of compounds that bind directly to DNA-topoisomerase II [53]. A recent study has shown that the binding of etoposide to topoisomerase was dependent on the presence of a specific tyrosine residue found in the active site of the enzyme involved in DNA scission [54]. More detailed knowledge than is available today about the enzyme's catalytic site and the binding site of the epipodophyllotoxins is needed to advance drug development. By and large, efforts to rationally design a better antineoplastic drug based on the aryltetralin structure have failed; this fact, together with the large number of possible structural variations, makes the accidental discovery of etoposide all the more remarkable. Antiviral Activity Lignans were first shown to possess antiviral activity over 50 years ago when the successful use of Podophyllum extracts in the treatment of venereal warts, caused by the human papilloma virus, was reported (2). Since then, numerous studies involving the evaluation of podophyllotoxin and its congeners and derivatives as antiviral agents have been performed. The results of these studies and the antiviral activity of other lignans have been reviewed recently [55]. Podophyllum lignans have been shown to inhibit the pathogenic effects of both DNA and RNA viruses. Podophyllotoxin, in particular, showed significant inhibitory activity against herpes simplex virus [56], vesicular stomatitis virus [57], and murine cytomegalovirus [58] at concentrations of 24, 100, and 241 nM, respectively. However, the potency of these lignans as antiviral agents is considered to be insufficient, and their cytotoxicity too pronounced, for therapeutic use. The similarity between the mode of action of aryltetralin lignans as anticancer and antiviral agents is noteworthy. Because of their ability to bind tubulin, these lignans disrupt the cellular cytoskeleton and thus interfere with viral replication. The development of successful

170

podophyllotoxin-based antiviral drugs is likely to require further derivatization work, as was the case for etoposide. Analysis of the results obtained thus far point to structural features, such as the presence of the lactone ring and the a configuration at C2, that positively influence antiviral activity. In addition to tubulin binding, synthetic podophyllotoxin analogs have shown inhibition of reverse transcriptase [59], which may be exploited to selectively combat RNA viruses such as the human immunodeficiency virus (HIV). These studies showed that aryltetralins having a methoxy group at C-8(B) and a 4-benzyloxy-5-demethoxy substituted E-ring had highest selectivity indices against HIV-1. Other Potential Uses The administration of some podophyllotoxin-based drugs causes complex physiological reactions beyond inhibition of tubulin polymerization and DNA-topoisomerase. Benzylidinated podophyllotoxin glucosides are currently in late clinical trials for the treatment of psoriasis and rheumatoid arthritis. Arthritis patients treated with CPH82, a mixture of two semisynthetic benzylidinated podophyllotoxin-based glucosides show reduction of the inflammatory process within three months of therapy [60]. CPH82 stimulates macrophage growth and inhibits lymphokine, this immune response being mediated by levels of cyclic AMP. The results of phase-I and -II trials on CPH82 for rheumatoid arthritis were promising regarding safety and efficacy, however some gastrointestinal inconveniences in 28% of patients were reported. As inhibitors of mitosis, podophyllotoxins have various potential medicinal applications, including uses as immunosupressive, antimalarial and antifungal agents [61]. The biological activity of podophyllotoxins may also have agricultural applications. Podophyllotoxin has been shovm to inhibit larval development [62] and root growth [63]. Phytotoxicity assays carried out in our labs have shown that rye seedUngs are more sensitive than lettuce seedlings to the presence of Podophyllum extracts in the soil, podophyllotoxin being the most active among the lignans [Moraes, unpublished data].

171

CURRENT DEMAND FOR PODOPHYLLOTOXIN AND THE SEARCH FOR ALTERNATIVE SOURCES While the preferred source of podophyllotoxin becomes scarcer, the demand for the compound continues to increase. US sales of etoposide tripled in 1995, after the commercial patent covering the drug expired, and have risen at an annual rate of more than 10% since 1997 [64]. Etoposide is used in combination therapy in refractory testicular, lymphoid and myeloid leukemia, stomach, ovarian, brain, breast, pancreatic, and small and non-small cell lung cancers. Approximately 60 clinical trials are under way to test etoposide for new indications, and another 140 trials use the drug as positive control. In addition, numerous new podophyllotoxin derivatives are currently under development and evaluation as topoisomerase inhibitors and potential anticancer drugs. Some of these compounds have shown improved therapeutic properties over etoposide [30, 65]. Podophyllotoxin-based pharmaceutical products have also been formulated for the treatment of rheumatoid arthritis, showing both efficacy and low toxicity [60]. Furthermore, the application of creams and solutions containing podophyllotoxin is still a common and effective treatment for genital warts, the incidence of which is rapidly increasing [66]. The amounts of podophyllotoxin and Podophyllum rhizomes required to satisfy the current demand of the pharmaceutical industry are difficult to estimate because of lack of published data. Demand is apparently increasing and will probably continue to do so as new podophyllotoxin derivatives become useful drugs and new therapeutic uses are approved for older ones. The destructive collection of wild-growing rhizomes of P. emodi has drastically reduced the size of natural populations. Even a rapidly growing plant such as mayapple may become endangered if the harvest of natural stocks to satisfy the current and anticipated need for podophyllotoxin occurs without proper replenishment. Consequently, scientists have considered it essential to develop alternative or more reliable sources of aryltetralin lignans, including chemical synthesis, bioprospecting for plant species that produce aryltetralin lignans, in vitro culture, and domestication of Podophyllum. Even though a long-term solution appears to have been found in the utilization of leaves of P. peltatum, alternative sources may still be useful for the identification of novel lignans and biological activities.

172 Chemical Synthesis The therapeutic success of semisynthetic podophyllotoxin derivatives such as etoposide has prompted researchers to explore synthetic routes to podophyllotoxin and analogs, both as an alternative to reUance on natural sources and a direct approach to alternative aryltetralin structures. Synthetic work reported up to 1991 has been categorized into four general approaches according to the key intermediate (the oxo ester and dihydroxy ester routes) and the key step (the tandem conjugate addition and DielsAlder reaction routes) involved [67]. With few exceptions, the synthesis of podophyllotoxin and related compounds leads to products having undesired relative configurations at CI, C2, and C3. Lengthy procedures are usually required to obtain the 2,3-trans configuration. More recently, the 8-step total synthesis of podophyllotoxin, with an optical purity of 98% and in an overall yield of 15% was reported [68]. Biotechnological and enzymatic approaches have also been tried. Cell cultures of P. peltatum transformed a synthetic precursor mostly into a podophyllotoxin analog with the targeted 2,3'trans configuration [69]. As an extension of the use of biological systems to establish the stereochemistry of the resulting lignan, cell-free enzyme preparations were used to catalyze the oxidative cyclization [70] and the selective deacetylation [71] of synthetic substrates. Although interesting, these approaches still involve lengthy and low-yielding multi-step procedures, and suffer from limitations typical of biotechnological processes, namely the high costs of enzymes and in vitro culture, as well as the instability of the enzymes. Alternative Natural Sources Aryltetralin lignans are widely distributed in the plant kingdom. In addition to plants of the Berberidaceae, most saliently in the genera Podophyllum, Diphylleia, and Dysosma, these compounds have been found in plants of numerous genera of other families, such as Hyptis, Teucrium, Nepeta, Thymus, Phomis and Salvia, in the Lamiaceae [72, 73], and Linum and Hugonia in the Linaceae [74, 75, 76]. These compounds are also present in gymnosperms of the genera Juniperus and Thujopsis [11, 78, 79]. For the most part, however, the quantities of aryltetralin

173

lignans obtained from plants other than Podophyllum are quite small (Table 1). With the exception of some species of Linum that are widely cultivated, the productive potential of these other sources appears to be very limited. The biotechnological approach to podophyllotoxin production, namely in vitro culture, has not been limited to biotransformation of complex intermediates, but has included attempts to obtain the compound from sugars and other simple precursors. Podophyllum callus has been induced to differentiate into tissues containing 1.6% podophyllotoxin, based on dry weight [85]. Callus, cell, and root cultures of Podophyllum have been shown to accumulate small amounts of podophyllotoxin [86, 87, 88]. Cell cultures of Linum flavum [80] and Linum album [89] have also been tested. The characteristically low productivity of undifferentiated cultures can be improved by the addition of precursors to the culture medium. For example, feeding 0.8 to 2.1 mM coniferin to cell suspension cultures of Indian Podophyllum resulted in an increase in podophyllotoxin production of up to 12.8 times relative to that obtained from control cultures [21]. Coniferin is not commercially available and earlier precursors such as phenylalanine, caffeic acid, and ferulic acid did not improve production. The use of coniferyl alcohol in feeding experiments is limited by its poor solubility, a problem that has been addressed by complexation with pcyclodextrin, which increases the water solubility of the monolignol and results in higher podophyllotoxin production [20]. Leaves of P. peltatum as a Renewable Source of Podophyllotoxin Until recently, the rhizomes and roots of P. emodi were considered the richest source of podophyllotoxin, reportedly containing 4.3% of the compound on a dry-weight basis [81]. It is now known that, if properly extracted, the leaves of P. peltatum may yield similar or larger quantities of podophyllotoxin [26]. Our research group chose to investigate the productive potential of P. peltatum because it is a known source of podophyllotoxin and its natural habitat covers a large area where it grows vigorously and abundantly. In April 1998, a survey of much of the natural range of P. peltatum was carried out to evaluate the biosynthetic capacity of the plants. Highyielding accessions were identified and collected both for extraction and propagation purposes. Encouraged by the identification of naturally

174 occurring chemotypes of mayapple that accumulate large amounts of lignan glucosides [90] our group recently developed an extraction method that takes advantage of the presence of highly active lignan y^Dglucosidases in dried tissues of Podophyllum [26]. This extraction method consists of rehydrating powdered tissues to allow the endogenous glucosidases to convert podophyllotoxin glucoside into the aglycone and thus makes it available for extraction using a water-immiscible organic solvent such as ethyl acetate. Lignan glycosides, including podophyllotoxin 4-y5-D-glucopyranoside were first extracted from Podophyllum rhizomes in 1954 by researchers who hoped that these compounds, in analogy to cardiac glycosides of Digitalis, would exhibit pharmacological properties superior to those of the aglycones [91]. Enthusiasm for the natural glucosides soon waned because they showed less potency than the aglycone as well as poor bioavailability. The leaves of P. peltatum were sufficiently rich in /?-Dglucosides to permit labeling studies, but the possibility of using endogenous enzymes to produce the aglycones was not recognized. Instead, the leaves were macerated with water and treated with almond figlucosidase [22]. Similarly, to liberate the aglycone from tissues of F. intermedia, the plant material was first extracted with methanol, and the crude extract then reacted with almond y5-glucosidase in acetate buffer [19]. The new extraction procedure does not require the addition of catalytic agents, can be carried out at ambient temperature and makes use of inexpensive buffering agents. Aqueous extraction of leaves of glucosiderich chemotypes of P. peltatum yielded over 5% of podophyllotoxin, based on dry weight, exceeding levels previously reported from any source. The significance of these developments cannot be overstated. Leaves are entirely renewable and their harvest does not destroy the plant. The use oi Podophyllum leaves opens the door for the managed utilization of a natural resource, avoiding the destruction of wild populations and virtually guaranteeing the long-term availability of raw material. Cultivation of American Podophyllum

The cultivation of mayapple as a medicinal cash crop was proposed a quarter of a century ago on the basis of its high demand as a source of

175 bioactive phytochemicals [6]. The demonstration that leaves of mayapple can be a highly productive source of podophyllotoxin has made the cultivation of this plant rather attractive. We are currently pursuing the domestication of P. peltatum in order to develop a reliable supply of extractable raw material of consistently high quality at a reasonable and predictable cost. Much work is still needed, however, to propagate highly productive chemotypes and establish the agronomics of mayapple cultivation. To this end, we are carrying out work on several fronts: 1) Identification of highly productive genotypes, 2) Establishment of efficient propagation protocols, and 3) Determination of the environmental factors that affect productivity. Chemical analysis of leaves harvested from 17 naturally occurring mayapple colonies suggested that, if collected randomly from the wild and extracted with an aqueous agent, the podophyllotoxin yield of the combined leaf biomass would exceed 3% of dry weight. Notwithstanding the costs of research and the initial investment needed to plant the required acreage, cultivation of mayapple genotypes that produce over 5% podophyllotoxin may be an economically superior alternative than the managed utilization of natural populations. We have thus far collected six wild mayapple accessions, the leaves of which can accumulate between 4 and 6% podophyllotoxin [82]. Specimens from these colonies are being propagated in our laboratories. We have considered three options for the propagation of mayapple; i.e., by seed, by rhizome cuttings, and micropropagation. The use of seeds is hampered by a number of factors such as low seed availability, poor germination rates, high seedling mortality, and a long juvenile stage. Assuming that the rate of germination and seedling establishment in mayapple can be improved, there may still be a delay of four to five years, before rhizome growth and colony expansion occur. Micropropagation offers the advantage of bypassing the difficult germination and seedling stages. A protocol for the in vitro propagation of P. peltatum has been established [92]. Following this protocol, the formation of adventitious buds can be easily induced, and a large number of rooted buds that adapt well to soil conditions under a controlled environment can be generated. Success rates for the establishment of micropropagated buds are as high as 85%. Unfortunately, micropropagated buds follow a developmental pattern similar to that of buds produced sexually, taking four to five years to initiate rhizome branching. In contrast, rhizome cuttings derived from a mature rhizome system retain their adult character in terms of their ability

176

to grow vigorously and branch. Rhizome cuttings containing a node or a terminal bud root readily in sandy soils. Roots grow at the base of the terminal bud. For cuttings containing a node formed during past growing seasons, the rooting process begins after a new branch has developed and given rise to a new terminal bud. Cuttings should therefore be made in early fall at leaf senescence, forcing the new terminal bud to develop before the temperature drops. Although the number of propagules that can be generated by micropropagation is far larger than that obtained from rhizome cuttings, the latter option appears to offer the most direct route to the replication of highly productive genotypes. Besides bypassing the juvenile stage of development, propagules derived from rhizome cuttings are more likely to be true to type than those obtained sexually or from in vitro culture. Currently, our group is carrying out multisite and multiyear evaluation experiments to determine to what extent highly productive phenotypes are the result of genotypic factors, and how environmental factors affect the productivity of our accessions. Some basic agronomic issues are also being addressed. Mayapple requires a well-drained soil, but can grow in a variety of different soil pHs. Our accessions were collected in sites where soil pH ranged from 4.3 to 7.2. No correlation was found between soil pH and podophyllotoxin content. Being a shade-tolerant plant and one of the first plants to grow in early spring, weed competition may not affect P. peltatum as much as other crops. Issues related to pest and disease control are very important. The presence of podophyllotoxin may afford protection from herbivores, but is not effective against the specialist fungus Puccinia podophylli. The incidence of this pathogen, to which sexual progeny are more vulnerable than are shoots produced vegetatively [93], is not only a contributing factor to seedling mortality, but also greatly decreases leaf area and therefore causes the loss of productive biomass and inhibition of rhizome growth. Preliminary observations suggest that there is variability among our accessions regarding their susceptibility to the fungus. In addition, plants growing in the shade appear to be more susceptible than those growing in open spaces. Shade conditions may also affect the onset of leaf senescence, which appears to occur earlier in the shade, and rhizome branching, which seems to be promoted under full sun. The determination of the optimal time of leaf harvest is considered essential. On the one hand, leaves should be harvested when podophyllotoxin content and leaf area are greatest, but on the other, leaves

177

should remain in place sufficiently long to allow the development of new rhizome segments. Manipulation of biosynthetic pathway Efforts to increase the production of a particular metabolite by manipulation of the biosynthetic pathway primarily target those biochemical branch points at which competition for precursors is likely to occur. Availability and partitioning of coniferyl alcohol is of direct relevance for production of podophyllotoxin. In addition to its importance for lignan biosynthesis, coniferyl alcohol is also a key intermediate in lignin production [94, 95]. Lignin biosynthesis is a temporally and spatially regulated process that may involve the action of pathway-specific enzymes and specialized subcellular structures [96]. For instance, in nonwoody plants, the bulk of lignin biosynthesis is associated with the formation of vascular tissues. Therefore, even though both lignan and lignin biosynthesis utilize monolignol precursors, the two pathways may not be directly competing for these. Nevertheless, the demonstration that suppression of lignin biosynthesis does not compromise structural integrity in transgenic aspens [97], offers the possibility to explore the effect of down-regulating the lignin pathway on lignan biosynthesis. The possible use of species outside the genus Podophyllum for podophyllotoxin production must take into account the characteristic stereospecificity of the monolignol coupling mechanism, which is known to vary among plant taxa [98]. The reduction of pinoresinol is thought to be a key branch point in lignan biosynthesis since it gives rise to precursors for various types of 88' lignans. A prerequisite for the genetic manipulation of the pathway at this point has been fulfilled by the recent cloning of the gene encoding the NADPH-dependent enzyme (+)-pinoresinol/(+)-lariciresinol reductase in F. intermedia [99]. Rhizomes and leaves of P, peltatum show different patterns of lignan accumulation that point to other regulatory controls within the lignan biosynthetic pathway. The ratio of podophyllotoxin to peltatin content ranged widely in leaves of various genotypes of wildgrowing P. peltatum, revealing a strongly negative correlation between the quantities of these compounds [82]. This observation suggests that precursor utilization at the matairesinol level and branch points downstream from matairesinol are differentially regulated in roots and

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leaves. Directing metabolic flux away from peltatin end products by controlling these key biochemical steps, either genetically or by modifying growth conditions, may bring about an increase in podophyllotoxin production. CONCLUDING REMARKS More than fifty years after the first medicinal application of the antimitotic activity of podophyllotoxin was proposed, the aryltetralin lignans of Podophyllum continue to be subjects of intense interest and research. Derivatization of podophyllotoxin has produced the potent and widely used antineoplastic drugs etoposide and teniposide. Numerous other analogs have been synthesized in the hope of increasing the usefulness of these drugs, but little improvement has been achieved. These efforts, nevertheless, have generated large amounts of data that may facilitate the design of podophyllotoxin-derived drugs having novel and improved pharmacological properties. On the other hand, significant progress has been made towards securing a reliable source of podophyllotoxin. This compound is, still today, primarily obtained from wild-harvested rhizomes of P. emodi, a species considered in danger of extinction due to overcollection and loss of habitat. The discovery that the leaves of P. peltatum, a North American species, produce large amounts of podophyllotoxin not only promises to make this plant the preferred source of the compound, but also offers the possibility to establish it as a new high value cash crop. ACKNOWLEDGEMENTS This work was partly funded by USDA grant 97-35501-4886. We thank Dr. Daneel Ferreira for his help in the nomenclature of some of the compounds. REFERENCES [1] [2] [3] [4]

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 26 © 2002 Elsevier Science B.V. All rights reserved.

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NEW FINDINGS ON THE BIOACTIVITY OF LIGNANS JOSE LUIS RIOS, ROSA M^ GINER, JOSE M^ PRIETO Departament de Farmacologia, Facultat de Farmdcia, Universitat de Valencia. Vicent Andres Estelles. 46100 Burjassot, Valencia, Spain ABSTRACT: This chapter reviews the lignans with biological and pharmacological activity, and includes new natural products and some synthetic or semisynthetic compounds. Lignans are widespread in plants and in many cases are tlieir effective principles. They play an important role as phytoestrogens in preventing menopausial symptoms, osteoporosis, cancer and heart diseases. Lignans possess anticancer and antiviral properties and specifically inliibit certain enzymes and mediators involved in inflammation and immunity processes. Tliey affect the cardiovascular system by different mechanisms including tlie modification of phosphodiesterase activity and platelet activating factor function. Lignans can modify liver function, inhibit hpid peroxidation and free radical-mediated processes and exhibit antioxidant activity. Toxicity to fiingi, bacteria, parasites and insects has also been documented. In several cases the most relevant structure-activity relationship established for certain synthetic lignans in different activities have been summarised.

FOREWORD Lignans represent a widespread, structurally diverse class of phenylpropanoid derivatives of considerable pharmacological interest. Increasing research on new^ structures and interesting biological activities has greatly enlarged our knowledge of this area of phytochemistry and pharmacognosy. An overview of the traditional importance of these metabolites offers diverse examples of crude drugs containing lignans as effective constituents. These crude drugs have been used extensively for their medicinal properties including antiviral, antitumor, anti-inflammatory and immunosuppressive actions among many others. The Podophyllum species (Berberidaceae) include antitumor lignans such as podophyllotoxins, from which synthetic analogues currently in clinical use in the treatment of many cancers are obtained. They also exhibit strong activity as potent anti-HIV agents. Schisandra (Schisandraceae) fruits, used successfully for the treatment of hepatitis, contain lignans effective against chronic viral hepatitis and with protective action against liver damage by hepatotoxins. Several of them also affect the cardiovascular system, decrease heart rate

184 and display platelet activating factor antagonist activity. Sesame seed lignans, used to prevent ageing, possess antioxidative properties and those contained in flaxseed, modified by intestinal bacteria and found in relatively large amounts in urine and other physiological fluids, afford protection against hormone dependent cancers. A great many reviews focusing on the distribution [1-4], biosynthesis pathways [1,2], chemistry and synthesis [5-8] and biological activities of lignans [9-11] have been published. Several refer to antiviral [12,13] and cardiovascular [14] activities, while others centre on podophyllotoxins [15,16], which are particularly important in cancer chemotherapy. Periodic reviews covering most of these aspects are published in Natural Product Reports [5-8,17-19]. The present review covers lignans whose biological properties have been reported from 1995 to 1999. In addition, the relevant activities and semisynthetic and synthetic analogues and the established structure-activity relationships are included. AN INTRODUCTION TO LIGNANS The lignans are secondary plant metabolites biosynthetically derived from the shikimate pathway. They possess diverse structures, usually dimers of phenylpropanoid units, although compounds containing three, four or even five such units, have been reported. The number of higher oligomers identified is currently increasing. Two classes of these natural products arising from the coupling of two cinnamate-derived units can be distinguished: lignans and neolignans. The term lignan, coined by Harworth, is used to define those metabolites formed via direct coupling of at least two C6-C3 units, exclusively derived from phenylalanine or tyrosine, and linked by the central carbon atoms (P,P' or 8,8') of their side chains. The term neolignan, introduced by Gottlieb, implies dimeric and oligomeric products of C6-C3 units linked otherwise than P,p'. Neolignans have been redefined as the products of oxidative coupling of allyl- or propenyl-phenols, while lignans are those resulting from the coupling of cinnamyl alcohols. Some authors [20] have recognised that in addition to simple lignans, there are different structural classes of oxygenated lignans: the lignanolides, which possess a lactone moiety, monoepoxy and bisepoxy lignans; cyclolignans, which contain a third six-member ring; and neolignans, obtained by unsymmetrical carboncarbon binding in the side chains. Lewis et al, [2] examined dimeric and

185 oligomeric products containing lignan or neolignan linkages classified into subgroups based on basic structural parameters. In view of this multiplicity it seems necessary to adopt a rational system of nomenclature. Occurrence Lignans are fairly widespread throughout the plant kingdom and have been documented in species belonging to up to seventy plant families. They have been identified in pterydophytes, gymnosperms and angiosperms. Their functions and ubiquitous distribution evidences their role in plant evolution, as the structure of lignans increases in complexity with the evolution of gymnosperms and angiosperms. They have been considered one of the earliest forms of defence to evolve in vascular plants [21]. Even within the bryophytes, the simple 8,8' linked lignans (+)megacerotonic and (+)-anthocerotonic acids, lacking regiospecific (9methylation at C3, have been identified in some hornworts (Anthocerotae). However, the lignans found in the Blechnaceous tree ferns, within the Pterydophytes, have 0-specific methylation and reductive modifications [2]. Castro et al [3] reviewed the distribution of lignans in the six families of the Coniferae order of Gymnosperms. They found different structural types in each, with the exception of Cephalotaxaceae for which no lignans had been reported. Cupressaceae and Pinaceae contain the largest variety of compounds, including neolignans, which are not detected in the other families. Norlignans are common in Taxodiaceae but are absent in Pinaceae. Further relevant features were the presence of dibenzylbutanediols in all the families except Taxodiaceae. Kadsuranin, identified in Larix leptolepis (Pinaceae) is the unique dibenzocyclooctene reported in conifers. Lignans are also found in Taxaceae, Ephedraceae and Ginkgoaceae [2]. Angiosperms constitute the most varied source of lignans. In the dicotyledons, 8,8'-linked lignans containing oxygenated functional groups at C9/C9' and lignans possessing methyl or methylenic groups at C9/C9' are common constituents. The structural variety in the monocotyledons is quite restricted, represented essentially by cyclobutanes and 5,5' or 3,3'-linked dimers [2]. The angiosperm lignans, as well as the gymnosperm lignans, show structural progressions in the superorders and their skeletal type is closely correlated with certain superorders.

186 Lignans occur in all parts of plants, such as heartwood, bark, roots, rhizomes, stems, leaves, flowers, fruits and seeds, as well as in secreted products such as resins. They may be found in variable content in both the free state and glycosidic combination. Lignans have also been detected in mammalians. They are either of dietary origin or are the metabolic products of the gut microflora. The first two lignans identified from these sources were the dibenzylbutyrolactone enterolactone and the dibenzylbutane enterodiol. Both arise from gut microflora action on the lignans matairesinol and secoisolariciresinol contained in whole grains, vegetable fibre, vegetables and finaits. Physiological significance The lignans play an important part in the defence mechanism of many plant species, against pathogens and predators [22]. Various roles in chemical defence, such as fungicidal, bactericidal and insecticidal, have been demonstrated for these secondary metabolites. In their defence strategy some Asteraceae and Piperaceae species include lignans that are inhibitors of the polysubstrate monooxygenases, polyphagous insect enzymes responsible for the biotransformation of plant metabolites. The planar methylenedioxyphenyl ring binds to the heme of the polysubstrate monooxygenases forming stable adducts that are not easily displaced by carbon monoxide or by most other ligands. Diasesartemin, dillapiol, sesamolin and cubebin inhibit polysubstrate monooxygenases in the European corn borer, Ostrinia nubilalis, an important crop pest and highly polyphagous insect [23]. The inhibition of the Phanerochaete chrysosporium lignin peroxidase by bis(methylenedioxy)cinnamic acid lends support to the hypothesis that the accumulation of lignans at the site of a wound may be a plant strategy to inhibit the fungal enzymes involved in lignin degradation [24]. Lignans from the Myristicaceae Virola sebifera and Otoba parvifolia were toxic to the symbiotic fungus of leaf-cutting ants of the species Atta sexdens rubropilosa [25]. Lignans together with other allelochemicals may play a role in synergising antifeedant or insecticidal properties. Sesamin and asirin showed synergistic properties with pyrethrum insecticides [2]. Pinoresinol, bis-epipinoresinol, the hemicetal and the diacid isolated from the Brazilian

187 Melia azedarach (Meliaceae), displayed phago-inhibitory activity against XhtYitmvpttvdiRhodniusproUxus [26]. Relatively few lignans have been recorded as allelopathic agents, and it is therefore interesting to find that dibenzylbutane nordihydroguairetic acid (NDGA), isolated from the creosote bush Larrea tridentata (Zygophyllaceae), is responsible for the growth inhibition of surrounding grasses [27]. A tetrahydrofurofuran derivative from Aegilops ovata (Gramineae) showed inhibitory activity on achene germination of Lactuca sativa (Compositae) in white light [2]. Various podophyllotoxin derivatives reduce the root growth oiBrassica campestris (Cruciferae). This effect has been ascribed to the presence of the methylenedioxy group and the configuration of the lactone ring. However, the activity of matairesinol was enhanced by methylation of the two phenolic groups [7]. CHEMISTRY Biosynthesis The biosynthesis of lignans is a rather unexplored area. Experimental evidence of all the postulated logical biosynthetic sequences has still not been found. Research has focused on enzymatic transformations occurring in lignan biosynthesis, with particular emphasis on the stereoselectivity and enantiospecificity of such conversions. Lignans arise by oxidative coupling of two coniferyl alcohol units. It has been accepted that a monolignol such as coniferyl alcohol is dehydrogenated to phenoxy radicals, and coupling of these radicals or mesomeric forms of them leads to quinone methide structures, which suffer nucleophil additions to give dilignols (lignans and neolignans). It has been demonstrated that two achiral molecules of ^-coniferyl alcohol suffer direct stereoselective coupling to give (+)-pinoresinol, which undergoes a highly enantiospecific NADPH-dependent reduction that sequentially yields (+)lariciresinol and (-)-secoisolariciresinol, the precursor of (-)-matairesinol [28,29]. 0-Methylation reactions of (+)- and (-)-matairesinol catalysed by Forsythia intermedia (Oleaceae) cell-free extracts, are neither highly enantioselective nor regiospecific. Regiospecific glucosylation first occurs to yield matairesinoside, and subsequent methylation yields actiin, which is then converted into arctigenin via the action of a p-glucosidase [30]. The first natural benzylic ether reduction (or quinone methide analogue) in plant

188 systems was demonstrated in Forsythia cell-free extracts, which catalyse the conversion of pinoresinol into secoisolariciresinol via lariciresinol [29]. The biosynthesis of (+)-lariciresinol via stereospecific reduction of (+)pinoresinol has also been demonstrated in Zanthoxylum ailanthoides (Rutaceae). This lignan is an intermediate on the pathway from coniferyl alcohol to arctigenin and is formed by reductive cleavage of one of the benzylic ether linkages in (+)-pinoresinol in presence of NADPH [31]. The biosynthetic pathway to the furoforan lignans from quinone methides by stereospecific nucleophilic additions was investigated in feeding experiments with Forsythia species. Dibenzylbutyrolactones require reduction and formation of the lactone ring. Methylation and glycosylation reactions occur late in the sequence [32]. Podophyllum aryltetralin lactone lignans are produced biosynthetically by oxidative cyclization of dibenzylbutyrolactone lignans. Feeding experiments with matairesinol in three different Podophyllum systems demonstrated this dibenzylbutyrolactone to be the common precursor of the 3',4',5'trimethoxy and 4'-hydroxy-3',5'-dimethoxy groups oiPodophyllum lignans [33]. Most lignans are obtained optically active and, presumably, enantiomerically pure. However, the phenolic coupling processes catalysed by H202-dependent peroxidases, 02-requiring laccases or phenol oxidases yield racemic products. A protein without an active centre has been isolated that in presence of an oxidase produces stereoselective bimolecular phenoxy radical coupling reactions in in vitro lignan biosynthesis. Its mechanism of action is presumed to involve capture of E'-coniferyl alcoholderived free radical intermediates [34]. Two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from Thuja plicata (Cupressaceae) that encoded a pinoresinol-lariciresinol reductase with different enantiospecificity have been reported. One of the proteins converted (+)-pinoresinol into (-)secoisolariciresinol, whereas the other utilised the opposite (-)-enantiomer to give the corresponding (+)-form [35]. Classification In the present review, we classify these metabolites following closely the classification based on structure designed in previous lignan research [5,6,7,8], but we cover only those with pharmacological activity.

189 These natural products are divided into two major groups, lignans and neolignans. Oligomeric lignans and neolignans, hybrid lignans and norlignans constitute three minor groups. The first of them comprises oligomeric phenylpropanoids. Hybrid lignans refer to a single C6-C3 unit bound to another structure and thus yielding flavanolignans, isoflavanolignans, xanthonolignans, coumarinolignans, stilbenolignans, sesqui-, di- and triterpenelignans. The lignans are classified into six subgroups based on general structures: the dibenzylbutanes and dibenzylbutanediols (formed by two P-P' linked C6-C3 units); dibenzylbutyrolactones (with two P-p' linked C6-C3 units and an additional y-y' lactonic bridge); substituted tetrahydrofurans (two P-P' linked C6-C3 units and an oxygen bridge); 2,6-diaryl-3,7dioxabicyclo[3.3.0]octanes, also called tetrahydrofiirofurans, (two p-P' linked C6-C3 units and two oxygen bridges); arylnaphthalene derivatives (two P-P' linked C6-C3 units and a C2,Ca' bond that yields a tetralin or a naphtalene); and dibenzocyclooctadiene derivatives (two P-p' linked C6-C3 units and a €2,02' bond that forms a biphenyl). The neolignans possess many different structures and are divided into more than fifteen subgroups. They include benzoftiran, dihydrobenzofuran, diarylethane, benzodioxin, alkyl aryl ether and bicyclo[3.2.1]octane derivatives among others The principal skeletons of lignans are listed in Table 1 and the chemical structures of the most relevant pharmacologically active lignans are listed in Tables 2-10.

190 Table 1.

General Skeletons of Lignans Cited in This Review

Dibenzylbutane

Arylnaphthalene

Tetrahydrofiiran

Dfcenzylbutyrolactone

Dibenzocycboctadiene

Aryltetrahydronaphthaleiie

Cycbbutane

Furo&ran

Table 2.

Active Dibenzylbutane Lignans

B

.A

Burseran 2,3-Dibenzylbutane-l,4-diol Dihvdrocubebin ,~~eso-Dihvdroguaiarelic acid (-)-3,4-Divanillyltetrahydrofuran Enterodiol Enterohran hIachilin .A Nordihydroguaiaretic acid (NDGA) Phyllantin Rhinacanthin E Rhinacanthin F -)-Secoisolariciresinol (-)-Secoisolariciresinol diglucoside 2,6,8,10-Tetra-O-rnethyl NDG.A

.A .-I .-I

B A

RI Rz -0CH:OH H -0CH:OOCH3 OH OCH3 OH OH H OH H -0CH;OOH OH OCH3 OCH3 -OCH;.O-0CH:OOCH3 OCH3 0-Glu OCH, OCH,

R3

OCHp H H H OCHs OH OH H H H OCH, OCH3

C R4

Rs

OCH3 OCH3 H H -0CH:OOH OCH3 OH H H H H H -0CH:OOH OH OCH3 OCHS -0CH:O-0CHzOOH H 0-Gh H OCH, OCH,

R-

Rs -CH:OCHz-

OH OH CHtOH CH:OH CH3 CHI -CH:OCHy CHzOH CHPH -CHzOCH:CHI CH3 CH3 CH3 CHiOCHt CH20CH3 COOCH3 COOCH, COOCHj COOCH3 CH-OH CH:OH CH:OH CH:OH CH3 CH3

-A

.A

B C

.A

1’ X B .A,B .A .A

B

OCH3 H

*

2

192

Table 3.

Active Dibenzylbutyrolactone Lignans

XN

r^^^

R5 R4

R4

Arctigenin Arctigenin methyl ether (-)-Arctiin Bursehemin 4'-Demethylyatein (-)-Deoxypodorhizone Enterolactone Epipodorhizol (-)-Hinokinin (-)-6'-Hydroxyyatein (-)Matairesinol (-)Matairesinol dimethyl ether Nortrachelogenin Podorhizol Prestegane B Trachelogenin (-)-rra«5-2-(3",4",5"-trimethoxybenzyl)3-(3\4'-ethylenedioxybenzyl)butyrolactone (-)-traAw-2-(3",4"-dimethoxybenzyl)-3-(3' 4' -methylenedioxybenzyl) butyrolactone Wikstromol (-)-Yatein

A A A A A B B A A B B B

Ri R2 OCH3 OH OCH3 OCH3 OCH3 0-Glu OCH3 OCH3 -OCH2O. -OCH2OOH H -OCH2O-OCH2O-OCH2OOCH3 OH OCH3 OCH3 OH OCH3 -OCH2OOH OCH3 OH OCH3 -OCH2O-

OCH3 OCH3 OH OCH3 H OCH3 OCH3 OCH3 OCH3 OCH3 OH OCH3 OCH3

R4 Rs H OCH3 H OCH3 OCH3 OCH3 -OCH2OOH OCH3 OCH3 OCH3 H H OCH3 OCH3 -OCH2OOCH3 OCH3 OH H H OCH3 OH H OCH3 OCH3 H OCH3 H OCH3 OCH3 OCH3

B

-OCH2O-

OCH3

OCH3

B A

OCH3 OH -OCH2O-

H OCH3

OH OCH3

B B B B A

R.. OCH3 OCH3

H H

R7

R6 H H H H H H H H H OH H H H H H OH H

H H H H H H H aOH H H H H H pOH H H H

R8 H H H H H H H H H H H H OH H H H H

H

H

H

H

OCH3 OCH3

OH H

H H

H H

193 Table 4.

Active Aryltetrahydronaphtalene Lignans ?5

Ri-

R2

R5

R^_

R2

0CH3

Austrobailignan-1 p-Apopi cropodophyll in 4 '-Demethyl deoxypodophy 11 otoxin 4'-Demethylepipodophyllotoxin 4'-DemethyIpodophyllotoxin Deoxypi cropodophyll in Deoxypodophyllotoxin (anthricin) 4-Epipodophyllotoxin Isopicropodophyllone Isodeoxypodophyllotoxin 4-0-Methylpicropodophyllotoxin 5-Methoxypodophyll otoxin 5-Methoxypodophyllotoxin-4-P-D-glu Morelensin a-Peltatin p-Peltatin p-Peltatin A methyl ether Picropodophyllone Picropodophyllotoxin Podophyll otoxin Po^ophyllotoxin-p-D-glucoside

A A A A A B A A D C B A A A A A A B B A A

Ri H H(A') H H OH H H H

=o

R2 H H H OH H H H OH

H H H OCH3 OH H 0-Glu H H H H H H H H H =0 OH H OH H OGlu H

R3 R4 -OCH2OOCH3 OCH3 OH OCH3 OH OCH3 OH OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 H OCH3 OH OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3

R5 H H H H H H H H H H H OCH3 OCH3 H OH OH OCH3 H OCH3 H H

194

Table 5.

Active Arylnaphtalene Lignans

R3 Ri R2 A -OCH2OH A -OCH2OH A -OCH2OOCH3 A -OCH2OH A -OCH2OH -OCH2OH A Deoxydehydropodophyllotoxin A OCH3 OCH3 Diphyllin H A OCH3 OCH3 H Diphyllin acetate A OCH3 OCH3 H Diphyllin-0-apioside Diphyllin-O-apioside-5-acetate A OCH3 OCH3 H A 0CH3 0CH3 Diphyllinin H A OCH3 0CH3 Diphyllinin monoacetate H A 0CH3 OCH3 Diphyllinin crotonate H B Helioxanthin H -OCH2OA OCH3 0CH3 Justicidin A H A OCH3 OCH3 Justicidin B H B H -OCH2OJusticidin D (Neojusticin A) H -OCH2OB Justicidin E (Taiwanin C) 5-M ethoxy dehydropodophyllotoxin -OCH2OA OCH3 B Neojusticin B H OCH3 OCH3 Phyllamyricin E A OCH3 OCH3 H B Retrojusticidin B H OCH3 OCH3 -OCH2OH Taiwanin E A A -OCH2OH Taiwanin E methyl ether H Tuberculatin A OCH3 OCH3 c a =-O-apiose-S-acetate; b =-OC4H40(OH)2CH20H; d =-OC4H40(OH)2CH20COCHCHCH3 Chinensinaphthol Chinensinaphthol methyl ether Dehydro-p-peltatin methyl ether Dehydropodophyllotoxin 4'-(9-Demethyldehydropodophyllotoxin

R6 R? R8 R5 R4 H H OH OCH3 OCH3 H H OCH3 OCH3 OCH3 H H OCH3 OCH3 OCH3 H OH OCH3 OCH3 OCH3 OH H OCH3 OH OCH3 H H OCH3 OCH3 OCH3 H -OCH2OH OH H OAc -OCH2OH H -OCH2OO-api H -OCH2OH H a b H -OCH2OH c -OCH2OH H d H -OCH2OH H -OCH2OH H -OCH2OH H OCH3 -OCH2OH H H H -OCH2OH OCH3 H -OCH2OH H H OH OCH3 OCH3 OCH3 H -OCH2OH OCH3 H -OCH2OH OCH3 H -OCH2OH H -OCH2OH H OH H -OCH2OH OCH3 H -OCH2OO-api H =-OC4H40(OH)2CH20COCH2

Table 6.1.

Active Aryltetralin Lignans

RI (+)-Dimethylisolariciresinol-2-a-~lose H>pophyllantin lsolariciresinol (+)-Lvoniresinol3a-O-P-D-glucop\ranoside hlethyl9-deo~-9-oso-a-apopicropodoph~llate Nirtetralin Nudiposide 2,2,2-Trifluoroethylhydrazoneof methyl -9-deosy-9-0x0-aapopicropodophvllate Phenylhydrazone of methyl 9-deo!q+OXO-~~apopicropodophvllate Podophyllic acid (+)-Tsugacetal

.\

B B

B .A B A .A

R2

R3

OCH3 OH H OCH3 -0CHzOOCH, OH H OCH3 OH OCH, -0CH20H -0CH-0OCH3 OCHl OH OCH3 -0CH20H

Rs

R6

R4 H H H OCH, OCHJ H OCH3 OCH,

OH CH3 OH OH OCH, CHI OH OCH3

CH2O-Xy1 CH2OCH3 CHzOH aCH20glu PCOOCH, CHzOCHi PCHZOXYI PCOOCH, PCOOCH,

.A

-0CHzO-

H

OCH3

OCH3

.A B

-OCH20OCH3 OH

H H

OCH3 H

OCHa OH

RCHzOH CHzOCH, CHzOH PCHzOH CHO CHzOCH, aCH2OH CH=N-NHCH:CF, CH=N-NH-Ph

aCOOH BCH20H -CH2OCH(POCH,)-

R8

H H H H

HJH H HJ’ H.1aOH

H

196 Table 6.2.

Active Aryltetralin Lignans H3C0.

H2C0

2,3-Bis(methoxycarbonyl)-l -(3,4dimethoxyphenyl)-4-hydroxy6,7,8-trimethoxynaphthalene Phyllamycin B Phyllamyricoside A Phyllanthostatin A

Table 7.1.

Ri

R2

R3

R4

R5

R6

R7

OH

CO2CH3

CO2CH3

OCH3

OCH3

H

OCH3

H 0-GIu H

CH2OCH3 CH2OH CH2OCOCH3

CHO CH3 COOGlu

H

-OCH2OH -OCH2C>H -OCH2O-

Active Dibenzocyclooctadiene Lignans

H3C0

0CH3

B R2

Ri Episteganangin

H

A 00c

CH3

Isopicrostegane Steganacin

B A

H H

Steganangin

A

H

H OAc

OOC

CH3

II H H

197 Table 7.2.

Active Dibenzocyclooctadiene Lignans CH3 R,0.

R9O

Angel oylgomi sin H

Ri

R2

CH3

CH3

R3

HjC

Benzoyl gomisin O Deoxygomisin A Gomisin A Gomisin B

R4

R5

Re

R7

Rs

R9

CH3

CH3

H

H

OH

CH3

OCOC6H5 H H

H H H

H H OH

CH3 CH3 CH3

H

CH3

OH

H H H H H H H

H H H H H H H

CH3 CH3 CH3 CH3 CH3 CH3 CH3

H

CH3

OH

H H H H

OH H OH CH3

CH3 CH3 CH3 OH

OH

CH3

H

CH3

CH3

CH3 CH3 -CH2-CH2-

CH3 CH3 CH3

-CH2CH3 CH3 CH3 CH3

-CH2-

CH3

CH3

CH3 H3C

Gomisin J Gomisin K3 Gomisin L2 Gomisin Mi Gomisin N Kadsuranin Interiotherin A

H CH3 CH3 CH3 H CH3 CH3 CH3 -CH2CH3 CH3 -CH2-

CH3 H CH3 H CH3 CH3 CH3

Interiotherin B

-CH2-

CH3

CH3 H CH3 CH3 -CH2-CH2CH3 CH3 -CH2-CH2-

CHj

H H H H H H OCOC6H5

-CH2H,C

Schizandrin Schisandrin A Schisandrol A Schisantherin D

CH3 CH3 CH3 CH3 CH3 CH3 -CH2-

CH3 CH3 CH3 CH3

CH3 CH3 CH3 CH3 CH3 CH3 -CH2-

Tigloylgomisin P

-CH2-

CH3

CH3

CH3

H

H H H OCOC6H5 H H5C

Wuweizisu C

-CH2-

CH3

-CH2-

H

CH3

H

198

Active Tetrahydrofuran Lignans

Table 8.1.

0CH3

0CH3

Grandisin L-652,731 L-659,989 L-662,025 Nectandrin B (+)-Neoolivil

C A A A B C

Ri

R2

R3

R4

OCH3 OCH3 SO2CH3

OCH3 OCH3 OCH2CH2CH3 OCH2CH=CH2 OH OH

OCH3 OCH3 OCH3 OCH3 OH OH

OCH3 OCH3 OCH3 OCH3 H H

N3

H H

R5 CH3

R6 CH3

H H H

H H H

CH3

CH3

CH2OH

CH2OH

199

Active Tetrahydrofuran Lignans

Table 8.2.

H3C0,

CH2OH

HO

R2

Ri Acuminatin Lariciresinol LariciresinoI-4p-D-glucoside

-OCH2OOH O-Glu

OCH3 OCH3

HOHjC

CH30' OCH3

(-)-Hernone 7-S,8i^,8'/?-(-)-Lariciresinol-4,4'-bis-0-|3-D-glucopyranoside Magnone A Magnone B 75,8/?,8'/?-(-)-5-Methoxylariciresinol-4,4'-bis-(9-p-D-gIu (-)-Nymphone

A B A A B A

Ri OCH3 H H H H OCH3

R2 H H H OCH3 OCH3 H

R3 R4 OCH3 OCH3 O-Glu OCH3 OCH3 OCH3 OCH3 OCH3 O-Glu OCH3 -OCH2O-

200

Table 9.1.

Active Furofuran Lignans

(+)-1 - Acetoxypinoresinol (+)-l -Acetoxypinoresinol dimethylether (+)-l -Acetoxypinoresinol-p-D-glu (-)-Eudesmin (+)-Eudesmin = (+)-Pinoresinol dimethyl ether (+)-l -Hydroxypinoresinol (+)-Isogmelinol Kobusin (+)-Magnolin (+)-Medioresinol (-)-Pinoresinol (-)-Pinoresinol-p-D-glucoside (+)-Pinoresinol (+)-Pinoresinol-p-D-glucoside (+)-Pinoresinol di-p-D-glucoside (+)-Pinoresinol monomethyl ether (-)-Prinsepiol (+)-Sesamin (-)-SyringaresinoI (-)-Syringaresinol di-p-D-glucoside (-)-Syringaresinol diacetate (+)-Syringaresinol = (+)-Lirioresinol B (+)-Syringaresinol di-p-D-glucoside = Liriodendrin (+)-Syringaresinol dimethyl ether = (+)-Yangambin

B B B A B

Ri OCH3 OCH3 OCH3 OCH3 OCH3

R2 OH OCH3 OH OCH3 OCH3

R3 H H H H H

R4 OCH3 OCH3 OCH3 OCH3 OCH3

R5 OH OCH3 O-Glu OCH3 OCH3

R6 H H H H H

R7 OAc OAc OAc H H

Rs H H H H H

H H OCH3 H H H H H H H H H H OCH3 OCH3 OCH3 OCH3

OH OCH3 OCH3 OCH3 -OCH2OOCH3 OCH3 OH OCH3 OH OCH3 OH OCH3 OH OCH3 OH OCH3 OCH3 O-Glu OH OCH3 OH OCH3 -OCH2OOH OCH3 OCH3 O-Glu OAc OCH3 OH OCH3

H H H OCH3 OCH3 H H H H H H H H OCH3 OCH3 OCH3 OCH3

OH

H

A A A B

OH OCH3 0CH3 OCH3 H OCH3 OCH3 OCH3 OH OCH3 OH OCH3 OCH3 O-Glu OH OCH3 OCH3 O-Glu OCH3 O-Glu OCH3 OCH3 OH OCH3 -OCH2OOH OCH3 OCH3 O-Glu OAc OCH3 OH OCH3

OH H H H H H H H H H OH H H H H H

H H H H H H H H H H OH H H H H H

B

OCH3

O-Glu

OCH3

OCH3

O-Glu

OCH3

H

H

B

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

H

H

B B B B B A A B B B B B

B

201

Table 9.2.

Active Furofuran Lignans

Hi'

(-)-Asarinin = Episesamin (+)-Epiaschaiitin (-)-Epieudesmin (+)-Epimagnolin (+)-Epipinoresinol (+)-Epipinoresinol dimethyl ether (+)-Epiyanganibin (+)-Fargesin Isomagnolin (-)-Phylligenin Phillyrin

A C B C A A C C A B A

Ri R2 -OCH2O-OCH2OOCH3 OCH3 OCH3 OCH3 OCH3 OH OCH3 OCH3 OCH3 OCH3 -OCH2OOCH3 OCH3 OCH3 OH OCH3 OCH3

R3 H H H H H H OCH3 H H H H

R5 R4 -OCH2OOCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OH OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 O-Glu H

R6 H OCH3 H OCH3 H H OCH3 H OCH3 H OCH3

202 Table 10.1.

Active Benzofuran Neolignans

Conocarpan Dehydroconiferyl alcohol 3 ',4-(9-Dimethylcedrusin Licarin A Licarin B Licarin D Obovatifol Perseal C

A B A B B B B B

Ri CH=CHCH3 CH=CHCH20H CH2CH2CH2OH CH=CHCH3 CH=CHCH3 CH=CHCH3 CH=CHCH3 CHO

R2 H OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3

R3 R4 H OH OCH3 OH OCH3 OCH3 OCH3 OH -OCH2OOCH3 OCH3 OH OH -OCH2O-

R5 H H H H H H OCH3 H

OH

Eupomatenoid-5 Obovaten Perseal D

Ri CH=CHCH3 CH=CHCH3 CHO

R2 H OCH3 OCH3

R3 OCH3 OH OH

R4 H OCH3 OCH3

R6 CH3 CH2OH CH2OH CH3 CH3 CH3 CH3 CH2OH

203

Table 10.2.

Active Benzofuran Neolignans OCH

HsCO-

4'-Demethoxy-3',4'-methyIenedioxyniethylrocaglate 4'-Demethoxy-3' ,4'-methylenedioxyrocaglaol l-0-Formyl-4'-demethoxy-3',4'-methylenedioxymethylrocaglate Methylrocaglate 1 -Oxo-4'-demethoxy-3 ',4'-methylenedioxyrocaglaol

Ri

R2

R3

OH OH

H H H H

COOCH3

OCHO

OH

=o

H COOCH3 COOCH3

H

R4

R5

-OCH2O-OCH2O-OCH2OOCH3 H -OCH2O-

Isolation and identification Research is increasingly focused on developing adequate techniques for analysing lignans from plant sources and physiological fluids. Four reviews contain a section dedicated to analysing and identifying lignan mixtures [58]. The efficiency and selectivity of supercritical fluid extraction (SFE) make this method an alternative to conventional extraction of lignans with organic solvents. Supercritical carbon dioxide was used to extract lignans from Schisandra chinensis (Schisandraceae) fruit, seeds and leaves [36,37]. Choi et al [38] have established the optimal SFE conditions of temperature and pressure for the main bioactive lignans schisandrol A, schisandrol B, schisandrin A, schisandrin B, and schisandrin C. The bioactive neolignans from Magnolia virginiana (Magnoliaceae) flowers were extracted sequentially with supercritical CO2 alone or modified with solvents, and

204

were quantified by high performance liquid chromatography (HPLC) using photodiode array detection (DAD) [39]. Magnolol and honokiol were extracted from Magnoliae cortex using supercritical-fluid chromatography (SFC) on-line coupled with SFE by an amino column trapping [40]. Wolf ^/ al [41] resolved the atropisomers of arylnaphthalene lignans by cryogenic subcritical fluid chromatography. Both normal phase and reverse phase HPLC with an UV detector or in combination with DAD were found to be rapid, reliable methods for qualitative and quantitative analysis of lignans. Quantification of the antioxidant lignan glucosides sesamin, sesamolin, sesaminol and sesamolinol in some varieties of sesame was achieved by HPLC determination [42]. On-flow liquid chromatography/proton nuclear magnetic resonance (LC/^H-NMR) and LC/UV/MS analysis was performed to identify the antioxidant lignans (-)-phylligenin, (-)-eudesmin and (-)epieudesmin from Orophea enneandra (Annonaceae) [43]. LC/mass spectrometry (MS), fast-atom bombardment (FAB)-MS and FAB-tandem MS techniques were applied to the rapid detection of lignans with radical scavenging activity from Krameria triandra (Krameriaceae) roots [44]. The isolation of the lignans eudesmin, magnolin, yangambin and kobusin from Polygala gazensis (Polygalaceae) and subsequent analysis by thermospray LC-MS and HPLC-DAD of two other related Polygala species have been reported [45]. This analysis was also applied successfully to the identification of lignan constituents from Schisandra chinensis fruit [46]. HPLC coupled with either ionspray or continuous-flow FAB-MS or gas chromatography (GC)-MS using single ion monitoring allowed rapid detection and identification of secoisolariciresinol diglycoside in flaxseed. Two diastereoisomers of secoisolariciresinol diglycoside were detected by this method [47,48]. The application of a solid-state fluorodensitometric technique after thin layer chromatography allowed highly reproducible, sensitive quantification of the toxic constituents of Cleistanthus collinus (Euphorbiaceae), the arylnaphthalene lignan lactone diphyllin, and its glycosides cleistanthin A and cleistanthin B in samples of forensic and clinical value [49,50]. Cleistanthin A was determined by an enzyme-linked immunoassay in cases of C. collinus poisoning [51]. Analytical high-speed countercurrent chromatography (CCC) has also been used to study lignans [52]. Interfacing CCC coupled with thermospray MS provides an analytical methodology for identifying bioactive lignans

205 from Schisandra rubriflora [53]. Anti-HIV lignans from Larrea tridentata were isolated by assay-guided CCC and their chemical structures determined by GC-MS and NMR [54]. More recently Ma et al [55] purified these compounds by pH-zone-refming CCC. LC-MS and GC-MS have been used to analyse samples containing the mammalian lignans enterolactone and enterodiol and their precursors matairesinol and secoisolariciresinol. Fotsis et al [56] described a capillary GC method for quantifying lignans in human urine after appropriate derivatization. The plant lignans lariciresinol, isolariciresinol and secoisolariciresinol were identified by GC/MS in human urine [57]. A quantitative method based on ion-exchange chromatography and isotope dilution GC-MS has been described for the determination of plasma lignans [58]. Secoisolariciresinol and matairesinol in plant-derived foods were measured by an isotope dilution GC-MS method [59] that was also applied to quantify enterolactone and enterodiol in human and animal sources [6062]. Schisandrin from Schisandra fruits has been determined in human plasma by selected-ion monitoring with GC-MS using a ftised-silica capillary column [63]. Atkinson et al [64] presented a GC coupled with an ion mobility spectrometric (IMS) method that makes it possible to quantify selected mammalian lignans in biological fluids. The need for selective, sensitive analytical techniques to measure these metabolites in human plasma, tissue and urine has led to the development of an HPLC with coulometric array detection, which uses a series of flow-through electrochemical sensors, each providing 100% electrolytic efficiency, with satisfactory resolution [65]. A method based on time-resolved fluoroimmunoassay using a europium chelate as a label has been described to measure plasma enterolactone [66]. Yoo and Porter [67] developed a sensitive immunoassay for the natural Podophyllum lignan, podophyllotoxin. Reverse-phase KPLC separation followed by radioimmunoassay (RIA) for human plasma samples and immunoaflfinity extraction (lAE) followed by RIA for human urine samples were used to investigate a lignan-related hypocholesterolemic agent. This analysis proved to be very usefiil in quantifying extremely low drug concentrations in body fluids [68].

206 PARMACOLOGICAL ACTIVITIES Introduction The principal pharmacological activity of lignans can be summarised in the following relevant groups. The lignans play an interesting role as phytoestrogens in mammals, including humans; they have anticancer and antiviral properties; they modify the cardiovascular function by different mechanisms including the modification of phosphodiesterase activity or platelet activating factor function; they have antioxidant properties and can modify the liver function; and some of them modify the activity of enzymes and mediators implicated in inflammation and immunity process. Lignans as phytoestrogens Phytoestrogens are a group of naturally occurring diphenolic compounds present in legumes, whole grains, fruits and vegetables that exert estrogenic effects on the central nervous system (CNS). The physiological effects are similar to those of endogenous estrogens mediated by binding to the estrogen-receptor (ER), followed by gene activation and its specific generation of products and physiological effects, like induction of estrus and stimulation of the female genital tract [69]. High consumption of phytoestrogen-rich foods has been linked to a reduced incidence of estrogen hormone-dependent cancers. Phytoestrogens were described for the first time by Setchell, Axelson et al [70,71], and their effects have been widely studied by Adlercreutz et al since 1980, and reviewed by them [72-77] and by Potter and Steinmetz [78], Knight et al [79], Kurzer and Xu [69], Rickard and Thompson [80], Mazur and Adlercreutz [81] and Tham et al [82]. They described three main classes of phytoestrogens, one of which are lignans. These compounds are not natural products present in plants but are produced by bacterial metabolism of dietary components in the animal and human gut. Matairesinol, secoisolariciresinol and its diglycoside are the major precursors of the mammalian lignans enterodiol and enterolactone and they are present in oilseeds, such as Linum usitatissimum (Linaceae) seeds (flaxseed), soybeans, sunflower seeds and peanuts but are also present in foods such as whole dried legumes (lentils, kidney, navy and pinto beans), whole grain cereals (triticale, wheat, oats, brown rice, corn, rye, barley),

207

cereal brans (oat, wheat and rice bran), vegetables (garlic, asparagus, carrots, sweet potatoes, broccoli, ...) and fruit (pears, plums, ...). In fact, enterolactone excretion may be an indicator of consumption of grains and legumes. In the case of glycosides, metabolism occurs after a previous hydrolysis of the sugar moiety by the gastric juice and p-glucosidases from foods or bacteria gut. After absorption in the small intestine, lignans are conjugated in the liver and eliminated in the kidney by urine and in liver by bile, after which they undergo enterohepatic circulation with a later reabsorption after new bacterial hydrolysis. In humans, the principal lignans identified are enterolactone, enterodiol and matairesinol in serum, semen, urine, faeces and bile. In addition, they have been identified in cow's milk. The values of excretal lignans increase 3- to 285-fold depending on the organic fluid. However, the urinary content of enterodiol and enterolactone originating from secoisolariciresinol diglycoside-fed rats represented only twenty per cent of the levels of flaxseed-fed rats. This indicates the presence of other precursors or the incomplete conversion of secoisolariciresinol diglycoside to enterodiol and enterolactone [83]. The most relevant physiological effects described for the phytoestrogens are: a) effects on human reproductive hormones, including sexual differentiation, fertility, binding to the estrogen receptor, synthesis and availability of estrogens, and effects on grov^h of estrogen dependent cells; b) tumor cell differentiation and mitogenesis, focusing on protein tyrosine kinase activity and DNA topoisomerase; c) angiogenesis; and d) antioxidation. In addition, other physiological processes may be modified, such as menstrual cycles and menopausal symptoms. On the other hand, the pathological effects are principally those related to sexual hormone dependent diseases, such as polycystic ovary syndrome, osteoporosis, sex organ cancer, and cardiovascular disease as well as menopausal symptoms. In the case of cardiovascular disease, the phytoestrogens decrease lowdensity lipoprotein (LDL) cholesterol and increase high-density lipoprotein (HDL) cholesterol. Gansser et al [84] described a test system designed to search for compounds that interfere with human sex hormone-binding globulin (SHBG) even in complex plant extracts, and report that the lignan secoisolariciresinol reduced the binding activity of human SHBG. After studying the effects of a short-term phytoestrogen-rich diet on menopausal symptoms and serum levels of phytoestrogens and SHBG, Brzezinski et al [85] concluded that a 12-week partial substitution of an omnivorous diet for

208 phytoestrogen-rich food increases the SHBG semm levels in postmenopausal women, and may alleviate symptoms such as hot flashes and vaginal dryness. However, the long-term effects on bone density and heart disease remain to be determined. Except (-)-pinoresinol, all the compounds tested by Schottner et al [86] had binding affinity to human SHBG in the in vitro assay. Whereas (±)enterodiol, (+)-neoolivil, (-)-isolariciresinol and dehydrodiconiferyl alcohol developed only weak affinities, (-)-secoisolariciresinol and enterolactone were moderate, enterofuran showed higher affinity (73% displacement of specific binding), and the affinity of (-)-3,4-divanillyltetrahydrofuran was the highest of all, with 95% ["^H]-dihydrotestosterone displacement. As lignans may displace active steroid hormones from the SHBG binding site, they may influence the blood level of these hormones and have potential beneficial effects on benign prostatic hyperplasia. In a complementary study, the same authors [87] investigated, in the same experimental system, a wide variety of synthetic lignans derived from the most active one, (-)3,4-divanillyltetrahydrofuran, with different substitution patterns in the aromatic and aliphatic part of the molecule. They concluded that (±)diastereoisomers are more active than meso compounds, that the 3methoxy-4-hydroxy substitution pattern in the aromatic part is the most effective, and that activity increased with the decrease in polarity of the aliphatic part of the molecule. Finally, they reported the effects of the previously studied natural lignans cited above, and also NDGA, on the binding of [^H]-5a-dihydrotestosterone to SHBG [88]. The lignan (-)-3,4divanillyltetra-hydrofiiran showed the highest binding affinity again {Kdi = 3.2 luiM). The results suggest a dose dependent competitive inhibition of the SHBG-dihydrotestosterone interaction. When hydrophobicity increases in the aliphatic part of the butane-1,4-diol-butanolide-tetrahydrofuran lignans, these compounds have higher binding affinity, whereas the 3-methoxy-4hydroxy substitution in the aromatic ring is the most effective for binding to SHBG. Enterolactone had been described as a moderate competitive inhibitor of human estrogen synthetase (aromatase) and it binds to or near the substrate region of the active site of the P-450 enzyme [61]. In another study [89], seven lignans were evaluated for their abilities to inhibit aromatase enzyme activity in a human preadipose cell culture system. The lignan enterolactone and its precursors, didemethoxymatairesinol and 3'-demethoxy-3-0demethylmatairesinol, decreased aromatase enzyme activity, with K\ values

209 of 14.4, 7.3 and 5.0 |iM, respectively, whereas the aromatase inhibitor aminoglutethimide had a K\ value of 0.5 |LIM. On the other hand, the lignan enterodiol and its precursors, 0-demethylsecoisolariciresinol, demethoxysecoisolariciresinol, and didemethyl-secoisolariciresinol, were less active. The inhibition of human preadipocyte aromatase activity by lignans suggests a mechanism by which consumption of lignan-rich plant foods may contribute to a reduction in estrogen-dependent disease. In addition, the endogenous lignans are inhibitors of 5a-reductase. This can justify the effects on the development of prostatic carcinoma and the inhibition of other growth-promoting steroid hormones. In fact, it was reported [90] that 5a-reductase and 17P-hydroxysteroid dehydrogenase are inhibited by enterodiol and enterolactone in human genital skin fibroblast monolayers and homogenates, in benign prostatic hyperplasia tissue homogenates, and in genital skin fibroblast monolayers, but enterolactone was the most potent inhibitor. To elucidate whether enterolactone has antiestrogenic properties Mousavi and Adlercreutz [91] studied the w vitro effect of relatively low concentrations of enterolactone (0.5-2 |Limol/L) added both alone and in combination with estradiol (1 nmol/L), using MCF-7 breast cancer cells in culture. By itself, enterolactone stimulated the proliferation of MCF-7 cells, but in combination with estradiol it either resulted in lower stimulation or had no effect at all as compared with the control. The concentrations of enterolactone needed for the elimination of the proliferative effect of estradiol are physiological and similar to those used in experiments with the antiestrogen tamoxifen. Recently [92], the antiestrogenic effects of the enterolactone precursor secoisolariciresinol diglycoside was compared with tamoxifen by monitoring rat estrus cycling, and it proved to be antiestrogenic but without having gross tissue toxicity. Phytoestrogens may modulate the activity of the human sex steroid binding protein and so influence the role of this protein in the delivery of hormonal information to sex steroid-dependent cells, as reported by Martin et al [93] in research with NDGA, enterolactone and enterodiol. These phytoestrogens had different dose-dependent inhibitory effects on the steroid binding protein. It is not clear whether there is a positive association between the rate of bone loss in postmenopausal related osteoporosis and the enterolactone level in human [94]. Experimental results on thirty-two selected women with an annual rate of radial bone loss did not demonstrate that a low.

210 unsupplemented dietary intake of phytoestrogens has a preventive effect on postmenopausal cortical bone loss. However, no conclusions can be drawn about effects of higher doses of phytoestrogens. Tou et al [95] studied the effects of the mammalian lignan precursor secoisolariciresinol diglycoside as an estrogen agonist or antagonist, and examined whether feeding flaxseed or lignan to rats during a hormonesensitive period can have effects on reproduction. The results demonstrated that flaxseed had no effect on pregnancy outcome, as compared with other treatments. The female offspring had shortened anogenital distance, higher uterine and ovarian relative weights, began puberty at an earlier age and weighed less at puberty, had lengthened estrus cycle and persistent estrus. The male offspring had reduced postnatal weight gain, greater sex gland and prostate relative weights, suggesting estrogenic effects. On the other hand, when compared with the basal diet, lignans reduced immature ovarian relative weight by 29%, delayed puberty, and tended to lengthen diestrus, indicating an antiestrogenic effect. Because flaxseed affects the reproductive development of offspring, caution is suggested when consuming flaxseed during pregnancy and lactation. Anticancer and cytotoxic effects The resin of Podophyllum, called podophyllin, has been known for its antitumoral properties since the end of the last century. Podophyllotoxin was isolated from it, and during the present century its pharmacological properties have been established. Its gastrointestinal toxicity, antimitotic properties, binding to tubulin and relationships with colchicine have been studied and developed over the last few years. Other closely related compounds were isolated from species of Podophyllum, such as the podophyllotoxin-P-D-glucoside and 4'-demethylpodophyllotoxin, 4'-demethylepipodophyllotoxin, a-peltatin and P-peltatin. In addition, some semisynthetic derivatives of 4-epipodophyllotoxin, such as etoposide and teniposide, are used in therapy. In 1998, Imbert [15] reviewed the history and isolation of the naturally occurring podophyllotoxins, hemisynthesis and use in cancer chemotherapy of this interesting group of lignans. He compiled all the information on the mechanism of action and cited the possibility of obtaining new hemisynthetic derivatives with different mechanisms of action to treat cancer. The podophyllotoxins are inhibitors of microtubule assembly with a capacity to inhibit topoisomerase II by

211

inducing double-stranded breaking of DNA molecules. The structural features responsible for this activity were broadly determined. Two hundred and seventy-four natural and semisynthetic derivatives of podophyllotoxins are reviewed by Damayanthi and Lown [16], including synthesis, pharmacological activity, clinical studies, mechanism of action, structureactivity relationships and data on cytotoxicity, inhibition of DNA topoisomerase II activity and cellular protein-DNA complex formation of many of them. Recently, Kinghorn et al [96] reviewed the anticancer agents discovered by activity-guided fractionation and they include seventy compounds from twenty-two plants. Only one lignan is cited in the review; guaiacylglycerolp-0-6'-(2-methoxy)cinnamyl alcohol ether isolated from Brucea javanica (Simaroubaceae) exhibited weak activity against the induction of cell differentiation of human promyelocytic leukemia (HL-60) cells [96,97].

0CH3

H3C0

Etoposide (R=CH3) Teniposide (R=tienyl)

Guaiacylglycerol-p-0-6'-(2-methoxy)cinnamyl alcohol ether

All the antitumor and antimitotic lignans discovered before 1984 have been compiled by MacRae and Towers [9]: NDGA, (+)-dimethylisolariciresinol-2a-xyloside, burseran, podophyllotoxins and peltatins, austrobailignan-1, diphyllin and its acetate, justicin-D, diphyllinin and its derivatives, stegnacin, stegnangin, stegnanol, stegnanone, (-)-/ram'-2(3",4"-dimethoxybenzyl)-3-(3',4'-methylenedioxybenzyl)butyrolactone and the 3",4",5"-trimethoxy derivative. Other interesting compounds, however, are not included in it, such as phyllanthostatin A (ED50 4 |Ltg/mL) isolated from Phyllanthus acuminatus (Euphorbiaceae) [98]; megaphone acetate.

212 dysodanthin A and dysodanthin B from Endlicheria dysodantha (Lauraceae), which showed activities in the brine shrimp lethality test, inhibited the growth of crown gall tumors on potato discs and were cytotoxic to human tumor cells in culture [99]; 3',4-(9-dimethylcedrusin from Croton spp. (Euphorbiaceae), which did not stimulate cell proliferation, but rather inhibited thymidine incorporation while protecting cells against degradation in a starvation medium [100]; schiarisanrin C from Schisandra arisanensis, which showed cytotoxic activity against tumor cells, with ED50 values between 0.36-7.1 |Lig/mL [101]; (-)syringaresinol from Annona montana (Annonaceae) and its acetate, which showed significant cytotoxicity (ED50 values of 0.67 and 3.78 M-g/mL, respectively) against P-388 cells [102]; brevitaxin, the first terpenolignan isolated from Taxus brevifolia (Taxaceae), which showed a selective cytotoxicity against the prostate cancer cells, with an ED50 value of 6.8 |LIM [103]; asarinin and xanthoxylol from Asiasarum heterotropoides var. mandshuricum (Aristolochiaceae), which exhibited remarkable inhibitory effects on a two-stage carcinogenesis test of mouse skin and pulmonary tumors [104]; (-)-6'-hydroxyyatein, (-)-hernone and (-)-nymphone isolated from Hernandia nymphaeifolia (Hernandiaceae), which exhibited cytotoxic activities against four cell lines with ED50 values were < 4 |ig/mL [105]. The in vitro cytotoxicity of 5-methoxypodophyllotoxin, obtained from a root culture derived from Linum flavum, against EAT and HeLa cells was determined and compared with those of podophyllotoxin, etoposide, teniposide and 5-methoxypodophyllotoxin-4-3-D-glucoside [106]. The tested lignans had about the same cytotoxic potency as podophyllotoxin (ED50 of 32 and 22 |Lig/mL versus 42.8 and 20.5 M^g/mL, respectively against EAT and HeLa cells). However, in comparison with etoposide and teniposide they were clearly less potent (1.1 and 7.9 |Lig/mL, and 0.06 and 0.3 iLig/mL, respectively). Nine lignans were isolated from Hyptis verticillata (Lamiaceae) by bioactivity directed fractionation using the brine shrimp lethality test [107]. 5-Methoxydehydropodophyllotoxin (P-388, ED50 4 |ig/mL) and the dehydro-P-peltatin methyl ether (P-388, ED50 18 |Lig/mL) were reported for the first time as isolated natural products. All the isolated compounds were evaluated against a panel of cell lines comprising a number of human cancer cell types (breast, colon, fibrosarcoma, lung, prostate, KB, and KBVI) and murine lymphocytic leukemia (P-388). 5-Methoxydehydropodophyllotoxin, dehydro-P-peltatin methyl ether, dehydropodophyllotoxin and

213 deoxydehydropodophyllotoxin showed marginal cytotoxic activity against the human cell lines tested. In contrast, (-)-yatein, 4'-demethyldeoxypodophyllotoxin, isodeoxypodophyllotoxin, deoxypicropodophyllin and Papopicropodophyllin demonstrated a general nonspecific activity comparable to that of podophyllotoxin (ED50 < 10 ng/mL).

0CH3 H3C0, '''0CH3

H3C0 OCH3

0CH3

Megaphone acetate

Dysodanthin A(Ri+R2=-OCH20-) Dysodanthin B (Ri=R2=OCH3)

Brevitaxin

Etoposide is the most potent lignan knov^n in chemotherapy. However, it is often applied to treat tumor cells that have developed resistance to previously used chemotherapeutic agents. It is still limited in its applications and common problems found in chemotherapy such as multidrug resistance and toxicity problems are also apparent [108]. Middel et al [109] synthesised eleven lignans and studied their cytotoxicities in a human small cell lung carcinoma cell line (GLC4) using the microculture tetrazolium assay. Furthermore, five naturally-occurring podophyllotoxin related compounds were tested and were compared with the clinically applied anticancer agents etoposide, teniposide and cisplatin. Most of the compounds showed moderate to high activities against GLC4, and two of

214

them containing a menthyloxy group showed activities comparable to the reference cytotoxic agents. Podophyllotoxin, 4'-demethylpodophyllotoxin, 5-methoxypodophyllotoxin, and compound-1 showed IC50 values in the same range as the reference drugs etoposide and teniposide (0.03-0.83 JLIM), except in the case of deoxypodophyllotoxin, which had an IC50 of 8 nM. A large number of cyclolignans isolated from Juniperus sahina (Cupressaceae) leaves and some semisynthetic derivatives were evaluated for their antineoplastic activity against P-388 murine leukemia, A-549 human lung carcinoma, and HT-29 colon carcinoma [110,111]. Some of them were active in both types of assays at concentrations below 1 |Lig/mL; deoxypodophyllotoxin (2.5-4 ng/mL) and (3-peltatin A methyl ether (4 ng/mL) were the most potent compounds in all cases, for the three neoplastic systems, and /raw5-tetralinelactones were clearly more potent than the c/^-form or non-lactonic compounds. In a complementary study, the same authors synthesised a series of fused pyrazole derivatives of cyclolignans, evaluated them for their cytotoxic activity against the same cell lines, and showed similar ranges of potency [112]. In addition, several cyclolignans lacking the lactone moiety were prepared from podophyllotoxin and deoxypodophyllotoxin by simple chemical transformations, and their cytotoxicity was studied. Most of the compounds show similar effects in all the. neoplastic systems tested, except methyl 9-deoxy-9-oxo-aapopicropodophyllate (IC50 12 ng/mL), 2,2,2-trifluoroethyl-hydrazone of methyl 9-deoxy-9-oxo-a-apopicropodophyllate (IC50 48 ng/mL), and phenylhydrazone of methyl 9-deoxy-9-oxo-a-apopicropodophyllate (IC50 39 ng/mL), which showed highly selective cytotoxicity towards HT-29 human colon carcinoma [113]. On the other hand, 4*-demethyldeoxypodophyllotoxin, P-apopicropodophyllin, and the isoxazopodophyllic acid demonstrated immunosuppressive activity [114]. The same group [115] synthesised a new type of cyclolignans with an isoxazoline ring fused to the cyclolignan core from 7-ketolignanolides. The eleven compounds prepared were evaluated for their cytotoxic activities on four cell lines and showed an IC50 range of 23-0.2 |iiM, clearly lower than the reference drug tested, podophyllotoxin (IC50 12 nM). Only one synthetic lignan, 7-oxime, obtained from podophyllotoxone, gave an IC50 of 0.2 |LIM.

215 OCHj

O

O Menthyl

OCH3

Compound-1 =((37?,4i?,5/?)7a/?)-3-[(3,4,5-TrimethoxyphenyI)[(ethoxyethyl)oxy]methyl]-4-[2-methoxy-3,4(methylenedioxy)-phenyl]oxomethyl)-5-(l-menthyloxy)dihydro-2(3//)-furanone

H3CO

H3CO' '•'COOH H3CC)

H3C0

y

0CH3

0CH3

Isoxazopodophyllic acid

Nemerosin

Several D-rings (the lactonic group) were obtained from podophyllotoxin by semisynthesis, and their in vitro anti-cancer activity was determined. Most of the analogues showed activity against breast, ovarian, prostate, colon, renal and lung cancer cell lines, and some of them were active in the nM range [116]. Compounds with an open D-ring had increased activity, and the podophyllic acid methyl ether was the most interesting, with higher potency than podophyllic acid and etoposide against different cell lines. The 50% growth inhibition (GI50) range was 10 to 60 nM (breast cancer cells), 10 to 40 nM (colon cancer cells) and 10 to 40 nM against other human cell lines. Deoxypodophyllotoxin (anthricin), (-)-deoxypodorhizone, nemerosin, morelensin, and (-)-hinokinin from Anthriscus sylvestris (Umbelliferae) showed antiproliferative activities in vitro against MK-1, HeLa, and

216 B16F10 cells. The ED50 range was 0.5-1.0 ng/mL for anthricin, 0.3-0.8 |Lig/mL for deoxypodorhizone and 0.4-07 \xg/mL for nemerosin [117], 1.672.72 \xg/mL for hinokinin and 53-87 ng/mL for morelensin [118]. Hattalin is a synthetic lignan with affinity for the ouabain receptor. A study of the effect of twelve mammalian lignans on the growth of human mammary tumor ZR-75-1 cells revealed that the suppressive effect on tumor cell growth did not occur through inhibition of DNA synthesis but rather partly by inhibition of the plasma membrane ATPase other than Na^ and K^-dependent ones [119]. The effect of hattalin, which inhibited growth at an EC50 of 2.1 |Lig/mL, was the strongest of all the assayed lignans. Hirano et al [120] demonstrated its specific activity against ATPase from cancer tissue, which was inhibited by more than 50% by 2.0 mM hattalin, whereas at the same dose there was only 33% inhibition of the specific activity of ATPase from normal gastric mucosa. Hattalin inhibited both enzymes more strongly than did ouabain. From these data it is evident that the sensitivity of plasma membrane ATPase associated to lignan increased in gastric cancer. The effects of steganacin analogues and the stereochemical requirements for them to be active against KB cells were established after studying nineteen isomer lignans in their (+)-, (-)- and (±)-forms [121]. The compounds showing higher activity were that of the (-)-series, with the same R absolute configuration around the pivotal biphenyl bond. In addition, a-acetoxy seems to be clearly better than the P-acetoxy form. Steganangin, steganacin, steganolide A, episteganangin, and steganoate B from Steganotaenia araliacea (Umbelliferae) demonstrated cytotoxic activity against a panel of eleven human tumor cell lines, with an ED50 range between 0.4 and 17.3 |Lig/mL for active compounds [122]. However, they were clearly less potent than colchicine, which had an ED50 range between 1 ng/mL and 3.5 |ig/mL, against the same cell lines tested. In a second paper they studied the effects of deoxypodophyllotoxin and Ppeltatin methyl ether, from Bursera permoUis (Burseraceae). In this case the lignans showed strong cytotoxic effects against the eleven human cancer cell lines assayed, with a ED50 range from 0.2 ng/mL to 11.3 |Lig/mL; deoxypodophyllotoxin was the most active and potent of the lignans tested [123].

217

H3C0

H3C0

HjCO

Steganoate B

H3CO

OCH3

Steganolide A

Phyllanthin and hypophyllanthin from Phyllanthus amarus had no cytotoxic activity against different cultured mammalian cells, but both enhanced the cytotoxic response mediated by vinblastine with multidrugresistant KB cells, and phyllanthin displaced the binding of vinblastine to membrane vesicles derived from this cell line, probably by an interaction with the P-glycoprotein [124]. Hirano et al [125] studied the anti leukemic cell efficacy of eleven naturally occurring lignans against the human promyelocytic leukemic cell line HL-60, and four of them showed considerable suppressive effects, with IC50 ranging from 10-940 ng/mL. However, they showed little inhibitory activity on mitogen-induced histogenesis of human peripheral blood lymphocytes. Of the active compounds, honokiol, machilin A, matairesinol and arctigenin had the strongest effects. Their IC50 values of less than 100 ng/mL were close to those of current anti-cancer agents. The lignans, however, had little or no cytotoxicity against the tested cells as assessed by dye exclusion tests (LC50 > 2.9 |Lig/mL), whereas the standard anti-cancer agents had potent cytotoxicity. These lignans inhibited HL-60 cell growth with a non-toxic mechanism, probably by interfering with the DNA, RNA and/or protein synthesis of the leukemic cells, because they strongly suppressed the incorporation of [^H]-thymidine, [^H]-uridine, and [^H]leucine into HL-60 cells. In addition, machilin A and arctigenin inhibited the growth of human T lymphocytic leukemia cells. Arctiin and arctigenin, isolated from Arctium lappa (Compositae), showed strong cytotoxicity against HepG2 cells, but little toxicity against Chang liver cells [126]. The cytotoxicity of arctigenin against Chang liver cells was markedly potentiated by treatment with the glutathione (GSH) synthesis inhibitor L-buthionine-(5',/?)sulfoximine, and the cytotoxicity of arctigenin against HepG2 cells increased in an exposure time-dependent

218 manner, but was hardly changed by the GSH inhibitor. Arctigenin and some of its aliphatic esters, especially ^/-decanoate, transformed more than half of the mouse myeloid leukemia M 1 cells into phagocytic cells at a concentration of 2 |iM. The y-lactone ring appears to have a relevant role in this activity, whereas cyclohexene and cyclooctadiene skeletons showed negative effects [127]. In another study, Kato et al [128] demonstrated that arctiin has weak protective potential against hepatocarcinogenesis in rats induced by 17p-ethinyl estradiol and 2-acetylaminofluorene. Only two of the five lignans isolated from Hernandia nymphaeifolia showed significant cytotoxic activities against P-388, KB 16, A549 and HT29 cell lines. (-)-Deoxypodophyllotoxin and (-)-yatein had ED50 values < 5 ng/mL, whereas in the rest of the isolated lignans, (+)-epimagnolin, (+)epiaschantin, (+)-epiyangambin, the activity range was 2.777-0.389 |Lig/mL [129]. Medarde et al [130,131] obtained a new class of heteroaromatic analogues of lignans, called heterolignanolides, with diverse heterocyclic rings instead of the trimethoxyphenyl ring found in the natural lignans yatein and podorhizol. They showed moderate antineoplastic activity against the tumor cell lines P-388, A-549 and HT-29 (IC50 > 2.5 |Lig/mL) when compared with yatein (IC50 range 25-50 ng/mL) [130]. The same authors [131,132] synthesised several bromo-, chloro- and iodolignanolides from yatein and 4'-0-demethylyatein, which were assayed as antineoplastics but only showed a modest antineoplastic activity. These results confirm that demethylation and halogenation decrease the activity of the dibenzylbutyrolactone type lignans. Five lignans isolated from Aglaia elliptica (Meliaceae) inhibited the growth of twelve human cancer cells in culture [133,134]. The IC50 values for methyl rocaglate, 4'-demethoxy-3',4'-methylenedioxy-methyl rocaglate, 4'-demethoxy-3 ',4'-methylenedioxyrocaglaol, 1 -oxo-4'-demethoxy-3 ',4'-methylenedioxyrocaglaol and 1 -(9-formyl-4'-demethoxy-3 ',4'-methylenedioxymethyl rocaglate were in the 0.8-30 ng/mL range. In a further assay, 4'demethoxy-3',4'-methylenedioxy-methyl rocaglate (IC50 = 0.9 ng/mL) induced accumulation in the Gi/Go phase of the cell cycle afler 24 or 32 h of incubation and inhibited cell proliferation in a dose-dependent manner, but colony formation was not reduced. In addition, the lignan strongly inhibited the protein biosynthesis (IC50 = 25 ng/mL), but did not affect the nucleic acid biosynthesis, even at a high concentration (1 |uig/mL). The growth of BCl in athymic mice was delayed by treatment with 10 mg of

219 lignan per kg three times per week, and there were no toxic effects nor was the body weight affected. The effects of dihydroguaiaretic acid on the initiation stage in a rat multiorgan carcinogenesis model were examined by Takaba et al [135]. Histological examination revealed no inhibitory effects in terms of the multiplicities and/or incidence of neoplastic lesions in any of the organs examined at the experimental doses assayed. The same lignan was isolated and identified as the cytotoxic principle of Pycnanthus angolensis (Myristicaceae), which inhibited the growth of six human tumor cell, with an ED50 range of 1.63-3.10 |Lig/mL [136]. Dihydroguaiaretic acid and NDGA inhibited complex formation of the fos'jun dimer and the DNA consensus sequence, with IC50 values of 0.21 \iM and 7.9 nM, respectively, and suppressed in vitro leukemia, lung cancer and colon cancer cell lines [137]. The growth and metabolism of four prostate cell lines were inhibited with different doses of NDGA over 3 days in culture. Differences in the pattern of the results suggest that different mechanisms were operating, but there was no evidence of any synergistic activity on the inhibition of cell proliferation [138]. As part of a search for chemopreventive agents from natural products, nearly 400 plant extracts were tested for their potential to induce human promyelocytic leukemia (HL-60) cell differentiation. Seventeen of the plant extracts were active, with ED50 values of 4 |j.g/mL [139], One of most potent was an extract derived from Dirca occidentalis (Thymelaeaceae), which gave an ED50 of 0.14 |Lig/mL, and from it genkwanin, (±)lariciresinol and sitoindoside II were isolated as active principles by a bioassay-guided fractionation. The ED50 values of the compounds were 18.3, 1.1 and 0.069 |j.M, respectively. Three new formyl neolignans, obovatinal, perseal A and perseal B, isolated from the leaves of Persea obovatifolia (Lauraceae) were assayed as cytotoxic agents by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric method against mouse lymphocytic leukemia P-388, human nasopharyngeal carcinoma KB 16, human lung adenocarcinoma A549, and human colon adenocarcinoma HT-29 cell lines [140]. The activity range measured by the ED50 values was 0.266 to 1.493 |ig/mL. Perseal A was five fold more potent against A549 adenocarcinoma than its isomer, perseal B. Continuing with this research, the same authors [141] completed the study with four additional neolignans isolated from the same source, obovatifol, obovaten, perseal C and perseal D. They showed

220

significant cytotoxic activities against the same cancer cell lines used in the former experiment. Obovatifol was the most active compound, with ED50 values close to those of the reference drug, mithramycin, against the KB 16 cell line. The acetylation of obovatifol and obovaten gave two compounds that were more potent than the reference drug against the KB 16 cell line (ED50 = 0.075 and 0.049 |Lig/mL, for obovatifol diacetate and obovaten diacetate, respectively). The bis-glucoside derivatives isolated from Galinum sinaicum (Rubiaceae), IS, 8/?, 8'i?-(-)-lariciresinol-4,4'-bis-0-p-D-glucopyranoside and IS, Si?, 8'/?-(-)-5-methoxylariciresinol-4,4'-bis-0-p-D-glucopyranoside, exhibited weak cytotoxic activity against the P388 cell line, with IC50 values of 100 and 42 |itg/mL, respectively [142]. Rocaglaol, pyrimidinone and aglaiastatin isolated from Aglaia odorata (Meliaceae) were potent inhibitors of the growth of K-ra^-NRK cells, with IC50 values of 1-10 ng/mL, and induced normal morphology in K-ra^-NRK cells at 10-30 ng/mL. They also specifically inhibited protein synthesis. Aglaiastatin was the most effective in inhibiting cell growth. Aglaiastatin reduced the amount of Ras, possibly by inhibiting its de novo synthesis [143]. OH

H3CO

Phytoestrogens have a direct relationship with the reduction in the risk of breast, colon and prostate cancer, but these effects are due to mammalian lignans that reach the human body from a vegetable diet. These effects are extensively dealt with in the corresponding section. We now focus on the direct cytotoxicity of mammalian lignans on cancer cells, their effects on cell growth and cell metabolism. The anticancer mechanisms of phytoestrogens, lignans included, seem to be due to the antagonism of

221 estrogen metabolism, antioxidant activity, and modulatory effects on key control points of the cell cycle [80]. Landstrom et al [144] demonstrated that soy flour inhibited implanted prostate cancer growth and suggest that the lignans present may participate in the delayed prostate tumor growth. They observed a significant increase in daily urinary excretion of enterolactone and enterodiol during the metabolic period. These findings support the relevance of phytoestrogens in the prevention of sex cancer. On the other hand, flaxseed reduces metastasis and inhibits the growth of the metastatic secondary tumors in animals, and it may be a useful nutritional adjuvant to prevent metastasis in cancer patients [145]. Sung et al [146] investigated whether enterolactone and enterodiol have growth inhibitory effects against colon tumor cells and whether these effects are mediated through their antiestrogenic activity. At 100 |LiM concentration both lignans significantly reduced the proliferation of all cell lines, though enterolactone was more than twice as effective as enterodiol at this concentration. Growth was not affected by the presence of 17P-estradiol, which implies that these cells are not estrogen-sensitive. Thompson et al. [147] demonstrated that secoisolariciresinol diglycoside has an antitumor effect when taken at the early promotion stage of tumor genesis and may contribute to the health benefits of high-fiber foods. This lignan reduced pulmonary metastasis of melanoma cells in mice and inhibited the growth of metastatic tumors that formed in the lungs [148]. However, while secoisolariciresinol diglycoside appears to be beneficial throughout the promotional phase of carcinogenesis, the oil component seems to be more effective at the stage when tumors have already started to develop [149]. In an in vivo study, Jenab et al. [150] demonstrated the protective effects of flaxseed lignans against colon cancer. This was associated with an increase in p-glucuronidase activity. The total activity of this enzyme correlated positively with total urinary lignan excretion, especially secoisolariciresinol diglycoside, and negatively with the total number of aberrant crypts and the total number of aberrant crypt foci in the distal colon. Different lignans, including some known phytoestrogens, were evaluated for their effects on DNA synthesis in estrogen-dependent and independent human breast cancer cells. At 0.1-10 \iM, enterolactone induced DNA synthesis by 150-235%, and at 20-90 |iM it inhibited DNA synthesis by 50%. Inhibition of estrogen-dependent and independent breast cancer cells

222

at high concentrations suggests additional mechanisms independent of the ER [151]. To study the mechanisms of phytoestrogen effects on estrogen action and tyrosine kinase activity, a number of phytoestrogens and related compounds were evaluated for their effects on DNA synthesis, estimated by thymidine incorporation analysis, in estrogen-dependent cells in the presence of estradiol, tamoxifen, insulin or epidermal growth factor. At 10 |LiM, enterolactone enhanced estradiol-induced DNA synthesis [152]. The lignans enterodiol, enterolactone and NDGA and the lignan metabolite methyl /?-hydroxyphenyllactate interfere with mitogenic and tumor promotional signal transduction pathways [153]. NDGA and methyl p-hydroxyphenyllactate did not inhibit 12-0-tetradecanoylphorbol-13acetate (TPA) mediated c-fos transcription, and enterolactone and enterodiol had only a weak inhibitory effect. NDGA at 0.1-10 |Limol/L increased c-fos mRNA levels. A potential mechanism of dietary anticarcinogenesis involves the induction of detoxifying phase II enzymes such as NADPH:quinone reductase. Wang et al. [154] examined the ability of six prominent phytoestrogens, including enterolactone, to affect cellular expression of NADPH:quinone reductase in colonic cells. The concentrations required to double the specific activity of the reductase for enterolactone was 0.04 \xM. The results demonstrated that these phytoestrogens are capable of inducing the synthesis of the enzyme in Colo205 cells by promoting its specific mRNA expression, and suggest a novel mechanism by which dietary phytoestrogens may be implicated in colorectal cancer chemoprevention. Antiviral eflfects MacRae et al [12] and more recently Charlton [13] reviewed the antiviral activity of lignans. Since the effect of resin containing lignans (podophyllin) against venereal warts {Condyloma acuminatum), an ailment caused by a papilloma virus, was first described, a large number of studies on the antiviral activity of these compounds have been reported. Charlton reviews forty-seven lignans up to 1997, belonging to the aryltetrahydronaphtalenes (or aryltetralins), arylnaphtalenes, dibenzocyclooctadienes and dibenzylbutanes. The first group is the most relevant and best known, and its compounds have the most potent antiviral effects, because some of them are active at nM concentrations, such as podophyllotoxins and peltatins. Several viruses have been assayed; there are reports on the

223

herpes simplex virus (HSV), measles virus, Sindbis virus, murine cytomegalovirus (MCMV), vesicular stomatitis virus (VSV), human papilloma virus (HPV) and human immunodeficiency virus (HIV). The mechanism of actions involves tubulin binding, reverse transcriptase inhibition, integrase inhibition and topoisomerase inhibition. Podophyllotoxins bind to tubulin and are able to disrupt the cellular cytoskeleton and interfere with some vital virus processes. There is no relationship between the inhibition of reverse transcriptase (RT) and chemical structure in the case of lignans, because all the chemical antiviral structures are able to bind to the enzyme. As to the rest of the mechanism, there is not much information available. The effects of rabdosiin may be due to its topoisomerase inhibitory effects. Charlton concluded that the antiviral activity of lignans is not strong and that except for podophyllotoxin, which is used topically to treat various warts caused by HPV, none of them are of interest for commercial application.

HO'

Rabdosiin

Many compounds of plant origin have been identified that inhibit different stages in the replication cycle of HIV. Some of them are lignans and have been reviewed by Vlietinck et al [155] and Matthee et al [156], along with their possible mechanism of action. The lignans (-)-arctigenin from Forsythia intermedia and (-)-trachelogenin from Ipomoea cairica (Convolvulaceae) suppressed the integration of proviral DNA into the cellular genome, and interiotherin A and schisantherin D from Kadsura interior (Schisandraceae) inhibited HIV replication at |j.g/mL range. Nine anti-HIV lignans were isolated from K. interior, of which gomisin-G was the most potent, with EC50 and therapeutic index values of 6 and 300

224

ng/mL, respectively. Schisantherin-D, kadsuranin, and schisandrin-C showed good activity, with EC50 values of 0.5, 0.8, and 1.2 |ag/mL, and therapeutic index values of 110, 56, and 33.3, respectively [157]. Interiotherin A and schisantherin D had EC50 values of 3.1 and 0.5 |Lig/mL, and a therapeutic index of 13.2 and 50.6 respectively [158]. Sometimes the antiviral activity is not due to a single lignan but to a mixture of them. This is the case of anolignan A and anolignan B, which were isolated and identified by bioassay-guided fractionation as the active HIV-1 RT inhibitory constituents of Anogeissus acuminata var. lanceolata (Combretaceae). Anolignan B had weak activity when tested alone (IC50 = 60.4 M 88 |LiM for the fourth passage of HSV-2, thus indicating a rapid build-up of drug resistance against acyclovir. The selective index (relationship between toxicity and activity, TC50/IC50) remained constant for tetramethylO-NDGA and no cross-resistance between the lignan and acyclovir in their anti-HSV effects was detected. Justicidins A and B, diphyllin, diphyllin apioside and diphyllin apioside5-acetate from of Justicia procumbens var. leucantha (Acanthaceae) showed strong antiviral activity, with a minimum inhibitory concentration (MIC) of 0.13, > 0.06, 0.25, 0.25 and 0.13 |Lig/mL, respectively, against VSV, and they had low cytotoxicity [167]. The lignans asarinin and xanthoxylol isolated from Asiasarum heterotropoides var. mandshuricum [104], inhibited Epstein-Barr virus (EBV) early antigen activation induced by TPA. Rhinacanthin E and rhinacanthin F isolated from the aerial parts of the plant Rhinacanthus nasutus (Acanthaceae) showed significant antiviral activity against influenza virus type A but no effect on HSV-2. The EC50 obtained in a hemadsorption-inhibition assay were 1.7 and < 0.94 |ig/mL, and in a viral cytopathic effect assay were 7.4 and 3.1 |Lig/mL, respectively [168]. From a water extract of the fruits of Helicteres isora (Sterculiaceae), Tezuka et al [169] isolated three new neolignans, helisorin, helisterculins A and B, with weak inhibitory activity against RT from avian myeloblastosis virus (IC50 0.46, 1.6 and 1.0 mM, respectively). Antibacterial, Antifungal, Parasiticidal and Insecticidal EtTects Antibacterial Effects

Myristica Jragrans (Myristicaceae), Magnolia obovata and M officinalis had antibacterial activity against the cariogenic bacterium Streptococcus mutans. The active principles from M jragrans were dihydrodiisoeugenol and 5'-methoxydehydrodiisoeugenol [170] which were bactericidal and

227

inhibited Streptococcus mutans growth at an MIC of 12.5 |Lig/niL. When the authors compared the effects of these compounds with the activity of isoeugenol, they observed that dihydrodiisoeugenol is much more potent than isoeugenol (MIC > 200 |ag/mL). The active principles from Magnolia species were magnolol and honokiol, the MIC of which were of 6.3 |ug/mL, but they did not inhibit the adherence of the heat-treated bacteria to glass or smooth surfaces in the presence of sucrose and glucosyltransferase, the synthesis of soluble and insoluble glucans from glucose, and the agglutination of bacteria in the presence of high molecular weight dextran [171]. In a research program on biologically active plants, the extract of Sassafras randaiense (Lauraceae) roots was active against Staphylococcus aureus, Mycobacterium smegmatis, Saccharomyces cerevisiae and Trichophyton mentagrophytes. El-Feraly et al [172] isolated two active neolignans, magnolol and isomagnolol from this species. Isomagnolol was slightly more potent than magnolol, with MICs of 3.12, 1.56, 25 and 1.56 l^g/mL, respectively for each microorganism tested.

xX

R2' HO

CH2—CH==CH2 Magnolol (Ri=H, R2=OH) Honokiol (R,=OH, R2=H)

Antifungal Effects

Two aryltetralin lignans, 4'-(9-demethyldehydropodophyllotoxin and picropodophyllone isolated from Podophyllum hexandrum showed strong antifungal activity against Epidermophyton fuccosum, Curvularia lunata, Nigrospora oryzae, Microsporum canis, Allescheria boydii and Pleurotus ostreatus. Picropodophyllone also showed activity against Drechslera rostrata [173].

228

Lignans isolated from Terminalia hellerica inhibited the growth of Penicillium expansum. The activity range was 1-5 fig with a direct bioautographic method, but against Candida albicans the activity on an agar overlay technique was 10 \ig for termilignan, 80 |ig for termilignan and > 200 |Lig for anolignan B [160]. Eighteen synthetic racemic 8.0.4'-neolignans with six different substitution patterns in rings A and B, in their ketone and in their erythro/threO'forms, and three hydroxy-derivatives were evaluated for antifungal activity by the agar dilution method against the dermatophytes Microsporum canis, M. gypseum, Tricophyton mentagrophytes, T. rubrum, and Epidermophyton floccosum [174]. Only the hydroxy lignans exhibited a broad spectrum of activities against the five fungus strains assayed. The neolignan compound-2 (Table 11) was the most active against E, floccosum, with an MIC of 5 |Lig/mL. The neolignans compound-3 to 5 (Table 11) were the compounds that inhibited the assayed fungi, with MIC under 100 |ig/mL. None of the tested compounds had activity against the yeasts Candida albicans, Saccharomyces cerevisiae and Cryptococcus neoformans, nor against the filamentous fiingi Aspergillus niger, A. fiimigatus and A. flavus. The racemic antifungal alcohols of the 8.0.4'neolignan type were evaluated for inhibitory activity on the fungal cell wall using the whole cell Neurospora crassa hyphal growth inhibition assay [175]. The experimental results suggest that these compounds could act by inhibiting cell wall polymer synthesis or assembly. Verrucosin, oleiferin-B, 3,4,3',4'-tetramethoxylignan-7-ol, oleiferin-F and oleiferin-G (Table 12) isolated from Virola oleifera showed antifungal activity against Cladosporium sphaerospermum and C cladosporoides at 25 lag, but only oleiferin-B and oleiferin-G inhibited C. cladosporoides at 10|Lig[176]. HO,

H3CO'

OC:H3

Verrucosin

229 Parasiticidal Effects The toxic lignans isolated from Bupleurum salicifolium (Umbelliferae) were assayed against Globodera pallida and G. rostochiensis, and bursehernin and matairesinol inhibited the hatching of the two nematodes tested. The authors conclude that lignans may play a role in the defence mechanisms of potato plants, as allelopathic substances acting against cystforming nematodes [177]. The in vitro antileishmanial activity of lignans from Doliocarpus dentatus (Dilleniaceae) against amastigotes of Leishmania amazonensis was reported by Sauvain et al [178]. The lignans isolated were (+)-pinoresinol, (+)-medioresinol and (-)-lirioresinol B. Table 11.

Chemical Structure of Compounds 2-5 ^5

()CH3

^ ' \ ^ , ^ ^ \ ^ ^ ^ OH

K^^^Y 2 (±)-£'rvr/7ro-3,4-methylenedioxy-7-hydroxy-1' allyl-3',5'-dimethoxy-8.0.4'-neolignan 3 (±)-r/7reo-3,4,5-trimethoxy-7-hydroxy-l'-allyl3 ',5 '-dimethoxy-8.0.4' -neolignan 4 (±)-r;2reo-3,4-dimethoxy-7-hydroxy-l '-allyl-3'niethoxy-8.0.4'-neolignan 5 (±)-£'rvr/?ro-3,4,5-trimethoxy-7-hydroxy-l '-(£)propenyl-3'-methoxy-8.0.4'-neoIignan

Ri R2 -OCH2O-

R3 H

R4 OCH3

Rs -CH2CH=CH2

OCH3

OCH3

OCH3

OCH3

-CH2CH=CH2

OCH3

OCH3

H

H

-CH2CH=CH2

OCH3

OCH3

OCH3

H

-CH=CHCH2

230

Table 12.

Active Dibenzylbutane Lignans OH

Oleiferin B Oleiferin F Oleiferin G 3,4,3 ',4'-Tetramethoxylignan-7-ol

Ri Veratryl(3,4-dimethoxyphenyl) Pip erony 1(3,4-m ethyl enedi oxypheny 1) Veratryl(3,4-dimethoxyphenyI) Veratryl(3,4-dimethoxyphenyl)

R2 Piperonyl(3,4-methylenedioxyphenyl) Guaiacyl(4-hydroxy-3-methoxyphenyl) Guaiacyl(4-hydroxy-3-methoxyphenyl) Veratryl(3,4-dimethoxyphenyl)

The lignan (H-)-nyasol, isolated from the roots of Asparagus africanus (Liliaceae), proved to be an antiprotozoal agent. It potently inhibits the growth of Leishmania major promastigotes, with an IC50 of 12 |LIM, and moderately inhibits Plasmodium falciparum schizonts, in this case with an IC50 = 49 |LiM. At the assayed concentrations, the proliferation of human lymphocytes was only moderately affected [179]. Although the potency was clearly lower than that of the reference drug used in the experiment, the lignan inhibited both the chloroquine-sensitive and the chloroquineresistant strains. This feature indicates a non cross resistance between the two drugs, and for this reason the authors recommend using this compound to develop antiprotozoal drugs. Among the compounds isolated from Virola surinamensis, the lignans presented the highest trypanosomicidal activity in vitro against the trypomastigote form of Trypanosoma cruzi [180]. They were identified as veraguensin and grandisin, two tetrahydrofiiran lignans. At 5 [ig/mL they produced total parasite lysis after 24 h, without red cell damage. After reinoculation in healthy mice, nofiirtherinfections were observed. Whereas burchellin was only partially active, NDGA drastically reduced the number of epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi in the excreta of infected Rhodnius prolixus, the insect vector feeding on epimastigotes of T cruzi. However, the number of parasites in the gut decreased in both cases [181].

231

Termilignan and anolignan B isolated from Terminalia bellerica inhibited the chloroquine-susceptible strain of Plasmodium falciparum assayed, with IC50 of 9.6 and 20.5 ^iM respectively [160].

0CH3

0CH3 H3C0

0CH3

Veraguensin

(+)-Nyasol

Insecticidal A ctivity

In their review of lignans MacRae and Towers [9] cited the activity of kobusin and sesamin against silkworm larvae, sesamin and sesamolin against juvenile hormone activity in the milkweed bug, and podophyllotoxins against insect larval growth. On the other hand, sesamin, asarinin, savinin and hinokinin enhanced the toxicity of a wide variety of insecticides. The powdered dried leaves and leaf extracts of Libocedrus bidwilli (Cupressaceae) were toxic to the larvae of the housefly (Musca domestica) and the codling moth (Laspeyresia pomonella), but the powdered material was not toxic to the light-brown apple moth {Epiphyas postvittana). The most active compound present in the leaves was the p-peltatin-A methyl ether, which at a concentration of 100 ppm in a chemically defined diet gave 98% mortality among housefly larvae [182]. The neolignan (+)haedoxan A present in Phryma leptostachya (Phrymaceae) and (+)haedoxan D, its synthetic demethoxy derivative, exhibited insecticidal activity against Musca domestica, whereas (-)-haedoxan D was inactive [183].

232

H3C0.

OCH3 (+)-Haedoxan A (R=OCH3) (+)-Haedoxan B (R=H)

Four known neolignans isolated from Piper decurrens (Piperaceae) showed insecticidal activity against mosquito larvae. Eupomatenoid-6 was the toxic lignan, followed by conocarpan and eupomatenoid-5, whereas decurrenal showed weak activity [184]. The neolignans magnolol, 4methoxyhonokiol and 4,4'-diallyl-2,3'-dihydroxybiphenyl ether, from Magnolia virginiana were toxic to brine shrimp and mosquito larvae and showed strong fungicidal and bactericidal activities [185], and helioxanthin from Taiwania cryptomerioides (Taxodiaceae), inhibited the yellow fever mosquito larvae growth with an LC50 of 3.0 |ig/mL [186]. Cardiovascular effects Recently, in 1997, Ghisalberti [14] published a review on the cardiovascular activity of naturally occurring lignans, including the effects on cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE), Ca^^ channel movement, hypertension, and as platelet activating factor (PAF) and endothelin antagonists. Antioxidant and vasorelaxant effects were also covered. The most relevant finding on this subject fi*om 1996 until 1999 are dealt with below.

233

Myocardial Ischemia

Human correlation and animal studies suggest that consuming of foods rich in phytoestrogens and lignans may result in reduced risk of cardiovascular disease. The postulated antiatherogenic mechanisms are hypocholesterolemic effects as well as inhibition of platelet activation [80]. Pretreatment with a lignan-enriched Sheng-Mai-San, a, traditional Chinese formulation used for the treatment of coronary disease, was effective in protecting against isoproterenol-induced myocardial injury in rats, and in ischemia-reperfusion injury in isolated perfused hearts prepared from pretreated animals [187]. The major myocardial protective compound seems to be the lignan-enriched extract of Fructus Schisandrae. In a complementary study with the same crude drugs, Li et al. [188] studied the effects of Sheng'MaUSan and the lignan-enriched extract of Fructus Schisandrae, its antioxidant component, in different experimental models of myocardial infarction and myocardial ischemia-reperfusion injury in rats. Their results indicate that the myocardial protection afforded by Sheng-MaUSan pretreatment may be mediated by the antioxidant activity of the Fructus Schisandrae lignans. Calcium Channel Blocker

Arctigenin, matairesinol, trachelogenin, nortrachelogenin and their glucosides isolated from the Compositae Trachelospermum jasminoides and Arctium lapa, hypophyllanthin from Phyllanthus niruri, and liriodendrin (but not syringaresinol-p-D-glucoside) from Boerhaavia diffusa (Nyctaginaceae) exhibited significant calcium channel antagonistic effects. Experimental data on these substances are compiled by Ghisalberti [14]. In addition, fargesone A, B and C, denudatin B, pinoresinol dimethyl ether, lirioresinol-B dimethyl ether, magnolin and fargesin from Magnolia fargesii were tested by Chen et al, [189] and were found to possess Ca^^antagonistic activity in taenia coli of the guinea pig. Denudatin B and fargesone A and B had over 50% antagonism in the 10"^ M range, whereas the rest of the substances needed concentrations of over 10""* M. Denudatin B seems to be the most active: 100% inhibition at 2.8 x 10'^ M and 50%) at 1.4 X 10"^ M. Graminone B from Imperata cylindrica (Gramineae) at 0.1

234

|j-M inhibited the KCl-induced contraction on rabbit aorta without affecting NA-induced contractions [190], but its mechanism was not elucidated. 2,3-Dibenzylbutane-l,4-diol is a Hgnan found in human urine, but its physiological significance is unknown. Abe et al [191] studied the effects of this Hgnan on vascular smooth muscle of rabbit aorta. It had no influence on the tension and the "^^Ca^^ uptake on the contractile response in vascular smooth muscle but completely inhibited the "^^Ca^^ uptake induced by high KCl or noradrenaline (NA). In the resting state, it inhibited high KCl- and CaCb-induced contractions in the partially depolarised muscle strip. The lignan inhibited NA- and angiotensin Il-induced contractions, but not the transient contraction evoked by NA in Ca^^-free solution. The results indicated that 2,3-dibenzylbutane-l,4-diol induces the relaxation of vascular smooth muscle by inhibiting Ca^^ influx but not Ca^^ release from the intracellular stores. In later research [192], the same authors studied the effects of this lignan on high K^-induced contraction in rabbit femoral artery. The lignan inhibited both the transient and the sustained contraction induced by high K^ and inhibited the transient contraction remaining in the verapamil- or the nifedipine-pretreated preparation in a concentrationdependent manner. Moreover, 2,3-dibenzylbutane-l,4-diol inhibited sustained contractions elicited by the Ca^^ channel activator Bay K 8644. As the most relevant feature of these experimental results, the mammalian lignan affected both the sensitive and the insensitive calcium channels. When 2,3dibenzylbutane-l,4-diol was assayed in rat aorta, it inhibited NA-induced contractions in a concentration-dependent manner [193]. This inhibitory effect was similar to that obtained in the high K^ solution containing verapamil. The lignan also inhibited the contractions and intracellular Ca^^ elevation induced by NA with the same potency and increased the cyclic guanine monophosphate (cGMP) levels. From these results the authors conclude that the inhibitory effects of 2,3-dibenzylbutane-l,4-diol on NAinduced contraction in rat aorta may be caused by an increase in cGMP levels.

235

OCH3

Graminone B

Antihypertensive Effects

Various reports on the antihypertensive activity of lignans were published before 1997 [14]. (±)-Pinoresinol diglucoside from Eucommia ulmoides (Eucommiaceae), pinoresinol monoglucoside from Forsythia spp., sesamin from sesame oil, neojusticin B from Justicia procumbens, ephedradine B (a hybrid neolignan-alkaloid) from Ephedra spp.(Ephedraceae), danshensuan B from Salvia miltiorrhiza (Labiatae) and magnolol from different sources were all reported to be hypotensives in different animal models. The antihypertensive effect of sesamin was studied by Matsumura et al [194] using hypertensive and unilaterally nephrectomised rats. The sesamin feeding group showed an improved development of deoxycorticosterone acetate salt-induced vascular hypertrophy in both the aorta and mesenteric artery. In a second study [195], these authors examined the antihypertensive effect of sesamin using two-kidney, one-clip renal hypertensive rats. In this experiment, hypertension was markedly reduced by the sesamin-containing diet. There were significant increases in the left ventricle plus septum weight to body weight ratio in the control group compared with the shamoperated rats. This rise was also significantly reduced in the sesamin group. The histochemical analysis of the thoracic aorta indicated that vascular hypertrophy occurred in the control group but not in the sesamin group. The sesamin diet tended to ameliorate this vascular hypertrophy, although its effect was not statistically significant. A third study [196] was performed

236 using salt-loaded and unloaded stroke-prone spontaneously hypertensive rats. Systolic blood pressure, cardiovascular hypertrophy and renal damage were evaluated. In the salt-loaded group, sesamin feeding suppressed the development of hypertension, and an efficient suppression was maintained. The left ventricle plus septum weight-to-body weight ratio was slightly but significantly lowered by sesamin feeding. In the histochemical evaluation of the degree of vascular hypertrophy, the aorta and superior mesenteric artery, wall thickness and wall area of these vessels decreased significantly in the sesamin feeding group. On the other hand, in the salt-unloaded group, only a slight, nonsignificant suppressive effect of sesamin on the development of hypertension was observed. Sesamin feeding was, then, more effective as an antihypertensive regimen in the salt-loaded strokeprone spontaneously hypertensive rats than in unloaded ones, which suggests that sesamin is more useful as a prophylactic treatment in the malignant status of hypertension and/or hypertension followed by water and salt retention. Digoxin-like activity

Some lignans with estrogenic properties have been described as "endogenous ouabain-like" factors. They were studied by Braquet et al [197-199], Hirano et al [200] and Fagoo et al [201]. The animal lignans (enterolactone, prestegane B and 3-(9-methyl enterolactone) inhibited the Na^/K^-ATPase pump activity in human red cells and human and guineapig heart cell membranes, with IC50 ranging from 5 to 9 x 10'"^ M, but the IC50 for ouabain (7 x 10'^ M) was not modified by addition of the lignans. These results suggest a non-competitive inhibition of ouabain receptor. In addition, the apparent affinity for internal Na^ and the maximal rate of cation translocation diminished, and the inhibition of the NaVK^-pump was obtained at doses higher than those required for ouabain. In a complementary study [201], enterolactone displaced [^H]-ouabain from its binding sites on cardiac digitalis receptor and inhibited dose dependently the Na^/K^-ATPase activity of human and guinea-pig heart in the 10""^ M range. The cross-reactivity of enterolactone with antidigoxin antibodies was low, even in man. In conclusion, lignans may contribute to the putative digitalis-like activity found in tissues, blood and urine of several mammals and may be endogenous digitalis-like substances.

237

Sixteen mammalian-type lignan derivatives, including enterolactone and enterodiol, were studied for their potential endogenous digoxin-like activity [200]. M(^5'o-2,3-dibenzylbutane-l,4-diol showed the most potent crossreactivity against the antidigoxin antibody, but many of the lignans had activity in the three experimental models tested, inhibiting NaVK^-ATPase activity with IC50 lower than 5 x 10"^ M and [^H]-ouabain displacement activity with an IC50 range of lO""* to 10*^ M. No study on the digoxin-like activity of hattalin has been reported though it has affinity for the ouabain receptor. Endothelin antagonism

Hussain et ah [202] isolated three lignans from Phyllanthus niruri which were identified as phyllanthin, hypophyllanthin and nirtetralin. They were also found to inhibit [^^^I]-ET-1 binding to the recombinant human ETA receptor expressed in Chinese hamster ovary cells (CHO-ETA), but were inactive against the recombinant ETB receptor. Hypophyllanthin was the most potent compound, with an IC50 value of 40 |LIM. Cyclic AMP phosphodiesterase (PDE) inhibition The cAMP-PDEs are a group of enzymes capable of hydrolysing cAMP to non-cyclic AMP (5'-AMP). Inhibition of PDE activity increases the cAMP levels, which produces stimulation of cardiac contractility and vasodilatation, and decreases vascular resistance and arterial pressure. Different subtypes of PDEs have been described, with different and selective effects. Since Nikaido et al [203] first described the inhibition of cAMP-PDE by lignans, different synthetic analogues have been described [204-206]. On a screening with 250 aqueous extracts, thirty-four of them gave inhibitory activity, and the active principles of the Anemarrhena asphodeloides (Anthericaceae) were norlignans such as m-hinokiresinol [203]. The same year, Nikaido etal [207] studied different anti-inflammatory plants, such as Forsythia suspensa, F. viridissima and F. koreana. (-f)Pinoresinol, (+)-pinoresinol-P-D-glucoside, (-)-matairesinol and arctigenin showed strong inhibitory effects against PDE, with IC50 values of 7.5, 14.2, 9.8 and 13.9 x 10"^ M respectively, whereas phillygenin, phyllirin, arctiin

238 and (-)-matairesinol-p-D-glucoside had no activity at 50 x 10"^ M. Stmctural modifications of these active principles showed that in general the synthetic derivatives did not improve the activity. However, some special cases should be mentioned. The presence of two/7-hydroxyl groups or substituted hydroxyl groups is essential for the PDE inhibitory activity of lignans. 1 - Acetoxy-(+)-pinoresinol and 1 -acetoxy-(+)-pinoresinol-(3-Dglucoside were the most potent of all the assayed lignans, with IC50 values of 3.2 and 4.4 x 10"^ M respectively. Other interesting compounds were (+)pinoresinol monomethyl ether, (+)-pinoresinol-di-P-D-glucoside and syringaresinol-di-P-D-glucoside. Different kind of lignans that inhibit PDE activity have been described, and are reviewed by Ghisalberti [14]. They are dibenzylbutyrolactones, fliranofurans and tetrahydroftirans. In the first group, (+)-matairesinol is the main principle, with an IC50 of 9.8 x 10"^ M. (+)-Pinoresinol is the most potent in the second group, which has an IC50 of 7.5 x 10"^ M, but acetylation clearly increases the potency. In addition, the stereochemistry of the fiiran-phenyl bond can clearly modify the activity, and the C-TS / C-IS compounds or series (+) are more active than the C-TSI C-7R compounds or series (-). A third group, the tetrahydrofiirans, is less relevant because there is a clear loss of activity. The dibenzocyclooctadiene lignans have a biphenyl characteristic, similar in part to that of the bipyridin group present in the standard inhibitors amrinone, milrinone and others. The activity of these lignans improves with the introduction of a methylenedioxy group or a bromide, and decreases with the introduction of a hydroxyl radical, whereas the enantiomers have the same range of activity. Other interesting compounds are (+)-dehydrocaffeic acid dilactone (from fungal origin), schisandrin and NDGA. NDGA is included in the dibenzylbutane lignan group, which can adopt a conformation similar to that of the alkaloid papaverine, according to the three dimensional modelling studies [14]. Inhibition of PDE IV contributed to relaxation of airway smooth muscle and the prevention of proinflammatory cell activation. However, important side effects limited their therapeutic effects. In order to obtain new data on lignans as PDE inhibitors, Iwasaki et ai [204] synthesised a series of 1aryl-2,3-bis(hydroxymethyl)naphthalene lignans and evaluated their ability to selectively inhibit PDE IV isolated from guinea pig and the histamineinduced and antigen-induced bronchoconstriction in the guinea pig. The more potent and selective compounds were the N-alkylpyridone derivatives (ring at C-1), and the most potent of these was 6,7-diethoxy-2,3-bis

239 (hydroxymethyl)-1 -[ 1 -(2-methoxyethyl)-2-oxo-pyrid-4-yl]naphthalene with ED50 values of 0.08 and 2.3 mg/kg /.v., against histamine-induced and antigen-induced bronchoconstriction in the guinea pig respectively. In addition, replacement of the 1-phenyl ring by a pyridone ring led to marked improvement in their selectivity for PDEIV over PDE III. Ukita et al [206] studied the structural requirements of a 1-pyridylnaphthalene series, and the best compound was compound-6 (PDE IV inhibition IC50 = 0.13 nM, selectivity PDE III/PDE IV ratio = 14000). This compound showed potent antispasmogenic activities, reducing the bronchoconstriction in guinea pigs induced by antigen and histamine, with ED50 values of 63 and 33 |Lig/kg, respectively, with few cardiovascular effects. Moreover, this synthetic lignan induced significantly weaker emetic effects than the reference drug after p.o, and /.v. administration in dogs, which is compatible with the lower affinity for the high-affinity rolipram binding site (2.6 nM). This may imply that the synthetic lignan has an improved therapeutic ratio because of a broad margin between the K\ value of binding affinity and the IC50 value of PDE IV inhibition (ratio = 0.050). In a complementary work, Ukita et al. [205] studied the structural requirements for potent, specific PDE V inhibition (cGMP-specific PDE) by a 1-arylnaphthalene lignan series. CH3CH20.

CH3CH2O

2HC1

Compound-6 2,3-Bis(hydroxymethyl)-6,7-diethoxy-l-{2[l(2//>.phthalazinon-4-(3-pyridyI)-2.yl]-4pyridyl} naphthalene hydrochloride

Compound-7 l-(3-bromo-4,5-dimethoxyphenyl)-5-chloro-3-[4-(2hydroxyethyl)-l-piperazinylcarbonyl]-2(methoxycarbonyl)naphthalene

This enzyme is distributed in different smooth muscle tissues, and its inhibition may be involved in the treatment of hypertension, angina, congestive heart failure and impotence. In this study, the synthetic lignan

240

compound-7 was the most potent specific inhibitor, with a PDE V inhibition of IC50 = 6.2 nM, and selectivity for PDE V against PDE I, II, III, and IV > 16,000. This compound had the best selectivity against the different enzyme isoforms among the known PDE V inhibitors. In addition, it showed relaxant effects on rat aortic rings, with an EC50 = 0.10 |LAM, and should be evaluated for use in the treatment of cardiovascular diseases. Platelet activating factor antagonism and coagulation Platelet activating factor (PAF) is a biologically active phospholipid released from activated basophils to induce platelet aggregation. It is a potent mediator that acts through a specific membrane receptor and its effects are exerted as nM concentrations. It is involved in physiological and pathological processes such as asthma, allergies, inflammation, respiratory and cardiovascular events, blood coagulation, immunity, etc. The isolation of specific PAF antagonists may be of interest in lignan research because some of them are clearly active. Ghisalberti [14] reviewed many of the lignans described before 1997. Kadsurenone from Piper futokadsura seems to be the most active as an inhibitor of PAF binding and of PAF-induced platelet aggregation, and kadsurenin H is the most potent of the bicyclo [3,2,l]octane neolignans. Different structural modifications have been made, and their principal features are included in the Ghisalberti review [14]. Other natural lignans of interest as PAF antagonists are honokiol and magnolol from Magnolia species; (-)-denudatin B from Piper kadsura, cinnamophilin and /wt^^o-dihydroguaiaretic acid from Cinnamomum philippinense (Lauraceae), and gomisin M from Kadsura heterocollita.

0CH3

H3C0 0CH3 HO

^.^^ OCH3 OH

Kadsurenone

Cinnamophilin

241 Shen and Hussaini [208,209] analysed the role of kadsurenone and other related natural and synthetic lignans as antagonists of PAF. Kadsurenone did not inhibit platelet aggregation induced by ADP, collagen, AA or thrombin even at 50 |LIM, but it inhibited the platelet aggregation induced by PAF. Among the structural analogous studied only the 9,10-dihydro derivative was similar in potency and site of activity to the original lignan. In addition, futoquinol, fiitoenone and fiitoxide, present in the same source, showed inhibitory effects, and the synthetic diaryltetrahydrofurane lignans, called L-652,731, L-662,025 and L-659,989, and the diaryltetrahydrothyophene called L-653,150, seem to be active in the same range and have the same mechanism as the natural lignans. The direct displacement of [^H]-PAF by the antagonists is usually used to study the specific PAF receptor antagonist. The dose-response curves of displacement of ["^H]-PAF showed a competitive antagonism in all cases, with an increase in potency from futoenone (less active), L-652,731, kadsurenone to L-659,989 (more potent). As inhibitors of PAF-induced platelet aggregation, the range of potency goes from the IC50 = 0.80 |LIM of L-659,989 to IC50 = 9.50 JLIM for futoenone. These values are in agreement with the previous results. Kadsurenone gave an IC50 = 3.50 |iM in this assay. These results demonstrate that kadsurenone and related lignans are specific, potent and reversible inhibitors of PAF receptor and PAF-induced platelet aggregation. In addition, they induce PAF-aggregation and degranulation of neutrophils. Thirty lignans from extracts of Forsythia suspensa and Arctium lappa were tested for their inhibitory effects on PAF binding to rabbit platelets, and nine of them were active [210]. Tetrahydrofurans had no activity. The active compounds as inhibitors of PAF receptor binding were bistetrahydrofiiran and butanolide type. Bistetrahydrofurans also inhibited in vitro platelet aggregation induced by PAF, which indicates that lignans of this type are PAF antagonist. The IC50 (l^iM) values for PAF binding obtained were 1.2 for (+)-pinoresinol dimethyl ether, 0.67 for (+)-acetoxypinoresinol dimethyl ether, 2.8 for (+)-isogmelinol, 1.6 for phillygenin, 0.91 for (+)epipinoresinol dimethyl ether, 0.66 for fargesin, 0.42 for isomagnolin, 2.9 for arctigenin and 0.56 for arctigenin methyl ether. When assayed for their capacity to inhibit the platelet aggregation induced by PAF the IC50 (|LAM) values were 31 for pinoresinol and 38 for fargesin, whereas for the reference drugs the value was 9.8.

242

H3C0.

H3C0'

MachilinG

L-653,150

The furofiiran lignan, epiyangambin, competitively inhibited in a dosedependent manner PAF-induced rabbit platelet aggregation in vitro, without modifying the amplitude of the maximal response, but it had no effect upon the platelet aggregation induced by collagen, thrombin or ADP [211]. The IC50 value for epiyangambin was 6.1 x 10'^ M for PAF (10'^ M)-induced aggregation, and the Schild analysis provided a pA2 of 6.91 with a slope of 0.98 and a pKb of 6.94. In vivo administration of the lignan at 20 mg/kg significantly inhibited PAF-induced thrombocytopenia in rats. These results confirm that epiyangambin is a potent and selective antagonist of PAF both in vitro and in vivo. In an analogous study, the same authors [212] demonstrated that (+)-yangambin, an isomer of isoyangambin, is an antagonist that selectively blocks PAF receptors on platelets. They studied its activity on rabbit platelet aggregation and binding of [^H]-PAF to rabbit platelet plasma membranes. Yangambin inhibited PAF-induced platelet aggregation competitively in platelet-rich plasma in a dose-dependent manner (pA2 of 6.45 with a slope of 1.17) and at 10"^ M did not affect the platelet aggregation induced by ADP, collagen, or thrombin. In a complementary study, yangambin competitively displaced [^H]-PAF binding, with an IC50 of 1.93 |iM, and did not prevent PAF-induced in vitro chemotaxis at 10"^ M. The results confirm that this lignan is an antagonist of PAF receptors on platelets, but as it had no effect on PAF-induced neutrophil chemotaxis, the authors hypothesise the presence of differences between PAF receptors expressed in platelets and neutrophils. In a later experiment, Herbert et al [213] demonstrated that yangambin can discriminate between two different types of PAF receptors on platelets and PMNL and can be considered the first PAF receptor antagonist described to date that exhibits this effect. Yangambin competitively displaced [•^H]-PAF from its high affinity binding sites on washed human platelets, with a K\ value of 1.1 (iM, and inhibited PAF-induced aggregation of human platelets in vitro (IC50 = 10 |LIM), but it had no effect on

243

PAF-induced oxidative burst in human PMNL. In guinea pigs, yangambin inhibited PAF-induced thrombocytopenia but did not affect leukocytopenia. The bistetrahydrofuran lignan (-)-syringaresinol isolated from the stem of Annona montana was tested on antiplatelet aggregation [102]. It showed significant inhibitory effects on the aggregation of rabbit platelets induced by collagen and PAF, and especially against the aggregation induced by AA. However, when the aggregation induced by thrombin was studied, no effect was observed. Puberulins A, B, and C (Table 13) from Piper puberulum were tested for in vitro inhibition of PAF binding on isolated rabbit plasma platelet membrane [214]. Only puberulins A and C inhibited specific binding of [^H]-PAF to its receptor site, with IC50 values of 7.3 and 5.7 |LIM, respectively. On the other hand, piperbetol, methylpiperbetol, piperol A, and piperol B (Table 13), isolated from Piper betle, selectively inhibited the washed rabbit platelet aggregation induced by PAF in a concentrationdependent manner [215]. The IC50 values of the four lignans were 18.2, 10.6, 114.2, and 11.8 |imol/L, respectively, whereas for ginkgolide B (reference drug) the IC50 was 4.8 |amol/L. The concentration-response curve of PAF-induced platelet aggregation was shifled to the right by 50 |Limol/L of lignans, and the EC50 of PAF was increased by these compounds from 1.5 nmol/L to 14.3, 23.1, 2.4 and 20.6 nmol/L respectively, and 47.2 nmol/L for the reference drug. The compounds also inhibited the binding of ["^H]PAF to washed rabbit platelets, with IC50 values of 8.7, 5.3, 8.8, and 6.2 |imol/L, and 1.8 |Limol/L for the reference drug. However, the aggregation of washed rabbit platelets induced by threshold ADP and AA were not affected by the lignans. Furthermore, piperbetol, methylpiperbetol, piperol A, and piperol B had no effects on the cAMP contents in the rest of the washed rabbit platelets. Neojusticin A, justicidin B, taiwanin E methyl ether and taiwanin E isolated from Justiciaprocumbens inhibited platelet aggregation induced by AA with an IC50 range of 1.1-8.0 inM. These values are higher than that of aspirin (20.3 |LIM) but lower than that of indomethacin (0.21 |LIM) [216]. Based on this structural model of active lignans, Tanabe et al. [217] synthesised and studied the expected anti-platelet activity of justicidin E, but using a ^H-C16-PAF binding assay. The IC50 value of justicidin E was 100-150 |LiM, whereas the reference drug gave an IC50 of 3.2 |LIM.

244

Table 13.

Active Neolignans

Puberulin A Puberulin B Puberulin C

Ri OCH3 OH

R2 OCH3 OCH2O OCH3

R3 OCH3 OCH3 CH2CH=CH2

R4 CH2CH=CH2 CH2CH=CH2 OCH3

H2C

CH30,

OCH3

OCH3

CH3O

RiO

Methylpiperbetol Piperbetol Piperol A Piperol B

A A B A

Ri CH3 H CH3 CH3

R2 Ac Ac H H

From the fruits of Schisandra chinensis Jung et al [218] isolated three lignans with antagonistic activities of ["^H]-PAF to washed rabbit platelet receptor binding. Schisandrin A was the most potent isolated compound. From the same source, Lee et al. [219] isolated pregomisin and chamigrenal, two lignans with PAF antagonistic activity. The IC50 values were 4.8 x 10"^ M and 1.2 x 10'"^ M, respectively. Recently, the same authors [220] studied the structure-activity relationships of Schisandra chinensis' lignans and their derivatives as PAF antagonists, and some general features were observed. The introduction of an ester group at C-6, a methylene dioxy moiety or a hydroxyl group at C-7 tends to decrease the activity, and lignans with an /?-biphenyl configuration are more active than

245

those with an iS-form. A derivative of schisandrol A, 6(7)dehydroschisandrol A, was the most potent of the compounds tested, with an IC50 of 2.1 |LIM, whereas schisandrol A had an IC50 of 0.25 mM. The authors suggested that these Hgnans could be the active principles of this crude drug in PAF-related inflammatory disorders, such as asthma, allergy, atopic dermatitis and other inflammatory diseases. Two new Hgnans isolated from the flower buds of Magnolia fargesii and called magnone A and magnone B showed antagonistic activity against PAF in the [^H]-PAF receptor binding assay, with IC50 values of 3.8 x 10"^ M and 2.7 x 10"^ M, respectively [221]. Although the antagonistic activities of both Hgnans were weaker than that of the reference drug (ginkgolide B IC50 1.9 X 10'^), they are in the range of other related compounds.

H3C0'

Futoquinol

Futoenone

Effects on Metabolism and Cholesterol Level Sesame oil affects cholesterol mobility in the human organism. To study the potential activity of other kinds of compounds, Hirose et al [222] analysed the effects of sesamin, a lignan present in the oil, on various aspects of cholesterol metabolism, and they observed that a diet with sesamin reduced the concentration of serum and liver cholesterol except in the group free of cholesterol. Sesamin decreased lymphatic absorption of cholesterol and increased the fecal excretion of the neutral but not the acidic form. At the liver level, there was a significant reduction in the activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase, but the activity of hepatic cholesterol 7a-hydroxylase and alcohol dehydrogenase were not affected. Microscopic histological examination did

246

not show any abnormalities, and the activity of serum transaminases (GOT and GPT) remained unchanged. The authors proposed further studies to ratify the possible use of sesamin as a hypocholesterolemic agent. In 1995, Ogawa et al [223] completed the previous study, and analysed the effects of sesamin and its epimer, episesamin, on cholesterol and lipid metabolism using normo- and hypercholesterolemic stroke-prone spontaneously hypertensive rats. In normocholesterolemic rats, both lignans increased the concentration of total cholesterol in serum by increasing HDL and decreased the serum concentration of very low density lipoprotein (VLDL). In the liver, only episesamin affected the microsomal enzymatic activity. In hypercholesterolemic rats fed a high-fat, high-cholesterol diet, only episesamin improved serum lipoprotein metabolism. In the liver both sesamin and episesamin significantly suppressed cholesterol accumulation, but only episesamin increased the activity of microsomal cholesterol 7ahydroxylase. These results confirm that sesamin may be effective in preventing cholesterol accumulation in the liver and episesamin may be effective in regulating of cholesterol metabolism in serum and liver. On the other hand, sesamin exerts its hypotriglyceridemic effect at least in part through an enhanced metabolism of exogenous-free fatty acid to oxidation at the expense of esterification in rat liver, as was demonstrated by Fukuda et al [224] working on ketone body production and lipid secretion in isolated perfused liver from rats. In fact, ketone body production increased in the livers perfused with oleic acid, and sesamin feeding caused a stimulation of ketone body production. In addition, the ratio of Phydroxybutyrate to acetoacetate, an index of mitochondrial redox potential, was increased in the oleic acid-perfused livers with respect to free fatty acid, though it was lowered by dietary sesamin. The cumulative secretion of triacylglycerol, but not of cholesterol, by the livers from sesamin-fed rats decreased, especially in the oleic acid administered group; this suggests an inverse relationship between the rates of ketogenesis and triacylglycerol secretion.

247

H3Q

.H

H3CO,

OCH3

H3CO

H3CO" OCH3

HsCC)

H3CO

Pregomisin

OCH3

6,7-Dehydroschisandrol A

Fifteen lignans isolated from the fruits of Schisandra chinensis, the leaves of Machilus thunbergii (Lauraceae), and the flower buds of Magnolia denudata were studied for their capacity to inhibit the microsomal acyl-coenzyme A (CoA):cholesterol acyltransferase. They inhibited the enzyme activity at different ranges of concentration. In order of decreasing potency the IC50 (l^M) values were gomisin N (25), gomisin K3 and licarin D (37), gomisin L2 (38), benzoylisogomisin 0 (47), gomisin J (51), schisantherin D (57), wuweizisu C (65), licarin A (75) and machilin G (81). The rest of the tested compounds had IC50 values higher than 100 |iM (gomisin A and B, angeloylgomisin H, tigloylgomisin P and schisandrin), and in the case of the reference drug used in this experiment the IC50 was 42 |aM. Some of these lignans may be useful lead compounds for the design of new acyl-CoA:cholesterol acyltransferase inhibitors, but further studies are necessary to verify the in vivo cholesterol lowering activity of these principles [225]. Sixteen synthetic arylnaphthalene lignans were evaluated for hypolipidemic activity in diet-induced hypercholesterolemia in rats. The total cholesterol and HDL cholesterol in plasma were measured as a reference of the activity [226]. 2,3-Bis(methoxycarbonyl)-l-(3,4dimethoxyphenyl)-4-hydroxy-6,7,8-trymethoxynaphtalene was the most active of the series assayed; it reduced serum cholesterol and increased HDL cholesterol. The effect was about 100 times higher than that of cholestyramine. The comparative studies of the results obtained with all of the assayed lignans made it possible to establish that the most relevant structure-activity relationships are as follows: 1) the introduction of a methoxycarbonyl group at the C-3 position increases the activity; 2) the conversion of the 2,3-dimethoxycarbonyls to a lactone diminishes the activity; 3) the elimination of a methoxy in A-ring decreases activity; 4) the

248 hydroxy aromatic group at C-4 of the B-ring is necessary for activity; 5) modifications of the 3,4-dimethoxy groups at the C-ring decreases the activity; and finally 6) the presence of a biphenyl group is essential for activity. A mechanistic study indicated that 2,3-bis(methoxycarbonyl)-l(3,4-dimethoxyphenyl)-4-hydroxy-6,7,8- trimethoxynaphtalene inhibits the intestinal absorption of both cholesterol and bile acids. The same research group [227] completed the study with a series of diesters of the arylnaphthalene lignan and their heteroaromatic analogues. Modifications at C-3 in the diesters improve the hypocholesterolemic and HDL cholesterol-elevating activities. Structure-activity analysis indicated that the 2-pyridylmethyl ester of 2-(methoxycarbonyl)-1 -(3,4-dimethoxy-phenyl)-4hydroxy-6,7,8-trymethoxy-3-naphtoic acid has the optimum activity both in hypocholesterolemic and HDL cholesterol-elevating properties. Flaxseed is the richest source of co-3 fatty acid and lignans and can reduce the levels of oxygen free radicals and hence prevent the development of hypercholesterolemic atherosclerosis. Prasad [228] studied the effects of flaxseed on a high cholesterol diet, which increases the serum level of total cholesterol without altering the levels of serum triglycerides. These changes are associated with a marked development of atherosclerosis in the aorta. Flaxseed reduced the development of aortic atherosclerosis by 46% without significantly lowering the serum cholesterol. Modest dietary flaxseed supplementation was effective in reducing hypercholesterolemic atherosclerosis markedly without lowering serum cholesterol. Its effectiveness against this disorder could be due to suppression of enhanced production of oxygen free radicals by PMNL in hypercholesterolemia. As the anti-atherogenic activity of flaxseed could be due to its a-linolenic acid and/or lignan content, Prasad et al [229] studied the different effects of two kinds of flaxseed: type I flaxseed with 51-55% a-linolenic acid in its oil and rich in plant lignans, and type II flaxseed, with similar oil and lignan content but a very low a-linolenic acid content (2-3% of the total oil). The results indicate that the anti-atherogenic activity of the type II flaxseed is not due to a-linolenic acid. For this reason it is possible to hypothesise that the role of lignans is more important than that of the unsaturated fatty acid. In a complementary study [230], the same authors investigated the effects of secoisolariciresinol diglycoside on various blood lipid and aortic tissue oxidative stress parameters and on the development of atherosclerosis in rabbits fed a high-cholesterol diet. The results are in agreement with the hypothesis about the effects of lignans, because secoisolariciresinol

249 diglycoside reduced hypercholesterolemic atherosclerosis and this effect was associated with a decrease in serum cholesterol, LDL-cholesterol, and lipid peroxidation products and an increase in HDL-cholesterol and antioxidant reserve. Antioxidant role of Lignans and Their Effects on Liver Function Antioxidative Properties and Antiperoxidative Effects.

In an interesting review on antioxidants and free radical scavengers of natural origin, Potterat [231] covers the principal antioxidative lignans. The exceptional stability of sesame oil from Sesamum indicum (Pedaliaceae) to oxidative deterioration led to the isolation of antioxidative lignans such as sesamol, together with several bisfuranyl lignans such as sesamolinol, sesaminol and pinoresinol. In addition, other antioxidative lignans were isolated and identified from other sources, such as NDGA from Larrea tridentata, dihydroguaiaretic acid, guayacasin and isopregnomisin from Porlieria chilensis (Zygophyllaceae), gomisin N from Schisandra chinensis, cinnamophilin from Cmnamomum philipense, kadsurin, kadsurenone and burchellin from Kadsura heteroclita, and magnolol and honokiol from Magnolia officinalis. The antioxidant activity of a series of lignans was studied by Faure et al [232] in a rat brain homogenate autoxidation test, and lipid peroxidation was evaluated from the luminescence intensity and thiobarbituric reactive product accumulation. Isopregomisin (0.7 JLLM) was the most active lignan, followed by guayacasin (I.IJLIM) and dihydroguaiaretic acid (2.8 |LtM). The difference in the effect can be explained by the degree of methylation, which increases the activity. Enterolactone, prestegane B and 2,3-dibenzylbutane-l,4-diol [233,234] were studied as antioxidatives on superoxide production and luminoldependent chemiluminescence response in human PMNL. None of the three lignans had a direct effect on the responses of human PMNL. Prestegane B and 2,3-dibenzylbutane-l,4-diol enhanced the superoxide production and luminol-dependent chemiluminescence response induced by formyl-Met-Leu-Phe (fMLP), but the effects of the latter were stronger than those of the former. Enterolactone inhibited fMLP-induced effects. The results suggest that activation of phospholipase A2 (PLA2) and Ca^^-

250 calmodulin-pathways may be involved in the effect of 2,3-dibenzylbutanel,4-d^ol but that activation of Hpoxygenase (LOX), cyclooxygenase (COX) or protein kinase C (PKC), and PAP release are not [235]. CH3 HaCO,,^^^

y

1 HI-

H3CO

X/-^"^

i /-=r\ H3C0

H3CO

o •"•

o

""-yj H3C0

OCH3

HO Sesamolinol

Schisanhenol

Seven of the nine lignans isolated from Schisandra chinensis, S. rubriflora and Kadsura longipedunculata inhibited the ascorbic acid/ NADPH induced lipid peroxidation of rat liver microsomes, iron/cysteineinduced lipid peroxidation of rat liver microsomes and superoxide anion production in the xanthine/xanthine oxidase system [236,237]. Schisanhenol, iS'-(-)-schizandrin C and iS'-(-)-schizandrin B were more potent than vitamin E at the same concentration (1 mM). Schisanhenol and schizandrin B inhibited gossypol-induced superoxide anion generation in rat liver microsomes. Oral administration of schisanhenol and schizandrin B reduced liver malondialdehyde (MDA) formation induced by ethanol and increased superoxide dismutase (SOD) and catalase activities in rat liver cytosol. The authors concluded that this kind of lignan has strong antioxidant activity. The effects of schisanhenol and schizandrin on the peroxidative damage of ageing and ischemic rat brain were studied by Xue et al. [238]. Schisanhenol at 10""* M completely inhibited the peroxidative damage to rat brain mitochondria membrane induced by Fe^^-cysteine, and prevented swelling and disintegration of brain mitochondria as well as reduction in brain membrane fluidity. On the other hand, schisanhenol significantly impeded production of MDA and loss of ATPase activity induced by reoxygenation following anoxia in an experiment on ischemia and reperfusion of brain mitochondria and membrane in vitro. In vivo, it increased cytosol GSH-peroxidase in mouse brain during reoxygenation following anoxia. Schisandrin had similar activity but was less potent.

251 These results demonstrate that both compounds have protective action against oxidative stress. In a later study [239], schisanhenol was tested in rat pancreatic islets against the inhibitory action of alloxan on glucosestimulated insulin release and had partially protective effects. In islets reincubated for 30 min in the presence of schisanhenol, the ratio between islets exposed to alloxan during the last 15 min of pre-incubation and those not exposed to the diabetogenic agent with respect to insulin output over 90 min incubation in the presence of D-glucose averaged 142% of that recorded under the same experimental conditions in islets pre-incubated in the absence of schisanhenol. The results demonstrated the potential benefit of schisanhenol as a preventive or therapeutic tool in situations characterised by oxygen radical-induced damage. Wuweizisu B from Schisandra chinensis showed strong protective effects on lipid peroxidation damage to the surface of cultured hepatocytes of rats treated with Fe^Vcystein and also scavenger free radical properties [240]. On the other hand, the oral administration of wuweizisu B increases the activities of antioxidant enzymes. Three new benzofiiran lignans were isolated from Schizonepeta tenuifolia (Labiatae) and called schizotenuins A, Ci and C2. Based on these chemical structures, Maeda et al [241] synthesised a series of related compounds that were then tested for their inhibitory effects on lipid peroxidation in rat brain homogenate and rat liver microsomes. Three of the twelve compounds tested, showed prominent inhibitory activity in rat brain homogenate, with IC50 values of 1.20 (compound-8), 0.70 (compound-9) and 0.77 ^iM (compound-10), respectively (Table 14). Compounds 8 and 10 were tested in rat liver microsomes and gave IC50 of 3.66 |j,M and 4.49 |LIM respectively, and they were found to be more potent than schizotenuin A (IC50 = 36.26 |j,M) and much more potent than that of (±)-a-tocopherol (IC50 = 976 |LiM). In a second study [242], the same authors obtained a new series of related compounds by an oxidative coupling reaction of methyl (£)-3-(4,5-dihydroxy-2-methoxyphenyl) propenoate and obtained four active principles, compounds 11-14. The IC50 obtained on lipid peroxidation in rat liver microsomes were 1.13, 1.20, 0.95 and 0.89 |j.M respectively, and in rat brain homogenate were 0.32, >1.00, 1.02 and 0.58 |iM respectively, which means that all of them are more potent than the compounds previously described.

252

OH Schizotenuin A

HOOC.

OH

OH Schizotenuin Ci HO.

COOH

OH

OH

OH

Schizotenuin C2

Gomisin C has an inhibitory effect on the respiratory burst of rat neutrophils in vitro [243]. The mechanism of action may be mediated partly by the suppression of NADPH oxidase and partly by the decrease in cytosolic Ca^"^ released from an agonist-sensitive intracellular store. In fact, gomisin C attenuated the activity of TPA-activated neutrophil particulate NADPH oxidase in a concentration-dependent manner and reduced the increase in cytosolic free Ca^^ in neutrophils stimulated by fMLP in presence or absence of ethylenediaminetetraacetic acid (EDTA). In addition, this study suggests that the gomisin C mechanism is not mediated by changes in cellular cAMP or in inositol phosphates, or by scavenging

253

superoxide anion O2" released from neutrophils, because it had no effect on O2' generation and uric acid formation in the xanthine-xanthine oxidase system and failed to alter O2" generation during dihydroxyfumaric acid autoxidation. Table 14.

Active Benzofuran Lignans C00CH3 H3C00C.

8 9

10

Methyl (£)-3-[2-(3,4-diacetoxyphenyl)-7-acetoxy-3methoxycarbonyl-benzofiiran-5-ylJpropenoate Methyl (£)-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-3methoxycarbonyl-benzofuran-5-yl]propenoate Methyl (£)-3-[2-(3,4-dihydroxyphenyl)-7-methoxy-3methoxycarbonyl-benzofiiran-5-yl]propenoate

Ri OCOCH3

R2

R3

OCOCH3

OCOCH3

OH

OH

OH

OCH3

OH

OH

The lignan glucosides obtained from germinated sesame seeds inhibited in a dose-dependent manner the lipid peroxidation reaction of linoleic, linolenic and arachidonic acids induced by an H202/FeCl2 system, and also suppressed enzymatic (ADP/NADPH/FeS04) and nonenzymatic (H202/FeCl2) lipid peroxidation by microsomes [244]. In an in vivo experiment, the lipid peroxidation activity measured as 2-thiobarbituric acid reactive substances v^as significantly lower in the kidneys and liver of sesamolin-fed rats than in the controls [245]. In addition, the amount of 8hydroxy-2*-deoxyguanosine excreted in the urine was significantly lower in the sesamolin-fed rats. These results suggest that sesamolin and its metabolites, sesamol and sesamolinol, may contribute to the antioxidative properties of sesame seeds and oil and support our hypothesis that sesame lignans reduce susceptibility to oxidative stress. In a recent study Ashakumary et al [246] demonstrated that dietary sesamin increase both mitochondrial and peroxisomal palmitoyl-CoA oxidation rates. In addition, sesamin increased the hepatic activity of fatty acid oxidation enzymes, including carnitine palmitoyltransferase, acyl-CoA dehydrogenase, acyl-

254

CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase. Dietary sesamin also increased the activity of 2,4-dienoyl-CoA reductase and A^,A"^-enoyl-CoA isomerase, enzymes involved in the auxiliary pathway for P-oxidation of unsaturated fatty acids [246]. On the other hand, the results obtained by Fukuda et al, [247] suggest that increased fatty acid oxidation by dietary sesamin leads to decreased esterification of fatty acids and reduces the synthesis and secretion of triacylglycerol. 0CH3

^COOCHj

CH3COO"

^

>r

"^COOCHs

OCOCH3 OCOCH, Compound-11 (R=H) Compound-12 (R=CH3CO) CHaOOC^ ^ ^ ' V .

Compound-13

^^N.

^0^.

^C00CH3

OCOCH3

CH3O

^-^

OCOCH3

Compound-14

Microsomal and mitochondrial lipid peroxidation induced by Fe^^ADP/NADH were inhibited by honokiol and magnolol, neolignans isolated from Magnolia obovata. Honokiol was more potent than magnolol as an antioxidative (IC50 8.7 versus 53.4 |iM on microsomal lipid peroxidation, and 7.0 and 11.3 |LIM on mitochondrial lipid peroxidation, respectively). The neolignans protected mitochondrial respiratory chain enzyme activity against NADPH-induced peroxidative stress and protected red cells against oxidative hemolysis [248].

255

From an active extract from Betula platyphylla var. japonica (Betulaceae), Matsuda et al [249] isolated four known lignans together with other compounds. They were tested as hepatoprotective agents on the liver injury induced by D-galactosamine/lipopolysaccharide or CCI4 and for O2 "-scavenging and antioxidative activities. The glucoside of (+)lyoniresinol and nudiposide had marked inhibitory activity on lipid peroxidation, giving inhibition values of 97 and 87% at 200 |Lig/mL, respectively, and 67 and 59% at 20 |Lig/mL, respectively. In the hepatoprotective test, nudiposide gave a weak effect only at 100 \xM (27% inhibition), whereas (+)-lyoniresinol 3a-0-P-D-glucopyranoside gave a weak effect but it was not dose-dependent (22-27%o inhibition at doses of 3100 laM). Other antioxidative compounds of interest are syringaresinol and medioresinol, which were determined by the ferric thiocyanate and thiobarbituric acid methods [235]; gomisin J inhibited Fe^Vascorbic acid and ADP/NADPH-induced lipid peroxidation in rat liver mitochondria, with IC50 of 5.5 and 4.7 |Limol/L, respectively [250]; vibsanol and 9'-0methylvibsanol isolated from the wood of Viburnum awabuki (Caprifoliaceae) had antioxidative properties in rat brain homogenate [251]; (-)-phylligenin from Orophea enneandra displayed antioxidative and radical scavenging properties against the 2,2-diphenyl-l-picrylhydrazyl radical in bioautographic TLC assays [43]; 3,4-/>/.v(4-hydroxy-3-methoxybenzyl) tetrahydrofiiran from Pandanus odoratissimus (Pandanaceae) exhibited strong antioxidative activities in the thiocyanate method [252]. Effects of Lignans on Liver Function

The flavolignans of Silybum marianum (Compositae) fruit are known antihepatotoxic agents used therapeutically. Hikino et al [253] used CCI4and galactosamine-induced cytotoxicity in primary cultured rat hepatocytes and a transaminase activity inhibition test to demonstrate the hepatoprotective effects of silandrin, silybin, silychristin, 3-deoxysilychristin, silydianin and silymonin. All of them reduced the cytotoxic effects of CCI4 and galactosamine at 1 mg/mL, but only 3-deoxysilychristin was significantly active at 0.01 mg/mL. Using the same experimental model, Hikino et al, [254] studied twenty-two lignans isolated from Schisandra chinensis and Kadsura japonica for their antihepatotoxic activity.

256

Wuweizisu C and schisantherin D were the most active on the CCU-test, with significant activity at 0.01 mg/mL, and deoxygomisin A, gomisin N, wuweizisu C, gomisin C and schisantherin D proved to be the most active on the galactosamine test at the same dose. The authors concluded that a methylenedioxy group in the dibenzocyclooctane skeleton is the principal feature in the antihepatotoxic activity of this kind of lignan. None of the tested compounds modified transaminase activity directly. In addition, gomisin A showed activity at 1 mg/mL on the CCU test and at 0.01 mg/mL on the galactosamine test. Maeda et al [255] reported the effects of gomisin A on liver functions in various experimental liver injuries and on bile secretion in CCU-induced liver injury. Gomisin A facilitated the liver function in normal and liver injured rats, and protected the liver against CCU-induced cholestasis, maintaining the bile flow and biliary output of each electrolyte nearly at the level of the vehicle-treated group, but did not affect biliary output of total bile acids. On the other hand, gomisin A inhibited the increase in the serum bile acid concentration induced by administration of deoxycholic acid, but hardly influenced the serum bile acids in the phenobarbital combined group [256]. The authors conclude that the inhibitory effect of gomisin A on the promoting action of deoxycholic acid is due to its improving bile acid metabolism, but the effect could not be elucidated from the metabolism of bile acids. In another study Ohkura et al [257] explained that the inhibitory effect of gomisin A on liver injuries is the result of its preventing the AA release and decreasing the LT production (see section about inflammation). In a study on immunologically induced liver injuries in vivo and in vitro [258], gomisin A inhibited dose-dependently the hepatotoxic chemicalinduced liver injuries and the mortality of mice with acute hepatic failure. It also suppressed necrosis, but the infiltration of non-specific inflammatory cells was not affected. It inhibited the isolated liver cell injuries induced by antibody-dependent cell-mediated cytotoxic reactions or activated macrophages in vitro. The results suggest that gomisin A can be protective against immunological liver injuries. In a related work, Nagai et al. [259] studied the hepatoprotective effect of gomisin A on three immunological liver injury models in mice, and they demonstrated that gomisin A inhibited the elevation of transaminase (GOT and GPT) activities and showed a tendency to inhibit histopathological changes in the liver in all the models. Moreover, the lignan inhibited deoxycholic acid-induced release of transaminase from cultured rat hepatocytes in vitro, but did not affect the

257

formation of hemolytic plaque forming cells in immunised mice spleens and the hemolytic activity of guinea pig complement in immunohemolysis reactions. Therefore, the hepatoprotective effect of gomisin A could have to do With the protective effect of hepatocyte plasma membrane rather than the inhibition of the antibody formation and complement activity. Gomisin A stimulated liver regeneration after partial hepatectomy by enhancing ornithine decarboxylase (ODC) activity [260], which is an important biochemical event in the early stages of liver regeneration. In addition, gomisin A enhanced the mitotic index and the level of DNA synthesis increased after partial hepatectomy. Moreover, the ODC activity increased in the early stages of liver regeneration, as did hepatic putrescine. On the other hand, in the liver from rats simultaneously treated with 3'-methyl-4dimethylaminoazobenzene (3*-MeDAB), gomisin A inhibited both the increase in number and size of glutathione S-transferase placental form (GST-P)-positive foci, a marker enzyme of preneoplasm, and the population of diploid nuclei, as a proliferative state of hepatocytes. The lignan inhibited the hepatocarcinogenesis induced by 3'-MeDAB by enhancing the excretion of the carcinogen from the liver and by reversing the normal cytokinesis [261]. Two years later, the same authors [262] investigated the relationship between the serum concentration of bile acids, the appearance of preneoplastic change, GST-P-positive foci in the liver of rats fed 0.06% 3'-MeDAB, and the effects of gomisin A. The increase in serum bile acids, especially deoxycholic acid, and the appearance of preneoplastic lesions, and the number and area of GST-P-positive foci in the liver were significantly inhibited by simultaneous oral administration of gomisin A (30 mg/kg). The results confirmed that deoxycholic acid is an endogenous risk factor for hepatocarcinogenesis and suggest that the antipromoter effect of gomisin A is based on its improving the metabolism of bile acids, including deoxycholic acid. These results were corroborated by Nomura et al [263,264], who suggested that the effects of gomisin A are related to improved liver fimction and reversal of abnormal ploidization, and that gomisin A may be a candidate for a chemopreventive drug that would inhibit the promotion process in hepatocarcinogenesis. Gomisin A protects the liver from injury by acetaminophen. One of its possible mechanisms involves the suppression of lipid peroxidation [265]. It inhibited not only the elevation of serum aminotransferase activity and hepatic lipoperoxide content, characteristic of acetaminophen administration, but also the appearance of histological changes such as degeneration

258

and necrosis of hepatocytes. However, gomisin A did not affect the decrease in liver GSH content. A histological analysis indicates that the massive necrosis and vacuolisation in the liver of rats treated with both acetaminophen and gomisin A were reduced in comparison with rats treated with acetaminophen only [266]. Gomisin A quickly induced the hepatocyte growth factor mRNA expression through mechanisms different from those involved in acetaminophen-induced liver injury. In three experiments, Ko et al [267-269] demonstrated the protective effect of a lignan-enriched extract of Fructus Schisandrae {Schisandra chinensis fruits) against physical exercise-induced muscle damage in rats and the liver damage induced by different agents. Pre-treatment of rats with the lignan-enriched extract caused a moderate enhancement of hepatic GSH regeneration capacity in control rats treated with /-butyl hydroperoxide, but this effect was greatly increased after CCU challenge. This was manifest in the increase in the hepatic GSH level and activities of hepatic glucose-6phosphate and GSH reductase, as well as by a decreased susceptibility of hepatic tissue homogenates to in vitro peroxide-induced GSH depletion. This effect was more evident after CCU challenge, because the lignanenriched extract caused a dose-dependent protection against the CCUinduced impairment in hepatic GSH status, thus decreasing the activities of MDA levels in tissue and alanine aminotransferase plasma activities. On the other hand, pre-treatment with the lignan-enriched extract protected against physical exercise-induced muscle damage in rats. This protection was associated with enhancement of the hepatic antioxidant status, whereas pre-treatment with a-tocopherol acetate decreased the MDA level in skeletal muscle but did not protect against exercise-induced muscle damage or improve hepatic antioxidant status in exercised rats. The protective effect that pre-treatment of rats with the lignan-enriched extract has on the muscle damage produced by physical exercise may be due to an enhancement of hepatic GSH status that provides GSH for effective antioxidant protection of skeletal muscle during exercise. Li [240] reviewed the activity of lignans from the Schisandraceae family {Schisandra and Kadsura) and cited the effects of eighteen lignans against CCI4 induced hepatotoxicity, including the relationship between the functional group and the stereostructures. The activities of ^S-enantiomers of wuweizisu B and C were stronger than their i?-forms, where the twist boat chair configuration of cyclooctadiene seems to be more active than the twist boat conformation. The compound with two methylenedioxyl groups is

259 more active than the ones with only a methylenedioxyl group. The compound with a hydroxyl in the cyclooctadiene ring showed only weak inhibition of MDA formation. H3CO.

Tribulusamide A (R=H2) Tribulusamide B (R=0)

Tribulusamides A and B, new lignanamides isolated from the fruits of Tribulus terrestris (Zygophyllaceae) prevented cell death induced by Dgalactosamine/tumor necrosis factor-a (TNFa) after addition to primary cultured mouse hepatocytes [270]. This experimental model is known to cause fatal liver failure, and in the case of the lignamide groups the cell survival rate rose to 70-106% that of the normal group at a concentration range of 50-200 |LIM. However, at higher doses the lignamides did not improve the cell survival rate because of their cytotoxicity. Two new dibenzocyclooctadiene type lignans called heteroclitins F and G and related compounds isolated from the stems of Kadsura heterodita inhibited lipid peroxidation in the rat liver homogenate stimulated by Fe^^ ascorbic acid, CCI4, NADPH and ADP-NADPH [271]. 7>a«5-kielcorin and ^ram-isokielcorin B were effective in preventing perturbation of cell GSH homeostasis, as revealed by measuring reduced and oxidised GSH, lipid peroxidation and cell viability after inducing toxicity with or without the studied compounds at different concentrations [272]. The lignan (+)-sesamin and related lignans present in sesame seeds or its oil are specific inhibitors of A^-desaturase in polyunsaturated fatty acid biosynthesis in both microorganisms and animals [273,274]. The results obtained in experiments with both a cell-free extract of the fungus Mortierella alpina and rat liver microsomes demonstrated that (+)-sesamin

260

is a non-competitive inhibitor of A^-desaturase at low concentrations (K\ for rat liver, 155 |iM), but does not inhibit A^-, A^- and A^^-desaturases. (+)Sesamolin, (+)-sesaminol, (+)-episesamin, (-)-asarinin and (-)-epiasarinin also inhibited only A^-desaturase of the fungus and liver. In an analogous study Umeda Sawada et al [275] demonstrated that sesamin inhibits the A^desaturation of n-6 fatty acid (dihomo-y-linolenic acid to AA), but not that of n-3 fatty acid to eicosapentaenoic acid in rat livers. Sesamin administration decreased incorporation of eicosapentaenoic acid and simultaneously increased the AA content in the liver. Changes in the hepatic concentration of eicosapentaenoic and linolenic acids (n-3) were significantly reduced by their simultaneous administration with sesamin, but sesamin had no effect on the n-6 and n-9 fatty acid concentrations. However, no significant differences in lymphatic absorption between eicosapentaenoic acid (n-3) and AA (n-6) were observed in presence or absence of sesamin [276]. In addition, sesamin, reduced the A^-desaturase activity in non-neoplastic cell cultures and could be used to manipulate the content of series 1 and 2 PGs derived from dihomo-y-linolenic acid and AA. However, it had little effect on the content and ratio of dihomo-ylinolenic acid and AA in neoplastic cells, which means that A^-desaturase activity was low in these cells [277]. In a complementary study, Umeda Sawada et al. [278] demonstrated that dietary sesame lignans promote ketogenesis and reduce polyunsaturated fatty acid esterification into triglyceride. They demonstrated that sesame lignans inhibited changes in the n-6/n-3 ratio by reducing the hepatic polyunsaturated fatty acid content, and this reduction may occur because of the effects of sesame lignans on polyunsaturated fatty acid degradation (oxidation) and esterification. Sesamin and episesamin can improve liver function. In studies on experimental models in rodents, Akimoto et al. [279] demonstrated the protective effects of these lignans against liver damage caused by alcohol and e c u . They improved mouse blood parameters, such as aspartate aminotransferase and alanine aminotransferase activities, and the concentrations of total cholesterol, triglycerides and total bilirubin that had been pathologically increased as a result of continuous inhalation of ethanol. In addition, sesamin showed a significant protective effect against the accumulation of fat droplets and vacuolar degeneration in the mouse liver, as confirmed by histological examination. The effects of a mixture of sesamin and episesamin (1:1) on ketone body production and lipid secretion were studied by Fukuda et al. [224] in isolated perfused liver from rats.

261 They demonstrated that dietary lignans exert their hypotriglyceridemic effect at least in part through enhanced metaboHsm of exogenous free fatty acid in rat Hver. The cumulative secretion of triacylglycerol, but not of cholesterol, by the liver rats decreased, especially when exogenous oleic acid was provided, suggesting an inverse relationship between the rates of ketogenesis and triacylglycerol secretion. Sesame seed contains y-tocopherol, a compound that has vitamin E activity 20% lower than that of a-tocopherol, but a sesame seed diet has high vitamin E activity. Yamashita et al [280] studied this paradox using changes in red blood cell hemolysis, plasma pyruvate kinase activity, and peroxides in plasma and liver as indices of vitamin E activity, and groups of rat which were fed with four different diets: vitamin E-free, a-tocopherol, Y-tocopherol and sesame seed. In a second series, two diets containing sesame lignan (sesaminol or sesamin) and y-tocopherol were tested. The global results of the experiments indicate that the vitamin E activity of ytocopherol in sesame seed is equal to that of a-tocopherol because of a synergistic interaction with sesame seed lignans. In a second paper and using the same methods, Yamashita et al [281] demonstrated that sesame seed and its lignans induce a significant increase in a-tocopherol content in the blood and tissue of rats. Supplementation with even 5% sesame seed increased the a-tocopherol content of plasma and liver produced by a low a-tocopherol diet. These results indicate that lignans enhance vitamin E activity in rats fed a low a-tocopherol diet and increase the concentration of a-tocopherol in blood and tissues. In a complementary study, Kamal et al [282] demonstrated that the bioavailability of y-tocopherol is enhanced in sesamin-containing diets as compared with purified diets. Sesamin feeding increased y-tocopherol and the y-/a-tocopherol ratios in plasma, liver and lung, but the increase in a-tocopherol was non-significant. Thus, sesamin appears to conserve y-tocopherol in rat plasma and tissues, and this effect persists in the presence of a-tocopherol, a known competitor of ytocopherol.

262 Effects on Inflammation, Immunity and Their Mediators Anti-inflammatory Effects

Inflammation involves a series of events that can be elicited by physical injuries, infectious agents, antigen-antibody reaction, and other agents. There are different phases in the inflammatory process, depending on the agents, time or intensity: the acute phase characterised by vasodilatation and edema, the subacute phase with leukocyte and phagocytic cell infiltration, and the chronic phase with tissue degeneration and fibrosis. Depending on the phase, there are different kinds of mediators: metabolites of AA such as prostaglandins (PGs) and leukotrienes (LTs), lipidic mediators like PAF, peptides such as interleukines (ILs), TNF, granulocyte/macrophage colony stimulating factor (GM-CSF), and others. Some of these increase gene expression, probably by the activation of transcription factors, such as the nuclear factor KB (NF-KB). Anti-inflammatory compounds can act at different levels. They can, for example, decrease vasodilatation and edema, as in the case of antihistaminic drugs; inhibit PGs synthesis as do the non-steroidal antiinflammatory drugs; modify synthesis or block the receptors of mediators such as PAF or LTs; interfere with the functions of peptide mediators; block the transcription factors or scavenge free radicals generated in the process. Natural products as anti-inflammatory agents may act at different levels, some by non specific pathways as antioxidants do or by a specific mechanism via receptor antagonism. The lignans can act in both cases. Few reports on the experimental in vivo anti-inflammatory activity of lignans have been published. In their 1984 review of the pharmacological activities of lignans MacRae and Towers [9] included no references to antiinflammatory activity. Kimura et al [283] studied the effects of lignans and neolignans on adjuvant-induced inflammation in mice. When administered into the pouch, the neolignans magnoshinin and magnosalin inhibited the granuloma tissue formation, but had no effect on the fluid volume. However, the lignans assayed had no activity. These results suggest that neolignans may act on the mechanism of granuloma tissue formation rather than on blood vessel permeability. Magnoshinin had stronger activity than the other neolignan, but its effects were not enhanced when the dose was increased. After oral administration, magnoshinin showed a stronger effect.

263 similar to that obtained with hydrocortisone at 60 mg/kg/day, but the neolignan only affected granuloma formation. 0CH3

H3C0.

H3C0.

^()CH3

H3C0 0CH3

OCH3

H3C0'

OCH3

Magnosalin

Magnoshinin

From the methanol extract of Haplophyllum hispanicum (Rutaceae) two topical anti-inflammatory aryl naphthalide lignans were isolated and identified as diphyllin acetyl apioside and tuberculatin [284]. The methanol extract was only active against the acute TPA-induced ear edema, whereas it was not active against the chronic inflammation induced by TPA or oxazolone-induced contact-delayed hypersensitivity in mouse ears. Diphyllin acetyl apioside was the most active of the isolated compounds on the acute TPA, with an ID50 of 0.27 |amol/ear, whereas tuberculatin gave an ID50 of 1.23 |Limol/ear. Yasukawa et al [285] tested seven dibenzocyclooctadiene lignans against inflammation induced by application of 1 \ig of TPA/mouse ear, and gomisin A, gomisin J, and wuweizisu C inhibited the edema. The ED50 range of these compounds for TPA-induced inflammation was 1.4-4.4 jiimol, and gomisin A was the most active compound. Furthermore, at 5 lamol/mouse, gomisin A suppressed the promoter effect of TPA on skin tumor formation in mice following initiation with 7,12-dimethylbenz[a]anthracene. The authors concluded that the inhibition of tumor promotion by gomisin A is related to its anti-inflammatory activity. Baba et al [286,287] studied the structure-activity relationships of some lignans as anti-inflammatory agents and in a second paper gave detailed information on the bone resorption inhibitory effects of these compounds. Chemical modification of the potent bone resorption inhibitor justicidin was performed and various naphthalene lactones, quinoline lactones and

264 quinoline derivatives bearing an azole moiety at the side chain were prepared. Antiallergenic Effects

In a study published in 1991 it was reported that Magnolia salicifolia flower bud extracts had an antiallergenic effect on the passive cutaneous anaphylaxis test. By monitoring their activities with an in vitro bioassay system measuring inhibitory effects on induced histamine release from rat mast cells, Tsuruga et al [288] isolated different active constituents, including lignans, which were evaluated for their biological activities using the in vitro bioassay. Although magnosalicin was not so active as an inhibitor of histamine release, the authors tested a large number of lignans from different structural types. Bistetrahydrofiirans and butanolides had activity, whereas tetrahydrofuran type lignans had no activity. The IC50 range was between 18 and 70 ^iM, but no chemical structure and pharmacological relationship was established. HjCO^

^^N.

.OCH3

H3CO ^0CH3

H3CO"

""^

*'0CH3

Magnosalicin

In an experiment on the ethanol-induced modulation of immune indices related to food allergy, sesamin and sesaminol affected the plasma levels of immunoglobulins (Ig) and eicosanoids. Sesamin increased the IgG level, but IgE was not affected. On the other hand, sesaminol decreased the plasma PGE2 concentration [289]. Yangambin is a flirofuran lignan with PAF antagonist properties that seems to also have anti-allergic properties. In a study with actively sensitised or normal rats, Serra et al [290] demonstrated that yangambin inhibited the pleural neutrophil and eosinophil infiltration evoked by injection of PAF or antigen and the pleural neutrophil infiltration triggered

265 by LTB4 in both group of rats, as well as the blood thrombocytopenia and intestinal anaphylaxis elicited by antigen in rats. However, plasma exudation evoked by both stimuli was unaffected. In addition, yangambin attenuated the hemoconcentration, thrombocytopenia, and leukocytosis observed after /.v. administration of PAF. At 10" and 10""^ M this lignan inhibited the anaphylactic contraction of longitudinal jejunal segments in response to antigen challenge and the contraction of jejunal segments from normal rats to PAF, but the response to serotonin (5-HT) was not affected. These findings indicate that yangambin is an antagonist of receptors other than those of PAF, such as LTB4, and it may be an important therapeutic tool in the management of some allergic diseases. Arachidonic Acid Metabolism

Gomisin A, a lignan present in Schisandra chinensis fruits, suppressed the production of LTB4 but did not affect 5-LOX or PLA2 activities [257]. The release of AA from macrophages stimulated with fMLP or the Ca^^ ionophore A23187 was suppressed by treatment with gomisin A. The results obtained in this work indicate that gomisin A inhibits the biosynthesis of LTs by preventing the release of AA, and the authors explain its inhibitory effect on liver injuries as the result of its preventive effect on the AA cascade due to the role of LTs in inflammatory liver diseases. Justicidin E from Justicia procumbens is a potent non-redox inhibitor of 5-LOX activity that inhibits the biosynthesis of LTB4 by human leukocytes with an IC50 = 70 nM [291]. Of the different semisynthetic principles, only the 5-phenylpyridyl derivative was more potent (about three times more) than justicidin E as a LOX inhibitor. The lignan compound-15 inhibits the oxidation of AA to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) by 5-LOX (IC50 = 14 nM) and the formation of LTB4 in human PMNL (IC50 = 1 5 nM) as well as in human whole blood (IC50 = 50 nM). This lignan is a selective, direct nonredox 5LOX inhibitor (FLAP) showing no significant inhibition of human 15LOX, porcine 12-LOX or binding to human 5-LOX-activating protein up to 10 |iiM. The open form of the lactone (carboxylate) obtained by the lignan is well absorbed in the rat and is transformed into the active form. In addition, the carboxylate is orally active in the rat pleurisy model (ED50 =

266 0.6 mg/kg) and in the functional model of antigen-induced bronchoconstriction in allergic squirrel monkeys. Pre-treatment at 0.3 mg/kg of active carboxylate produced a 95% inhibition of the increase in airway resistance and a 95% inhibition of the decrease in dynamic compliance [292].

^-'"X

Compound-15 7-[[3-(4-Methoxy-tetrahydro-2//-pyran-4-yl)phenyl]methoxy]-4-phenylnaphtho[2,3-c]fijran-l(i//)-one

The tetrahydropyranyl naphthalenic lignan lactone called L-702,539 is a potent, selective 5-LOX nonredox inhibitor transformed in vivo from the hydroxy acid L-702,618 by an enzymatic process [293]. Studies with microsomes from genetically engineered human cell lines expressing individual cytochrome P-450 indicate that the isozyme responsible for the metabolism at the tetrahydropyran ring was P-450(3 A4). Another derivative was synthesised from this compound and called L-708,780 [294]. It inhibited the oxidation of AA to 5-HPETE by 5-LOX (IC50 = 190 nM) and the formation of LTB4 in human PMNL (IC50 = 3 nM) as well as in human whole blood (IC50 = 150 nM). In addition, it was orally active in the functional model of antigen-induced bronchoconstriction in allergic squirrel monkeys (95% inhibition at 0.1 mg/kg). Cinnamophilin proved to be a selective thromboxane A2 (TXA2) receptor antagonist especially in rat aorta with voltage-dependent Ca^^ channel blocking properties [295]. Cinnamophilin (10 |LIM) protected against the irreversible vasoconstriction of rat aorta caused by 9,11dideoxymethanoepoxy-9a,lla-PGF2a (U-46619) at 0.05 |j,M and showed voltage-dependent Ca^^ channel blocking action. At 30 |LIM it produced a slight relaxation of NA-induced tonic contractions, but this relaxation was abolished in presence of nifedipine. At 100 |j,M, cinnamophilin did not

267 affect the aortic contraction induced by endothelin-1, angiotensin II, carbachol or serotonin, but cAMP and cGMP levels did not increase in rat aorta. In an in vitro study [296] with human platelets, rat isolated aorta and guinea-pig isolated trachea and in vivo in mice and guinea-pigs, cinnamophilin inhibited in a dose dependent manner the human plateletrich plasma aggregation induced by AA, collagen and U-46619, with IC50 of 5.0, 5.6 and 3.0 |LIM, respectively. Cinnamophilin blocked the TXA2 receptor of human platelets, rat aorta and guinea-pig trachea in a study on the competitive antagonism of U-46619-induced aggregation of human platelet-rich plasma, contraction of rat aortic rings and guinea-pig tracheal rings. The pA2 values were 7.3, 6.3 and 5.2, respectively. The lignan also suppressed ["^HJ-inositol monophosphate formation and the rise of intracellular Ca^^ induced by U-46619 in human platelets, induced a dosedependent inhibition of TXB2 formation, and increased PGE2 formation. The authors conclude that cinnamophilin is a novel dual TX synthase inhibitor and TXA2 receptor antagonist and propose its use as a tool in research and treatment of diseases involving TXA2. Tetrahydrofurofiiran lignans have been described as 5-LOX inhibitors in addition to having the previously reported anti-COX properties. In fact, ten lignans were isolated from Zanthoxylum armatum and tested against COX from sheep seminal vesicles and 5-LOX from pork leukocytes [297]. (+)Spinescin, (+)-sesamin and (+)-asarinin were active, with IC50 of 55, 17 and 56 |iM, respectively, when assayed against 5-LOX. These values are clearly higher than that of NDGA used as the positive control (IC50 =1.5 |aM). Only (+)-fargesin and planinin inhibited COX activity and the inhibition was not significant. In order to establish more data on structure activity relationships, these authors tested other synthetic-related lignans. The lignan (±)-pinoresinol was the most active inhibitor of 5-LOX. Other active compounds against 5-LOX were (+)-epiashantin, (±)-syringaresinol, (-)prenylpiperitol and (-)-prenylpluvia-tilol. On the other hand, (-)prenylpiperitol was the most active lignan in inhibiting COX, with an inhibition percentage of nearly 50%, while the rest gave only about 30% inhibition or less. As the compounds had no activity in the peroxidation assay, the mechanism of inhibition may have to do with a direct inhibition of the enzyme. The authors summarise some of the structural features for the anti-LOX activity of these lignans: a) diequatorial-substituted derivatives are more active than the endo-exo substituted one; b) piperonyl moieties are more active than veratryl substituted ones; c) methylenedioxy

268 function enhances their activity, but the methoxy groups and hydroxy substituents at the bridge carbons decrease it. Eudesmin, magnoHn and lirioresinol-B dimethylether, the three Hgnans isolated from the flower buds of Magnolia fargesii by means of bioassayguided isolation, showed inhibitory effects on TNFa production in lipopolysaccharide (LPS)-stimulated murine macrophage cell lines. Eudesmin showed the strongest activity of the three isolated compounds, with an IC50 of 51 |iM [298]. Using the same experimental protocol, Cho et al [299] isolated a series of lignans from the rhizomes of Coptis japonica var. dissecta (Ranunculaceae), which were assayed as inhibitors of TNFa production. The active lignans, pinoresinol, woorenoside-V and lariciresinol glycoside, with an activity range from 37 to 55% at the concentration of 25 |J,g/mL, may partly participate in the anti-inflammatory and the antiallergenic effect of this species by inhibiting TNF-a production. Seven lignans and two neolignans were assayed on the concanavalin Ainduced proliferation of human peripheral blood lymphocytes in vitro [300]. All the tested compounds showed inhibitory activity, with IC50 ranging from 0.02 to 4.30 \x§JrvL. Machilin A was the strongest inhibitor, with an IC50 of 1.6 x 10'^ M, which is lower than that of the immunosuppressive glucocorticoid prednisolone (1.7 x 10"^ M). Other interesting lignans are /wt^w-dihydroguaiaretic acid and (-)-sesamin, which showed IC50 values of 4.8 x 10"^ and 9.3 x 10"^ M, respectively. The viability of lymphocytes before and after treatment indicated no changes, and therefore the lignans are not toxic against lymphocytes but may inhibit DNA synthesis. The authors suggest the possible value of plant lignans as immunosuppressive agents. Other Effects Effects on Smooth and Skeletal Muscle

The lignans of (+)-pinoresinol, (+)-epipinoresinol, (+)-lariciresinol and (+)isolariciresinol isolated from Fagraea racemosa (Loganiaceae) were tested using a bioassay of the relaxation effect on NA-induced contraction in rat aortic strips [301]. The analgesic properties of the plant extract in the acetic acid-induced writhing and tail pressure tests in mice are due to the lignan

269 fraction. (+)-Pinoresinol showed dose-dependent analgesic effect on writhing symptoms in mice, and local anaesthesia in guinea pigs. Cinnamophilin has protective activity against reperflision injury of the ischaemic skeletal muscle in rats. Ischaemia was induced in one hind limb by application of a tourniquet on the proximal thigh; the contralateral limb served as an internal control. Of the four-reperfiision groups, only the cinnamophilin group had a smaller triphenyltetrazolium chloride reduction and lower muscle weight gain [302]. The dihydrobenzofliran lignan, 3',4-0-dimethylcedrusin improved wound healing in vivo by stimulating the formation of fibroblasts and collagen [303]. Renal and Diuretic Effects

Hall et al [304] tested the effects of a diet supplemented with flaxseed, rich in a-linolenic acid and plant lignans, on a murine model of lupus nephritis, and observed that the percentage of flaxseed-fed mice with proteinuria was lower than the control and that spleen lymphocyte proliferation was significantly higher in the control group than in the flaxseed group. In addition, mortality was lower in the flaxseed-fed mice versus the control. The diuretic properties of arctigenin and hattalin were studied by Hirano et al. [305], who demonstrated that arctigenin had no effect on urine volume, but that hattalin increased it in rats and mice. This compound decreased Na^, K^ and CI' excretion in rats, but serum Na^ and K^ levels did not change. However, serum CI' levels in these animals decreased with respect to the control group. The authors propose that the mechanism of the diuretic effects of hattalin was different from that of known diuretics. The natriuretic effect of prestegane B observed in vivo [306] could have to do with the inhibition of NaVK^-ATP activity demonstrated in vitro in previous studies. This synthetic lignan probably acts beyond the proximal tubule, as urinary phosphate was not altered. Prestegane B mimics the effects of other endogenous diuretic and natriuretic hormones, but its site of action and its effect on renal hemodynamics are obviously different.

270

Central Nervous System Effects

Few studies on the activity of lignans on CNS have been reported. In 1979 was reported the central depressive effects in rabbit of (+)-nortrachelogenin from Wikstroemia indica [9]. Watanabe et al [307] isolated magnolol and honokiol from Magnolia officinalis and studied their central depressant effects. In the animal experiments both compounds produced this kind of effect as well as muscle relaxation by a central action. In addition, magnolol depressed both the hypothalamic and the reticular formation ascending activating system in rat, according to the results obtained in the EEG experiments. Yamazaki et al [308] studied the effect of (+)- and (-)-syringaresinol, (+)-syringaresinol glucosides and syringin on neurite outgrowth of a cultured cell line of paraneuron, PC12h cells, in order to know if they can induce the neuronal differentiation in cells. Only (+)-syringaresinol diglucoside was found to be a promoter of the neurite outgrowth and stimulated responses to a high concentration of KCl and to carbachol in the cells, as demonstrated by the increase in the concentration of cytosolic free calcium. Schisandrin has extensive inhibitory effects on the CNS, which is characteristic of neuroleptic drugs [240], and isoamericanol A, americanol A and americanin A enhanced choline acetyltransferase activity at 10"^ M in a cultured neuronal cell system derived from fetal rat hemisphere [309]. Four lignans isolated from the roots of Valeriana officinalis (Valerianaceae) were identified as (+)-l-hydroxypinoresinol, (+)-pinore-sinol-p-Dglucoside, (+)-pinoresinol and (-)-prinsepiol. They were tested as potential ligands of the 5-HTIA-, GABAA-, benzodiazepin- and |Li-opioid-receptors, but only (+)-l-hydroxypinoresinol showed affinity for the 5-HTiA-receptor, with an IC50 of 2.3 |LIM [310]. Genotoxicity and Mutagenicity

The effect of NDGA on the production of sister-chromatid exchanges and on the level of the mitotic index in cultured human lymphocytes and in mouse bone marrow cells in vivo was evaluated by Madrigal Bujaidar et al [311], and they observed that in both models NDGA produced genotoxic and cytotoxic effects. In a later study [312], the same author observed

271 contradictory effects when they tested the capacity of NDGA to inhibit the rate of sister chromatid exchanges induced by methyl methanesulfonate. They used cultured human lymphocytes from two female donors for the experiment, and the results indicated that NDGA may be an antigenotoxic agent in mammalian cells in vitro and in vivo. The methanol extract from Machilus thunhergii [313] showed a suppressive effect on the Salmonella typhimurium test against the mutagen 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole which requires liver metabolising enzymes. The fractionation of the methanol extract gave a suppressive compound in the chloroform fraction that inhibited the activity of the mutagenic agent and was identified as w^^o-dihydroguaiaretic acid. In addition, it suppressed the gene expression and aflatoxin B-1 effects. Kulling et al [314,315] studied the potential genotoxicity of enterolactone and enterodiol, and their precursors, matairesinol and secoisolariciresinol, at concentrations of 200 mM in the cell-free system and 100 mM in cultured cells, and the effects were compared with those of the aneuploidogen diethylstilbestrol and the clastogen 4-nitroquinoline-A^oxide. As none of the four lignans had any activity at the endpoints studied, the authors concluded that the four lignans are devoid of aneuploidogenic and clastogenic potential under the experimental conditions used in this study. CONCLUSIONS The lignans are an interesting group of natural products than can serve as a reference in the search for pharmacologically active drugs in different therapeutical groups. In addition, they have phytoestrogen properties which justify their interest in dietetic. Lignans present in food have no direct effects as phytoestrogens but some of their metabolites do. They are not potent as phytoestrogens, but some of them play a relevant role in sex differentiation and function, including different pathologies related with sexual hormone dependent diseases such as postmenopausical troubles, osteoporosis, estrogen dependent cancers, cardiovascular and liver diseases. A diet rich in proestrogenic lignans, such as flaxseed or soybean or whole dried legumes and cereals, can be a complementary treatment in the above-cited diseases. Lignans have been described as the active principles in different antiviral and anticancer medicinal plants and crude extracts. However, few of them

272

are potent enough to be used as therapeutical agents. Only some podophyllotoxins and their derivatives showed clear efficacy and potency as anticancer and antiviral agents. Of the cited properties, the activity on PAF and cAMP-PDE can be of interest in the selection of series to synthesise new and potent lignans. Isolation of compounds with activity in the 10'^ M range as PAF inhibitors can provide a good starting point for obtaining new compounds. On the other hand, the selectivity as inhibitors of different PDEs can serve to obtain new synthetic derivatives in future research. ABBREVIATIONS AA ADP AMP cAMP ATP CCC CNS CoA COX DAD DNA ED50 ER FAB FLAP FMLP GC OCR GMP cGMP GSH GST-P HDL HIV ^H-NMR

= Arachidonic Acid = Adenosine Diphosphate = Adenosine Monophosphate = Cyclic Adenosine Monophosphate = Adenosine Triphosphate = Counter Current Chromatography = Central Nervous System = Coenzyme A = Cyclooxygenase = Diode Array Detector = Deoxyribonucleic Acid = Effective Dose-50 = Estrogen Receptor = Fast-Atom Bombardment = 5- Lipoxygenase Activating Protein = Formyl-Met-Leu-Phe = Gas Chromatography = Glucocorticoid Receptor = Guanine Monophosphate; = Cyclic Guanine Monophosphate = Glutathione = Glutathione iS-Transferase Placental Form = High-Density Lipoprotein = Human Immunodeficiency Virus = Proton Nuclear Magnetic Resonance

273

= High Performance Liquid Chromatography = Human Papilloma Virus = Herpes Simplex Virus = Serotonin, 5-Hydroxytriptamine = Inhibitory Concentration 50 IC50 = Immunoglobulin Ig IL;IL-1 = Interleukin; Interleukin-1 /.v. = Intravenously LC = Liquid Chromatography = Lethal Dose 50 LD50 LDL = Low-Density Lipoprotein LOX = Lipoxygenase LPS = Lipopolysaccharide LTs; LTB4 = Leukotrienes; Leukotriene B4 MDA = Malondialdehyde 3'-MeDAB = 3'-Methyl-4-dimethylaminoazobenzene MIC = Minimum Inhibitory Concentration MPO = Myeloperoxidase MS = Mass Spectrometry = Noradrenaline, Norepinephrine NA NADPH = Nicotinamide Adenine Dinucleotide Phosphate NDGA = Nordihydroguaiaretic Acid ODC = Ornithine Decarboxylase PAF = Platelet Activating Factor PDE = Phosphodiesterase PGs; PGE2 = Prostaglandins; Prostaglandin E2 PKC = Protein Kinase C PLA2 = Phospholipase A2 PMNL = Polymorphonuclear Leukocytes P.O. ^^ per OS, orally RT = Reverse Transcriptase SFC = Supercritical Fluid Chromatography SFE = Supercritical Fluid Extraction SHBG = Sex Hormone-Binding Globulin SOD = Superoxide Dismutase TNF;TNF-a = Tumor Necrosis Factor; Tumor Necrosis Factor -a TPA = 12-0-Tetradecanoylphorbol-13 -Acetate TX; TXA2 = Thromboxane; Thromboxane A2 HPLC HPV HSV 5-HT

274

VLDL VSV

= Very Low Density Lipoprotein = Vesicular Stomatitis Virus

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

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CROCUS SATIVUS'mOl.OGlCA\. ACTIVE CONSTITUENTS *M. LIAKOPOULOU-KYRIAKIDES^ AND D.A. KYRIAKIDIS^ ^Dept. Chem, Engineering, Section of Chemistry and ^Faculty of Chemistry, Aristotle University ofThessaloniki, Greece 54006 ABSTRACT: The recent advances in separation and characterization of the volatile and non-volatile components of the dried stigmas of Crocus sativus (saffron) are presented. The volatiles with a very strong odor are consistent of more than 34 components that are mainly terpenes, terpene alcohols and their esters. Non-volatiles include crocins 1,2,3 and 4 that are responsible for the red or reddish brown color of stigmas together with carotenes, crocetin, picrocrocin (a glycosidic precursor of safranal) the bitter substance and safranal the major organoleptic principle of the stigmas. The methodology and techniques developed for the analysis of saffron metabolites including various chromatographic and spectroscopic techniques (TLC, HPLC, GC-MS, LC-MS, NMR.) are described. An extended study of the biological effects of either extracts or specific constituents of saffron (crocetin, picrocrocin and safranal) or extracts from its roots, from experiments in vivo and in vitro are discussed. A growing body of evidence indicates that carotenoids possess anticarcinogenic, antimutagenic and immunomodulating effects. Other effects such as lowering of blood pressure, stimulation of respiration, sedative effects, inhibition of human platelet aggregation in vitro and the dietary effects are reported as well. In addition the use of its coloring agents (crocins and carotenes) in dying cotton and wool fabrics and/or other uses in industry as well as attempts for biotechnological production of saffron and its metabolites are mentioned.

INTRODUCTION Crocus sativus L (Family Iridaceae) a perennial herb, native to the Eastern Mediterranean region, is cultivated in Spain, France, Greece, Italy, India, Turkey, Iran, e.t.c. Saffron-the yellow-orange stigmas from a small purple Crocus sativus flower- is the world's most expensive spice. According to the Greek mythology a gorgeous mortal named Crocos fell hard for the nymph Smillax who rebuffed Crocos' overtures and then Crocos became a lovely purple flower, the well known Crocus sativus. Greek myths and poetry exhibit an extravagant admiration of the color

294 and perfume from saffron. Homer sings the "Saffron mom" whereas gods and goddesses, heroes and nymphs and vestals are clothed in robes of saffron hue. To the nations of Eastern Asia, saffron's yellow dye was the perfection of beauty and its penetrating odor a perfect ambrosia. From historical records, saffron yellow shoes formed part of the dress of the Persian Kings. Crocus sativus plants (schemes 1,2) require strict agroclimatic conditions for their growth, that influence the quality of the spice. Samples obtained from different geographical locations and from different processing methods show variations with respect to color, flavor and the bitter principle.

Scheme 1. Crocus flowers. CH3

CH3

OR2

all-trans CH2OH Q

OH HO HO

\. 'CH2

CH2OH Q

H0--^Y"^V-^^ \

^ OH X Y Crocetin (I) R„ R2=H, Crocins (II) R„ R2=X, Y

OH

295 Crocins (II) that are glycosyl esters of crocetin (I) are the major components of saffron. Crocins {trans and cis isomers) belong to the family of carotenoids that are water-soluble. Safranal (III), a monoterpene aldehyde and picrocrocin (IV) precursor of safranal are also main components.

CH3 Safranal (III) H3C

CH3

CH2OH Q

"''^^^^^W^''

-

^CH3

Picrocrocin (IV)

Volatile constituents Analysis of saffron volatile components has been reported by various investigators [1-7]. Volatiles with a very strong odor are consistent of more than 34 components, that are mainly terpenes, terpene alcohols and esters. The major organoleptic principle present (60-70% of the volatiles) is safranal (2,6,6-trimethylcyclohexa-l,3-dien-l-carboxaldeyde) and is formed by de-glucosylation of picrocrocin, the bitter compound. Zarghami and Heinz [2,3] have identified volatile compounds similar or different than safranal. The structure of picrocrocin has been established by Buchecker and Eugster [8]. Safranal has been determined by TLC, GC and spectrophotometry [4]. Thin layer chromatography plates impregnated with 0.25 mm thick silica gel G were used with n-butanol-acetic acidwater (4:1:1) solvent system. Crocins and crocetins were visible in white light whereas picrocrocin can be detected under UV lamp (254 nm) as a dark brown fluorescent spot. For determination of safranal [4], crude ethanolic extract was extracted with diethyl ether. Ether layers were pooled, air dried and the volume made up to 0.1 ml. TLC of the extract

296 developed in the solvent system hexane-ethyl acetate (9:1) gave a spot that became visible on spraying with 2,4-dinitrophenylhydrazine (DNPH) reagent. Safranal gives a UV spectrum with X^max 308 nm.

Scheme 2. Crocus sativus plant. 1, roots; 2, bulbs; 3, corm; 4, leaves; 5, close flower; 6 open flower.

Sujata et al [4] have also reported the use of SE-30 and Carbowax columns for gas chromatographic analysis of the saffron volatile constituents. Isothermal run with SE-30 column resolved safranal into a sharp single peak at a retention time of 3.6 min. Catwallander et al [9] and Tarantilis and Polissiou [7] have reported, from the analysis of saffron volatiles, a minor component 2-hydroxy4,4,6-trimethyl-2,5-cyclohexadien-l-one that is considered the most powerful aroma constituent of saffron followed by safranal. Monoterpene aldehydes, isophorone and isophorone-related compounds have been also found in saffron. Zarghami and Heinz [3] have identified six new isophorone-related compounds listed in Table 1 from ether extracts of saffron by IR, NMR, UV and MS. In the same table are listed the most common volatiles of safron. The structures of carotenoid derived volatiles found by various researchers are shown below (scheme 3). Quantitative determination of picrocrocin and crocetin by High Performance Thin Layer Chromatography (HPTLC) is reported by Corti et al [\Q]. In addition, Kanasawud and Crouzet [11,12] and Crouzet and

297 Kanasawud [13] have proposed a mechanism for the formation of volatile compounds of saffron by thermal degradation of carotenoids including acarotene (VI) and lycopene (VII) degradation.

Scheme 3. Crocus carotenoid-derived volatiles isolated from stigmas.

Non-volatile constituents Qualitative methods that are used for determination of the color and flavor profiles of saffron have been reviewed by Sampathu et al{\A]. Separation of crocetin glycosyl esters by HPLC is referred by Pfander and Rychener [15], Pfander and Wittwer [16,17], Pfister [18], Sujata et al [4], Tarantilis et al [19] and Solinas and Cicheli [20]. A pretreatment of crude ethanol extract of stigmata by gel filtration on a G-50 column prior to HPLC is reported by Pfander and Rychener [15]. This treatment is necessary to

298 remove carbohydrates, that disturb the separation of pigments. HPLC analysis was performed using I) Lichrosorb SI 60 column (Merck, Darmstadt) with eluent ethyl acetate-isopropanol-water (56:34:10) and II) Lichrosorb RP-18 (Merck) with methanol-water (60:40) and UV detector in both cases at 440nm. It was found that, the main pigment was crocin (40-45%) followed by the mixed ester (35%), the diglucosyl ester (10%) and the two monoesters (2% each). Reverse-phase systems were used for the separation of the diglycosyl esters (polar compounds) including digentiobiosyl, diglucosyl- and the mixed ester with Lichrosorb RP-18 column. Crocin, crocetins and picrocrocin can be separated according to Sujata et al [4] either on a gradient run from 20 to 80% (v/v) acetonitrile/water in 20min at a flow rate of 0.5 ml/min and detection at 308nm or isocratic run with 76% (v/v) using a Shimadzu 15cmx4.9 mm ID CLC-ODS column. Table 1. The most volatile components of saffron Compound 1. Picrocrocin 2. Safranal 3. Isophorone 4. Isophorone related compounds

Chemical name

Formula weight

4-(P-D-glycopyranosyloxy)-2,6,6-trimethyl-1 -cyclohexen-1 carboxaldehyde

C,6H2607

2,6,6-trimethylcyclohexa-1,3-dien-1 -carboxaldehyde

C.OHHO

3,5,5-trimethyl-2-cyclohexen-l-one

C9HJ40

3,5,5-trimethyl-4-hydroxy-l-cyclohexanon-2-ene

C9H,402

5.

»

3,5,5-trimethyl-l,4-cyclohexadione

C9H,402

6.

»

3,5,5-trimethyl-l,4-cyclohexadion-2-ene

C9H,202

7.

»

3,5,5-trimethyl-2-hydroxy-1,4-cycIohexadion-2-ene

C9H12O3

8.

»

2,6,6-trimethyl-4-hydroxy-1 -cyclohexene-1 -carboxaldehyde

C10H16O2

9.

»

2,4,4-trimethyl-3-formyl-6-hydroxy-2,5-cycIohexadien-1 -one

CloH,203

As it has been reported previously [21] direct spectrophotometric determination of the crude ethanol extract at A^max values corresponding to crocin, crocetin, picrocrocin and safranal respectively, does not give reproducible results. Nevertheless, use of crude plant extract directly for determination of the various components is referred by Tarantilis et al [19] using HPLC with UV-Visible, Photodiode-Array Detection (UV-Vis-DAD) and mass

299 spectrometry coupled in line. ES (electrospray) instead of TS (thermospray) interfaces combined with HPLC-UV-Vis-DAD can also be used for characterization of other unknown compounds as pointed out by the same authors. UV-Vis spectra of saffron components are characteristic; picrocrocin [4-(a-D-glycopyranosyloxy)-2,6,6-trimethyl-1 -cyclohexen-1 carboxaldehyde] exhibits a characteristic broad absorption band at 250nm Oxidative decarboxylation of picrocrocin gives the precursor isophorone (3,5,5-trimethyl-2-cyclohexen-l-one) that is also a minor component of saffron. Crocus derivatives show characteristic UV-Vis spectra of the carotenoid moiety in the molecule. Carotenoids absorb in the visible region with double peaks between 400 and 500nm, that vary with the position and number of cis double bonds, and a single peak between 320 and 340nm. These characteristic peaks are presented by the cis isomer. The UV-spectra of dlX-trans glycosidic carotenoids show two bands one at 256nm (glycosyl esters bonds of crocins) and the other one between 400 and 500nm with A^ax at 437nm. The above data show that UV-Vis spectra can be used for identification of cis and trans isomers of carotenoids. The cis isomer shows 3 absorption bands whereas two absorption bands correspond to trans isomers. A large number of carotenoid compounds have been isolated from saffron. Crocins and crocetin have been already mentioned above and are listed in Table 2. a-Carotene (V), p-carotene (VI), lycopene (VII), zeaxanthin (VIII), phytoene (IX), phytofluene (X) are the minor ones [22]. Straubinger et al [23] have reported the identification of four novel glycoconjugated carotenoid breakdown products of saffron that are the PD-glucosides of (4R)-4-hydroxy-3,5,5-trimethylcyclohex-2-enone, (4S)-4hydroxy-3,5,5-trimethylcyclohex-2-enone and (4S)-4-(hydroxymethyl)3,5,5-trimethylcyclohex-2-enone as well as the P-D-gentiobiosyl ester of 2-methyl-6-oxohepta-2,4-dienoicacid. It should be mentioned that, in saffron all crocin derivatives, except crocin-1, occur as pairs of cis-trans isomers [24,25]. Minor carotenoids such as isorhamnetin-4-0-a-L-rhamnapyranosyl(l-^2)-p-D-glucopyranoside and P-(p-hydroxyphenyl) ethanol-a-0-a-L-rhamnopyranosyl(l-->2)p-D-glucopyranoside have been identified by chemical and spectroscopic analysis. Preparative HPLC and TLC have been used by Castellar et al

300

[26] and Iborra et al [27] respectively, for the separation of saffron secondary metabolites.

a-Carotene (V) HsC^

P-Carotene (VI)

OH

"CHs

Zeaxanthin (VIII)

Responsible for the flavor of saffron, as it has been mentioned, is the bitter tasting glycoside picrocrocin, and its degradation product, safranal.

301 An oxidative cleavage of zeaxanthin has been proposed [28] for the formation of carotenoid degradation. Table 2. Crocetin and crocins of saffron Chemical name

Compound Crocetin Crocin-1

Formula weight

2,6,11,15-tetramethylhexadeca-2,4,6,8,10,12,14heptaenedioic acid

C20H24O4

Ri= P-D-glucosyl (Y),

C26H34O9

R2=H Crocin-2

Ri= p-D-gentiobiosyl (X),

C32H44O14

R2=H Crocin-2'

Ri=R2= P-D-glucosyl (Y)

C32H44O14

Crocin-3

Ri= P-D-gentiobiosyl (X),

C3SH54O19

R2= p-D-glucosyl (Y) Crocin-4

Ri=R2= P-D-gentiobiosyl (X)

C44H64O24

Crocin-5

Ri= Three P-D-glucosyl (Z), R2= P-D-gentiobiosyl (X)

C50H74O29

Extractions and pre-separations Various methods have been applied for the recovery of saffron volatiles including steam distillation (SD), micro-simultaneous steam distillation extraction (MSDE) and vacuum head space (VHS) analysis. Simultaneous distillation-extraction with diethyl ether-pentane is reported by Romer and Rennel [29] and head space analysis by Mookherjee et al [30] and Joulain et al [31]. As reported by Godefroot et al [32], Nickerson and Lickens [33] and Schmitt [34] in some cases MSDE method gives the volatiles in low concentrations whereas high boiling point components are obtained in high concentrations. The use of the three mentioned methods for isolation and identification of the aroma components of saffron has been reported [7]. Soxhlet extraction is mainly used for the preparation of saffron crude extract and isolation of coloring agents using methanol or ethanol in the dark and under nitrogen atmosphere [4,27]. Pre-separation of the various components is achieved by chromatographic techniques, including silica gel or gel filtration column chromatography [16], Multilayer Coil Coulter Current Chromatography (MLCCC) [10]. A pre-separation of methanolic extract of saffron by MLCCC method is reported by Straubinger et al

302

[23]. Fractions containing picrocrocin were re-chromatographed on the same column using CHCh/methanol (1.75:0.3) and the fraction corresponding to picrocrocin was further identified by ^"^C-NMR and MS. Of the various analytical methods described, the ISO recommended a thin-layer chromatographic technique [35] for qualitative analysis of saffron, whereas quantitative HPLC methods have been described for the analysis of commercial saffron products [36]. Other reports on chemical analysis of saffron constituents including combination of the reported methods, crystal structure analysis, Fourier Transform-Infra Red (FT-IR) and Raman analysis are worth mentioned [37-40]. Very interesting is also the review article by Rios et al[A\] on chemical analysis of saffron. Flavonoids and other constituents Flavonoids occur in plants as mono- and di-glycosides and derive from 2phenyl-y-benzopyrone. Their UV spectra depend on the number and substitution of the hydroxy groups in the phenol nucleus. Flavonoid derivatives have been also found in stigmata of Crocus sativus. Among them kaempherol diglycoside has been isolated and identified by mass spectra [38]. Four isolectins have been isolated from bulbs of Crocus sativus with approximately molecular weight 48 KDa as determined by gel filtration chromatography [42]. Rivoflavine and thiamin are also constituents of saffron [43]. Very recently, anthocyanins were identified in the flowers of Crocus sativus [44]. Pharmacology and Medicine Carotenoids have been studied extensively and their biological effects including antioxidant, antitumor and in general their immunomodulating effects are known [45-52]. It has been reported that extracts of saffron show various pharmacological activities including antiviral [50] and antitumor activity [53-56]. The effect of saffron on cell colony formation and cellular and intracellular DNA, RNA and protein synthesis in malignant and non-malignant cells has been studied by Abdullaev and Fenkel [57,58]. Abdullaev and Frenkel detected a dose-dependent decrease in colony formation of A549 lung adenocarcinoma, cervical

303

epitheliod carcinoma and HeLa cells using saffron extracts and isolated crocetin respectively. Inhibition of DNA and RNA synthesis in isolated nuclei and suppression of the activity of the purified RNA polymerase II is also reported [59-60] The inhibitory effect of Crocus sativus in chemical carcinogenesis in mice and the modulatory effects on cis platininduced toxicity in mice have been reported as v^ell [61,62]. The protective effect of crocetin against oxidative damage in rat primary hepatocytes has been further pointed out [63]. In vitro cytotoxic analysis of the main saffron compounds (crocins, crocetin, picrocrocin and safranal) using HeLa (human cervical epitheliod cancer) cells was carried out by Escribano et al [64]. It was found that in terms of LD50 values (dose inducing 50% cell growth inhibition on these cells) safranal has 0.8mM, crocin and picrocrocin 3mM respectively. Furthermore, Escribano et al [65] have also isolated from corms of Crocus sativus a glycoconjugate with cytotoxic activity as well. Experiments with carotenoids from Crocus sativus in HL60 cells were conducted by Tarantilis et al [66] where inhibition and induction of differentiation of these cells was studied. Furthermore, cytotoxicity experiments of dimethylcrocetin extracted from saffron were carried out on sarcoma-180, Ehrlich ascites carcinoma, P388 leukemia, Dalton's lymphoma ascites and primary cells from surgical excised tumor samples [53]. LD50 values were 9 mg/ml for Dalton's lymphoma and L1210 and a little higher for the other series with the maximum value in P388 cells. Dimethylcrocetin and crocin were also found to inhibit DNA and RNA synthesis [53]. These experiments were carried out using ^[H] thymidine or ^[H] uridine and evaluating DNA or RNA synthesis in the presence of saffron compounds. The protective effects of crocetin on bladder toxicity induced by cyclophosphamide has been reported by Nair et al [67]. The effect of saffron on vitamin A levels and the inhibition of growth of solid tumors in mice has been studied [68]. The reports of Lin and Wang [69] on the protection of crocin in the acute hepatic damage in rats induced by aflatoxin-Bl and dimethylnitrosamine and Wang et al [70,71] on the effects of crocetin on the hepatotoxicity and hepatic DNA binding of aflatoxins Bl in rats. We have recently shown (unpublished data) the antitumor activity in mice and the gonotoxic effect of a fraction isolated from methanol extract of saffron, that had been purified on a silica gel column using chloroform/methanol as eluent. P388 tumor cells were transplanted in

304

BDFi mice and the tumor static factor (T/C%, the percent of survival increase of mice) after the administration of the specific saffron extract was found to be 140 and 149 (blank 125).The same fraction w^as also tested against P388 cancer cells for its gonotoxic effect. The high SCE (sister chromatide exchange) values in combination with the significant decrease of PRI values are an indication of DNA damages caused by this fraction. SCEs values have been frequently used as a highly sensitive indicator of DNA damages and/or subsequent repair. The ability to excise and repair various types of damage to DNA seems to be a general property of living cells [72]. Lowering of blood pressure of anesthetized dogs and cats, stimulation of respiration, and reports for pulmonary oxygenation, cerebral oxygenation, alveolar increased oxygenation are among the other biological properties of saffron [73-75]. In a recent report, the reduction of hypervalent iron in myoglobin by crocin fiirther indicates the antioxidant action of carotenoids and saffron in general [76]. Saffron stimulates uterus and in ancient times it had been used for abortions. Crocus sativus bulbs are toxic to young animals and the stigmas in high doses are narcotic [77]. Hypoxia and in general the effect of saffron in cardiovascular diseases is reported by Grisola [78]. Inhibition of growth and induction of differentiation of promyelocytic Leukemia HL60 by the carotenoids of Crocus sativus has been studied by Tarantilis et al [19]. The IC50 values found, were 0,8 muM for dimethyl crocetin, 2 muM for crocetin and crocin respectively, and 5 muM induction of differentiation of HL60 cells. We have reported that bulbs of Crocus sativus contain a poly-histidine protein factor, which aggregates human platelets in vitro. The molecular weight of this factor as found by gel filtration chromatography on a Sephadex G-75 column and sodium dodecylsulfate (SDS) polyacrylamide slab gel electrophoresis is 42,000Da [79]. In addition, another low molecular weight protein factor isolated from the same crude extract was found to inhibit human platelet aggregation in vitro [80]. The effect of Crocus sativus on blood coagulation has been also examined by Nishio et al[Sll The antitumor activity of saffron was also evaluated against a variety of murine tumor models. Ascites tumors were induced by i.p. transplantation of one million cells in mice and appeared in 7-14 days depending on the type of tumor cells used [82]. Saffron was administered

305 orally (200ml/kg) for 9 days continuously, one day after tumor inoculum. It was found that saffron, at that dose delayed ascites tumor growth and increased the life span of the treated mice compared to untreated controls The role of saffron from dietary sources in chemoprevention and in general in modifying cancer has been nicely reviewed by Nair et al [82] and by Rios et a/ [41] where hypolipidemic and tissue oxygenation effect of active constituents of saffron are reported as well. Chemopreventive agents have been widely used in pharmacology in the treatment of various diseases including cancer [83]. The role of chemopreventers in human diet is also discussed by Stavric [84]. Alcohol extract from saffron has been also examined for learning and memory, in step through (ST) and step down (SD) tests, in normal and in leaming-and memory-impaired mice. The results showed that this extract ameliorates the impairment effects of ethanol on learning and memory processes and possesses a sedative effect [85-87]. In addition, recent experiments have shown that crocin and crocetin derivatives inhibit promotion of tumor in mice [88]. Other activities Dyeing of fabrics Recent reports have shown that saffron extracts were used for dyeing cotton and wool fibbers [89-92]. As it has been found, both crude methanol or aqueous extracts of saffron and the isolated crocin fraction give satisfactory results in terms of color fastness and color brightness. Crocin or saffron extracts together with other natural pigments such as berberine, azulen, curcumin, betanin, etc have been used as a hair dye [93]. In a US Patent [94] a scalp treatment composition is described for scalp and hair health and growth containing saffron, among the other plant extracts. Food coloring The use of natural pigments for food applications is gaining soil from day to day [95]. Curcumin, betaine, amarathine, anthocyanins and p-carotene are the most common and widely used pigments [95,96]. Saffron's coloring properties attributed mainly to water-soluble carotenoids are used for coloring of foods. The stability of these saffron pigments in aqueous

306 extracts and data on the condition of storage and applications as well as kinetic studies of caronenoid loss and changes under various conditions of water activity (aw) and temperature have been reported [97,98]. Changes in pigments (crocins) and volatiles of saffron {Crocus sativus L) during processing and storage have been reported by Raina et al [99]. According to these Authors, drying of stigmas between 35-50° C leads to highest percentage in pigments 15-17% and about 60% safranal.. A quality evaluation of saffron by sensory profile and gas chromatography has been presented by Narasimhan et al [100] where, by using a modified Steven's equation, the dose-response relationship for saffron flavor (safranal) can be estimated. Another interesting article on the various uses of saffron is that of Basker and Negbi [101]. Biosynthesis of C2o-carotenoids in Crocus sativus As Pfander and Schurtenberger have reported [28] the absence of C20hydrocarbon precursors of crocetin in saffi'on supports a degradation pathway for the biosynthesis of crocetin by the occurrence of picrocrocin and safranal. Two different pathways have been proposed for the biosynthesis of the C2o-aglycon of the main pigments a) oxidative degradation of a C40carotenoid such as zeaxanthin [102,28] or b) dimerization of two Ciocompounds such as geranylpyrophosphate followed by dehydrogenation and oxidation as reported for the formation of Cso-carotenoides from the famesylpyrophosphate and of C4o-carotenoids from geranyl geranylpyrophosphate (C20) [103]. The interesting article by Armstrong and Hearst [104] on the molecular biology of carotenoid pigment biosynthesis should be mentioned. Biotechnological production of Saffron and its metabolites Tissue culture of saffron including somatic embryogenesis and shoot regeneration has been first reported by George et al [105]. Induction of crocin, crocetin, picrocrocin and safranal synthesis in callus cultures of saffron-Crocw^* sativus L has been reported by Visvanath et al [106]. Callus cultures were obtained from floral buds on Murashige and Skoog's medium supplemented with 3% sucrose, 2,4-dichlorophenoxy acetic acid

307

(2 mg/1) and crocetin (0.5 mg/1). The experiments showed that red global callus (RGC) and red filamentous (RFC) callus cultures can produce crocin, crocetin, picrocrocin and safi-anal. The yield of safi-anal obtained fi*om RGC cultures was comparable to that obtained fi'om natural dried saffron. Picrocrocin content in both cultures was higher than that of stigmas and picrocrocin was less in both cultures. It was also shown in this study that C sativus callus tissues have the biosynthetic capability to produce the above reported metabolites that is considered an important step in developing a biotechnological process for saffron production. Enhanced crocetin glucosylation by encapsulation into maltosyl-Pcyclodextrin was reported by Cormier et al [107] as an attempt to overcome the difficulties of the previous method, where the use of DMSO inhibited the enzymatic glucosylation of crocetin catalyzed by cell free extracts of saffron. Furthermore, in vitro production of stigma-like cultures from stigma explants and synthesis of crocin, picrocrocin and safranal by saffron stigma-like structures proliferated in vitro and synthesis of aroma compounds by microbial transformation of isophorone with Aspergillus niger cultures have been reported [108-112]. General conclusions The biological effects and uses of saffron, extracts of saffron and its specific constituents show that the plant must achieve more attention from Scientists as a real "active plant". Biotechnological preparation of saffron and its active metabolites will bring more light to the above investigations and establish Crocus sativus as a "multipurpose" plant. In addition, the above data support the consideration that folklore is still a useful tool in predicting sources with various biological activities e.g. antitumor, antiviral activity, etc. Furthermore, future screening of plant extracts from folklore medicine might be more profitable, in terms of curing human infectious diseases.

308 ABBREVIATIONS TLC HPLC GC-MS LC DNPH TS ES DAD SD MSDE VHS MLCCC ISO FTIR T/C SCEs PRI aw RGC RFC DMSO

= Thin layer chromatography = High pressure liquid chromatography = Gas chromatography - Mass spectrometry = Liquid chromatography = 2,4 dinitrophenylhydrazine = Thermospray = Electrospray = Diode array detection = Steam distillation = Micro-simultaneous steam distillation extraction = Vacuum head space = Multilayer coil coulter current chromatography = International standardization organization = Fourier-transform infra red = Tumor static factor = Sister chromatide exchange values = Proliferation rate index = Water activity = Red global callus = Red filamentous callus = Dimethylsulfoxide

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

313

CHEMICAL COMPONENTS OF FRAXINUS ORNUS BARK - STRUCTURE AND BIOLOGICAL ACTIVITY IVANKA N. KOSTOVA AND TANYA lOSSIFOVA Institute of Organic Chemistry with Centre ofPhytochemistry, Bulgarian Academy of Sciences, Bg-1113 Sofia, Bulgaria ABSTRACT: This review describes the investigations carried out by the authors and coworkers on the chemical composition and biological activity of Fraxinus ornus. Its stem bark has been used in the traditional medicine for treatment of wounds, inflammation, arthritis and dysentery. Our results support the claims of the folk medicine by presenting scientific proofs for the antimicrobial, antiinflammatory, immuno-modulatory, skinregenerating, antioxidant, photo-dynamic damage prevention and antiviral properties of the bark extract and its components. The ethanolic extract of the stem bark and its main constituent esculin were found practically non-toxic. They inhibited the classical and alternative pathways of complement activation. The extract and esculin displayed antiinflammatory activity in both zymosan- and carrageenan-induced paw edema in mice. The extract exhibited a pronounced antioxidative activity and caused intense wound epithelization. The antimicrobial and photodynamic damage prevention properties of the extract and its fractions were dependable on their hydroxycoumarin composition. The isolated secondary metabolites belong to the groups of hydroxycoumarins, secoiridoids, phenylethanoids and lignans. Most of the coumarins and secoiridoids possess the ability to affect the complement activity. A clear correlation between the structure and the biological activity (antimicrobial, antioxidative and photodynamic damage prevention) of the studied hydroxycoumarins was observed. Isolation of new biologically active compounds and finding of new biological properties of already known bioactive substances has been achieved.

INTRODUCTION Throughout the ages plants have been the source of medicinal agents. Today a revival of thousand-years-old herbal remedies and a return to the ancient form of medicine is observed. Herbal remedies are common in Asia and Europe, particularly in Germany, France and Italy. More and more Americans are supplementing and replacing prescription medicines with various medicinal herbs. However, in many cases the claims of the folk medicine are still to be scientifically proved. Some claims may not be accurate. The herbal medicines should also meet the contemporary requirements for safety and effectiveness. The expansion of the market for herbs demands strict standards for ingredients and manufacturing. The standardization of the herbal preparations requires a detailed study of their chemical composition and finding of the active components. Herb remedies that

314 enjoy the greatest popularity are generally those that have been the most thoroughly investigated. Fraxinus ornus (L.) is a small tree belonging to the Oleaceae family widely found in Bulgaria. Its stem bark is used in the Bulgarian folk medicine for treatment of infected wounds, inflammation, arthritis and dysentery [1,2]. However, the claims of the traditional medicine were not scientifically confirmed, the toxicity of the extract was not examined and the chemical composition of the bark was not completely investigated. In order to explain and confirm the biological activities claimed by the traditional medicine, and to search for new biologically active compounds we studied the antimicrobial, antioxidative, immunomodulatory, antiinflammatory, skin-regenerating and antiviral properties of Fraxinus ornus bark extract and its components. In a parallel detailed phytochemical investigation of the extract we isolated and determined the structures of many hydroxycoumarins, secoiridoid glucosides, caffeoyl esters of phenylethanoid glycosides, lignans and other phenolic compounds. In this review we present the results from our phytochemical and biological investigations. PREVIOUS INVESTIGATIONS Previous investigations on bark, flowers and leaves of Fraxinus ornus have shown the presence of the hydroxycoumarins esculin (1), esculetin (2), fraxin (3 ), fraxetin (4), cichoriin (5) and of some phenolic acids [36]. The isolation of the flavonoids rutin, quercetin, quercetin-3-glucoside, quercetin-3-galactoside, quercetin-3,7-digalactoside and rhamnetin from leaves and flowers has also been reported [7-9]. GENERAL APPROACH The bark of Fraxinus ornus was subjected to systematic phytochemical investigation. The isolated compounds and some bark extracts were fiirther investigated for their biological activities. In some cases synthetic derivatives of the natural compounds were prepared and their biological properties studied.

315

CHEMICAL STUDIES Extraction Extraction of the dried and well ground plant material was carried out with hot ethanol. The extract was concentrated to a small volume and the deposited solid (a mixture of the coumarin glucosides 1 and 3) filtered. The mother liquor was concentrated to obtain the total ethanol extract 1 (TEl). In some cases the ethanolic extract was concentrated without removal of the deposited solid to give the total ethanol extract 2 (TE2). TEl and TE2 were further used in the biological studies and for isolation of pure components. Hydroxycoumarins A characteristic feature of Fraxinus species is the presence of simple hydroxycoumarins having OH or/and OMe groups only in the benzene ring. In Fraxinus ornus we found derivatives of 6,7-dihydroxy-, 6,7,8trihydroxy- and 5,6,7-trihydroxycoumarins. Isolation of Hydroxycoumarins

For isolation of hydroxycoumarins the TEl was subjected to liquid vacuum chromatography (LVC) with PE, CHCI3, EtOAc and MeOH to yield the corresponding fractions [10]. No coumarins were found in the PE fraction. The CHCI3 fraction was chromatographed over a silica gel column with a dichloroethane (DCE) - MeOH gradient. A TLC study of the DCE fractions on silica gel yielded the coumarins esculetin (2), fraxetin (4), scoparone (6), isoscopoletin (7), scopoletin (8), fraxidin (9) and fraxinol (10). Identification of all coumarins was achieved by UV, IR, ^H NMR and mass spectra, and direct comparison with authentic samples. NOE experiments confirmed the structures of 9 and 10. RP' HPLC Analysis of Hydroxycoumarins in Fraxinus ornus Bark Extract

The hydroxycoumarin content of plants varies significantly depending on the stage of the plant growth and development, and on the climatic conditions. Investigations of these variations are of scientific and practical interest. High pressure liquid chromatography (HPLC) is the most promising method for separation and detection of coumarins because it allows high resolution and a rapid and reproducible determination even of

316 trace compounds [11-13]. However, the very complex hydroxycoumarin composition of many plants requires further improvement and development of the HPLC procedure.

R'

R^

R^

R'

1

H

OGlc

OH

H

la

H

OGlc 4Ac

OAc

H

2

H

OH

OH

H

2a

H

OAc

OAc

H

3

H

OMe

OH

OGlc

4

H

OMe

OH

OH

4a

H

OMe

OAc

OAc

5

H

OH

OGlc

H

6

H

OMe

OMe

H

7

H

OH

OMe

H

8

H

OMe

OH

H

9

H

OMe

OMe

OH

OMe

OH

OMe

H

10 11

H

OGIc

OMe

H

12

H

OMe

OMe

OMe

13

H

H

H

H

Glc == glucose Ac = acetate

In this connection we attempted a reverse phase - high pressure liquid chromatography (RP-HPLC) determination of 11 naturally occurring hydroxycoumarins 1-8 and 11-13 [14]. Conditions were found for best resolution of the standard mixture - mobile phase H20-MeOH, detection at A.= 220nm, and appropriate gradient profile and flow rate. These conditions were used for the HPLC analysis of the total extract of Fraxinus ornus bark.

317 2

3

30

w. 40 Mm.

Fig. (1). HPLC profile of the ethanolic bark extract of F. ornus collected from region 1 (see Table 1): peaks are labelled with the corresponding compound numbers.

The HPLC profile of the extract showed a good resolution of the main constituents. Esculin (1), esculetin (2), and fraxin (3) are the major components of this species, while the others are present in smaller amounts, Fig (1). Coumarin (13) was not detected in the extract. In addition 7-methylesculin (11) and 6,7,8-trimethoxycoumarin (12 ) were detected. This was the first report of the occurrence of 11 and 12 in the Oleaceae family. In Bulgaria Fraxinus ornus bark is a major source for the industrial preparation of esculin (1), an antiinflammatory and vitamin-P-like agent. The selection of appropriate plant material, i.e. of higher esculin (1) and of lower esculetin (2), fraxin (3) and fraxetin (4) content is economically important. For this reason a quantitative determination of 1-4 in commercial samples of Fraxinus ornus bark from five different regions in Bulgaria was carried out and the results presented in Table 1. The table shows that the samples from regions 1-4 are characterized by higher esculin (more than 8%) and total coumarin content (more than 9%). These values are also higher than those reported for the Chinese species F. chinensis, F. bungeana and F. stylosa [12]. Region 5 exhibited lower

318 esculin (6.3%) and higher esculetin, fraxin and fraxetin content (total coumarins 7.8%). Table 1. Concentrations of Hydroxycoumarins 1-4 in Samples of F. ornus Bark from Different Regions of Bulgaria

Regions 1 2 3 4

1

5

Esculin (I) 8.06 8.48 8.83 8.53 6.27

Esculetin (2) 0.25 0.26 0.29 0.29 0.50

Concentration % w/w) Fraxin Fraxetin (4) (3) 0.79 0.05 1.02 0.04 0.05 1.03 0.94 0.05 0.07 1.25

1

Total

1

9.15 9.80 10.20 9.81

7.79

1

The proposed method is applicable to the analytical control of 1-4 for scientific and industrial purposes. This method has been applied for determination of the hydroxycoumarin composition of all extracts in our further biological studies. It could be successfully used for phytochemical investigation of many hydroxycoumarin-bearing plants belonging to different families. Secoiridoids and Phenylethanoids Isolation of Secoiridoids and Phenylethanoids

The general scheme which we have employed for isolation of pure secoiridoids and phenylethanoids is schematically described below. Solvent-solvent partitioning of the TEl with PE and EtOAc yielded the corresponding PE, EtOAc and MeOH-H20 extracts. The EtOAc extract was further subjected to LVC with DCE-MeOH gradient yielding residues Rl and R2 (DCE-MeOH, 10:1); R3, R4 and R5 (DCE-MeOH, 5:1), and R6 and R7 (DCE-MeOH, 3:1). From Rl ligstroside (14), omoside (15) and caffeic acid (16) were isolated by LVC and TLC. R3 was subjected to CC over silica gel and HPLC to give hydroxyomoside (17). R4 after repeated CC and RP-HPLC afforded secoiridoids 14, 17-20a,b and the lignan 21. RP-HPLC of R5 gave the coumarin glucosides esculin (1) and fraxin (3), and the caffeoyl esters of phenylethanoid glucosides 22 and 23. RP-HPLC of R6 and R7 yielded the caffeoyl esters of phenylethanoid glycosides 24- 27.

319 Secoiridoid Glucosides

Secoiridoid glucosides of oleoside (28) type frequently occur in Oleaceae family. Usually, in Fraxinus species they are present as mono- or diesters of p-hydroxyphenylethanol (29) derivatives. Our phytochemical studies on Fraxinus ornus resulted in the isolation of ligstroside (14), oleuropein (18), framoside (19) and of the new compounds ornoside (15), hydroxyornoside (17), hydroxyframoside A (20a) and hydroxyframoside B (20b) [15-17]. The FAB spectrum of ornoside (15) exhibited [M+H]^ at m/z 629. Its ^H and ^"^C NMR spectra (Table 2) revealed the typical signals of an oleoside (28) nucleus and suggested the presence of one 1,4-disubstituted benzene ring, one 1,2,4-trisubstituted benzene ring and two sets of OCH2CH2Ph moieties. The ^H NMR spectrum of its acetate 15a exhibited signals for only four alcoholic and one phenolic acetyl group in the trisubstituted benzene ring, and suggested no free PhCH2 CH2 OH groups. Alkaline hydrolysis of ornoside (15) afforded glucose and the new phenolic compound, named omosol, whose structure was elucidated as 30 on the basis of its spectral data and chemical behavior. These findings implied that in ornoside, the ornosol (30) moiety is linked to the oleoside (28) through the nonphenolic CH2CH2OCO ester bonds at C-7 and C-11. The position of attachment was unambiguously established by detailed NOE experiments on ornoside. The most characteristic NOEs are presented in Fig. (2). On irradiation of CH2-2", an enhancement of the H-3 singlet, the doublet for H-4" and H-8" and CH21" was observed. Irradiation of CHa- T" leads to enhancement of CH2-2*" and H-4'". This confirms the assignment of CH2-I", 2", 1'" and 2'" and proves, that the disubstituted (tyrosol) moiety is linked to C-11. Therefore, the structure 15 was assigned to ornoside.

0-Glc

Fig. (2). Most important NOEs observed for 15

320

14

14a

CH2CH2—(

. 7 R^OOC

11 , C00R2

8

\ OGIcR^

>—OH

-OAc

CH2CH2-

R^

R^

R^

Me

OH

H

Me

OAc

8" T

CH2CH2-¥ T-OH \ / 5" 4' ^ _ ^ ^ C ' H / H 2

15

OH

r~8" CH2CH2—/

\—OAc

15a

OAc Cn2Cn2

CH2CH2—f

17

^

>—OH ^

CHjCHz-

/

\

^O—
lOOO^g/ml). This in contrast to the higher potency of the corresponding aglucones esculetin (2) and fraxetin (^4) (MIC = 500 and 125 |^g/ml, respectively) and indicates, that glucoside bonding of one of the phenolic hydroxyls leads to a significant weakening of the inhibitory properties. As already established by the method of Heiss, fraxetin (4), a 6-OMe,7,8dihydroxycoumarin, is a more potent inhibitor of the growth of S. aureus than esculetin (2), a 6,7-dihydroxycoumarin derivative. Compound 2 inhibits both the gram-positive and gram-negative organisms, while 4 is an inhibitor only of the gram-positive. This suggests that the position of the phenolic hydroxyls is essential for the activity. Isoscopoletin (7), a 7- methyl derivative of esculetin is less inhibitory than esculetin itself, while scoparone (6), a 6,7-dimethyl derivative of esculetin, is completely inactive. The 7-0-methylesculin (11) and 6,7,8trimethoxycoumarin (12) are totally deprived of activity. Evidently, a methylation of the phenolic OH decreases the activity. The MIC of the acetates esculetin 2 Ac (2a) and fraxetin 2 Ac (4a ) were found to be equal to those of the parent compounds 2 and 4. Most probably, acetylation of the phenolic OH does not alter the antibacterial activity of the hydroxycoumarins. Compounds 4 and 4a are the most potent inhibitors of S. aureus among the tested Fraxinus ornus components.

333

Tabie 8. Antibacterial Activity of Hydroxycoumarins, Secoiridoids and Tyrosol Derivatives

1 Compound 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Inhibitory zone (d jmrnl) S. aureus E. coli

1 Hydroxycoumarins 16 18 Esculin (1) Esculetin (2) 17 19 16 17 Esculetin 2Ac (2a) 17 20 Fraxin (3) 21 Fraxetin(4) 0 20 0 Fraxetin 2AC (4a) 0 0 Scoparone(6) 14 0 Isoscopoletin (7) 0 0 Methylesculin(ll) 0 0 6,7,8-Trimethoxy-coumarin (12) Secoiridoids 28 Ligstroside (14) 26 26 0 Ligstroside 5 Ac (14a) 24 Insularoside (15) 16 23 0 Insularoside 5 Ac (15a) Tyrosol derivatives 6 0 Tyrosol (29) 24 0 Omosol(30) ' MIC was determined only for compounds with inhibitory zone more than

MIC

1

S. aureus

£. coli

> 1000 500 500 >1000 125 125 -

> 1000 500 500 > 1000

500 500 500 1000

500

1 1 1 1 1 1 500

1 500 16mm.

1

-

1 1 1 I

The resistance of some plants to attacks of insects and microbes has been attributed [37] to the presence of the bitter secoiridoid ligstroside (14), which predominantly occurs in Fraxinus species. In this investigation ligstroside (14), its acetate (14a), insularoside (15) and the tyrosol derivative omosol (30) exhibited equal inhibition (MIC = 500 jag/ml) on the growth of S. aureus. The natural secoiridoids 14 and 15 inhibited both the gram-positive and the gram-negative microorganisms (MIC = 500 |Lig/ml), while their respective acetates 14a and 15b, and tyrosol derivatives 29 and 30 inhibited only the gram-positive. Our preliminary screening of the secoiridoids 14, 14a, 15, 15a and omosol 3 Ac (30a), using the method of Romans et al. [38], showed clearly visible zones of inhibition of the growth of Cladosporium cucumerinum and suggested them to be fungitoxic. The antibacterial activity of the secoiridoids 14,15, 17-19, 20a, b, 28a and 38, and the caffeoyl esters of phenylethanoid glycosides 22 -27 has also been tested using the direct bioautographic TLC assay as published by Hamburger and Cordell [39]. Bacillus subtilis spp. and Pseudomonas fluorescens were the representatives of the gram-positive and gramnegative bacteria, respectively. The minimum inhibition amount (MIA) was determined. Cefotaxime was used as a positive control.

334

Most of the tested secoiridoids displayed an inhibition of the growth of B. subtilis and P, fluorescens (Table 9). Insularoside (15) and hydroxyornoside (17) are more active against B. subtilis. The mixture of hydroxyframoside A, B (20a, b) showed the best activity against Pseudomonas fluorescens. Table 9. Minimum Inhibition Amount (MIA) of Secoiridoids Against B, subtilis and P. fluorescens Compound Ligstroside (14) Insularoside (15) Hydroxyornoside (17) 1 Oleuropein (18) Framoside (19) Hydroxyframoside A,B (20a,b) 7,11 -Dimethyloleoside (28a) 10-Hydroxyligstroside (38) 1 Cefotaxime ^NA- not active at a concentration of 50 |ig/spot

B. subtilis [|ag/spot] 0.5 0.2 0.2 1.0 1.0 2.5 NA 40 0.01

P. fluorescens [|ag/spot]

10

1

10 20 10 10 2.5 40 NA

0,01

1

The caffeoyl esters 22-27 showed no activity against Pseudomonas fluorescens at a concentration of 50 |uig/spot. The MIA of 22-27 against Bacillus subtilis is the following: 22- 20; 23 - 10; 24 - 2.5; 25 - 2.5; 26 20; 27 - 50 |ig/spot [40]. Skin - regenerating Properties of Esculin and Fraxinus ornus Bark Extract We investigated the skin regenerating properties of esculin (1) and Fraxinus ornus bark extract on male white Wistar rats having standard oval wounds [41]. The rats were divided into four groups of 6 animals and treated as follows: I. (control) group, destined for spontaneous recovery; II. (control) group, treated with propylene glycol; III. (test) group, treated with extract. A solution (18.2%) of the total ethanol extract of the bark (TE2) in propylene glycol was applied. RPHPLC analysis of the extract revealed the following hydroxycoumarin composition: esculin (27.1%), esculetin (0.1%), fraxin (0.3%), fraxetin (0.4%) and minor components scoporone (6), isoscopoletin (7), scopoletin (9), methylesculin (11) and 6,7,8- trimethoxycoumarin (12). The presence of coumarins 10 and 11, and of compounds 14 - 27 was confirmed by TLC and HPLC.

335

IV. (test) group, treated with a 3.45% solution of esculin in propylene glycol. The percent of epithelization with respect to the beginning of the experiment (zero day) was calculated (Table 10). Table 10. 1

Group

i II III

1

IV

Epithelization (%) of Wounds with Respect to the Zero Day

V

3^*^ day

•^th

Day

day

lO*" day

10.0 9.4 39.8 38.6

30.7 31.1 55.8 50.4

43.9 45.3 84.9 67.8

89.9 88.8 96.9 87.4

day

i^

11

963

1

96.5 100.0

98.9

1

The III group of animals exhibited a more intense epithelization of the wounds in comparison with the control groups at every stage of the investigation. A weaker regenerating effect was found in the IV group of animals treated with esculin. The application of propylene glycol alone ( II group) did not result in an epithelizing effect. On the 7^^ day the biopsy of the I and II (control) groups established mostly a massive leucocytic necrotic swellings on the wound surface. A less pronounced leucocytic necrotic elevation was established in the III group, where definite zones of the wounds were almost completely filled with granular tissue. The amount of the riper collagen fibrils exceeded the amount of young fibrils. The epithelial regenerate consisted of ten and more cells. The basal prismatic layer, which was rich in mitoses, was separated from the overlying layers consisting of larger cells with round light nuclei. The III and IV groups of animals exhibited similar developments. These investigations have shown that the alcoholic extract and esculin obtained from Fraxinus ornus bark exercise moderate skin regenerating effects, no toxicity or local irritation being observed. The experimental results are in line with the antimicrobial properties of the extract and its constituents, and with the use of the bark in the traditional medicine for wound treatment and against inflammation. In addition, we studied the acute toxicity of the total ethanolic extract TE2 and its main component esculin (1) and found that applied p.o. to white mice and white Wistar rats in doses from 50 to 8000 mg/kg they were practically non-toxic. No lethality was observed up to 21 day and with the highest doses used. No significant changes were found both in the behavior and the reflexes of the animals. No pathological deviations from the physiological values were found in all hematological and clinical -chemical indices studied.

336 Antioxidative Action of the Ethanolic Extract Hydroxycoumarins from Fraxinus ornus Bark

and

some

The need to prepare lipids and lipid-containing products that are stable with respect to the effect of atmospheric oxygen has increased the interest in finding suitable natural sources of harmless antioxidants. The high biological activity of extracts from such sources is often due to the presence of phenolic compounds, many of them exercising a pronounced antioxidative effect on lipids [42,43]. In the literature there was no information about the antioxidative action of Fraxinus ornus extract and the data on hydroxycoumarins were scarce [44]. We examined the antioxidative action of the total ethanolic extract (TE2) of Fraxinus ornus bark as well as of its main hydroxycoumarin components esculin (1), esculetin (2), fraxin (3) and fraxetin (4) [45]. Quantitative reverse phase HPLC analysis [14] of the extract used in this study established the following hydroxycoumarin content: esculin 53.7%, esculetin 0.5%, fraxin 8.7% and fraxetin 0.3%. The investigations were performed at 100^ with kinetically pure triacylglycerols of lard and sunflower oil (TGL and TGSO), which represent models of two types of natural lipid unsaturation. The stabilization factor F as a measure of the effectivity and the oxidation rate ratio (ORR) as a measure of the strength of the tested antioxidants were estimated. The parameters F and ORR for TGL and TGSO oxidation in the presence of 0.05 and 0.10% ethanolic extracts oi Fraxinus ornus bark are presented in Table 11. The data indicate that the ethanolic extract of Fraxinus ornus bark has a pronounced antioxidative activity during the oxidation of both lipid substrates. This activity is commensurate with the inhibiting effect of the same concentration of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) during TGL oxidation [46]. In order to elucidate the contribution of the main phenolic components [esculin (1), esculetin (2), fraxin (3) and fraxetin (4)] in the extract to its stabilizing action, we studied the autoxidation kinetics of the two natural lipid systems (TGL and TGSO) in the presence of different concentrations of 1-4. For TGL autoxidation in the presence of 0.04% esculin and 0.04% fraxin the following data for F and ORR were obtained: esculin, F = 2.5, ORR = 0.7; fraxin, F = 2.1, ORR = 1.0. In TGL addition of 0.02% esculin and 0.02% fraxin resulted in practically no stabilizing action. In TGSO these glucosides exhibited no antioxidative action. The kinetic parameters characterizing the inhibitor action of esculetin and fraxetin are also presented in Table IL The experimental data demonstrate that the fraxetin possesses a higher effectivity and greater strength than does the esculetin. Both substances are less effective

337

inhibitors in the lipid system with a higher oxidizability (TGSO). Both hydroxycoumarins are antioxidants of a relatively high effectivity and of a great strength because, under the same oxidation conditions for 9.1x 10' "•M B H T in TGL F = 4.8, ORR = 0.33; for 4.6 x 10"* M a-tocopherol F = 6.5, ORR = 0.13; for 1.0 x 10"^M ferulic acid F = 3.1, ORR = 0.59; for 1.3 x lO'^M 3,4-dihydroxybenzoic acid F = 13.4, ORR = 0.07 and for 1.1 x lO'^M caffeic acid F = 62.1. For 1.0 x 10"^ M ferulic acid in TGSO F = 2.6, ORR = 0.60, ; for 1.3 x lO'^M 3,4-dihydroxybenzoic acid F = 3.6, ORR = 0.60 and for 1.1 x 10"^M caffeic acid F = 33.6, ORR = 0.07 [46,47]. Table 11. Stabilization Factor F and Oxidation Rate Ratio ORR for the Inhibited Oxidation of TGL and TGSO at 100°C in the Presence of Ethanolic Extract, Esculetin (2) and Fraxetin (4) from Fraxinus omus bark 1 Antioxidant 1 Extract

Esculetin (2)

Fraxetin (4)

Inhibitor concentration M % 0.05 0.10 0.56x10-^ 0.01 0.02 1.12x10-^ 0.05 2.81x10-' 0.10 5.56x10-' 0.48x10-' 0.01 0.02 0.95x10"' 0.05 2.38x10-' 0.10 4.76x10-'

TGSO

TGL F 4.8 6.1 22.7 42.7 64.5 73.1 38.2 86.3 208.0 340.0

ORR 0.28 0.28 0.07 0.06 0.05 0.05 0.05 0.03 0.02 0.01

F 3.6 4.0 14.8 20.8 37.6 41.2 13.2 27.2 72.0 125.0

1 ORR 1 0.60 0.50 0.15 0.09 0.06 0.05 0.15 0.09 0.04

0.02 1

It is worth mentioning that when ORR is larger than 1, the oxidation proceeds faster in the presence of an inhibitor than in its absence. The lower the ORR, the stronger the inhibitor. Comparison of the F and ORR values for the glucosides with the values of the same parameters for the aglucones at almost the same molar concentrations (0.02% aglucones) shows that the glucoside bonding of one of the phenolic OH groups leads to a significant weakening of the inhibiting properties. The results obtained indicate blocking of the more active phenol group in fraxin, due to which the difference in antioxidative activities of glucosides esculin and fraxin is, in contrast to aglucones esculetin and fraxetin, negligible. Considering the antioxidative effects of coumarins 1-4 in TGL and TGSO and the content of these compounds in the extract, and eliminating the possibility of antagonism or synergism between them, it was calculated that the four compounds under consideration determined twothirds of the antioxidative effectivity of the extract in TGL and half of the effectivity in TGSO.

338 TLC analysis of the antioxidatively acting compounds [48] in the ethanoHc extract from Fraxinus ornus bark revealed the presence of additional antioxidative acting compounds. Among them calceolarioside B (23) demonstrated a significant activity. Most probably, caffeic acid (16) and the caffeoyl esters of phenylethanoid glycosides 22, 24-27 also contribute to the antioxidative properties of the extract. Photodynamic Damage Prevention by Extracts Hydroxycoumarins from Fraxinus ornus Bark

and

Some

The antioxidants or quenchers of free radicals are knovm to minimize skin photoaging and the protective action of sun screens correlates closely to their free radical scavenging activity [49,50]. This prompted us to use the prevention of photodynamic yeast cell damage to comparatively investigate the protective activity of the four hydroxycoumarins esculin, esculetin, fraxin and fraxetin, and a widely used sun screen paminobenzoic acid (FABA). We applied the same test to characterize the protective activity of four Fraxinus ornus bark preparations containing these coumarins in different concentrations and to examine the activity of caffeic acid (16), a minor constituent of these preparations [51]. Table 12. Preparation 1 Total extract 1 Fraction A 1 Fraction B 1 Fraction C

Protective Effect of the Fraxinus ornus Preparations Protectio n factor in% 9.8 2.3 54.6 6.1

Hydroxycoumarin composition in % EsculinCl) 40.5 0.0 28.4 45.3

Esculetin (2) 1.0 0.0 6.3 0.3

Fraxin (3) 6.9 0.0 2.5 9.0

Fraxetin (4) 0.2 0.0 1.6

|

0.2

1

The total extract of Fraxinus ornus bark v^as prepared and further subjected to solvent-solvent partition to obtain the fractions A, B, and C w^ith the hydroxycoumarin composition given in Table 12. The test for protective effect evaluation was performed using yeast cells (strain Kluyveromyces fragilis 129-1) according to the procedure described by Lazarova and Ignatova [52] and the protection factor determined. All of the tested pure compounds showed protective activity (Table 13). The protection achieved was higher for esculetin (2) and fraxetin (4) as compared to the corresponding glucosides esculin (1) and fraxin (3). This finding is in accordance with the data obtained by Bakalova et al [53] concerning the effect of the same coumarins on the lipid peroxidation of liver microsomes, as well as with our own results on their antioxidative action presented above.

339 The protective effects of the four Fraxinus ornus preparations depend on their hydroxycoumarin composition (Table 12). Fraction B, which is enriched in aglucones, exerts the highest protective activity. Fraction C, with lower aglucone and higher glucoside concentration, demonstrated a protective activity lower than that of the total extract. The negligible protective activity of fraction A correlates very well with its composition, i.e. no measurable concentration of the coumarins under investigation. Protective Effect of the Pure Compounds

Table 13. 1

Compound Esculin (1) Esculetin (2) Fraxin (3) Fraxetin (4) PABA

Caffeic acid (16)

Concentration in mg/1

Protection factor in %

20 50 20 50 20 50 20 50 2.5 5 25 2.5 5 25

38.7 26.3 97.6 99.8 23.1 72.8 94.7 98.2 47.2 943 92.6 49 1 91.0

98,2

1

Our results suggest that the caffeic acid (16) and the aglucones esculetin (2) and fraxetin (4) approximate the skin protective effect exerted by the conventional sun screen PABA. The Fraxinus ornus bark preparations enriched in the compounds 2 and 4 seem to be effective protectors too. Complement Inhibition and Antiinflammatory Activity of Hydroxycoumarins, Secoiridoids and Extracts from Fraxinus ornus Bark The use of Fraxinus ornus bark in the Bulgarian folk medicine for treatment of inflammation, arthritis and dysentery [1,2] suggests the presence of some active principles with anti-inflammatory activity. Our phytochemical investigations have shown that the bark contains hydroxycoumarins, secoiridoid glucosides, caffeoyl esters of phenylethanoid glycosides and other phenolic compounds [10,14-17,28]. RP-HPLC analysis of commercial samples of Fraxinus ornus bark revealed high esculin (1) content (6-9%) [14].

340

Since the complement system is highly involved in an inflammatory response [54,55] many substances exhibiting anticomplementary activity have proved to be effective antiinflammatory agents [56]. In this connection we studied and compared the effects of the ethanolic extract of Fraxinus ornus bark and its main component esculin (1) on some in vitro and in vivo reactions related to acute inflammatory processes [57]. Quantitative RP-HPLC analysis of the total extract used in this study showed the following hydroxycoumarin composition: esculin 40.0%, esculetin 2.4%, fraxin 7.8% and fraxetin 0.4% [14]. The inhibitory effects of TE2 and 1 on the classical pathway (CP) and the alternative pathway (AP) of the complement activation in mouse serum were estimated at final concentrations varying from 1 to 50)ag, Fig. (5). The TE2 caused a more pronounced reduction of CP hemolysis compared to esculin. In the AP assay they exhibited nearly equal dosedependent inhibition of complement - mediated lysis.. inhibition (%)

Concentration (|ig)

Inhibition (%)

Concentration (|ig)

Fig. (5). Inhibition of CP (A) and AP (B) complement activity in mouse serum by different concentrations of TE2 (o) and esculin (•)

341 The comparison between the effects obtained with TE2 and 1 in the hemolytic inhibitory assay indicates that the anticomplementory action of TE2 is not due only to esculin. The effect of esculin on CP activity was less pronounced than that of the total extract, although it represents 40% of the content of TE2 and in these experiments it was tested in equal concentrations with TE2. The full inhibition of AP activity was achieved at concentration of 50)ig for both esculin and TE2. This also suggested that, excepting esculin, some other extract constituents contribute to its anticomplementary action. The esculin concentration causing 50% inhibition of complement activity in vitro is about 10"^ M which appears to be similar to that established for chemically similar compounds. Carrageenan- and zymosan- induced paw edema were chosen as suitable models for evaluation of the antiinflammatory activities of TE2 and esculin (Table 14). The results showed that both TE2 and esculin significantly reduced formation of the zymosan-induced paw edema in mice. In the case of carrageenan-induced edema, only TE2 at a dose of 15 mg/kg significantly reduced the inflammation, while esculin was ineffective. This indicated that the extract contains active components with different mode of action. Evidently, TE2 and esculin possess the ability to influence complement activity in vitro and to suppress some complement-mediated reactions after in vivo application. Coumarin derivatives are known to possess antiinflammatory and antimetastatic properties. The mode of action of coumarins is mainly attributed to their direct action on cells participating in the inflammatory process [58]. The influence of coumarins on the complement system, which is involved in the different stages of inflammatory response, has not been thoroughly investigated. Table 14. Effect of Esculin (1) and Total Extract from F. ornus Bark on Zymosan- and Carrageenan- Induced Paw edema in Mice Carrageenan- induced Zymosan-induced oedema oedema | Paw volume" Paw volume' 1 Control 42.6 ±5.5 89.0 ±9.5 15 30.3 ±5.4* 15.7 ±1.3* Total extract 5 28.6 ±10.0* 21.0 ±2.0 15 38.0 ±6.2* 36.0 ±2.6 Esculin (1) 5 38.2 ±2.0 1 40.6 ±3.5* ® Difference (mg) between the weight of a zymosan or carrageenan treated paw and the concentrated saline treated paw. Significant from respective control: * P< 0.05. Test material

Dose (mg/kg)

For this reason we examined the in vitro effect of 12 hydroxycoumarins on classical and alternative complement activity in

342

normal human serum and the consumption of the key components CI and C3 [59]. The coumarins 1-4, as well as their acetylated and methylated derivatives la, 2a, 4a, 6-8,11 and 12 have been investigated at different concentrations. The effect of the substances at concentrations of 1.2x10"'^ and 5.0 xlO'^^M, as the most representative, are shown in Table 15. All the substances tested had a moderate or weak ability to affect at least one of the complement pathways. The effect was not strictly dose-dependent. Esculin 5Ac (la), esculetin 2Ac (2a) and 7-methylesculin (11) exhibited good inhibition on CP activity. Scoparone (6) strongly reduced AP activity in normal human serum (NHS). Scopoletin (8), esculin (1) and esculetin (2) enhanced complement mediated hemolysis. Some of the compounds exhibited combined effect - activated one of the pathways and inhibited the other. Table 15. Inhibition (-) or Activation (+) of CP and AP Activity by Some Hydroxycoumarins

Hydroxycoumarins 1 1

1 1 1 1 1 1 1

Esculin (1) Esculin 5Ac (la) Esculetin (2) Esculetin 2Ac (2a) Fraxin (3) Fraxetin (4) Fraxetin 2Ac (4a) Scoparone (6) Isoscopoletin (7) Scopoletin (8) Methylesculin(ll) Trimethoxycoumarin (12) NA = not active

CP + 28.2 -21.5 + 7.1 + 8.2 + 28.2 -8.2 -8.2 + 5.9 + 8.2 + 22.4 -28.2 -16.5

Concentration 1.2xlO-^M Activity (%) AP NA -28.2 -5.9 -28.2 NA NA -20.6 -45.9 NA NA -34.1 -25.9

CP + 27.1 -40.0 + 24.3 -32.9 NA -17.5 -28.6 -11.4 + 24.3 + 10.0 -51.4 + 13.6

Concentration 5.0xlO"*M Activity (%)

|

AP

1

+ 10.7 + 17.9 + 15.7 -28.6 + 15.0 -15.0 -12.1 -57.1 NA + 20.0 -8.6

-28.6

1

Seven hydroxycoumarins were further tested at a single concentration (5.0x10"^ M) for their ability to influence CI and C3 functional activities after preincubation with undiluted NHS. 7-Methylesculin (11) had a good effect on reducing total, CI, and C3 hemolysis via both pathways. Scoparone (6) strongly inhibited C3 alternative activity but in the case of the classical pathway only the total hemolysis was diminished without influence on CI and C3. Esculin (1) slightly increased C3 classical activity but caused exhaustion of alternative C3 activity. In subsequent experiments 1 and 8 altered the effect of other complement activators (heat aggregated IgG, suramin and zymosan) when applied with them simultaneously in vitro.

343

The experimental data presented above suggest that hydroxycoumarins may counteract with some of the complement proteins and thus inhibit their functional activity. Also, it is possible that the substances form complexes with the serum proteins which are able to activate complement system. It is difficult to make conclusions about the relationship between the structure of the coumarins and their action on complement mediated reactions. It might be concluded, however, that the methylated hydroxycoumarins 6 and 11 are the most potent inhibitors of AP and CP activities and deserve attention as a possible antiinflammatory agents. A series of pure secoiridoid glucosides isolated from different Fraxinus species was compared in vitro for anticomplement action as well as for their ability to prevent cobra venom-induced complement activation in normal human serum [60]. Table 16 shows that most of the secoiridoids possess the ability to suppress CP and AP activities. The most effective inhibitors of CP in guinea-pig serum (GPS) were ligstroside (IC50 33 |Lig/ml) and insularoside (IC50 62 |iig/ml). With regard to NHS the most pronounced decrease of CP was caused by 7,11-dimethyloleoside (28a) at a concentration of 250 |ag/mL Altemative pathway hemolysis was slightly altered, although the substances were used in a higher concentration (Img/ml) than in the CP assay (250 |ag/ml). Evidently, secoiridoid glucosides exhibit a greater effect on CP activity. It makes them interesting for further investigation as there is a need for selective inhibitors of the complement system for possible therapeutic use. Table 16. Effect of Some Secoiridoids and Fraxinus ornus Bark Extracts on CP and AP Activity in GPS and NHS 1 1

Product

GPS ICP5o(Mg/ml)*

?3HS Inhibition (%)

CP**

AP'

1

Extract TEl 302 ±10 1 Extract TE2 1438 ±20 12.7 15.5 1 Ligstroside (14) 33 ±4 38.2 18.2 1 Insularoside (15) 62 ± 8 28.2 1 Hydroxyornoside (17) 5.5 185 ±10 23.6 18.2 1 Oleuropein (18) 130 ±8 29.1 1 Framoside (19) 4.5 160 ±6 96.4 7,11 -Dimethyloleoside (28a) 8.2 180±4 1 10-Hydroxyligstroside (38) 34.5 >1000 4.5 1 ' The concentration giving 50 % inhibition of CP (ICP50). ^ Measured at concentration of 250 ^g/ml. Measured at concentracion of l^ig/ml 1

1

In our further experiments the secoiridoids 15, 17-19 expressed the ability to prevent CP and AP activation, caused by cobra venom [61]. This

344

suggested that among the active secoiridoid constituents may exit one with C3- convertase inhibitory property. Our investigations revealed the contribution of secoiridoid glucosides to the antiinflammatory action of the extract. Antiviral Activity of Some Hydroxycoumarin Derivatives Studies on the antiviral effects of 6,7-dihydroxy- and 6,7,8trihydroxycoumarin derivatives were very limited [62,63]. This prompted us to study the antiviral properties of the structurally related compounds 1, 2, 4, 6, 7,11,12 and acetates la, 2a, 4a [64]. Primary screening for antiviral activity was carried out using the agardiffusion plaque-inhibition method with cylinders [65]. The compounds were tested against one representative in each of the four taxonomic viral groups; namely, picoma-, orthomyxo-, paramyxo- and herpes viruses, which represent a few of the most important families of the human patogens. The viruses used were poliovirus 1 (PVl), influenza virus A (FPV), Newcastle disease virus (NDV) and pseudorabies virus (PsRV). Two of the ten hydroxycoumarin derivatives tested showed activity against NDV, namely esculetin (2) and its diacetate (2a), when applied in cylinders at a dose of 2.8 and 1.9 mM/0.1 ml, respectively. Their activity was significant, although inferior when compared to that of ribavarin (used as a reference paramyxovirus inhibitor) at a dose of 2.0 mM /O.l ml. The remaining compounds tested were without effect on the replication of the four viruses studied. Evidently, no definitive conclusion could be drawn regarding structure-activity correlations. It could be noticed, however, that methylation and glucosidation of esculetin (2) lead to a loss of activity. SUMMARY AND CONCLUSIONS The work presented in the preceding pages described the antimicrobial, antioxidative, antiinflammatory, immunomodulatory, skin regenerating, photodynamic damage prevention and antiviral properties of Fraxinus ornus bark extract and its constituents. The total ethanolic extract of the stem bark and its main constituent esculin (1) were found practically non-toxic. They inhibited the classical pathway and alternative pathway of complement activation. The total extract and 1 displayed antiinflammatory activity in both zymosan- and carrageenan-induced paw edema in mice. The extract exhibited a pronounced antioxidative activity and caused intense wound epithelization. The antimicrobial and photodynamic damage prevention properties of the extract and its fractions were dependable on their

345

hydroxycoumarin composition. These results to some extent explained the traditional use of the bark for treatment of wounds, inflammation, dysentery and arthritis. The isolated secondary metabolites belong to the groups of hydroxycoumarins, secoiridoid glucosides, phenylethanoids and lignans. Of them 6 are new compounds. Isolation of new biologically active compounds and finding of new biological properties of already known bioactive substances has been achieved. Most of the secoiridoids and the coumarins possess the ability to affect at least one of the complement pathways. A clear correlation between the structure and the biological activity (antimicrobial, antioxidative and photodynamic damage prevention) of the studied hydroxycoumarins was observed. Our investigations provide more data on the chemical composition and biological properties of Fraxinus ornus and support the claims of the traditional medicine by presenting scientific proofs for the biological activity of the bark extract and its components. It was concluded that the total ethanolic extract of the bark can be included in the group of the practically non-toxic substances and may be used in the therapeutic practice for treating wounds, bums etc., as well as in the perfumery and cosmetics [66]. ABBREVIATIONS Ac AP BHT BHA Caff CGI CP DCE FPV Glc GPS HPLC IR LVC MIA MIC MS NA NAS NDV

Acetate (COCH3) Alternative Pathway Butylated Hydroxytoluene Butylated Hydroxyanisole Caffeoyl Ester Unit Contact Growth Index Classical Pathway Dichloroethane Influenza Virus A Glucose Guinea-pig Serum High Pressure Liquid Chromatography Infra-red Liquid Vacuum Chromatography Minimum Inhibition Amount Minimum Inhibitory Concentration Mass Spectrometry Not Active Normal Human Serum Newcastle Disease Virus

346 NMR ORR PABA PhG PsRV PVl Rha RP TEl TE2 TGL TGSO TLC UV

= = = = = = = = = = = = = =

Nuclear Magnetic Resonance Oxidation Rate Ratio p-Amino Benzoic Acid Phenylethanoid Glykoside Pseudorabies Virus Poliovirus 1 Rhamnose Reverse Phase Total Ethanol Extract 1 Total Ethanol Extract 2 TriacylglycerolsofLard Triacylglycerols of Sun Flower Oil Thin Layer Chromatography Ultra-violet

ACKNOWLEDGEMENTS The phytochemical investigations described in this review were carried out at the Institute of Organic Chemistry, Bulgarian Academy of Sciences and partially in a collaboration with Prof. W. Kraus, Dr. B. Vogler and Mrs. L Klaiber from the University of Hohenheim, Stuttgart. We are grateful for their participation. The work carried out at the Institute of Organic Chemistry was made possible thanks to the skillful efforts of Dr. B. Mikhova, Dr. G. Stoev, Dr. E. Vassileva, Mr. N. Nykolov, and for the antioxidative action thanks are due to Dr. N. Yanishlieva and Dr. E. Marinova. The biological investigations are a result of an effective collaboration with our following co-workers from the Institute of Microbiology, Bulgarian Academy of Sciences: Prof A. Galabov, Dr. N. Ivanovska, Dr. A. Kuyumgiev, Dr. G. Lazarova, Dr. H. Neychev, Dr. Z. Stefanova and Mrs. A. Ignatova. We gratefully acknowledge their valuable contribution. We also acknowledge the collaboration with Dr. L. Chipilska from the National Centre of Hygiene and Medical Ecology and Dr. E. Klouchek and Dr. A. Popov from the Higher Medical Institute. Dr. I. Kostova thanks Mrs. A. Ivanova for the technical assistance in the preparation of the manuscript.

REFERENCES [1] [2]

Stoyanov, N. In Our Medicinal Plants (Bulg.); Ularova, K., Ed.; Nauka i izkustvo: Sofia, 1973; Vol./, pp. 321-322. Asenov, I.; Nikolov, S. In Pharmacognosy (Bulg.); Popova, M., Ed.; Medizina i Phizkultura: Sofia, 1988, p. 41.

347

[3] [4]

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[32] Jurd, L.; Gorse, J.; King, A.D.; Bayne, H.; Mihara, K.; Phytochemistry, 1971, 10, 2971-2974. [33] Kostova, I.N.; Nikolov, N.M.; Chipilska, L.N.; J. Ethnopharmacol, 1993, 39, 205208. [34] HQiss,F.; Aerosol report, 1975, 12,540-553. [35] lossifova, T.; Kujumgiev, A.; Ignatova, A.; Vassileva, E.; Kostova, I.; Pharmazie, 1994, 49, 298-299. [36] Bankova, V.; Kujumgiev, A.; Ignatova, A.; Dyulgerov, A.; Pureb, O.; Zanyansan, Z.; Popov, S.; Proc. Vint. Conf. Chem. Biotechnol Biol Active Nat. Prod, Varna, 1989,2,239-343. [37] Kubo, I.; Matsumoto, A.; Takase, I. J. Chem. Ecol, 1985, 11,251-263. [38] Romans, A.L.; Fuchs, A.; J. Chromatogr, 1970, 51, 327-329. [39] Hamburger, M.O.; Cordell, G.A.; J. Nat. Prod, 1987, 50, 19-22. [40] lossifova, T.; Vogler, B.; Klaiber, I.; Kostova, I.; Kraus, W.; 46^*^ Ann. Congress Soc. Med. Plant Res., Vienna 1998, Abstract and Poster 034. [41] Klouchek, E.; Kostova, I.N.; Popov, A.; Compt. Rend. Acad Bulg. Sci, 1994, 47, 125-128. [42] Houlihan, H.M.; Ho, C.-T.; Chang, S.S.; J. Am. Oil Chem. Soc, 1985, 62, 96-100. [43] Torel, J.; Cillard, J.; Cillard, P.; Phytochemistry, 1986,25, 383-385. [44] Dziedzic, S.Z.; Hudson, B.J.F.; Food Chem, 1984, 14,45-51. [45] Marinova, E.M.; Yanishlieva, N.Vl.; Kostova, I.N.; Food Chem, 1994, 51, 125132. [46] Yanishlieva, N.VL; Marinova, E.M.; Fat Sci. Technol, 1992, 94, 374-379. [47] Marinova, E.M.; Yanishlieva, N.; Fat Sci. Technol, 1992, 94, 428-432. [48] Marinova, E.M.; Yanishlieva, N.; Comm. Dept. Chem. Bulg. Acad. Sci, 1986, 19, 524-527. [49] Pathak, M.A.; Photochem. Photobiol, 1990,51,68-69 8. [50] Cesarini, J.P.; Msika, P. Photochem. Photobiol, 1988,47, 73-74 S. [51] Lazarova, G.; Kostova, I.; Neychev, H.; Fitoterapia, 1993, LXIV, 134-136. [52] Lazarova, G.; Ignatova, M.; Biotechn. BioE, 1991, 5, 37-42. [53] Bakalova, R.; Somleva, T.; Serebinova, E.; Ognianov, A.; Kagan, V.; Stoichev, Z.; Free Radicals and Biostabilizers, Sofia,19S7, Abstract from the Symposium, p.9. [54] Dias da Silva, W.; Lepow, I.H.; J. Exp. Med, 1967,125,921-946. [55] Frank, M. M.; Frues, L.F.; Immunology Today, 1991,12, 322-326. [56] Engleberger, W.; Hadding, U.; Etschenberg, E.; Graf, E.; Leyck, S.; Winkelman, J.; Pamham, M.J.; Intern. J. Immunopharmacol, 1988, 10, 729-737. [57] Stefanova, Z.; Neychev, H.; Ivanovska, N.; Kostova, I. J. Ethnopharmacol, 1995, 46, 101-106. [58] ?\\\QX,'^B.\Lymphology, 1980, 13, 109-119. [59] Ivanovska, N.; Yossifova, T.; Vassileva, E.; Kostova, 1.; Meth. Find. Exp. Clin. Pharmacol 1994, 16, 557-562. [60] Ivanovska, N.; lossifova, T.; Kostova, I.; Phytotherapy Research, 1996, 10, 555558. [61] Van Dijk, H.; Rademaker, P.M.; Klerx, J.P.A.M.; Willers, J.; J. Immun. Meth, 1985,85,233-243.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

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NATURAL COMPOUNDS FOR THE MANAGEMENT OF UNDESIRABLE FRESHWATER PHYTOPLANKTON BLOOMS KEVIN K. SCHRADER, AGNES M. RIMANDO, STEPHEN O. DUKE United States Department ofAgriculture, Agricultural Research Service, Natural Products Utilization Research Unit, P.O. Box 8048, University, MS 38677-8048, United States ofAmerica ABSTRACT: Significant losses occur every year in aquaculture, farm livestock, and waterfowl due to problems with toxin and "off-flavor" compound production from microalgae blooms. In addition, an overabundance of algae can clog streams and create flavor problems for municipal drinking water suppHes. At present, application of synthetic compounds to the affected aquatic ecosystem is one management method used to control and prevent the growth of noxious phytoplankton. Unfortunately, many of these synthetic compounds have limitations for their usefulness in controlling phytoplankton blooms including restricted use (by government), broad-spectrum toxicity towards non-target organisms, high toxicity to non-target organisms, and public perception of adverse health risks associated with their use. The discovery, characterization, and use of natural compounds for phytoplankton control would provide environmentally safe alternatives to chemical compounds. This chapter focuses on past and present research in natural algicides including isolation and characterization of bioactive lead compounds, toxic selectivity and mechanism of action of lead natural compounds (and materials), and efficacy in aquatic ecosystems in terms of impact on target phytoplankton blooms and non-target organisms. The economic cost of using natural algicides is also discussed.

INTRODUCTION Freshw^ater phytoplankton blooms commonly occur in reservoirs, lakes, canals, and ponds under eutrophic and other physicochemical conditions that are favorable for bloom formation. Among the different types of phytoplankton blooms that can occur in freshwater ecosystems, cyanobacterial (blue-green algal) blooms are usually the most undesirable for the following reasons: 1) certain species of cyanobacteria can produce toxins that kill aquatic and terrestrial animal life; 2) some species of cyanobacteria produce "off-flavor" compounds that can impart an undesirable taste to cultured fish; 3) cyanobacteria are a poor base for

352 aquatic food chains in aquaculture ponds [1]; 4) cyanobacteria are poor oxygenators of water in food-fish production ponds [1]; and 5) filamentous cyanobacterial blooms can block filters used in drinking water supply systems. In this chapter, we will focus on freshwater cyanobacterial blooms. Cyanobacterial toxins are increasingly associated with human and animal acute and chronic health problems [2]. Occurrences of toxic cyanobacterial blooms are worldwide [2,3], and toxin production has been confirmed in bloom-forming cyanobacteria such as members in the genera Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nodularia, Nostoc, Oscillatoria (Planktothrix), and Umezakia [2,3]. Of the dozens of cyanobacterial toxins identified, most are classified as cytotoxic, hepatotoxic, neurotoxic, or causing allergic or irritation-type reactions. Examples of some of the health-related incidents in humans associated with cyanobacterial toxins include the following: 1) severe hepatoenteritis in 149 persons exposed to Cylindrospermopsis raciborskii [4]; 2) skin irritations and gastroenteritis after bathing in freshwater containing cyanobacteria [5]; 3) rashes and fever after contact with a microcystin-containing bloom oi Oscillatoria agardhii [2]; 4) 2000 cases of gastroenteritis and 88 deaths linked to Microcystis and Anabaena bloom in a Brazilian reservoir [2]; 5) 60 deaths in haemodialysis patients in Brazil linked to microcystin from cyanobacteria [6]; and 6) irritation and bhstering due to a toxic bloom of Microcystis [7]. Water filtration through activated carbon in a filter bed at the end of the water treatment process is one method used by some municipal water systems to help remove cyanobacterial toxins [8]. The oxidants chlorine and ozone also help degrade some types of cyanobacterial toxins [8]. Management options that involve removal of cyanobacteria or prevention of their growth in water supply reservoirs and lakes include the use of artificial mixing to reduce water stratification which favors the growth of cyanobacteria [1,9], reduction of nutrient loading to the water body, and the use of algicides, although release of toxins due to lysis of cyanobacterial cells from an established bloom can result in a short term increase in dissolved toxins levels [8]. There are numerous reports attributing cyanobacterial blooms to poisoning and death incidents in wild animals and domestic livestock. Wild and domestic animals will usually avoid drinking water contaminated with cyanobacteria. However, fences that restrict the movements of farm animals and limit their access to cyanobacteria-free

353 drinking water has probably contributed to more cases of poisoning incidents involving farm and livestock animals than for wild animals [7]. The most prevalent reports of cyanobacterial toxicosis involving livestock have been in sheep [8] and in cattle [10]. Other cyanobacterial poisonings of domestic animals include lambs [11-13], horses [14], pigs [14], and dogs [14,15]. Waterfowl have also been poisoned lethally by ingestion of a filamentous species of cyanobacteria [16]. There are also other repeated cases of wildUfe poisonings after ingestion of water containing cyanobacteria [17]. In aquaculture ponds, fish kills due to toxic cyanobacterial blooms are infrequent, and there is little direct evidence of harmful consequences of toxin-producing cyanobacteria in aquaculture ponds [18]. At least one report [19] attributes large losses of channel catfish {Ictalurus punctatus L.) in a catfish production pond in the southeastern United States to the toxic cyanobacterium Aphanizomenon flos-aquae. Fish may avoid ingesting harmful quantities of toxic cyanobacteria to help reduce the adverse consequences posed by such blooms. In any case, prolonged exposure to toxin-producing cyanobacteria may stress fish and cause them to become more susceptible to adverse conditions, thereby reducing growth [18]. Numerous off-flavor compounds have been identified and implicated as the cause of noxious taste and odor episodes in drinking water supplies and in cultured fish. The compounds of microbial origin most often implicated in such episodes are geosmin (^ran5-l,10-dimethyl-^ra«5-9decalol) (lA) and 2-methyHsobomeol or MIB ((1-R-exo)-1,2,7,7tetramethyl bicyclo-[2,2,l]-heptan-2-ol) (IB).

H3C

B

Fig. (1). Geosmin (A) and 2-methylisobomeol (B)

CH3

354

Geosmin is often described as having an earthy odor, similar to the odor encountered from freshly plowed soil. MIB has a musty odor, similar to the odor from a damp cellar. Planktonic, filamentous species of cyanobacteria are most commonly associated with geosmin and MIB production in freshwater ecosystems. In water, the sensory threshold odor concentration (concentration recognized as earthy or muddy by 75% of judgements) for geosmin is 18-20 ng/L [20] and 40 ng/L for MIB [21]. In fish, sensory threshold concentrations differ from species to species, and the range for geosmin is 6-10 |Lig/kg [20,22] and for MIB is 0.1-0.7 |ig/kg [20,23]. Other off-flavor compounds of microbial origin have been isolated and identified from cultured fish and drinking water supplies. P-Cyclocitral has a tobacco-like odor and has been isolated from blooms of the cyanobacterium Microcystis spp. [24] and the flesh of farm-raised channel catfish [25]. P-Cyclocitral does not appear to be a large off-flavor problem in aquaculture [18]. Isopropylmercaptan, a compound with an oniony odor, has been isolated from cultures of Microcystis sp. [26], and 1-octen3-one, a mushroom-like odor-producing compound, has been isolated from cultures of an Anabaena sp. [27]. In addition, offensive sulftircontaining compounds are released into the water during cyanobacterial cell decomposition [26,28]. In catfish aquaculture, offensive odors can be rapidly absorbed in the flesh of the fish. The major uptake route for geosmin and MIB is across the gills [18], and these compounds are primarily stored in the adipose tissue [29]. Elimination of these odorous compounds is much slower than the uptake rate, and elimination rate decreases as water temperature decreases and adipose tissue content of the catfish increases [30]. The problems with off-flavor episodes in municipal drinking water systems (reservoirs) are frequent and can occur worldwide. Earthy and musty off-flavor incidences in water supply systems have been attributed to cyanobacteria in CaHfomia, USA [31-36], Florida, USA [37], Pennsylvania, USA [38], Austraha [39,40], Canada [41], Japan [42-47], and Norway [48]. In 1989, 63% of 388 water utilities surveyed in the United States reported earthy- or musty-odor episodes in their drinking water supply [49]. Such episodes usually result in consumer (customer) complaints to the local water utility board or company but do not represent a health hazard to humans. Re-occurrences of off-flavor episodes erode consumer confidence in the quality and safeness of public drinking water

355 supplies [50] and have contributed to an increase in the consumption of bottled water [51]. Water utilities usually rely on filtration of the water supply through powdered or granular activated carbon at the treatment plant to remove the undesirable earthy and musty odors [49-51]. However, during cyanobacterial bloom die-offs in the water supply ecosystem, lysis of the dead or dying cyanobacterial cells releases such large amounts of the off-flavor compounds that carbon filtration usually can not completely remove the earthy and musty odor compounds. Ozonation is another method used by some municipal water districts to reduce undesirable tastes and odors in drinking water. The addition of the oxidant ozone to water at alkaline pH levels will significantly reduce levels of geosmin and MIB [49,51]. Conditions that favor development of cyanobacterial blooms are high nutrient loading rates, diminished vertical mixing of the water column, and warm water temperatures [1]. Cyanobacteria are able to out-compete green algae and other types of phytoplankton for light and nutrients due to their ability to regulate their position in the water column by changing cell buoyancy. In addition, conditions in which nitrogen is limited relative to phosphorus favor growth of nitrogen-fixing species of cyanobacteria. Unfortunately, cyanobacteria are able to compete well for resources but have slow growth rates compared to most species of eukaryotic phytoplankton [55]. Slower growth rates of cyanobacteria translate into reduced aquaculture yields for types of fish or crustaceans that depend on in-pond primary production. Cyanobacteria are poorly utilized as a food source by herbivorous crustacean zooplankton, an early step in the aquatic food chain, thereby reducing transfer efficiency of nutrients from primary production to aquaculture crop [1]. Biomass-specific rates of net carbon fixation by cyanobacteria are lower than for eukaryotic phytoplankton, and the slower growth rates of cyanobacteria also translate to their poor oxygenation of water. The formation of surface scums of cyanobacteria followed by their massive die-offs can endanger fish crops due to acute dissolved oxygen depletion [56]. The worldwide economic impact of undesirable cyanobacterial blooms in municipal water systems is unknown. Increased costs can occur due to increased amounts of activated carbon used in water treatment facilities to counteract higher levels of off-flavor compounds in the water. In addition, filters and filtration equipment can become clogged with certain types of filamentous cyanobacteria and result in significant "down" time to correct these problems. Certain species of cyanobacteria (e.g., Lyngbya wollei)

356 can also form floating mats of filaments that clog waterways, ponds, and lakes, thereby impeding navigation and recreation. Although cyanobacterial-derived off-flavors can occur in many aquatic animals and in aquaculture throughout the world, the only economic impact studies of such off-flavor problems have been in channel catfish aquaculture in the southeastem United States. Off-flavor catfish are held until flavor quality improves, and these delays in harvest result in economic losses from inventory management problems and increased production costs [57]. Based on off-flavor problem added costs [57-60], catfish producers may have lost as much as U.S.$60 million in 1998. Approaches to the management of undesirable cyanobacterial blooms in freshwater ecosystems include managing around the bloom, treatment of the water or aquatic animal (depuration) to purge undesirable microbial metabolites, and prevention of the development of cyanobacterial blooms. Several prevention methods and strategies utilized in drinking water reservoirs and lakes are the following: 1) water shed protection including the control of point sources of nutrient inputs; 2) hypolimnetic aeration to destratify the water column; 3) control of inorganic chemical composition (e.g., alkalinity) that promotes cyanobacterial growth; and 4) the application of chemicals to kill or prevent the growth of cyanobacteria [52]. In aquaculture pond management, several approaches have been used to help prevent cyanobacterial growth. These approaches include the following: 1) the apphcation of alum (aluminum sulfate) to precipitate (remove) phosphorus from the water; 2) nutrient removal from pond water by macrophytic plants; 3) manipulating the ratio of total nitrogen to total phosphorus in the water; 4) water exchange to reduce nutrient availability; 5) biomanipulation using planktivorous fishes; and 6) application of synthetic algicides [18]. The use of algicides is an approach similar to that used for dealing with weeds in terrestrial agriculture, i.e., application of phytotoxic chemicals (herbicides). Many commercial aquaculturists favor this approach. Currently, only copper-based products are approved by the United States Environmental Protection Agency for widespread use as an algicide in food-fish production ponds and municipal drinking water reservoirs in the United States. These copper-based products, such as copper sulfate and chelated copper compounds, are commonly used in aquaculture and in municipal drinking water systems. Unfortunately, copper-based algicides do not provide a large degree of toxic selectivity towards noxious

357

cyanobacteria [61]. In addition, strains within species of cyanobacteria can vary considerably in their tolerance to copper [62,63]. Copper sulfate toxicity towards non-target organisms such as plants and fish is very susceptible to environmental variables. Environmental factors can decrease the toxicity of copper sulfate so that it is ineffective in controlling the growth of target cyanobacteria or increase the toxicity towards nontarget organisms [18]. Copper from copper sulfate will rapidly disappear from the water column after application and accumulate in the aquatic ecosystem sediments. Copper in chelated copper algicides does not rapidly precipitate out of solution and is claimed to be more effective than copper sulfate since chelated copper provides a higher concentration of total dissolved copper in the water for a longer period of time [64]. However, these benefits may be outweighed by the increased cost of using chelated copper products compared to copper sulfate. Although repeated applications of copper algicides at low dosages reduces the overall abundance of cyanobacteria and off-flavor problems in catfish production ponds [65], the broad spectrum toxicity of copper reduces water quality in terms of depressed dissolved oxygen levels and increases in ammonia concentration [66]. Such deterioration in water quality can endanger fish health and reduce aquaculture productivity. The unacceptable risks that synthetic herbicides pose to the environment and human health prevent their approval for use in drinking water supply systems and aquaculture ponds. The discovery of environmentally safe compounds that have a greater degree of toxic selectivity towards noxious species of cyanobacteria than copper algicides currently available would greatly benefit commercial aquaculturists and municipal drinking water suppliers and consumers. The use of natural products to selectively control cyanobacteria is desirable due to environmental safety issues and consumers' negative perceptions of the use of synthetic compounds (herbicides). Cyanobacterial Inhibitory Compounds and Sources Compounds reported to have inhibitory effects on cyanobacteria have been isolated from a diverse range of organisms including terrestrial plants, aquatic plants, fungi, bacteria, actinomycetes, protozoa, and even from some species of green algae and cyanobacteria. The structural types

358 of the secondary metabolites identified from these organisms are equally diverse, with a number being phenolic compounds. Barley straw has been shown to be effective in controlling general algal blooms [67-70]. Cyanobacteria inhibited by barley straw include Anabaena sp., Aphanizomenon sp., Microcystis sp., and Oscillatoria sp. [71]. Barley straw introduced into the water inhibited algal growth during its decomposition [72]. In a study using an artificial rumen reactor, decomposing barley straw was shown to contain lignin-derived phenolic compounds [73]. Derivatives of coniferyl, coumaryl, and sinapyl alcohols were identified by UV and mass spectrometry. Phenol, /7-cresol, and biphenyl-2-ol, compounds of known toxicity to algae, were present in close proximity to rotting straw at concentration ranges of 10-100, 10-100, and 1-10 |J,g/L, respectively [74]. In their studies. Barret and coworkers [75] also found a relatively high concentration of phenol, /?-cresol, and additionally, 4-ethyl-phenol released from the straw. The phenolics tannic acid, propyl gallate, and gallic acid were found to inhibit growth and pigment synthesis of Nostoc sp. strain MAC and Agmenellum quadruplicatum PR-6 [76]. In the same study, methyl gallate was found to also inhibit the growth of ^. quadruplicatum PR-6, but not of Nostoc sp. Further studies showed that these tannin compounds, except for gallic acid, inhibited chlorophyll a and c-phycocyanin synthesis in Nostoc sp. strain MAC, and, except for methyl gallate, also had an effect on electrolyte efflux [77]. Tannins have been reported to be the algicidal constituents of several Acacia species [78,79]. The algicidal activity was lost after acetylation and methylation of the plants' methanolic extracts indicating that the activity was due to the free phenolic hydroxyl groups present as galloyl residues [79]. Tannins from the extracts of the fhiits of Acacia nilotica, in a spray-dried form, controlled algal populations including Oscillatoria and Microcystis [78]. Leachate from the hardwood tree aspen (Populus tremuloides) inhibited algal growth [80] and was found to be rich in phenols. However, experimental data obtained do not support toxicity attributable only to the phenolic compounds. No further work has been reported on the algal toxicity of aspen wood leachate. Theoretically, aquatic plants, both higher plants and algae, might be expected to be a source of algicides since these organisms compete with cyanobacteria. There is some evidence to support this theory. Cattails {Typha latifolia) are rich in p-sitosterol, which has algicidal activity against Phormidium autumnale, Naviculla pelliculosa, Chlorella emersonii, Chlorella vulgaris, and Stichococcus bacillaris [81]. In a

359

bioassay-directed study of the components of water hyacinth {Eichhornia crassipes), two highly algicidal compounds identified as A^-phenyl-Pnaphthylamine and 7V-phenyl-a-naphthylamine were found [82]. Few such bioassay-directed studies directed toward algicide discovery have been made. Species of the watermilfoil genus Myriophyllum (Haloragaceae) have been reported to release phenohc cyanotoxins [83]. Three 1 '-0-caffeoyl-6'-0-galloyl-p-Dphenylpropanoid glucosides: glucopyranose (2), r-0-coumaroyl-6'-0-galloyl-P-D-glucopyranose (3), and r-0-sinapoyl-6'-0-galloyl-P-D-glucopyranose (4) were found to be toxic to the common freshwater cyanobacterium Synechococcus leopoliensis [84]. Phenylpropanoid 2 was found to undergo hydrolysis under experimental conditions, yielding gallic and caffeic acids. Observation of higher activity of the intact molecules of compounds 2, 3, and 4 compared to the individual phenolic acid hydrolytic products led the authors to believe that synergism was occurring between the phenolic acids. Synergism was confirmed with mixtures of gallic-p-coumaric and gallic-sinapic acids. Ellagic acid, (+)-catechin, gallic acid, and pyrogallic acid released by M spicatum were shown to synergistically inhibit the growth of Microcystis aeruginosa [85]. In a separate study, tellimagrandin II (eugeniin) (5) was found to be the main inhibitory compound in M. spicatum following a bioassay-directed fractionation [86]. Tellimagrandin II strongly inhibited Anabaena sp. PCC7120, Synechococcus sp. PCC 6911, Synechocystis sp. CB-3, and Trichormus var. P-9 in agar-diffusion assays. Investigation of M alterniflorum and M, verticilatum from northern Germany did not reveal the presence of tellimagrandin II, but several other hydrolyzable polyphenols were found. Proserpinaca palustris (another Haloragaceae plant) also inhibited Trichormus var. P-9. Hydrolysis of the crude extract of M. alterniflorum yielded gallic and ellagic acids, while tannase treatment of the crude extracts of M, verticilatum and of P. palustris yielded mainly gallic acid. In M. brasiliense, tellimagrandin II was also identified as the most toxic components to Microcystis aeruginosa and Anabaena flos-aquae, along with 1-desgalloyleugeniin (6) [87]. Additionally, a mixture of epicatechin-3-gallate and catechin-3-gallate, gallic acid, and the flavonoids quercetin, quercitrin, and avicularin were reported as anti-algal compounds from this aquatic plant.

360

R1 2 OH 3 H 4 OMe

HO

R2

H H OMe

OH

5 R = galloyi 6R = H

361 Fatty acids appear to also have activity against cyanobacteria. Oxygenated fatty acids resembling prostaglandin structures and with selective inhibition against cyanobacteria were isolated from the aquatic plant Eleocharis microcarpa [88]. The prominent components from the active chromatographic fraction were identified as C^^ trihydroxycyclopentyl (7) and C^g hydroxycyclopentenone (8, 9) fatty acids. These compounds were also found in other higher aquatic plants (Potamogeton sp., Najas sp.) as well as in the waters in which these plants grow. Long chain fatty acids were also found in the waters around decomposing barley straw [74]. Analysis of the waters by GC-MS showed the presence of 9-eicosene, octadecanoic acid, hexadecanoic acid, heptanoic acid, and hexanoic acid in high concentrations [74]. From the ether extract of the aquatic plant Typha latifolia, the compounds linolenic acid, 6-linolenic acid, and an unidentified CI8:2 fatty acid were isolated, along with three steroidal compounds; these compounds exhibited selective inhibition of T625 Synechococcus leopoliensis and T1444 Anabaena flos-aquae in vitro [89].

COOH

8 R = .CH=CHCH2CI^ 9 R = -(CH2)3CH3

Ricinoleate is in the same chemical class of compounds as those allelochemicals isolated from Eleocharis microcarpa and was determined

362 to be selectively toxic towards cyanobacteria [90]. Potassium ricinoleate, the active constituent of the commercial algicide Solricin 135®, was shown to inhibit the growth of cyanobacteria in laboratory tests [90]. However, when its effectiveness was evaluated in ponds, potassium ricinoleate did not inhibit the growth of cyanobacteria [91]. The authors could not explain the loss of activity. Phenylpropanoid-type compounds have been isolated as the algal inhibitory constituents from the aquatic plant Acorus gramineus [92] using a filter paper-dish bioassay. It was noted that l,2-dimethoxy-4-(£'-3'methyloxiranyl) benzene (10), l,2,4-trimethoxy-5-(Z-r-propenyl)benzene (11), l,2,4-trimethoxy-5-(£'-3'methyloxiranyl)benzene (12), the three most abundant phenylpropanes isolated, were either inhibitory or not in each of the cyanobacterial strains tested which included Anabaena flos-aquae, Nostoc commune, and Synechococcus leopoliensis.

MeO OMe R1

R2

10 H C - C H - C H o

H

O 11 HC = CH-~CH3

OMe

12 H C - C H - C H o

OMe

\ / O

^

A number of structurally unrelated compounds were tested in a general screen using microtiter plates to discover natural products with selective growth inhibition towards off-flavor-compound producing cyanobacteria [93]. In this study, ^raw5-ferulic acid was found to be the most selectively toxic to Oscillatoria cf chalybea among the phenolic compounds tested. However, it was not toxic to Anabaena sp. LP 691. Cinnamic acid, one of the compounds released from decomposing barley straw, was also selectively toxic to O. cf chalybea. Among the naturally-occurring

363 selectively toxic to O. cf. chalybea. Among the naturally-occurring quinones tested anthraquinone (13) was the most toxic, and also selectively toxic, to O. cf. chalybea. Anthraquinone, 2methylanthraquinone, and juglone had the highest differential activity to cyanobacteria compared to green algae tested. Artemisinin (14), the antimalarial sesquiterpenoid lactone peroxide from Artemisia annua, was shown in this screening study to be highly toxic with selectivity towards O. cf chalybea. The use of A. annua leaves and/or flowers in ways similar to the use of barley straw may be of great potential.

Some cyanobacteria produce secondary metabolites that inhibit the growth of other cyanobacteria. Cyanobacterin (15), bearing a y-ylidene-ybutyrolactone structure, was isolated from the freshwater cyanobacterium Scytonema hofmanni and was found to inhibit Synechococcus cultures and other microorganisms [94]. Cyanobacterin appears to act as an allelopathic substance by allowing the survival of the slow-growing Scytonema while among the more prolific-growing species. Studies with Synechococcus sp. and Euglena gracilis indicate cyanobacterin acts on the thylakoid membranes [95]. Hapalosiphon intricatus and Hapalosiphon fontinalis were reported to be inhibitory to Anabaena sp., but the extracellular substance(s) secreted was not identified [96]. Subsequent work with H. fontinalis led to the isolation of a tetracyclic indole alkaloid, hapalindole A (16), the compound responsible for antialgal acitivity [97]. The isothiocyanate form of hapalindole A (17) was also isolated, among other minor constituents.

364

OCHo 15

16 R = - N ^ C 17 R=:_N=C=S A structurally unique group of compounds (those with a long hydrocarbon chain bearing a conjugated enediyne and a heterocyclic ring at the end) was isolated from the fresh-water cyanobacterium Fischerella muscicola UTEX 1829 [98,99]. The compounds, named fischerellins A (18) and B (19), were also found in Fischerella ambigua [99,100]. Fischerellin A inhibited other cyanobacteria {Anabaena variabilis P9, Phormidium sp. UTEX 1540, Synechococcus sp. PCC 6911, Synechocystis sp. CB-3), as well as several chlorophytes, but not eubacteria [100]. Fischerellin B was also found to have algicidal properties but is produced in smaller quantities than fischerellin A [99]. Fischerellin A is a potent photosystem II (PSII) inhibitor [98] and affects the fluorescence transients and O^ evolution by the cyanobacterium Anabaena P9 and the green alga Chlamydomonas reinhardtii, as well as higher plants [101]. An algicidal compound from Oscillatoria late-virens was found to be toxic towards

365 Microcystis aeruginosa via a mechanism that appears to be an inactivation of PSII-mediated electron flow [102]. In an earUer study, the active fractions of an ether extract from O. late-virens were found to contain long-chain saturated fatty acids as major components [103].

18

Metabolites from heterotrophic bacteria have also been reported to inhibit cyanobacteria. Pseudomonas aeruginosa inhibited the growth of several test species, which included the cyanobacteria Anabaena sp., Phenazine Phormidium bohneri, and Oscillatoria agardhii [104]. pigments released by the bacterium mediated inhibition of algal growth. Oxychlororaphine (20) and 1-hydroxyphenazine (21) showed strong antialgal activity while pyocyanine completely inhibited algal growth.

O II C-NH2

^-^

N^ ^ ^ ^ 20

OH

^ ^

N^ ^ ^ \ ^ 21

366 Antibiotics have also been studied as useful compounds to selectively inhibit the growth of cyanobacteria. Matsuhashi et al. [105] found several antibiotics (e.g., bacitracin, D-cycloserine, novobiocin, ristocetin, penicillin G, and vancomycin) that inhibited the growth of Anabaena variabilis at concentrations ranging from 10-1000 |Lig/mL while several green algal strains tested were unaffected at the same concentrations. The semisynthetic P-lactam antibiotic amoxicillin showed strong inhibition of Microcystis aeruginosa as indicated by a decrease in chlorophyll concentration measured fluorometrically [106]. The use of antibiotics to selectively control the growth of cyanobacteria is not a favorable approach due to the broad-spectrum toxicity of certain antibiotics toward bacteria and the subsequent potential for the selection of antibiotic-resistant strains of catfish disease-producing bacteria. Yeast extract was reported to contain components that are toxic to cyanobacteria [107]. L-lysine and malonic acid completely killed Microcystis viridis at a concentration of 1 and 40 ppm, respectively. Lysine malonate was found to be more toxic than DL-lysine. A few other types of microorganisms have been reported to inhibit the growth of cyanobacteria; however, no work has been performed on the structural characterization of the responsible constituents. In the screening of 65 cyanobacterial strains for production of phages and/or antibiotics, seven N^-fixing strains of cyanobacteria {Anabaena doliolum, Fischerella muscicola, and five strains of Nostoc) were found to produce metabolites which caused clearing of growth of indicator cyanobacterial strains on sohd media [108]. The metabolite produced by Nostoc sp. 78-11 A-E appears to be a proteinaceous, low molecular weight compound. None of the other metabolites were structurally characterized. In another study, cultures (whole cell) of 198 cyanobacterial strains isolated from soil and freshwater samples were screened for inhibitory activity against green algal species [109]. Twenty isolates found to be active against green algae were also tested for activity against cyanobacteria. Anabaena doliolum was inhibited by Fischerella JAVA 94/20. Fischerella NEP 95/1 inhibited Anabaena sp.; the toxicity was lost with the inclusion of proteinase K in the media, suggesting that the active agent was a peptide. In the same study, three Fischerella strains (JAVA 94/10, NEP 95/1, LOM 95/17) were inhibitory to Synechocystis PCC 6803 and Nostoc sp. from the plant Macrozamia communis (Cycadaceae). Also, Calothrix WA 96/8 inhibited only A. doliolum, and three strains of Calothrix, 9 strains of Fischerella,

367 and Nostoc NSW 95/10 showed toxicity toward Anabaena circinalis. Microcystis aeruginosa, and Nodularia spumigena. These cyanotoxins were neither isolated nor characterized. Several other viral, fungal, bacterial, and protozoal agents were reported to inhibit cyanobacteria but no phytochemical studies on the inhibitory components from these organisms have been done [110]. The diversity of the classes of compounds reported to be toxic towards cyanobacteria reflects the potential for many sites and mechanisms of actions for growth inhibition (to be discussed in the next section). The diversity of the sources that has been reported so far substantiates the fact that natural products are a rich source of cyanobactericidal compounds. Modes of Action Knowing the mode of action of pesticides is valuable for several reasons. For example, this information can be useful in the design of better pesticides, in anticipating toxicological problems, and in predicting the evolution of resistance. Algae and cyanobacteria are photosynthetic and share with higher plants many of the biochemical target sites of synthetic herbicides and other phytotoxins. There is considerable literature on the mode of action of synthetic herbicides [e.g., 111-114] and, to a lesser degree, on that of phytotoxins produced by plant pathogens [e.g., 115,116]. Many herbicides are as toxic to algae as to higher plants [e.g., 61], and, because of their ease of manipulation, are used by some scientists in studies for indication of mode of action of herbicides in higher plants [e.g., 117]. Relatively few studies have been done to identify compounds that might be used as algicides, and even fewer studies have been done to determine the mechanism of action of those compounds with useful activity as algicides. However, just as algicide mode-of-action studies have been extrapolated to higher plants, the reverse can also be done. Two points should be kept in mind when studying mode or mechanisms of action of biocides. First, a compound that kills will eventually affect every physiological system in the target organism. Most of the literature on the mode of action of herbicides deals with these secondary effects. Proof of a primary molecular target site is often very difficult, especially when the target is one that has not been previously described. Usually, the first papers on a biocide describe secondary effects, and are then followed by more definitive studies. For example.

368 the first paper on the biological effects of cyanobacterin (15) described its effects on growth [94], to be followed by a study showing its effects on PSII [118]. A strategy for determination of molecular target sites of herbicides or algicides has been recently published [119]. A second important aspect of modes of action that one should be aware of is that a compound can have more than one molecular site of action. For example, fischerellin A (18) is both a PSII inhibitor and an antifungal agent [98]. Clearly, the molecular target as a fungicide is not PSII. If both sites are of equal value in killing the target organism, determination of the molecular sites of action can be challenging. There are only about fifteen well-defined molecular target sites for commercial herbicides and about twenty for natural phytotoxins (Table I). Most of these have been documented to be toxic to green algae or cyanobacteria. Furthermore, we are not aware of any studies showing that any of these compounds are not toxic to green algae or cyanobacteria. There has been no systematic study of the toxicity of these compounds to algae or cyanobacteria. The closest attempt at this has been the work of our laboratory [61,93]. There is relatively little overlap between the molecular target sites of commercial herbicides and those of natural compounds (Table I) [116,120,121]. We will describe what we know of the mechanism of action of a few selected compounds that are discussed in other parts of this chapter. The largest class of commercial herbicides are those that inhibit photosynthesis by inhibition of PSII by stopping electron transport [112]. This phenomenon is easily monitored by measuring increases in variable fluorescence or decreases in oxygen evolution. A vital component of PSII is a quinone-binding protein called D-1 in eucaryotic plants and L in cyanobacteria. A quinone (plastoquinone) is necessary as a redox compound to transport electrons. PSII inhibitors compete for the quinonebinding site on D-1. PSII inhibitors can be divided into two groups: those that depend more on binding the Ser264 amino acid of D-1 and those that bind the His2i5 moiety. The Ser264 inhibitors include triazines, substituted ureas, and carbamates, whereas the His2i5 family of inhibitors include benzoquinones, naphthoquinones, and acridones. There is apparently enough homology between D-1 and L for similar activity of all PSII inhibitors studied against electron flow in both eucaryotic plants and cyanobacteria. However, this cannot be said for all photosynthetic bacteria [101].

369

Table I. Modes of Action of Commercial Herbicides and Natural Phytotoxins (Adapted in part from references 116 and 120). Physiological site

Molecular site

Herbicide or natural product^

Amino acid synthesis aromatic amino acids branched chain amino acids

EPSP synthase acetolactate synthase

glutamine synthesis

glutamine synthetase

glutamate synthesis general amino acid synthesis ornithine synthesis methionine synthesis

aspartate amino transferase many transaminases ornithine carbamoyl transferase P-cystathionase

glyphosate imidazolinones, sulfonylureas, others many, including phoshinothricin, oxetin, and tabtoxinine gostatin gabaculin phaseolotoxin

Photosynthesis electron transport

D-1, quinone-binding

photophosphorylation

CF| ATPase

many, including triazines, substitued ureas, etc.; sorgoleone, cyanobacterin, fischerellin A ten toxin

electron transport diverters plastoquinone synthesis

photosystem 1 4-hydroxyphenylpyruvate dioxygenase

bipyridiliums {e.g., paraquat) isoxazoles, pyrazoles, and triketones; leptospermone

protoporphyrinogen oxidase ALA synthase causes accumulation phytoene desaturase

p-nitrodiphenyl ethers, oxadiazoles, etc. gabaculin ^-aminolevulinic acid many, including pyridazinones, etc. isoxazolidinones

Pigment synthesis porphyrins

carotenoids

a prenyl transferase

rhizobitoxin

Cell division mitotic disruptors

P-tubulin

dinitroanilines, phosphoric amides; vinca alkaloids, colchicine, etc.

Vitamin synthesis folate synthesis

dihydropteroate synthase

asulam

370

Table 1 - cont'd Lipid synthesis acetyl-Co A carboxylase acetyl-Co A transacylase 3-oxoacyl-ACP synthase ceramide synthase

Nucleic acid synthesis plastid nucleic acid synthesis

aryloxyphenoxypropanoates, cyclohexanediones thiolactomycin cerulenin AAL-toxin and analogues, australifungin

RNA polymerase adenylsuccinate synthase AMP deaminase

tagetitoxin hydantocidin carbocyclic coformycin

unknown at present

rose bengal and many others; cercosporin, hypericin, and many others

H"^-ATPase

syringomycin

Direct photodynamic action

Plasma membrane function

Cell wall synthesis cellulose synthesis? unknown site ^compounds in italics are natural products

dichlobenil isoxaben

Caution should be exercised in assuming that plastoquinone analogues will exert most of their phj^otoxicity through PSII inhibition because some are also excellent inhibitors of mitochondrial respiration [122]. In some cases, this may be because they bind to the electron transport protein that normally binds to ubiquinone, a redox compound involved in mitochondrial electron transport in a similar way that plastoquinone is involved in photosynthetic electron transport. Thus, some inhibit respiration by a very similar mechanism to that of PSII inhibitors. Some natural quinones, such as sorgoleone and fischerellin A (18) are apparently sufficiently close analogues of plastoquinone that they are excellent PSII inhibitors [123,124]. Juglone (22) is phytotoxic to algae and has been reported to inhibit both photosynthesis and respiration in higher plants [125,126], although exactly how it inhibits photosynthesis is not clear. An analog of juglone, anthraquinone (13), is highly phytotoxic to certain

371 species of cyanobacteria [93] and is a strong inhibitor of photosynthesis [127].

OH

O 22

One potential problem with the use of PSII inhibitors as algicides is that resistance may evolve relatively quickly. In higher plants, resistance has evolved to certain classes of PSII inhibitors, but not to others [128]. In all but a few cases, amino acid substitutions in D-1, resulting in reduced affinity for the inhibitor, account for the evolved resistance [129]. These alterations in D-1 reduce photosynthetic efficiency under some environmental conditions, but under strong selection pressure of the herbicide, herbicide resistance is favored. Another potential drawback of PSII inhibitors is that there is little likelihood of such an algicide being effective at very low doses because of the very large amount of target site in each cell. Compared to some of the "low dose" herbicides, all PSII herbicides are applied at relatively high doses. Artemisinin (14) and several of its analogues are quite toxic to both cyanobacteria and higher plants [93,130]. However, its mode of action is still a mystery. It affects a large number of physiological parameters [130], but no primary site of action has been estabhshed. Like another sesquiterpene lactone, dehydrozaluzannin C, it may act by binding SH groups of proteins [131]. Effects of artemisinin can be reversed with cysteine [132]. If this is the mechanism of action, resistance is unlikely to evolve by alterations in binding sites. Photodynamic compounds, that is those that generate singlet oxygen in the presence of light and molecular oxygen, are toxic to all living things. Singlet oxygen causes rapid membrane disruption by peroxidation of membrane lipids [112]. Hypericin (23), a photodynamic product of several species of Hypericum, is an effective biocide on higher plants [133] and cyanobacteria [134]. However, photodynamic compounds have

372

the inherent problem of being toxic to everything. There are both natural and synthetic compounds that cause the accumulation of toxic levels of Some porphyrins are particularly porphyrins in plants [135]. photodynamic, and normally do not accumulate in healthy cells.

The natural glutamate synthetase (GS) inhibitor, tabtoxinine, is toxic to the green alga Chlorella vulgaris, and this green alga was used in elucidating its mode of action [136]. There are a number of other highly active natural GS inhibitors [137] that should be examined for their effects on both green algae and cyanobacteria. There are other natural compounds that are known to be toxic to algae for which we do not have knowledge of a specific molecular target. These include many of the phenolic acids, such as ferulate, and hydrolyzable polyphenols such as tellimagrandin II (5). One mode-of-action theory for these compounds as algicides is that they non-competitively inhibit algal exoenzymes such as alkaline phosphatase [83,138]. In fact, these compounds do bind enzymes non-competitively, often inhibiting their activity. Furthermore, these compounds can be taken up by cells and inhibit intracellular enzymes. For example, tannic acid, gallate, and two analogues of gallate were found to lower levels of glutamate synthase and nitrate reductase activity extracted from Nostoc sp. [77]. However, whether this was a direct or indirect effect was not clear because no in vitro effect of the compounds was reported. If non-specific inhibition of enzymes is the mode of action of many simple phenohcs and hydrolysable

373

phenolics, they have no specific molecular site of action, although each compound might be expected to have a slightly different spectrum of enzymes that it preferentially inhibits. An advantage to such a mode of action is that evolution of resistance to such a compound at molecular target sites is highly unlikely. An alternative or complimentary theory for the mode of action of simple phenolic compounds is that they are converted to much more toxic quinones. Pillinger et al. [69] found that various phenolic decomposition products of barley straw were most toxic under conditions favorable for oxidation of the compounds to quinones, and that quinones were up to one thousand-fold more toxic to algae than the parent compounds. The most likely route to conversion to a quinone is enzymatic. Peroxidases and polyphenol oxidases can perform such a reaction, depending on the substrate. However, polyphenol oxidase cannot be detected in most green algae [139] and has not been reported in cyanobacteria. There are studies that indicate a particular mode of action as a natural algicide, without elucidating that it is a primary site. For example, Kida [140,141] reported that the Streptomyces purpeofuscus product 7-deoxyD-glycero-D-glucoheptose inhibited chlorophyll synthesis in the green alga Scenedesmus obliquus. Yet, only chlorophyll accumulation was measured. Phytotoxins with many other sites of action could cause a similar effect. Ideally, a biocide should selectively kill the target organism(s) at doses that have little or no effect on non-target organisms. In aquaculture, a compound that would selectively remove the cyanobacteria while leaving the more desirable green algae is much more desirable than non-selective algicides. A selectivity factor of greater than ten-fold is most advantageous because of uncertainties in efficacy due to environmental factors and human error. Selectivity can be based upon differences in target sites, in movement to the target sites, and/or in metabolic detoxification of the biocide or toxicants generated by the biocides (e.g., singlet oxygen). To our knowledge, we are the only laboratory that has searched for selective cyanobactericides [61,93], although others have noted differences in activity of specific compounds to different algae and algal groups [e.g., 118,142]. In our studies, we found several synthetic herbicides to have 100- to 1000-fold greater activity in causing complete inhibition of growth of the filamentous cyanobacterium Oscillatoria perornata (formerly referred to as Oscillatoria cf. chalybea) than the unicellular green alga Selenastrum capricornutum: diquat, paraquat.

374

diclofop, and bromoxynil [61]. Diquat and paraquat act by diverting electrons from photosystem I (PSI) to generate toxic levels of superoxide radical, resulting in similar damage as that caused by photodyamic compounds. Diclofop inhibits lipid synthesis via specific inhibition of acetyl CoA carboxylase. Bromoxynil is a PSII inhibitor. Only diquat and paraquat were effective on O. perornata at economical rates; however, these herbicides are perhaps the most acutely toxic of all herbicides to animals. We are unaware of any commercial herbicides that have the desired levels of selectivity and safety for use in aquaculture. However, our limited experience with natural products indicates that there may well be a natural product that has the optimal properties. Among a very limited number of natural products, we found anthraquinone, 2-methylanthraquinone, juglone, and sorgoleone to be two to three orders of magnitude more toxic to 0. perornata than to S. capricornutum [93]. These compounds are either known [128,143] or suspected PSII inhibitors. Only the first two of these compounds were active enough to be of interest as cyanobactericides. Whether there is a large difference in binding affinities of these compounds for D-1 versus L or some other mechanism of selectivity is unknown. A strategy can be derived for selective cyanobactericide discovery, based on physiological or biochemical differences between cyanobacteria and eucaryotic photosynthetic organisms. For example, a compound that would specifically inhibit synthesis of phycobilins (photosynthetic pigments peculiar to cyanobacteria) or phycobiliproteins (a phycobilinprotein complex of cyanobacteria) could be expected to be selective. This sort of biorational approach has not been attempted for discovery of algicides and has been unsuccessfiil in the discovery of herbicides. Those antibiotic cyanobactericides, such as bacitracin and penicillin G mentioned previously, probably act on cyanobacteria via a mode of action similar or identical to that by which they kill pathogenic bacteria. The selectivity of these cyanobactericides found by Matsuhashi et al. [105] supports the view that they are procaryote-specific. A variation of this approach is to target more general processes that might differ in the target organismfi-omother organisms. Cyanobacteria are more primitive photosynthetic organisms than eucaryotic plants, and thus, evolved when atmospheric oxygen was at much lower concentrations than today. Thus, they might be more sensitive to toxic oxygen species than more recent evolutionary arrivals. One could argue that the evolution of the chloroplast was in response to the toxic effects of oxygen. Our

375 finding that O. perornata was 1000-fold more susceptible to paraquat and diquat than the green alga S. capricornutum and the result of Brody et al. [134] that cyanobacteria are highly sensitive to hypericin supports this view. Thus, concentrating on natural compounds that might directly or indirectly cause toxic oxygen species to increase might be a viable strategy for the discovery of algicides and, perhaps even selective cyanobacterial algicides. Knowledge of the site of action can be valuable in designing a better algicide through structure-activity relationship (SAR) studies. This approach works quite well with synthetic compounds. However, if the biological activity of interest is the function of a natural compound in nature, its activity may have already been optimized by evolution. Examples are summarized by Duke et al. [144]. In the only case of a SAR study of a natural product algicide of which we are aware, Gleason et al. [145] could not produce a more active cyanobacterin (15) molecule than the parent molecule. There have been exceptions to the natural compound being the optimal pesticide, such as the pyrethroid insecticides. Modifications are often most needed to increase the environmental halflife or to improve uptake by the target organism. Our knowledge of the mechanisms of action of algicidal compounds is fragmentary. We still have much to do. Bioassay Methods for Discovering Selective Algicidal Compounds A variety of approaches have been used in the development of rapid bioassay systems to determine the toxicity of compounds towards algae and cyanobacteria. The variables that have been measured to determine algal response to a particular compound include rate of chlorophyll fluorescence and ^"^C-uptake to measure photosynthesis, measurement of growth inhibition zones on solid agar, growth rate, biomass, cell number at a given time, alkaline phosphatase activity, and motility. In most cases, the toxicity of the compound can be expressed as the reduction of the response relative to the control response as percentage of inhibition [146]. Several algal toxicity methods are discussed, with an emphasis on a rapid bioassay developed specifically for screening a large number of compounds to determine their usefulness as selective cyanobactericides [147].

376 An initial consideration of algal and cyanobacterial toxicity testing involves selecting a microcosm-type screen versus a screen using individual species. Microcosm-type screening involves the use of natural water containing many different species of phytoplankton in addition to a target species of algae or cyanobacteria. The argument against using single-species screens is that assumptions are made concerning the organism's responses at levels of biological organization above a single species [148]. Although microcosm-type screening may provide a more reliable indication of the target organism's response toward a toxicant while in its natural environment [148], it may limit the ability to rapidly screen compounds due to the difficulty in measuring responses of a separate individual target species amongst a mixture of phytoplankton. Therefore, microcosm-type screening should be reserved for later use, after lead algicidal compounds have been discovered using rapid, singlespecies bioassays and as an intermediate step in the progression from smaller bioassays to large full-scale lake or pond testing. Chlorophyll fluorescence, ^"^C-uptake, and growth rate measurements provide an indication of viable cells, whereas biomass and cell number measurements do not distinguish between the living and dead cells. In photosynthetic algae and cyanobacteria, the reaction centers referred to as photosystem I (PSI) and photosystem II (PSII) are special chlorophyll molecules used to dissipate excitation energy derived from the deexcitation of an excited pigment molecule that has absorbed the energy of a photon. Chlorophyll fluorescence occurs when the excited pigment returns to the ground state by the route of emitting a fluorescence photon, of a wavelength longer than that of the photon initially absorbed [149]. Most of the chlorophyll fluorescence from algae is associated with PSII [150], while in cyanobacteria, most of the chlorophyll fluorescence emanates from both PSII and phycobiliproteins [149], the major lightPhycobiliprotein harvesting pigments in cyanobacteria [151]. fluorescence overlaps with the spectrum of chlorophyll emission [149]. Chlorophyll fluorescence measurements and analyses for cyanobacteria must be modified from those used for algae due to the difference in the pattern of chlorophyll fluorescence signals between the two groups of microorganisms [149]. For certain compounds, i.e., those inhibiting electron flow in photosynthesis, fluorescence measurement can help evaluate the toxicity of a compound towards algae and cyanobacteria. Fluorescence measurement is more sensitive than measuring culture absorbency (optical

377

density). However, in vivo chlorophyll fluorescence measurement bioassays do not provide as broad a screening method of toxic compounds as with the use of growth inhibition assays. Only those compounds adversely affecting photosynthesis are best suited for such an assay. In addition, in vivo chlorophyll a fluorescence measurements are very complex and have been discouraged as a method for conducting screening bioassays [150]. Radiocarbon-uptake, such as ^"^C-assimilation, has been used for species of green algae, and this test essentially measures the rate of photosynthesis. In addition, ^"^C-uptake is mainly used with phytoplankton in natural water and rarely used with cultured algae as the test organisms [152]. The duration of the ^"^C-assimilation test is a few hours, and compounds that are phytotoxic towards algae adversely affect the rates of ^^C-uptake. Although radiocarbon-uptake testing is reproducible, this toxicity testing method is less sensitive than growth inhibition toxicity testing [153], and, therefore, it is not applicable for a thorough, rapid screening of compounds to determine their toxicity towards algae and cyanobacteria. For growth inhibition bioassays using hquid media, large-scale laboratory and batch-culture toxicity [Algal Growth Inhibition Toxicity Physical/chemical (AGIT)] tests have been widely used [154]. experimental parameters cause variability between batch culture tests, and these parameters need to be understood and controlled. The most important experimental parameters include light, temperature, carbon dioxide availability, pH, and the test organisms used [155]. Continuous light is recommended over a lightidark cycle because it is more practical and most algae grow well in continuous light [155], Temperature needs to be maintained near the optimum for the particular test species and near uniform among batch-culture flasks [155]. Algal laboratory cultures can become carbon limited very quickly and carbon dioxide availability can be maintained by adding bicarbonate to the medium [155]. Also, buffered media will reduce any effect of pH on the toxicity of the test compound. Different groups of algae (cyanobacteria, diatoms, green algae) and different species within the same group of algae should be used in the bioassay to adequately determine the toxicity of the test compound and its potential usefulness as a selective algicide. The most commonly used freshwater green alga species used in toxicity tests is Selenastrum capricornutum [155]. Of course, the other species included in the bioassay must include the target species, i.e., noxious species to be

378 selectively eliminated from the natural aquatic ecosystem. Algal and cyanobacterial bioassays should be run for relatively short periods (i.e., no more than four days) before the growth of the control cultures becomes adversely affected by growth-limiting factors such as nutrient depletion or the build-up of toxic cataboHc metabolites excreted from the cells [156]. For growth bioassays using sohd media, modification of the agar diffusion assay used by Flores and Wolk [108] has been shown to provide reliable results [100]. Gross et al. [100] spotted methanol and ethanol extracts on 1% agar plates, dried the extracts in sterile air, and then overlaid the plate with a suspension of indicator cells in 1% agar. After several days of incubation, areas (zones) of no growth were measured and quantification of the inhibitory effects of the test extracts was estimated by comparison with/to the serial dilutions and controls. The most widely measured response variables in growth inhibition toxicity tests are specific growth rate and biomass. Specific growth rate is determined by calculating the linear regression of logarithmic biomass data from an exponential growth curve [157]. Biomass is usually determined by measuring either cell biomass (dry weight or cell volume counting using a electronic particle counter) or optical density (absorbance) using a spectrophotometer [155]. In certain types of toxicity testing [e.g., determination of EC50 (50% effective concentration) values], monitoring growth rate is better than biomass measurements [156,157]. The measurement of alkaline phosphatase activity (APA) of target phytoplankton is a recently developed bioassay that has been used to determine the algicidal effects of polyphenols from Eurasian watermilfoil {Myriophyllum spicatum) [80]. Phytoplankton produce extracellular enzymes, such as alkaUne phosphatase, to provide additional sources of nutrients. Fluorescence spectrometry is used to measure APA, with methylumbeliferyl-phosphate used as substrate and mixed with the algal or cyanobacterial suspension and the suspected inhibitor. Rapid bioassays using the response of cyanobacterial motility as an indication of the toxicity of a compound have been used [158]. However, this type of bioassay is impractical when using non-motile species of cyanobacteria and algae such as Anabaena spp. and Selenastrum capricornutum, respectively. An alternative to batch algal bioassays is the use of cage-culture turbidostats (CCT) [154]. The CCT is computer controlled and will monitor algal growth responses to the addition of test compounds. Although easy and quick to set up, the CCT limits the number of

379 concentrations of a particular compound that can be tested during an experimental run, thereby reducing data essential for statistical analysis. Microbiotests (microtiter plate bioassays) offer a much more rapid, simpler, and low cost alternative to batch-culture toxicity testing of compounds [147,159-162]. Microtiter plate bioassays require the use of continuous culture (chemostat) systems to provide a source of cells (inoculum) in continuous, steady-state growth. The continuous culture system maintains a particular growth rate of the culture by the addition of fresh medium at a set rate [163]. The volume in the continuous culture vessel is kept constant by an overflow apparatus so that the rate of fresh medium entering the vessel is equal to the removal rate of medium and cells. Cells growing at a constant rate should adsorb test compounds more consistently, thereby reducing the potential influence of certain test compounds (e.g., lipophihc organics) on distorting test results [155]. In addition, minor changes in the growth of the test organism toward a particular compound are easier to observe and analyze. Schrader et al. [147] developed a rapid, inexpensive bioassay to screen a large number of natural compounds for selectivity as cyanobactericides. In this bioassay, continuous culture systems provided a source of cells (inoculum), and a representative green alga, Selenastrum capricornutum, and the MIB-producing cyanobacterium Oscillatoria perornata were used. O. perornata is a filamentous plankton that has been attributed as being the major cause of musty off-flavor in farm-raised channel catfish in west Mississippi [164]. This organism was previously identified as Oscillatoria cf chalybea [165]. It does not conform very well to the classic description of that species [166] and, therefore, a more appropriate designation of this organism is Oscillatoria perornata f. attenuata [167]. The bioassay utilizes 96-well cell culture plates (microtiter plates). Test compounds at various concentrations and pre-dissolved in either water, ethanol, or methanol are placed in wells of the culture plate, followed by the addition of either unialgal or cyanobacterial culture to the wells. Absorbance (650 nm) is measured at 24-h intervals for four to five days using a microplate reader, and growth of the treated cultures (wells) relative to untreated cultures is used to determine the relative toxicity of the test compounds to the two microorganisms. The supplementation of culture inoculum placed in the microplate wells with sodium bicarbonate as a means to provide an additional source of carbon dioxide was found to be unnecessary. Pediastrum simplex and Anabaena sp. LP-691 are other

380 species of green algae and cyanobacteria, respectively, that have been used as test organisms with this bioassay [93]. One of the problems encountered in the bioassay developed by Schrader et al. [147] was the limitation in the type of compound amenable for the screening procedure. Only compounds that are soluble in water, ethanol, or methanol could be screened since these solvents will not react with the polystyrene in the microplates. However, the substitution of a quartz glass microplate for a polystyrene microplate allows solvents such as pentane, hexane, and acetone to be used in the bioassay. Therefore, natural compounds and crude extracts that may only be soluble in one or several of these types of solvents can now be screened. The potential change in toxicity of a compound towards a particular organism should be considered in the progression from a 96-well microplate (300 \iL capacity per well) to a large aquatic ecosystem, such as a lake or pond. Scale-up bioassays from 96-well to six-well microplates discovered a reduction in the toxicity of the natural compound ferulate towards O. perornata [168]. Other scale-up studies from microplate to flask bioassays have also found a reduction in phytotoxicity of the test compound [161]. In addition to small-scale laboratory studies, dose-response experiments as a prelude to full-scale lake or pond testing should be performed in an intermediate-scale system using a much larger volume (liters) of culture or natural water containing the target microorganism(s). Any pond or lake testing should include close monitoring of the persistence of the test compounds in the aquatic environment to help determine their effectiveness as a selective algicide and any potential threat that they might pose to the environment. Efficacy Testing of Natural Algicides in Freshwater Ecosystems Lead cyanobactericidal compounds from laboratory screening must undergo efficacy testing in appropriate freshwater habitats to ultimately determine their usefulness as selective algicides. In catfish aquaculture, efficacy testing using whole ponds has its drawbacks. For example, a large amount of lead compound is required when testing in several ponds. The cost of synthesis or direct purchase from an appropriate supplier can be high. Also, there is a need to test in several ponds at the same time to obtain a large number of replicate observations, i.e., to determine if any statistically significant treatment effect occurs. The use of several ponds

381 to obtain such information introduces high pond-to-pond variabihty of phytoplankton community structure with inconsistent estabhshment of target cyanobacterial populations. In addition, high pond-to-pond variability of pond water chemistry could affect the stoichiometry of the test compound after application. An alternative to fiill-scale lake or pond (real or artificial) studies is the use of "limnocorrals." Limnocorrals are enclosures that isolate a volume of water for study and are also referred to as experimental enclosures or in situ mesocosms. Their use provides an intermediate step between laboratory-scale studies, which are often too artificial, and full-scale lake or pond studies. In addition, a much smaller amount of test compound is required, and the initial environmental conditions are the same within all of the enclosures. Limnocorrals can be made of translucent fiberglass, polyvinyl chloride, or some other flexible, inert material. These enclosures can be open-bottomed so that they will sink into the bottom sediment to help form a watertight seal to separate the intemal environment fi*om the outside. In larger-scale (lake) studies using limnocorrals, divers have been used to help ensure that a proper seal in the sediment is obtained [169]. Vertical mixing of the water column inside small-scale limnocorrals is necessary in order to help achieve environmental conditions more similar to those in the lake or pond outside of the enclosure. One method used to achieve water mixing/aeration inside limnocorrals is by pumping air through submerged airstones placed within the limnocorrals. Some of the earliest studies using limnocorrals have been to determine the effects of mercury on algae [170], to study phytoplankton growth [171,172], and to study the fates of insecticides in aquatic ecosystems [173]. Other studies using limnocorrals have assessed the short-term impacts of various chemicals and pesticides on aquatic ecosystems [174177]. However, limnocorrals are not suitable for long-term impact studies due to deterioration of environmental conditions within the enclosures over longer periods of time (e.g., weeks). In addition, the intensive labor demands required in setting up the limnocorrals often limit the number of units that can be used for a study. Dose-response studies can be performed using as few as eight enclosures [178]. Although limnocorrals do not provide an exact duplication of the lake or pond environment, their use does permit an adequate determination of the impact of a test compound on the phytoplankton community.

382 CONCLUSION Although previous research concerning the use of natural compounds as selective cyanobactericides is not very extensive, past and present research indicate that such an approach has merit and should continue to be investigated. The vast majority of natural compounds in nature remain unscreened as to their potential for use as environmentally-safe, selective compounds to help control noxious species of cyanobacteria in freshwater ecosystems. The discovery of such compounds that are cost-effective would be very beneficial to industries such as catfish aquaculture and municipal drinking water systems by reducing their dependence on synthetic compounds (herbicides) to control cyanobacterial blooms. The use of synthetic herbicides carries with it environmental safety concerns and a negative perception by the public. ABBREVIATIONS APA OCT EC50

= = =

GS MIB PSI PSII SAR

= = = = =

Alkaline Phosphatase Activity Cage-culture Turbidostat 50% Effective Concentration (toxicant concentration affecting a specific response in 50% of exposed test organisms) Glutamate Synthetase 2-Methyhsobomeol Photosystem I Photosystem II Structure-activity Relationship

ACKNOWLEDGEMENTS We have been fortunate to work with Dr. Craig Tucker who has contributed greatly to current knowledge about water quality and phytoplankton communities in catfish aquaculture ponds. We thank two reviewers, Drs. Casey Grimm and Mario Tellez, for their many helpful suggestions during preparation of this chapter.

383

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Devine, M.D.; Duke, S.O.; Fedtke, C; Physiology of Herbicide Action, Prentice Hall: Englewood Cliffs, New Jersey, 1993. Cobb, A.; Herbicides and Plant Physiology, Chapman and Hall: London, 1992. Roe, R.M.; Burton, J.D.; Kuhr, R.J., Eds.; Herbicide Activity: Toxicology, Biochemistry and Molecular Biology, lOS Press: Amsterdam, 1997. Gilchrist, D.G. In Toxins and Plant Pathogenesis; Daly, J.M.; Deverall, B.J., Eds.; Academic Press: New York, 1983; pp. 81-136. Duke, S.O.; Abbas, H.K.; Amagasa, T.; Tanaka, T. In Crop Protection Agents from Nature. Natural Products and Analogues; Copping, L.G., Ed.; Royal Chemical Society: Cambridge, 1996; pp. 82-113. Boger, P. In Target Assays for Modern Herbicides and Related Phytotoxic Compounds; Boger, P.; Sandmann, G., Eds.; Lewis Publishers: Boca Raton, Florida, 1993; pp. 83-91. Gleason, F.K.; Paulson, J.L.; Arch. Microbiol, 1984,138, 273-277. Dayan, F.E.; Romagni, J.G.; Duke, S.O.; J. Chem. EcoL, 2000, (In press). Duke, S.O.; Dayan, F.E.; Hernandez, A.; Duke, M.V.; Abbas, H.K.; Brighton Crop Protection Conference, Weeds, 1997, 1, 83-92. Dayan, F.E.; Romagni, J.G.; Tellez, M.R.; Rimando, A.M.; Duke, S.O.; Pestic. Outlook, 1999, JO, \S5ASS. Moreland, DE. In Weed Physiology, Vol. II Herbicide Physiology; Duke, S.O., Ed.; CRC Press: Boca Raton, Florida, 1985; pp. 37-61. Czamota, M.A.; Weston, L.A.; Dayan, F.E.; WeedSci. Soc. Amer. Abstr., 1998, 38, 52. Duke, S.O.; Dayan, F.E.; RIKENRev., 1999, 21, 9-10. Hejl, A.M.; Einhellig, F.A.; Rasmussen, J.A.; 7. Chem. EcoL, 1993, J9, 559568. Koeppe, D.E.; Physiol. Plant., 1972, 27, 89-94. Schrader, K.K.; Dayan, F.E.; Allen, S.N; de Regt, M.Q.; Tucker, C.S.; Paul, R.N.; Internal J. Plant Sci., 2000, (In press). De Prado, R.; Jorrin, J.; Garcia-Torres, L., Eds.; Weed and Crop Resistance to Herbicides, Kluwer: Amsterdam, 1997. Gronwald, J. In Herbicide Resistance in Plants; Powles, S.B.; Holtum, J.A.M., Eds.; CRC Press: Boca Raton, Florida, 1994; pp. 27-60. Dayan, F.E.; Hernandez, A.; Allen, S.N.; Moraes, R.M.; Vroman, J.A.; Avery, M.A.; Duke, S.O.; Phytochemistry, 1999, 50, 607-614. Galindo, J.C.G.; Hernandez, A.; Dayan, F.E.; Tellez, M.R.; Macias, F.A.; Paul, R.N.; Duke, S.O.; Phytochemistry, 1999, 52, 805-813. Duke, S.O.; Paul, R.N.; Lee, S.M.; Amer. Chem. Symp. Soc. Ser., 1988, 380, 318-334. Knox, J.P.; Dodge, A.D.; Plant Cell Environ., 1985, 8, 19-25. Brody, S.S.; Papageorgiou, G.; Alygizaki-Zorba, K.Z;. Naturforsch., 1997, 52c, 165-168. Duke, S.O.; Rebeiz, C.A., Eds.; Porphyric Pesticides, ACS Symp. Ser. 559, American Chemical Society: Washington, DC, 1994. Braun, A.; Phytopathology, 1955, 45, 659-664.

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Lydon, J.; Duke, S.O. In Plant Amino Acids; Singh, B.K., Ed.; Marcel Dekker: New York, 1999; pp. 445-464. [138] Wetzel, R.G.; Verk Int. Ver. Theor. Angew. LimnoL, 1993, 25, 122-128. [139] Sherman, T.D.; Vaughn, K.C.; Duke, S.O.; Phytochemistry, 1991, 30, 24992506. [ 140] Kida, T. In Novel Microbial Products for Medicine and Agriculture; Demain, A.; Somkuti, G.; Hunter-Cevera, J.; Rossmoore, H., Eds.; Elsevier Science B.V.: Amsterdam, 1989; pp. 195-202. [141] Kida, T; Shibai, H.; Agric. Biol Chem., 1986, 50, 483-484. [142] Gleason, F.K.; Baxa, C.A.; FEMSMicrobiol. Lett., 1986, 33, 85-88. [143] Rimando, A.M.; Dayan, F.E.; Czamota, M.A.; Weston, L.A.; Duke, S.O.; / Nat. Prod, 199S, 61,921-930. [144] Duke, S.O.; Dayan, F.E.; Romagni, J.G.; Rimando, A.M.; Weed Res., 2000, 40, 99-111. [145] Gleason, F.K.; Thoma, W.J.; Carlson, J.L. In Progress in Photosynthesis Research; Biggens, J., Ed.; Martinus Nijhoff Publishers: Dordrecht, 1987; pp. 763-766. [146] Nyholm, N.; Sorensen, P.S.; Kusk, K.O.; Christensen, E.R.; Environ. Toxicol. Chem., 1992,11, 157-167. [147] Schrader, K.K.; de Regt, M.Q.; Tucker, C.S.; Duke, S.O.; Weed Technol., 1997, 11,161-11 A. [148] Cairns, Jr., J.; Hydrobiologia, 1983, 100, 47-57. [149] Campbell, D.; Hurry, V.; Clarke, A.K., Gustafsson, P.; Oquist, G.; Microbiol. Mol Biol. Rev., 1998, 62, 667-683. [150] van der Heever, J.A.; Grobbelaar, J.U.; Arch. Environ. Contam. Toxicol., 1998, 55,281-286. [151] Fay, P.; Microbiol. Rev., 1992, 56, 340-373. [152] van der Heever, J.A.; Grobbelaar, J.U.; J. Appl. Phycol, 1996, 8, 65-71. [153] Nyholm, N.; Damgaard, B.M.; Chemosphere, 1990, 21, 671-679. [154] Clarkson, N.; Leftley, J.W.; Meldrum, D.T., Watson, J.W.; Water Res., 1998, 32, 1162-1168. [155] Nyholm, N.; Kallqvist, T.; Environ. Toxicol. Chem., 1989, 8, 689-703. [156] Nyholm, N.; Water Res., 1985,19, 273-279. [157] Nyholm, N.; Arch. Environ. Contam. Toxicol, 1990, 19, 518-522. [158] Zullei, N.; Benecke, G.; Bull Environ. Contam. Toxicol, 1978, 20, 786-792. [159] Blaise, C; Legault, R,; Bermingham, N.; van Coillie, R.; Vasseur, P.; Tox. Assess., 19^6,1,26\'2%\. [160] Blaise, C; Environ. Toxicol Water Qual, 1991, 6, 145-155. [161] St. Laurent, D.; Blaise, C; MacQuarrie, P.; Scroggins, R.; Trottier, B.; Environ. Toxicol Water Qual, 1992, 7, 35-48. [162] Cerejeira, M.J.; Pereira, T.; Silva-Femandes, A.; Chemosphere, 1998, 37, 29492955. [163] Tempest, D.W. In Methods in Microbiology; Norris, J.R.; Ribbons, D.W., Eds.; Academic: London, 1970; Vol. 2, pp. 259-276. [164] van der Ploeg, M.; Tucker, C.S.; J. Appl Aquacult., 1993, 3, 121-140. [165] Martin, J.F.; Izaguirre, G.; Waterstrat, P.; Water Res., 1991, 25, 1447-1451.

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Gomont, M.; Ann. Sci. Nat Bot. VIL, 1892, 75, 263-368 [Reprinted in 1962 by Wheldon & Wesley, Ltd. and Hafner Publishing, New York]. Skuja, H.; Nova Acta Reg. Soc. Sci. Ups., Series 4,1949, J4(5), 1-188. Schrader, K.K.; Rimando, A.M.; Tucker, C.S.; Duke, S.O.; Pestic. Sci, 1999, 55, 726-732. Liber, K.; Solomon, K.R.; Carey, J.H.; Environ. Toxicol. Chem., 1997,16, 293305. Blinn, D.W.; Tompkins, T.; Zaleski, L.; J. Phycol, 1911,13, 58-61. Lund, J.W.G.; Verh. Int. Ver. Theor. Angew. Limnol, 1972, 18, 71-77. Lack, T.J.; Lund, J.W.G.; Freshwater Biol, 1974, 4, 399-415. Solomon, K.R.; Smith, K.; Guest, G.; Yoo, J.Y.; Kaushik, N.K.; Can. Tech. Rep. Fish. Aquat Sci., 1980, 975, 1-9. Stephenson, R.R.; Kane, D.F.; Arch. Environ. Contam. Toxicol, 1984, 13, 313326. Solomon, K.R.; Yoo, J.Y.; Lean, D.; Kaushik, N.K.; Day, K.E.; Stephenson, G.L.; J. Fish. Aquat. Sci, 1985, 42, 70-76. Solomon, K.R.; Bowhey, C.S.; Liber, K.; Stephenson, G.R.; J. Agric. Food Chem., 19SS, 36, 1314-1318. Solomon, K.R.; Stephenson, G.L.; Kaushik,; N.K. Environ. Toxicol Chem., 1989, 8, 659-669. Liber, K.; Kaushik, N.K.; Solomon, K.R.; Carey, J.H.; Environ. Toxicol Chem., 1992,77,61-77.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

391

PHARMACOLOGICAL ACTIVITIES AND APPLICATIONS OF SALVIA SCLAREA AND SALVIA DESOLEANA ESSENTIAL OILS *ALESSANDRA T. PEANA, MARIO D.L. MORETTI Dipartimento di Scienze del Farmaco, Universita degli Studi di Sassari 1-07100 Sassari, Italy ABSTRACT: Pharmacological properties of S. sclarea and S. desoleana oils are discussed in relation to their chemical composition. After systemic administration, these oils had a depressant action on the CNS in mice and a hydrocholeretic effect in rats. Other studies demonstrated also a good anti-inflammatory activity in rats as well as a peripheral analgesic action in mice. These essential oils possess in vitro antimicrobial properties against some human pathogen strains and their activity is comparable to S. officinalis oil, well known for its antiseptic properties. In vitro studies carried out on mucoadhesive preparations showed the ability of the oil components to permeate the oral mucous. This could be of interest in the treatment of human inflammatory diseases of mucous tissues, frequently associated with microbial infections. These oils are also able to inhibit the growth of some phytopathogenic fungi and could therefore be useful in the agronomic field as an alternative to synthetic compounds, with a view to reducing environmental pollution. Some biological effects were correlated with the chemical composition and the kind of the formulations utilized in order to examine some possible applications of these oils in human medicine. All pharmacological activities seem to be attributable to the content of some oxygenated compounds, like alcohols (mainly linalool and alphaterpineol) and esters (linalyl and alpha-terpinyl acetate). Experimental observations point the hypothesis of a synergic action between the different components, even if the oils in toto were more active than their fractions or single components.

INTRODUCTION A remarkable variety of aromatic plants has long been used in folk medicine, cosmetics, and the food industry. Most of the properties of aromatic species can be attributed to their essential oil content, that is the volatile component of the secondary metabolism of the plant, whose biological function is still not completely understood. It accumulates in the secretory gland system and is released into the environment after various kinds of stimulation, such as a change in the weather (rain, wind, a rise in temperature), or the intervention of animal species like pollinator insects, etc.

392 Several studies analyzing essential oils and describing new techniques to optimize yield while retaining the characteristics of the oil during extraction have recently appeared in the literature [1-5]. When aromatic plants and essential oils are used as additives to food, besides giving a particular aroma and taste, they aid preservation since it is likely that they inhibit the proliferation of microorganisms affecting the organoleptic quality of the food. They are also used in phytotherapy to relieve symptoms and cure a variety of ailments, both acute and chronic. In alternative medicine, particularly aromatherapy, numerous preparations are based on aromatic plants and their essential oils. Many claims are made in non-specialist magazines and on television about the healing properties of the plants, but these are not supported by data from a sufficient number of specific studies. Only in a few cases, has research been carried out according to clear protocols, with results in line with the authors' objectives. Different research groups have studied various species of aromatic plants to assess their use in different sectors, with the further aim of creating a source of income for the local population by utilizing the most promising native species of plants [6-9]. Moreover, several studies have been initiated to discover why some species are used in folk medicine and examine the possibility of using the essential oils and their fractions in new, potentially profitable sectors. SALVIA SCLAREA L. General and Botanical description This plant (named also clary sage) is a member of the Labiatae (lipped flowers) or more correctly the Lamiaceae family, which gets its name from Lamium, one of the genera of this family. Lamiaceae (and Lamium) are thought to have been given the name because it is akin to Lamia, a man-devouring monster with the head and breast of a woman and the body of a serpent. According to Willis [10], there are about 700 members of the Salvia genus that can be found in tropical and temperate parts of the world. According to Briquet [11] the genus Salvia, which contains both Old world and New world species, is subdivided into subgenera and sections [8]. S. sclarea (2n=22) grows wild in countries around the Mediterranean (Southern Europe, North Africa and Western Asia). It is also wide-

393 spread from Iran to the Transcaucasican region [8]. In Italy it is found mainly in Sardinia and Sicily [12]. S. sclarea is a perennial herbaceous plant, very occasionally biennial, rhizomatous, with typical quadrangular stems and opposite leaves. It grows to a height of about 100 cm, although in very long photoperiods and dry summers it can reach more than 150 cm. Although the steams are erect, they are densely haired and much branched. The leaves are simple petiolate, broadly ovate-cordate and pubescent, possessing numerous glands. The inflorescence comprises verticillasters generally possessing 4-6 flowers with the bracts exceeding the 20-30 corolla which can be purple, lilac or white in color with the upper lip strongly falcate. The calyces are about 10 mm with spinase pubescent teeth which are glandular peltate. The 2-3 mm long nutlets are dark brown. The plant flowers between mid-May and August. It blooms from the second year and the flowers are protandrous. It grows in sunny dry coastal and submontane areas in stony or loose soil, usually calcareous, and along dry slopes, particularly in rocky places [12-14]. Commercial production of 5. sclarea crops has greatly increased around the world, mainly in some Soviet countries (Russia, the Crimean region of the Ukraine, Georgia and Uzbekistan), the USA, Bulgaria, France, China, Hungary and India [8]. The reason for this expansion is related to production of the essential oil, which is still used as a major ingredient in perfumes because of its good fixative properties and characteristic pleasant, intense smell of ambergris. The essential oil is also used in flavoring foodstuffs, particularly confectionery, giving a pineapple taste, besides being added to liqueurs and aromatic wines, not only for flavor but also for its eupeptic and digestive properties [15-16]. Phytochemical investigations From the economic and pharmaceutical point of view the main components of S. sclarea are the essential oil and a diterpene alcohol named sclareol. The former was first produced by Schimmel [17] and is usually obtained by steam distillation of fresh plant material. The latter is usually obtained by counter current extraction of spent steamed plant material using hexane as the solvent. After concentration by solvent evaporation a "partial concrete" is obtained and sclareol is selectively removed by a second counter current extraction with a polar solvent. In the USA, most of the S. sclarea is grown for the production of this non -volatile compo-

394 nent while the oil is produced as a by-product [18]. Sclareol is mainly utilized in the production of sclareolide, a diterpene lactone obtained by a two-stage oxidation process of sclareol [19] or by its fermentation with various soil-borne microorganisms [20-22]. Sclareolide and its precursor sclareol are of value as a tobacco flavor additive and fragrance fixative. The former is also the precursor of norambrienolide ether (know as "ambrox" or "ambroxan"), an extremely valuable amber-like odored compound which has a low threshold as well as fixative qualities [23]. Oil yield and composition Many workers have determined that the maximum oil content is obtained when all the flowers have reached full maturity because the calyces contain by far the most essential oil glands per unit area [24-28]. In contrast to some other oil rich Lamiaceae species, the leaves of the plant in question are fairly poor in oil. Table 1. Yield date w/v on a fresh weight basis Part of plant:

Yield %

stems

0.002 - 0.06

leaves

0.002-0.15

flowers spikes

0.11 -0.51

calyces

0.50-1.20

new buds 1 whole above ground plants

0.04 - 0.09 0.06 - 0.30

Like many aromatic plants, a diurnal fluctuation in oil content has been found in 5. sclarea with the maximum reached during the early morning. The most important contribution to an understanding of the composition of the oil came from the studies of Teisseire et al [29-31]. Further information on the oil composition can be found in the publications of Bulgarian researchers [32-33] and, more recently, Lawrence and Moretti [3435]. Comparative studies on the composition of the oil have come from the major commercial sources (USA, France, Italy and Russia); the main

395 differences between these oils are in the linalool, linalyl acetate and alpha terpinyl acetate contents. Table 2. Comparative chemical composition of commercial S. sclarea oil Compounds:

USA

France

Italy

Russia

alpha-pinene

0.1 -0.2

0.1 -0.3

trace - 0.9

0.2-0.3

myrcene

1.2-1.7

0.1 -0.2

0.1 -0.1.6

0.3-0.5

limonene

0.4-0.8

0.1-0.2

0.1 -0.9

0.1-0.2

(Z)- beta-ocimene

0.4-0.7

trace

trace

trace - 0.2

(E)- beta-ocimene

0.4-1.4

0.1-0.2

0.1-0.3

0.1-0.4

44.9-53.4

49.0-73.6

23.3-29.0

45.3-61.8

0.9-1.3

1.4- 1.6

0.2 - .03

1.1-1.8 10.4-19.3

linalyl acetate beta-caryophyllene linalool

20.3 - 28.6

9.0- 16.0

10.6- 14.6

alpha-terpineol

1.0-3.0

0.2-0.6

0 . 1 - 1.12

1.2- 2.5

neryl acetate

1.0- 1.7

0.2-0.3

1.2-2.0

0.4 - 0.6

geranyl acetate

1.9-3.2

0.3-0.5

1.2-3.2

0.8-1.2

nerol

0.6-1.1

trace - 0.1

0.9 - 2.7

0.3 - 0.5

geraniol

1.7- 3.3

0.1 - 0.3

2 . 5 - 3.1

0.6- 1.2

germacrene D

2.6-3.5

1.6-2.0

0.6- 1.4

0.7-2

caryophyllene oxide

0.2-0.3

0.3 - 0.5

trace

0.5

sclareol

0.2-0.4

0.1 -0.2

-

0.1-0.2

n.r.

n.r

30.7-38.5

n.r.

alpha-terpinyl acetate

Differences in linalool and linalyl acetate content were ascribed to the state of the plant material on distillation. In the USA and Italy, the plant material is freshly harvested, chopped and distilled within a few hours, whereas in France and the Russian countries, the harvested material is airdried prior to distillation. The excess moisture which is associated with processing of fresh plant material could lead to the major constituent, linalyl acetate, hydrolyzing to linalool, probably facilitated by an increased pH of the medium during distillation [8]. The presence of a large amount of alpha-terpinyl acetate in the essential oil extracted from 5. sclarea plants of Italian origin was reported [6]. As can be seen in the following table, the composition of an essential oil obtained from S. sclarea plants growing wild in a typical calcareous soil [35], contained high levels of methyl chavicol.

396

Table 3. Yield and composition of 5. sclarea oil obtained from Sardinian plants Compounds:

Cultivated

Wild

alpha-pinene

0.1

0.2

j alpha-thujene

n.d.

v. ^^^"^^^

ff^^^^^^

SCoA

« o ^

p-coumaric acid

/>-coumaroyl-CoA

Fig. (1). Bio synthesis of resveratrol (1)

Cardioprotective Properties of Resveratrol Oxidative processes are believed to play a significant role in the development of vascular tissue damage and promotion of cardiovascular disease. Several studies [11,12,13,14] have now demonstrated that 1 possesses potent in vitro antioxidant effects; however, its in vivo activity is questionable [15]. Once oxidative tissue damage has occurred, blood platelet accumulation at the site of injury can lead to localized blood vessel occlusion or facilitate the development of plaques that may breakfree resulting in the formation of blockages in the peripheral vasculature. Therefore, drug therapies targeted at inhibiting platelet aggregation have been employed to help reduce the chance of clot formation in high-risk patients. Research now indicates that 1 may play a role in stemming this

510

risk as an inhibitor of the blood clotting process [16]. Furthermore, 1 has been shown [17,18,19] to be a potent in vitro inhibitor of platelet aggregation induced by arachidonic acid, ADP, and collagen; however, its in vivo activity appears to be more modest [16]. Vascular tissue damage and subsequent platelet clotting processes are also influenced to a great extent by leukocyte activity. Some leukocytes are responsible for the release of a host of pro-inflammatory substances that lead to additional vascular tissue damage and promote further platelet aggregation. In vitro tests [20] employing isolated leukocytes have demonstrated that 1 inhibits both the expression and subsequent release of several pro-inflammatory substances associated with the development of vascular tissue damage. In addition, studies have shown that 1 interferes with blood vessel endothelial cell adhesion molecule expression [21] and other associated tissue factors [22], thus providing further protection from leukocyte induced vascular injury. Furthermore, recent evidence now indicates that 1 possesses estrogenic properties that may convey additional cardioprotective effects [23,24,25]. In light of these cardioprotective properties exhibited by 1, it is conceivable that 1 may help to reduce the risk of developing cardiovascular disease in humans. Cancer Chemopreventive Properties of Resveratrol Research has also shown that 1 possesses potent cancer chemopreventive activity as exhibited by the inhibition of the initiation, promotion, and progression of cancer cell growth [26,27]. Several in vitro studies have demonstrated that 1 prevents the initiation of tumor formation through antimutagenic effects [28], inhibition of the expression of the phase I enzyme CYPlAl that is responsible for procarcinogen activation [29], and induction of the carcinogen-detoxifying phase II enzyme quinone reductase [26]. Further research has demonstrated that 1 inhibits the promotion of cancer cell growth through the inhibition of the enzymes responsible for the production of cancer promoting agents. For example, 1 appears to interfere with prostaglandin synthesis by directly inhibiting cyclooxygenase I and II activity and by further suppressing COX II expression [26,30]. In addition, 1 blocks the progression of cancer cell growth as illustrated in in vitro studies employing human estrogen dependent and independent epithelial breast carcinoma [31] and promyelocytic leukemia cell lines [26]. In these studies, 1 was noted to

511 alter cancer cell growth by preventing exponential growth, reducing cell viability, and promoting cellular differentiation to a nonproliferative phenotype. In related in vivo studies employing animal models, 1 has also been shovm to be a potent inhibitor of tumor development. In studies using both mice [26] and rats [32], 1 selectively inhibited tumor growth while failing to produce any noted systemic toxicity. Additional studies attempting to further elucidate the mechanisms behind the cancer chemopreventive and inhibitory effects of resveratrol have noted several possible mechanisms of action. For example, the cancer inhibitory effects of 1 may be mediated in part through the inhibition of protein tyrosine kinase and protein kinase C signal transduction pathways [33,34]. Further studies lending support to the anticancer activity of 1 demonstrate that it can inhibit ribonucleotide reductase activity [35], cleave DNA through the formation of an oxidative copper complex [36], and induce apoptosis in growing cancer cells [37,38]. Considering the promising pharmacological properties of 1, it is conceivable that 1, or one of its analogs, could serve as an important cancer chemopreventive agent for humans. In light of the important role that 1 plays in plants and its potential pharmacological applications for humans, significant interest has developed to identify new analogs of 1 that may possess interesting biological activity. One group of derivatives of 1 that have received significant attention in recent years is the resveratrol oligomers. To date, a host of resveratrol oligomers have been described. These compounds have demonstrated an array of promising pharmacological properties. In addition, new research is providing valuable insight into the in vivo roles that these compounds play in plants.

THE RESVERATROL OLIGOMERS The resveratrol oligomers are composed of a diverse assemblage of polyphenolic compounds derived from the trihydroxystilbene monomer resveratrol (1). Previously, in a review by Sotheeswaran and Pasupathy [7], the authors proposed a simple dichotomous classification scheme for categorizing these compounds based on a single structural feature. Under

512

this system, 'group A' oligomers consisted of those compounds that contain one or more five-membered oxygenated heterocyclic ring systems as typified by a dihydrobenzofuran moiety. In contrast, all compounds that lacked this moiety were assigned to 'group B'. While this categorization method does provide a simple means for gross structural classification, it is readily apparent that this system lacks any biogenic foundation. Therefore, due to this organizational scheme's limited applicability, it will not be utilized in this review. Instead, we have chosen to present the resveratrol oligomers based on their biogenic origin. Resveratrol oligomers have been isolated from a relatively small assemblage of plant families. More recently, resveratrol oligomers have also been identified as fungal detoxification products of resveratrol metabolism. In Table (1), a summary of all the reported occurrences of resveratrol oligomers based on their biogenic origins is presented. This information was compiled following a thorough review of the available literature. Several computerized databases were employed for this purpose including AGRICOLA®, AGRIS®, Biological Abstracts®, Chemical Abstracts®, Current Contents®, MEDLINE®, and TOXLINE®. In Table (1), the names of the resveratrol oligomers and their sources are provided followed by a description of the oligomer type (i.e. dimer, trimer, tetramer, or pentamer), molecular formula, and reported optical rotation. In almost all cases, the resveratrol oligomers have been isolated from their natural sources in an optically active form. There are several cases reported in which both the (+) and (-) enantiomeric forms of a resveratrol oligomer have been isolated from different plant families. In some instances, researchers have been able to assign the absolute stereochemistry of each enantiomer; however, there are still several resveratrol oligomers whose absolute configuration remains unknown. In order to avoid confusion regarding the numbering of the resveratrol oligomers in this text, we have adopted a simple classification scheme in

Table 1.

Sources of Resveratrol Oligorners Source

iPlACEAE beniculirm vulgure Miller

Part Examined

fruit

Compound

foeniculoside I (2a)

fruit

foeniculoside I1 (3a)

fruit

foeniculoside I11 (4a)

fruit

foeniculoside IV (5a)

fruit

miyabenol C (6a)

fruit

cis-miyabenol C (7a)

NA'

(+)-a-viniferin (8a)

NA

kobophenol A (9)

NA

pallidol (10)

roots and rhizomes roots and rhizomes

.c-viniferin (1 1 )

Oligomer Type (Molecular Formula) trimer + 1 Glc (C4xH420I 4) trimer + 1 Glc (C48H42014) trimer + 2 Glc (C54HS2019) trimer + 3 Glc (C60H62024) trimer (C42H3209) trimer (C42H3209)

Optical Rotation (Concentration, Solvent)

+46.8" (c 0.9, MeOH)

+89.1" (c 0.9, MeOH) +4 1.9" (c 1.0, MeOH)

+40.7" (c 0.8, MeOH)

+22 I .4" (c 1.1, MeOH)

+98.5" (c 1.0, MeOH)

YPERACEAE arex ciliuto-marginuta Nakai

arex fedia Nees ex Wight

var. miyabei (Franchet) T. Koyama

miyabenol A (12b)

trimer (C42H3009) tetramer (C56H440 13) dimer (C2XH2206) dimer (C28HZ206) tetramer (cS6H42012)

NA NA NA

NA -91" (c 0.18, MeOH)

Table 1.

Sources of Resveratrol Oligorners (Continued) Part Examined

Compound

Oligomer Type (Molecular Formula)

roots and rhizomes roots and rhizomes NA

miyabenol B (13b)

(+)-a-viniferin (82)

NA

kobophenol A (9)

NA

pallidol (10)

roots

(+)-a-viniferin (8a)

tetramer (C56H400I 2 ) trimer (C42H3209) trimer (C42H3009) tetramer (CS6H440 13) dimer (C28H22°6) trimer (C42H3009) dimer (C28H2206) tetramer (C56H44013) trimer (C42H3209) trimer (C42H3009) tetramer (CS6H44013) dimer (C28H2206)

miyabenol C (6a)

roots and rhizomes

(-)-&+inifwin ( l l b )

roots and rhizomes roots and rhizomes NA

kobophenol A (9a)

(+)-a-viniferin (8a)

NA

kobophenol A (9)

NA

pallidol (10)

miyabenol C (6a)

Optical Rotation (Concentration, Solvent) -126" (c 0.23, MeOH) +69" (c 0.07, MeOH)

NA NA NA +S I .4" (c 1 0 , EtOH)

-46" (c 0.5, MeOH) +227" (c 0 17, MeOH)

+206" (c 0.070. MeOH)

NA NA NA

Table 1.

Sources of Resveratrol Oligomers (Continued) Source

Part Examined

Compound

Oligomer Type (Molecular Formula)

Optical Rotation (Concentration, Solvent)

NA

(+)-a-viniferin (8a)

NA

NA

kobophenol A (9)

NA

pallidol (10)

roots and rhizomes roots and rhizomes

(-)-c-viniferin ( I l b )

roots and rhizomes roots and rhizomes roots and rhizomes

kobophenol B (16b)

trimer (C42H3009) tetramer (CS6H44013) dimer (C28H2206) dimer (c281.12206) tetramer (C56H42012) tetramer (C56H40O 12) tetramer (cS6H42012) trimer (C42H3209)

heartwood

(-)-hopeaphenol(14b)

alanocarpus zeylanrcus Trimen

bark

balanocarpol(17b)

opea brevipetiolarrs (Thwaites) P.S. Ashton

bark

copalliferol A ( H a )

hrex multifolia Ohwi

'arexpumila Thunb

NPTEROCARPACEAE alanocarpus heimii King

hopeaphenol(14b)

miyabenol A (12) miyabenol C ( 6 )

tetramer (C56H42012) dimer (C28H2207) trimer (C42H3209)

NA NA

(c

-27.9" 0.843, MeOH)

-370" (EtOH) - 199" (c 0.17, MeOH) NA

NA

-407" (EtOH) -17" (c 0.005, MeOH) +115.6" (MeOH)

Table 1.

Sources of Resveratrol Oligomers (Continued) Source

Hopea cordfolio Trimen

Part Examined

Compound

bark

copalliferol A (18a)

Oligomer Type (Molecular Formula)

Optical Rotation (Concentration, Solvent)

trimer

+115.6" (MeOH) -5.6" (MeOH) -17" (c 0.005, MeOH)

(C42H3209)

bark

stemonoporol (19a)

trimer (C42H3209)

Hopeajucunda Thwaites

bark

balanocarpol (17b)

dimer (C28H2207)

Vopea malibato Foxworthy

leaves

balanocarpol (17b)

dimer (C28H2207)

leaves leaves

dibalanocarpol (ZOb) maliabatol A (21b)

Yopea odorala Roxb.

heartwood

malibatol B (22b) (-)-hopcapheno1 (14b)

tetramer

-227" (c 0.73, MeOH)

dimer

bark bark

distichol (23b) E-viniferin (11)

-38" ( c 0.37, MeOH)

dimer

-77"

(C28H2009)

(c 0.06, MeOH)

tetramer

(C4ZH3209)

-407" (EtOH) -44" (MeOH)

dimer

NA

(CS6H42012)

ihorea disticha (Thwaites) P.S. Ashton

0.52, MeOH)

(CShH42014)

(C28H2207)

leaves

-18" (c

trimer

(C28H2206)

;horea robusra A. DC

heartwood

hopeaphenol(14b)

tetramer

-402.9"

(C56H42012)

;horea lalura Roxb.

heartwood

hopeaphenol(14b)

tetramer (C~6H42012)

-402.9"

Table 1.

Sources of Resveratrol Oligomers (Continued)

I Source

Part Examined

Compound

Oligomer Type (Molecular Formula) trimer (C4ZH3209) trimer (C42H3209) trimer (C42H3209) trimer (C4ZH3209) tetramer (CS6H12012) tetramer (CS6H42012) trimer (C42H3209) trimer (C42H3209) trimer (C42H3209) trimer (C42H3209) tetramer

I Shorea stipularis Thwaites

bark

copalliferol A (18a)

Istemonoporus affinis

bark

copalliferol A (18a)

bark

stemonoporol (19b)

bark

canaliculatol (24b)

bark

vaticaftinol (25b)

bark

vaticaffmol (25b)

bark

copalliferol A (18a)

I

Thwaites

Stemonoporus cordfolius (Thwaites) Alston Stemonoporus elegans (Thwaites) Alston

Stemonoporus kannelryensis A.J.G.H. Kostermans

Stemonoporus lancijolius (Thwaites) P.S. Ashton Stemonoporus oblongifolius Pierre

bark

stemonoporol (19b)

bark

copalliferol A (18a)

bark

stemonoporol (19b)

bark

vaticaftinol(25b)

bark

copalliferol A (18a)

(C56H4201

2)

trimer (C42H3209)

Optical Rotation (Concentration, Solvent)

+ I 15.6" (MeOH) + I 15" (MeOH) -5.58" (MeOl-I) -25.5" (c 0.003, MeOH) -22" (MeOH) -22.5" (MeOH) +115"

(MeOH) -5.58" (MeOH) + I 15" (MeOH) -5.58"

(MeOH) -22.5" (MeOH) + I 15" (MeOH)

Sources of Resveratrol Oligomers (Continued)

Table 1.

I'atica af/inis Thwiates

Vatica diospyroides Symington

Valeria copallifera (Retz.) Alston

Valeria copallifera (Retz.) Alston

FABACEAE Caragana chamlagu Lam

Part Examined

Compound

Oligomer Type (Molecular Formula)

Optical Rotation (Concentration, Solvent)

bark

stemonoporol(19h)

bark

6-viniferin (11)

-5.58" (MeOH) NA

bark

vaticaffinol(25b)

trimer (C42H3209) dirner (C28fi2206) tetramer (C56H42012) tetramer (C56H4.2012) tetramer (C56H42012) trimer (C42H3209) trirner (C42H3209) trimer (C42H3209) trinier (C42H3209)

stem

vatdiospyroidol (26b)

stem

vaticaphenol A (27h)

bark

copalliferol A (18)

bark

copalliferol B (28h)

bark

copalliferol A (18a)

bark

stemonoporol(19b)

roots

(+)-a-viniferin (8a)

roots

caraganaphenol A (29a)

-22.5' (MeOH) -67" ( c 0.1, MeOH) -29" (c 0.1, MeOH) NA -186" (pyridine)

+ I 15.6" (MeOH) -5.6" (MeOH)

trimer (C42H3009) tetramer

+188.5"

(C56H42013)

(c 0.785, MeOH)

+50.7" (EtOH)

Table 1.

Sources o f Resveratrol Oligomers (Continued) Part Examined

Compound

roots

kobophenol A (9a)

roots

(+)-a-viniferin (8a)

roots

kobophenol A (9a)

roots

miyabenol C (6)

roots

davidiol D (30b)

roots

(-)-E -vini ferin (1 1b)

roots

hopeaphenol(14b)

roots roots

roots roots roots

leachianol A (31b) leachianol B (32a) leachianol C (33b) leachianol D (34b) leachianol E (3Sb)

Oligomer Type (Molecular Formula) tetramer (c56H44°13) trimer (C42H3009) tetramer (C5~3~44~13) trimer (c42113209) pentamer (C70H53015) dimer (C2SH2Z0G) tetramer (C56H42012) trimer (C4ZH3209) trimer (C42H3209) tetramer (C56H44012) trimer (C42H3209) trimer (C4ZH3209)

Optical Rotation (Concentration, Solvent) +199.4" +52.9" (c 0.52, EtOH)

+250.6" (c 0.39, MeOH) NA

-135" (MeOH) -52.4" (c 0 057, MeOH) -312.0" (c 0.13, MeOH)

-159.8" (c 0.25, MeOH)

+147.4" (c 0.25, MeOH)

-30.3" (c 0.10, MeOH)

-18.7" (c 0. I 1, MeOH)

-84.8" (c 0.12, MeOH)

Reference

Part

Compound

Examined

roots

leachianol F (36)

roots

leachianol G (37)

roots

pallidol (lob)

roots

davidiol B (38)

roots

(-)-E-viniferin (1 1 b)

roots

stenophylla A (39b)

roots roots

1Gnerum hainanense C.Y. Cheng

Gnetum ieyboldi Tul.

wl

Sources of Resveratrol Oligomers (Continued)

Table 1.

lianas

stenophylla B (40b) stenophylla C (41b)

(-)-z-viniferin ( l l b )

N 0

Oligomer Type

Optical Rotation

(Moleeular Formula)

(Concentration, Solvent)

dimer (C28H2407) dimer (C28H2407) dimer (C28H2206) trimer (c42h4O 13) dimer (C28H2206) tetramer (C&tr1012) trimer (C42H3209) tetramcr (C56H42012)

NA NA

-36.3" (c 0.13, MeOH) NA NA

-345" (c 0.7, MeOH) (c

-2" 0.1, MeOH)

(c

-66" 0. I , MeOH) -38.6"

(c 0.12, MeOH)

lianas

resveratrol trans dehydrodimer (42)

NA

bark

gnetin A (43)

NA

rable 1.

Sources of Resveratrol Oligomers (Continued) Source

Part Examined

Compound

bark

gnetin B (44)

Oligomer Type (Molecular Formula)

Optical Rotation (Cnncentration, Solvent)

dimer

NA

(C28H2406)

bark

gnetin C (45)

dinier

NA

(C28H2206)

bark

gnetin E (46)

trimer

NA

(C42H320d

hefumparvfolium (Warb.) Cheng

lianas

hetum venosum

seeds

c-viniferin (11)

dimer

NA

(C2RH2206)

gnetin C (45)

Spruce ex Benth.

dimer

NA

(C28H2206)

seeds

gnetin E (46)

trimer

NA

(C42H3209)

Vehvitschia mirabilis

wood

gnetin F (47)

J.D. Hook

dimer

NA

(C28H2406)

wood

gnetin G (48)

dimer

NA

(C28H2207)

wood

gnetin H (49)

trimer

NA

(c4203209)

wood

IAEMODORACEAE nigoranfhospreissii Endl.

gnetin I (SO)

trimer

NA

(c4203209)

root cell culture

anigopreissin A (51)

dimer (C28H2006)

NA

Sources of Resveratrol Oligomers (Continued)

Table 1.

VI

N

N

I

Source

Musa cavendishii Lambert ex Paxt. PAEONIACEAE Paeonra suffruticosa Andrews

VITACEAE Ampelopsis brevipedunculala Maxim. ex Trautv.

Part

Compound

Optical Rotation (Concentration, Solvent)

Examined

rhizome

anigopreissin A (51)

NA

seeds

suffruticosol A (52)

NA

seeds

suffruticosol B (53)

NA

seeds

suffruticosol C (54)

NA

roots

ampelopsin A (SSa)

+167" (c 2.12, MeOH)

roots

ampelopsin B (56a)

+ 123" (c 0.93, MeOH)

roots

ampelopsin C (S7a)

+24" (c 1.04, MeOH)

roots

ampelopsin D (58)

roots

/ram -ampelopsin E (59)

NA 0" (c 2.06, MeOH)

roots

cis -ampelopsin E (60)

0"

(c 1.OO,MeOH)

Reference

Sources of Resveratrol Oligomers (Continued)

Table 1. Source

Part

Compound

Examined

roots roots

ampelopsin F (61a) ampelopsin G (62a)

Oligomer Type

Optical Rotation

(Molecular Formula)

(Concentration, Solvent)

dimer

+14.0"

(C28H2206)

( c I .98, MeOH)

trimer (C42H3209)

roots

ampelopsin H (63a)

tetramer (CS6H320

roots

miyabenol C (6)

12)

trimer

+32.0" ( c 0.23, MeOti)

+ I050 (c 0.83, MeOH) NA

(C42F13209)

roots

pallidol (10)

dimer

NA

(C28H2206)

'issuspallida Planch. 'issus quadrangularis L

stemwood stems

pallidol (10) pallidol (10)

dimer

0"

(C2nH2206)

(MeOH)

dimer

NA

(C28H2206)

stems

parthenocissin A (64)

dimer

NA

(C28H2206)

stems yphosfemmacrotalarioides (Planch.) Descoings

roots

quadrangularin A (65b) cyphostemmine A (66)

dimer

-2"

(C2nH2206)

(MeOH)

dimer

NA

(C2aH2206)

roots

cyphostemmine B (67)

dimer

NA

(C28H2206)

roots

trans- z-viniferin (11)

dimer (C28H2206)

NA

Sources of Resveratrol Oligomers (Continued)

Table 1.

~

Source

Part

Compound

Examined

roots

cis -&-viniferin (68)

Oligomer Type

O p z a l Rotation

(Molecular Formula)

(Concentration, Solvent)

dimer

NA

(C28H2206)

roots

gnetin C (45)

dimer

NA

(C28H2206)

roots

gnetin E (46)

trimer

NA

(c42133209)

roots

pallidol (10)

dimer

NA

(c28f42206)

roots

parthenocissin A (64)

dimcr

NA

(C2RH2206)

'arthenocissusquinquefolia Planch.

stems stems

parthenocissin A (64b) parthenocissin B (69b)

dimer

-25"

(C28H2206)

(c 0.13, MeOH)

trimer (C42H3209)

arthenocissus tricuspidata (Sieb. & Zucc.) Planch.

stemwood stemwood

isoampelopsin F (70b) (+)-&-viniferin ( I l a )

(c

-39" 0.13, MeOH)

dimer

-56.7"

(C28H2204

(c 0.23, MeOH)

dimer

NA

(C2RH2206)

sternwood

(-)-pallidol (lob)

dimer

NA

(C28H2206)

stemwood

tricuspidatol A (71)

dimer

NA

(C28132407)

itrs

betulrfolia Diels. & Gilg.

stems

arnpelopsin A (55)

dimer (C2RH2Z07)

NA

Sources of Resveratrol Oligomers (Continued)

Table 1. ~

Source

Part Examined

Compound

Oligomer Type (Molecular Formula)

Optical Rotation (Concentration, Solvent)

stems

ampelopsin C (57)

NA

stems

betulifol A (72a)

trimer (C42H3209) dimer (C28H2006) dimer (C28H240x) dimer (cZ8i tetrarner (C56H42012) tetramer (C56H42012) tetramer (C56H4201 2) dimer (C282207) trimer (C42H3209) dimer (C28H2206) dimer (C28H22Od dimer (C28H2206)

stems

betulifol B (73a)

stems

(+)-s-viniferin ( I l a )

stems

heyneanol A (74)

stems

hopeaphenol (14)

stems V i m corgneliae Pulliat ex Planch.

vitisin A ( 7 ~ ) ~

NA

(+)-ampelopsin A (55a)

NA

ampelopsin C (57a)

NA

ampelopsin F (61a)

leaves

(+)-s-viniferin (Ila)

NA

(+)-E-viniferin (1l a )

+28.9" (c 0.28, MeOH)

+8 1.6" (c 0.19, MeOH) NA NA NA NA

+167" (c 2.0, MeOH) +24" (c 1.0, MeOH) +I40 (c 2.0, MeOH) NA

+38" (c I .83, MeOH)

Sources of Resveratrol Oligomers (Continued)

Table 1. Part

Compound

Examined

stems roots roots stems stems bark bark

E-viniferin diol (78a) (+)-trans-vitisin A (76a)

(+)-cis -vitisin A (77a) (-)-trans-vitisin B (79b) (-)-cis-vitisin B (80b)

(+)+itisin D (82a) (+)-vitisin E (83a)

stems

(+)-E-viniferin ( l l a )

stems

flexuosol A (84b)

stems

gnetin A (43)

stems

hopeaphenol(l4)

stems

vitisin A ( 7 ~ ) ~

Oligomer Type

Optical Rotation

(Molecular Formula)

(Concentration, Solvent)

dimer (C28H2408) tetramer (CS6H42OI2) tetramer (C56H42012) t et r am e r (CS6H42012) tetramer (C56H420I 2) tetramer (CS6~~42012) trimer (C42H3209) dimer (C28H2206) tetramer (C56H42013) dimer (C28H2206) tetramer (CS&WI~) tetramer (CS6H420 12)

+136.0" (c O19,MeOH)

+195.1' (c 1.1, MeOH)

+ 184.0" (c 0.5, MeOH)

-90.0" (c 2.28, MeOH)

-4 I .9" (c 0.72, MeOH)

+222.0" (c 0.20, MeOH)

+94.5" (c 0.20, MeOH) NA

-99.6" (c 0.15, MeOH)

NA NA

NA

Table 1.

Sources of Resveratrol Oligomers (Continued) -Source

'rtrs heyneana Roem. & Schult.

Part Examined

Compound

stems

ampelopsin A (55)

Oligomer Type (Molecular Formula)

Optical Rotation (Concentration, Solvent)

dimer

NA

(C28H22O7)

stems

ampelopsin C (57)

trimer

NA

(C42H320Y)

stems stems ills

sp

roots

(+)-&-viniferin ( I l a ) heyneanol A (74b) E-viniferin (11)

dimer (C~Hzz0d tetramer

(c 0.40,

(C56I142O12)

(c 0.31, MeOH)

dimer

NA

+39" MeOH) -53"

(C28H2206)

roots

r-viniferin (8Sb)

tetramer (C56H42012)

roots

r-2-viniferin (86a)

tetramer (C56H42012)

itis vinifera L

leaves

a-viniferin (8)

trimer

-86.4" (c 1.1, MeOH)

+132.9" (c I . I , MeOH)

NA

(C42H3009)

UV-irradiated leaves leaves

(-)-a-viniferin (8b)

trimer

NA

(C42H3U09)

&-viniferin (11)

dimer

NA

(C2gH2Z06)

stalks

c-viniferin (11)

dimer

NA

(C28H2206)

leaves

resveratrol trans dehydrodimer (42)

dimer (C28H2206)

NA

Table 1.

Sources of Resveratrol Oligomers (Continued) Part Examined

Compound

roots

ampelopsin A (55)

Oligomer Type (Molecular Formula) dimer

Optical Rotation (Concentration, Solvent) NA

(C28H2207)

roots

hopeaphenol(l4)

tetramer

NA

(C56H42012)

roots wood

(+)-ampelopsin A (55a) (+)-E-viniferin (1 l a )

dimer

+167.0"

(C28H2207)

(c 2. I, MeOH)

dimer

+49.1" (c 1.9, MeOH)

(c28t12206)

wood

(+)-hopeaphenol(14a)

tetramer (C56H420

wood

(-)-isohopeaphenol (15b)

12)

tetramer (C56H42012)

wood

(-)-viniferal (87b)

from a trimer (C3SH2608)

wood

viniferifuran (88)

dimer

+374.8" (c 0.78, MeOH)

- 1 14.5" (c 0.44, MeOH)

- 1 3 1.7" (c 1.6, MeOH)

NA

(C28H2U06)

wood wood

(+)-vitisifuran A (89a) (-)-vitisifuran B (90b)

tetramer

+236.1"

( C 5 6 H 4 0 0 12)

(c 0.44, MeOH)

tetramer (C56H40012)

wood

(-)-from-vitisin B (79b)

tetramer (CS6H42012)

wood

(+)-vitisin C (81a)

-133.7" (c 0.12, MeOH)

-90" (c 2.3, MeOH)

tetramer

i239.9"

(C56H42012)

(c 0.5, MeOH)

Sources of Resveratrol Oligomers (Continued) I

Source

Part

Compound

Examined

MICROBIAL CULTURES AND ENZYMES Botrylis cinerea ATCC 1 1542

Bottytyliscinerea isolate Horseradish peroxidase

growing incubations growing incubations growing incubations growing incubations growing incubations growing incubations culture medium filtrate NA NA NA

a

not applicable or data not available

leachianol F (36) pallidol (10) restrytisol A (91b) restrytisol B (92b) restrytisol C (93b) resveratrol trans dehydrodimer (42b) resveratrol trans dehydrodimer (42) resveratrol trans dehydrodimer (42) (-)-trans-vitisin B (79b) (+)-vitisin C (81a)

Oligomer Type (Molecular Formula)

Optical Rotation

dimer (C28H2407) dimer (C28H2206) dimer (C2UH2407) dimer (CZRH2407) dimer (C2RHZZ06) dimer (C2UH2206) dimer (CZSH2206) dimer (C28H2206) tetramer (Cj6H42012) tetramer (C56H42012)

NA

(Concentration, Solvent)

NA -2 20" (c I .8, acetonc)

-0.42" (c

4.7, acetone) -0.92"

(c 1. I , acetone)

-1.15" (c 7.3, acetone)

NA NA -90" (c 2.3, MeOH)

t239.9" (c 0.5. MeOH)

Reference

530

which the (+) enantiomer has been assigned a number followed by the letter 'a', while the (-) enantiomer has been assigned the letter 'b'. Furthermore, for cases in which the stereochemistry of the reported oligomer was not defined, no letter designation was given. For example, Ito and Niwa [103] reported isolating (H-)-6'-viniferin from Vitis vinifera, Kurihara and colleagues [44] obtained (-)-6'-viniferin from Carex kobomugi, and Lin and colleagues [76] acquired an unspecified form of ^•-viniferin from Gnetum parvifoUum. Under our classification scheme, sviniferin has been assigned the number 11, (+)-6'-viniferin the number 11a, and (-)-£•-viniferin the number lib. The structures of these and all the other resveratrol oligomers can be found in Appendix (1). It should be noted that 1 and its glucoside conjugates, piceid (94) and resveratroloside (95), have been isolated from a large number of plant families; however, only a portion of these have been examined as a source of oligomeric stilbenes. Table (2) provides a brief listing of all the plant families known to produce 1, glucoside conjugates of 1, and resveratrol oligomers. This information was compiled based on the distribution of these compounds as reported by Gorham [3] and those references cited in Table (1). Future examination of many of the plant families presented in Table (2) will likely reveal many new sources of resveratrol oligomers. Biosynthesis of Resveratrol Oligomers Biosynthetic processes leading to the formation of the resveratrol oligomers have been hypothesized by several authors [3,4,44,51,52,57,62,84,89,103]; however, conclusive evidence supporting these conjectures is still lacking. In general, the plant derived resveratrol oligomers are believed to be the products of a successive series of oxidative couplings of resveratrol radicals (Fig. (2)). Recent efforts to identify the enzymes responsible for the biotransformation of 1 in Vitis spp. have uncovered at least two stilbene-metabolizing peroxidases as likely catalysts. These peroxidases have been shown to carry out reactions similar to that of horseradish peroxidase that has been documented for its role in the formation of structurally analogous neolignans [78,104, 109]. It has been shown that grapes contain several peroxidases that vary both temporally and spatially with regards to their in vivo expression in grape tissues indicating that these enzymes may

Table 2.

Distribution of Resveratrol and Its Derivatives by Plant Family Resveratroloside (94)

532

OH

Fig. (2). Depiction of a proposed biosynthetic scheme for the dimerization of resveratrol (1) to (+)-£--viniferin (11a)

have highly specific functional roles such as the polymerization of 1 [110]. Morales and colleagues [111] have identified a possible candidate peroxidase isozyme that is expressed constitutively in grape vacuoles. This isozyme has demonstrated a high affinity for 1 in an acidic medium in which it readily oxidizes 1. It is likely that additional peroxidases also play a role in the formation of defensive resveratrol oligomers such as those produced by plants during periods of microbial infection [112,113]. Based on the proposed free-radical mechanism for the formation of the resveratrol oligomers (Fig. (2)), it is likely that accessory proteins are involved in the biosynthesis of these compounds. It has come to light in recent years that auxiliary or dirigent proteins play an important role in the free-radical coupling of monolignols in lignin biosynthesis [114,115]. These proteins are believed to capture the oxidized free-radical substrate providing a scaffold upon which radical coupling can occur yielding an optically active product. Due to the optical activity generally exhibited by the resveratrol oligomers (Table (1)), it is a distinct possibility that a form of dirigent protein may be involved in the biosynthesis of these compounds. It is readily apparent that a great deal of research is needed to elucidate the biosynthetic pathways of the resveratrol oligomers. It is interesting to note that in vitro incubations of 1 with horseradish peroxidase [108] as well as with growing B. cinerea cultures [106] and B. cinerea culture medium filtrates [107] have yielded a unique resveratrol oligomer known as the resveratrol rraw^'-dehydrodimer (42). This dimer shares a similar structural design with the more common plant-derived resveratrol dimer, 6"-viniferin (11). It has been proposed that the B. cinerea mediated transformation of 1 to 42 may be facilitated by other stilbene oxidizing enzymes such as copper-containing laccases [106,107].

533 In another study, Pezet [116] provided evidence that a putative laccase isozyme from a B. cinerea isolate was capable of transforming resveratrol into 11 under in vitro conditions. ISOLATION OF RESVERATROL OLIGOMERS Resveratrol oligomers have been obtained from a wide variety of plants (Table (1)). These compounds have been isolated primarily from the roots, wood, and bark; however, they have occasionally been extracted from the seeds, fruits, and leaves of some species. In addition, a resveratrol oligomer has been obtained from a plant cell culture. Other resveratrol oligomers have also been obtained following the incubation of 1 with fungal cultures and isolated enzyme systems. Extraction Only a few solvents have been used to successfully extract resveratrol oligomers from both fresh and dried plant materials. Most isolation schemes utilize a single solvent extraction with methanol [43,86,92,99,103], ethanol [77,84,89,97], or acetone [51,62,65,93,96,105] at room temperature. Many resveratrol oligomers contain an ethylene bridge in a trans configuration; however, this double bond has been shown to be highly susceptible to ultraviolet induced isomerization [93,107]. In an effort to avoid isomerization of the double bond, several research groups have reported incorporating measures to shield the plant materials from light during the extraction process [54,59,72,98,99]. This has helped in some cases to prevent the formation of contaminating cis isomer artifacts. Partitioning Following extraction, the crude plant extracts are often subjected to one or more liquid-liquid phase partitioning steps. Ethyl acetate and water are most frequently utilized with the resveratrol oligomers readily accumulating in the organic phase [45,71,84,86,92,96,106]. In other cases, more elaborate partitioning schemes have been used such as

534

defatting with hexanes and/or chloroform to help remove unwanted highly non-polar substances before proceeding with ethyl acetate [39,103]. Still others have reported success by directly partitioning their crude extracts between a non-polar solvent such as hexanes and aqueous methanol [80]. Isolation A host of methods has been used to facilitate the isolation of single chemical entities from the complex polyphenolic mixtures obtained following their initial extraction and partitioning. The most common procedure is a simple two-step process beginning with silica gel column chromatography to yield a crude resveratrol oligomer mixture. This is followed by preparative thin-layer chromatography (PTLC) to separate the individual oligomers from each other. Often times the same or a similar solvent system is employed for both steps. The most common solvent system that has been employed successfully to isolate resveratrol oligomers is a chloroform-methanol solution [46,62,87,89, 96,103,104,106]. Other useful solvent systems for siHca gel chromatography include hexane-acetone [72], chloroform-benzenemethanol [41,43,44,45], and toluene-ethyl acetate-acetic acid [106]. Occasionally, some resveratrol oligomers have proven difficult to isolate using the previously outlined methods. In these cases, reversephase Cg and Ci8 bonded silica gel materials were invaluable for isolation [42,84,92,98,102,106,107]. This stationary phase material has been widely used in both preparative HPLC and PTLC. The most commonly employed solvent systems in these applications are aqueous methanol and aqueous acetonitrile solutions. Other less frequently used stationary phases include both Sephadex LH-20 and a small assortment of resins [39,43,55,65,66,71,98,99,106]. Another less commonly used, but promising technique that has been employed in the isolation of cis and trans isomers of resveratrol oligomers is recycled HPLC. Using an aqueous methanol solvent system, Ito and colleagues [93] were able to separate both the cis and trans isomers of the tetrameric resveratrol oligomer (+)-vitisin A (77a and 76a, respectively).

535

STRUCTURE ELUCIDATION OF RESVERATROL OLIGOMERS Structure elucidation studies of resveratrol oligomers have benefited greatly from the development of NMR spectroscopy. However, other spectroscopic methods such UV, IR, and MS still remain important tools in this research. UV Spectroscopy The extended conjugated stilbene moiety of 1 has been examined using UV spectroscopy. This chromophore typically presents a UV absorption spectrum with a X^max of approximately 310-320 nm. However, it has been noted that for resveratrol oligomers in which the ethylene bridge has been reduced, such as that seen in (-)-a-viniferin (8b) [100], kobophenol A (9) [43], and tricuspidatol A (71) [89], the A^max is shifted to a lower wavelength of approximately 280-290 nm. Most resveratrol oligomers present relatively similar UV absorption spectra; however, Mattivie and Reniero [117] have examined the spectra of an assortment of resveratrol oligomers and noted several key UV spectral features that they in turn have applied as a tool for predicting some basic structural features of these compounds. Using these methods, Mattivie and Reniero have reported that it is possible to determine the presence and stereochemistry of an ethylene bridge and predict the number of resveratrol monomers incorporated in an oligomer. IR Spectroscopy Infrared absorption spectroscopy has been used to aid in determining the structures of resveratrol oligomers. Resveratrol oligomers present several characteristic peaks such as a broad 0-H stretch and C=C stretching and bending vibrations. However, some of the most useful information obtained from the IR spectra of resveratrol oligomers result from some of the less commonly seen structural features in these compounds. The most informative of these is the presence or absence of a carbonyl stretch as was noted for caraganaphenol A (29a) [65], kobophenol B (16b) [45], gnetin A (43) [74], and leachianol C (33b) [70].

536 Mass Spectrometry Mass spectrometry has proven to be an important tool for quickly and reliably identifying the number of monomeric units of 1 that are incorporated into an oligomer. A variety of ionization techniques have been used on resveratrol oligomers with widely varying results. Standard electron impact techniques have occasionally resulted in failure to observe a molecular ion [100]; however, the use of other 'soft' ionization methods has helped circumvent this problem. Some of the more commonly employed techniques that have repeatedly been used with a high degree of success include electrospray [106], field desorption [47,65], and fast atom bombardment [39,42,53,67,68,92,99,103] ionization. In addition the utility of mass spectrometry for identifying the molecular weight of resveratrol oligomers, this technique has been used in a few limited applications for direct structural analysis. This type of application of mass spectrometry can be seen in a portion of the work regarding the structure elucidation of selected resveratrol oligomers [45,52,59,63]. More recently, Senda and colleagues [40] have used a combination of electron impact and secondary ionization mass spectrometry to investigate the fragmentation of three resveratrol oligomers. It is interesting to note that in some cases a greatly enhanced molecular ion has been observed using negative ion analysis [70,86,98,99,106]. *H and ^^C NMR Spectroscopy The development of modern NMR techniques has had a significant impact on structure elucidation studies of resveratrol oligomers. Highresolution ^H NMR spectra of resveratrol oligomers present several key features that aid in the structure elucidation of these compounds. For example, the presence of para-substituted phenolic moieties are readily discernible as a pair of AA'XX' doublets, each integrating to two protons, between 6.4-7.6 ppm with an approximate coupling constant of J=8 Hz. The 1,3,5-trisubstituted resorcinol moiety also presents some readily recognizable features including a doublet integrating to two protons at

537

approximately 6.2 ppm (J=2 Hz) and a triplet integrating to one proton around 6.0 ppm (J=2 Hz). Most resveratrol oligomers also possess two or more aliphatic proton spins between 3.0-5.5 ppm depending on the degree of unsaturation. A great deal of important information can also be gleaned from the ^"^C NMR spectra of the resveratrol oligomers. These compounds contain several characteristic spins that aid in the rapid determination of the presence or absence of certain key structural features of the resveratrol oligomers. For example, the presence of a signal between 95-103 ppm is a key indicator of a dihydrobenzofuran system, which is a common structural moiety observed in approximately 80% of the resveratrol oligomers. Most other spins appear at a lower field between 105-160 ppm. The high-field portion of the NMR spectra of resveratrol oligomers (approximately 35-95 ppm) contain, on average, about 10% of the ^^C spins, but these are exceptionally useful for rapidly identifying (or excluding) many of the structural moieties incorporated in the resveratrol oligomers. The ^H and ^"^C NMR data for the majority of the resveratrol dimers are presented in Tables (3) and (4), respectively. The data presented in these tables represent the typical range of chemical shift values and coupling constants that are encountered with all known resveratrol oligomers. Since most resveratrol oligomers can be considered as a dimer to which additional resveratrol monomers are fused (i.e. the trimer, leachianol D (34b), is the dimer pallidol fused with a third resveratrol moiety), these tables can be used to quickly help identify which structural moieties may be present in a new resveratrol oligomer. Other ID and 2D NMR Techniques Several ID and 2D NMR techniques have become standard tools for structure elucidation studies of the resveratrol oligomers. The most commonly employed of these include the one- bond ' ^ C - ' H H E T C O R and HMQC, as well as, the two- and three-bond '^C-*H FLOCK [106], COLOC [67,72,88,106], and HMBC [53,71,78] pulse sequences. Typical ^"Vc-H coupling constant values used for these experiments range between J=8-12 Hz. Other commonly used ' H - ' H correlation methods include standard COSY [64] and NOESY [67,72,78] pulse sequences; however, the most widely employed experiment concerning proton-proton

Table 3.

H N M R Data of Selected Resveratrol Dimers (6, multiplicity, J in Hz)

1

Reference

ampelopsin D (58)

ampelopsin F (61a)

(acetone-d,)

(acetone-d,)

1821

1831

anigopreissin A (51) (acetone-d,)

1791

Position 2

6.90 d (8.3)

6.99 d (8.5)

6.97 d (7.5)

7.46 d (8.8)

3

6.65 d (8.3)

6.62 d (8.5)

6 63 d (7 5)

6.80 d (8.8)

5

6.65 d (8.3)

6.62 d (8.5)

6.63 d (7.5)

6.80 d (8.8)

6

6.90 d (8.3)

6.99 d (8.5)

6.97 d (7.5)

7.46 d (8.8)

7 8

5.45 d (5.0)

4.15 brs 4.02 brs

4.07 brs

5.42 brs

3.23 brs

5.97 d (2.0)

10

6.49 d (2.2)

12

6.16 d (2.3)

5.99 t (2.0)

5.94 d (2.5)

6.41 t (2.2)

14

6.62 d (2.3)

5.97 d (2.0)

6.39 d (2.5)

6.49 d (2.2)

2‘

7.12 d (8.3)

7.05 d (8.5)

6.66 d (7.5)

7.45 d (8.5)

3’

6.78 d (8.3)

6.52 d (8.5)

6.44 d (7.5)

6.85 d (8.5)

5,

6.78 d (8.3)

6.52 d (8.5)

6.44 d (7.5)

6.85 d (8.5)

6’

7.12 d (8.3)

7.05 d (8.5)

6.66 d (7.5)

7.45 d (8.5)

7’

5.77 d ( I 1.7)

6.91 brs

3.52 brs

7.13 d (16.3)

8’

4.17d(11 7)

4.01 brs

7.06 d (16.3)

10’

7.25 d (0.8)

12’

6.43 d (2.3)

6.17 d (2.0)

6.03 d (2.5)

14’

6.24 d (2.3)

6.68 d (2.0)

6.32 d (2.5)

6.87 d (0.8)

1

Table 3. Compound (Solvent)

H N M R Data of Selected Resveratrol Dimers (6, multiplicity, J in Hz) (Continued)

balanocarpol (17b)’ (chloroform-d ,)

Reference

betulifol A (72a)

betulifol B (73a)

(pyridine-d,)

(acetone-d6)

cyphostemmin A (66) (acetone-d6)

1901

1901

i861

Position 2

7.59 d (8.7)

7.73 d (8.3)

7. I5 d (8.4)

7.29 d (9)

3

7.02 d (8.7)

7.25 d (8.3)

6.83 d (8.4)

6.91 d (9)

5

7.02 d (8.7)

7.25 d (8.3)

6.83 d (8.4)

6.91 d (9)

6

7.59 d (8.7)

7.73 d (8.3)

7.15 d (8.4)

7.29 d (9)

7

5.79 d (9.9)

5.79 d (10.3)

5.35 d (5.1)

8

5.28 d (9.9)

4.81 d(10.3)

4.40 d (5.1)

10

6.22 d (2.4)

6.76 brs

6.04 d (2.1)

6.32 d (2 5)

12

6.28 d (2.4)

6.83 brs

6.04 d (2.1)

6.28 d (2.5)

a 4.13 bd (15) b 4.02 bd (15)

6.04 d (2.1)

14 2’

6.77 d (8.7)

7.73 d (8.3)

6.93 d (8.4)

7.08 d (9)

3’

6.57 d (8.7)

7.25 d (8.3)

6.65 d (8.4)

6.79 d (9)

5’

6.57 d (8.7)

7.25 d (8.3)

6.65 d (8.4)

6.79 d (9)

6’

6.77 d (8.7)

7.73 d (8.3)

6.93 d (8.4)

7.08 d (9)

7‘

4.88 d (1.8)

5.79 d (10.3)

4.93 d (8.7)

8’

5.40 d (1.8)

4.81 d (10.3)

4.58 d (8.7)

10’

6.03 d (2.2)

6.76 brs

6.67 d (2.0)

12’

6.21 d (2.2)

6.83 brs

6.37 d (2.0)

14’ a

pentarnethyl ether derivative

6.49 d (2.5) 6.3 I t (2.5) 6.49 d (2.5)

Table 3.

1

H NMR Data of Selected Resveratrol Dimers (6, multiplicity, J in Hz) (Continued)

(Solvent)

cyphostemmin B (67) (acetone-d6)

Reference

1861

Compound

(+)-E-viniferin ( I l a ) (methanol-d4)

11051

E-viniferin diol (78a) (acetone-d,)

gnetin A (43) (acetone-d6)

1921

1751

Position 2

7.06 d (9)

7.13d(8.8)

6.77 d (8.6)

7.56 d (8.5)

3

6.80 d (9)

6.76 d (8.8)

6.62 d (8.6)

6.88 d (8.5) 6.88 d (8.5)

5

6.80 d (9)

6.76 d (8.8)

6.62 d (8.6)

6

7.06 d (9)

7.13 d (8.8)

6.77 d (8.6)

7.56 d (8.5)

7

4.28 brs

5.36 d (6.6)

4.35 d (4.0)

7.28 d (16.0)

4.51 d (4.0)

7. I5 d (16.0)

8

4.41 brs

4.34 d (6.6)

10

6.45 d (2.5)

6.15 d (2.2)

12

6.33 t (2.5)

6. I7 t (2.2)

6.34 d (2.0)

3.68 ddd (7.0, 1.5, 1.0)

14

6.45 d (2.5)

6.15 d (2.2)

6.68 d (2.0)

3.87 dd (1.5, 1.0)

2‘

7.35 d (9)

7.03 d (8.8)

7.17 d (8.5)

7.09 d (8.5)

3,

6.81 d (9)

6.64 d (8.8)

6.83 d (8.5)

6.74 d (8.5)

6.26 t (1.0)

5’

6.81 d (9)

6.64 d (8.8)

6.83 d (8.5)

6.74 d (8.5)

6‘

7.35 d (9)

7.03 d (8.8)

7.17d(8.5)

7.09 d (8.5)

7,

7.19 brs

6.56 d (16. I )

5.37 d (4.0)

3.77 t (7.0)

6.81 d (16.1)

4.40 d (4.0)

3.43 d (7.0)

6.18 d (2.5)

6.19 d (2.0)

8’ 10’

6.92 d (2.5)

6.62 d (2.2)

12’

6.41 d (2.5)

6.24 d (2.2)

14’

6.32 t (2.5)

6.26 t (2.0)

6. I8 d (2.5)

6. I9 d (2.0)

rable 3.

1

H NMR Data of Selected Resveratrol Dimers (6, multiplicity, J in Hz) (Continued)

Compound

gnetin B (44)

gnetin C (45)

gnetin F (47)

gnetin G (48)

(Solvent)

(acetone-d,)

(acetone-d6)

(acetone-d,)

(pyridine-d,)

Reference

1751

1751

1781

1781 7.48 d (8.5)

Position 2

7.38 d (8.5)

7.45 d (8.5)

6.82 d (8.5)

3

6.82 d (8.5)

6.85 d (8.5)

6.66 d (8.5)

7.00 d (8.5)

5

6.82 d (8 5)

6.85 d (8.5)

6.66 d (8.5)

7.00 d (8.5)

6

7.38 d (8 5)

7.45 d (8.5)

6.82 d (8.5)

7.48 d (8.5)

7

6.91 d (16.5)

7.11 d(16.5)

2.4-2.6

6.41 d (5.0)

8

6.76 d (16.5)

6.97 d (16.5)

2.4-2.6

6.16 d (5.0)

10

6.19s

6.60 brd (1.0) 6.26 d (2.0)

6.78 d (2 0)

12

3.15 d(14.0) 3.83 d (14.0)

14

3.02 s

6.70 brd (1 .O)

6.30 d (2.0)

7 45 d (2.0)

2.

6.92 d (8.5)

7.22 d (8.5)

7.14 d (8.5)

7.35 d (8.5)

3‘

6.65 d (8.5)

6.84 d (8.5)

6.89 d (8.5)

6.97 d (8.5)

5’

6.65 d (8.5)

6.84 d (8.5)

6.89 d (8.5)

6.97 d (8.5)

6’

6.92 d (8.5)

7.22 d (8.5)

7.14 d (8.5)

7.35 d (8.5)

7‘

3.15 d (5.5)

5.39 d (4.5)

5.34 d (6.0)

6.02d(11.5)

8’

3.02 d (5.5)

4.39 d (4.5)

4.19 d (6.0)

4.95 d (1 1.5)

6.20 d (2.0)

10’

6.14 d (2.0)

6.17 d (2.0)

12’

6.12 t (2.0)

6.24 I (2.0)

6.28 I (2.0)

7.09 d (2.5)

14’

6.14 d (2.0)

6.17 d(2.0)

6.20 d (2.0)

6.70 d (2.5)

Table 3. Compound

I

H N M R Data of Selected Resveratrol Dimers (6, multiplicity, J in Hz) (Continued)

leachinol G (37) (acetone-d,)

malibatol A (2Ib) (acetone-d,)

parthenocissin A (64b)

(Solvent)

(acetone-d6)

restrytisol A (91b) (acetone-d6)

Reference

1711

1531

1871

11061

2

7.07 d (8.0)

7.45 dd (8.5,2.5)

6.99 d (8.8)

7.17 d (8.4)

3

6.72 d (8.0)

6.80 d (8.8)

6.64 d (8.4)

5

6.72 d (8.0)

6.80 dd (8.5, 2.5) 6.80 dd (8.5, 2.5)

6.80 d (8.8)

6.64 d (8.4)

6

7.07 d (8.0)

7.45 dd (8.5,2.5)

6.99 d (8.8)

7.17 d (8.4)

7

4.48 dd (8.0, 4.0)

4.26 d (1.8)

5.82 d (4.4)

Position

8

3.40 dd (8.0,4.0)

3.45 brs

3.84 dd (4.4, 6.2)

10

5.75 d (2.0)

6.51 d (2.5)

6.19 bn

6.1 I d (2.3)

12

6.22 d (2.0)

6.30 d (2.5)

6.19 brs

5.95 t (2.2)

6.19 brs

6. I I d (2.3)

6.87 d (8.0)

7.02 dd (9.0, 2.5)

7.20 d (8.3)

7.28 d (8.4)

14 2’ 3’

6.7 I d (8.0)

6.33 dd (9.0, 2.5)

6.72 d (8.3)

6.77 d (8.8)

5’

6.71 d (8.0)

6.33 dd (9.0, 2.5)

6.72 d (8.3)

6.77 d (8.8)

6’

6.87 d (8.0)

7.02 dd (9.0, 2.5)

7.20 d (8.3)

7.28 d (8.4)

7’

4.26 d (4.0)

5.46 dd (2.5, 1.0)

6.31 brs

5.51 d (10.3)

8’

3.48 t (4.0)

5.28 ddd (2.5, 1.0, 1.0)

10’

6.14 d (2.0)

7.01 dd (2.0, 1.0)

12’

6.16 t (2.0)

6.57 dd (2.0, 1.0)

14’

6. I4 d (2.0)

3.95 dd (6.4, 10.4) 6.08 d (2.2) 6.26 d (1.5)

6.05 t (2.2)

6.52d(1.5)

6.08 d (2.2)

Table 3.

I

H NMR Data of Selected Resveratrol Dimers (6, multiplicity, J in Hz) (Continued)

Compound

restrytisol B (92b)

restrytisol C (93b)

tricuspidatol A (71)

viniferifuran (88)

(Solvent)

(acetone-d6)

(acetone-d6)

(acetone-d6)

(methanol-d,)

Reference

11061

11061

1891

11051

Position

2

7.09 d (8.4)

6.34 d (8.0)

7.21 d (8.5)

7.42 d (8.8)

3

6.62 d (8.4)

6.41 d (8.0)

6.87 d (8.5)

6.69 d (8.8)

5

6.62 d (8.4)

6.41 d (8.0)

6.69 d (8.8)

6

7.09 d (8.4)

6.34 d (8.0)

6.87 d (8.5) 7.21 d (8.5)

7

5.49 d (8.8)

7.42 d (8.8)

5.25 dd (5.0, 1.5)

8

3.97 t (9.2)

6.84 brs

3.50 dd (5.0, 1.5)

10

6.06 d (2.2)

6.00 brs

6.25 brs

12

5.96 t (2.0)

6.28 d (2.2)

6.25 brs

6.47 t (2.2)

14

6.06 d (2.2)

6.25 brs

6.40 d (2.2)

2’

7.31 d (8.4)

7.26 d (8. I )

7.21 d (8.5)

6.98 d (8.8)

3’

6.82 d (8.4)

6.84 d (8.0)

6.87 d (8.5)

6.65 d (8.8) 6 65 d (8.8)

6.40 d (2.2)

5’

6.82 d (8.4)

6.84 d (8.0)

6.87 d (8.5)

6’

7.31 d (8.4)

7.26 d (8.1)

7.21 d (8.5)

6.98 d (8.8)

7,

5.00 d (9.5)

4.79 d (4.0)

5.25 dd (5.0, 1.5)

6.94 d (16.3)

8‘

3.41 t (9.5)

4.62 d (4.4)

3.50 dd (5.0, 1.5)

6.85 d (16.3)

10‘

6.26 d (2.2)

6.50 d (2.2)

6 25 brs

6.99 d (2.0)

12’

6.18 t (2.0)

6.28 t (2.2)

6.25 brs

6.80 d (2.0)

14’

6.26 d (2.2)

6.50 d (2.2)

6.25 brs

VI P

W

544

X NMR Data of Selected Resveratrol Dimers (5)

Table 4. Compound (Solvent) Reference Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14

|

r 2 3 4 5 6 7 8 9

loll

12 13

1

1£

J

ampelopsin D (58)

ampelopsin F (61a)

anigopreissin A (51)

{sLCttont-d 6)

(acetone-fiffi)

f821

(acetone-flffi) f83]

137.7 129.2 116.4 157.0 116.4 129.2 59.0 59.9 149.7 106.8 159.6 101.7 159.6 106.8 130.0 131.4 116.7 157.6 116.7 131.4 123.0 143.4 147.9 124.2 156.4 98.8 160.0

138.7 130.3 116.0 156.4 116.0 130.3 47.5 58.5 147.7 128.2 153.4 102.3 158.8 104.6 135.7 129.6 115.9 156.4 115.9 129.6 50.8 50.1 147.9 113.8 158.1 102.3 157.3

123.0 128.6 116.1 158.4 116.1 128.6 150.6 116.3 136.5 109.7 159.5 102.9 159.5 109.7 130.0 128.9 116.4 158.1 116.4 128.9 128.7 126.8 136.4 101.4 156.5 118.6 152.4

104JI

J

106_J

™

J

107_4

545

Table 4.

"C NMR Data of Selected Resveratrol Dimers (6) (Continued)

Compound

betulifol A (72a)

betulifol B (73a)

(+)-e-viniferin (11a)

(Solvent)

(pyridine-£?5)

(acetone-c?6)

(methanol 4)

Reference

[90]

1901

1105] 133.9

Position

1

1

130.5

134.2

2

130.3

128.7

128.2

3

116.7

116.5

116.3

4

159.7

159.1

1584

5

116.7

116.5

116.3

6

130.3

128.7

128.2

7

92.8

93.7

94.8

8 9

48.3 136.8

56.9 148.0

58.3 147.4

10

104.5

106.9

107.5

11

159.7

160.0

160.1

12

97.3

102.0

102.2

13 14

160.8

160.0

160.1

121.8

106.9

107.5

r

130.5

131.2

130.4

2

130.3

127.5

128.8

3

116.7

115.4

116.4

4

159.7

158.0

158.5

5

116.7

115.0

116.4

6

130.3

127.5

128.8

7

92.8

74.5

123.7

8

48.3

75.3

130.4 136.9

9

136.8

134.6

10'

104.5

108.2

104.4

ir 12

159.7 97.3

159.1 96.7

159.8 96.9

13

160.8

mj

161.9 n92

162.9

1£

120J

546

Table 4.

1

"C NMR Data of Selected Resveratrol Dimers (5) (Continued)

Compound

gnetin A (43)

gnetin C (45)

(Solvent)

(acetone-flffi)

(acetone-i/g)

(acetone-fifs)

^75]

[75]

1781

127.8

133.0

132.2

130.1

127.9

130.2

115.8 161.8 115.8

115.5

115.6 155.9

130.1

127.9

115.6 130.2

138.8

126.0

36.7

122.7

128.4

35.7

156.9

140.4

140.7

126.7

98.7

120.0

195.5

162.1

161.7

72.8

114.3

95.4

203.6

154.3

159.1

Reference

|

1

gnetin F (47)

Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14

56.4

107.3

109.2

r

129.3

129.2

133.5

2 3 4 5 6 7 8 9 10'

130.3

127.0

116.4

115.7

127.8 116.0

159.3 116.4

156.9 115.7

157.8 116.0

130.3

127.0

127.8

54.2 51.7

92.8 55.0

93.8 56.8

146.9

145.3 106.2 158.4

147.1

105.5 159.7 102.1

101.4

101.9

159.7

158.4

ir 12 13

1

156.9 115.5

]£

J

105_5

J

1062

107.0 159.7 159.7

J

107^0

1

547

Table 4.

"C NMR Data of Selected Resveratrol Dimers (6) (Continued)

Compound (Solvent) Reference Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10' 11 12' 13

1

1£

1

gnetin G (48)

leachianol G (37)

(acetone-^/g) [78]

(acetone-rffi) [71]

129.8 127.8 114.7 157.1 114.7 127.8 69.8 42.5 138.8 116.9 158.6 95.9 157.6 104.2 131.2 128.6 114.7 155.9 114.7 128.6 87.1 48.2 141.7 117.5 154.7 100.3 157.6

135.6 129.4 115.2 157.2 115.2 129.4 77.3 62.5 147.2 105.6 158.5 102.4 154.8 123.1 137.9 129.2 115.6 157.1 115.6 129.2 55.9 59.0 151.3 106.2 159.2 101.0 159.2

124.7 130.9 116.4 159.1 116.4 130.9 151.2 117.3 135.8 109.7 156.7 102.2 157.5 121.3 133.4 130.6 114.7 155.2 114.7 130.6 48.8 74.8 139.7 109.9 155.4 95.9 156.2

1^2

n9A

1094

1

malibatol A (21b) (methanol-£f4) i

153]

1

548

Table 4.

C NMR Data of Selected Resveratrol Dimers (5) (Continued)

Compound (Solvent) Reference Position 1 2 3 4 5 6

|

"7

1

parthenocissin A (64b) (acetone-c^g)

restrytisol A (91b) (acetone-f/g)

187]

[1061

[106]

137.4 128.9 115.8 156.6 115.8 128.9 54.9 64.4 145.6 106.9 159.3 101.5 159.3 106.9 129.9 130.6 115.9 157.2 115.9 130.6 125.2 149.9 142.9 128.0 155.4 104.1 158.5

131.1 127.3 114.4 155.6 114.4 127.3 84.2 57.6 139.8 109.4 157.5 100.6 157.5 109.4 134.5 127.5 115.0 156.8 115.0 127.5 82.3 60.4 139.8 107.7 157.8 100.9 157.8

1317 128.1 114.2 155.9 114 2 128.1 83.6 59.2 142.6 108.0 157.7 100.5 157.7 108.0 131.5 128.1 115.0 157.0 115.0 128.1 86.6 59.8 142.5 106.8 158.5 101.2 158.5

8 9 10 11 12 13 14

r 2 3 4 5' 6 7 8' 9' 10 11' 12 13

1

MJ

J

mA

1

1077

|

restrytisol B (92b) (acetone-^g)

J

1_06^8

1

549 Table 4.

"C NMR Data of Selected Resveratrol Dimers (5) (Continued)

Compound (Solvent) Reference Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9'

loll 12 13

1

1£

1

restrytisol C (93b) (acetone-^/g)

tricuspidatol A (71) (acetone-fi^g)

[106]

189]

130.0 130.4 113.9 155.8 113.9 130.4 138.7 123.6 148.9 105.2 156.3 101.4 150.9 124.7 134.5 129.7 114.9 155.6 114.9 129.7 49.8 54.0 145.3 105.2 158.7 101.2 158.7 105^2

133.7 128.3 115.6 157.3 115.6 128.3 87.9 63.3 145.4 107.4 158.9 102.9 158.9 107.4 133.7 128.3 115.6 157.3 115.6 128.3 87.9 63.6 145.4 107.4 158.9 102.9 158.9 107_4

viniferifuran (88) (methanol-£^4) !

11051 123.8 128.4 116.2 158.4 116.2 1284 150.6 117.4 138.7 110.2 160.6 103.1 160.6 110.2 130.7 128.8 116.3 158.2 116.3 128.8 123.3 129.3 133.3 107.4 156.5 97.3 156.4

1

122^5

550

correlations is the difference NOE pulse sequence [65,72,87,106]. This experimental technique has proven to be extremely useful for assigning the relative stereochemistry of many resveratrol oligomers since coupling constants are notoriously poor predictors of stereochemistry in many of the five-, six-, seven-, and eight-membered ring systems encountered in these compounds [87,106]. Most NMR experiments with resveratrol oligomers are performed in deuterated acetone. This solvent has proven itself ideal for this class of compounds due to the high degree of solubility of the resveratrol oligomers and the lack of overlap between the solvent and analyte peaks. The second most commonly employed solvent is deuterated methanol, with deuterated dimethyl sulfoxide and pyridine used only occasionally. In some cases the NMR spectra of some 0-methyl and 0-acetyl resveratrol oligomer derivatives have been collected in deuterated chloroform [51,57,93,103]. Despite the exceptional analytical power afforded by the combination of these previously outlined techniques, discrepancies still remain regarding the structures and trivial names of some resveratrol oligomers (for example, see [2], [71,78], [82,86,118], [85], and [92,97,98]). Further comparative studies are needed to critically evaluate these incongruities. X-ray Crystallography In most cases, resveratrol oligomers have proven recalcitrant to crystallization preventing the widespread application of X-ray crystallographic techniques. Most resveratrol oligomers reportedly precipitate as amorphous off-white to reddish powders. This is likely the reason why there has only been one study published to date reporting the X-ray crystal structure of an 0-methyl, dibromo-derivative of hopeaphenol (14b) [50]. Circular Dichroism Due to the resistance of most resveratrol oligomers to forming suitable crystals for X-ray analysis, alternative measures have been sought in order to define the absolute stereochemistry of these compounds. As a result, circular dichroism (CD) studies have been applied to a large

551 number of resveratrol oligomers yielding important clues regarding the absolute configuration of many of these compounds. The first reported application of CD analysis to determine the absolute configuration of a resveratrol oligomer was published by Lins and colleagues [78]. In this study, the CD spectra of two neolignans, one possessing a 7-S, 8-5 absolute stereochemistry (96) and the other a 7-7?, 8-i? absolute stereochemistry (97), were compared with the CD spectra of gnetins F-I (47-50). In each case, the gnetins exhibited a negative Cotton effect at approximately 300 nm which was comparable to the negative Cotton effect exhibited by neolignan 96 at 280 nm. Therefore, the authors concluded that gnetins F-I each possessed an absolute stereochemistry of 7'S, S-S as shown for structures 47-50. In the years subsequent to this study, a number of research groups have utilized the absolute configuration assignments proposed for 47-50 in order to determine the absolute stereochemistry of a number of resveratrol oligomers. For example, Kurihara and colleagues [46] observed that a 7',8'-dihydroderivative of (-)-6'-viniferin (lib) obtained from Carex pumila exhibited identical EIMS and NMR spectral data to that obtained for gnetin F (47). However, the CD spectrum of this compound possessed a completely opposite pattern as compared to 47 indicating that the 7',8'-dihydroderivative of l i b was the enantiomer of 47. Based on these data, the authors were able to infer that the l i b found in the Cyperaceae possessed a 7-i?, 8-7? absolute configuration while the enantiomer, (+)-6'-viniferin (11a), obtained from the Vitaceae shared the same absolute configuration (7-iS', 8-iS) of the dihydrobenzofuran moiety as that observed in the Gnetaceae. More recently, Ito and colleagues [93] were able to independently assign the absolute stereochemistry of another resveratrol oligomer using spectroscopic methods. A sample of (-f)-ampelopsin A (55a) from Ampelopsis brevipedunculata var. hancei was converted by chemical means to yield a pentamethyl ketone derivative. Difference NOE spectroscopy was then used to assign the relative stereochemistry and conformation of the molecule in solution. CD spectroscopy of the derivatized (+)-ampelopsin A gave a positive Cotton effect at 357 nm; thereby, the authors were able to apply the 'octant rule' and deduce the absolute stereochemistry of (+)-ampelopsin A as shown for 55a.

552

Chemical Methods of Structural Analysis There have been no reports of the total synthesis of any resveratrol oHgomer. However, numerous chemical derivatization methods have been utilized to help elucidate the structures of these compounds. Two of the most commonly employed methods include simple methylation and acetylation procedures [81,92,103,105]. Methylation is generally performed in acetone with dimethyl sulfate and potassium carbonate under reflux for 12 h at approximately 80 °C. Acetylation is commonly carried out in pyridine with acetic anhydride for 24 h at room temperature. The 0-methyl and 0-acetyl derivatives of the resveratrol oligomers have been utilized to help deduce the number and type of hydroxyl groups present due to the resistance of aliphatic hydroxyl groups to methylation while remaining susceptible to acetylation [81]. In addition, the methylated and acetylated derivatives of the resveratrol oligomers provide a new source of non-exchangeable protons as targets for conformational NOE studies and also serve to protect labile hydroxyl groups when further chemical derivatization methods are employed. Some of the other chemical methods used to help deduce the structures of resveratrol oligomers include ozonolysis [92,93,105], hydrogenation [91], and enzyme-mediated catalysis [39,103]. In addition, ring-closure procedures have been performed on both r-viniferin (85b) [99] and miyabenol A (12) [119] to yield r-2-viniferin (86a) and miyabenol B (13), respectively, as a means of confirming their assigned structures and illustrating their biogenic origins. BIOLOGICAL OLIGOMERS

ACTIVITIES

OF

THE

RESVERATROL

Resveratrol oligomers are known to exhibit a wide-array of biological activities. Current evidence indicates that many resveratrol oligomers play a variety of important roles in plants ranging from inhibition of the spread of microbial infection to prevention of insect predation. It has also come to light in recent years that a number of resveratrol oligomers possess a host of promising pharmacological properties. Some of these resveratrol oligomers may ultimately serve as lead compounds in the development of new drug entities.

553 The Role of Resveratrol Oligomers in Plants In some plant species, the production of stilbene monomers and oligomers has been shown to be associated with a variety of stress factors [2,112,120,121,122,123,124]. The stilbene 1 is one of the most commonly encountered of these stress metabolites. While 1 has been shown to exhibit rather modest antimicrobial properties, some of its oligomeric derivatives have demonstrated more potent phytoalexin activity [2,8,125,126]. The biochemical events that unfold in plants following microbial infection and the subsequent production of defensive stilbenoids have been previously reviewed [2]. These events will be briefly outlined here with further consideration for the potential deleterious effects that the resveratrol oligomers may have on the host plant (Fig. (3)). Upon infection by an invading microorganism, plants respond by up-regulating the enzyme, stilbene synthase, resulting in a rapid and localized accumulation of 1 surrounding the area of infection. The phytoalexin 1 is then metabolized by the plant yielding a variety of stilbene monomers and oligomers. These compounds possess varying degrees of antimicrobial activity thereby preventing the spread of the microbial invader. For example, several resveratrol oligomers such as a-viniferin (8) [8], canaliculatol (24b) [57], ^-viniferin (11) [8], and vaticaffinol (25b) [58] have been shown to inhibit the growth of the fungal plant pathogen Cladospohum cladosporioides. In addition, 8 and 11 have demonstrated antifungal activity against other plant pathogens such as Botrytis cinerea, Plasmopara viticola, and Piricularia oryzae [8]. Some microbial pathogens can circumvent the defensive response of plants by biotransforming the antimicrobial stilbenoids in a multi-step oxidative detoxification process [106]. Research has shown that the pathogenicity of B. cinerea strains is positively correlated with these fungi's production of blue-copper oxidases known as stilbene oxidases or laccases [127,128]. These enzymes are polyphenol oxidases capable of catalyzing the oxidation and polymerization of numerous phenolic substrates [129,130,131,132]. It has been shown that 1 is readily transformed in the presence of B. cinerea culture medium filtrates that contain laccases [107]. Recently, six resveratrol dimers (restrytisols A-C

stilbene synthase transcription and translation

'\' @

resveratrol synthesis #

cellular fragments

invading fungal pathogen

inhibition

photosynthesis

resveratrol oligomers translocation to non-photosynthetic tissues Fig. (3). Biosynthesis of resveratrol oligomers in plants

555 (91b-93b), resveratrol trans-dehydrodimer (42b), leachianol F (36), and pallidol (10)) were reported as the products of the oxidative detoxification of resveratrol by growing B. cinerea cuhures [106]. Additional evidence suggests that these metabolites of 1 may be sequestered in the conidia vacuoles and subjected to further metabolic transformations by B. cinerea [133]. Further studies have indicated that 1 and its oligomers may be capable of inhibiting insect predation by serving as ecdysteroid antagonists [80]. In vitro studies have shown that cz^-resveratrol (98) and suffruticosols AC (52-54) may interact with insect ecdysteroid receptors in an antagonistic fashion, thereby preventing proper growth and development of the insect to occur. It is also important to consider any possible deleterious effects that resveratrol and its metabolites may have on plants. Production of defensive stilbenes in plants results in resource competition between parallel biosynthetic pathways. For example, one group of metabolites that may be severely impacted by this competition is the chalcones and their metabolites that play a variety of important roles in plants. These compounds share a pool of basic precursors with the stilbenoids [134,135]. Alterations in stilbenoid production could have far reaching effects on the health of the plant. The severity of this problem has been highlighted by Fischer and colleagues [136] who reported that overexpression of a stilbene synthase gene in transgenic tobacco and petunia plants resulted male in sterility. Stilbenes can cause a variety of other physiological problems for plants. Stilbenes alter ion transport by inhibiting ATPase activity [137,138,139]. Other stilbenes, including 1, have been shown to be rather effective in vitro inhibitors of photosynthesis [140,141]. As a result, the production of defensive stilbenes could have severe consequences on normal metabolic activities in plants. Accordingly, it is imperative that plants maintain guarded regulation over the synthesis of 1. Therefore, following the synthesis of 1 and other stilbenoids, it is important that these compounds are sequestered in storage vesicles in order to reduce their negative impact upon basic physiological functions in the plant [140]. As a feasible alternative, plants may transform 1 into oligomers as a means of detoxification (Fig. (3)). By forming oxidized resveratrol oligomers, the levels of the 'free' monomer are reduced, thereby sparing the plant from the deleterious effects of 1 on normal metabolic processes.

556 The resveratrol oligomers could then be stored by the plant in nonphotosynthetic tissues where they could act as defensive tannins [77] in the stems and roots. Alternatively, the resveratrol oligomers may be incorporated as part of the protective lignified tissues that are typically produced as a barrier at the site of infection [142]. Pharmacological Properties of Resveratrol Oligomers The diversity of biological properties associated with polyphenolic compounds is unquestionable [135,142,143,144]. These compounds have demonstrated great potential as pharmacological agents for treating a plethora of diseases. This includes the resveratrol oligomers that have been shown in recent years to exhibit an array of promising biological properties (Table (5)). Several resveratrol oligomers have been tested for antimicrobial properties. Many have shown moderate inhibitory activity against bacterial pathogens such as Escherichia coli and Staphylococcus spp. [41,43^44,45,52,54,59,62]. Other resveratrol oligomers have been evaluated for anti-HIV activity. In vitro testing of two resveratrol oligomers, balanocarpol (17b) and dibalanocarpol (20b), showed that both possessed moderate inhibitory activity against the HIV virus with EC50 values of 20 //g/mL and 46 //g/mL, respectively [53]. Several other resveratrol oligomers have been tested; however, these compounds did not inhibit HIV-viral replication [106]. Free-radical generation and inflammatory processes are associated with the development of a host of diseases such as arthritis, cancer, cardiovascular disease, and immune disorders. Several resveratrol oligomers have been screened for their ability to inhibit selected steps associated with the development of these diseases. A number of resveratrol oligomers have been shown to exhibit antioxidant, antihyaluronidase, anti-inflammatory, and cyclooxygenase inhibitory activities. For example, Ono and colleagues [145] reported that several resveratrol trimers including miyabenol C (6a), c/^-miyabenol C (7a), and foeniculosides I-IV (2a-5a) exhibited potent antioxidant effects, while two of these oligomers, miyabenol C (6a) and cz5'-miyabenol C (7a), also

Table 5. ~

~~

Pharmacological Properties of Resveratrol Oligomers ~

Pharmacological

Compound

Property

Reference

Pharmacological

malibatol B (22b)

copalliferol B (28b) (-)-E-viniferin ( l l a )

cytotoxic

dehydrodimer (42b)

distichol (23b)

pallidol (10) vatdiospyroidol(26b)

kobophenol B (16b)

cyclooxygenase I

miyabenol A (12b)

inhibition

-

stemonoporol(19a)

(+)-c-viniferin (I l a ) hepatoprotective

balanocarpol(17b)

ampelopsin E (59) cis -ampelopsin

E (60)

ampelopsin C (57) hepatotoxic

cis /trans -vitisin A

(+)-a-viniferin @a)

tumor necrosis

(-)-/runs -vitisin B (79b)

foeniculoside I (2a) foeniculoside II (3a)

factor inhibition prostaglandin H,

(+)-vitisin C (81a) (+)-a-vinifcrin (8a)

foeniculoside 111 (4a)

synthase inhibition

foeniculoside IV (5a)

protein kinase C

(+)-a-viniferin (8a)

[66,146]

inhibition

kobophenol A (9)

166,1461

miyabenol C ( 6 )

[66,146]

miyabenol C (6a) cis -mivabenol C (7a)

antioxidant

resveratrol-tram

dehydrodimer (42b)

dibalanocarpol(20b)

anti-inflammatory

restrytisol C (93b)

miyabenol C (6a) vaticafinol (25b)

antihyalurouidase

resveratrol-trans -

c-viniferin (11) kobophenol A (9a)

anti-HIV

Reference

malibatol A (21b)

copaiifero~A (18a)

antibacterial

1

Compound

Property

miyabenol C (6a) cis -miyabenol C (7a)

mixture (77a/76a)

558 inhibited hyaiuronidase activity with IC50 values of 135 and 60 juM, respectively. Another resveratrol trimer, (+)-a-viniferin (8a) has been reported to possess anti-inflammatory properties [64]. In a mouse carragenininduced paw edema model, ;7.o. injections of 10 mg/kg of 8a reduced paw edema by approximately 26% after five hours. One of the important groups of enzymes associated with inflammatory processes is cyclooxygenases. It has been shown that 8a can also inhibit the prostaglandin H2 synthase activity of cyclooxygenase with an IC50 value of 7 juM [42]. More recently, other resveratrol oligomers such as resveratrol trans-dohydrodirnQv (42b) and restrytisol C (93b) have also demonstrated moderate cyclooxygenase I inhibitory activity with IC50 values of 26 and 47 //M, respectively [106]. Some resveratrol oligomers have demonstrated toxicity toward mammalian cells. It has been reported that malibatols A (21b) and B (22b) (IC50 13 and 21 jug/mL, respectively) [53], as well as, pallidol (10) and resveratrol trans-dohydrodirnQv (42b) (IC50 32 and 49 juM, respectively) [106] exhibited moderate cytotoxicity toward human lymphoblastoid cells. The resveratrol tetramer, vatdiospyroidol (26b), has shown an increased degree of cytotoxicity against several cancer cell lines including oral epidermal carcinoma (ED50 1.0 jug/mL), colon cancer (ED50 1.9 jug/mL), and breast cancer (ED50 3.8 jug/mL) [61]. Other resveratrol oligomers have been evaluated for their hepatotoxic properties [91]. Mice treated with 30 mg/kg of ampelopsin C (57a) or a mixture of CIS' and rra^^-vitisin A (77a and 76a, respectively) expressed elevated levels of plasma alanine transaminase relative to control animals while the same dose of 11a exhibited hepatoprotective effects in mice treated with carbon tetrachloride. Two other resveratrol oligomers, transampelopsin E (59) and c/^-ampelopsin E (60), were also found to possess hepatoprotective properties based on in vitro studies utilizing primary cultured rat hepatocytes treated with carbon tetrachloride [82]. In the search for new anticancer agents, many compounds have been evaluated for their abilities to interrupt normal intracellular signal transduction pathways such as those mediated by protein kinase C. Xu and colleagues [146] reported that three resveratrol oligomers, (+ysviniferin (11a), miyabenol C (6), and kobophenol A (9), inhibited the activity of rat protein kinase C with IC50 values of 62.5, 27.5, and 52.0 juM, respectively. In addition, Ito and Niwa [103] reported that both

559 (-)-rra^5-vitisin B (79b) and (+)-vitisin C (90a) were able to inhibit the activity of tumor necrosis factor; however, the full experimental details of these tests were not described. In light of the many promising biological properties associated with these compounds, it is conceivable that many new pharmacological applications of the resveratrol oligomers await discovery. CONCLUSIONS The resveratrol oligomers are a unique class of biologically active polyphenolic compounds. Compelling evidence clearly indicates that the resveratrol oligomers are involved in a number of important physiological processes in plants. The most important of these appears to be the phytoalexin role of the resveratrol oligomer; however, studies suggest that this benefit may come at a price to the plant. Additional research is needed to further define the roles that the resveratrol oligomers play in plants and to discern how these compounds impact the physiological status of a plant. Moreover, the enzymes and cofactors involved in the formation of the resveratrol oligomers are of particular interest due to their value as tools for providing a greater understanding of the stereochemical precision involved in the formation of natural products by enzymes that utilize free-radical chemistry. Due to the wide array of promising pharmacological properties exhibited by resveratrol oligomers, these compounds may serve as leads for the development of new drugs. The resveratrol oligomers show intriguing potential for serving as agents that can inhibit inflammatory processes. Further research is needed to screen additional oligomers in order to deduce meaningful structure-activity relationships for this class of compounds. In addition, many plants that produce 1 have not yet been examined for the presence of resveratrol oligomers. It is conceivable that these and other plant species may contain many new resveratrol oligomers. The future of resveratrol oligomer research appears bright as scientists continue to discover new oligomeric entities and to further define the pharmacological potential of these compounds.

560

Appendix 1.

Structures of Resveratrol Oligomers HO

(

V-OR,

2a 3a 4a 5a l2i

foeniculoside I foeniculoside II foeniculoside III foeniculoside IV c/>miyabenol C

Rl D-Glc H D-Glc D-Glc H

R2 H D-Glc D-Glc D-Glc H

OH

1 /m^T5-resveratrol

8 or-viniferin 8a (+)-a-viniferin 8b (-)-cif-viniferin

^-r^< 10 pallidol

9 kobophenol A ^OH

m-r\ H-7 H-8 11 f-viniferin 11a (+)-//'7) linked P-caryose, another new unusual monosaccharide characterised as below described as the 4,8-cyclo-3,9-dideoxy-L-^r);rroD-/(io-nonose [32, Fig. (7)]. When the ^H and ^^C NMR spectra of the hydrolysed LPS fraction were recorded, the presence of a further anomeric signal, not attributable to the completely hydrolysed caryophyllan, was observed. The 0-chain structure of the new LPS, called caryan, was determined by applying the

603 same techniques used for caryophyllan. In particular, the p-configuration for the interglycosidic hnkage between the different caryose units was deduced from the value of 170 Hz measured for the Vc,H of the anomeric carbons. This bond proved to be of (l->7) type by analysis of the NMR, GC and FAB MS spectra of the product obtained by the total permethylation of the polysaccharide fraction containing caryose [103].

32

Fig. (7). Caryose (32).

The latter fraction was subjected to methanolysis and the product [33, Fig. (8)] analysed using one- and two-dimensional NMR techniques. The caryose proved to possess a uncommon new carbocyclic structure with a fiirane ring spirofused at C-4. The relative configuration of all carbinolic hydroxy groups was deduced from the evidence of several NOEexperiments. When the methylglycoside 33 was subjected to acid hydrolysis a mixture of three compounds was obtained. They proved to be the two anomeric furanose forms [34a and 34b, Fig. (8)] of the hydrolysed 33 and the pyranosidic isomer in its a-configuration [35, Fig. (8)]. On the basis of these results the structure 32 [Fig. (7)] is defined for this novel monosaccharide [105]. The absolute configuration of caryose was determined applying the Exciton Chiral CoupUng to 36 [Fig. (8)], which is the di-O-pbromobenzoyl derivative, obtained as the main product of the isopropylidenation of the monosaccharide equilibrium mixture (34a, 34b and 35) followed by esterification with /^-bromobenzoyl chloride. The results obtained have suggested for caryose the absolute configuration

604 depicted for 32 in Fig. (7). Caryose was named, in accordance with the systematic nomenclature used for carbohydrate, as the 4,8-cyclo-3,9dideoxy-L-erytrO'D-ido-nonosQ, derived from the formal cleavage of the C-4/C-8 bond by the addition of two hydrogens [105].

HOy^W. 4 linl 9 the A1(0H)3 dissolves again. Kiel found that fixation of the textile at 40°C for 30 min or 1 min at 70°C in 3 ml sodium silicate solution (or 10 g chalk/L) are the best conditions [20].

669 6. Dyeing; The stained cloth is dyed with madder or synthetic ahzarin. A colour complex is formed between the dye (alizarin), the mordant (aluminium) and the calcium. The water bath that contains the textile and the dye is slowly heated. Finally the cloth is briefly boiled in the dye bath [20]. 7. Washing; The excess dye and other used compounds are removed from the cloth. The cloth is rinsed thoroughly in flowing water, or the cloth is washed with soap [20]. Over the years a lot of dyeing procedures have been proposed to speed up the dyeing process. Most of these procedures combine two or more steps of the Turkish red procedure. Up to now all these procedures give a qualitatively less dyed product. A lot of other mordants have also been used in the past to change the dyeing result. Most of these mordants do not increase the quality of the dyed product but merely gave a different coloured product. For example the use of the alternative mordant FeS04 -7 (-)

O(-) HO—^/^---HoO

Fig. (14). Structure of the dye complex suggested by Kiel [20]

Ca""^' 2H2O

670 H2O results in a darker and blacker colour. Copper gives a warmer and deeper colour. If the textile is placed in a tin bath after drying, the colour turns more yellow. It is assumed that the Sn(II) ions displace some of the calcium ions. This process is named "aviveren" in Dutch [20]. In the past a lot of authors have proposed a structure for the coloured complex that will be formed during the dyeing process. It was already known before 1868 that besides dye and mordant also calcium is essential for the complex. The pKa values of the alizarin hydroxyl groups at position 1 and 2 are respectively 12.0 and 8.2 making the 2-hydroxyl group the more acidic one. During the formation of the dye complex a calcium ion reacts with the 2-hydroxyl while an aluminium ion forms a complex between the 1-hydroxyl and the carbonyl group. The ratio between both alizarin and aluminium and alizarin and calcium is 2:1 [20]. After analysing IR spectra of alizarin-metal ion complexes Kiel suggested the structure depicted in Fig. (14). The model showed a complex of A P ^ chelated in the li-ketol (C-1 or C4) position of the alizarin molecule. Thirty-five years later Soubayrol et al

Azi

0\

4Na'

Fig. (15). Structure for the dye complex suggested by Soubayrol et al. [140]. For an explanation of the dashed lines see Fig. (16) and main text.

671 showed with ^'^Al NMR in the soHd state that the complex is a binuclear co-ordination complex (Fig. (15)) of the structure proposed by Kiel [140]. The binuclear species consists of a tetra anion skeleton formed around two Al^^ ions linked by two hydroxyl bridges [140]. Depending on the nature of the cation and the drying conditions the complex is surrounded by a variable number of water molecules, [Al2(|Li-OH)2Na2(Ci 41^504)4 (H20)4](H20)2(Na)2 and [Al2(^-OH)2Ca2(Ci4H604)4(H20)4](H20)2 respectively. The four-alizarinate ligands are probably bound with their eight free oxygen atoms to the four H2O molecules as shown in Fig. (15).

Fig. (16). Three-dimensional structure of one half of the binuclear dye complex suggested by Soubayrol e^ Of/. [140]

Alizarin molecules Azl and Az3 partially overlap each other with two water molecules in front of the complex. Similarly molecules Az2 and Az4 overlap each other with the two remaining water molecules at the rear of the complex. The alizarin entities, which overlap each other with two benzene rings form a sandwich structure inside of which two of the Na^ or two Ca^^ cations are probably confined (Fig. (16)). The ionic radii of Na"^ and Ca^"^ fit nicely into a benzene hexagon of 2.4 A diameter, which therefore supports the formation of (C6H6)2Na sandwich subunits. Deformation of the four 0-Al bonds in the core is probably responsible for the decrease in the apparent co-ordination number of Al in the closed complex, (a lot of strain for including the bulk of the ligands) leading to an atypical chemical shift for ^^Al near 24 ppm. If instead K"^ or Ba^"^ are used as ions the complexes [Al2(|i-OH)2(Ci4H604)4(H20)]

672 (H20)5(K)4 and [Al2(^-OH)2(Ci4H604)4(H20)3] (H20)3(Ba)2 are formed. These are called binuclear open structure complexes. The bulkier K"^ or Ba^"^ cations are not able to enter the alizarin sandwich structure and rather than having Al-O-Al bridges that are too strained, the structures remain open and the water molecules are no longer necessary. In this case, the co97

ordination number six gives standard Al chemical shifts near zero and probably standard 0-Al bond lengths near 1.86 A as in [(Al2)^-OH)2]'^^ core complexes [140]. In the case of Ba^"^, the stable complex is a trihydrate, with probably two alizarinates connected by two water molecules, the other two ligands remaining free [140]. Inspection of molecular models shows that the two water molecules of each dibenzene sandwich may be replaced by a cellobiose entity representing a unit of the cellulose fibre. This possibility provides a good explanation of the strong fixation of this dye on cotton fabric [140]. BIOLOGICAL ACTIVITY AND NON-DYEING APPLICATIONS Biological and Pharmacological Activities of Rubia Extracts Antioxidant activity of alizarin in vitro and in vivo

The antioxidant activity of alizarin was established in four different assays: (1) suppression of light emission in the /^-iodophenol enhanced chemiluminescent assay, (2) scavenging of superoxide anion (02*~) in a hypoxanthine-xanthine oxidase system, (3) protection of rat liver microsomes from lipid peroxidation by ADP/iron(II) ions, and (4) protection of bromobenzene-intoxicated mice from liver injury in vivo [141]. Alizarin was compared with Trolox (water soluble vitamin E), the flavonoid baicalin and green tea proanthocyanidins. In assay (1) the activity of alizarin was 76% of that of Trolox. In assay (2) the inhibition of 02'~-induced chemiluminescence was 40%, 32%), 23%) and 14%) for Trolox, alizarin, green tea polyphenols and baicalin respectively. Alizarin was not significantly active in the lipid peroxidation assay but after baicalin the most active compound in the in vivo assay. This shows again the difficulty in the evaluation of antioxidant activity and the differences between in vitro and in vivo assays [141].

673 Antimicrobial activity

Rath et al. have studied the antifungal activity of ten anthraquinones occurring in Morinda lucida [142]. Several of these anthraquinones also occur in madder. As bioassay during the fractionation they used the activity against Cladosporium cucumerinum and Candida albicans in TLC bioautography. Of the compounds tested only 3-hydroxy-2-formylanthraquinone, nordamnacanthal, damnacanthal and alizarin 1-methyl ether showed significant activity against both fungi. Of these four compounds the latter one was the most active with detection limits at 0.5 and 1 |ig against Cladosporium cucumerinum and Candida albicans respectively. Alizarin 1-methyl ether was the only active compound in agar dilution assays when tested against the human pathogenic fungi C albicans, Aspergillus fumigatus and Trichophyton mentagrophytes. The minimum inhibitory concentrations were 10, 100 and 50 |Lig/ml respectively [142]. The antibacterial activity of aqueous (glycosides) and ether (aglycones) extracts of madder was assessed against Salmonella typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. enteritidis, S. typhi-murium. Shigella flexneri 2a, Sh sonnei, Sh. largei-sachsii, Sh. boidii, Sh. ambigua, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli O55, Staphylococcus aureus. Streptococcus haemolyticus. Neisseria gonorrhoeae and Mycobacterium tuberculosis var. hominis [143]. The diffusion and dilution methods were used. The aqueous extract at 2% showed only activity against Sh largei-sachsii. The ether extract had a broader and stronger activity. It was active against Sh. largei-sachsii, Staphylococcus aureus and Streptococcus haemolyticus. Also the antifungal activity was determined. Both madder powder and the total aglycones showed activity against Candida albicans, Geotrichum candidum, Geotrichum louberi, Rhodotorula rubra, Rhinoclaviella sp. and Saccharomyces cervisiae in an inliibition zone assay. At 0.4 mg inhibition zones against all species varied from 11-25 mm. An aqueous extract as well as the aglycones in sunflower oil were not active against any fungus except the Rhinoclaviella species [143]. Activity against flagellates

Chagas's disease caused by the flagellate Trypanosoma cruzi is currently becoming more and more of a problem due to its wider occurrence in

674 Latin America and the lacic of good drugs. With the aim of developing new safe and efficient chemotherapy, 45 compounds were tested. Of the natural compounds tested, purpurin was the most effective one with an ID50 of 358 ± 35 |LiM. It was active against bloodstream forms also at 4°C and will be further tested for its usefulness for chemoprophylaxis in donated blood [144]. Antilithiasis activity

In rats oral intake of fresh madder root (10% of the food) decreased stone formation in bladder and kidney induced by 3% calcium carbonate [145]. In rabbits, oral intake of madder root extract (150 mg/kg) caused decreased calcium oxalate crystallization in the kidney. An increased death rate was observed in feeding experiments of rats. Furthermore, feeding experiments with rabbits showed hepatotoxic effects. Genotoxic effects were observed in bacterial in mammalian cell test systems [145]. Medicinal Uses of Rubia Extracts Phytopharmaceutical Uses

Extracts of madder root {Rubia tinctorum) contain some compounds of pharmacological interest. Crude extracts have been used for the treatment of bladder and kidney stones, especially those consisting of calcium oxalate and calcium phosphate in the urinary tract. In vitro experiments showed that ruberythric acid prevented the formation of calcium phosphate and calcium oxalate [146]. Extracts of madder roots have been used as ingredients of phytopharmaceuticals [4,39,46,57,61,63,64, 66,68,71,146]. Rubia Teep® tablets from Madaus (Koln, Germany) are an example of a madder phytopharmaceutical. With regard to the effect against stones no clinical trials exist [145]. Thus in view of the potential toxicity a negative advice about the medicinal use of madder was released by the German Commission E. Madaus withdrew Rubia Teep® from the market in 1990 due to the possible mutagenic properties of lucidin {vide infra).

675 Use as Diagnostic Tool

Alizarin can be used to stain calcium deposits in soft tissues. Dermatopathologists use it to detect dermal calcium in disorders such as pseudoxanthoma elasticum and calcinosis cutis [147]. It harmlessly stains also living tissues. Bones of animals that ate madder turned pinlc or red. The active compound responsible for the staining is supposed to be pseudopurpurin [147]. Alizarin is also used diagnostically as a marker for the study of bone growth. Alizarin and radioactive calcium are deposited similarly in growing bones, including those of the skull. The results that can be obtained by the use of alizarin or radioactive calcium are fully complementary. The hazard of the radioactive calcium to both the investigator and the patient does not arise from the use of alizarin [1,148]. Mutagenic and Carcinogenic Activity of Rubia tinctorum Extracts Because of the application of Rubia tinctorum extracts in phytopharmaceuticals and as food colourants studies on the safety of these products have been carried out. Brown and Dietrich were the first to report lucidin to be mutagenic [79]. Later different authors confirmed that this anthraquinone shows mutagenicity in several strains of Salmonella typhimurium [57,65,79,84,149-152]. The mutagenicity of lucidin was also tested in a battery of genotoxicity assays: mutagenicity in bacterial cells [65] and mammalian cells [65,68,79,84,153], induction of DNA repair in primary rat hepatocytes [68] and in vivo transformation of C3H/M2-mouse fibroblasts [68,79,154,155]. Lucidin induced unscheduled DNA synthesis in primary rat hepatocytes and transformed C3H/M2 mouse fibroblasts [57,79,84,153]. The genotoxic effect of lucidin is a matter of some concern in products containing Rubia extracts. For example, Rubia Teep® tablets contain small amounts of lucidin and lucidin primeveroside [66] and products with madder root extract are used as a food colourant. The glycoside lucidin primeveroside (a compound of madder root) is metabolised in rats to the genotoxic lucidin [57,84,150,153] and rubiadin [156,157]. In a thorough study Kawasaki et al (1992) elaborated that the mutagenicity of madder is not exclusively due to lucidin. Dried roots of

676 madder were extracted with different solvents and these extracts were fractionated by chromatography. Twenty compounds were isolated from the roots of Rubia tinctorum and these compounds were tested for their mutagenicity in Salmonella typhimurium strain TA 100 and/or TA98. 1Hydroxy-2-methylanthraquinone, lucidin-co-methylether, rubiadin, xanthopurpurin, 7-hydroxy-2-metliyl-antliraquinone, lucidin, lucidin-coethylether, lucidin primeveroside and the non-antliraquinone compound mollugin showed mutagenicity [52,84]. Mollugin is a direct mutagen. It is suggested that the phenolic hydroxyl group of mollugin reacts with oxygen and forms a phenoxyl radical and superoxide anion. Oxygen radicals react with guanine at the C-8 position [52]. Kawasaki et al further studied the mutagenicity of 25 anthraquinones to determine the structure-mutagenicity relationship. Lucidin and the alkoxy derivatives (lucidin-co-methylether and lucidin-co-ethylether) as such showed mutagenicity. Rubiadin that can be regarded as lucidin reduced at the hydroxymethyl group, showed mutagenicity only after metabolic activation. They concluded that 1,3-dihydroxyanthraquinones possessing a methyl or hydroxymethylgroup on carbon-2 show mutagenicity. An oxygenated state of the benzylic carbon-2 is essential for direct mutagenicity [52]. Lucidin-3-O-primeveroside showed mutagenicity towards Salmonella typhimurium TAIOO in the absence of metabolic activation [158]. When the glycoside was treated with hesperidinase during the preincubation period, it became more active, hi that case it was active both in the presence and absence of the S9 metabolic activation mix. This pointed in the direction of lucidin as the direct and indirect mutagen. When the preincubation period was prolonged, higher mutagenicity was found confirming the hypothesis about lucidin as the responsible compound [158]. The data confirmed earlier findings about the mutagenicity of lucidin. These mutagenic studies showed that lucidin can be metabolised to a reactive compound which forms covalent adducts with DNA and possibly other macromolecules. It was reported that lucidin forms ethers and esters upon heating with alcohol or acids. This supports the reactive character of lucidin [57,97,153]. Kawasaki et al. (1994) proved that lucidin forms adducts with the nucleic acids adenine and guanine under physiological conditions [159]. These adducts were identified as condensed reactants at the benzylic position of lucidin with a nitrogen atom of a purine base. This

677 indicated the formation of an exomethylenic compound as an electrophilic intermediate. Poginsky et al (1991) also suggested the formation of an electrophilic intermediate (Fig. (17)) that could react with DNA [57]. 0

OH CH2OH

sulphotransferase P

OH .CHsOSOaH

Fig. (17). Suggested formation of electrophilic intermediates out of lucidin that could form adducts with DNA [57,159]

To elucidate the possible carcinogenicity of madder roots, three groups of rats received either a normal diet or a diet supplemented with 1% or 10% madder for more than two years [160]. After this period all surviving animals were sacrificed and their organs studied. Weight gain and morbidity were not different among the three groups. Non-neoplastic lesions related to the treatment were evident in the liver and kidneys of both sexes. Moreover, dose-dependent increases in benign and malignant tumour formation were observed in the liver and kidneys of treated animals. ^^P-Post-labelling analysis showed an increase in the overall level of DNA adducts observed in the liver, kidney and colon of rats treated with 10% madder root in the diet for two weeks. HPLC analysis of 32p-labelled DNA adducts revealed a peak co-migrating with an adduct obtained after in vitro treatment of deoxyguanosine-3'-phosphate with lucidin. These observations implied that the long-term medicinal use of madder by humans is associated with the risk of formation of malignant tumours [160]. Use of Rubia Extracts as Food Colourants Another application of madder extract is its use as food colourant. Natural food colourants are used rather than synthetic ones, because of a consumer preference for natural products. It is widely believed that natural

678 colourants are generally safer than synthetic ones. Madder root extract has been used as a food colourant in confections, boiled fish and soft drinks in Japan due to their colours with distinctive heat and light resistant properties. Madder root anthraquinones turn purple after reacting with proteins in foods [66,71,154,161]. ABBREVIATIONS 2,4-D AD ATCC B.A.S.F. CPC DCCC DMSO DNA E.T. G.I. GLC HPLC lAA I CI IR L.E. MeOH MHz MS MS-medium NAA NMR 0V-17 SE-30 Si02 SPE TLC TMS UC-W98 UV

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

(2,4-dichlorophenoxy)acetic acid anno domini fungal strain code chemical company in Germany centrifugal partition chromatography droplet counter cliromatography dimethyl sulphoxide deoxyribonucleic acid electron transfer growth index gas liquid chromatography high pressure liquid chromatography indoleacetic acid chemical company in England infrared local excitation methanol megahertz mass spectrometry Murashige and Skoog medium 1-naphthaleneaceticacid nuclear magnetic resonance type of stationary phase for gas chromatography type of stationary phase for gas chromatography silica gel solid phase extraction thin layer chromatography trimethylsilyl typeof stationary phase for gas chromatography ultraviolet

679 VIS VOC

= visible = Dutch East India Company

ACKNOWLEDGEMENTS The authors thank Prof. R. Verpoorte of the division of Pharmacognosy, State University of Leiden, The Netherlands for his comments on the biosynthetical and cell culture sections and Prof. JE. de Groot of the Laboratory of Organic Chemistry of Wageningen University, The Netherlands for his comments on the nomenclature of compounds.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 26 © 2002 Elsevier Science B.V. All rights reserved.

685

THE CHEMISTRY AND TOXICOLOGY OF BIOACTIVE COMPOUNDS IN BRACKEN FERN (PTERIDIUM SSF), WITH SPECIAL REFERENCE TO CHEMICAL ECOLOGY AND CARCINOGENESIS MIGUEL. E. ALONSO-AMELOT Grupo de Quimica Ecologica, Departamento de Quimica, Universidad de Los Andes, Merida, Venezuela. [email protected]

ABSTRACT: The Pteridium taxonomic complex grouped around the general denomination of "Bracken Fern" is a factory of obnoxious secondary metabolites, which in combination with a series of remarkable biological traits, contribute to its status as one of the five worst weeds in the world. Bracken excludes competing vegetation effectively, fends off the great majority of herbivorous insects, poisons numerous farm animal species, and resists fire and repeated cuttings. Evidence is also accumulating that it may affect people, not only economically but also by eliciting the onset of gastric cancer by direct ingestion of boiled croziers or consumption of animal products exposed to feed on bracken fronds. The present chapter reviews the xenobiotic chemistry of bracken by structural types, including all those compounds derived from the shikimic acid and mevalonic acid biosynthetic routes, and describes recent developments in the chemical behavior, toxicology, and chemical ecology of the most notorious of these compounds. Particular reference is made to the mechanisms that bring about the various cancer scenarios that the ingestion of this plant may cause, and the molecular processes involved.

INTRODUCTION Bracken fern {Pteridium ssp) is both an evolutionary w^onder and an agricultural scourge. The characters that endow this plant with its tremendous ecological success [1] are the same that powerfully influence managed ecosystems for forestry [2-4], conservation [5-7], agriculture [8] and farm animal production [9]. Some of these characters are primitive traits preserved over the ages, and others are idiosyncratic of modem plants [10]. Among the outstanding biological features, there is the extraordinary ability of bracken to colonize new territory through dispersion of enormous numbers of microscopic spores [11-13], which may amount up to 10 g per frond in the arachnoideum genotype [14]. Carried by wind, these spores travel long distances [15-16]. Bracken also takes over new land by the rapid expansion of its profuse rhizome system [17-18]. The

686 rate of growth of rhizomes and biomass above the ground, especially in the spring months in temperate climates [19-20] and during most of the rainy season in tropical regions [18,21], is frequently greater than that of potentially competing plants. On land cleared by slashing and/or fire, not only spores germinate abundantly [22] but if the ground previously harbored bracken rhizomes that grev^ unnoticed under the existing vegetation with all but a modest aerial representation of dispersed fern blades, rapidly growing fronds are usually the first to emerge from the bare ground surface. Numerous croziers appear during the first weeks after fire or clearing, achieving high densities, Fig. (1-A). The blades swiftly cover the area with its expanding pinnae, thus retarding or inhibiting the germination and growth of other heliophile, early succession plants for critically long periods of time. Areas of periodical wildfires are especially prone to bracken growth. Pteridium perpetuation results from the survival of fire-resistant rhizomes, located deep underground sometimes, in combination with the denudation of the ground surface from competing vegetation. In the neotropics, it has been recorded that the time from frond emergence to a fully expanded blade may be less than 45 days. Fig. (1-B) [18]. In such a short time pinnae spread out to a structure of considerable dimensions, measuring more than a meter long and occasionally up to 2.5 meters. The rapid growth of individual sporophytes and their longevity make Pteridium ssp one of the largest plants loiovm [23,24]. In addition, if not flattened by snow or severe wind storms, the wilted fi-onds will remain standing and providing mechanical support for new frond growth. The buildup of litter is such that the undercroft shade at ground level becomes equivalent to the darkness of the tropical rain forest [14]. (A)

687 (B) 100 -r

u w

u

80 1 60 1

cd

ROOH -f Fl-0* HO* + Fl-OH -^ H2O + Fl-0*

(1) (2)

metals, through complexation, thereby suppressing the superoxide-driven Fenton reaction (Eq 3 and 4), which is currently believed to be the most important route to activate oxygen species [145]. 02*" + FeIII ^ 0 2 + F e l l Fe II + H2O2 ~> Fe III + HO* + HO"

(3) (4)

So far it has been widely believed that the antioxidant ability of flavonoids resides mainly in their ability to donate hydrogen atoms and thereby scavenge the free radicals generated during lipid peroxidation. In spite of some flavonoid structures which allow them to form heavy metal complexes, metal chelation has been regarded to play a minor role in the antioxidant activity of these compounds and has not been studied much by researchers Notwithstanding, some recent studies point in this direction. When erythrocytes are in the presence of oxidizing agents, iron is released in a free form (as chelatable desferrioxiamine) and does not bind to specific proteins as it is naturally occurring in living systems. This iron can easily lead to membrane lipid peroxidation and hemolysis, this being the effect of iron inside the cell [146]. Recent studies show that certain flavonoids, such as quercetin, can penetrate the cell and can bind iron, thus preventing lipid peroxidation and hemolysis. Rutin and other flavonoids can also chelate iron, but their penetration inside the cell is lower than that of quercetin [147]. When comparing the effectiveness of several flavonoids (quercetin, rutin, luteolin, chrysin, naringenin and hesperetin) at inhibiting metal-ion-induced peroxidations with peroxidations induced by the water

763 soluble free radical generator AAPM (2, 2'-azobis (2-amidino propane) dihydrochloride), researchers found that these compounds were more effective at inhibiting metal-induced peroxidation than AAPM-induced peroxidation [148]. In this study, quercetin, rutin and luteolin exhibited higher antioxidant activities than in the other flavonoids. This provides more evidence for the highly significant contribution of the 3', 4 dihydroxy phenyl substitution pattern on the B-ring, as will be discussed below with the structural requirements for these chelating properties. As has been mentioned above, flavonoids can protect the DNA from damage by acting as free radical scavengers. In this respect Noroozi et al [60] carried out a study in which the protective effect of vitamin C and several flavonoids (luteolin, myricetin, kaempferol, quercetin, apigenin, quercetin-3-glycoside and rutin) against DNA damage was compared. At the assayed concentrations the protective effect of vitamin C against DNA damage was significantly lower than that of all the flavonoids except apigenin, quercetin-3-glycoside and rutin (all of them glycosilated structures). The data suggest that the free flavonoids are more protective than the conjugated flavonoids. These data are also consistent with the hypothesis that antioxidant activity of free flavonoids is related to the number of hydroxyl groups [137, 138]. Nevertheless, the tremendous lack of antioxidant activity when compounds are conjugated suggests that polarity, as cell permeability, may play a very important role. However, so far the protective mechanism of flavonoid is not entirely clear. When prevention of DNA single strain breakage is evaluated, the chelating mechanism is shown as the main factor. By using an experimental approach based on the notion that iron chelators suppress DNA strand scission and cytotoxicity caused by tert-butylhydroperoxide (a free radical generator), whereas radical scavenging antioxidants prevent only the latter responses, Sestili et al [149] provide experimental evidence indicating that the most prominent activity of the flavonoid quercetin resides in its ability to chelate iron. Further research points in this direction. Thus, when Jurkat T cells are supplemented with green tea (very rich in epigallocatechin gallate) and treated with Fe^^ as prooxidant, they suffered much less oxidative DNA damage (single strand breaks) than those of the control group[150]. Another healthy property of flavonoids related to their antioxidant ability may be the prevention of atheroma formation. Within the last several years, the oxidative theory of atherogenesis has provided another avenue of therapy using antioxidants [151]. According to this theory.

764 antioxidants should protect lipoproteins against oxidative modification and reduce the biological consequences. Vitamin C is the major chainbreaking antioxidant against free radicals and protects LDL (Low Density Lipoproteins) from oxidation in vitro, ahhough it does not bind to LDL [152]. Flavonoids are powerful antioxidants and they act against cupric ion-induced in vitro LDL + VLDL (Very Low Density Lipoproteins) oxidation [153] and also bind to LDL [154]. When citrus extracts, containing flavonoids with and without ascorbic acid^ were tested against oxidation-susceptible lipoprotein, the authors found that citrus extracts containing flavonoids but not ascorbate did not significantly change any lipid contents. However^ citrus extracts containing flavonoids, plus ascorbate^ had a strong beneficial effect on hamster lipids (lower triglycerides and significantly decreased cholesterol and LDL+VLDL compared to the control) [155]. Thus, for the authors there appears to be an in vivo synergism of the flavonoids and ascorbate with respect to the cholesterol, LDL+VLDL, triglycerides, and the atherogenic index. However, no correlations with chelation metal ions were found [155]. Further evidence obtained from in vivo experiences supported the former theory. Thus, when rats fed with PUFA (Polyunsaturated Fatty Acids) or MUFA (Monounsaturated Fatty Acids) were supplemented with flavonoids (a mixture of quercetin and catechins) the amount of dienes produced was significantly reduced and consequently there was lipid peroxidation [156]. Structural requirements for scavenging and chelating properties Bor et al performed studies based on the generation of flavonoid radicals (aroxyl radical) by pulse radiolysis [138, 157, 158]. The observations of fast absorption changes with kinetic spectroscopy allowed the determination of the ratio of the constants, which were obtained with several oxidizing radicals of flavonols such as kaempferol and quercetin. These studies revealed on the one hand that B-ring-localized semiquinones are the major radical species observed after univalent oxidation of dihydroflavonols, flavanones, and flavanols. Simultaneously revealed on the other hand was that a saturated 2,3 bond leads to a break of the 7c-electron system between the carbonyl group and the B-ring. The presence of a 2,3-double bond (flavones and flavonols) led to a dislocation of the scavenged electron across the flavonoid structure. Such

765 results led Bor et al [138] to postulate that it was basically the presence of a B-ring catechol group which stabilized the aroxyl radical. In summary, in the chemical skeleton of the flavonoids there are three basic groups which determine the ability to scavenge radicals: (I) The o-dihydroxy (catechol) structure in the B ring which confers great stability to the aroxyl radicals and which participates in electron dislocation; (II) The double bond between carbons 2 and 3 in conjugation with a 4-oxo function, which is responsible for electron dislocation from the B ring; and (III) The presence of both 3 and 5 hydroxyl groups for maximal radical scavenging capacity and strongest radical absorption [137, 159]. When antioxidant activity of flavonoids is evaluated in liposomal systems, the 3-hydroxy group becomes less importance and an ohydroxylation is considered at A-ring as a requirement for antioxidant activity [148] Fig. (5). (I)

OH

I

OH

OH

HO.

OH

O

(IV)

(III)

OH

HO

OH--- O

OH

O

Fig. (5). Structural criteria tliat enliances the antioxidant activity of flavonoids. 1,11, III: Structural groups proposed by Bor et al [137,138, 157]. IV: Stmctural group proposed by Arora et al [148].

Antioxidant activity of flavonoids and in non-processed foodstuff which are present has been extensively studied [13, 29, 155, 160, 161,

766 162]. Concerning these, and those previously reported, there results a hierarchy of antioxidant activity that has been proposed in which flavanone 4'-hydroxylated keeps the lowest activity and flavonol with catechol structure in the B-ring the highest one. Fig. (6). Table 2 shows the antioxidant activity of some flavonoid families obtained by different researchers. OH

x/^

OH OR


rutin > luteolin > apigenin « hesperetin « naringin > diosmin « hesperidin > naringin. The flavonoids with the highest activity are quercetin, catechins and anthocyanins^ which have an antioxidant activity up to three times higher than vitamin C (TEAC (Trolox equivalent antioxidant activity): 1,12 mM) and slightly higher than licopen (TEAC: 2,9 mM) [29]. Others, such as naringin, have a similar, or even lower, antioxidant activity than vitamin C. As discussed previously flavonoids have the property of chelating bivalent metals. This ability requires a special pattern of substitution which can be supplied by an environment of bivalent negative charges. In the flavonoid structure this requirement is present in the o-hydroxyl substitution, which is often present in the B-ring, and may exist in the

769 simultaneous presence of 3-hydroxyl and 4-oxo groups under some conditions. The flavonoids present a characteristic UV/V (Ultraviolet/Visible) spectra defined by two bands. Band I (320-385 nm) relates to ring B absorption and Band II (250-285 nm) relates to ring A absorption [164]. Brown et al [165] studied the interaction between several flavonoids (quercetin, rutin, kaempferol, and luteolin) with Cu^^ ions finding spectral shifts for all the compounds. However, these shifts were greater and more intense in Band II (ring B) for those compounds with catechol structure (quercetin, rutin and luteolin) than in Band I (ring A) for those with a 4-oxo and 3-hydroxyl substitution (quercetin and OH

Low pH, M +2

Fig. (7). Chelating mechanism proposed by Samia et al [167] for a lone 3-hydroxyl substitution on flavylium cation (B) besides on catechol group (A). M^^: Bivalent metals.

kaempferol). This suggests that chelating properties of flavonoids seem to be based on catechol structure rather than on the 4-oxo group, with either

770

of its adjacent hydroxyl groups [165]. However, another class of flavonoids can chelate bivalent metals with a unique 3-hydroxyl substitution, the anthocyanins. Anthocyanins at low pH ranges exist mainly in the form of flavylium cations and because of the charge distribution, they are susceptible to nucleophilic attack on positions 2 and 4 [166]. According to this and based on spectral shift studies^ it has been postulated that the hydroxylation of an anthocyanin at these positions enhances its chelating ability [167], Fig. (7). Ascorbic acid preservation by flavonoids The antioxidant ability of flavonoids may affect several physiological processes in which redox reactions are involved. In the sixties, it was proposed that the flavonoids might act as protecting agents of ascorbic acid oxidation. Ascorbic acid is the main naturally occurring antioxidant in biological systems and therefore in foodstuffs. Besides this, its importance takes root in the fact that synergistically it may regenerate vitamin E, resulting in an enhancement of antioxidant activity in membranes and non-polar environments [168, 169, 170]. Moreover, it has been proposed that a recycling chain might exist between thiols, glutathione and lipoic acid, in which ascorbic acid and flavonoids might be involved. Thus, flavonoids may play a very important role in preserving antioxidant capacity in biological systems as in foodstuff by working on the recycling chain of antioxidants by protecting the ascorbic acid from its oxidation, for those flavonoids with a lower redox potential than the ascorbate radical/ascorbate couple [171], Fig. (8). Interaction between ascorbic acid and flavonoids has been demonstrated by Bor et al [172] when pulse radiolytic generation of azide radicals was used, which led to the formation of both ascorbate and flavonoid radicals. The same group also investigated the interactions between acorbate radicals and flavonoids when ascorbic acid was oxidized enzimatically with tyrosinase [173] while Cossin et al performed this research by enzimatically oxidizing the ascorbic acid with ascorbate oxidase [171]. This last group reported also that someflavonoids^with a triphenol structure such as myricetin, epicatechins and epicatechins gallates, compete with ascorbic acid for the ascorbate oxidase and therefore are preserving in a double way the ascorbic acid from oxidation

771 [171]. Another way to protect ascorbic acid from oxidation consists of the chelating mechanism. As has been discussed above, flavonoids with ortho-phenol structure have the ability to chelate bivalent metals^ which may undergo the oxidation of ascorbic acid, and this mechanism of protection for anthocyanins is being proposed by Sarma et al [167]. These former reports might explain what was proposed by Rusznyak and SzentGyorgyi more than six decades ago when they observed that the administration of lemon juice decreased the fragility and permeability of the capillary wall, where ascorbic acid alone had no protective effect, and led them to propose flavonoids as vitamin P.

VitE

a-lipoate and othei>>

VitE liliydrolipoate and others

Dehydroascorbate

Fig. (8). Involvement of flavonoids (Fla) in tlie ascorbate recycling pathw^ay. Proposed by Cossins etal. [171].

ABBREVIATIONS ABTS AhR AAPM BPV-4 CAMP CGMP DNA EGRFR LDL

= 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid = Aril Hydrocarbons Receptor. = 2, 2'-azobis (2-amidino propane) dihydrochloride. = Bovine Papilloma Virus type 4. = Cyclic Adenosin Monophosphate. = Cyclic Guanosine Monophosphate. = Deoxyribonucleic acid. = Epidermal Growth Factor Receptor. = Low Density Lipoproteins.

772

MUFA PCA PUFA RTK TEAC TPO UV UV/V VLDL

= Monounsaturated Fatty Acids. = Prostate Carcinoma. = Polyunsaturated Fatty Acids. = Receptor of Tyrosine Kinase. = Trolox equivalent antioxidant activity. = Thyroid Peroxidase. = Ultraviolet radiation. = Ultraviolet/Visible. = Very Low Density Lipoproteins.

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Bors, W., Heller, W.; Michel, C; Saran, M. In Free Radicals and the Liver, Csomos, Q; Feher, J., Eds.; Springer: Beriin, 1992, pp. 77-95. Rice-Evans, C.A.; Miller, N.J.; Paganga, G. Tren. PlantSci., 1997, 2, 152. Cano, A.; Hernandez-Ruiz, J.; Garcia-Canovas, F.; Acosta, M.; Amao, M.B. Phytochem. Analysis, 1998, 9, 196. Amao, M.B.; Cano, A., Acosta, M. Recent Res. Devel. In Agric. Food Chem., 1998,2, 893. Chen, Z.Y.; Chan, P.T.; Ho, K.Y.; Fung, K.P.; Wang, J. Chem. Phys Lipids, 1996, 79, 157. Markham, K.R. Techniques of Flavonoid Identification, Acadecim Press Inc.: London, 1982. Brown, J.E.; Khodr, H.; Hider, R.C.; Rice-Evans, C.A. Biochem. J., 1998, 330, 1173. Brouillard, R. Phytochem., 1983,22,1311. Sarma, A.J.; Sreelakshimi, Y.; Sharma, R. Phytochem., 45,671. Constantinescu, A ; Han, D.; Packer, L. J. Biol. Chem., 1993, 268,10906. Sharma, M.K.; Buettner, G.R. Free Rad Biol. Med., 1993,14,649. Hamre, K.; Waagbo, R.; Berge, R.K.; Lie, O. Free Rad Biol. Med., 1997, 22, 137. Cossins, E.; Lee, R.; Packer, L. Biochem. Mol Biol. Int., 1998,45, 583. Bors, W.; Michel, C; Schikora, S. Free Rad Biol. Med., 1995,19, 45. Bors, W.; Michel, C; Stettmaier, K. J. Mag. Res. Anal, 1997,6.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

779

Natural Products as potential antiparasitic drugs OLIVER K A Y S E R \ A L B R E C H T F . K I D E R L E N ^ SMON L. CROFT^

^Freie Universitdt Berlin Institutfiir Pharmaziey Pharmazeutische Biotechnologie Kelchstrafie 31, 12169 Berlin, Germany Robert Koch-lnstitut Nordufer 20 13353 Berlin, Germany ^London School of Hygiene and Tropical Medicine Department of Infectious and Tropical Diseases Keppel Street London, WCIE 7HT, United Kingdom

ABSTRACT: Pharmaceutical research in natural products represents a major strategy for discovering and developing new drugs. The use of medicinal plants for the treatment of parasitic diseases is well known and documented since ancient times e.g. by the use of Cinchona succiruba (Rubiaceae) as an antimalarial. This chapter provides a comprehensive review of the latest results in the field of antiparasitic drug development from biologic sources (plants, bacteria, fungi and marine organisms) focussing on the treatment of protozoal infections (Plasmodium, Leishmania, Trypanosoma spp.). The status of validated in vitro and in vivo assays is reviewed, discussing their different features, problems and limitations. Because of the high number of natural products tested against the aforesaid protozoa in the last years, we limit the discussion to lignans, phenolics, terpenoids, and alkaloids as defined natural product classes. The review also covers essential research topics of recent publications on specific natural products (e.g. licochalcone A, benzyl- and naphthylisoquinoline alkaloids, and artemisinin) and gives an outlook to semisynthetic approaches of drugs already introduced in clinics or in clinical trial studies.

780

1. INTRODUCTION The fascination of natural products, mostly as used as a preparation from a plant with known medicinal properties, goes back to ancient times. The discovery of pure compounds as active principles in plants was first described at the beginning of the 19^^ century, and the art of exploiting natural products has become part of the molecular sciences. The discovery of quinine (1) from Cinchona succiruba (Rubiaceae) and its subsequent development as an antimalarial drug represented a milestone in the history of antiparasitic drugs from nature for the treatment of all parasitic diseases - not just infections caused by only Plasmodium, Leishmania and Trypanosoma spp.. Early studies on plant products were followed by an era of organic chemistry that led to the development of arsenical and antimonial (melarsoprol, sodium stibogluconate), diamidine (pentamidine) and nitroheterocyclic (metronidazole) antiprotozoal drugs. In the past decades natural products have attracted renewed interest, especially with bacteria and fungi as important sources of biologically active compounds. Recently marine organisms have also been recognized as attractive source of antiparasitic compounds, and it can be expected that in the future other living organisms (for example, insects and amphibians) will provide additional sources. It is, therefore, not surprising that one of the most rewarding frontiers in modem science is the study of the chemistry and biology of natural products. Discovering untapped natural sources of novel antiprotozoal compounds from nature remains a major challenge and a source of novelty in the era of combinatorial chemistry and genomics. Since plants contain a high variety of constituents it is often claimed that the use of a whole plant rather than one single purified product may be more effective therapeutically. Because of the limited space in this contribution we restrict ourselves to defined natural products that have been tested in standard in vitro and in vivo assays.

781 2.

PRESENT SITUATION AND CHEMOTHERAPY OF MALARIA, LEISHMANIASIS AND TRYPANOSOMIASIS

2.1 MALARIA It is estimated that there are 300-500 million cases of malaria annually with 1.75 to 2.5 million deaths. Malaria is a particularly important disease in sub-Saharan Africa, where about 90% of cases and deaths occur, but is also a serious public health problem in certain regions of southeast Asia and South America [1]. Human malaria is caused by four species of Plasmodium, P. falciparum, P. vivax, P. ovale and P. malariae, which are transmitted by female Anopheles mosquitoes. The majority of cases of malaria and deaths are caused by P. falciparum [1]. The life cycle, immunological defence mechanisms, and clinical development of malaria in humans is complex [2, 3]. The sporozoites that develop in the salivary glands of the female mosquito are inoculated into the human when the insect bites to acquire a bloodmeal. The sporozoites travel in the bloodstream to the liver where they invade the hepatocytes, differentiate and undergo asexual division (the exoerythrocytic cycle) and form a schizont (a multiple division form). Mature tissue schizonts release thousands of merozoites after 5-15 days (depending on species). The merozoites invade erythrocytes where they appear initially as a ring stage, followed by a growing trophozoite stage, which develops into a dividing asexual schizont stage. During the erythrocytic stage, which lasts from 48-72 hours depending upon species, the parasite develops in a parasitophorous vacuole surrounded by host cell membrane. The Plasmodium parasite adapts to life within the erythrocyte, depending mainly on glycolysis for energy and altering the erythrocyte membrane with transporters that enable increased uptake of hexoses, amino acids and lipid precursors. During the growth cycle up to 80% of host cell haemoglobin is ingested by and digested in the food vacuole of the Plasmodium trophozoite. The trophozoite divides (schizogony) and the erythrocyte lyses releasing more merozoites to invade further erythrocytes. At some point during the infection the intraerythrocytic stage develops to form sexual stages, male or female gametocytes, rather than merozoites. These sexual stages are taken up

782 in a blood meal by another Anopheles mosquito where fertilisation occurs and the life cycle is completed. Clinical malaria is characterised by periodic fever, which follows the lysis of infected erythrocytes, and caused mainly by the induction of cytokines interleukin-1 and tumour necrosis factor. P. falciparum infection can have serious effects, for example anaemia, cerebral complications (from coma to convulsions), hypoglycaemia and glomerulonephritis. The disease is most serious in the non-immune, including children, pregnant women and tourists. Humans in endemic areas, who have survived an attack of malaria, are semi-immune and disease can be characterised by headache and mild fever. Infection by the other species of Plasmodium is normally self-limiting although relapses may occur, particularly in P. vivax infections. The species of parasite and the age and immune status of the patient are important in considerations of treatment and interpretation of the effects of all medicines. The chemotherapy and prophylaxis of malaria have been undermined by the development of worldwide resistance of P. falciparum to the 4-aminoquinoline chloroquine, first observed in the 1960s, as well as resistance to the antifolates pyrimethamine and cycloguanil. Resistance to quinine and other more recently developed drugs, for example mefloquine, have also been reported [4, 5]. The search for alternative antimalarials is one of the main themes of this chapter. The re-emergence of malaria as a public health problem is due mainly to the development of resistance of P. falciparum to cheap highly effective drugs like chloroquine and pyrimethamine. As a consequence of this problem over 300,000 compounds were tested for antimalarial activity by the Walter Reed Army Institute of Research, USA between 1965 and 1986. The 4-quinolinemethanol mefloquine and the 9-phenanthrenemethanol halofantrine emerged from this programme. Mefloquine (see also section 6.2) has been registered for little over 10 years, but there is already resistance in South East Asia, concern over cross-resistance with quinine and controversy over toxic side effects. Chloroquine is still used in some low resistance areas in Africa and South America and quinine is used for the treatment of cerebral malaria (see section 6.2). The most important recent discovery for the therapy of P. falciparum malaria has been the identification of the sesquiterene lactone artemisinin (qinghaosu) from

783 Artemisia annua (Asteraceae) (see section 6.1). Artemisinin and its' derivatives, for example artemether and artesunate, are rapid acting antimalarials, effective against multidrug resistant P. falciparum, that have been used to treat over 3 million cases in South East Asia [6]. Another new drug, the hydroxynaphthoquinone atovaquone, identified as an antimalarial in the eariy 1980s [7], has proved to be highly effective in clinical trials but has to be used in combination with proguanil (as Malarone®) to prevent the development of resistance. The use of combinations to combat the development of resistance is a current strategy as demonstrated in the clinical use of other combinations for example mefloquine - artemisinin, co-artemether (lumefantrine/ benflumetol - artemether) and Lapdap® (chlorproguanil - dapsone) [8]. A new 8-aminoquinoline, tafenoquine, is on clinical trial as a potential replacement for primaquine to treat P. vivax malaria; it also holds promise as a prophylactic against P. falciparum [9].

2.2 LEISHMANIASIS Protozoa of the genus Leishmania are obligate intracellular parasites of mononuclear phagocytes. Leishmaniasis is a spectral disease, depending on the Leishmania species involved and genetic potential and acute predisposition the hosts defense system. It ranges from selfhealing ulcers (cutaneous leishmaniasis, CL) to progressive nasopharyngal infections (mucocutaneous leishmaniasis, MCL) to disseminating visceral leishmaniasis (VL). While CL poses essentially cosmetic problems, and MCL leads to painful disfiguration, social stigmatization and often highly severe secondary infections, VL is generally lethal if left untreated. Leishmaniasis occurs from tropical to Mediterranean regions where the parasite is transmitted by female sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World [10]. In the insect gut as well as in tissue culture media, the parasite exists as extracellular, elongated, flagellated promastigotes. Promastigotes are injected into the skin during a blood meal and rapidly taken up by mononuclear phagocytes where they reside in the parasitophorous vacuole. In contrast to other intracellular pathogens, for example Toxoplasma, Leishmania do not inhibit fusion

784 of lysosomes with the parasitophorous vacuole. Within the phagolysosome the parasites transform into and multiply as ovoid amastigotes, which are specially adapted to the elevated temperatures within the mammalian host and the hostile environment within these potent effector cells [11]. Massive amastigote multiplication leads to host cell disruption and release of parasites to infect freshly recruited host cells. While "resting" macrophages support parasite multiplication and thus spread of infection, these cells can also be activated by elements of the natural and the specific cellular immune response to kill intracellular parasites leading to cure. The prime signal for macrophage activation is the cytokine interferon EFN-y, a glycopeptide released by Natural Killer (NK) cells and T lymphocytes. In the murine model for CL caused by L. major it was shown that distinct populations T cell populations exist with antagonizing effects. While T helper 1-type cells secrete IFN-y and promote cure, Th2-type cells secrete interleukin IL-10, inhibiting macrophage activation and exacerbating disease. The exact factors promoting each type of immune response remain obscure but are in part genetically determined. According to the World Health Organization (WHO) there are 2 million new cases of leishmaniasis per year. With the advent of ADDS, which currently affects over 30 million people in geographical regions largely overlapping with leishmaniasis, the nature of the disease has changed. In Mediterranean countries, where infant VL is endemic, adult VL is now considered a genuine AIDS-related opportunistic disease largely due to reactivation of latent infections by immunosuppression [12]. Although the three disease complexes leishmaniasis, human African trypanosomiasis (HAT, sleeping sickness) and South American trypanosomiasis (Chagas disease) are caused by closely related trypanosomatid parasites, the diseases are treated with different drugs and the parasites themselves have varying sensitivities to many compounds [13, 14]. The recommended drugs for both visceral and cutaneous leishmaniasis are the pentavalent antimonials sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime). Both drugs have been used for over 50 years, require long courses of parenteral administration, and have toxic side effects. In addition there has been a dramatic increase in the number of cases

785 of VL in northeastern India that do not respond to antimonials. Alternative treatments for VL include the polyene antibiotic amphotericin B [15] that has highly effective less toxic lipid formulations [9]. A parenteral formulation of the aminoglycoside paromomycin (aminosidine), and the orally available alkylphospholipid miltefosine [16] are also potential treatments for VL. Interest in a new treatment for CL has focussed on different topical formulations of paromomycin. The increasingly observed partial resistance to antimonials has been overcome by higher dose regimens but in general therapy of MCL and VL is becoming increasingly problematic. Experimental studies and treatment of AIDS patients has revealed that successful treatment with some drugs requires the active participation of the immune system [12]. Antimonial drugs have been successfully combined with EFN-y, although costs of such treatmenr render it useless for third world countries.

2.3 AFRICAN TRYPANOSOMIASIS Flagellated protozoa of the genus Trypanosoma infect humans (sleeping sickness; T. brucei rhodesiense, T. b. gambiense) and domestic animals (nagana; T, b. brucei, T. congolense, T. vivax) causing major health and commercial problems in subSaharan Africa. In the 1960s African trypanosomiases was under control, mainly due to eradication of its insect vector, the tsetse fly. According to the WHO, sleeping sickness is again endemic in 36 African countries with over 250 thousand newly infected cases per year [17]. Following the bite of an infected tsetse {Glossina spp.) parasites multiply in the skin for one to three weeks before invading the haemolymphatic system. Early symptoms include high fever, swelling of lymph nodes (neck), hepatosplenomegaly, oedema and diarrhoea. Generalized inflammation of blood and lymph vessels may lead to myocarditis and encephalitis and often to death due to heart failure. Days or many weeks later, parasites invade the central nervous system causing the typical symptoms of sleeping disease: failures in speech and coordination of movement, epileptic episodes, somnolence, apathy, cachexy, and finally - if left untreated - coma and death [18].

786 Most Trypanosoma spp. are found only in wild and domestic animals. While for some species (e.g. antelopes, goats, pigs) infection with T. brucei produces no apparent symptoms, others (e.g. dogs, horses, cattle) often succumb to disease. Cattle show reduction in weight gain, milk yield, reproduction, and general performance. Laboratory strain rats and mice can also be infected, providing useful experimental models. In West African sleeping sickness, especially during epidemics, there is direct human-to-human transmission of T, b. gambiense, whereas the other species that infects humans, T. b. rhodesiense, is mainly transmitted between reservoir mammals. T, brucei spp. are extracellular parasites living in blood and lymph as elongated trypomastigotes. Here, they obtain their energy by glycolysis, whereas in their insect vector they switch to oxidative metabolism using mainly proline. In contrast to intracellular parasites such as Trypanosoma cruzi and Leishmania sp., T brucei are vulnerable for humoral defense mechanisms. Complement-activating antibodies may bind to their surface, facilitating recognition, phagocytosis and destruction by the monocytic phagocyte system. Trypanosoma brucei evade eradication by antigenic variation, sequentially producing new clones differing in their surface glycoproteins which are not susceptible to the prevailing antibody population. This strategy of antigen variation effectively neutralizes the hosts' immune defense mechanisms and has confounded all efforts of vaccine development. Furthermore, T. brucei organisms have been found to activate immunosuppressive macrophage functions. Obviously, any successful strategy to combat the African trypanosomiases must be two-pronged: controlling the insect vector and combating the parasites by prophylactic and therapeutic measures. Chemotherapy of nagana has been reliant for over 40 years on diminazene (berenil), isometamidium and homidium. Due to the intensive use and structural similarities of these drugs, trypanosomes have developed multiple drug resistance in Ethiopia, Kenya, Somalia, and many other African countries. Drugs for the treatment of human African trypanosomiasis are also inadequate. The diamidine pentamidine and the sulphonated naphthylamine suramin have been used for over 50 years, require parenteral administration and are only effective against the early haemolymphatic stage of the disease. The only drug currently available for the treatment of the late stage CNS infection is the

787

trivalent arsenical melarsoprol. Melarsoprol requires parenteral administration, has unacceptable side effects including 5 % mortality due to encephalopathy, and there are an increasing number of patients who are no longer responding to treatment. Eflomithine, an inhibitor of polyamine biosynthesis, proved to be an effective treatment in trials in the late 1980s and was registered in 1990 for the treatment of late stage infections caused by T. b. gambiense (but not T. b. rhodesiense which is refractory). Limited supplies of the eflomithine are available as the drug is no longer manufactured [21]. 2.4 SOUTH AMERICAN TRYPANOSOMIASIS South American trypanosomiasis (or Chagas disease) is caused by Trypanosoma cruzi and is widespread thoughout the subcontinent with an estimated 18 million people infected. The parasite is transmitted by triatomine insects, not directly by by contamination: the insect defaecates whilst taking a blood meal and parasites in the faeces can invade wounds, the eye or mucosal tissues. In mammals the parasite exists in two forms: the extracellular trypomastigote that is an invasive haemolymphatic form and is non-dividing, and the amastigote that divides in the cytoplasm of cells. 7. cruzi trypomastigotes will invade many cell types, in particular macrophages, muscle cells and nerve cells. Chagas disease is characterised by three stages: (a) an early symptomatic acute phase during which the infection spreads throughout the body and up to 30% of deaths occur, (b) an asymptomatic indeterminate phase that may last for many years, and (c) a symptomatic chronic phase when immunopathological reactions to low numbers of parasites in heart and nerve cells cause illness and death. Some cases of transmission of Chagas disease in urban areas have resulted from blood transfusion. Two drugs have been available for the treatment of Chagas disease since the early 1970s: the nitrofuran derivative nifurtimox (now no longer manufactured) and the 2-nitroimidiazole derivative benznidazole. Both drugs are only effective in the acute phase of the disease, have variable efficacy against different strains of T, cruzi, require long courses of (oral) treatment, and have high levels of side effects. Few drugs have proved effective against all stages of the

788 disease. The antifungal sterol biosynthesis inhibitors have shown the most promising activity against T. cruzi inexperimental models. Recently Urbina and colleagues [20] have identified several antifungal triazoles that are active against both acute and chronic T. cruzi infections in rodent models. 2.5 CHEMOTHERAPY OF OTHER PROTOZOAN DISEASES For several other protozoan diseases there is adequate chemotherapy: the 5-nitroimidazoles (for example, metronidazole) for the treatment of amoebiasis, giardiasis and trichomoniasis, the hydroxynaphthoquinone bupravaquone for theileriosis in cattle and other ungulates, and the polyene ionophores (for example monensin, lasalocid, narasin and salinomycin) for the prophylaxis of avian coccidiosis. However, improved therapies are required for some opportunistic parasites that cause disease in immunocompromised humans. Paromomycin and nitazoxanide have some effect in the treatment of cryptosporidiosis and albendazole appears to be effective for microsporidiosis caused by Encephalitizoon intestinalis.

3. IN VITRO AND IN VIVO ASSAYS FOR DETERMINATION OF DRUG ACTIVITY 3.1 ANTIPLASMODIAL ACTIVITY Prior to 1976, when the asexual cycle of P. falciparum was successfully cultured in human erythrocytes, antimalarial drug screening was dependent upon in vivo avian and rodent models, using non-human Plasmodium species. Since 1976 P. falciparum has been used extensively in in vitro screens; techniques to culture the other three human Plasmodium species have been less successful. A semiautomated microdilution assay, in which parasite viability and drug activity is related to the uptake of a nucleic acid precursor [^H]hypoxanthine, was described by [22] and is still widely used in modified 96-well format versions [23]; 384 well format assays are also now being used. The sensitivity (and resistance) of a wide range of P. falciparum strains to the standard antimalarial drugs has been reported using this assay. The technique has some limitations but

789 modifications using synchronous cultures and altering the medium have enabled studies on differential drug effects on trophozoite stages and schizonts [24] or improved sensitivity to antifolates [25]. A colorimetric assay based on the reduction of nitroblue tetrazolium to formazan by lactate dehydrogenase (a glycolytic enzyme essential in Plasmodium) has been used to test drugs against P. falciparum in vitro [26], and is simpler and cheaper to use but less sensitive than the [^H]hypoxanthine assay. Drug sensitivity testing for P. vivax and P. ovale remains a problem as continuous culure of these species is difficult due to nutritional and host cell requirements; only short term cultures have been used in drug tests [27]. In vitro culture of the exoerythrocytic liver stages of malaria infection have been achieved using hepatocytes and hepatomas to culture P. berghei, P. vivax and P. falciparum [28] but these models have not been used widely in drug evaluation studies. None of the four Plasmodium species that cause disease in humans can infect rodents or other animals used in in vivo screens. For the past 40 years in vivo screens have based on rodent malaria models, in particular those using P. berghei, P. yoelii and P. chabaudi infections in mice. Rodent malaria has proved to be an essential part of the process of drug development for detecting blood schizonticidal, tissue schizonticidal and repository activity. The activity of standard antimalarials against a number of P. berghei and P. yoelii strains has been well characterised [29]. The development of drug-resistant strains of these parasites has added to their use in the identification of novel drugs and drug combinations. However, there are differences between the biology of these rodent species and that of P. falciparum and the pharmacokinetic properties of a drug in mouse and humans can be different. Candidate antimalarial drugs are therefore often tested in primate models of P. falciparum infection using Aotus and Saimiri monkeys. 3.2 ANTILEISHMANIAL ACTIVITY In their mammalian hosts, Leishmania parasites exist primarily as amastigotes within phagolysosomes of macrophages. Extra- and intracellular promastigotes occur only during a few hours after infection and extracellular amastigotes appearing between disruption

790 of one host cells and uptake by the next. Antileishmanials must be also be accumulated by infected macrophages enter phagolysosomes and be active under the specific conditions within this compartment (e.g. low pH) and either kill amastigotes or reduce viability. The relative ease of mass cultivation of most Leishmania isolates in the laboratory and the availability of relevant animal models for human CL and VL facilitate adequate screening for novel antileishmanials at different levels of complexity. Toxicity assays for extracellular promastigote are easy to perform and have been used in drug screening. However, they have limited as the intracellular amastigote has different biochemical and molecular properties. Promastigote Leishmania are cultured in a variety of liquid or two-phase semi-synthetic or fully synthetic media generally at 23-27 °C and direct cytotoxic effects can be assessed microscopically as reduced motility, altered morphology (rounded and bloated) or reduced numbers. Growth inhibition assays involving incorporation of radioactive nucleotides (e.g. [^H]-thymidine) by proliferating cells or metabolization of chromophores (e.g. MTT) [30]. Assessments of effects on the intracellular survival of amastigotes is more complex. Primary macrophage cultures or monocytic cell lines are parasitized in vitro with promastigote Leishmania cultures or freshly isolated amastigotes. The former should be cultured at 37 °C at least over night to allow infection and intracellular transformation to amastigotes [31]. After exposure to test compounds, the cultures ells are stained and the average number of intracellular parasites/host cell determined in comparison to untreated controls. For mass screening, parasitized macrophages can be seeded into microtiter plates. After exposure to test substances, the host cells can be selectively lysed releasing intracellular parasites. The relative numbers of surviving transformed parasites can then be determined by the radiometric or colorimetric methods described above [31]. Indirect antileishmanial activity through activation of host macrophages needs to be assessed separately in adequate toxicological and inmiunological assays. For many Leishmania spp. that cause visceral or cutaneous leishmaniasis, rodents are natural hosts and provide excellent laboratory models. Furthermore, genetically defined mouse strains vary according to their spontaneous healing/non-healing capacity and immune response patterns thus providing means for specific experimental design. For CL, highly susceptible, non-curing BALB/c

791

mice can be inoculated subcutaneously with L. major into a footpad or the shaven rump. Progression of disease is monitored by measuring the diameter of the developing lesion. Antileishmanial drugs can be given orally, as topical ointments, or injected into the lesion. As tissue response does not necessarily correlate with parasite numbers, impression smears of tissue samples taken from the periphery of the ulcer should be stained and evaluated microscopically. Alternatively, viable parasite numbers can be estimated from limiting dilution cultures of tissue homogenates. For VL, hamsters or mice should be inoculated intravenously or intracardically with L. donovani. Disease progression can be monitored by the extent of cachexia or development of ascites. Visceral leishmaniasis affects all internal organs, and parasite counts are performed at least on spleen, liver and bone marrow, again using impression smear or limiting dilution culture methods. Sensitive molecular techniques such RT-PCR - while providing little advantage when quantifying an acute infection - are useful for detection of latent infections after parasitological cure.

3.3

ANTITRYPANOSOMAL BRUCEI)

ACTIVITY (TRYPANOSOMA

T. b. bruceiy T.b. rhodesiense and T.b. gambiense extracellular bloodstream form trypomastigotes can be grown axenically in supplemented standard media. Trypanocidal activity may be tested by culturing a constant number of parasites (lOV ml) in serial dilutions of substances for 24-72 hours in microtitre plates at 37 "^C and 5 % CO2. For reference, standard drugs such as melarsoprol (Arsobal®), pentamidine (Pentacarinat®) or suramin (Germanin®) should be included. The % surviving trypomastigotes indrug treated cultures can be estimated colorimetrically using a p-nitrophenyl phosphate as substrate for acid phosphatase [32], the MTT assay (described in section 3.2) or a fluorochrome such as Alamar blue [33]. Plotting % growth inhibition {= [1 - Ssample - 5max kill) / (5max growth " Smax kill)] X 1 0 0 } againSt drUg

concentration, dose-response curves can be generated and EC50 and EC90 values calculated for better comparison. It should be noted, that for some drugs, the two major variants T. b. gambiense and T. b.

792

rhodesiense exhibit in vitro differences in drug sensitivity and some compounds, for example eflomithine are poorly active in vitro . Most animal models are restricted to testing compounds against T. b. brucei or T. b. rhodesiense. In vivo tests against. T. b. gambiense are restricted to Mastomys rats or scid mice. However, some strains of T. brucei can establish chronic CNS infections in mice thus providing models for late-stage sleeping sickness. An effective new drug for sleeping sickness must also be capable to cross the blood/ brain barrier. Monkey models, available for testing of candidate drugs, can also provide data on the penetration of drugs into the CNS.

3.4

ANTITRYPANOSOMAL CRUZI)

ACTIVITY (TRYPANOSOMA

The dividing T. cruzi amastigote can be cultured in vitro in a variety of host cells, most commonly macrophages, fibroblasts and myoblasts. Trypomastigotes used to infect these cells can also be cultured in vitro. Two points about the design and interpretation of the assay: (a) it is normally limited to 3-4 days, as intracellular amastigotes transform to trypomastigotes after this time, and (b) as drug activity is measured by determining the number of amastigotes/host cell in treated and untreated cultures, the effects of compounds on the division rate of host cells as well as amastigotes has to be considered. The standard drugs nifurtimox or benznidazole should be included for comparison, but as mentioned above their activity is variable depending upon the strain of T. cruzi used. This also underlines the importance of testing lead compounds against a number of strains of this parasite. Recently the in vitro amastigote assays have been automated through the use of beta-galactosidase transfected T. cruzi Tulahuen strain [19]. Active compounds can be tested further against the extracellular trypomastigotes over 24 hours at 4°C as a new drug for sterilizing blood transfusions is also required. Inbred mouse strains offer the most useful in vivo models, either for simple suppressive tests or more complex curative tests. Mice are infected by trypomastigotes and treatment starts when the parasitaemia is detectable in tail blood. To determine cure sensitive techniques that can detect low numbers of parasites (haemoculture, serology, immunohistopathology, PCR) have to be performed on

793

blood, muscle and other tissues [20]. Again it is important to include standard drugs and a number of strains of T. cruzi in the process of identifying a lead compound. Chronic infections can be established by infecting with a low number of parasites (10"^) and monitoring the infection by sensitive techniques during and after treatment [20]. 4.

ANTIPROTOZOAL DRUGS FROM NATURE

41 LIPIDS AND RELATED ALIPHATICS 4.1.1 ORGANIC ACIDS, LIPIDS AND ACETOGENINS Long chain hydrocarbons and fatty acids are best known as constituents of waxes and lipophilic compounds. Some representatives of this natural product group show high antiprotozoal activity but mostly combined with a high levels of toxicity to mammalian cells. One example is trans-dicomiic acid (2) that was used in combination with sodium stibogluconate, allopurinol, or pentamidine for experimental visceral leishmaniasis to determine synergistic effects [34]. When these three drugs (50, 15, 8 mM/kg/day, respectively) were used with fmn^-aconitic acid (5 mM/kg/day) the parasite load in BALB/c mice was inhibited by 100, 88, and 100%, respectively. At tested concentration rmn^-aconitic acid itself showed an inhibition of 59.2 %. Four acetogenins from Rollinia emarginta (Annonaceae) were identified with antiprotozoal activity by bioassay-guided screening. Against different Leishmania and T. cruzi strains at 250 fig/mL inhibitory activity up to 89% and 67% was reported for rolliniastatin-1 (3) and squamocin (4) [35]. The leishmanicical activity was related to the number of hydroxy groups on these acetogenins. Maximum activity activity was observed in compounds that possessed three hydroxy groups, for example rolliniastatin-1 and squamocin, while activity was low in acetogenins having four or more hydroxy groups, e. g. sylvaticin and rollidecin.

794

y-WCOOH HOOC COOH

(2) trans-siconiiic acid

H,C»""

(3)rolliniastatin-l QH _

OH

OH CH,

H,C»»*'

(4) squamocin

4.1.2 POLYENES This class of antibiotics is well known because amphotericin B (5) is used as second line drug for the therapy of visceral and mucocutaneous leishmaniasis. Different polyene analogues, related to amphotericin B, also inhibit parasite growth. Polyene antibiotics can be divided into a non-aromatic group, which includes amphotericin B, and an aromatic group, which includes hamycins A and B another potent antiprotozoal agents. Hamycin (6), a polyene antibiotic, now in extensive use in the treatment of candidiasis and otomycosis, is found to be remarkably effective in killing Leishmania donovani promastigotes in a liquid medium at a concentration of 0.1 /xg/mL. Glucose stimulated respiration and the uptake of 2-deoxy-D[U-^^C]-glucose was inhibited in cells treated with the drug at a growth inhibitory concentration. The primary site of action of hamycin on L. donovani promastigote cells appears to be similar to amphotericin B, binding to membrane sterols, disrupting membrane stability with the loss of the permeability barrier to small metabolites. The lower minimum inhibitory concentration (MIC) of hamycin compared to other established drugs warrants further study in the

795 context of increasing reports of clinical resistance to pentavalent antimonials [36]. Despite the high activity of this class of compound there has been no discovery of new potent but less toxic polyene antibiotics in the past decade. Most interest has been focussed on the formulation of amphotericin B in colloidal drug carriers like liposomes, emulsions, micro-, and nanoparticles to improve bioavailability and reduce toxicity (AmBisome®, Ambicer, Amphocil®).

COOH

(5) amphotericin B

^NHCH

COOH

(6) Hamycin

4.1.3 AJOENES Some simple fatty acid or aliphatics show antiprotozoal activity. Ajoenes metabolites (7) are a good example with the naturally

796 occuring allicin that has proved to be active against rodent malaria and Trypanosoma cruzi. These sulfur containing aliphatics, initially isolated from garlic {Allium sativum, Liliaceae), showed significant suppression of Trypanosoma parasitemia in vivo with daily doses of 50 mg/kg over 12 days [37]. Gallwitz et al. [38] identified ajoenes also as potential drugs effecting thiol metabolism by acting as a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and secondly of the Trypanosoma cruzi trypanothione reductase (TR). The interactions between the flavoenzymes and ajoene lead to increased oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of antioxidant thiol metabolism. Urbina et al. also demonstrated an effect on the phospholipoid biosynthesis of Trypanosoma cruzi with an alteration of the lipid composition of parasites from phosphatidylcholine to phosphatidylathanolamine [39]. Ajoenes also inhibited de novo synthesis of neutral lipids and sterols in T cruzi epimastigotes, but these effects are not sufficient to explain antiproliferative effects of the drug.

ft (7) ajoene

4.2

PHENOLS

4.2.1

SIMPLE PHENOLS, COUMARINS

PHENOLIC

ACIDS

AND

Simple phenols that are widely distributed in plants have been tested for their ability to inhibit parasite growth. For example, gallic acid (8) and its derivatives inhibit the proliferation of Trypanosoma cruzi trypomastigotes in vitro with an EC50 value of 15.6 jLtg/mL [40], Higher activities were observed for the gallic acid esters ethyl-gallate and n-propyl-gallate which had EC50 values of 2.28 and 1.47 /ig/mL, possibly due to increased lipophilicity. No in vivo data has been published. It seems unrealistic that such compounds, which form part of the daily diet, will have significant effects. The mechanism of

797 action remain obscure and the authors suggest that the formation of reactive oxygen species might be involved in the galhc acid induced apoptotic cell death [41]. Interestingly, Kayser et al. could not demonstrate any direct toxic effect of gallic acid and related gallotannins on L. donovani in infected macrophages [42], Further studies should be conducted to clarify immunstimulatory activity of gallic acid [43]. Ascofuranone (9), an isoprenoid prenylphenol antibiotic, derived from the fungus Ascochyta visiae, specifically inhibits mitochondrial glycerol-3-phosphate (G-3-P)-dependent electron transport in T. b. brucei [44]. Ascofuranone strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial oxygen consumption of T. b. brucei bloodstream form trypomastigotes. This inhibition was suggested to be due to inhibition of the mitochondrial electron-transport system, composed of glycerol3-phosphate dehydrogenase and a plant-like alternative oxidase. Ascofuranone noncompetitively inhibited the reduced coenzyme Q l dependent O2 uptake of the mitochondrion with respect to ubiquinol (Ki = 2.38 nM). The site of action was deduced to be the ubiquinone redox machinery that links the two enzyme activities. Further, ascofuranone in combination with glycerol completely blocked energy production, and potently inhibited the in vitro growth of the parasite. Other simple phenols include the hydroquinone derivatives miconidin (10) and espintanol (11), formed from its biosynthesis of a monoterpene to a phenolic, and pholoroglucinol derivatives from Hypericum calycinum (Hypericaceae). Quantitative data are not available for miconidin, but espintanol exhibited an IC90 in the 25-100 jLtg/mL range against twenty different T. cruzi strains [45], and a prenylated phloroglucinol derivative (12), inhibited P. falciparum growth in vitro with an EC50 of 0.88 /xg/mL [46]. The mode of action of these semiquinones is unclear. It is attractive to speculate that the sensitivity of Trypanosoma and Plasmodium is due to oxidative stress resulting from the metabolic oxidation of semiquinone radicals or benzoquinones. The presence of antiplasmodial phloroglucinol derivatives has already been mentioned. Sarothalen B was found to be active in vivo in the 1950s [47] and the biogenetically related phloroglucinol derivative (12), which lacks the cyclohexadienone moiety of sarothalen B, is active in in vitro assays, indicating, that it conserves essential chemical features for this antiprotozoal activity.

798

Oketch-Rabah et al. reported the antiprotozoal activity of 2"epicycloisobrachycoumarinone epoxide (13) and its stereoisomer that has been isolated from Vemonia brachycalyx (Asteraceae) [48]. Both stereoisomers show similar in vitro activities against chloroquinesensitive (CQ-S) and chloroquine-resistant (CQ-R) strains for P. falciparum as well as L. major promastigotes with EC50 values of 0.11/ig/mL and 0.15 /xg/mL for Plasmodium and 37.1 jLtg/mL and 39.2 /xg/mL for L. major, respectively.

v (8) gallic acid

(9) ascofurane H,

(10) miconidin

HsC^As^OM .OMe

OMe CH,

(11) espintanol

(12) phloroglucinol-derivate

(13) 2'-epicycloisobrachy-coumarinone epoxide

799 4.2.2 LIGNANS Lignans are a potent group of natural products with many toxic side effects, best represented by podophyllotoxin derivatives and the antineoplastic drug etoposide. Despite their known biological activities few lignans have been tested against parasitic protozoa. Lopes et al. demonstrated for the potential of the tetrahydrofuran lignans grandisin (14) and veraguensin (15) to prevent the transmission of Chagas disease by blood transfusion [49]. The activity of these terahydrofuran lignans (62 % and 87 % growth inhibition at 2.5 /xg/mL, respectively) was forty times higher than that of the reference drug gentian violet. Lignans isolated from the hexane extract of the leaves of Zanthoxyllum naranjillo (Rutaceae) were tested in both in vitro and in vivo against two strains of Trypanosoma cruzi [50]. The compound (-)-methylpluviatolide (16) was highly effective for chemoprevention in the in vitro assay and healthy animals injected with the tested samples did not develop infection. Moreover, (-)-methylpluviatolide was also highly active against the bloodstream forms of two strains of T. cruzi in an in vivo assay [48].

OCH, 0CH3

HXO H3C0

0CH3

(14) grandisin

OCH, HXO

OCH,

(15) veraguensin

(16) (-)-methylpluviatolide

800 4.2.3 CHALCONES AND AURONES Phlorizidin (17), a naturally occurring dihydrochalcone glycoside from Micromelum tephrocarpum (Rutaceae), was one of first chalcones shown to possess antiparasitic activity. In ethnomedicine it was used for the treatment of malaria because of the bitter taste, a property shared with quinine and other antimalarials. Recent studies provide a rational basis for its antiplasmodial activity. Phlorizidin inhibits the induced permeability in Plasmodium infected erythrocytes to various substrates including glucose. The most promising compound out of this natural product class is licochalcone A (18). This compound was first isolated from Glycyrrhiza glabra (Fabaceae) and is the subject of intensive preclinical studies. The activity of licochalcone A is well documented in vitro and in vivo against a panel of different parasites including P.falciparum L donovani and L. major. The antileishmanial mechanism of action in through the inhibition of the electron transport in the mitochondrion [51]. Further biochemical studies were employed in L. donovani to demonstrated that growth inhibition is mitochondrial specific, and that the main targets are the fumarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, all essential for parasite viability [51]. Starting with licochalcone A as a lead structure, a large number of chalcones have been synthesized and structure-activity relationships determined with regard to their antiplasmodial, antileishmanial and trypanocidal activity [52]. Kayser et al. [53] demonstrated that structurally related aurones (19) share similar antiparasitic activites with chalcones. It is not kown, if this biogenetically related natural product group inhibits the same target sites as chalcones, but they have a similar size, integrated threecarbon linkers, and similar substituents on both aromatic rings. The main difference lies in the conjugation of the three-carbon linker that in aurones is linked to the B-ring, giving a two-member ring system. A planar structure is typical for all aurones and this conformation has a high similarity to compounds that Li et al. proposed as optimal lead structure of chalcones as protease inhibitors [54]. From molecular modeling studies it appears that chalcones are not only rigid but also adopt an extended structure due to the nature of the conjugated linker. The resulting linear, nearly planar structure, fits perfectly into the active site of Trypanosoma and Plasmodium cysteine proteases [54].

801

These findings suggest that both aurones and chalcones might interact with similar sites in essential parasite enzymes and thus have similar mechanisms of antiparasitic activity.

GIcO

O

(17) phlorizidin (Glc = Glucose)

(18) licochalcone A

(19) aurones (R = H or CH3)

4.2.4 FLAVONOIDS Flavonoids are widespread in the plant kingdom. In contrast to some ethnomedicinally reports up to 1986, there was no scientific evidence of their activity against Leishmania spp., T.cruzi and P, falciparum [55]. However, following the detection of antiplasmodial flavonoids from Artemisia annua (Asteraceae) this natural product group has attracted renewed interest. Elford et al. demonstrated that methoxylated flavonones artemetin (20) and casticin (21) act synergistically with artemisinin against P. falciparum in vitro [56]. The exact mechanism is unclear but tested flavonoids did inhibit the influx of L-glutamine and myoinositol into infected erythrocytes [57]. As a part of a multidiscipHnary research programme in Thailand on antiplasmodial drugs, additional Artemisia species have been screened [58] and exiguaflavanone A (22) and B (23) isolated from Artemisia indica (Asteraceae) exhibited in vitro activity against P. falciparum

802 with EC50 values of 4.6 and 7.1 jLCg/mL, respectively. The flavonoids sakuranetin (24) and 7-methoxyaromadendrin (25) were also reported to be antiprotozoal natural products with inhibition rates of 100 % and 86 % at 500 jLtg/mL in vitro against T. cruzi, respectively. Despite the limited data of the antiprotozoal activity of flavonoids, it can be speculated that the mode of action is linked to the unusual antioxidant pathway. Ribeiro et al. discussed, that the lack of defense mechanisms against oxidative stress makes the parasites susceptible to drugs having an effect on the generation of reactive oxygen species [59]. Recently, Perez-Victoria et al. [60] suggested that specific flavonoids could effect transport mechanisms in Leishmania. The C-terminal nucleotide-binding domain of a P-glycoprotein-like transporter, encoded by the Itrmdrl gene in L. tropica and involved in parasite multidrug resistance (MDR), was overexpressed in Escherichia coli as a hexahistidine tagged protein and purified. The L. tropica recombinant domain efficiently bound different classes of flavonoids with the following affinity: flavone > flavanone > isoflavone > glucorhamnosyl-flavone. The affinity was dependent on the presence of hydroxyl groups at positions C-5 and C-3 and was further increased by a hydrophobic 1,1-dimethylallyl substituent at position C-8. When flow cytometry was used to measure daunomycin accumulation in a L. tropica line, a reversing effect was observed with flavones such as dimethylallyl-kaempferide at low concentrations or apigenin at higher concentration, but not with the glucorhamnosyl derivative rutin nor with the isoflavone genistein (26). The in vivo reversing effect of dimethylallyl-kaempferide was correlated with a high inhibition of the Leishmania cell growth in the presence of daunomycin. The results suggest that flavone inhibition of both daunomycin efflux and parasite growth in the presence of the drug correlates to direct binding of the compound to cytosolic domain of the P-glycoprotein-like transporter [60]. Gale et al. also identified genistein (26) as potent natural compound as modulator of protein phosphorylation with effect on the SPK89 protein kinase in trypanosomes [61]. 4,2.5 NAPHTHOQUINONES Naphthoquinones and other related quinoid compounds are one of the major natural product classes with significant activity against

803

OCH,

(22) exiguaflavanone A (R = H) (23) exiguaflavanone B (R = CH3)

(20) artemetin (R = CH3) (21) casticin (R = H)

R.0

OH

O

(24) sakuranetin (Ri = CH3, R2 = H) (25) 7-methoxyaromadendrin (R = OH) (26) genistein

Leishmania, Trypanosoma and Plasmodium. Many naphthoquinones have been isolated but frequently their potential use has been limited by low bioavailabihty and high toxicity. Wright and Phillipson [62], Sepuvelda-Boza and Cassels [63], Foumet et al. [64] and Akendengue et al. [65] have reviewed much of the literature on naphthoquinones, we focus here on the latest developments and some new structures and their biological activities. The plant derived product hydrolapachol (2-hydroxy-l,4-' naphthoquinone) (27) [66] was shown to have activity against Plasmodium lophurae in ducks in the 1940s (see [67] for details). This observation provided the stimulus for the synthesis of hundreds of analogues including a series of 2-hydroxy-3-alkyl-l,4-naphthoquinones. One of this series, lapinone, synthesised in 1948, showed high activity in experimental models and was used to treat P, vivax infected patients in 1951. Due to high doses required and pharmacological problems interest in naphthoquinones faded. In the 1960s interest was revived and a new compound, menoctone, proved

804 to be highly active in the P. berghei mouse model but disappointing in chnical trials. In the late 1970s and 1980s a series of hydroxynaphthoquinones (HNQs) was synthesised at the Wellcome Laboratories, UK that overcame the problems of poor oral absorption, rapid metabolism and protein binding associated with previous series. These HNQs, with a cyclohexyl ring at the 2-position showed, showed activity against the apicomplexan parasites, Eimeria, Toxoplasma, Theileria and Plasmodium species [68]. This work resulted in the development of parvaquone and buparvaquone for the treatment of theileriosis in cattle and other ungulates and atovaquone for the treatment of malaria (in combination with proguanil) and Pneumocystis carinii pneumonia [69]. Besides this synthetic route, new and structurally interesting naphthoquinones have been isolated. A dimeric naphthoquinone diospyrin (28) from Diospyros montana (Ebenaceae) was found to be active against L. donovani [70]. The inhibition of Type I DNATopoisomerase in this parasite has been suggested as a mechanism of action [70]. Plumbagin and related HNQs have activity against Leishmania spp. in vitro and in vivo [64]. These compounds have been used as the basis for the synthetisis of naphthoquinones designed as subversive substrates of trypanothione reductase. Other rare naphthoquinones have been identified as potential antiparasitic drugs. From Psychotria camponutans (Rubiaceae) the benzisoquinoline-510-dione (29) has been isolated and tested against P. falciparum with an EC50 of 0.84 jLtg/ mL associated with significant cytotoxicity (EC5o= 1.62 jLCg/ mL, KB cells) [71].

o (27) 2-hydroxy-1,4-naphthoquinone

o (28) diospyrin

(29) benzisoquinoline5,10-dione

805 4.2.6 ANTHRAQUINONES AND XANTHONES This natural product group is related to naphthoquinones in structure and biological activity. The main chemical difference between the groups is the tricyclic aromatic system with a para-quinoid substitution. Schnur et al. [71] and Foumet et al. [64] demonstrated that some derivatives have activity in vitro against Leishmania species, but few naturally occuring anthraquinones have been tested. Anthraquinones isolated from the tropical tree Morinda lucida (Rubiaceae) were tested for their antiplasmodial and antileishmanial activtity [72, 73]. In vitro, some (30-32) were more active against L. major promastigotes (EC50 = 9.6 to 185 /xM) than amastigotes and also acive against CQ-R strains of P. falciparum (EC50 = 21.4 to 82.9 jLtM in vitro). The most active compounds have an aldehyde group at C-2, well known as a cytotoxic substructure in other natural products. The activity may also be explained by the cyclic planar structure that makes them potential DNA-intercalators. From the toxicological point, tested compounds showed moderate effects in the lymphocyte proliferation test with all EC50 values over 175 /xM [75]. Antiprotozoal anthraquinones are known from microbial sources [76]. Mycotoxin MT81 (33) and some of its derivatives from Penicillium nigricans showed only moderate antileishmanial activity against L. donovani promastigotes (46% growth inhibition at 250 /xg/mL in vitro). In parallel to the inhibitory effects of naphthoquinones on mitochondria, Majumdar et al. determined the effect of the drugs on the respiration of L. donovani cells [76]. The oxygen uptake was significantly inhibited (inhibition rate > 50 %) by all mycotoxins indicating a similar mode of actions as known for other naphthoquinones. Antiplasmodial xanthones have been isolated from Garcinia cowa (Guttiferae) [77]. Preliminary screening of five prenylated xanthones dmonstrated significant activity against P. falciparum in vitro with EC50 concentrations between 1.5 and 3.0 /xg/mL. Cowaxanthone (34) displayed an antiplasmodial potential (EC50 =1.5 /xg/mL) comparable to that of pyrimethamine (EC50 = 2.8 /xg/mL). Although a number of biological properties are known about xanthones (antibacterial, antifungal and cytotoxicity) there have only been three reports on the

806 antiplasmodial activity of these and only one dealing with a pure natural product.

HX,

0

R3

(30) digitoiutein; Rl = H; R2 = CH3; R3 = OCH3 (33)mycotoxinMT81 (31) rubiadin-1-methyl ether; Rl = OCH3; R2 = CH3; R3 = H (32) damnacanthal; Rl = OCH3; R2 = CHO ; R3 = OCH3

(34) cowaxanthone

4.3 TERPENOIDS 4.3.1 IRIDOIDS The secoiridoid amarogentin (35) isolated from the upper parts of Swertia chirata (Loganiaceae) is a promising compound with leishmanicidal activity. This compound inhibited DNA-Topoisomerase I activity from Leishmania donovani at 30 /xM [79]. This principle is quite interesting as most trypanocidal drugs target type H topoisomerases. There are a few compounds that specifically alter biological functions of toposisomerase I with the enzyme or DNAenzyme complex, an exception being camptothecin an antineoplastic drug [80]. Other natural compounds with the iridoid parent structure also show significant activity against Leishmania parasites. A series of

807 iridoids isolates from Nyctanthes arbortristis (Oleaceae), arbortristosides A, C (36, 37), have antileishmanial activity. Unfortunately, although parasite growth was reduced in vitro at 100 jLtg/mL by 64.5% and 67.4%, respectively, cytotoxicity was noted at 60 jUg/mL [81].

4.3.2 MONOTERPENES Monoterpenes are examples of simple antiprotozoal drugs. Espintanol (11) has already been discussed above and piquerol A (38) was active about 100 /xg/mL [82]. Espintanol, isolated from the bark of Oxandra espinata (Annonaceae) and synthesised [83], is active against Leishmania promastigotes and Trypanosoma epimastigotes. In view of the sensitivity of pathogens from the genus Trypanosoma and Leishmania to oxidative stress, as discussed under section 4.2.1, piquerol A activity may also be due to interaction with the parasite redox cycling system leading to enzyme inhibition and parasite killing.

HO

9OOCH3

H,C OH

(35) amarogentin

"OH HO'

HO

^"

.X^.

(36) arbortristosides A; (38) piquerol A R= p-methoxy cinnamoyl (37) arbortristosides C; R= coumaroyl

4.3.3 SESQUITERPENE LACTONES The antiprotozoal potential of sesquiterpenes is well established since artemisinin (39) and a second endoperoxide sesquiterpene yinghaosu A, were identified as new drugs with high clinical relevance. In addition to artemisinin and other sequiterpene endoperoxides,

808 described in detail in Section 6.1, other series of sequiterpenes with antiprotozoal activity have been described. The sequiterpene lactone parthenin (40) has an EC50 value of 1.29 /xg/mL against P. falciparum in vitro. Although parthenin is described as highly toxic, rats treated with 100 mg/kg/day did not show any signs of toxicity. From these results a series of parthenin derivatives have been synthesized and retested and those with an exocyclic methylene lactone have been identified as active. Exocyclic methylene lactones are well known as the allergic principle in medicinally used plants (mostly Asteraceae, e.g. Arnica montana (Asteraceae)). Pieman et al. [84] demonstrated that parthenin (40) is capable of blocking parasite specific targets responsible for glutathinonylspermidine- and trypanothione synthesis from cysteine and glutathione precursors in Leishmania and Trypanosoma [38]. The sequiterpene lactones brevilin A (41) from Centipeda minima (Asteraceae) and dehydrozaluzanin C (42) from Munnozia maronii (Asteraceae) were discovered from ethnopharmacological screening. From Neuroleaena lobata (Asteraceae), a medicinal plant used in Guatemala for the treatment of Plasmodium infections, activity was documented for germacranolide sesquiterpene lactones as well for furanoheliangolides [85]. These compounds are also active against Leishmania promastigotes and Trypanosoma epimastigotes in vitro. From preliminary structure-activity relationship analysis based on in vitro EC50 data, germanocrenolide sesquiterpenes, like neurolenin A (EC50 = 0.92 /iM) and B (43, 44) (EC50 = 0.62 JLCM), were found to be more potent, than furanoheliangolides as represented by lobatin A and B (EC50 = 15.62 /iM, 16.51 jitM, respectively) (45, 46) [85]. The main reason for the lower activity of the former could best be explained by the shift of the double bond from the 2,3 (neurolenin) into the 3,4 (lobatin) position, suggesting that one of the structural requirement in sesquiterpenes is an a/6-unsaturated keto function. Another approach for discovering antiprotozoal natural products is described by Koshimizu et al. [86]. Wild chimpanzees were observed to chew young stems of Vemonia amygdalina (Asteraceae) from which antiplasmodial sesquiterpenes (vemodalin (47), vemolide, hyroxyvemiladin) have been isolated. Also unusual sesquiterpenes (48, 49) with significant antiplasmodial activities (EC50 < 4 /ig/mL) were isolated from marine red algae {Laurencia implicata.

809

Rhodomelaceae) and brown Rhizophylladaceae) [87].

o (39) artemisinin

algae

6

T

homemannii,

^

(40) parthenin

OH N ^ ^ ^ T T ^ ^

(Portiera

o

(41) brevilin A

T

T

T

(42) dehydrozaluzanin C

. OH

(43) neurolenin A; R = H (44) neurolenin B; R = OAc

(45) lobatin A

(46) lobatin B

"XSO ? (47) vernodalin

(48) 5-isopropyl-3,3,9-trimethylbicyclo-nona-5-en-4ol

(49) 9,10-trisepoxypentadec-12-1,2-diene

4.3.4 DITERPENES Diterpenes from many species are well known for their biological activity and are amongst the most widely distributed terpenes in the plant kingdom. However, most of them combine both high antiparasitic activity as well as high cytotoxicity to mammalian cells. Both jatrogrossidione (50) and jatrophone, isolated from Jatropha

810 grossidentata and J. isabelli (Euphorbiaceae) respectively, showed significant activity against Leishmania promastigotes in vitro (EC50 values of 0.75 /xg/mL (2.4 mM) and 5 jLtg/mL (16 mM)) and L. amazonensis amastigotes in vivo where jatrophone reduced parasite growth at a dose of 25 mg/kg/day. Unfortunately, both proved to have toxic effects of therapeutic doses [88]. A series of 80 labdane derivatives showed significant antileishmanial activity (L. donovani L enriettii, L. major, L. infantum), but cytotoxicity increased in parallel with the antiprotozoal effect [89]. From structure-activity studies overbridged tri- or tetracyclic ring labdanes had more significant activity in comparison to bicyclic labdanes. These results confirmed other data cited in literature, for example the subtype of overbridged labdane derivates (e.g. kauran-, trachyloban-type), as displayed by ent-kaur-16a-ol-19oic acid (51) from Mikania obtusata, ent-kauran-16-en-19-oic acid from Wedelia paludosa (52), and (-)-trachyloban-19-oic (53) from Viguirea aspillioides (Asteraceae). Results of their extraordinary activity against Leishmania and Trypanosoma spp. have been published [90-92]. However, most antiprotozoal labdanes also show low EC50 values in vivo (EC50 < 3.0 /xg/mL), and high toxicity (EC50 < 10 jLtg/mL). One compound isolated from the sponge Acanthella klethra, axisonitrile (54), a sequiterpene derivative, showed potent antiplasmodial activity with no detectable cytotoxic properties [93]. A series of different terpenes have been isolated from marine organisms and are of considerable interest for their unique structural features in antiparasitic drug research. In contrast to plant metabolites most of these 100 plus isolated natural products contain isonitrile, isothiocyanate, and thiocyanate functionalities. Major interest has been focussed on these marine drugs by [94], doubling the information in the literature and identifying novel compounds with EC50 values below 1 jLCg/mL with high selective indices (SI > 50) [95]. From the point of antiparasitic research four diterpene subclasses are of interest: kalihinane diterpenes, e.g. kalihinol A (55) [96], amphilectanes, for example 7-isocyano-ll(20),14-epiamphilectadiene (56) [95], cycloamphilectanes, for example 7-isocyanocycloamphilect-10-ene (57) [95], and isocycloamphilectanes, for example 7,20-diisocanoisocycloamphilectane (58) [95]. The potent and

811 selective biological activities of these compounds represents an exciting advance in the search of novel antiplasmodial agents. In vivo studies are required now to validate the potential of these compounds. Not only novel compounds with unique structural features attract attention, but also well known compounds like macrocyclic terpenes used in other pharmacological fields, like taxol and epothilione (59) (potent antineoplastics) or highly toxic phorbol esters, have been tested in antiparasitic drug screens. Macrocyclic trichothecenes are known as mycotoxins, and a variety of biological activities have been reported. From the fungal culture of Myrothecium verrucaria BCC 112 (Hypomycetes) roridin E (60) show high antiplasmodial activity (EC50 = 0.15 ng/mL) but also significant cytotoxicity (EC50 = 0.5 ng/mL, KB cells (human epidermal nasopharyngeal cancer cells) [97]. It must also be noted that the selective index of roridin E (SI = 12) in comparison to artemisinin (SI > 7,100) was too low to make it useful for further in vivo investigation. The experience of research into antineoplastics with macrocyclic ring systems suggests that it maybe possible to find or synthesize new trichothecene derivatives with high antiplasmodial activity and low toxicity. Oketch-Rabah et al. showed that the macrocyclic germancrane dilactone 16,17-dihydrobrachycalyxolide (61), from Vemonia brachycalyx (Asteraceae), has both antileishmanial and antiplasmodial activity [98]. In in vitro tests the compound is strongly active against L. major (EC50 =17 /xg/mL) and P. falciparum (EC50 =17 /ig/mL), but also inhibits the proliferation of human lymphocytes at the same concentration indicating general toxicity [98].

COOH

(50) jatrogrossidione (51) ent-kaur-16a-ol-19-oic acid

^

COOH

(52) ent-kauran-16-en19-oic acid

812

COOH

(53)

(-)-trachyloban-19-oic

(56)7-isocyano-ll(20),14epiamphilectadiene

CI

(54)

axisonitrile-3

(57) 7-isocyanocycloamphilect-10-ene

O

OH

(55)

kalihinol

(58) 7,20-disiocanoisocycloamphilectane

O

(59) epothilone A

(60) roridin E

A

(61) 16,17-dihydrobrachylocalyxolide

813 4.3.5 TRITERPENES Triterpenes and saponins from plant sources are known for their biological activity (antineoplastic, anthelmintic and antiviral), but they exhibit some toxicity to humans and other mammals. Despite the fact that triterpene action in biological systems is well known, the first rational reports on their antiprotozoal activity were first described late 1970s. Tingenone (62) and pristimerin (63), from species of Celastraceae, have in vitro activity against T. cruzi amastigotes and P. falciparum, Tingenone could act through interaction with DNA or inhibition of DNA synthesis [45]. The lupane-type triterpene betulinic acid (64), also known for its antineoplastic effect, was identified by bioguided fractionation and identified as the antiplasmodial principle of Triphyophyllum peltatum and Ancistrocladus heyneanus (Dioncophyllaceae and Ancistrocladaceae, respectively) [99]. Against P. falciparum in vitro betulinic acid had an EC50 value of 10.46 jLtg/mL and with the exception of human melanoma cells (EC50 =1-5 jLig/mL), only moderate cytotoxicity (EC50 > 20 /xg/mL). Based on saponins isolated from ivy, Hedera helix (Araliaceae), MajesterSarvonin et al. gave a first insight in the structure-activity relationship of different antileishmanial saponin types [100]. Bidesmosides have no effects on the proliferation of either promastigote or amastigotes. In contrast, monodesmosides and hederagin (65) were highly active, especially the sodium salts of a- and 6-hederin (66, 67) are highly active at concentration similar to that of pentamidine. Monodesmosides from Hedera helix damaged macrophages host cells at concentrations between 5 and 25 ixg/mL, but this level of toxicity was in the same range as for Glucantime [100]. The use of saponins as drugs is limited due to poor bioavailability, reduced absorption in the gastrointestinal tract and their hemolytic toxicity when given by parenteral route. It is noteworthy that despite this fact medicinal plants that contain saponins are known. Oketch-Rabah et al. isolated a new steroidal saponin, muzanzagenin (68), from Asparagus africanus (Liliaceae) which had antileishmanial and antiplasmodial activity (EC50 = 70 /xM, L. major, EC50 = 61 /xM, P, falciparum K39) [101].

814

.xCOOCHg

(62) tingenone

(63) pristimerin

COOR, COOH

(64) betulinic acid

(65) hederagin; R,= H, R2 = OH, R3 = H (66) a-hederin; Rl= rham(l-^2)ara (l-^,R2 = OH,R3 = H (67) 6-hederin; Rl= rham(l->2)ara (1->,R2 = H,R3 = 0H

(68) muzanzagenin

4.3.6 LIMONOIDS Bitter terpenoids, known as limonoids, are biosynthetically related to the quassinoids that are produced by species of Meliaceae. One well known representative from this family is Azadirachata indica, the neem tree, widely used as an antiplasmodial plant in Asia. Rochanakij

815

et al. initially identified nimbolide (69) as the active antimalarial principle of the neem tree (EC50 = 0.95 ng/mL, P. falciparum Kl) [102]. Nimbinin, geduin (70) (EC50 = 0.39 ng/mL, P. falciparum D6) and its dihydroderivative were also found to be active in vitro against Plasmodium parasites in the range of EC50 values of 0.72 - 1.74 /xg/ml [103-105]. The mode of action of this natural product group is still unclear. The cytotoxic activity of gedunin was moderate (EC50 = 275 /xg/mL). Geduin derivatives, D-seco-limonoids, do not show significant antiplasmodial activities (EC50 > 100 ng/mL, P. falciparum D6) comparable to the parent structure of geduin [105]. Insufficient geduin-related limonoids have been tested to allow a proper evaluation of this group.

'"OAc

(69) nimbolide

(70) geduin

4.3.7 QUASSINOIDS Quassinoids are biosynthetically related to triterpenes and share the same metabolic precursors. Most of the presently known quassinoids were found in the family Simbaroubaceae, and extracts and isolated natural compounds have been widely tested [106]. As deduced from structure-activity relationship analysis most potent quassionoids have a pentacyclic ring systems with a lactone function and a methyleneoxygen ring bridge linking C-8 and C-13 (e.g. brusatol (71)) or C-11 (e.g. ailanthinone (72)) [107]. Most of the quassinoids do not have a sufficient selective index to be considered as lead structures for clinical drugs. The antiplasmodial activity is high with EC50 values around 0.02 jU-g/mL, but the most active compounds like simalikalactone D (73) from Simaba guianensis (Simaroubaceae) [108], 156-heptylchaparrinone [109] and different sergolide

816

quassinoids [110] were too toxic in vivo. The mode of action of quassinoids seems to be the inhibition of protein synthesis [111]. Quassin (74) is inactive due to the missing methylene/oxygen bridge. A current research aim is to modify the parent compound synthetically, to find semisynthetic quassinoids with reduced toxicity and to improve the toxic/therapeutic ratio. As a potential lead structure for this approach Francois et al. identified chaparrinone (75) derivatives with an improved selective index [112]. The in vitro activities of chaparrinone and 15-desacaetylundulatone (76) were lower than reported activities of certain other quassinoids (EC50 = 0.037 jLtg/mL and 0.047 /xg/mL, P. falciparum NF54, respectively). In vivo, however, when given at 50 mg/kg/day they produced significant reduction of parasitaemia with survival times similar to those of the control group with no signs of acute toxicity [112]. The contradictionary toxic/therapeutic ratio was explained by the occurence of the keto-function at C-2 and missing hydroxy] group at C-14 in a C8->C11 overbridged ring system improving its performance.

(71) brusatol

(72) ailanthinon

rr (13) simalikalactone D

817 0CH3

HXO

(75) chaparrinone, R = H (76) 15-desacaetylundulatone; R = 0-tiglate

4.4. N-CONTAINING NATURAL PRODUCTS (NONALKALOIDS) Besides the large group of alkaloids some nitrogen-containing natural products, do not fall under the definition of alkaloids, and are therefor discussed separately.

4.4.1 STEROIDAL ALKALOIDS The literature contains numerous reports on biological activities of nitrogen-containing steroids of the Solanum-iypQ. Most are quite common derivatives that occur in vegetables and thus in the daily diet; a-solanine (77), tomatine (78) which have been tested for their toxicological potential but their antiparasitic activity has not fully tested. Chataing et al. tested a series of Solanum-iype steroid alkaloids against Trypanosoma cruzi in vitro in comparison to ketoconazole [113]. Glycoalkaloids containing a chacotriose sugar moiety showed trypanocidal activity against epimastigote and against metacyclic trypomastigote forms. The mechanism of action probably is the membrane, followed by structural changes of internal compartments, resulting in destruction of organelles such as mitochondria and glycosomes. The data indicate that steroid alkaloids containig 6chacotriose trisacharide moiety, e.g. a-chaconine (79) and asolarmargine (80), posses antitrypanosomal activity in the range of EC5o = 6.0/xM[113].

818

(77) a-solanine (solatriose)

(79) a-chaconine (chacotriose)

819

(80) a-solarmargine (chacotriose)

4.4.2 OTHER N-CONTAINING COMPOUNDS Piperine (81), a major constitutent of pepper (Piper nigrum, Piperaceae), was tested against L donovani promastigotes [114]. However, as this compound has been part of the daily diet over centuries, is cytotoxic potential, and only the moderate antileishmanial activity, it has not been considered as a potential antiparasitic agent. Recent studies found three halogenated pyrrole-2-carboxylic acids in a Maltese sample of the marine sponge Agelas oroides (Agelasidae) (82-84) [115]. Activity was evaluated against P. falciparum (strains D6 and W2) with EC50 values between 3.3 and 5.3 /xg/mL. Parallel testing for against KB, Lul, LNCaP and ZR-75-1 cells showed cytotoxic activity (EC50 = 2.0 -14.5 /xg/mL).

'^ (81) piperine

(82) 4,5-Dibromopyrolle-2-carboxylic acid; R = OH (83) 4,5-Dibromopyrolle-2-carboxylic methylester; R = OCH3 (84) Oroidin; R = NH^ NH

820 4.5 ALKALOIDS Alkaloids are one of the most important classes of natural product providing drugs for humans since ancient times. Most alkaloids are well known because of their toxicity or use as psychodelic drugs (e.g. cocaine, morphine or the semisynthetic LSD), but many alkaloids have had a deep impact on the treatment of parasitic infections. The outstanding example is quinine (1) from Cinchona succirubra (Rubiaceae) used for the treatment of malaria for more than three centuries.

4.5.1 QUINOLINES Up to the middle of this century quinine (1) was used for the treatment of malaria, and with the widespread development of chloroquineresistant strains of Plasmodium falciparum it has become important again. Quinine has been used for the treatment of malaria for more than 350 years and has its origin in Peru in the early 17^^ century. Quinine was the lead structure in the discovery of new synthetic derivatives like mefloquine that have higher antimalarial activity. This section will focus on other new quinoline alkaloids. The mechanism of antiplasmodial action and resistance of quinolines is well described elsewhere [116]. As the result of an ethnopharmacological search for new antileishmanial drugs aryl- and alkyl-quinolines were isolated from Galipea longiflora (Rutaceae) [117]. These simple natural quinohne derivatives 2-n-propylquinoline (85), chimanine B (86), chimanine D (87), 2-n-pentylquinoline (88), 4-methoxy-2-phenylquinoline (89), and 2-(3,4-methylenedioxyphenyl)-quinoline (90) were tested against strains of parasites causing cutaneous leishmaniasis and exhibited activities of EC50 = 25-50 jLtg/mL or 150 - 300 /xM [117, 118]. Only chimanine B was active in vivo (50 mg/kg, BALB/c mice); twice daily oral treatment results in a decrease of parasite load by 95 %, a similar activity to that of the standard drug Glucantime [119]. No mechanism has been found yet to explain these effects. Two piperidino-4quinolinone alkaloids dictyolomide A (91) and B were identified from Dictyoloma incanescens (syn. D. vandellianum) and D, peruviana (Rutaceae), collected in South America (Bolivia). They induced a

821

lysis of various strains of Leishmania promastigotes in vitro at a concentration of 100 /xg/mL [120].

(85) 2-n-propylquinoline; Ri = C3H7, R2= H (86) chimanine B; Ri= CH=CH-CH3, R2= H (87) chimanine D; Ri XN>/ = , R2 = H

(88) 2-n-pentylquinoline; Ri = C5H11, R2= H (89) 4-methoxy-2-phenylquinoline; Ri = phenyl, R2= OCH3 (90) 2-(3,4-methylenedioxyphenyl)quinoline, \..-^%«^^ R2 = H Ri=

^'j

(91) Dictyolomide A

4.5.2 BENZYL- AND NAPHTHYLISOQUINOLINE ALKALOIDS The chemical structure of this alkaloid group is well known through the widespread and abundant berberine (92). Many antiprotozoal isoquinolines have been isolated from the families Annonaceae, Berberidaceae, Menispaermaceae and Hemandiaceae [65]. Berberine is active at EC50 = 10 /xg/mL against Leishmania amastigotes within murine peritoneal macrophages. Vennerstrom et al. tested berberine and several of its derivatives for antileishmanial activity against L. donovani and L. panamensis in golden hamsters [121]. Tetrahydro-

822 berberine is less toxic and more potent than berberine against L. donovani but was not as potent as meglumine antimonate (Glucantime). Only berberine, the natural product, showed significant activity (greater than 50% suppression of lesion size) against L. panamensis. Berberine was used for cutaneous leishmaniasis in India but was not effective when applied topically [122]. Recently catecholic berberines, (-)-pessione (93) and (-)-spinosine (94), have been isolated and tested for antileishmanial and trypanocidal {T. cruzi) activity in vitro. At a single concentration of 250 /xg/mL 50 % inhibition for T. cruzi is found, indicating low trypanocidal activity [123]. Naphthylisoquinoline alkaloids isolated from tropical llianas have been identified as new promising leads as antiprotozoals. They show remarkable activity against P. falciparum in vitro and in vivo, as well against Leishmania and Trypanosoma species [124]. Extracts from the single species of Triphophyllum peltatum (Dioncophyllaceae) dioncopeltine A (95) and, in particular, dioncophylline B (96) and dioncophylline C (97) exhibited high antiplasmodial activity [124]. Dioncopeltine A is able to suppress parasitaemia almost totally, while dioncophylline C cured infected mice completely after oral treatment with 50 mg/kg per day for 4 days without noticeable toxic effects. Analysis of the dose-response relationship of dioncophylline C revealed an ED50 dose of 10.71 mg/kg/day. Although four daily treatments with 50 mg/kg/day are needed to achieve parasitological cure, one oral dose is sufficient to kill 99.6% of the parasites. Intravenous application of dioncophylline C is even more effective, with an ED50 of 1.90 mg/kg/day and no significant toxic effects. The compound also suppressed more established P. berghei infections when applied orally at day three post infection. Both dioncopeltine A and dioncophylline C are active against the chloroquine-resistant P. berghei Anka CRS parasites. Structure-activity relationships indicate that the presence of a secondary amine function, and the absence of an oxygen substituent at C-6 and R-configuration at C-3 are important. Recently, a novel dimeric antiplasmodial naphthylisoquinoline alkaloid heterodimer, korundamine A (98), has been isolated from another species, Ancistrocladus korupensis belonging to the family Ancistrocladaceae that is biogenetically related to Dioncophyllaceae. Korundamide A is one of the most potent naturally occuring naphthylisoquinoline dimers

823 yet identified in antiplasmodial in vitro screening with an EC50 of 1.1 jLtg/mL against P. falciparum [125].

(93) (-)-pessione; R = H (94) (-)-spinosine; R = CH3

OH = HO- - < ^

(95) dioncopeltine A

>X%:^ -

-

OH =

[I

^OCH,

(96) dioncophylline B

0CH3

OH

(98) korundamine A

(97) dioncophylline C

824 4.5.3 BISBENZYLISOQUINOLINES A number of different bisbenzylisoquinolines with antiprotozoal activity have been identified. Although their antiparasitic activity has been recognised for years, particularly the antiplasmodial activity, the mechanism of action of these alkaloids is still unclear. So far in vivo activity has not been demonstrated. In vitro most bisbenzylisoquinolines exhibit activities in vitro far below 1 /xg, close to the EC50 value of chloroquine (EC50 - 0.2 /iM). Some bisbenzylisoquinolines like gyrocarpine (99), daphnandrine (100) and obaberine (101) are more potent than antimonials and nifurtimox and benznidazole, respectively, against Leishmania and Trypanosoma parasites (EC50 < 50 jtig/mL) [123, 127]. Despite the fact that a large number of bisbenzylisoquinolines has been tested, a clear structureactivity relationship is not clear. Some structural features that appear to be important include the linkage of the heteromers and the number of ether bonds. Studies on Triclisia alkaloids showed that those compounds with two ether bridges (e.g. pycnamine) (102) are more potent than those with three ether bridges such as cosculine (103) (EC50 values of 0.15 /xg and 15.56 ng/mL, respectively) [128]. Recently, Angerhofer et al. published an intensive study on structureactivity/toxicity-relationship of a series of 53 structurally different bisisoquinolines [129]. More than half of the compounds tested against KB cells for cytotoxicity and P. falciparum strains W2 and D6, however, showed selective antiplasmodial activity, with > 100-fold greater toxicity toward one or both of the P. falciparum clones, relative to cultured mammalian cells. The most selective alkaloids were (-)-cycleanine (104), (+)-cycleatjehine (105), (H-)-cycleatjehenine (106), (+)-malekulatine (107), (-)-repandine (108), and (+)temuconine (109). As a result of these studies, an understanding of the relationships between the structures, the stereochemistry, the substitution patterns of these alkaloids and their in vitro antiplasmodial and cytotoxic activities are beginning to emerge. The quatemarization of one or two nitrogen atoms, presence of an acetyl function at N-2', and N-oxide formation leads to a loss of toxicity and antiplasmodial activity. The decrease in lipophilicity (membrane permeability) of all of these alkaloids probably contributes to the lower toxicity observed. Within each subgroup of bisbenzyliso-

825 quinolines a change of configuration of the chiral center, as well as modification of substituents, may lead to independent changes in cytotoxicity and antiplasmodial activity. However, except for the three one-bridged compounds, (+)-neothalibrine (110), (+)-temuconine, and (+)-malekulatine, which show low toxicity and appreciable antiplasmodial activity, the current results do not reveal any clear structure-activity relationship between subgroups of bisbenzylisoquinoline alkaloids. With the exception of the onebridged bisbenzylisoquinolines, all possess a large heterocycle of 18 to 20 atoms, which confers flexibility to the molecule. A study of the conformations assumed by compounds of the same subgroup (e.g., modification of conformation with the change of configuration at C-1 and C-H should give more information on the structure-activity relationship. As the therapeutic index of the most antiplasmodial alkaloids is around 100 and those of chloroquinine, quinine and artemisinin, are 5460, >285 and >4680 respectively, the bisbenzylisoquinolines do not appear to be promising candidates as antimalarial agents. Monomeric benzylisoquinolines do not appear to have potential. The activity of some aporhinoids, like isoguattouredigine (111) (from Guatteria foliosa, Annonaceae) argentinine (112), unonopsine (113) and hydroxynomuciferine (114) show only minor activity against T. cruzi in vitro (EC50 > 250 ixM) [130, 131]. The isoquinoline derivate camptothecin (115), a well known antineoplastic drug and a topoisomerase I inhibitor, showed antiprotozoal activity when tested against L. donovanU T. cruzi and T. b. brucei with EC50 values of 1.5, 1.6 and 3.6 /xM [132, 133]. For these parasites, camptothecin is an important lead for much-needed new chemotherapy, as well as being a valuable tool for further study of topoisomerase I activity.

826 OCH,

PCH, PCH,

R.0

(99) (+)-gyrocarpine

(100) (+)-daphnandrine; Rj = CH3, R2 = H (101) (+)-obaberine; R, = R2= CH3 (108) (-)-repandine; Ri = H, R2 = CH3

.OCH, PCH, PCH,

HO

(102) (+)-pycnamine

o-

(103) (+)-cosculine

^OCH, OCH,

H3CO'

(104) (-)-cycleanine

(105) (+)-cycleatjehine; R = H (106) (+)-cycleatjehinine; R = CH3

827

OCH,

/

"^V—OH OCH3

OCH3

(109) (+)-temuconine; Ri = H, R2 = CH3 (110) (-f )-neothalibrine; Ri = CH3, R2 = H

(107) (+)-malekulatine

H,CO'

(111) isoguattouredigine

(112) argentinine

(114) hydroxynornuciferine

(113) unonpsine

(115) camptothecin

4.5.4 INDOLES Indoles comprise another group of alkaloids with high biological activity. The indole sub-structure is widely distributed in the plant kingdom. Some indole derivatives have been reported to possess antiprotozoal activity. Indoles are biosynthetically derived from

828 tryptophan metabolism, which appears to be important in protozoa such as Leishmania and Trypanosoma, The end products of the tryptophan metabohsm are thought to be involved in carbohydrate metabolism [134]. A simple derivative with antileishmanial activity (L. amazonensis amastigotes) is harmaline (116), often found in indole containing plants, e. g. Peganum harmala (Rutaceae). Harmaline, a harmane-type (117) indole alkaloid, is active at EC50 = 24 jitg/mL, but too toxic for human use. The relevant pharmacological and antiprotozoal action of harmaline and related tryptamine derivatives is intensively discussed by [135]. Other monomeric indole derivatives are olivacine (118) and ellipticine (119). Both were identified as antiprotozoal compounds in the 1970s [136], and both showed in vitro activity against T, cruzi epimastigotes with EC50 values of 2.5 and 5.0 jLtg/mL, respectively. In contrast, both were inactive in vivo, maybe because of inactivation through first pass metabolism. Cryptolepine (120) and related alkaloids, indole-quinolines, have been isolated from Cryptolepis sanguinolenta (Periplocaceae) and were active in vitro against P. falciparum in vitro (EC50 = 27-41 ng/mL, P. falciparum^!, D6, and Kl) but failed in vivo (only 10.8 - 19.4 % suppression of P. yoelii at 100 mg/kg/day) [137]. Among the group of "dimeric" indole alkaloids the tubulin polymerisation inhibitor and antineoplastic agent vinblastine (121) is of experimental interest. In the therapy of protozoa infections its use is limited because of the poor therapeutic ratio against Trypanosoma gambiense, L donovani and P. falciparum [138, 139]. Conodurine (122) and conoduramine from Peschiera van heurkii (Apocyanaceae) (123) showed antileishmanial activity with EC50 value of 50 jiig/mL against L. amazonensis promastigotes in vitro [140]. Conodurine was less active than Glucantime (EC50 = 40 mg/kg/day, BALB/c mice) in vivo against L. amazonensis, and doses of conodurine at 200 mg/kg were toxic [140]. The mechanistically unusual antineoplastic product taxol (124), a diterpene-alkaloid, inhibits depolymerization of tubulin also in Plasmodium, Trypanosoma and Leishmania parasites, acting at concentrations as low as 0.1 /xM. Because of toxicity reasons, this compound does not seem to be a particular attractive candidate for further development as antiparasitic agent.

829

(116) harman; Ri = H, R2 = CH3 (117) harmaline; Ri = OCH3, R2 = CH3

(118) olivacine; R, = CH3, R2 = H (119) ellipticine; Ri = H, R2 = CH3

\ ^ w ^

OH

OCOCH3

(121) vinblastine; R = COOCH3

(120) cryptolepine

C00CH,PH

HjCOOC-

H3COOC

(122) conodurine

(123) conoduramine

830

(124) taxol (paclitaxel)

4.6 OTHER NATURAL PRODUCT CLASSES 4.6.1 NUCLEOSIDES Sinefungin (125), a natural nucleoside isolated from cultures of Streptomyces incamatus and S. griseolus, is structurally related to Sadenosylhomocysteine and S-adenosylmethionine (SAM) (126) [141]. Sinefungin has been shown to inhibit the growth of various fungi and viruses, but its major attraction resides in its potent antiparasitic activity. This natural product has attracted renewed interest since the synthetic S-adenosylmethionin-decarboxylase inhibitor 5-([(Z)-4amino-2-butenyl]-methylamino)-5-deoxyadenosine (MDL 73811), a decarboxylated S-adenosyl-L-methionine analog, was introduced in experimental studies as a new drug for the treatment of Leishmania and Trypanosoma [142, 143]. Sinefungin does not inhibit Sadenosylmethionin-decarboxylase, but its action is focussed on SAMsynthases affecting methylation of macromolecules as nucleic acids and blocking of DNA polymerase by reduction of dATP. NH,

NH

H3

»j»"^N^

NH,

NH^

NH,

Ij^'^^N^

HO

OH

HOOC HO

OH

(125) sinefungin

(126) SAM

831 4.6.2 AMINOGLYCOSIDES The aminoglycoside antibiotic, aminosidine (127), also known as paromomycin and monomycin, was first shown to be active against experimental cutaneous leishmaniasis in the early 1960s [144]. Later studies showed that is was the most potent among a series of tested compounds derived from microbiological sources [145]. Interest in the antileishmanial properties of this compound has been revived by the development of topical formulations for the treatment of cutaneous infections. It was found that topical application of either paromomycin or gentamicin, together with a transdermal enhancing agent, cured the parasite lesion, and that combined treatment with the two compounds had an additive effect [146]. The pharmacology and antiparasitic mechanism of these drugs formulations is discussed in detail by [147].

HO

Q

HO OH OH

(127) aminosidine (paromomycin)

5.

FROM MEDICINAL HERB TO THE DRUG MARKET

5.1 FROM ARTEMISININ TO ARTEMETHER (PALUTHER®), ARTEMETHER (ARTENAM®) AND ARTESUNATE (ARSUMAX®) History. For thousands of years Chinese herbalists treated fever with a decoction of the plant called "qinghao", Artemisia annua, "sweet wormwood" or "annual wormwood" belonging to the family of Asteraccae. In the 1960s a program of the People Republic of China re-examined traditional herbal remedies on a rational scientific basis including the qinghao plant. Early efforts to isolate the active principle

832 were disappointing. In 1971 Chinese scientists followed an uncommon extraction route using diethyl ether at low temperatures obtaining an extract with a compound that was highly active in vivo against P. berghei in infected mice. The active ingredient was febrifuge, structurally elucidated in 1972, called mostly in China "qinghaosu", or "arteannuin" and in the west "artemisinin". Artemisinin, a sesquiterpene lactone, bears a peroxide group unlike most other antimalarials. It was also named artimisinine, but following lUPAC nomenclature a final "e" would suggest that it was a nitrogencontaining compound that is misleading and not favoured today. Chemistry and Pharmacology. The chemistry and pharmacology of artemsinin has been reviewed in detail by Klayman [149], Luo and Shen [150], Woerdenbarg et al. [151], and van Agtmael et al. [152]. The limited stability as well as the poor solubility of artemisinin in water and oil, the two commonly used and approved media for parenteral administration, prompted scientist to prepare semisynthetic derivatives leading to improved solubility in water or higher chemical stability in oil formulations. Artemisinin is poorly soluble in water and decomposes in other protic solvents, probably by opening of the lactone ring. It is soluble in most aprotic solvents and is unaffected by them at temperatures up to 150°C and it shows a remarkable thermal stability. This section will focus on biological and pharmaceutical aspects; synthetic routes to improve antimalarial activity and to synthesize artemisinin derivatives with different substitutiuon patterns are reviewed elsewhere [151, 153]. Most of the chemical modifications were conducted to modify the lactone function of artemisinin to a lactol. In general alkylation, or a mixture of dihydroartemisinin epimers in the presence of an acidic catalyst, gave products with predominantly 6-orientation, whereas acylation in alkaline medium preferentially yields a-orientation (128-132). Artemether (128) as the active ingedient of Paluther® is prepared by treating a methanol solution of dihydroartemisinin with boron trifluoride-etherate yielding both epimers. The main goal was to obtain derivatives that show a higher stability when dissolved in oils to enable parenteral use. The a-epimer is slightly more active (EC50 = 1.02 mg/kg b.w.) than the 6 epimer (EC50 = 1.02 mg/kg) and artemisinin itself (EC50 = 6.2 mg/kg) [154]. Synthesis of derivatives

833 with enhanced water solubility has been less successful. Sodium artesunate, Arsumax®; (132) has been introduced in clinics, is well tolerated and less toxic than artemisinin. The synthetic routes start with dihydroartemisinin treated with succinic anhydride in the presence of DMAP.

(128) (129) (130) (131) (132)

dihydroartemisin; R = H (a + 6) artemether; CH3 (6) arteether; CH2CH3 (6) artelinate; CH2C6H4COONa (B) artesunate; COCHjCHsCOONa (a)

Pharmacokinetics and Pharmacodynamics. A characteristic of artemisinin and its related endoperoxide drugs is the rapid clearance of parasites in the blood in almost 48 hours. Titulaer obtained pharmacokinetic data for the oral, intramuscular and rectal administration of artemisinin to volunteers [155]. Rapid but incomplete absorption of artemisinin given orally occurs in humans with a mean absorption time of 0.78 h with a absolute bioavailability of 15 % and relative bioavailability of 82%. Peak plasma concentrations at a given dose are reached after 1-2 h and the drug is eliminated after 1 to 3 hours. The mean residence time after intramuscular administration was three times that when given orally. Other routes of adminstration, for example rectal or transdermal, are of limited success, but for the treatment of convulsive malaria in children artemeether in a rectal formulation is favoured. Artesunate acts as a prodrug that is converted to dihydroartemisinin. When given orally the first pass mechanism in the gut wall takes places metabolizing half of the administered dose. Oral artemether is rapidly absorbed reaching maximum blood levels (Cmax) within 2-3 hours. Intramuscular artemether is rapidly absorbed reaching Cmax within 4-9 hours. It is metabolized in the liver to the demethylated derivative dihydroartemisinin. The elimination is rapid, with a half-life time

834 (Ti/2) of 4 hours. In comparison, dihydroartemisinin (128) has a Tm of more than 10 hours. The degree of binding to plasma proteins varies markedly according to the species considered. The binding of artemether to plasma protein was 58% in mice, 61% in monkeys and 77% in humans. Radioactive labeled artemether was found to be equally distributed in plasma as well as in red blood cells indicating an equal distribution of free drug between cells and plasma.

Haem (Fe^^)

Haem (Fe^^)

HXi

HX-

Alkylation of proteins Formation of

Fig. (1). Mechanism of action of artemisinin drugs, Active metabolites and formation of reactive epoxide intermediates (according to van Agtmael et al., 1999)

From the toxicological point of view artemisinin seems to be a safe drug for the use in humans. In animal tests neurotoxicity has been documented, but as yet this side effect has not been reported in

835 humans [156]. A major disadvantage of the artemisinin drugs is the occurrence of recrudescence when given in short monotherapy. So far no resistance has been observed chnically although it has been induced in rodent models in vivo. The mechanism of action is different from the other clinically used antimalarials. Artemisinin drugs act against the early trophozoite and ring stages, they are not active against gametocytes, and it affects blood- but not liver-stage parasites. The mode of action is explained by haem or Fe^"*", from parasite digested haemoglobin, catalysing the opening of the endoperoxide ring and forming free radicals. Malaria parasites are known to be sensitive to radicals because of the lack of enzymatic cleaving mechansims. The mechanism of action and the metabolism of reactive artemisinin metabolites is shown in Fig. 1. Prospects. Other indications for malaria for the artemisinin drugs are currently under investigations. Without a final proof, other erythrocyte persisting parasites like Babesia are maybe another interesting target parasite. But also Toxoplasma gondii, Pneumocystis carinii infections in mice have been treated successfully with artemisinin drugs [157159].

6.2 FROM QUININE TO MEFLOQUINE History. Quinine has been listed as one of the six most important plant products that have influenced human history [160]. Cinchona, or "Quinine Bark" is one of the most famous plants from South America and most important discoveries. Legend says that the name "cinchona" comes from the Countess of Chinchon, the wife of a viceroy of Peru, who was cured in 1638 of a malarial type of fever by using the bark of the Cinchona tree. The legend starts with a misspelled name, continues with an extract named mistakenly by Linnaeus in 1742 as "quinquina", and maintains the reputed traditional use of plant extract for a disease probably introduced to that continent by Europeans and their African slaves [see 161, 162, 6]. Quinine bark was used by the Jesuits very early in its history, first advertized for sale in England in 1658 and was made official in the London Pharmacopoeia in 1677.

836 Several years after "Countess's powder" arrived in England, it arrived in Spain where the virtues of the bark were rapidly recognized, from this "tree of fever of the region of Loxa". Rapidly and due to the influence of the Company of Jesus, the "Jesuits' powder" became known all over Europe. Physicians gave credit to the drug, and because of the specificity of its action on malaria, it was recognized officially even when the identity of the producing species remained unknown. Despite the confused history quinine still provides an important treatment for malaria, in particular cerebral malaria, with formulations of interest such as Quinimax, containing quinine and other active isomers, quinidine and cinchonine. Perhaps of greater significance to the history of malaria treatment, by a contorted route, is the work of William Perkins who in 1856 while trying to synthesise quinine discovered the first synthetic dye "mauve". His work led to analine dyes that in turn led to methylene blue, the first compound rationally used for the treatment of malaria by Guttman and Ehrlich in 1891. Methylene blue provided the template for the design of the aminoquinoline drugs mepacrine, primaquine and chloroquine in the 1920s and 1930s [161]. Research on quinine also led to other drugs. In 1944 scientists were able to synthesise and structure elucidate (Fig. 2) the quinine alkaloid in the laboratory in Germany. This led to various synthesized quinine drugs to treat malaria and the use of the common bark and the natural quinine extracted from the bark and sold as antimalarial drugs fell out use. Indonesia and India still cultivates the Cmc/iona-tree, but Zaire has become the top supplier of a world market which is also supplied by other African countries (Burundi, Cameroon, Kenya), and much lower on the list of producers are the South American countries of Peru, Bolivia and Ecuador. Plant products have also played a significant role in the treatment of another parasitic disease, amoebiasis. The root and corm extracts of Cephaelis ipecuanhana were traditionally and empirically for the treatment of dysentry and the active ingredient emetine was isolated in 1817. It was not until 1912 that the antiamoebic activity of the alkaloid emetine was placed on a rational basis. Another product cepahaeline proved to be less active than its methyl ester emetine. In the early 1960s the derivative dehydroemetine was shown to be more active against E. histolytica and less toxic than the parent compound.

837 This compound is still in use. Traditional uses of other plant products led to the identification of other compounds with activity against Entamoeba in the 1940s and 1950s, including the steroidal alkaloid conessine from Holarrhena sp. (Apocyanaceae), glaucarubin from Simarouba amara (Simaroubaceae), and henna from Lawsonia alba (Lythraceae) [163].

\

1 R = H: cinchonine 2 R = OCH3: quinidine ^

R5

3 R = H: epi-cinchonine 4 R = OCH3: epi- quinidine

R

5 R = H: cinchonidine 6 R = OCH3: quinine

7 R = H: epi- cinchonidine 8 R = OCH3: epi-quinine

Fig. (2). The four principal Cinchona-d\ka\oids and their stereochemistry

Chemistry and Pharmacology. Quinine acts as a blood schizonticide although it also has gametocytocidal activity against F. vivax and P, malariae. Its effect is probably because of its properties as a weak base. As a schizonticidal drug, it is less effective and more toxic than chloroquine. However, it has a special place in the management of severe P. falciparum malaria in areas with known resistance to chloroquine [164]. Quinine is readily absorbed when given orally or intramuscularly. Peak plasma concentrations are achieved within 1-3

838 hours after oral dose and plasma half-life is about 11 hours. In acute malaria, the volume of distribution of quinine contracts and clearance is reduced, and the elimination half-life increases in proportion to the severity of the illness [165]. Therefore, the maintenance dose of the drug may have to be reduced if the treatment is continued for more than 48 hours. The drug is extensively metabolised in the liver and only 10% is excreted unchanged in the urine. Quinine is also a potentially toxic drug. The typical syndrome of quinine side effects is referred to as cinchonism and severity is related to size of dose. Mild cinchonism consists of ringing in the ears, headache, nausea and disturbed vision. Functional impairment of the eighth nerve results in tinnitus, decreased auditory acuity and vertigo. Visual symptoms consist of blurred vision, disturbed colour perception, photophobia, diplopia, night blindness, and rarely blindness. These changes are due to direct neurotoxicity, although vascular changes may contribute to the problem. Rashes, sweating, angioedema can occur. Excitement, confusion, delirium are also seen in some patients. Coma, respiratory arrest, hypotension, and death can occur with over dosage. Quinine can also cause renal failure. Massive hemolysis and hemoglobinuria can occur, especially in pregnancy or repeated use. Quinine has little effect on the heart in therapeutic doses and hence regular cardiac monitoring is not needed. However it can cause hypotension in the event of overdose. Quinine reduces the excitability of the motor end plate and thus antagonises the actions of physostigmine. It can cause respiratory distress and dysphagia in patients of myasthenia gravis. Quinine is administered orally at 10 mg/kg 8 hourly for 4 days and 5 mg/kg 8 hourly for 3 days, intravenous at 20 mg of salt/kg in 10 ml/kg isotonic saline or 5% dextrose over 4 hours, then 10 mg of salt/kg in saline or dextrose over 4 hours, every 8 hours until the patient is able complete oral administration or for 5-7 days, and finally intramuscularly at 20 mg/kg stat, followed by 10 mg/kg 8 hourly by deep intra muscular injections for 5-7 days [165]. Mefloquine (133) was developed during the Vietnam war, during a programme to find new antimalarials, to protect American soldiers from the multidrug resistant P. falciparum infection. The Walter Reed Army Institute for Research started preclinical development of mefloquine in 1972, filed and started human studies in the same year. After a successful evaluation of the drug potential further

839 development was done in a cooperation with Hoffman LaRoche leading to a final FDA-approval in 1988. Since then, it has been used worldwide for the treatment [166] and prophylaxis [167] of P. falciparum malaria, known under the trade name Lariam®. The antiplasmodial activity and mechanism of action is unknown. It probably affects the membranes of the parasites. It is effective against the blood forms of P. falciparum malaria, including the chloroquine resistant types. Mefloquine is available for oral administration only. It is absorbed rapidly and is extensively bound to plasma proteins [166]. The elimination half-life is about 2-3 weeks. It is mainly excreted in the faeces. It is generally well tolerated in therapeutic doses up to 1,500 mg. Nausea, vomiting, abdominal pain and dizziness can occur in doses exceeding 1 g [166]. Less frequently it can cause nightmares, sleeping disturbances, dizziness, ataxia, sinus bradycardia, sinus arrhythmia, postural hypotension, and an 'acute brain syndrome' consisting of fatigue, asthenia, seizures and psychosis. It is given at 25 mg/kg in a single dose.

(133) mefloquine (Lariam )

6.

FUTURE DIRECTIONS - DOES NATURE PROVIDE LEADS FOR NEW ANTIPARASITICS?

The widespread opinion that parasitic diseases no longer pose a problem thanks to antibiotics and vaccines is wishful thinking rather than reality. Today we are further away from controlling parasitic diseases than we were 20 years ago. Parasitic protozoa remain a major

840 threat to the health of human population throughout the world. Despite this fact, there are few effective drugs for the treatment of many protozoal diseases. However, the therapies for malaria, leishmaniasis and trypanosomiasis, diseases that threaten more than two billion people in mostly underdeveloped countries, are inadequate. This is now being recognised in multinational programmes such as "Roll back malaria". Traditionally, medicinal plants have already provided valuable leads for potential antiparasitic compounds, including naphthoquinones, terpenoids and alkaloids. The renewed interest in plant products has been stimulated in part by the identification of the antiplasmodial activity of the sesquiterpene lactone artemisinin (qinghaosu). This experience is not to be ignored as plants have frequently provided the template molecules on which to base further novel structures. For more than fifty years important antimicrobial and antiparasitic drugs have been identified from the products of fungi and bacteria and we should look to this source for future novel leads. In recent years marine organisms have been actively investigated and basic information has been made available to evaluate their potential. Although discovery of antiparasitic active compounds was not expected in the first pharmacological studies, promising leads have already been identified with new chemical types and active principles. So what is the potential of natural products as a source of new antiparasitics? From the experiences with artemisinin (qinghaosu) it is wishful thinking to suppose that parasitic diseases can be treated with a single compound. Pure natural products are useful as lead structures, but in most cases high toxicity has restricted their use in humans. Many natural products with the desired activity and low toxicity have been identified, as reviewed here, but they did not progress through preclinical studies for evaluation as a potent drug because of low bioavailability and/or poor solubility. These pharmaceutical problems point towards the need for a rational, preferably mechanistically based, structural modification of chemical leads from nature. Natural products give new inputs to medicinal chemistry to develop new safe and effective drugs. By classical synthetic strategies organic chemists may create safer compounds close to lead to reduce toxicity, side effects or to improve bioavailability. This has been bone very successful for artemether from artemisinin and atovaquone from simple naphthoquinones.

841 Natural products have made an important contribution to antiparasitic drug research and despite all problems there is every indication that they will continue to make a contribution to the efforts to develop new and urgently needed drugs for the future.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

849

NATURAL INSECTICIDES: STRUCTURE DIVERSITY, EFFECTS AND STRUCTURE-ACTIVITY RELATIONSHIPS. A CASE STUDY

A. GONZALEZ-COLOMA ^\ M. REINA^ C. GUTIERREZ\ B.M. FRAGA^ ^Centro de Ciencias Medioambientales, CSIC, Serrano 115-dpdo., 28006 Madrid, Spain. Institute de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico F. Sanchez, 38206 La Laguna, Tenerife, Spain. Mailing address, e-mail: [email protected], telephone: 34-91-5625020, fax: 34-915640800 ABSTRACT: For the past ten years our research group has been working on bioactive natural products with insecticidal effects. We have focused mainly on the flora of the Canarian Archipelago because of its rich biodiversity (Lauraceae, Compositae, Boraginaceae). Additionally, we have carried out comparative studies of species belonging to these plant families from different geographical origins (Japan, Chile). As a result of these studies we have isolated bioactive compounds belonging to several chemical classes (sesquiterpenes, diterpenes, lignans, diterpenoid alkaloids, pyrrolizidine alkaloids) with selective modes of action and low toxicity. The structure-activity relationships of these compounds have also been established. In this chapter we will present the structures and biological effects of these compounds according to their chemical classes as follows: Diterpene ryanodanes and isoryanodanes from Lauraceae species Lignans from Lauraceae species Sesquiterpenes from Compositae species Diterpene alkaloids from Ranunculaceae species Pyrrolizidine alkaloids from Compositae and Boraginaceae species

INTRODUCTION During the past decades the excessive use of synthetic pesticides has given raise to several problems including the selection of resistant pest

850 populations [1], negative side effects on beneficial parasites and predators and deleterious effects on human health [2]. Therefore, the search for alternative pest control agents with reduced environmental consequences is of great interest [3]. In this context we are studying the phytochemistry of the flora of the Canarian Archipelago [4], searching for environmentally-friendly pesticides. As a result of this ongoing study we have identified several bioactive compounds belonging to different chemical classes. These compounds have been isolated from plant species of the families Lauraceae, Boraginaceae, Ranunculaceae and Compositae. Species of the Lauraceae and Boraginaceae families endemic to Japan and Chile have also been included in this study for comparison purposes. These results emphasize the potential added value of local biodiversity given its wide range of possible agrochemical applications. Diterpene ryanodanes and isoryanodanes from Persea indica (Lauraceae) The Canarian laurel forest is a relic of the Tertiary Mediterranean flora. This forest has suffered from human activity, and is nowadays restricted to small protected areas of the Canarian Archipelago. The uniqueness of the this forest [5] prompted us to investigate the biological value of the dominant species {Laurus azorica Seub., Ocotea foetens Alton, Apollonias barbujana Cay, y Persea indica L.) [6]. Following an insecticidal activity screening, we found that P. indica was a strong antifeedant against several insect species in choice assays (Spodoptera litura, S. littoralis, Macaronesia fortunata, Heliothis armigera, Leptinotarsa decemlineata andMyzus persicae) (table 1). P. indica also inhibited the growth of the lepidopteran species tested (table 1), suggesting a broad range of insecticidal action and emphasizing the phytochemical importance of this plant. The chemical study of the antifeedant fractions of P. indica resulted in the isolation of the following ryanodane-type diterpenes: Ryanodol (1), isolated for the first time as a natural product, the known ryanodanes cinnzeylanol (3) and cinnzeylanine (4) [7, 8] and the new ones cinnzeylanone (6), ryanodol-14-monoacetate (2) and epi-cinnzeylanol (5) [9, 10]. In addition to ryanodanes 1-6, the new isoryanodane type diterpenes indicol (9), vignaticol (7) and perseanol (8) were also isolated [11].

851

Table 1. Antifeedant and growth inhibition effects of P, indica ethanolic extract on several insect species Insect Family

Insect species

FI/Sl'(100|^g/cm^)

% Growth inhibition^

Lepidoptera

1

S. VUura

100

26

S. lUtoralis

100

83

M.fortunata

25

H. armigera

43

Coleoptera

L. decemlineata

93

Homoptera

M. persicae

47

-

1

' %FI/%SI= [1-(T/C)]xl00, where T= consumption/settling of treated disks and C = consumption /settling of control disks. ^ Larval growth inhibition observed in diet-incorporation bioassays (n=20, 0.1%w/wt)

1 R=H 2 R = Ac

3 R=a-OH, H 4

R=a-OAc,H

5 R=p-OH. H 6 R= 0

7 R=:H 8 R = OH

852 Ryanodine-type compounds act primarily at the Ca^^ release channel in both mammals and insects [12]. Ryanodol-type compounds, however, are more selective toxicants for insects than they are for mammals, suggesting a different mode of action for these compounds [13, 14]. To further asses such hypothesis we carried out a comparative study on the antifeedant and insecticidal effects of the ryanodol/isoryanodol-type diterpenes from P. indica (non-alkaloidal type ryanoids 1-9) [9, 10, 11] and ryanodine-type (alkaloidal type) ryanoids (ryanodine/spiganthine ryanoids 10-18), isolated from Spigelia anthelmia (Loganiaceae) [15, 16]. We studied their effects on the feeding behavior, survivorship and performance (biomass gain and food ingestion) ofS. littoralis larvae and L. decemlineata (Colorado potato beetle, CPB) aduhs [17].

^OH

H

10 R1=CH3

R2 = H

R3 = 0 H

11 R1=CH3

R 2 = R3 = H

O

14

12 R1 =CH20H R2 = R3 = H 13 Rl=CH20H R2 = 0 H R3 = H

15 R=CH3 16 R=CH20H

17

853

OH

18

The activity of the test compounds varied depending upon the insect species and type of treatment. In general, the ryanodol/isoryanodol diterpenes are more effective antifeedants and less toxic than the ryanodine/spiganthine ones. Epicinnzeylanol (5), cinnzeylanine (4) and the epoxide 15 were the most promising molecules against S, littoralis, with strong antifeedant (4 and 5, table 2) and/or postingestive effects (5 and 15) Figure (1). Cinnzeylanone (6) was the most active against L decemlineata, with antifeedant and knock-down effects as well as oral toxicity (tables 2 and 3) [17]. The structure-activity study of the ryanoids showed that both C-1 and C14 substituents play an important role in their antifeedant and toxic activity against S. littoralis, as previously shown for their antifeedant effects on S. litura [10]. The (i-stereochemistry at C-1 (5 versus 3) and its O- acetylation (4), increased the toxic and/or antifeedant activity of these compounds. Hydroxylation (1), O- acetylation (2) and pyrrolcarboxylate-esterification (10) at C-14 along with the hydrophobicity of the cyclohexane ring (6) resulted in intermediate activities. Additionally, the presence of a C-2/C-3 epoxide (15,16) increased the toxicity against this lepidopteran (table 2, Figure (1)) [17].

854

S O

U

0^

1 1—\—i \ 10 11 12 13 14 15

r 16 18

Compound Figure (1). Relative consumption (RCR) and growth (RGR) rates of 5". littoralis L6 larvae orally injected with 10 fig of compounds 1-18 with or without PBO pre-treatment (5 jig). Data is expressed as % of control (average+SE). *Denotes a significant difference from the control, p A

•

100 ^g

e

o

-^

T

• 0.0 h^

i •

^ • -0.5 -0.5

1

0.0 1

0.5

1.0

'

1

1

1.5

2.0

2.5

Relative Consumption Rate

Figure (2). Plot of the relative growth rate (RGR) on the relative consumption rate (RCR) for L. decemlineata L4 larvae fed for 24 h on leaf-disks treated with compound 23. The line represents the calibration curve (y= 0.371x-0.220, r=0.84, p100

12.86(0.16,25.56)

28

>100

-

29

>100

27.25(22.95,31.55)

30

5.48 (3.04,7.92)

22.50(19.73,25.27)

31

23.67(19.37,27.97)

108.30(9.97,116.9)

Aconitine

32.35(19.65,45.05)

178(>39)'

Concentration required to give an antifeedant inhibition of 50%. 'From [19].

These alkaloids were not toxic to S. littoralis, while aconitine negatively affected both feeding indexes (RCR and RGR, table 15). Their toxicity on L. decemlineata was inversely correlated with their antifeedant effects, the amino alcohol (31) derivative being the most toxic (table 15). None of the test compounds proved to be as toxic as aconitine on this insect species [20]. Table 15. Oral and hemolymph injection effects of compounds 27-31 on S. littoralis L6-larvae (72h., RCR and RGR) and L. decemlineata adults (3 days % mortality) respectively [20]. Treatment (10 (xg/insect) Control 27 28 29 30 31 Aconitine

S, littoralis RCR*

RGR^

L. decemlineata % mortality^

100 99.57+4.67 97.17+11.79 113.90+19.61 103.32+4.26 104.54+4.08 62.80+6.24a

100 100.10+4.24 105.89+20.32 87.98+6.65 110.32+6.22 106.46+5.68 42.30+11.22a

0.00 7.85 18.08b 25.16b 15.75b 72.20b 100b

^ RCR=I/(BI)xT, I=mg food consumed, T=feeding period (days), BI=initial insect weight (mg). Data represented as %control. ^ RGR=AB/(BI)xT, AB=change in insect body weight (mg). ^ Corrected according to Abbott [70]. ^'^ Denote a significant difference from the control. 95% LSD test and Contingency table analysis (p50 1.3(0.2,7.9) >50 S glaber^ 13.2(5.7,30.7) E. wildprettf >50 H. megalantum^ 31.6(27.7,35.9) H. bovef -50 H. sinuatum^ -35 H. floridunr' H. floridunv'

>50 >50

Toxic effects S.I.' L.d:

Ld..

RCR*

RGR^

%M'

89.9 84.7 94.4 48.6*

88.2 77.3 113.25 42.0*

0 15 0 9

1.4(0.8,2.1)

118.2

165.5

>100 >50 20.5 (8.2,51.5) 1.8(0.9,3.4) >50

109.0

100.6

0 12

-

-

-

91.6

82.4

3

-

-

5

^ Reina etal, [63] ^ Oral injection (20|ig/insec t) ^ Hemolymph 1njection(10 Insect) ^As in table 15 Percent mortality (%M) corrected according to Abbott [70]

-

875 These results indicate that antifeedant PAs studied here are structure- and species-dependent. The PA-tolerant S. littoralis was deterred by all the unsaturated monoesters tested, and the secopyrrolizidinic diester otosenine (35). Therefore, esterification of the hydroxyl group at C-9 in the presence (37 and 38) or absence (39) of the C-7 hydroxylation and the C-l/C-2 insaturation of the necine base determined the antifeedant action on S. littoralis. Additionally, the antifeedant and postingestive effects of otosenine (35) indicate that the presence of an electrophyllic ketone group in C-8 at the necine base could play a role in determining such activity. CPB sensitivity to these compounds followed a different pattern from that ofS, littoralis. The macrocyclic diester senecionine (34), the open diester echimidine (36) and the saturated monoester 40 were the strongest antifeedants to this insect with similar potencies but few structural features in common to conclude structure-activity relationships. Furthermore, the unsaturated monoester 39 had moderate-low antifeedant effect, suggesting that CPB taste receptors can bind to different PA chemical classes with high molecular selectivity. Similarly to CPB, larvae of the tortricid Chonistoneura fumiferana (a non PA-tolerant insect) were strongly deterred by the open diester lasiocarpine, moderately by the macrocyclic diester senecionine and the unsaturated monoester heliotrine, while otosenine was inactive The antifeedant effects of PAs are proposed to play a key role in plant defense from insect herbivores [59]. However, little is known on the molecular mechanisms modulating PA-insect taste reception. Several PAs, including echimidine, monocrotaline and senecionine, have shown significant binding activity to muscarinic and serotonin receptors, indicating that PAs can affect several molecular targets besides long-term toxicity through DNA alkylation by PA metabolites generated in the liver [66]. Therefore, the interference of PAs with neuronal signal transduction could mediate insect taste regulation as proposed for chrysomelid beetles [18, 19]. Tertiary PAs are deleterious for organisms with a microsomal cytochrome P450, but S. littoralis larvae tolerate PAs. These larvae prevent poisoning by rapid and efficient excretion of the absorbed tertiary alkaloid [56]. However, otosenine (35) had toxic post-ingestive effects on S. littoralis, indicating that the excretion of this compound is not efficient enough and therefore might undergo bioactivation by the insect's cytochrome P-450.

876 None of the PAs tested resulted toxic to the chrysomeUd L. decemlineata. Oreina (Chrysomehdae) beetles are able to take up plant alkaloid N-oxides and eliminate tertiary PAs, but are unable to N-oxidize tertiary PAs [55, 67, 68]. Similarly, L. decemlineata adults could eliminate tertiary PAs efficiently enough to avoid poisoning. In conclusion, PA antifeedant effects are species and structure-dependent with a broad range of action probably related to their ability to interfere with several molecular targets. However, their toxic effects on insect herbivores seem to be more restricted. Conclusions Among the compounds presented here, 34% were insect antifeedants, 22% toxicants and 44% had both effects. These compounds belong to very different chemical classes, have been produced by different plant species and are proposed to act on different molecular targets, suggesting high plasticity for these biological effects at the molecular level. The molecular diversity and multipurpose biological effects of alkaloids has been postulated as the result of "evolutionary molecular modeling" [69]. The data presented in this chapter supports such hypothesis but without restrictions in terms of chemical class as long as the molecule being considered is involved in the defensive strategy of a plant. ACKNOWLEDGEMENTS We gratefully acknowledge A. Santos, Director of the Botanic Garden at La Orotava, A. Fernandez, Director of Garajonay Natl. Park, and S. Carlin for language advice. This work has been partially funded by grants PI 91/17, PB 94-0020-B, PI 13/95, PB97-1226 and PI 1999/076 from the Canarian Government and CICYT-Spain. REFERENCES [1]

[2]

Georghiou, G.P. In Managing Resistance to Agrochemicals: From Fundamental Research to Practical Stratigies; Gree, M.B.; Le Baron, H.M.; Moberg, W.K., Eds.; American Chemical Society: Wasintong D.C., 1990. Metcalf, R.L. In The Pesticide Question. Environment, Economics and Ethics; Pimentel, D.; Lehman, H., Eds.; Chapman and Hall: New York, 1993, pp. 426-430.

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[30]

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[54] [55] [56]

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

881

OCCURRENCE, STRUCTURE AND BIOACTIVITY OF 1,7-DIARYLHEPTANOIDS

PER CLAESON*, UBONWAN P. CLAESON^ PATOOMRATANA TUCHINDA^ AND VICHAIREUTRAKUL^ Division of Pharmacognosy, Department of Pharmacy, Uppsala University, Biomedical Centre, Box 579, SE-751 23 Uppsala, Sweden ^Institute for Bioactive Natural Products, Uppsala Science Park, SE-751 83 Uppsala, Sweden ^Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand "^To whom correspondence should be addressed Tel: ^4618 471 44 79. Fax: +4618 50 91 OL E-mail: per. claeson @fkog. uu. se ABSTRACT: The 1,7-diarylheptanoids are a class of secondary plant metabolites characterised by the structural motif aryl-C7-aryl. Based on their chemical structures, the diarylheptanoids have been divided into five different groups: non-phenolic linear diarylheptanoids (Type I), phenolic linear diarylheptanoids (Type II), macrocychc biarylheptanoids (Type IE), macrocyclic diaryl ether heptanoids (Type IV) and diarylheptanoids with cyclised C7-chain (Type V). The literature up to October 1993 has previously been covered (Claeson ^r a/. J. hidian Chem. Soc. 1994, 71, 509-521), and presentiy the literature of October 1993 - September 1999 has been reviewed. During the period of review, reports on a total of 114 diarylheptanoid structures (including 75 new natural products) have been retrieved from the literature. These structures, together with pertinent information on plant sources, biological activities and references, have been compiled.

INTRODUCTION The 1,7-diarylheptanoids constitute a class of secondary plant metabolites characterised by the structural motif aryl-Cy-aryl. The diarylheptanoids appear in both linear and cyclic forms. Based on their chemical structures, the diarylheptanoids have been grouped in five major types [1]. The diarylheptanoids of both Type I and n are linear structures, but for practical reasons they have been separated into two groups. The nonphenolic diarylheptanoids (Type I) sometimes appear under the

882 consignation diphenylheptanoids and, historically, the phenolic linear diarylheptanoids (Type H) have also been called curcuminoids after curcumin, the orange pigment of turmeric, Curcuma longa. In the diarylheptanoids of Type HI and IV, the aromatic rings are meta-meta' or meta-para'COimcctQd to form macrocyclic structures with biaryl and diaryl ether moieties, respectively. In the Type V diarylheptanoids, the C7 chain is cyclised to form a pyran-like ring.

Typen

Type I

Type in

m

Type IV

TypeV

The first review of diarylheptanoids covered the literature up to October 1993, and a total of 117 substances were listed [1]. Another review on diarylheptanoids, particularly informative on synthetic aspects, subsequently appeared [2]. In the present review, covering reports appearing in major journals during October 1993 - September 1999, information on 114 diarylheptanoids has been found. Out of these, 75 substances are new natiial products; and for the remaining 39 known compounds, studies on biological activities, re-isolation from other plant sources, synthesis, structure revision etc., have been pubUshed. Tables 1-5 list the diarylheptanoids with trivial names, occurrence in nature and pertinent

883 literature references. Information on biological activities is found in the respective sections. The appearance of reference [1] (the first review) in Tables 1-5 indicates that the substances are not isolated for the first time, but that additional information is reported. Non-Phenolic Linear Diarylheptanoids (Type I) Reports on 11 diarylheptanoids (1-11) of Type I have been published during the time period of review (Table 1). Four of the diarylheptanoids (3, 5,6, and 8) in Table 1 are new natural products. The occurrence of Type I diarylheptanoids is still confined to the families Betulaceae and Zingiberaceae (c/ [1]). Table 1. Non-Phenolic Linear Diarylheptanoids Compound Trivia] name

Occurrence"

Ref.

1

-

Curcuma xanthorrhiza (Z)

3^

2

Dihydroyashabushiketol

Alpinia conchigera (Z) Alnus maximowiczii (Be)

1, 4 5

3

-

Alpinia conchigera (Z) Alnus maximowiczii (Be)

4 5

4

-

Curcuma comosa (Z) Curcuma xanthorrhiza (Z)

1, 6 3

5

-

Curcuma comosa (Z) Curcuma xanthorrhiza (Z)

6

6

-

Curcuma comosa (Z) Curcuma xanthorrhiza (Z)

3" 6 3^

7

-

Curcuma comosa (Z)

1, 6

8

-

Alnus maximowiczii (Be) Alpinia qfficinarum(Z)

9

-

Curcuma comosa (Z) Curcuma xanthorrhiza (Z)

10

Alnustone

5 7 1, 6 3 1, 3

1 1 1 1 1

11

-

3^

1

i Curcuma xanthorrhiza (Z) Curcuma xanthorrhiza (Z)

1

*(Be)=Betulaceae; (Z)=Zingiberaceae ''Semisynthetic compound

A series of naturally occurring (4, 9 and 10) and semisynthetic (1, 5, 6

884 and 11) diaryIheptanoids from Curcuma xanthorrhiza have been evaluated for topical anti-inflanunatory activity in the murine model of ethyl phenyl propiolate-induced ear oedema. A distinct structure-activity relationship could be identified; in which compound 10 was the most potent agent and compound 1 virtually inactive [3]. Compounds 4, 5, 6 and 7 have been isolated from Curcuma comosa as the substances responsible for the nematocidal effect on Caenorhabditis elegans of a methanolic extract of the plant. Compound 9 was also isolated from the same plant, but it was found inactive in the same assay [6].

Rl

R2

Rl

R2

*^1

*^2

1

— H

— OH

4

—H

— OH

2

=

0

—m

5

—H

— OAc

3

=

0

=

6

—"H

=

0

7

— OH

=

0

9

— OH

0

10

=

0

11

—OAc

Phenolic Linear Diarylheptanoids (Type II) The phenolic linear diarylheptanoids (12-59; Table 2) constitute the largest subgroup with reports on 48 substances, of which 28 substances are

885

Table 2. Phenolic Linear Diaryllieptanoids Compd. Trivial

name

Occurrence*

JRef.

|l2

(-)-Centrolobol

Centrolobium sclerophyllum (F)

|l, 8

|l3

Aceroside VII

Betula platyphylla var. japonica (Be)

|l,9

|l4

Aceroside VIII

Betula platyphylla var. japonica (Be)

1,9,10

|l5

Aceroside XIII

Acer triflorum (A)

11

|l6

Aceroside XII

Acer triflorum (A)

11

|l 7

Platyphyllonol

Corylus sieboldiana (Be)

1, 12

|l8

Platyphylloside

Betula platyphylla var. japonica (Be)

|l9

1-

Betula platyphylla var. japonica (Be)

1» 10 1, 10

1 1

{20

Betulaplatoside la

Betula platyphylla var. japonica (Be)

10

1

|21

Betulaplatoside lb

Betula platyphylla var. japonica (Be)

10

1

22

-

Alpinia concigera (Z) Alpinia officinarum (Z)

1,4

1

7

1

|23

-

Alpinia officinarum (Z)

1, 7

1

|24

Rhoiptelol C

Rhoiptelea chilianta (R)

13

1

|25

-

Pinus flexilis (?)

14

1

|26

-

Pinus flexilis (?)

14, 15

1

[27

-

Alnus japonica (Be)

15

1

|28

Hirsutanonol

Pinus flexilis (?)

1, 14

1

|29

Oregonin

Pinus flexilis (?)

1, 14

1

jao

-

Alnus japonica (Be)

1, 15

1

|31

-

A/nw5 japonica (Be)

15

1

32

Oregonoside A

A/nw5 rw^ra (Be)

16

1

33

Oregonoside B

A/n«5 rubra (Be)

16

1

34

Demethoxyyakuchinone B

Alpinia oxyphylla (Z)

17'

35 36

1 Yakuchinone B 1 YPE-01

Alpinia oxyphylla (Z)

1, 17

1 Alpinia oxyphylla (Z)

ir

'(A)=Aceraceae; (Be)=Betulaceae; (F)=Fabaceae; (?)=?inaceae; (R)=Rhoipteleaceae; (Z)=Zingiberaceae ''Semisynthetic compound

1

1

886

Table 2. Phenolic Linear Diarylheptanoids (cont.) Compd. Trivial name

Occurrence'

Ref.

137

1-

Curcuma xanthorrhiza (Z)

18

|38

-

Curcuma xanthorrhiza (Z)

18

|39

-

Alpinia katsumadai (Z)

19

40

-

Alpinia concigera (Z) Alpinia officinarum (Z)

1, 4 7

|41

-

Betula platyphylla var. japonica (Be)

1, 9, 10

|42

-

Betula platyphylla var. japonica (Be)

9

43

Hirsutenone

Alnus japonica (Be) Pinus flexilis (P)

1, 15 14

44

Calyxin A

Alpinia blepharocalyx (Z)

20, 21, 22

45

Calyxin E

Alpinia blepharocalyx (Z)

21, 22

46

Calyxin H

Alpinia blepharocalyx (Z)

22, 23

47

Epicalyxin H

Alpinia blepharocalyx (Z)

22, 23

48

Calyxin B

Alpinia blepharocalyx (Z)

20, 22, 24 1

49

Epicalyxin B

Alpinia blepharocalyx (Z)

20, 22, 24 1

50

Calyxin C

Alpinia blepharocalyx (Z)

22, 24

1

51

Epicalyxin C

Alpinia blepharocalyx (Z)

22, 24

1

52

Calyxin D

Alpinia blepharocalyx (Z)

22, 24

1

53

Epicalyxin D

Alpinia blepharocalyx (Z)

22, 24

1

54

Blepharocalyxin A

Alpinia blepharocalyx (Z)

22, 23, 25 1

55

Blepharocalyxin B

Alpinia blepharocalyx (Z)

22, 23, 25 1

56

-

Alpinia blepharocalyx (Z)

22, 26

1

57

Curcumin

Curcuma longa (Z)

1, 27, 28

1

58

Demethoxy-curcumin Curcuma longa (Z)

1, 27, 28

[

1, 27, 28 26

1 1

59

Bisdemethoxycurcumin

Curcuma longa (Z) Alpinia blepharocalyx (Z)

'(A)=Aceraceae; (Be)=Betulaceae; (F)=Fabaceae; (P)=Pinaceae; (R)=Rhoipteleaceae; (Z)=Zingiberaceae ^'Semisynthetic compound

1

887 reported as natural products for the first time. The majority of these diarylheptanoids have been isolated from the families Betulaceae and Zingiberaceae, but compounds 24-26, 28, 29 and 43 have been isolated from the two families Pinaceae and Rhoipteleaceae, neither of which have previously been reported to contain diarylheptanoids. A novel series of compounds (44-55) with chalcone, flavanoid or chalcone-diarylheptanoid moieties attached to the diarylheptanoid backbone has been isolated from Alpinia blepharocalyx [22-24]. Compound 12 has shown significant anti-leishmanial activity in vitro against the extracellular forms (promastigotes) of Leishmania amazonensis [8]. A series of compounds from the bark of Betula platyphylla var. japonica, 13,14, 18, 19, 20 and 21, has been evaluated in a battery of tests for hepato-protective properties (prevention of CCI4- and D-GalN/LPSinduced liver damage, anti-oxidative activity, 02-scavenging activity). The diarylheptanoids showed varying degrees of activities in the assays [10]. Compounds 22 and 23 also exhibited antioxidant activity [7]. Bioassay-guided fractionation of a CH2Cl2-MeOH extract of Pinus flexilis led to the isolation of 25, 26, 28, 29 and 43. They were aU found to inhibit the enzyme protein kinase C, which has been implicated as a potential target for novel anti-cancer drugs [14]. Compounds 34 and 35 were found to be inhibitors of 5-lipoxygenase and cyclooxygenase-1 and -2. The closely related derivative 36, was, however, found to be a selective inhibitor of 5-lipoxygenase. Topical application of 36 significantly suppressed both arachidonic acid- and 12-0tetradecanoyl phorbol 13-acetate-induced ear oedemas in mice [17]. The two new diarylheptanoids 37 and 38 exhibited significant hypolipidemic action by inhibiting hepatic triglyceride secretion in hamsters [18]. Compounds 44-56 from Alpinia blepharocalyx have all been shown to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophages in vitro. Compound 55 was the most potent inhibitor [22]. Compounds 55 and 59 furthermore inhibited collagen-, arachidonic acid- and adenosine-induced platelet aggregation of human whole blood [26]. Curcunun (57) and its two derivatives 58 and 59 from Curcuma longa exhibited nematocidal activity against second-stage larvae of dog roundworm, Toxocara canis. The nematocidal activity increased remarkably when the curcuminoids were mixed, which was suggested to be due to a synergistic action between them [27].

12

..... OH

— OH

13

••"• 0-P-D-Glc/?

— OH

14

••" 0-p-D-Api/-(l-*- 6)-P-D-Glc/7

— OH

15

•""CM

— O-p-D-Glc/7

16

.....CH

""• o-3-D-Api/--(r

Rl

" 6)-3-D-Glcp

R2

17

=

0

— OH

18

=

0

— O-P-D-Glc/?

19

=

0

— 0-p-D-Api/-(l—»- 6)-P-D-Glc/?

20

•••"OH

— 0-P-D-Glc/?

21

— OH

— O-p-D-Glc/?

R2

R3 CMe

^1

K2

R3

22

—H

= 0

—OH

23

—H

= 0

— O^e

889 R2

R3 OMe

24

•«"0H

""(H

Rl

•••••OH

R2

25

•""(H

—-H

26

•••" 0-p-D'Glcp

—H

27

'""6-0-(3-D-Api-P- D-Glcp

—H

28

= 0

— (B

29

=

— 0-p-D-xylose

30

= 0

— 0-3-D-Glc

31

=o

— 6-O-galloyi-P-D-Glcp

0

32

— 0-I3-D-G1C.6-0-CO-

^ ^ OtAe

33

= 0

— O-P-D-Glc-6-O-CO-. ^

V-CH

890

Ri

R2

34

—H

— H

35

— CMe

— H

36

— OMe

— OMe

Rl

R2

*^1

*^1

K2

37

— H

-—OH

-^H

38

— H

— OH

— OH

39

..... CM

=

— H

0

OMe

40

891

R

41

— H

42

— OH

43

OH

44

45 HO

R

892 OMe

C-7

Rl 46

— H

S

47

— H

R

48

— OH

S

49

— OH

R

C-7

Rl

50

51

0

O^J^^OMe

(Me

893

Rl

52

53

54

55

C-7

894

56

Rj

R3

57

— (Me

— (Me

58

— (Me

—H

59

—H

—H

Macrocyclic Biarylheptanoids (Type HI) Scientific reports have appeared on 24 macrocyclic biarylheptanoids (60-83; Table 3), of which 17 are new natural products. A revised structure of porson (61) has been proposed [29]. Plants of the genera Betula (Betulaceae), Acer (Aceraceae) and Corylus (Betulaceae) are reported for the first time to contain Type III diarylheptanoids. Rhoiptelol A (79) is the first macrocyclic biarylheptanoid fi-om the family Rhoipteleaceae. Compounds 65 and 66 were reported in 1991, unfortunately missed in the previous review (1), but arbitrarily included in the present compilation. No biological activities have been reported for any Type IE diarylheptanoids.

895 Table 3. Macrocyclic Biarylheptanoids Trivial name

Occurrence'

Ref.

Myricanone

Myrica gale var. tomentosa (M)

1, 29

Person

Myrica gale var. tomentosa (M)

1, 29'

I 62

12-Dehydroporson

Myrica gale var. tomentosa (M)

29

1 63 1 64

12 -Hydroxy-myricanone Myrica gale var. tomentosa (M)

29

Myricatomentoside II

Myrica gale var. tomentosa (M)

30

1 65

-

Myrica rubra (M)

66

-

Myrica rubra (M)

3r 3r

1 ^^ '

-

Betula davurica (Be)

32

1 68

-

Betula davurica (Be)

32

69

Acerogenin E

Acer nikoense (A) Betula ermanii (Be) Betula platyphylla var. japonica (Be) 5^f«/fl maximowicziana (Be) B^m/

1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 20 21 22 23 OMe OMe

108.3 147.1 143.4 100.7 134.3 123.8 51.5 59.9 170.0 50.0 77.0 47.8 31.1 26.2 59.5 41.8 49.4 52.1 139.3 123.8 64.3 55.8

(90 MHz)s a): In CDCI3-CD3OD.

13'-> 120.5 124.3 128.3 114.6 142.3 134.8 52.3 67.5 168.5 46.3 120.5 141.1 34.7 25.9 63.5 36.9 52.9 54.0 137.7 126.5 58.0

b): In CDCI3.

14 "> 105.8 146.2 149.1 99.5 135.2 125.1 52.4 67.6 167.7 45.6 120.5 137.4 34.7 25.7 62.9 36.7 52.7 53.9 142.2 126.7 57.9 56.1 56.5

15-> 124.7 126.9 131.6 116.7 142.9 130.5 52.9 66.8 171.2 44.3 125.0 140.8 34.5 25.2 81.2 44.9 68.4 68.7 133.3 136.8 59.4

16'^ 108.6 149.2 152.0 101.5 136.7 125.4 53.3 67.7 170.7 44.3 124.0 141.8 34.9 25.1 81.5 42.6 69.8 69.9 132.5 135.1 59.4 57.9 57.5

c): In CD3OD.

Twelve alkaloids, seven of which are new, have been isolated from the root bark of Strychnos panganensis collected in Tanzania. The novel monoindole alkaloid are A^-desacetylspermostrychnine (17), 12-hydroxy11 -methoxy-nor-c-fluorocurarine (18), 12-hydroxy-11 -methoxy-A^acetyl-nor-C-fluorocurarimine (19) [33].

1053 Phytochemical investigation of the minor alkaloids of Strychnos myrtoides resulted in the isolation of four new alkaloids of the Nb-C(21) secocuran series, viz.,myrtoidine (20), 11-demethoxymyrtoidine (21), which showed an a-|3 unsaturated y-lactonic ring hitherto not found among Strychnos alkaloids, 12-hydroxy-19-epi-malagashanine (25) and malagashanol (26). Except for 26, the other Nb-C(21) secocuran alkaloids described have an uncommon H-3P configuration [36].

MeO

17

18

12-hycIroxy-11 -methoxy-nor-Cfluorocurarine R1=H; R2=CHO

19

12-hydroxy-11 -methoxy-N-acetyl-nor-Cfluorocurarimine Rl=Ac; R2=CHNH

N-desacetylspermostrychine

o3)2CO) ppm: 9.12 (IH, s), 6.09 (IH, d, 7=15.1 Hz), 5.35 (IH, dt, 7=7.3 & 14.7 Hz), 4.354.38 (IH, m), 3.81 (2H, t, 7=6.8 Hz), 2.66 (2H, t, 7=7.3 Hz), 2.42-2.53 (2H, m), 2.21 (3H, s), 2.11-2.19 (IH, m), 2.09 (3H, s), 1.97-2.03 (IH, m); 13C-NMR6 ((CD3)2CO) ppm: 184.6, 175.2, 127.4, 122.6, 58.6, 40.9, 32.1,

1090 31.5, 29.6, 14.9, 14.4) [79]. We studied the effect of pH on the formation of 2-thiohydantoins using allyl isothiocyanate instead of MTBI. The formation was best at alkaline pH's, but the 2-thiohydantoins could be formed at neutral and weakly acidic (pH 5.0-7.0) pH's, suggesting that a certain amount of 2-thiohydantoin derivatives can be formed in isothiocyanate-containing foods or seasonings, and even in the human digestive tract [79]. We are now investigating this finding to determine the time required for the 2-thiohydantoin derivative's formation.

Rv.

^NH, coo-

Ami no acid

R: -CH2-CH-(CH3)2 (1)

-CHrf^

(2)

-CH 2-CH2-S-CH3 (3) Fig. (10). Formation of 3,5-disubstituted 2-thiohydantoins from MTBI and amino acids.

1091 Biological activities of MTBI and MTBI-derived products A series of our studies has shown that the pungent principle of radish, i.e., MTBI, can be labile and reactive in food systems and in the human digestive tract to form various sulfur-containing compounds. We subsequently describe below some of the biological activities of the reaction products and their action mechanisms. Antimicrobial activity of MTBI There have been many investigations into the antimicrobial properties of naturally occurring and synthetic isothiocyanates [18-20]. Most of the investigations were done under such conditions as directly mixing the test compounds with a liquid or a moistened agar-medium. Esaki and Onozaki have reported their study of the antimicrobial activity of MTBI, in which this activity was evaluated using MTBI-containing agar-plates [80]. Considering the labile property of MTBI in the presence of water, the activity should be assayed in a manner that will minimize the reaction of MTBI with water and the components of the medium. Recent investigations have shown that the antimicrobial activity of volatile compounds was much higher when some microbial strains grown on agar plates came into contact with the compounds vaporized within the dishes [81, 82]. Accordingly, we assayed the antimicrobial activity of MTBI in a manner similar to the technique for gaseous contact, using five molds, three yeasts, and four Gram-negative and four Gram-positive bacteria [45]. In these experiments, MTBI was dissolved in acetone and added to a 10-mm diameter paper disk. The paper disk with MTBI was then placed on a small aluminum pipe which had been fixed on the inside of the dish's lid , on which the microorganism-containing agar-plate was placed and sealed tightly with a plastic film. The bacteria and fungi were then incubated at 37° C for 48-60 h and at 25"" C for 120-144 h, respectively. The resulting growth-inhibitory zones were measured. Simultaneously, the concentration of MTBI vaporized in the head space of the dish was monitored by FPD-GC (flame photometr}^ detector-gas chromatography) during the incubation. All the fungal growth examined was inhibited by MTBI at a dose greater than 2.5 [xmol (397.5 fxg), in which MTBI showed a highly inhibitory activity against the growth of Cladosporium colocasiae, Alternaria helianthi, Eurotium chevalieri, and Candida valida. As for the antibacterial activity, it was lower than that on the fungi, except in the case of Bacillus cereus and Enterobacter cloacae. On the other hand, the concentration of MTBI vaporized within the head space of the dishes was unexpectedly low. For example, the concentration in the vapor within the dishes added with 7.5

1092 nmol MTBI increased to a maximum after 12 h of incubation at 25° C, at which time the average amount estimated was ca. 12 nmol/dish (0.16% of the dose), which corresponded to approximately 30 ppb. This may be partly due to a high boiling temperature of MTBI. In the previous report by Esaki and Onozaki, the amount of MTBI required to completely inhibit the growth of microbes, including Escherichia coli, was over 23.5 [xmol /plate, although this value was obtained by using MTBI-containing media [80]. Obviously, MTBI vapor has a significantly high antimicrobial activity, being nearly comparable to that of allyl isothiocyanate [83]. The antimicrobial activity ofHMTP, MMTP, and dithiocarbamates On the basis of the inconsistencies between the labile properties of MTBI in moistened media and the results of Esaki and Onozaki, we presumed that the products formed from MTBI also had an antimicrobial activity. We next attempted to confirm this presumption [47, 84, 85]. For the antimicrobial studies, we conducted a preliminary examination in order to determine the pH-stability of the reaction products of MTBI by HPLC analysis of the remaining amounts of the reaction products in some media. Only HMTP, which has a weakly acidic hydroxy group within the structure, was unstable in a neutral or an alkaline medium. In the pH 5.0-6.0 media containing heart-infusion broth, soy bean-casein-digest broth or potato-dextrose agar, HMTP was stable in the pH 5.0 media for at least 196 h at 25° C, and a few percent of the initial amount was lost at pH 6.0 after 24 h at 37° C [85]. Thus, the minimum inhibitory concentrations (MICs) of the MTBI-derived 2-thioxopyrrolidines and dithiocarbamates were determined against fungi at pH 5.0 and against bacteria at pH 6.0 [47, 85]. The results are summarized in Table 4. Among the six MTBI-derived products, HMTP was found to be the most active toward a wide range of the microbes. The MICs of HMTP ranged from 50 to 400 [xg/ml, providing evidence that this degradation product of MTBI acts as an antimicrobial agent. Because HMTP has watersoluble properties but lacks any odor [84], it is possible that HMTP could be used for food processing and/or preservation. In contrast, a significantly reduced activity was observed with the structurally related products (£)- and (Z)-MMTP, suggesting that a hydrophilic hydroxy group contained within the HMTP molecule plays an important role in antimicrobial action. On the other hand, the three dithiocarbamates were more prominently active on the fungi than on the bacteria: their MICs toward the fungi ranged from 100 to 400 [Ag/ml, but those against the Gram-negative and Gram-positive bacteria were 400 |xg/ml or more. Dithiocarbamates have been known to be antifungal agents and are used for soil fungicides. With respect to the 2thioxopyrrolidines like HMTP and MMTP, however, no modes of action have been reported. We have studied the modes of antimicrobial action of

1093 HMTP using a fungal strain, E. chevalieri, and a bacterium, 5. epidermidis, because of their relatively higher sensitivities to HMTP [85]. The effects of HMTP on the growth of the fungal and the bacterial strains are shown in Fig. (11) and Fig (12). When the fungal spores were seeded in a medium with HMTP corresponding to the MIC, the growth was completely inhibited. Table 4. Antimicrobial activity the MTBI-derived products. (Modified from Matsuoka etal. [47]) MIC (mg/ml)^ of the Products Microorganism HMFP

(Z )-MMTP

{E )-MMTP

MBDC

(Z)-MBDC

{E )-MBDC

(Molds) ^ A. fumigatus

200

>800

800

200

200

200

C. colocasiae

200

400

800

200

200

100

E. chevalieri

200

400

400

200

100

100

-

-

-

-

M

racemosus

400

. d

(Yeasts)*' C. albicans

400

-

-

-

-

200

S.pombe

200

400

800

200

200

200

400

(Bacteria) ^ 50

400

400

>400

400

100

>800

>800

>400

400

400

S.typhimurium

200

-

-

-

-

>400

S. epidermidis

100

800

>800

>400

>400

>400

B. subtilis E. coli

a) Maximum dose was 800 \iglm\ for the thioxopyrrolidines and 400 MS^ml for the dithiocarbamates. b) Determined in glucose-peptone-broth at pH 5.0. c) Determined in nutrient broth at pH 6.0. d) Not determined.

However, neither delay in their growth curve nor the inhibition was observed when the same levels of HMTP were added to the medium after 48 h of growth, at which the fungal mycelia had apparently grown. When a half-MIC (100 fig/ml) of HMTP was added to the medium at the start of culture of the fungal spores, about 24 h delay in their growth curve was shown (data not shown). On the other hand, a significant reduction in the number of bacterium-survivors was observed after the addition of HMTP corresponding to the MIC (100 jxg/ml).

1094

60 4

40 4

20 4

^.

0 4

s "S 40 •c Q

20

Culturing (h) Fig. (11). Effect of HMTP on the growth of spores (A) and mycelia (B) of the fungal strain E. chevalieri. Modified from Matsuoka et al. [85], The fungal strain (lO"* spores) was grown in a glucose-peptone broth (pH 5.0) for 144 h at 25° C, and the growth was evaluated by measuring the weight of dried mycelium. Symbols: • , without HMTP; and O , with 200 fig/ml HMTP. The arrow indicates the time at which the corresponding amounts of HMTP were added.

These data clearly demonstrated that the antifungal activity of HMTP was due to its sporicidal action, and the antibacterial activity was attributed to its bactericidal property. We also investigated the effects on the cellular

1095 biosynthesis of DNA, RNA, proteins, lipids, and cell-wall peptideglycans using radioactive precursors, [methyl-^H]-thymidine, [5,6-^H]-uridine, [U14C]-leucme, [l,3-14c]-glycerol, and [l-l^CJ-glucosamine [85]. We used an HMTP-sensitive bacterium, S. epidermidis, for the experiment.

10»

-,

107

10^

u 10 5

J

104

J

10-^

1 0

1 1

1 2

r 3

Incubation time (h Fig. (12). Bactericidal effect of HMTP on the strain S. epidermidis. Modified from Matsuoka et al. [85]. The bacterial strain (10^ cells/ml) was incubated in a nutrient broth (pH 6.0) for 4 h at 37** C, during which the survivors were determined on nutrient broth-containing agar plates. Symbols: • , without HMTP; and O , with 200 ^ig/ml HMTP. The arrow denotes the time at which the corresponding amounts of HMTP were added.

A nearly identical pattern of inhibition was revealed in the biosynthesis of these biologically important molecules, suggesting that the biosynthetic systems of the molecules were damaged by the incorporation of HMTP (Fig. (13)). Also, oxygen uptake by the bacteria was completely inhibited within 15 min of the addition of 200 fxg/ml HMTP, in which little change in

1096 number of the bacterial survivors was observed; thus, the decrease in oxygen uptake was not attributable to bacterial death [85]. Although further studies are required to identify a more detailed mode of action, HMTP appears to be a nonspecific and multi-action growth-inhibitor for the microorganisms. 100

"5 o o o c

50

100

150

200

0

50

100

150

200

Dose of HMTP (mg/ml) Fig. ( 1 3 ) . Inhibitory effect of HMTP on incorporation of labeled compounds in S. epidemddis. Modified from Matsuoka et al. [85]. Symbols: • , [methyl-^H]-thymidine; • , [5,6-3H]-uridine; O , [U-l^CJ-L-leucine; A , [l,3-^^]-glycerol; and O , [l-^^C]-D-glucosamine. The incubation of the bacteria with these compounds lasted 40 min, and the incorporated radioactivity was measured by a scintillation counter.

The relationships between the structure and antimicrobial activity of 2thioxopyrrolidines The significant difference in the antimicrobial activities between HMTP and MMTP suggested that the side chain bound at the C3-position of the thioxopyrrolidine ring plays an important role in the antimicrobial activity. To compare the effects of differences in the side chain structure on antimicrobial activities, we synthesized fifteen HMTP- and MMTP-related thioxopyrrolidines consisted of seven 3-[(a-alkoxy)-a-methoxy]methyl-2thioxopyrrolidines (alkoxy group: methoxy; ethoxy; n-propoxy; n-butoxy; n-pentyloxy; n-hexyloxy; and n-heptyloxy), four 3-[(a-alkoxy)-amethylthio]methyl-2-thioxopyrrolidines (alkoxy group: methoxy; ethoxy; npropoxy; and n-butoxy), and four 3-(alkoxy)methylene-2-thioxopyr-

1097 rolidines (alkoxy group: methoxy; ethoxy; n-propoxy; and n-butoxy) [86]. MICs for these compounds were assayed against the fungal strains Eurotium chevalieri and Schizosaccharomyces pombe and the bacterial strain S. epidermidis [86]. The fungi (5 x 10^ colony forming unit (CFU) per ml) were cultured in a glucose-peptone broth (pH 5.0) for 72-120 h at 25"" C, and the bacteria (1 x 10^ CFU/ml) were grown in a nutrient broth (pH 5.0) for 24 h at 36° C. The compounds dissolved in dimethylsulfoxide were serially diluted so that the final concentrations were 0-800 [xg/ml in the media. The results are summarized in Table 5. Ikble 5. Relationship bet^veen structure and antimicrobial activitiy of the synthetic thioxopyrrolidines. (ModiHed from Matsuoka et al. [86]) M I C ( i mg/ml) Structure (A) 0CH3

L

y^oK

/' / ^

\

\ ^

R

E. chevarieli ^^

S, pombe^^

S. epidermidis"^

methyl ethyl n-propyl n-butyl n-pentyl n-hexyl n-heptyl

>800c) >800 800 800 400 100 50

>800 200 200 200 50 25 25

>800 >800 >800 800 800 400 200

methyl ethyl n-propyl n-butyl

>800 >800 800 400

800 200 400 >800

400 400 200 100

methyl ethyl n-propyl n-butyl

>800 >800 800 400

>800 >800 100 200

800 800 800 800

H SCH3

(B)

y^oR

/

\

( H

(C)

. - ^ ° ^ 1

/ \

\

v / ^

^r

a) Eurotium chevalieri and Schizosaccharomyces pombe were grown in a glucose-peptone broth at pH 5.0. b) Staphylococcus epidermidis was cultured in a nutrient broth at pH 6.0. c) Maximum dose was 800 mg/ml. (A): 3-[(a-alkoxy)-a-methoxy]methyl-2-thioxopyrrolidines;(B): 3-[(a-alkoxy)-a-methylthio]methyl-2-thioxopyrrolidines; and (C): 3-(alkoxy)-methylene-2-thioxopyrrolidines.

1098

Among the seven a-alkoxy-a-methoxy-substituted derivatives, dimethoxyderivative shov^ed no growth inhibition against the microorganisms tested at the highest dose examined. Similarly, ethoxy, n-propoxy, and n-butoxy derivatives had only weak antimicrobial activity against the fungal strain E, chevalieri and showed no inhibition of the growth of the bacterial strain, 5. epidermidis. The derivatives n-hexyloxy and n-heptyloxy showed a much higher growth inhibition activity against the fungi and bacteria. (A)-heptyloxy r-,

3.5

(A)-hexyloxy I

3.0

(B)-butoxy (Q-butoxy A^

I
oxynietliylene, and n-butoxymethylene derivatives of 3-(alkoxy)methylene-2-thioxopyrrolidines shown in Table 5. Salmonella typhimuriumTA9S was simultaneously treated with IQ (0.5 ^g), Trp-p-1 (0.5 \ig), and the test compounds (HMTP: 130-520 ^ig/ml; other compounds: 500 fxg/ml) in the presence of S9-mix for 20 min at 37° C. After 48 h incubation, the bacterial survivors and revertants were counted, and ratios of the numbers of revertants against those of the survivors were calculated. The ratio obtained from the control (treated with only IQ or Trp-p-1) is defined as 100% mutagenicity. A statistically significant difference between the control and the treating was determined by t-test (* (p < 0.05) and ** (p < 0.01).

1101 HMTP, which had been pretreated with S9 for 20 min at 37° C before contact with the bacterial strain TA98, had no mutagenic or bactericidal activity[, even at 500 ng/ml dose, but without the S9-treatment, this compound behaved as a slightly mutagenic agent at a dose over 390 ^g/ml dose, the level at which a cytotoxic effect was also shown. On the strain TAIOO, the spontaneous mutation frequencies that resulted with or without the S9 were 9- to 13-fold higher than those in TA98. Based on these results, we concluded that HMTP has a weak mutagenic property, but that it could be converted to form nonmutagenic and noncytotoxic product(s) by the action of the S9. MMTP and other 3-alkoxymethylene-derivatives had no cytotoxic or mutagenic activity against the bacterial strains with or without S9 at a 500 [xglml dose. The antimutagenic activities of the MTBIderived products against the heterocyclic amines in S. typhimurium TA98 are shown in Fig. (15).

100-

(B)

?

T-

-7 75a

s.

O

-r

A

LS

C

1 25-

1

15

1

30

1

45

1

n _

^ "

60 Incubation time (min)

1

1

0

15

30

45

60

Fig. (16). Inhibitory effects of HMTP and (£,Z)-MMTP on the S9-mediated metabolism of the heterocyclic amines IQ (A) and Trp-p-1 (B). Modified from Uda etal. [90, 91]. A mixture of 5.0 ^g of IQ or Trp-p-l in 25 ^.1 of dimethylsulfoxide (DMSO), 250 \ig of HMTP or (E,Z)-MMTP in 25 jxl of DMSO, and 450 ^il of S9-mix (S9 : co-factors = 1 : 9 ) was incubated for 60 min. The amounts of IQ and Trp-p-1 were periodically determined by a reverse phase-HPLC analysis. Symbols: w, IQ alone; O , Trp-p-l alone; B , IQ -i- HMTP; • , Trp-p-1 + HMTP; A, IQ + (£,Z)-MMTP; and A , Trp-p-l + {E,Z)-UUTP.

1102 HMTP showed a suppressing effect on the mutation frequency of IQ and Trp-p-1 at a dose over 390 fxg/ml. At a dose of 500 ng/ml, MMTP showed an antimutagenic activity against Trp-p-1, but its effect on the mutagenicity of IQ was much lower than on that of Trp-p-1, suggesting that MMTP behaves differently from HMTP against both heterocyclic amines and/or their S9-mediated metabolic activation system. The synthetic HMTP-related compounds exhibited antimutagenic activity against the heterocyclic amines at 500 \ig/ml dose, where the activity of the alkoxylated compounds appeared to decrease with a lengthening in the carbon chain of the alkoxy group. These results clearly demonstrated that HMTP as well as its structurally related 2-thioxopyrrolidines can act as an antimutagenic agent against the heterocyclic amines in the presence of S9. We subsequently studied the effects of HMTP and MMTP on the S9-mediated decline in the amount of heterocyclic amines and changes in the amounts of HMTP and MMTP during their incubations with the S9 mix. As shown in Fig. (16), IQ (5.0 fxg) and Trp-p-1 (5.0 ^ig) were largely reduced during the incubation with the S9 mix in the absence of HMTP. When the same amounts of the heterocyclic amines were incubated with the S9 mix in the presence of 250 ^ig HMTP, the reduction rates of both heterocyclic amines were considerably decreased. On the other hand, HMTP (250 \xg) was decreased to about 60% of the initial amount during 15 min of incubation with the S9 mix, but the reduction rate slowed thereafter. These results suggested that HMTP was partially metabolized with the S9 mix to form an inhibitor of the S9 fraction, by which HMTP inhibited the S9-mediated activation of the heterocyclic amines. This was considered to be the reason for the reduction of the mutation frequencies of IQ and Trp-p-1 in the presence of HMTP. MMTP also inhibited the S9mediated reduction of both IQ and Trp-p-1, though the reduction rates of IQ were lower than those of Trp-p-1. This may support the above results that MMTP showed a little effect on the mutagenicity of IQ. To investigate the mode of the inhibition of the S9-mediated reduction of the heterocyclic amines, Lineweaver-Burk plots were obtained for combinations of various amounts of IQ (1.25-20 ^ig) and HMTP (0-300 \xg) or MMTP (0-300 [ig). As Fig. (17) shows, an uncompetitive inhibition of the metabolic decrease of IQ was observed in the presence of 50-100 \ig HMTP. With 200-300 [ig HMTP, however, the IQ-metabolism was so strongly inhibited that no V'max value was obtained. In contrast, MMTP showed a noncompetitive inhibition at 50-300 fxg/ml doses. Since cytochrome P-450s are involved in the conversion of the heterocyclic amines into their activated Nhydroxy-forms, both HMTP and MMTP may be able to act as blocking agents for the P-450-mediated activation of the heterocyclic amines by such a mode of action as binding to the enzyme(s)-IQ complex or binding to the enzyme(s) [90,91].

1103 7n

60 80 1/ IQ (mM)

100

Fig. (17). Lineweaver-Biuk plots showing an uncompetitive and a noncompetitive inhibitions of S9-mediated reduction of IQ by addition of HMTP (A) and MMTP (B). Modified from Uda et al. [90, 91]. Symbols denote the amounts of HMTP and MMTP added • , 0 ^g ; O , 50 ^g; O , 100 ^g; A , 200 ^g; and ttj, 300 ^g. IQ was incubated with S9 in the presence or absence of represented amounts of HMTP or MMTP at 37° C for 15 min, and then the remained amount of IQ in the reaction mixtures was measured by an ODS-HPLC.

1104

The antimutagenic activity of 2-thiohydantoins Since the 3,5-disubstituted 2-thiohydantoins may be produced in isothiocyanate-containing foods and in the human digestive tract, we evaluated their antimutagenic activities with a Salmonella test using S. typhimurium TA98 [79]. The MTBI-derived 2-thiohydantoins, i.e., 3-[4(methylthio)-3-butenyl]-5-isobutyl-2-thiohydantoin, 3-[4-(methylthio)-3butenyl]-5-benzyl-2-thiohydantoin, and 3-[4-(methylthio)-3-butenyl]-5-[2(methylthio)ethyl]-2-thiohydantoin were studied for their antimutagenicity against the food bom-heterocyclic amine, IQ.

500 Dose (^ig/ml)

Fig. (18). Antimutagenic activities of the 2-thiohydantoins. Modified from Takahashi et al. [79]. (A), 3-[4-(methylthio)-3-butenyl]-5-isobutyl-2-thiohydantoin; (B), 3-[4-(methylthio)-3butenyl]-5-benzyl-2-thiohydantoin; and (C), 3-[4-(methylthio)-3-butenyl]-5-[2-(methylthio)ethyl]-2-thiohydantoin. Symbols: , Salmonella typhimurium TA9S was simultaneously incubated with IQ (0.5 \ig) and the 3,5-disubstituted 2-thiohydantoins (0-500 jAg/ml) for 20 min at 37° C and then cultured for 48 h at the same temperature; , the bacterial strain was treated with the same amount of IQ for 20 min at 37° C, rinsed with a buffered saline to eliminate the mutagen, and incubated with the test compounds for an additional 20 min. The bacteria were cultured for 48 h at 37° C. Mutagenicity is expressed in terms of the percentage in the revertant number found with or without the test compounds, where the mutagenicity of IQ is defined as 100%. Symbols: I—I and™, 3-[4-(methylthio)-3-butenyl]-5-isobutyl-2-thiohydantoin; A and A, 3-[4-(methylthio)-3-butenyl]-5-benzyl-2-thiohydantOLn; a n d O and w , 3-[4-(methylthio)3-butenyl]-5-[2-(methylthio)ethyl]-2-thiohydantoin.

1105 As Fig. 18 shows, the former two compounds, which were formed by the reaction of MTBI with Leu and Phe, were able to inhibit the IQ mutagenicity at concentrations of 125-500 ^ig/ml when incubated simultaneously with IQ in the presence of S9, and the compound derived from MTBI and Met showed an antimutagenic activity at a dose of 500 Hg/ml by simultaneous treatment with IQ. However, no inhibition of the IQ mutagenicity was observed when the test compounds were incubated with the bacteria that had been preincubated with IQ to induce mutation. This suggests that these 2-thiohydantoins are not involved in the repair of DNA lesions, but they are involved with the inactivation of IQ or the inhibition of S9-mediated metabolic activation of the mutagen. Dijferences in the mutagenic and antimutagenic properties of stereoisomers of tetrahydro-p-carbolines (PTCCs)

the

PTCC, which can be derived from MTBI and tryptophan, has two stereoisomers, (IS*, 3S*, 3'R*)-PTCC and (IR*, 3S*, 3'R*)-PTCC. In a fermented radish product, the amount of (IS*, 3S*, 3'R*)-isomer was much higher than that of the (IR*, 3S*, 3'R*)-isomer. We studied the difference in their mutagenic and antimutagenic properties using Salmonella typhimurium TA98 and TA100. In this study, both isomers were isolated from the reaction mixture of MTBI and L-tryptophan, and their FAB-MS, iH-lH-COSY, iH-l^C-COSY, and NOESY spectral data were compared along with their UV and IR spectra [60]. The spectral data showed that both PTCC-isomers were successfully obtained, and we were then able to carry out an Ames test. Neither PTCC had any bactericidal activity toward the bacteria at the highest dose (600 ^ig/plate) examined. An Ames test was then done in the presence or absence of S9. (IS*, 3S*, 3'R*)-PTCC had no mutagenic activity in a dose range of 0-600 ng/plate toward both tester strains, regardless of the presence or absence of S9. On the contrary, (IR*, 38*, 3'R*)-isomer showed obvious mutagenic activity, depending on its dose, toward S, typhimurium TA98 with S9, but not in the absence of S9. This isomer was not mutagenic toward S. typhimurium TAIOO in the same dose range with or without S9. However, the mutagenicity of (IR*, 3S*, 3'R*)-PTCC against TA98 was not considered strong, because the revertant number was below 100 even at a 600 jxg/plate dose. Since pcarboline derivatives enhanced the mutagenicity of IQ and Trp-p-1 [100], we examined the effect of PTCCs on the mutagenicity of IQ. Neither PTCC had an effect on the mutagenic or antimutagenic properties against IQ. In conclusion, the pungent principle of radish, MTBI, is a class of compounds that is extremely labile in the presence of water and can change into a variety of products having biological and physiological activities. There have so far been no studies on the activity that induces cancer-

1106 protective phase II enzymes like quinone reductase and glutathione Stransferase. Further investigations of this type should be conducted because of their potential importance to consumers and the food industry. ABBREVIATIONS (£)-MBDC (Z)-MBDC

= (£)-Methyl 4-(methylthio)-3-butenyldithiocarbamate. = (Z)-Methyl 4-(methylthio)-3-butenyldithiocarbamate. I H - I ^ C - C O S Y = Proton-Carbon Correlation Spectrometry. 1 H - 1 H - C 0 S Y = Proton-Proton Correlation Spectrometry. CFU = Colony Forming Unit. FAB-MS = Fast Atom Bombardment Mass Spectrometry. FPD-GC = Flame photometry detector-gas chromatography. HMTP = 3-(Hydroxy)methylene-2-thioxopyrrolidine. HR-EI-MS = High Resolution-Electron Impact lonization-Mass Spectrometry. IQ = 2-Amino-3-methylimidazo[4,5-f]quinoline. IR = Infrared Spectrometry. KBr = Potassium Bromide. LR-EI-MS = Low Resolution-Electron Impact lonization-Mass Spectrometry. MBDC = Methyl 4-(methylthio)butyldithiocarbamate. MeOH = Methanol. MIC = Minimum Inhibitory Concentration. MMTP = 3-(Methylthio)methylene-2-thioxopyrrolidine. MS = Mass Spectrometry. MTBI = 4-(Methylthio)-(£,Z)-3-butenyl isothiocyanate. NMR = Nuclear Magnetic Resonance Spectrometry. NOESY = Nuclear Overhauser Enhancement and Exchange Spectrometry. ODS = Chemically Octadecylated Silica Gel. PTCC = l-(2'-Pyrrolidinethione-3'-yl)-1,23,4-tetrahydro-b-carboline-3carboxylic acid. TMS = Tetramethylsilane. Trp-p-1 = 3-Amino-l,4-dimethyl-5H-pyrido[4,3-b]indole. UV = Ultra Violet Spectrometry. ACKNOWLEDGMENT The authors would like to thank Dr. H. Matsuoka, Gunma Women's Junior College, and Dr. Y. Yamada, Utsunomiya University, for their

1107 measurements of the NMR spectra. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]

Kjaer, A. In Progress in the Chemistry of Organic Natural Products; W. Hetz; H. Grisebach; G.W. Kirby, Ed.; Springer-Verlag: New York, 1960; pp. 122-176. VanEtten, C.H. In Toxic Constituents of Plant Foodstuffs; I.E. Liener, Ed.; Academic Press; New York, 1969; pp. 103-142. Fenwick, G.R.; Heaney, R.K.; MuUin, W.J. CRC Crit. Rev. Food Sci. Nutr., 1983, 18, 123. Roubiquet, P.J.; Boutron, F. J. Pharm. Chim., 1830, 96, 443. Ettlinger, M.G.; Kjaer, A. In Recent Advances in Phytochemistry; T.J. Mabry; R.E. Alston,; V.C. Runeckles, Ed.; Appleton-CenturyCrofts: New York, 1968; Vol. 1, pp. 59-144. Kjaer, A.; Skrydstrup, T. Acta Chem. Scand., 1987, B41, 29. Ettlinger, M.G.; Dateo, G.R; Harrison, Jr., B.W.; Mabry, T.J.; Thompson, C.P. Proc. Nat. Acad. Sci. USA., 1961, 47, 1875. Tookey, H.L.; Wolf, I.A. Can. J. Biochem., 1970, 48, 1024. Uda, Y.; Kurata, T.; Arakawa, N. Agric. Biol. Chem., 1986, 50, 2735. Uda, Y; Kurata, T; Arakawa, N. Agric. Biol. Chem., 1986, 50, 2471. Lee, C.-J; Serif, G.S. Biochim. Biophys. Acta, 1968, 165, 569. Kjaer, A.; Larsen, P.O. In Biosynthesis; T.A. Geissman, Ed.; The Chemical Society: London, 1973, Vol. 2, pp. 71-105. Rausch, T.; Butcher, D.N.; Hilgenberg, W. Physiol. Plant., 1983, 58,93. Astwood, E.B.; Greer, M.A.; Ettlinger, M.G. J. Biol. Chem., 1949, 181, 121. VanEtten, C.H; Gagne, W.E.; Robbins, D.J.; Booth, A.N.; Daxenbichler, M.E.; Wolff, I.A. Cereal Chem., 1969, 46, 145. Nishie, K.; Daxenbichler, M.E. Food Cosmet. Toxicol., 1980, 18, 159. Searle, L.M.; Chamberlain, K.; Rausch, T.; Butcher, D.N. J. Exp. Botany, 1982, 33, 935. Virtanen, A.I. Angew. Chem. Internat., 1962, 1, 299. Drobnica, L.;Zemanova, M.;Nemec, P.; Antos, K.; Kristian, P.; Stullerova,A.; Knoppova, V.; Nemec, Jr., P. Appl. Microbiol., 1967, 15,701. Chew, F.S. In Biologically Active Natural Products for Potential Use in Agriculture; H.G. Cutler, Ed.; American Chemical Society:

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Washington, DC, 1988; pp. 155-181. Talalay, P.; De Long, M.J.; Prochaska, H.J. Proc. Natl. Acad. Sci. USA., 1 9 8 8 , 8 5 , 8 2 6 1 . Zhang, Y.; Talalay, P.; Cho, C-G.; Posner, G.H. Proc. Natl. Acad. Sci. USA., 1992, 89, 2399 Zhang, Y.; Talalay, P. Cancer Res. Suppl., 1994, 54, 1976s. Wattenberg, L. W. Cancer Res., 1981, 4 1 , 2991. Wattenberg, L.W. Carcinogenesis, 1987, 8,1971. Morse, M.A.; Eklind, K.I.; Hecht, S.S.; Jordan, K.G.; Chok, CI.; Desai, D.H.; Amin, S.G.; Chung, F-L. Cancer Res., 1991, 5 1 , 1846. Verhoeven, D.T.H.; Verhagen, H; Goldbohm, R.A.; Van Den Brandt, P.A.; Van Poppel, G. Chemico-Biol. Interact., 1997, 103, 79. Brusewitz, G.; Cameron, B.D.; Chasseaud, L.F.; Gorier, K.; Hawkins, D.R.; Koch, H.; Mennicke, W.H. Biochem. J., 1977, 162,99. Gorier, K.; Krumbiegel, G.; Mennicke, W.H.; Siehl, H-U. Xenobiotica, 1982,12, 535. Mennicke, W.H.; Gorier, K.; Krumbiegel, G. Xenobiotica, 1983, 13, 203. Shapiro, T.A.; Fahey, J.W.; Wade, K.L.; Stephenson, K.K.; Talalay, P. Cancer Epidemiol., Biomarkers & Prevention, 1998, 7, 1091. Ashworth, M.R.F. The Determination of Sulfur-containing Groups, Academic Press: New York, 1972. Kawakishi, S; Namiki, M. J. Agric. Food Chem., 1982, 30, 618. Kawakishi, S.; Goto, T.; Namiki, M. Agric. Biol. Chem., 1983, 47, 2071. Kawakishi, S.; Muramatsu, K. Agric. Biol. Chem., 1966, 30, 688. Friis, P.; Kjaer, A. Acta Chem. Scand., 1966, 20, 698. Daxenbichler, M.E.; Spencer, G.F.; Carlson, D.G.; Rose, G.B.; Bnnker, A.M.; Powell, R.G. Phytochemistry, 1991, 30, 2623. Kjaer, A.; Madsen, J.0.; Maeda, Y.; Ozawa, Y.; Uda, Y. Agric. Biol. Chem., 1978, 42, 1715. Carlson, D.G.; Daxenbichler, M.E.; VanEtten, C.H.; Hill, C.B.; Williams, P.H. J. Am. Soc. Hortic. Sci., 1985, 110, 634. Ishii, G.; Saijo, R.; Nagata, M. Jpn. J. Food. Sci. Technol. {Nippon Shokuhin Kogyou Gakkaishi), 1989, 36, 739. Visentin, M.; Tava, A.; lori, R.; Palmieri, S. J. Agric. Food Chem., 1992; 40, 1687. Kawakishi, S.; Namiki, M.; Watanabe, H.; Muramatsu, K. Agric. Biol. Chem., 1 9 6 7 , 3 1 , 8 2 3 Kawakishi, S.; Namiki M. Agric. Biol. Chem., 1 9 6 9 , 3 3 , 4 5 2 .

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Chen, C.-W; Ho, C.-T. J. Agric. Food Chem., 1998, 46, 220. Uda, Y.; Matsuoka, H.; Kumagami, H.; Shima, H.; Maeda, Y. Jpn. J. Food Sci. Technol. {Nippon Shokuhin Kogyo Gakkaishi), 1993, 40, 743. Uda, Y.; Ozawa, Y.; Ohshima, Y.; Kawakishi, S. Agric. Biol. Chem., 1990, 54, 613. Matsuoka, H.; Toda, Y.; Yanagi, K.; Takahashi, A.; Yoneyama, K.; Uda, Y. Biosci. Biotech. Biochem., 1997, 61, 2109. Nakamura, N. Biochem. Z., 1925, 164, 31. Shibayama, F.; Yamada, S.; Kikuchi, H.; Yamada, Y. Jpn. Kokai Tokkyo Koho JF 81, 1981, 104, 864. {Chem. Abstr. 1982, 96, 35076p.) Hasegawa, K.; Shiihara, S.; hvagavva, T.; Hase, T. Plant Physiol, 1982, 70, 626. Harada, N.; Hagiwara, H.; Ono, H.; Uda, H.; Ohba, S.; Kubota, M.; Nishiyama, S.; Yamamura, S.; Hasegawa, K.; Sakoda, M. Tetrahedron Letters, 1991, 32, 6757. Kosemura, S.; Yamamura, S.; Hasegawa, K. Tetrahedron Letters, 1993. 34, 481. Matsuoka, H.; Uda, Y.; Mitani, K.; Yoneyama, K.; Maeda, Y. Biosci. Biotech. Biochem., 1996, 60, 914. Matsuoka, H.; Toda, Y.; Yoneyama, K.; Uda, Y. Phytochemistry, 1998,47, 975. Maeda, Y.; Ozawa, Y.; Uda, Y. Jpn. J. Food Sci. Technol. {Nippon Shokuhin Kogyo Gakkaishi), 1982, 29, 522. Ozawa, Y.; Kawakishi, S.; Uda, Y.; Maeda, Y. Agric. Biol. Chem., 1990,54, 1241. Ozawa, Y.; Uda, Y.; Kawakishi, S. Biosci. Biotech. Biochem., 1992, 56, 144. Ozawa, Y.; Uda, Y.; Kawakishi, S. Jpn. J. Food Sci. Technol. {Nippon Shokuhin Kogyo Gakkaishi), 1992, 39, 490. Ozawa, Y.; Uda, Y.; Kawakishi, S. Agri. Biol. Chem., 1990, 54, 1849. Ozawa, Y.; Uda, Y.; Matsuoka, H.; Abe, M.; Kawakishi, S.; Osawa, T. Biosci, Biotechnol. Biochem., 1999, 63, 216. Whaley, W.H.; Govindachari, T.R. In Organic Reactions Coll.; R. Adams Ed.; Wiley; New York, 1951;.Vol. 6, pp. 151-X. Buckholtz, N.S. Life Sci., 1980, 27, 893. Beck, O.; Holmstedt, B. Food. Cosmet. Toxicol., 1981, 19, 173. Beck, O.; Bosin, T.R.; Lundman, A. J. Agric. Food Chem., 1983,31,288. Wakabayashi, K.; Ochiai, M.; Saito, H.; Tsuda, M.; Suwa, Y.; Nagao, M.; Sugimura, T. Proc. Natl. Acad. Sci. USA., 1983, 80, 2912. Bosin, T.R.; Krogh, S.; Mais, D. J. Agric. Food Chem., 1986,

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34, 843. Adachi, J.; Mizoi, Y.; Naito, T.; Ogawa, Y.; Uetani, Y.; Ninomiya, I. J. Nutr. 1991, 121, 646. Herraiz, T. J. Agric. Food Chem., 1996, 44,3057. Chu, N.T.; Clydesdale, F.M. J. Food ScL, 1976, 4 1 , 891. Bobbitt, J.M.; Willis, J.P. J. Org. Chem., 1980, 45, 1978. Kato, S.; Nakase, A. Jpn. J. Food Sci. Technol. (Nippon Shokuhin Kogyo Gakkaishi), 1 9 8 6 , 3 3 , 659. Ozawa, Y.; Uda, Y.; Ohshima, T.; Saito, K.; Maeda, Y. Agric. Biol. Chem., 1990, 54, 605. Kj£er, A.; Madsen, J.0.; Maeda, Y.; Ozawa, Y.; Uda, Y. Agric. Biol. Chem., 1978, 42, 1989. Nielsen, J.K.; Olsen, O.; Pedersen, L.H. ; Sorensen, H. Phytochemistry, 1984, 23, 1741. Harborne, J.B.; Corner, J.J. Biochem. J., 1961, 81, 242. Edman, P. Acta Chem. Scand., 1950, 4, 283. Drobnica, L ; Augustin, J. Collect. Czech. Chem. Commun., 1965, 30, 99. Drobnica, L.; Augustin, J. Collect. Czech. Chem. Commun.,

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Takahashi, A.; Matsuoka, H.; Ozawa, Y.; Uda, Y. J. Agric. Food Chem., 1998, 46, 5037. Esaki, H.; Onozaki, H. Jpn. J. Food Nutr. (Eiyo Shokuryo Gakkaishi), 1982, 35, 207. Inoue, S.; Goi, H.; Miyauchi, K.; Muraki, S.; Ogihara, M.; Iwanami, Y. J. Antibact. Antifung. Agents, 1983, 11, 609. Goi, H.; Inoue, S.; Iwanami, Y. J. Antibact. Antifung. Agents, 1985, 13, 199. Issiki, K.; Tokuoka, K.; Mori, R.; Chiba, S. Biosci. Biotech. Biochem., 1992, 56, 1476. Uda, Y.; Matsuoka, H.; Shima, H.; Kumagami, H.; Maeda, Y. Jpn. J. Food Sci. Technol. (Nippon Shokuhin Kogyo Gakkaishi), 1993, 40, 801. Matsuoka, H.; Takahashi, A.; Yanagi, K.; Uda, Y. Food Sci. Technol. Int. Tokyo, 1997, 3, 353. Matsuoka, H.; Takahashi, A.; Y oneyama, K.; Uda, Y. Biocontrol Sci., 1 9 9 9 , 4 , 101. Graham, S. Cancer Res. SuppL, 1983, 43, 2409s. Caragay, A.B. Food Technol. 1992, April. 65. Block, G.; Patterson, B.; Subar, A. Nutr. Cancer, 1992, 18, 1. Uda, Y.; Shimizu, A.; Hayashi, H.;Ozawa, Y. Proceeding for 5th International Congress on Mechanisms of Antimutagenesis and Anticarcinogenesis (Okayama Univ. Japan), 1996, Page 119. Uda, Y.; Hayashi, H.; Takahashi, A.; Shimizu, A. Food Sci. Technol., 2 0 0 0 , 3 3 , 37.

nil [92]

Nhomi, T.; Matsui, M.; Ishidate, M. Toxicol. Forum, 1986, 9, 189. [93] Aeschbacher, H.U.; Turesky, R.J. Mutation Res., 1991, 259, 235. [94] Ohgaki, H; Takayama, S.; Sugimura, T. Mutation Res., 1991, 259, 399. [95] Ishii, K.; Yamazoe, Y.; Kamataki, T.; Kato, R. Cancer Res., 1980, 40, 2596. [96] Yamazoe, Y.; Shimada, M.; Kamataki, T.; Kato, R. Cancer Res., 1983, 43, 5768. [97] Soucek, P.; Gut, I. Xenobiotica, 1992, 22, 83. [98] Masson, H.A.; loannides, C.; Gorrod, J.W.; Gibson, G.G. Carcinogenesis, 1983, 4, 1583. [99] Aldrick, A.J.; Rowland, I.R. Mutation Res., 1985, 144, 59. [100] Friedman, M.; Cuq, J.L. J. Agric. Food Chem., 1988, 36, 1079.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 26 © 2002 Elsevier Science B.V. Allrightsreserved,

1113

STRUCTURE, OCCURRENCE AND ROLES OF CARBOHYDRATES WITH THE HEXO-D-MANNO CONFIGURATION

NORMAN K. MATHESON

Department ofAgricultural Chemistry and Soil Science, The University of Sydney N.S W, Australia, 2006 ABSTRACT: Aspects of the occurrence, structure, properties, metabolism, biological activity and uses of naturally-occurring carbohydrates based on the hexo-D-manno structure have been discussed. These include D-mannose, D-mannitol, oligosaccharides, a-mannoproteins of fungi, glycoproteins, p-mannan, p-glucomannan, a-galacto-Pmannan, galacto-P-glucomannan, alginic acid, as well as bacterial polysaccharides and plant gums and mucilages containing D-mannose. Some in vitro syntheses of mannans and manno-oligomers have also been described.

INTRODUCTION The most abundant naturally-occurring sugar is D-glucopyranose, with equatorial hydroxyl groups on C-2, 3 and 4 and an equatorial hydroxymethyl group on C-5 when in the usual ^Ci conformation. It is the thermodynamically most stable aldohexose structure. Manno- and galacto- configurations in the "^Ci conformation, which also occur extensively, have one axial OH - at C-2 in mannose - and both avoid a 1:3 diaxial interaction as either anomer. A six carbon chain is the shortest that can provide an unstrained hemi-acetal ring (six-membered) in which all carbons are substituted with hydrophilic -OH groups and a -CH2OH. The biosynthesis of D-mannose involves the conversion of the D-gluco isomer via the D-fructo (as the 6-phosphates) by isomerization at C-2; differing from the conversions of other sugars (including D-galactose) which proceed via nucleoside diphospho sugars, the formation of which requires a supply of nucleoside triphosphates. However, despite the widespread distribution of D-mannose and its utilisation in glycolysis and

1114

biosynthesis in most species, under some circumstances it can be deleterious to growth and even toxic at low concentrations. The related hexitol D-mannitol also occurs extensively. It is stored in fungi and plants and is readily utilised. Mannosyl glycosides and oligosaccharides, particularly lipid-linked, occur and phosphoric esters are participants in metabolism. Related monomeric structures, such as amino sugars, deoxy-sugars, ethers, esters and uronic acids have also been detected. Polysaccharides containing D-manno-hexo structures can be broadly grouped into those in which these monomers constitute the majority of the polymer and those in which they contribute a minority of residues, aMannans and a-phospho-mannans are found as proteoglycans in the cell walls of fungi, and a-mannans and a-glucomannans as fibrous material in the walls of algae. The glycoproteins, which are of general distribution, also contain a-mannosyl Unkages. Fungal mannoproteins and the glycoproteins are related structurally and biosynthetically. P(Gluco)mannans and their substituted polymers - a-galacto-p-mannan and galacto-P-glucomannan - are associated with plants. A major sugar in the seaweed polysaccharide alginic acid is D-mannuronic acid. The viscosity and gel-forming characteristics of a-galacto-P-mannan, p-glucomannan and alginic acid, which is also a polyelectrolyte, leads to their use in the food, pharmaceutical, paper and textile industries. D-mannose provides a hexose with many properties similar to those of D-glucose, even reacting with the same enzyme (hexokinase) in one instance, but clearly differentiated in other enzymic reactions. This allows the production of a different set of oligomeric and polymeric structures with (1-3), (1-4) and (1-6) inter-residue linkages, having many properties similar to glucans but quite separate modes of biosynthesis and cellular recognition. The conformations of (1-3), (1-4) and (1-6) linked mannans resemble those of the equivalent glucans. Structural polymers, although having many similarities to glucans with the same linkage, have somewhat different physical properties, and energy reserve polysaccharides are utilised (apart from hexokinase) by a separate set of enzymes. a(l-2) Mannan provides a linkage of two axial bonds which is not available for the gluco conformation. D-mannose is also present in smaller amounts in other polysaccharides, such as bacterial lipo- and exopolysaccharides, and the plant gum exudates and mucilages. Chemically synthesized oligomers and polymers have also been prepared.

1115

In this text, biochemical style abbreviations - the condensed system of symbolism - have been used for the formulae of carbohydrates [1]. Where more convenient M has also been used as a symbol for D-mannopyranose. Unless described otherwise, chiral compounds are D- and glycosidically linked sugars are pyranose. In oligosaccharide formulae a reducing-end sugar is italicised. Italicised reference numbers indicate reviews and these provide early information and lists of references. P represents a phosphate group. DISCUSSION 1. Metabolism of D-Mannose Man enters metabolism as Man 6-phosphate (Man 6-P) in a reaction catalysed by hexokinase [EC 2.7.1.1] (reaction A) in which the phosphate donor is MgATP and the other product of reaction MgADP [2,5,4].

Man+ MgATP Man 6-P Man6-P

A -> B ^ D ^

Man 6-P -f MgADP C Fru6-P ^ Glc6.P Manl-P

Man 6-P can then be converted into Fru 6-P by phosphomannoisomerase (PMI) [EC 5.3.1.8] [5,6] in an equilibrium reaction of 40% Man 6-P and 60% of Fru 6-P (reaction B). Isomerisation of Fru 6-P by phosphoglucoisomerase [EC 5.3.1.9] (reaction C) in another equilibrium reaction gives Glc 6-P and entry into glycolysis. Free Man can also be recovered through phosphorylation to Man 6-P by hexokinase. Man 6-P is converted to Man 1-P by phosphomannomutase [EC 5.4.2.8] [7-10a] (reaction D) for further metabolism in the D-manno configuration. Although some kinases specific for Glc have been detected [2,3], hexokinase generally phosphorylates both Glc and Man even when obtained from sources in which free Man is absent. A number of legume seeds (fenugreek, guar, carob and honey locust) contain a reserve agalacto-p-mannan. After germination, when depolymerisation occurs, the released Man is readily utilised. In honey locust {Gleditsia triacanthos) attempts to separate gluco- and manno-kinase activities were unsuccessful

1116

[4]: the rates of loss of activity of the purified enzyme by slow heat inactivation were similar with both substrates. The Michaelis constants (Km) were similar for both hexoses, as also were the specificities [Vmax/Km] with the P-pyranose anomers. Despite the general occurrence of Man in cellular glycoprotein and its widespread presence in structural polysaccharides of plants, as well as the ability of hexokinase to phosphorylate both Glc and Man - which extends to analogues such as 2deoxy-D-glucose (2dGlc), D-glucosamine and 1,5-anhydro-D-glucitol [4,11] - Man (and analogues) are deleterious to many organisms, such as mammalian cells [12], yeast mutants [13], bees [14] and a range of plants and plant parts [3]. Man and Glc analogues act at the same site on the enzyme as Glc, both as substrates and competitive inhibitors to Glc. Inhibitor constants for Man (Ki, inhibitor concentration giving 50% inhibition) versus Glc for the hexokinase from brain [15] and honey locust seeds [4] of .036 and .031mM have been estimated. Toxic effects are associated with undetectable or very low levels of endogenous PMI, leading to an inability to further metabolize the Man 6-P synthesized by hexokinase. Where Man and 2dGlc are deleterious their 6-phosphates accumulate [14, 16, 17]. When 2dGlc, for which there is no available further reaction for the 6-phosphate (except hydrolysis), is supplied even more species are affected [18, 19-21]. One proposal for the cause of this effect of Man and Glc analogues not further metabolised in species with undetectable or very low levels of PMI, is that the accumulation of Man 6-P (or analogue-P) sequesters inorganic phosphate (Pj), lowering endogenous concentrations of Pi, and so denying Pj for the synthesis of Glc 6-P. Reductions in Pi have been demonstrated in many cells [5, 12, 16-18, 20, 22, 23]. In 5 minutes [Pi] decreased markedly in ascites tumour cells [12] maintained in 1 mM 2dGlc and, in yeast [18] in 20 mM 2dGlc, dropped from 34 to 25 |amole per g in 4 hr. The Pi content of wheat leaf fragments [22] in 10 mM Man was lowered in 6 hr from 4.4 nmole per g fresh weight to 2.9. Depletion has also been noted in pear fruits [17] and spinach leaves [3], ^^P n.m.r. spectra of maize leaves [23] incubated in 25 mM Man for 1 hr had a lowered [Pi]. In spectra of maize root tips [20], incubated for 2 hr with 50mM 2dGlc, the peak attributable to cytoplasmic Pi virtually disappeared, while vacuolar Pi decreased slowly. In another study [16], at a much lower concentration of Man (2mM), after 30 hr, cytoplasmic [Pi] remained constant but Pi disappeared from the vacuole.

1117 The very sensitive inhibition of mammaUan hexokinase by the product Glc 6-P (vs Mg ATP, Ki 0.04 mM) suggests the possibility of inhibition by accumulating Man 6-P or analogue-P. However, although in yeast [24] mixed inhibition by Glc 6-P does occur, it is only at much higher levels (20-30mM). In wheat germ hexokinase [25], with Man as substrate, Glc 6-P inhibited non-competitively to MgATP (Kj 16.2 mM). With the enzyme from potato tubers [26], it acted non-competitively against Glc (Ki 4.1 mM) and, since a cellular concentration of Glc 6-P of approximately 3-7 mM was estimated, it was concluded that a capacity for a moderate effect may be present. When analogue inhibition occurs, although [Glc 6-P] decreases, there is an increase in the concentration of analogue-P, which may inhibit hexokinase. ^^P n.m.r. spectroscopy indicated large peaks ascribed to Man 6-P in potato slices after 12.5 hr in 10 mM Man [16] and in maize root tips after 30 hr in 2 mM Man [20]. When pear fruits [17] were infiltrated with 100 mM Man for 2 days, the [Man 6-P] rose to 491 nmole per g fresh weight, and in 300 mM Man to 550 nmole per g. Bees fed Man [14] accumulated 11 ^mole per g of 6phosphate after 4 hr. Similarly, in yeast [18] in 2 mM 2dGlc, the 6phosphate content rose to 50 |imole per g; P n.m.r. spectroscopy [19] indicated that in 25 mM Glc there was a 25% increase in the content of 2dGlc 6-P when the external concentration of 2dGlc was increased from 5 to 10 mM. A large spectral peak attributable to 2dGlc 6-P appeared in maize root tips [20] after 2 hr in 50 mM 2dGlc and rat heart perfUsed with 2dGlc attained a level of 55 mM 2dGlc 6-P[21]. With Glc as substrate, yeast hexokinase [15] was inhibited by Man 6P - with mixed inhibition versus ATP - with a Kjs value of about 2-3 mM. The enzyme from beef heart [21] with 2dGlc as substrate, was inhibited by 2dGlc 6-P against ATP and non-competitively to 2dGlc with a Ki of 1.4 mM. However, when phosphate transfer from ATP and phosphorylation of Glc were measured for hexokinase from honey locust seeds [27] in the presence of Man 6-P, negligible inhibition against Glc and a very slight inhibition against ATP were observed (Kjs 52 mM). The results indicate that, whereas there could be a possible effect after significant accumulation of analogue-P, it was unlikely that analogue phosphates were associated with the initiation of inhibition. The supply of Man to sensitive species also leads to decreases in phospho compounds such as ATP, polyphosphate, Glc 6-P and phosphoenolpyruvate, significant high-energy intermediates in fiirther metabolism. The decreases of these are more pronounced than those of

1118

Pj. Where measured, much smaller decreases in ADP have been noted [12, 22, 28]. In bees[14] fed Man, [ATP] changed from 1.6 to 0.3 ^imole per g in 4 hr. In maize scutellum slices [28] after 1 hr in 50 mM Man, [ATP] decreased from 0.40 to a(l-6)>a(l2)>Manal-OMe. Periodate-oxidised and then reduced a(l-3) pentasaccharide also inhibited, indicating that the lectin recognised internal a(l-3) linkages. The lectin from bulbs of Crocus vernus selectively interacted with terminal Manal-3Man- groups [158]. Garlic {Allium sativum) lectins [159] bound strongly to oligomannose chains, shoving a preference; Man9 (8 with asparagine replacing -P-P-Dol) > >Man5 with a(l-6) and a(l-3) links > Manal-6Manal-3A/a«>Manal3Man, Two GlcNAc residues at the reducing end of triose or pentaose enhanced activity. Glycoproteins with compound chains, like fetuin, were ligands: removal of terminal sialic acid residues increased binding. Mycobacteria contain a lectin that adheres to macrophages [160]. The fimbriae of more highly virulent strains oi Escherichia coli, which act as

1136

adhesive organelles, expressed a lectin [161] that interacted with single Man residues, whereas those of less virulent strains expressed a lectin specific for mannotriose. Arcelin-1 from wild varieties of the bean, Phaseolus vulgaris [162], a lectin that has insecticidal properties, does not interact with monosaccharides, but binds to complex type glycan chains. An insect-derived mutant of the toxin ricin, modified at three sites to give a 1,000-fold reduced galactoside avidity [163], when supplied to mouse macrophages was still lethal, showing that intracellular binding to galactose is not required for toxicity. The function of a biological macromolecule in solution is dependent on its conformation. The determination of three-dimensional structures in solution, in conjunction with the dynamic behaviour, contributes to understanding of the associations between the polypeptide and its substituent glycan chains, and between the glycoprotein and interacting molecules, as well as the significance of glycoforms. Restrictions in flexibility imposed by the hydrodynamic volume of chains and the possibility of interaction with the polypeptide are factors in the conformation. Molecular modelling [725, 750, 757], calculations of minimum energy conformations [164-166], n.m.r. spectroscopy [757,164, 167-169], molecular dynamics simulations [164, 169-171], molecular orbital calculations [164, 169] and fluorescence energy transfer measurements [172] have been used to study these aspects of glycoproteins. Molecular modelling [125] of a dianntenary complex structure suggested a compact pentasaccharide core, with the diequatorially linked P(l-4)-Man-GlcNAc-GlcNAc- segment as a flat ribbon, and the two antennae as helical chains with two possible orientations. Preferred positions have been proposed [165, 166] for the sugar residues in oligomannose chains and a bisected pentasaccharide. Time resolved fluorescence energy transfer measurements [172] on a dansyl-labelled triantennary complex glycan indicated two populations of conformers when two of the antennae were labelled, but only one when the third was labelled. N.m.r. spectroscopy, molecular orbital calculations and molecular dynamics simulations [169] of an oligomannose type heptasaccharide and an undecasaccharide indicated that the Manal3ManP linkage was highly restrained and the Manal-6Manp and Manal6Mana linkages disordered. The Man al-6ManP linkage was more restrained in the undecasaccharide. Comparison of a hybrid heptasaccharide with the bisected octasaccharide structure showed that the presence of the bisecting GlcNAc restricted the dynamic behaviour of the

1137

Manal-6ManP and Manal-3ManP bonds [170]. Molecular dynamics simulation of an oligomannose chain (Man5.GlcNAc2) attached to immunoglobulin M, and a complex chain in immunoglobulin G [171] indicated the availability of various conformational minima and differences in mobilities of the various glycosidic linkages. From these multiple minima and the presence of the same glycans in different proteins, it was suggested that the same chain sequence might have different dominant conformations in different protein environments. The calculations were consistent with results from n.m.r. spectroscopy and fluorescence energy measurements. Examination of the adhesion domain of a cell surface glycoprotein (CD2) of human T cell lymphocytes [173], which contains a single oligomannose type glycan, indicated that this chain increases rigidity and stability by counterbalancing a cluster of positive changes about lysine 61. Removal of this N-linked chain caused loss of binding activity, with unfolding of the protein. Reduction to a single GlcNAc unit reduced stability. Protein with lysine 61 replaced with glutamic acid required no glycan chain to be stable. It was found that through interaction with the polypeptide the conformational mobility of parts of the attached glycan can be restricted relative to the free oligosaccharide. Bovine pancreatic ribonuclease B has five glycoforms in which a series of high mannose oligomers (Mans GlcNAc2 - Man9 GlcNAc2) are attached to the single Nglycosylation site [174]. Exo-glycosidase digestion gave individual glycoforms. Varying glycosylation affected dynamic stability and the glycoforms showed increased resistance to proteolysis compared with the unglycosylated enzyme. There was a fourfold variation in enzymic activity among glycoforms and molecular modelling suggested that steric factors were responsible. Glycan chains-oligosaccharides MansGlcNAc and Man3(Xyl)GlcNAc(L-Fuc)G/cA^^c - at concentrations of 0.5 and 5|ag/mL (2|iL/g fresh weight) promoted fruit ripening when infiltrated into tomatoes [175]. (c) Seaweed a-Mannans Sulphated and xylo-a(l-3) mannans have been extracted from seaweeds. A water-soluble (1-3) mannan has been reported from Urospora penicilliformis [176], A sulphated a-mannan was isolated in more than 30% yield (dry weight) from Nemalion vermiculare by extraction with hot water. Cetavlon gave a precipitate containing 15.5% sulphate.

1138 Methylation analysis of the desulphated polymer showed an unbranched (1-3) linked mannan chain and the positive optical rotation indicated aglycosidic links [64, 65]. Water extraction of the red seaweed Nothogenia fastigiata, gave a sulphated xylomannan [177]. Methylation analysis and n.m.r. spectroscopy showed a structure with an a(l-3) mannan main chain, substituted at 0-2 with single Xylpl- groups. The Man residues were also substituted at 0-2 or 0-6 with sulphate, with some substituted at both. The polysaccharide inhibited viral replication and this activity was considered to depend on its ability to block virus receptors necessary for adsorption to target cells. 5. P-Mannans A number of plant [106, 178-183] and algal [64, 176] polysaccharides have structures based on a P(l-4)mannan main chain - P(l-4)maiman, a(l-6)galacto-P(l-4)-D-mannan and the related P(l-4)-D-glucomannan and galacto-P(l-4)glucomannan. They serve as cell wall structural material and as carbohydrate reserve polymers. An unbranched p(l-2) mannan of low molecular weight has been reported from the insect flagellate Herpetomonas samuelpessoai [184]. (a) J3(l'4)-D'Mannan P-Mannan is water-insoluble and solubilizing it requires very strong alkali, cuprammonium or derivatization. X-ray and electron diffraction show that mannan has a ribbon-like conformation, similar to cellulose, with the 2-OH axial instead of equatorial. In seeds (palm, coffee, caraway) a few (

II

M->M->M-^M->M ->

-^M-^M-^ M->M->M->M-^

L_

I

I Gal

11 Gal

12 Gal

I

I

->M ~ - > M - > M - ^ M - > M - > M - >

I

Gal 13 Simulated structures of galactomannan were computed with a chainextending sequence [196]. With substitution affected by the presence or absence of Gal groups on the nearest and next-nearest Man, four situations exist. Both or neither can be substituted, or either can be substituted and the other unsubstituted. Probabilities for substitution were assigned for each of these situations and a polymer generated. This structure was then subjected to simulated hydrolysis following the known action pattern of a p-mannanase and the degree of hydrolysis, amounts and structures of released oligosaccharides and galactose content of the generated polymer found. Probabilities were then adjusted until these parameters matched the experimental values. The distributions of

1142

substituents in carob galactomannan and its hot and cold-water-soluble fractions were quite different from regular, random or block patterns. They could be described as non-regular. There was a high proportion of two contiguous substituents (couplets) and single isolated Gal groups but few sequences of alternate substituted-unsubstituted units. There was a low amount of triplets, an absence of substituted blocks and a higher proportion of unsubstituted blocks of intermediate size than in a random structure. The probabilities of substitution were: nearest-neighbour only substituted > no substitutent > next-nearest-neighbour substituted > both substituted, indicating that steric hindrance was not the only factor. If the nearest-neighbour has just been substituted the enzymes and substrate are in proximity and the probability that the enzymes will continue to react with the same chain is elevated. The degree of substitution of guar galactomannan with two thirds of Man residues substituted makes the application of this procedure difficult, but it appeared to also have a nonregular structure. The non-regular but not random model also fitted the structures of the galactomannans of Ceratonia siliqua, honey locust and Caesalpinia vesicaria, but not of Sophora japonica and Caesalpinia pulcherima [197] whose structures did not show a significant difference from the random model. Those of honey locust and C vesicaria, although both non-random, differed in their distribution of substituents. Guar and Leucaena leucocephala galactomannans have similar Gal contents but the degree of hydrolysis of guar by p-mannanase is half that of L leucocephala. The hydrolysates of the latter indicated the presence of frequent regions of alternating substituted and unsubstituted residues, whereas the former appears to contain a high proportion of doublets. After the Gal content of fenugreek galactomannan had been reduced by hydrolysis with a-galactosidase, the oligosaccharides then produced by Pmannanase contained a high proportion of the branched trisaccharide Man-(Gal)Ma«, indicating that a-galactosidase removed substituents sequentially from one edge of the mannan ribbon-like chain and not in a random manner. Cell particulate preparations containing both mannosyl and galactosyl transferase activities from developing seeds of fenugreek and guar incorporated label from GDP [U-^ C] Man, giving a polymer with the properties of a p(l-4)mannan [190]. No transfer was observed with UDP [U-*^C] Gal, but with labelled UDPGal and GDPMan the product had the properties of a-galacto-P-mannan. The level of substitution by Gal could be varied by supplying saturating amounts of UDPGal and changing the

1143

concentration of GDPMan. Incorporation of Gal and Man was concurrent. Pre-formed Man sequences did not subsequently substitute either with UDPGal alone or mixed with GDPMan. The patterns of change in levels of Gal substitution of developing of seeds of Gleditsia triacanthos and Senna occidentalis differed from that of guar and fenugreek. In the first and second the Gal content decreased at later stages of development, wticreas in guar and fenugreek it remained constant [191, 198]. The first two plants belong to the Caesalpiniodeae, which contain galactomannans with lower levels of Gal than those of the Faboideae to which fenugreek and guar belong. The Caesalpiniodeae are more primitive than the Faboideae. The polysaccharide occurs as a thickening of the secondary wall and those in the Caesalpinioideae have more of the characteristics of structural polymers - low levels of substitution and solubility. The higher levels of Gal in the Faboideae may reflect an evolutionary change to a more soluble material with better properties for a storage polysaccharide [187]. From analysis of the oligomers released by P-mannanase from the galactomannans extracted fi-om G, triacanthos at different stages of development [198] it was considered that the observed decrease in Gal content in late samples was probably not due to hydrolysis by the small amount of a-galactosidase in seeds near maturity. This enzyme sequentially removes Gal groups that lie on one edge of the P-mannan ribbon [196], leading to an absence of doublets, whereas the oligosaccharides produced by depolymerization of the galactomannan of G, triacanthos revealed the polymer had a high level of doublets but not of single substituents [198]. A limiting supply of UDPGal or changes in levels of transferase near maturity provide possible causes of the observed decrease in Gal content. Another study [191] concluded the decrease was due to a-galactosidase action. In S. occidentalis, guar and fenugreek, the ratios of the two biosynthetic transferases were correlated with the Gal content. In the first (with a lower level of Gal) the ratio of mannosyl to galactosyl transferase in extracts was higher than in guar or fenugreek and increased during seed development. In guar and fenugreek the levels of activity of the two transferases were similar and remained constant with development. Isolated, membrane-bound preparations were presented [192] with a mixture of saturating levels of UDPGal and decreasing levels of GDPMan, when a series of galactomannans containing from 14-35% Gal were produced. The Gal contents of fenugreek and guar galactomannans are 48 and 38%. The high level in fenugreek galactomannan does not

1144

allow any structural analysis with p-mannanase and limited information can be obtained from guar. The synthetic polysaccharides, which undergo significant hydrolysis, allowed investigation of their biosynthesis. The polymers were hydrolysed with P-mannanase and the products matched to models derived from simulations of synthesis and hydrolysis. In S. occidentalis with a low GalrMan ratio, a low probability of substitution applied when there was a Gal on the next-nearest-neighbour or on both nearest and next-nearest-neighbours. In guar the probabilities were similar, except for a somewhat higher value if the nearest-neighbour carried a Gal residue; this would produce a preference for doublets. In fenugreek, when the next-nearest-neighbour or both Man residues were substituted,the probabilities were significantly higher. When the most recently added Man residue has just been substituted, bringing the enzyme-membrane complex and terminus of the polymeric substrate into proximity, and steric hindrance is not encountered, further substitution of the next Man residue added would be favoured over dissociation of the enzyme and reaction with another chain. The probabilities for biosynthesis of S. occidentalis galactomannan from mature seeds were similar to those for enzymically synthesized chains. If polymer in the seed undergoes a-galactosidase hydrolysis near maturity this would only be expected if hydrolysis were random. However, the oligosaccharides obtained from p-mannanase hydrolysis of fenugreek galactomannan, in which the Gal content had been decreased by a-galactosidase [196], indicated that hydrolysis was not random but proceeded sequentially along one edge of the ribbon-like mannan chain. (c) P(1'4) Glucomannan and 6a-Galacto-P(l-4) Glucomannan p(l-4) Glucomannan is a secondary wall component (hemi-cellulose) of wood [775, 199-201], and also occurs in tubers, bulbs, seeds, roots and leaves of monocotyledonous plants, such as Liliaceae, Amaryllidaceae and Orchidaceae [181-183, 202]. It may be partly acetylated and this increases solubility in water. Polymers with a small amount (3*

2-D-Rha4NH2al->2

* side chain compounds ^ side chain and main chain components numbers are side chain linkages

^ bracketed

1154

that are found, but much less frequently, are ManNAc, D-Rha, ManNAcA, ManA, D-Rha4NH2, D-Rha2NAc3NFo, as well as monomethyl Man substituted at 0-1, 0-2, 0-3, 0-4 or 0-6. 4,6-Pyruvyl Man derivatives have the asymmetric carbon in the S configuration. Acetyl esters are common. Exocellular polymers generally contain an acidic group - uronic acid, pyruvyl acetal (pyr) or phosphate. In contrast to plant heteropolysaccharides their structures have a regularly repeating unit and this is a consequence of the mode of biosynthesis [69, 256]. The number of sugars in the repeating unit usually ranges from two to eight. The polymers consist of a main chain with most having a repeating unbranched side chain. In some cases two repeating unbranched side chains are attached. These side chains vary in d.p. from one to four residues. Table 2 shows types of Man linkages to other sugars from a survey of 90 polysaccharides. Both a- and P- anomeric linkages are found and Man may be in the main chain or the side chain. Three illustrative examples of repeating structural units are 15,16 and 17. -4Glcpi-4Glcpi3

I al pyr4,6ManP 1 -4GlcAp 1 -2Man6 Ac Xanthomonas campestris extra-cellular polysaccharide

15 -3Mana 1 -3Mana 1 -2Mana 1 -2Mana 1 -2aMana 1 Klebsiella 03 lipopolysaccharide

16 -4FucNAca 1 -3 GlcNAcP 1 -4ManN AcP 1 3 3

I

I

al GalNAc

al Glcal-2Glc

Streptococcus pneumoniae XyxiQ 12A polysaccharide

17 The high number of linkage types of Man, combined with the number of other component sugars [83, 256] and the possible variations in

1155

numbers of sugars and sequence in the repeating oligosaccharide, provide a great diversity of polysaccharide structures. Conformational aspects of a nonasaccharide segment from the polysaccharide of a Bradyrhizobium sp. have been determined by molecular dynamics and n.m.r. spectroscopy [257]. A quite extended main chain with a significant amount of flexibility was indicated. The side chain was highly flexible. A few polysaccharides [82], such as the 0-antigens of Klebsiella 03 and 05 and Pseudomonas diminuta consist of a majority of Man residues. Micrococcus spp. produce an unbranched lipo-a-mannan in the cell wall, which is linked 0-2, 0-3, 0-6 (2:2:1) and esterified with succinic acid. Thermoplasma acidophilum has a lipoglucomannan composed of a repeating tetrasaccharide unit-3Manal-2Manal-4Manal-3Glcal. The cell wall of Mycobacterium smegmata and Streptomyces griseus contains a polymer of a(l-4) linked Man30Me. Mycobacterium tuberculosis and leprae and other Mycobacteria [258, 259] contain a lipo-arabinomannan interspersed in the cell wall. The mannan portion has an a(l-6) mannan chain to which are linked many single Manal~->2 groups. Also attached to 0-2 is a polymer of a(l-5)D-Ara/* chains branched through 0-3 and terminated by D-Ara/pi->2 groups, or with these further substituted by Manpi-^5, Manal-^2 Manpi-^5 or Man al-2 Man pi-»5. The reducing end the a(l-6) mannan chain is linked to wyo-inositol at 0-6, which in turn is linked at 0-2 to a Manal-group and through phosphodiester to glycerol lipid, showing similarities in structure to lipooligosaccharides from Mycobacteria spp. [71, 72] {section 4}. A lipomannan, consisting of the mannan section has also been described. Bacterial polysaccharides are antigenic and provide vaccines [260]. Some are used industrially [7(?6]. One of these, xanthan (15), contains Man. In Proteus mirabilis swarming is facilitated by a colony migration factor, which is a capsular polysaccharide containing ManA [261]. Biosynthesis by a block mechanism is usual [69, 256] although some are made by a regular sequence of addition from nucleoside diphospho sugars. In the block mechanism, the sugars are assembled sequentially as polyprenol diphospho diesters, to form a lipo-oligosaccharide, and then transferred as a block to the growing polymer, leading to a regularly repeating structure. Direct addition from nucleoside diphospho sugars, where it occurs, also leads to regularity, due to the strictness of the transferase enzymes for the donor nucleoside diphospho sugar and the acceptor sugar, as well as for the anomeric linkage produced and the OH in the acceptor to which it is joined [69, 256, 262],

1156 Methods of determination of structure include complete hydrolysis and identification of component sugars (which may be unusual) and any further substituents such as pyruvate or esters; methylation analysis; partial hydrolysis (by acid, acetolysis or enzymically) to oligosaccharides followed by n.m.r. spectroscopy, which has also been applied to whole polymers; modification of uronosyl residues - by reduction prior to or after methylation, and by p-elimination reactions; Smith periodate degradation. N.m.r. spectroscopy of whole polymers avoids problems associated with partial hydrolysis and acetolysis - incompleteness, low yields and differential loss of sugars. Enzymic partial depolymerization [106, 263] which may be by endo-hydrolase or lyase action, provides high yields of oligosaccharide related to the repeating unit and leaves intact acid-sensitive substituents such as pyruvyl acetal, as well as often cleaving at a different glycosidic bond to that split by hydrolysis or acetolysis. Many of the depolymerases are derived from bacteriophages some from other microorganisms - and they may be associated with the production of exo-polysaccharide. (b) Plant gum exudates and mucilages Plant gum exudates [264-267] are formed in response to disease, insect and mechanical damage and physiological stress such as drought. They appear to seal and protect damaged sections. The isolated gums form viscous solutions and find use in pharmaceutical and food preparations. The structures are highly branched, and characterised by the presence of uronic acids (GlcA, GlcA40Me and GalA) and the absence of Glc. They can be grouped into substituted arabinogalactans, glucuronomannans, galacturonorhamnans and glycanoxylans. The chains comprising the branched structure divide into the single basal chain, which contains the reducing-end sugar, those joined to it by glycosidic links - first tier chains - and then additional tiers joined sequentially. Their structures have some resemblance to polymers found normally in plant cells - arabinogalactan, glucuronomannan, pectic substances and xylans. Protein may be covalently linked. A diversity of component sugars, glycosidic linkages and branching increase the difficulty of depolymerisation by the enzymes of pathogens. Due to their highly branched structure, the number of constituent sugars and the limited regularity of structural elements, specific structures cannot be assigned - only generalised formulae. Methods of determining structural elements include methylation analysis,

1157 Smith periodate degradation, partial acidic hydrolysis, reduction of uronosyl residues, acetolysis, as well as base elimination reactions and other degradation procedures that can be applied to uronosyl residues, and n.m.r. spectroscopy [261]. Increasing availability of purified enzymes with well-defined action patterns should assist future studies [106, 262]. Structural features can also be recognised by monoclonal antibodies [268]. Table 3. Chain Structure of Gum Ghatti Chain position Basai First tier

Second tier

Structures (4GlcApi-2Manal)„ L-Ara/1L- Ara/1-2 (or 3 or 5) L-Ara/GicAal-(6Galpl)„-3L-Ara Gaipi-(6Gaipi)„-3L-Ara L-Ara/1L-Ara/1 -2 (or 3 or 5) L-Ara/1 -

Linkage to lower positioned chain

— -^6 Man ->6 Man ->3 Man ^ 3 Man ->3Gal ->3Gal

A number of plant gums contain Man, Anogeissus (gum ghatti), Combretum, Encephalartos (cycad) spp., Chohsia speciosa [269] and Grevillea robusta [270] have been extensively examined, but Man also occurs in Albizia, Virgilia, Hakea, Brabeium and Prunus spp. Determination of Man content in 16 species of cycads gave considerable variation [271] but it was always a minor component ( units, which can be further extended through 0-4 of the GlcA residues by (-L-Rha l-^)n

1158

where n=l-3, or branched at 0-3 of Gal residues by L-Ara groups. Some L-Rha residues are 2,4 disubstituted with L-Rha units. Encephalartos friderici-guilielmi gum [273] also contained P(l-3) linked Gal residues, L-Rha and L-Rha30Me residues and the same basal chain. An exudate of Ceratozamia spinosa (cycad) also had this basal chain [274] with L-Rha, L-Rha30Me or L-Ara/linked to 0-3 of some GlcA residues. The 0-4 of Man was substituted with GlcApi-. The 0-4 of these GlcA residues were substituted with L-Rhaal-, L-Arayfecl- or Xyipi-. 0-3 of the same GlcA residues could be substituted with these sugars to give a second tier. Plant mucilages are associated with normal metabolism and are seed components [275'] but are also found in fruits, leaves and inner bark. They are used medicinally. Polymers with a repeating 4GlcApl-2Manal unit, in which the amount of this aldobiuronosyl residue is higher than in gums, and sufficient in most to be described as a main chain, have been detected. A polysaccharide secreted by leaves of Drosera capensis contained 64% of Man plus GlcA with L-Ara/'and Xyl groups attached to 0-3 of GlcA, and Gal groups to 0-3 of Man residues [276]. Previously, an extra-cellular polymer from suspension-cultured cells of tobacco {N. tabacum) containing 49% of Man plus GlcA, with L - A r a / a l - groups linked to 0-3 of some GlcA and L-Ara/pi- joined to 0-3 of some Man residues [277, 278] had been described. The polysaccharide from the stem pith of Actinidia deliciosa consisted of less aldobiuronic acid (20%) [279]. It had (1-3) Gal chains joined to 0-3 of Man and L-Ara/to 0-3 of GlcA. The (1-3) Gal chains can be terminated with D-Aral-5-L-Ara/l->3 or carry a second tier of L-Fucl->2 (or 1-^6), L-Ara/1~>2 (or 1~>6) or Gal 1^6. The leaves of Dicerocaryum zanguebaricum - an African plant - have specialised mucilagenous hairs. Water extraction affords a polysaccharide [280] with a simple pattern of substitution of the glucuronomannan chain. This main chain is substituted at the 0-3 of Man and some GlcA units with single Xyipi- or Galal groups. The carboxylreduced polymer contained 56% of Man plus Glc. 8. Synthesis of Mannans and Manno-oligomers by in vitro Methods. The stereo-regular, chemical synthesis of a-manno-pyranans with (1-2), (1-3) or (1-6) linkages by a mechanism of chain growth from monomeric derivatives, as well as a singly branched a(l-6) polymer from a disaccharide derivative has been reviewed [209, 281] and the synthesis of

1159 heteropolymers discussed [282]. Oligomers with specific linkages have been prepared by sequential addition of appropriately protected monomers [283, 284] and also by enzymic addition with glycosyl transferases and nucleoside diphospho sugar. [J36, 262]. Block addition can also be used. The oligomers provide the glycan segment for the synthesis of neoglycoconjugates [96, 283] which consist of carbohydrate joined synthetically to other molecules such as proteins, lipids and synthetic polymers. The carbohydrate can also be derived from naturally occurring glycoconjugates. These compounds find a range of uses in biochemical separations and purification, assays and the study of cellular processes. An unbranched a(l-6) mannan was synthesized from 1,6-anhydro-Dmannopyranose, with benzyl ether protecting groups on 2, 3 and 4-OH, by polymerisation at -78° initiated by phosphorus pentafluoride, followed by debenzylation with Na in liquid NH3. Results from periodate oxidation and optical rotation were consistent with an a(l-6) structure, and from the viscosity average d.p. values of up to about 1,000 were estimated [285]. The polymer cross-reacted with an a(l-6) mannan from Trichophyton rubrum. Polymerising in the presence of dimethoxymethane reduced the average d.p. and allowed the synthesis of oligomers [286]. Cationic catalysts polymerised l,3-anhydro-2,4,6-tri-0-benzyl D-mannopyranose to a product with an average d.p. of 60-90, which, after debenzylation, was shown by n.m.r. and infra-red spectroscopy to be virtually completely an a(l-3) mannan with an average d.p. of 30-60 [287]. Polymerisation of the benzyl protected 1,2-anhydro sugar with potassium tert-butoxide complexed with a crown ether, followed by debenzylation, gave a stereoregular product with a low average d.p. [288]. Starting with a benzyl protected anhydro disaccharide - l,6-anhydro-2,3-di-0-benzyl-4(2,3,4,6-tetra-0-benzyl-a-D-mannopyranose)-p-D-mannopyranose - and polymerising at -60° with PF5, followed by debenzylation, gave an a(l-6) mannan with single Man groups on the 4-OH. The structure was supported by n.m.r. spectroscopy. The average d.p. was much lower (56) than that obtained for the unbranched a(l-6) linked polymer [289]. P(l4) Mannan (from ivory nut) and a glucomannan (konjac) have been branched by reacting them with the 3,4,6 acetylated 1,2-ethylorthoacetate of P-D-glucopyranose in the presence of 2,6-dimethyl-pyridine perchlorate [290]. Unbranched hetero-polysaccharides have been prepared by condensation of hetero-oligomers with a l,2-0-(lcyanoethylidene) group on the reducing-end sugar, a triphenylmethyl

1160 ether at the reacting OH, and non-reacting groups protected with benzyl (for OH) or as phthalimide (for NH2). Reaction of the protected repeating tetrasaccharide unit of Shigella flexneri with triphenylmethylium perchlorate in dichloromethane, followed by removal of the protecting groups gave a product with an average d.p. of about 40. Its n.m.r. spectrum was identical to that of the native 0-antigenic polysaccharide [291]. Earlier, polymerisation of the cyanoethylidene derivative of Manpi-4-L-Rhaal-3Ga/, in which the 6-OH of Man was substituted as a triphenylmethyl ether and all other free hydroxyls were acetylated gave, after removal of protecting groups, a fraction with an estimated d.p. of 30 [292]. The P(l-6) linked polymer from this trisaccharide is the Oantigenic polysaccharide of Salmonella newington, and in haemagglutin tests the synthetic polymer gave a positive reaction in which it was eightfold times as active as the trisaccharide. The synthetic polymer in which the anomeric linkage of Man was a was practically inactive. Unbranched a-D-mannopyranans with (1-6) and (1-4) linkages with an average d.p. of 10 and 15 have been prepared by this method [283]. A synthesis of a lipid-mannodisaccharide-phosphate, P-6Manal2Manal-0-octyl, a partial structure of phosphorylated glycoproteins that are targets for the Man 6-P transporter, consisted of the following sequence [293]. Octanol was reacted with 2-0-acetyl-3,4,6-tri-0-benzyla-D-mannosyl chloride. Removal of acetyl and glycosylation with the ethyl thioglycoside of 6-0-acety 1-2,3,4-tri-O-benzyl-a-D-mannopyranose gave a disaccharide, which, after removal of acetyl, was phosphorylated with chlorodiphenylphosphate. Hydrogenolysis gave the dimannosephosphate. The a(l-3), a(l-6) branched trisaccharide (linked to lipid) which is part of the common core of glycoproteins has been conveniently prepared [294] by reacting unprotected mannoside-0-lipid with 2 moles of the 2,3,4,6-tetra-O-benzoyl-l-trichloroacetamide, followed by debenzoylation. The unbranched pentasaccharide segment (containing DRha) of a antigenic glycopeptidolipid of Mycobacterium arium was made by block synthesis with the thioether of the protected disaccharide and the trisaccharide with one available OH: the promoter was N-iodosuccinimide and trifluoromethanesulphonic acid [295]. Synthesis of the pentasaccharide Mana 1 -2[Mana 1 -6]ManP 1 -4[GlcNAcp 1 -6]GlcNAcp 1 0-/7-methoxyphenyl and comparison of the n.m.r. spectrum with that of the core structure from a glycan of Chinese hamster ovary suggested that the latter had a GlcNAc (1-6) linked branch with a novel a-linkage [296].

1161 The P-Man residue was joined to the 4-OH of GlcNAc with silver alumina-silicate as promoter. The enzymic synthesis of oligosaccharides avoids the need for the addition and removal of protecting groups and, with Man residues, overcomes the difficulty of synthesising the (J-configuration. The core trisaccharide of Salmonella group Ei has been made in a reaction of GDPMan with L-Rhaal-3Ga/, catalysed by a recombinant P(l-4) mannosyl transferase. Reaction was rapid and the yield was high [297]. Glycoprotein oligosaccharides have also been prepared by removal of sugars by glycosidase hydrolysis of native oligomers [137]. Sequential addition of specifically substituted monosaccharides has given a tri-antennary undecasaccharide with the structure of a glycan chain of the N-linked glycoprotein of calf thyroglobulin (18). Synthesis started from the trisaccharide Manpi-4GlcNH2 ^\-4GlcNH2 [298]. Mal-2M al 6 Mal-2Mal-3M al

I 6 Ma 1-2 Ma 1-2 M a 1-3 MP 1-4 GlcNAcpi-^G/cA^^ic 18 The NH2 groups were protected as phthalimides, and all OH groups except the 3 and 6 on the terminal Man, as benzyl ethers. Selective protection of the more reactive primary 6-OH with chloracetyl chloride allowed glycosylation of 3-OH. Alternatively, the 6-OH could be selectively silylated with a bulky tert-butyl dimethyl silyl group. Glycosylation with the 1-trichloro-acetamide of 2-0-acetyl-3,4,5-tri-0benzyl-a-D-mannoside, with BF3 in ether as promoter, gave the tetrasaccharide with the first Man residue of the trimannosyl antenna in posifion. Removal of the silyl group with HF and reaction with 1-chloro3,6-di-O-acety 1-2,4-di-O-benzyl-a-D-mannoside added the second branched Man residue. De-acetylation and glycosylation with 3 molecules of 1 -chloro-2-0-acetyl-2,4,6-tri-0-benzyl-a-D-mannoside gave an octasaccharide and repetifion of de-acetylation and glycosylation gave the

1162 protected undecasaccharide. Hydrazine hydrate in ethanol and then acetic anhydride converted phthalimide into N-acetyl and hydrolysed 0-acetyl. Hydrogenolysis then gave an undecasaccharide, for which the n.m.r. and mass spectra were in agreement with those of the native glycan. A procedure for the rapid synthesis of Man glycans that removes the need for the unmasking of protecting groups between glycosylation steps, by utilising the difference in reactivity of seleno- and thio-glycosidic groups when reacted with N-iodosuccinimide and trifluoromethanesulphonic acid (combined with protection of the 3 and 4 OH groups as the cyclohexane di-acetal) has been described [299]. A nona-mannosaccharide containing a(l-2), (1-3) and (1-6) linkages, which is a glycan chain found in the viral coat of HIV-1, has been prepared. The synthesis involved only one operation with a protecting group and several glycosylations could be combined, removing the need for the isolation of intermediates. ABBREVIATIONS Cer Dol d.p. Fo P PMI Pyr

= = = = = = =

ceramide dolichyl degree of polymerisation formyl phosphate phosphomannoisomerase pyruvyl acetal

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[237] Parker, A.; Leiimousin, D.; Miniou, C; Boulenguer, P.; Carbohydr. Res., 1995, 272,91-96. [238] Ridout, M.J.; Cairns, P.; Brownsey, G.J.; Morris, V.J.; Carbohydr. Res., 1998, 309, 375-379. [239] Newman, R.H.; Hemmingson, J.A.; Carbohydr. Polym. 1998, 36, \61A12, [240] Mishima, T.; Hisamatsu, M.; York, W.S.; Teranishi, K.; Yamada, T.; Carbohydr. Res., 1998,56/(5,389-395. [241] Whitney, S.E.C.; Brigham, J.E.; Darke, A.H.; Reid, J.S.G.; Gidley, M.J.; Carbohydr. Res., 1998, 307, 299-309. [242] Percival, E.; McDowell, R.H. In Methods in Plant Biochemistry, Dey, P.M., Ed.; Academic Press: London, 1990; Vol. 2, pp. 523-547. [243] Shankar, S.; Ye, R.W.; Schlictman, D.; Chakrabarty, A.M.; Adv. Enzymol, 1995, 76/, 221-255. [244] Skjak-Br^k, G.; Biochem. Soc. Trans., 1992, 20, ll-^'i. [245] 0stgaard, K.; Larsen, B.; Carbohydr. Res., 1993, 246, 229-241. [246] Heyraud, A.; Colin-Morel, P.; Girond, S.; Richard, C; Kloareg, B.; Carbohydr. Res., 1996, 291, 115-126. [247] Ochi, Y.; Takeuchi, T.; Murata, K.; Kawabata, Y.; Kusakabe, I.; Biosci. Biotech. Biochem., 1995, 59, 1560-1561. [248] Heyraud, A.; Colin-Morel, P.; Gey, C; Chavagnat, F.; Guinard, M.; Wallach, J.; Carbohydr. Res., 1998, 308, 417-422. [249] Panikkar, R.; Brasch, D.J.; Carbohydr, Res., 1997, 300, 229-238. [250] Draget, K.I.; Skj^k-Brask, G.; Smidsrod, O.; Carbohydr. Polym., 1994, 25, 31-38. [251] Miller, I.J.; Phytochemistry, 1996, 41, 1315-1317. [252] Simpson, J.A.; Smith, S.E.; Dean, R.T.; Biochem. Mol. Biol. Inter., 1993, 30, 1021-1034. [253] Franklin, M.J.; Chitnis, C.E.; Gacesa, P.; Sonesson, A.; White, D.C.; Ohman, D.E.; J. BacterioL, 1994, 176, 1821-1830. [254] Quillet, M.; de Lestang-Bremond, G.; Phytochemistry, 1985, 24, 43-45. [255] H0idal, H.K.; Ertesv^g, H.; Skj^k-Brask, G.; Stokke, B.T.; Valla, S.; J. Biol. Chem., 1999, 274, 12316-12322. [256] Shibaev, V.N.; Adv. Carbohydr. Chem. Biochem., 1986, 44, 277-339. [257] Poveda, A.; Santamaria, M.; Bernabe, M.; Prieto, A.; Bruix, M.; Corzo, J.; y\m€v\QZ-BdivhQro,].', Carbohydr. Res., \991, 304,209-2X1. [258] Brennan, P.J.; Nikaido, H.; Ann. Rev. Biochem., 1995, 64, 29-63. [259] Chatterjee, D.; Lowell, K.; Rivoire, B.; McNeil, M.R.; Brennan, P.J.; J. Biol Chem., 1992, 267, 6234-6239. [260] Bishop, C.T.; Jennings, H.J. In The Polysaccharides; Aspinall, G.O., Ed.; Academic Press: New York, 1982; Vol. 1, pp. 291-330. [261] Rahman, M.M.; Guard-Petter, J.; Asokan, K.; Hughes, C; Carlson, R.W.; J. Biol. Chem., 1999,27^,22993-22998. [262] Matheson, N.K.; McCleary, B.V. \x\ The Polysaccharides', As^m?i\\,G.O., Ed.; Academic Press: Orlando, Florida, 1985, Vol. 3, pp. 1-195. [263] Sutherland, I.W.; Carbohydr. Polym., 1999, 55, 319-328. [264] Aspinall, G.O.; Adv. Carbohydr. Chem. Biochem., 1969, 24, 333-379. [265] Stephen, A.M. In The Polysaccharides', Aspinall, G.O., Ed.; Academic Press: New York, 1983; Vol. 2, pp. 97-193.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 26 © 2002 Elsevier Science B.V. All rights reserved.

1175

THE CHEMISTRY OF LITHISTID SPONGE: A SPECTACULAR SOURCE OF NEW METABOLITES M. VALERIA D'AURIA, ANGELA ZAMPELLA AND FRANCO ZOLLO* Dipartimento di Chimica delle Sostanze NaturalU Via D. Montesano 49 80131 Napolh Italy ABSTRACT: Among marine invertebrates, sponges belonging to the lithistid order are certainly the most spectacular source of many classes of compounds, some of them showing interesting pharmacological activities. The extreme diversity of lithistid sponge metabolites, macrolides and polyketides, cyclic and linear peptides, alkaloids, pigments and novel sterols, as well as their structural similarity to those reported from microorganisms, gave well founded the hypothesis that they may be produced by symbiotic bacteria and miaoalgae. The purpose of this review is to offer a comprensive survey of the chemistry of lithistid sponges with a particular emphasis to the pharmacological properties and to the mechanisms of action of their metabolites, and to report the total syntheses of representative macrolides and peptides isolated from the sponges belonging to this so fascinating order.

INTRODUCTION Some of the most exciting marine metabolites from a structural point of view have been isolated in recent years from sponges of order Lithistida: Theonella, Discodermia, Jereicopsis, Neosiphonia, Reidispongia, Callipelta, Corallistes, Aciculites and Microscleroderma. These natural products, encompassing a wide range of molecular diversity, such as linear and cyclic peptides, macrolides, lipids, alkaloids and steroids, possess a variety of pharmacological properties, including insecticide, antimicrobial, antiviral, antitumor and tumour promotive, antiinflammatory and immunosuppressive. Some of them represent lead compounds in drug design and in the study of human biochemical pathways. The presence of an extreme diversity of bioactive metabolites appears surprising taking into account that lithistid sponges have not only hard bodies, but also a shield of fused siliceous spicules which offers good physical protection against predators, and so they don't need chemical defence. The extreme diversity of Uthistid sponge metabolites as well as the resemblance between these metabolites and those isolated from micro-organisms made well founded the hypothesis of their symbiotic origin. To date, two excellent reviews have been published on the chemical composition of lithistid sponges [1,2], The 1993 review "Bioactive

1176 Sponge peptides" by Fusetani and Matsunaga contains a detailed description of the peptide derivatives isolated from lithistid sponges whereas the recent review "Lithistid sponges: star performers or hosts to stars" by Bewley and Faulkner reports a summary of the chemistry of lithistid sponges, with a particular emphasis to the relationship between these sponges and their symbionts. Our review constrains its scope to a more thorough discussion of the chemistry, total synthesis and biological activity of secondary metabolites from lithistid sponges. The material is organised in two main sections. A first section is devoted to the structure and the chemical distribution of the secondary metaboUtes isolated from each genus belonging to the Uthistid order, A second section will focus on the total synthesis of representative metaboUtes from Uthistid sponges. CHEMICAL STRUCTURES In this section the secondary metaboUtes from Uthistid sponges are described foUowing the taxonomic classification of extant Uthistid sponges reported by Faulkner [2]. Metabolites from Theonella sp. and Theonella swinhoei Lithistid sponges identified as Theonella sp. are often superficiaUy similar to Theonella swinhoei except for the orange colour of their interior tissue due to the presence of aurantoside A (1) and related polyenes.

loH

OH

O/,

't.

Aurantoside A (1)

'y—o OH

Sterols The pioneering paper on metaboUtes from sponges of Theonella genus appeared in Uterature in 1981 deaUng with the isolation of two new and unusual sterols with the unprecedented 4-methylene nucleus, conicasterol (2) and theoneUasterol (3) [3]. Subsequently, the 3-keto derivatives, conicasterone (4) and theoneUasterone (5), together with a Diels Alder type dimeric steroid, named bistheoneUasterone (6), were reported from the Okinawan marine sponge Theonella swinhoei [4]. It is noteworthy that

1177

Conicasterone (4)

o

Theonellasterone (5)

.-

Bistheonellasterone (6)

Bisconicasterone (7)

5 and 6 were seen under an optical microscope as crystals deposited in the tissue of fresh marine sponge. In 1994 bisconicasterone (7) was isolated from a Theonella swinhoei specimen collected off Hachijo-jima Island and its absolute stereochemistry determined by X-ray crystallography [5]. It is intriguing that no trace of theonellasterone (5) and its bis-derivative 6 has been found in this collection of Theonella swinhoei in contrast with

1178

the finding of Kitagawa who reported theonellasterone (5) as a major component of the same species collected at Okinawa [4]. This suggests a site dependence of the ingredients of marine organisms belonging to the same species. A further investigation of this specimen of Theonella swinhoei afforded in the isolation of seven oxygenated conicasterol derivatives along with the unusual sterol 8 with 7,8-epoxy functionaUty [6].

HO

R= f Swinhosterol A (9) R= I Swinhosterol B (10)

^0CH3

Swinhosterol C (11)

During the investigation of an Okinawan T. swinhoei, it was found that the apolar extract contained three new steroids, swinhosterols A-C (9-11) [7] which structures were determined by spectroscopic investigation. Compounds 9 and 10 combine the unusual 4-methylene and seco features. Cyclopeptides Small cyclopeptides, particularly rich in non-conventional aminoacids, are very promising and interesting to medicinal chemistry because of their unique features. In fact the presence of non-ribosomal aminoacids, including D-series and N-alkylated versions of the natural ones as well as strongly structural modified aminoacids, produces pronounced effects on the pharmacological activities of these metabolites. For example, Nalkylation and the lack of N and C termini enhance the hydrophobicity of depsipeptides, thus determining a more facile crossing of biological membranes and an improved stability to enzymatic degradation. In

1179 addition N-alkyl residues exhibit reduced preferences to form the trans conformation of the amide bond leading to biologically important P-tum structures. Cyclization reduces peptide conformational flexibility that results in a higher receptor binding affmity and often offers the possibility to determine their three dimensional structures. All these features make cyclopeptides promising lead compounds in drug discovery. In 1990 Fusetani et al. isolated from different collections of the same

A^C}~°"

HN—C

NH

>=0

°

)—NH

y

I

Cyclotheonamide A (12) R=H Cyclotheonamide B (13) R=Me

Specimens of a Theonella sponge collected off Hachijo-jima Island, a mixture of aurantoside A (1) and B together with an important family of cyclic peptides, named cyclotheonamides [8]. Cyclotheonamide A (12) is a cycUc pentapeptide containing the vinylogous tyrosine (V-Tyr), the a-

OH

V

NHCHO

HgN

^N

H2N NH

Cyclotheonamide C (14)

NH

Cyclotheonamide D (15)

%HCHO

1180

ketohomoarginine (k-Arg) and a diaminopropionic acid as unusual aminoacid residues. This peptide is a potent serine protease (thrombin and trypsin) inhibitor and its mechanism of action has been clarified by X-ray study of the complex with human a-trombin. This study disclosed that cyclotheonamide A (12) binds to the catalytic triad of the enzyme by forming a covalent bond between its a-keto group and the hydroxyl group of a serine residue of one of the triad residues [9]. Cyclotheonamide B (13) is the acetamido derivative of 12 whereas cyclotheonamide C (14) [10] possesses an additional insaturation in the V-Tyr residue. The aminoacid constitution of cyclotheonamide D (15) [10] is quite different from cyclotheonamide A (12) with a Leu residue in place of D-Phe. From a morphologically different sample of Theonella swinhoei, cyclotheonamide E (16) has been isolated [10]. This latter includes D-Ile and a phenylacetyl-Ala amide groups in place of D-Phe and formyl groups in 12. Recently, a recollection of Theonella sp. off Tanegashima Island, afforded cyclotheonamides E2 (17) and E3 (18) [11], closely related to 16 from which they differ in the N-acyl group of the alanyl side chain. These structural modifications affect the biological activity. Infact the side chain modified cyclotheonamides 16, 17 and 18 showed an enhanced specificity toward thrombin due to the bulky acylated alanyl R

Y^

Cyclotheonamide E (16)

Cyclotheonamide E2 (17)

HgN

IT

rr

Cyclotheonamide E3 (18)

NH

residue that increases the hydrophobic interaction with the enzyme. On the other hand the properties of the aminoacid residues present in cyclotheonamides A-D (12-15) gave more favourable interactions with trypsin than trombin [12]. Immediately after the publication of the first paper on the isolation of cyclotheonamides A (12) and B, these compounds attracted considerable synthetic interest. The first total synthesis of cyclotheonamide B (13) was reported by Schreiber and Hagihara in 1992 and led to a stereochemical reassignment of the stereocenters in V-Tyr and k-Arg residues for which

1181 the (S) configuration was assigned [13]. Further total syntheses of cyclotheonamides were reported by Wipf [14], Shioiri [15], Maryanoff [16], and Ottenheijm [17]. Recent reviews provide a comprensive survey on the syntheses of cyclotheonamides in particular [18] and on the coupling methods for the incorporation of noncoded aminoacids in peptides in general [19] thus these topics will not be discussed in the Total Syntheses Section. The presence of a-ketoaminoacids appears to be the main feature of the peptides isolated from sponges of the genus Theonella. The cytotoxic orbiculamide A (19) (IC50 4.7 |ig/ml), isolated from the same marine sponge, Theonella sp., that contained cyclotheonamides, represents another example of cyclic peptide containing three new aminoacid residues, theoleucine, theonsJanine and 2-bromo-5-hydroxytryptophan [20].

H

11

L

'^'^V V

NH

Orbiculamide A (19)

From a structural point of view, keramamides B-H (20-26) and J (27), a series of novel cyclopeptides isolated from Okinawan marine sponges Theonella sp. [21-23], are closely related to orbiculamide A (19). Keramamides B-E (20-23) [21,22] are composed by a cyclic moiety and a side chain. The cycUc hexapeptide portion contained three modified aminoacid residues linked each other to form a segment containing an a,P-unsaturated amide group conjugated with an oxazole as well as a aketo-P-aminoacid moiety constituting a homoleucine-like unit. In keramamides F-G (24-25) [22,23] and H-J (26-27) [23] a residue of Omethylseryl thiazole replaces the alanyl (or homoalanyl) oxazole present in keramamide B-E (20-23). Keramamides B-D (20-22) inhibited the superoxide generation induced by the chemiotactic peptide, N-formylMet-Leu-Phe whereas keramamides E (23) and F (24) showed cytotoxicity against human epidermoid carcinoma cells (IC50 of 1.6 and 1.4 |Ltg/ml, respectively) and against murine lymphoma L1210 with IC50 values of 1.55 and 2.0 |LLg/ml respectively. Keramamides G-J (25-27)

1182 showed weak cytotoxicity (IC50 >10 |xg/ml). Recently, the total synthesis of a molecule possessing the structure proposed for keramamide J (27) OH =

H

Keramamide B (20) Keramamide C (21) Keramamide D (22) Keramamide E (23)

n

-^

^

HN

R,

Rj=CrT2CH3 R2=Crl2CH3 Rj=CH2CH3 R2=CH2CH3 R2=Crl3 R2=CH2Cri3 Rj=Cri3 R2=CH2Crl2Cri3

was reported [24]. Spectroscopic data indicated that the synthetic compound is not the same compound reported by Kobayashi and coworkers [23]. A detailed analysis of ^H and ^^C NMR data would suggest that the two compounds are epimeric at the homoleucine chiral Ca.

.0CH3

"

Keramamide F (24) *S Keramamide G (25) *R

Keramamide A (29) was quite different in the aminoacid constituents and its most important feature was the presence of an ureido bond consisting of the a-NH of the lysine and the a-NH of the phenylalanine

1183 residues [25]. Keramamide A (29) showed no cytotoxicity against various human and murine carcinoma cells whereas exhibited inhibitor activity

.0CH3

Keramamide H (26) Keramamide J (27) Keramamide K (28)

Ri=Br, R2=0H, R3=H Ri=R2=R3=H Ri=R2=H, R3=CH3

against sarcoplasmic reticulum Ca^"^"ATPase (IC50 3 x 10"^ mol dm'^). Further investigation on the extracts of the Okinawan Theonella sponge resulted in the isolation of two new keramamide derivatives, K (28) and L (30) [26]. Keramamide K (28), closely related to 27, is a new

Me

COOH

Keramamide A (29) R=OH Keramamide L (30) R=H

thiazole containing cyclic peptide with the rare 1-Me-Trp residue while keramamide L (30) is the first peptide with a MeCtrp residue from natural origin. These compounds are cytotoxic in vitro against L1210 murine leukaemia cells (IC50 0.72 and 0.46 ^g/ml, respectively) and KB cells (IC50 0.42 and 0.90 |Lig/ml, respectively).

1184

Other cyclic peptides including konbamide (31), from the keramamide derivatives containing Theonella[21], and mozamides A (32) and B (33), from a Theonellid collected in the southern Mozambique [28], incorporate an ureido linkage. In the cyclic hexapeptides 32 and 33 an

Me

X 'NH

COOH

Konbamide (31)

isoleucine residue is joined to the a-amino group of a lysine giving the ureido moiety. In the konbamide's side chain the leucine takes isoleucine's place. Although keramamide A (29), mozamides (32-33) and konbamide (31) are structurally related, only 31 showed a calmodulin antagonistic activity in mammalian cells. Me I

COOH

Mozamide A (32) R=H Mozamide B (33) R=Me

The cytotoxic cyclic peptide, oriamide (34), containing the new 4propenoyl-2-tyrosylthiazole unit, has been isolated from the blue marine sponge, Theonella sp., collected in Sodwana Bay [29]. This unusual aminoacid is reminiscent of the thiazole moiety early reported as a constituent of keramamide F (24) [22]. In 1989, Fusetani group reported the isolation and the complete structure of theonellamide F (35), a novel antifungal peptide, from a

1185 OH

OH

I

O

^

/

^

'^^^ NH

\ NaOgS—^

/

-OH

]\

Oriamide (34)

Theonella sp. collected off Hachijo-jima Island [30]. Theonellamide F (35) is a bicyclic peptide containing twelve aminoacids. In this cyclic peptide a dehydroalanine residue is masked by intramolecular Michael addition, giving rise to the elaborate and unprecedented histidino-alanine bridge. Further separation of the antifungal fraction of the sponge extract afforded five related peptides, theonellamides A-E [31]. Theonellamide A HO. HOgC HN

AAA A L " T H

/ ^OH

O

Theonellamide F (35) Ri=R2=H, R3=R4=Br Theonellamide B (36) R^OH, R2=Me, R3=Br, R4=H Theonellamide C (37) Ri=R2=R3=H, R4=Br

(38), B (36) and C (37) are closely related to 35, from which they differ in three aminoacid residues. Additionally, 38 bears a p-D-galactose linked to the free imidazole nitrogen. Theonellamide D (39) and E (40) are the P-Larabinoside and the P-D-galactoside of 35, respectively. Theonellamides A-F were moderately cytotoxic against P388 murine leukaemia cells with IC50 values of 5.0, 1.7, 2.5, 1.7, 0.9 and 2.7 |Lig/ml, respectively. Moreover, theonellamide F (35) inhibited growth of various pathogenic

1186 fiingi at concentrations of 3-12 |Lig/nil. Indeed theonegramide (41), isolated from a Theonella swinhoei collected at Antolang, Negros Island, the FiMppines, represents the first glycopeptide from lithistid sponge to be fiiUy characterized [32]. The structure of this antifungal bicyclic glycopeptide closely resembles those reported for 35. HO

0°-^' H

l/^OH

Theonellamide A (38) RpOH, R2=Me, R3=H Theonellamide D (39) Ri=R2=H, R3=Br Theonellamide E (40) Ri=R2=H, R3=Br O

HO.

o ^V^''"r^N

•02C

?

X J

NH

/ ^ * ^ \ ^

X=p.D-Gal X=P-L-Ara X=p-D-Gal

L ^

\-'^^

yj ,

°

H2NOC

Theonegramide (41) X=3-D-Ara Theopalauamide (42) X=p-D-Gal

During their studies on symbiotic filamentous eubacteria found in the interior tissue of Theonella swhinoei from Palau, Faulkner et al. isolated a bicycUc glycopeptide, theopalauamide (42) [33]. Its molecular formula, deduced by HR mass measurements, differs from that of theonegramide

1187 (41) by addition of CH2O in agreement with the replacement of the pentose sugar (D-arabinose) in 41 by a hexose in theopalauamide (42). The sugar was identified as galactose by NMR analysis on the natural product whereas its absolute stereochemistry as D was determined by analysis of the hydrolysis products by chiral GC-MS. However the structure determination of 42 was complicated for the presence of a minor peptide, isotheopalauamide later identified as a stable conformational isomer occurring during the acid isolation procedure [33]. Both 42 and its isomer inhibited the growth of Candida albicans with a 10 ^,g/disk and a 50 ^ig/disk dose, respectively. The butanolic extract of a sponge of the genus Theonella, collected off Perth, Australia, was found to inhibit the binding of the ligand [^^^I]IL-ip to the intact receptor, the EL4.6.1 cells. Thus, the bioassay-guided purification of the active components was undertaken and the perthamide B (43) was isolated [34]. Perthamide B is a cyclic octapeptide that showed moderate activity in the above binding assay but this activity

could not be differentiated from the toxicity of the compound. During a period of five years, Kitagawa group reported the isolation and the structural elucidation of five cyclic depsipeptides, the theonellapeptolides, from an Okinawan collection of Theonella swinhoei [35]. Theonellapeptolide la, lb, Ic, Id, le (44-48) are tridecapeptide lactones particularly rich in N-methyl and D-aminoacids. In these peptides the N-terminus is protected with a methoxyacetyl group and the C-terminus is connected through a lactone linkage to the p-hydroxyl group of a threonine residue. Among these five peptides, 47 has iontransport activities for Na"", K"", and Ca^"" ions, 48 exhibited iontransporting activities for Na"^ and K"^ ions across human erythrocyte

1188 membranes. Theonellapeptolides 45-48 showed moderate cytotoxicity against L1210 cells (IC50 16, 1.3, 2.4 and 1.4 |ig/ml, respectively). Following the characterisation of theonellapeptolide la-Ie (44-48), another new tridecapeptide lactone, theonellapeptolide lid (49), was isolated from the Okinawan sponge Theonella swhinoei. From NMR and mass evidences, the structure of 49 has been determined as an L-Ala

R4

TheonellapeptoUde la (44) RpOCHa, R2=H, R3=CH3, R4=H, R5=CH3, R6=CH3 TheonellapeptoUde lb (45) RPOCH3, R2=H, R3=H, R4=CH3, R5=CH3, R6=CH3 TheonellapeptoUde Ic (46) Ri=OCH3, R2=H, R3=CH3, R4=CH3, R5=CH3, R^=H TheonellapeptoUde Id (47) Ri=OCH3, R2=H, R3=CH3, R4=CH3, R5=CH3, R^=CH3 TheoneUapeptoUde le (48) Ri=OCH3, R2=CH3, R3=CH3, R4=CH3, R5=CH3, R^=CH3 TheoneUapeptoUde lid (49) RPOCH3, R2=H, R3=CH3, R4=CH3, R5=H, R6=CH3 50 Ri=SOCH3, R2=H, R3=CH3, R4=CH3, R5=CH3, R6=CH3 51 R2=H, R2=CH3, R3=CH3, R4=CH3, R5=CH3, R^=CH3

analogue of theonellapeptoUde Id (47), which possesses L-MeAla as the tenth aminoacid from the N-terminus [36]. Despite their strong structural similarity, surprisingly, theonellaptoUde lid (49) didn't show Ca^^ ion binding and ion-transport activities. Infact, it seems that the lack of NMeAla produces a significant change in the three-dimensional peptide conformation, responsible for the biological activity mentioned above. TheonellapeptoUde lid (49) prevented fertilisation of sea urchin Hemicentrotus pulcherrimus at a concentration of 25 ^ig/ml while has no effects on early embryonic development of fertilised eggs up to the gastrula stage. This remarkable effect is restricted to 49 whereas 47 did not affect fertilisation of sea urchin gametes even at 100 ^g/ml. Investigation of another Theonella sp., collected off Kerama Island, Okinawa, resulted in the isolation of two new theonellapeptolides (50-51)

1189 [37]. These compounds are new congeners of theonellapeptolides and possess a methylsulfinylacetyl and an acetyl group, respectively, at their N-terminus, replacing the methoxyacetyl group found in all theonellapeptolides previously isolated [35,36]. Compounds 50-51 showed antimicrobial activity against some Gram-positive bacteria and against fungi with MIC in the range 8-60 |Lig/ml. It should be noted that in 1998 a new peptide lactone, theonellapeptoUde Ille (52) has been isolated

I

O

II

Ma

r

II

I

A"••'X.Jl heonellaDeDtolide Ille (52) Theonellapeptolide

I

from a sponge, Lamellomorpha strongylata [38], belonging to an order taxonomically distant from that of Theonella swinhoei. Theonellapeptolide Ille (52) possesses the same molecular formula as theonellapeptoUde le (48) [35] and a similar peptide backbone but differs in four aminoacid residues. In particular the substitutions of L-Ala for PAla results in a 36-, rather than 37-membered peptolide ring for 52. Isolation of closely related compounds from taxonomically remote species suggests the involvement of symbionts in the production of theonellapeptolides. Another evidence for the symbiotic origin of theonellapeptolide family has been reported in a recent Crews' paper [39] in which seventeen specimens of the Papua New Guinea collections of Theonella swinhoei and T. conica were examined. This study has resulted in the isolation of theonellapeptolide Id (47) [35] and motuporin (53), as

Motuporin (53)

1190

the two major metabolites of these collections of Theonella swinhoei and conica, both heavily infested with cyanobacteria. Interestingly one of these specimens of Theonella swinhoei afforded the new antifungal cycUc decapeptide cyclolithistide A (54) [39]. Its structure, which contains the unique aminoacids 4-amino-3,5-dihydroxyhexanoic acid, formyl-leucine and chloroisoleucine, was elucidated through a combination of CI

II

Me

-.

o H2N^

CycloUthistide A (54)

spectroscopic techniques. The cycUc pentapeptide motuporin, firstly isolated from a Theonella swinhoei [40] represents an extremely potent PPl protein phosphates inhibitor belonging to a family of structural related cyanobacterial toxins, including nodularins [41] and the heptapeptide microcystins [42] Specifically, these peptides all contain the unusual aminoacid Adda and an a,p-unsaturated aminoacid, judged, on the basis of X-ray studies [43] the essential features required for binding to PPl [44]. Although their potent and competitive inhibition of protein phosphatases PPl and PP2A [45,46], motuporin shows antineoplastic activity [40] whereas nodularins and microcystins promote tumour formation in mice [47,48]. This interesting paradox in the biochemical profiles of these peptides suggests that the biological outcome of phosphatase inhibition is dependent upon cellular localisation and the specific phosphatase that is targeted. The structural differences between microcystins and motuporin may contribute to the different localisation of these compounds in the cells. A new cycUc heptapeptide cupolamide A (55) was isolated from two samples of the sponge Theonella cupola collected in Indonesia and Okinawa [49]. Three of the aminoacid residues, fran5-4-hydroxyprohne, homoarginine and 2,4-diaminobutanoic acid are of uncommon occurrence; in particular the hydroxy-proline is a constituent of collagen and occasionally has been found in terrestrial biota [50] and in marine

1191

cyanobacterial metabolites [51]. Cupolamide A (55) was active against NH H2N—^ HN-

O O HO.

^NH ^,1

\

I O

//

O

Cupolamide A (55)

^ ^

^

Q^T)-..iOS03Na

,^^0^°"

P388 murine leukaemia cells with an IC50 value of 7.5 |ULg/ml. Analysis of the extracts obtained from Papua New Guinea collections of Theonella mirabilis and Theonella swinhoei resulted in the isolation of four novel cycUc depsipeptides, papuamides A-D [52]. The structures of papuamide A (56) and B (57) were determined from a combination of spectroscopic analysis, chemical degradation and derivatization studies.

O^NH Papuamide A (56) Ri=R2=H, R3=CH3 Papuamide B (57) Ri=R2=R3=H

O V ' " O

=

OH

1192

These peptides contain a number of unusual aminoacids including 3,4dimethylglutamine, P-methoxytyrosine, 3-methoxyalanine, 2,3diaminobutanoic acid or 2-aniino-2-butenoic acid residues. Papuamides are also the first natural marine-derived peptides reported to contain the 3hydroxyleucine and homoproline residues, as well as an unprecedented acid moiety N-linked to the terminal glycine residue. Papuamide A (56) was cytotoxic against a panel of human cancer cell lines with a mean IC50 value of 75 ng/ml; papuamide A (56) and B (57) inhibited the infection of human lymphoblastoid cells by HIV-1 in vitro with an EC50 of 4 ng/ml. Linear peptides Further examination of the same sponge that contained the thrombininhibitory cyclotheonamides [8], led to the isolation of a thrombininhibitory linear tetrapeptide, nazumamide A (58) [53]. It is the first naturally occurring peptide possessing the N-2,5-dihydroxybenzoate terminus. The discovery of thrombin inhibitors is of extreme interest due •\^^

OH

OH

^

^^

Nazumamide A (58)

to their role in promoting the coagulation process. Recently Nienaber et al [54] reported the crystal structure of 58 complexed with human thrombin. This structure demonstrated that 58 binds to thrombin with a novel retromanner but with low selectivity and specificity. Although the weak binding proprieties of this natural linear peptide make it a nonviable anticoagulant, 58 may serve as a usefiil template for the design of more potent and specific thrombin inhibitors via synthetic [55] and combinatorial methods. Thus a large library of NAZA analogous has been prepared using 25 natural and unnatural aminoacids [56]. These studies led to the identification of 2,5-dihydroxylbenzoyl-lysyl-isoleucylphenylalanyl-arginine as a novel thrombin inhibitor 25 times more potent (IC50 1.9 p,M) than the natural peptide 58 (IC50 53 |iM). In 1999, koshikamide Ai (59), a new cytotoxic (IC50 2.2 ^g/ml) linear decapeptide, has been isolated from a recollection of Japanese Theonella sp. [57]. Its aminoacid constituents have been determined by NMR analysis and chemical methods and the sequence was elucidated through

1193 an extensive HMBC analysis and supported by FAB-MS/MS, Unique features of 59 are the methoxyacetylation of the phenylalanyl N-temiinus, C0NH2 O

^ H

Me

Me O

O

z

" z » O ^y^^ Me

/

.CONH2 ^ O

H 5

O

I / . / ', ^

^

Koshikamide Ai (59) reminiscent of theonellapeptoUdes [35], as well as the unprecedented presence of five contiguous N-methyl aminoacids. Another class of linear peptides is represented by polytheonamides

PH

o=< w

NH

NHMe

° H SVK S^K'^^^K'J 0=< NHMe

%

HO

H,N^O

OH

o

0=< NH2

NH2

NHMe

NHMe

Polytheonamide A (60) R=H Polytheonamide B (61)R=Me (60-61), isolated from the Upophilic extract of Theonella swinhoei [58]. The structures of highly cytotoxic polytheonamides 60-61 were assigned to be linear 48-residue peptides with N-terminus blocked by a carbamoyl group.

1194 A recent reinvestigation of 1993 collection of Theonella swinhoei collected off Hachijo-jima Island has resulted in the isolation of six new peptides related to cyclotheonamides named pseudotheonaraides Aj, A2, B2, C, D (62-66) and dihydrocyclotheonamide A (67) [59]. HO.

HN

I

I

Q^iJs^NH HN / ^ ^

HN

„

HO / N//

^^

Pseudotheonamide A^ (62)

Pseudotheonamides 62-64 are linear pentapeptides embracing the rare piperazinone and piperidinoiminoimidazolone ring systems. Pseudotheonamide C (65) contains a residue of vinylogous tyrosine instead of a piperazinone ring. Pseudotheonamide D (66) is a linear tetrapeptide, which lacks the C-terminal a-ketohomoarginine unit whereas the dihydrocyclotheonamide A (67) is a reduction product of the above-mentioned cyclotheonamide A (12). These linear congeners of

H' -O O Pseudotheonamide A2 (63) cyclotheonamide family also showed a serine protease activity; in particular pseudotheonamides 62-66 and 67 inhibited thrombin and trypsin with IC50 values in the range of 0.33-3 |LiM and 4.5-10 |ULM, respectively. Since the power of cyclotheonamides as serine protease inhibitors is associated with the presence of the a-keto group in the k-Arg

1195 residue, it is not surprising that compounds 62-67, in which this group was modified or missing, showed moderate activity.

Pseudotheonamide B2 (64)

d^^

d

NH2 HN^

^1

^

0y \

,^^C02CH3 2- NaBH4 H3C 3. TBS-CI NHTs

/

LNaCN.

V ^ ^™o

2. HCI

CH2OTBS 149

N(Boc)Ts

N(Boc)Ts

D-Thr

^COgCHa

HOH2C'

^

^C02CH3

Swern

^ 150

146

NHBoc NHBoc

nr

H3CO2C

C02Pac

C02CHPh2 1. H2, Pd/C

HgCOgC^^^r^

2. PhCOCH2Br KF(H20)2

147

Scheme 1 - Schreiber synthesis of the C1-C4 subunit of the Adda residue (150) and of protected Me-Asp residue (147).

residue was obtained from benzyloxymethyl-protected mandelaldehyde through initial crotylstannane addition. The diene functionality was installed through sequential application of Wittig and Julia olefinations to give the protected Adda 143 (Scheme 2). 1.DIBAL-H

BOMO

MgBr20Et2 3. NaH,CH3l

C02Et

CH2TS

^

CO2CH3 1. BuLi, then 150 2. NaNaphthalene

Scheme 2. Schreiber synthesis of the Adda residue.

Both dipeptide N-Boc-valine-Adda and tripeptide 144 were obtained

1220

using corresponding protected amino acids and conventional coupling technology. The macrolactamization of the protected linear pentapeptide 142, Fig. (1), was achieved by a four-step sequence involving: a) reductive removal of the phenacyl group (Pac), b) activation of the Cterminus as pentafluorophenyl ester, c) N-terminal Boc group deprotection, d) neutralization and dilution. Treatment with Ba(0H)2 afforded concurrent methyl ester deprotection and N-methylthreonine dehydration. In their retrosynthetic plan Armstrong and co-workers proposed to effect the macrolactamization of the linear peptide at the Adda (iS')-valine

Motuporin (53) COgf-Bu'

y" 0^. .OMe

U H I r.Buo\^YT'

!f^?25,J^H OH NH

COoMe - '^ NHg

.. Me

O

OH

ZHN

'/OBn

OMe

152

153

fl

fl

COaMe

OH

°v^H„r^"°/r"™"-'V™' ™''-^' NHBoc

O^

OMe

^

156

o

o

155

OBn

157

Fig. (2). Armstrong's retrosynthesis of motuporin (53)

amide bond. Fig. (2). This is supposed to be the actual point of cyclization in the biosynthesis of the natural product based on the reported isolation of related linear peptides from cyanobacteria and subsequent feeding studies [156]. The linear peptide was further disconnected to give Bocprotected Adda 151 and the remaining tetrapeptide which was in turn disconnected into dipeptides 152 and 153. Installation of the diene moiety in Adda residue was anticipated to be performed via a Wittig or a related olefmation reaction between a nucleophile derived from bromide 154 and aldehyde 155. The p-methylaspartate residue may arise from the same

1221 aldehyde intermediate. The dipeptide 153 was envisioned to arise from an Ugi four-component condensation (4CC) of monoprotected glutamic acid 156, aldehyde 157, methylamine and cyclohexenyl isocyanide. The two contiguous stereocenters present in the aldehyde 155 were installed in a straightforward manner and with an extremely high stereoselectivity using the dihalomethyllithium insertion method of Matteson (Scheme 3) [157]. The (-h)-pinanediol boronic ester derivative

cte^

PMBOv^B,

158

CHaMgCI LiCHCl2 2. ZnCl2 P M B O ^ ^QV\^, 2. LDA, CHgBrg 01 159 3. ZnCl2

^•^160

LNaNg 2. BULC PMBO

CH2ICI

Scheme 3. Armstrong synthesis of aldehyde 155

158 which acts in the process as chiral director, was prepared according to Uterature [158] and subjected to the first insertion step with dichloromethyllithium followed by ZnCl2. The a-chloroborate ester 159, obtained as single diastereoisomer, was treated with methylmagnesium chloride to give the corresponding a-methyl borate ester subjected to the second insertion with dibromomethyllitium. After displacement of the bromide in 160 with sodium azide, a third insertion step was performed with chloroiodomethyllithium affording the azido borate 161, which was transformed in four steps with standard methods into the desired aldehyde 155. The remaining two stereocenters in the Adda residue were installed through Brown crotylboration method using phenylacetaldehyde and the reagent derived from (+)-5-(methoxy)diisopinocampheyl-borane and cis2-butene (Scheme 4). The obtained homoallylic alcohol was elaborated by using standard conditions to give the allylic bromide 154. The coupling of different Wittig or Homer-Emmonds reagents derived from bromide 154 with the aldehyde 155 was tested in different base and solvent conditions. Best results in terms of yield and stereoselectivity were obtained using the triphenylphosphonium yUde derived from bromide 154 and LDA as base. The diene 162 was easily converted in three steps to N-Boc protected Adda 151. The Y-(D)-glutamyl-N-Methreonine dipeptide 153 was prepared in a one-pot process which utilizes the Ugi technology (Scheme 5) [159]. This latter offers the advantage to form the synthetically challenging tertiary amide bond, usually obtained in low yields using standard peptide coupling techniques. Ugi reaction of the carboxylbenzyloxy (Z) protected glutamic acid derivative 156,

1222 aldehyde 157, methylamine and cyclohexenyl isocyanide afforded cyclohexenamide dipeptide 163, which was converted in the

ou.

1. a) (/Pc)2B\ = / BF30Et2

1.a)03;b)PPh3

b) H2O2, NaOH

'^^^^y^oEt

2. NaH, CH3I

2. a) LDA b)155 162 Scheme 4. Armstrong synthesis of Boc-Protected Adda residue (151).

corresponding free carboxylic acid 153 through acid hydrolysis. A 1:1

HCI 153 DBn Schemes. Armstrong synthesis of the Y-(D)-Glutamyl-N-MeThreonine residue (153).

mixture of diastereoisomers at the a-stereocenter of the threonyl residue was obtained, but this is inconsequential for purposes of synthesizing motuporin as this carbon becomes part of the dehydroamino residue. Panek and co-workers adopted a convergent approach in which the chiral aUylsilane bond construction methodology, elaborated by the same author [160] was extensively used for the introduction of the stereochemical relationships. Fig. (3). Disconnection of 53 at the

1223 indicated sites afforded two principal fragments: A^-Boc-valine-Adda dipeptide 164 and the remaining tripeptide fragment 165. The dipeptide 164 was further disconnected at the C5-C6 bond to give a vinyl metal species 166, and the (F)-vinyl iodide 167. In the synthetic Fragment Coupling

"

Macrocyclization CHaOaq BocHN

O^^^OH

O

HN

XX 164 AZ-Boc Valine-Adda

I

.OSIR3

HN^ ^ O

^O

Tripeptide fragment 165

N3 Tce02Cx^^^xV^

ZnCI

166 Left hand subunit

168

167 Right hand subunit

C02Me

C02Me Me2PhSi R=H, (S)-Silane 170 R=N3, (S,/?>SiIane 171

CO2MG

169 ^*g* (^)- Panek's retrosynthesis of motuporin (53)

1224

direction, a Pd(0)-catalyzed cross-coupling reaction of the C-6 vinyl zinc species and the C-5 vinyl iodide was planned to complete the carbone backbone of the dipeptide 164. Further analysis of the individual subunits produced two chiral silane synthons 170 and 171, of which the anf/-azido silane 171 is derived from 170 through diastereoselective azidation of the corresponding P-silyl enolate [161]. The 1,3-azido alcohol 169, a precursor of both dipeptide 167 and of the methyl aspartate derivative 168 was prepared in two steps from (»S',/?)-silane 171 via sequential diastereoselective crotylation and allyUc azide isomerization (Scheme 6) [162]. After protection of the primary alcohol as TBDPS ether, and reduction with SnCl2 of the azide group, the free amine was condensed with the pentafluorophenyl ester activated A^-Boc-L-valine to afford the dipeptide 172. The double bond was oxidatively cleaved to the corresponding aldehyde which was subjected to Takai's homologation protocol to give dipeptide 167. The C6-C10 subunit of the Adda residue was prepared in a high yielding short sequence involving a ^-yn-selective

C02Me Me

169

NHBoc

1.TBDPSCI 2. SnCl2

1.03,thenDMS

3. /V-Boc-(L)-Valine-OC6F5 aq. NaHCOa

TBDPSO

2. CrCl2/CHl3

-•167

COaMe Scheme 6. Panek synthesis of dipeptide 167.

crotylation reaction to install the correct C8-C9 stereochemical relationship (Scheme 7). Therefore, the phenyl acetaldehyde dimethyl acetal was condensed, under Lewis acid catalysis, with silane 170. Oxidative cleavage of trans double bond in 173 and direct dibromoolefmation of the resulting aldehyde gave the dibromoolefm 174. This latter was treated with n-BuLi to give the acetylene anion which was trapped with methyl iodide to afford the methyl substituted acetylene 175. The vinyl zinc intermediate 166 to be used in the Pd(0)-catalyzed cross coupling reaction with dipeptide 167 was obtained from acetylene 175 through hydrozirconation foUowed by in situ transmetalation with ZnCli. The one-pot cross coupling reaction between 166 and 167 afforded the configurationaUy pure trisubstituted (E,E)-ditne 176 in 80% yield. This latter was easily transformed into required A^-Boc-valine-Adda dipeptide

1225 164 through silyl deprotection of the primary alcohol and oxidation of the C02Me

1. O3, DMS

2. ZnCl2 166 OTBDPS 1.TBAF-SiOo HN^^O 176

2. TPAP/NMO

•^

Dipeptlde 164

- ^ , NHBoc

Scheme 7. Panek synthesis of the N-Boc Valine Adda dipeptide 164.

aforementioned functional group to a carboxyl group. The protected P-methyl aspartate fragment was prepared (Scheme 8) starting from the same azido alcohol 169 previously used in the synthesis of dipeptide 167 through a) Jones'oxidation of the primary alcohol, b) protection of the carboxyUc acid as trichloro-ethyl ester, c) oxidative cleavage of the double bond (RuCla, NaI04), d) protection of the resulting a-azido acid as methyl ester, e) reduction of the azido group in 1. Jones'Oxidation

_ • MeOgC

2. 2,2,2-trichloroethanol DCC, DMAP

(OTce

1.RuCl3(cat),Nal04 • 1168 I 2. CH2N2

Scheme 8. Panek synthesis of the p-methyl aspartate precursor 168.

168 with SnCl2. The obtained free amine was immediately used in standard peptide coupling conditions to eventually give the tripeptide 165. The approach to the key fragment iV-Boc-Adda methyl ester used by Toogood and co-workers in their total synthesis of motuporin (53) [155b]

1226 parallels that employed by other groups [163]. In fact, as already reported by Armstrong and co-workers, see Fig. (2), the diene functionality, present in this molecule, was envisioned to arise from the coupling of a nucleophile derived from bromide 154 (Scheme 9) and aldehyde 177

Ph' 178 1-Se02

,

,

179

180

„./-v^.xV^^^^/0'^ - • Ph J '

2.NaBH4. CeCl3

MeO

CBr4,Ph3P

•Ph

/s.

^^^

JL ^

MeO

^Br

^54

Scheme 9. Toogood synthesis of the C5-C10 fragment of Adda residue (154).

(Scheme 10). For controlling the chirality at C-8 and C-9 positions of Adda, the Evans' OH

9

1.LDA,-78°C

''//

2. CH3I, NaH 182

-

.

Q

BocHN

5 Steps BocHN^^A^

/

^

OCH3

183

CO2CH3

177

\l.Nal04,Rua3 2. DCC, DMAP

BocHN,^X^

'OCH3

^ ^

CeFsOH

^^2C6F5 184

Scheme 10. Toogood synthesis of the C1-C4 fragment of Adda (177) and of the a-methyl p-pentafluorophenyl-A^-Boc-methyl aspartate residue (184).

chiral oxazolidine template methodology was used, as already reported by Rinehart and Beatty [163]. The aldol 178 obtained from phenylacetaldehyde was easily converted to aldehyde 179 which was homologated under standard Wittig condition to alkene 180. This latter was regioselectively oxidated with Se02 to the corresponding £-allylic alcohol 181 which, in turn, was transformed to the corresponding allylic bromide 154 (Scheme 9). The C1-C4 fragment of Adda was prepared

1227 (Scheme 10) from (/?)-3-pentyn-2-ol through a highly efficient method previously reported by the same authors [164]. The /^-Boc-L-glycine ester 182, obtained in two steps from the above alkynol, was subjected to a Kazmaier's modification of the Ireland-Claisen rearrangement to give, after esterification, the methyl ester 183. Standard ftinctional groups elaboration afforded the aldehyde 177, which was used in the olefmation reaction to give A^-Boc-Adda methyl ester.The methyl ester 183 was also transformed in the a-methyl P-pentafluorophenyl-A^-Boc-methyl aspartate residue 184, ready for the subsequent fragment coupUng, through oxidative cleavage (RuCl3/NaI04) of the double bond and conversion of the resulting carboxylic acid to its pentafluorophenyl ester. After completion of the synthesis of the key monomeric fragments, several routes to motuporin were explored. They differ in the order of fragment assembly and in the timing of the dehydratation step to install the mdhb double bond. It was found that macrolactamization of linear peptides containing the A^-methyldehydrobutyrine residue proceeds primarily with epimerization, whereas, as reported previously by Schreiber, the same process was cleaner using the dehydroamino acid residue masked as a threonine. The synthesis of 5-[L-Ala]-motuporin, in which the dehydroamino acid residue was replaced by L-alanine, was also performed in order to explore the role of this dehydroamino acid to the biochemical activity of motuporin. Some epimerization was also observed during the cycUzation of the alanine-containing peptide. 5-[L-Ala]motuporin was found to inhibit PPl activity with IC50 values comparable to those found for motuporin itself so suggesting that the A^methyldehydrobutyrine residue is not essential for the inhibition of PPl [155b]. Swinholide A (68) and congeners The limited natural supply and the remarkable anticancer properties, together with the spectacular molecular architecture made the swinhoUde A (68) and its congeners popular targets for synthetic efforts which cuhninated with two excellent and flexible total syntheses of swinholide. The first total synthesis of swinholide was achieved by Paterson and its group at Cambrige in 1994 [165] shortly after the completion of the synthesis of the monomeric seco acid pre-swinholide A (73) [166] Nicolaou and co-workers reported an alternative total synthesis of swinholide A (68) in 1996 [167]. Paterson total synthesis was already reviewed in 1995 by Paterson itself in the review 'Total Synthesis of Bioactive Marine MacroUdes" [168], that also included detailed accounts of the alternative strategy and the synthesis of significant segments of swinholide A at that time reported by Nicolaou.Rather than duplicate this review, we will not report detailed analysis of the two synthetic planes but we will show only the general strategies, focusing our discussion on the

1228 key steps which allowed the stereospecific construction of a so complex molecule. As reported in Fig. (4), the synthesis by Paterson et al was based on the selective deprotection and regiocontroUed dimerization of 185, a fully

Fig. (4). Paterson's retrosynthesis of swinholide A (68)

protected version of the monomeric seco-acid preswinholide A. The carbon skeleton of this derivative was constructed by the union of the ClC15 aldehyde segment 186 [169] and the C19-C32 aldehyde segment 187 [170] using a suitable butanone synton as a linking unit [171]. Owing to the polypropionate nature of the skeleton of preswinholide A, the stereocontrolled synthesis of this derivative was achieved through a correct choice of different types of asymmetric aldol reactions which allowed the stereoselective formation of the C6-C7, C12-C13, C15-C16, C18-C19 and C22-C23 bonds. The final macrolactonization steps

1229 affording swinholide A from the monomelic secoacid were proved to be very regioselective and highly yielding (Scheme 11).

ester at C-23 (191) 190:191=2:1 r- 190 P^=H, P^=Me. P^=Steu2 U-192 P^=TBS, p2=P^=H P^O "O

OMe Protected Swinholide 193 a) 2,4.6-Cl3(C6H2)COCI EtaN, DMAP, Yamaguchi b) DCC, DMAP, DMAPHCI, Keck

+

1 1

1

Protected 1 isoswinholide 1 194

Scheme 11. Paterson swinholide A and isoswinhoUde A synthesis (final steps).

The resulting ring size (44- vs 46- membered) was simply controlled by the macrolactonization conditions employed without differential C21 and C23 hydroxyl groups protection. Hydrolysis of the terminal methyl ester in 185 gave the acid 188 which selectively esterified, under Yamaguchi conditions [172], the C21 hydroxyl group of the diol 189 obtained by removal of the silylene group in 185. A 2:1 mixture of the desired C21

1230 ester 190 and its C23 regioisomer 191 was obtained. This latter by-product could be recycled by methanolysis to give back diol 189 and the fully protected methyl ester 185. Then a silyl C23 hydroxyl protection was made before sUylene removal and selective hydrolysis of the terminal methyl ester to give the dimeric secoacid 192. This protection was needed in order to prevent cleavage of the C21 ester linkage. The regioselective macrolactonization of 192 to generate the desired 44-membered ring was facile and high yielding. Under Yamaguchi conditions [172] [a) conditions in Scheme 11], a 86:14 mixture of desired 44-membered ring 193 over the 46-membered ring 194 was obtained. This ring-size selectivity may be reversed by changing to the Keck DCC protocol [b) conditions in Scheme 11] [173]; in facts in this case a 9:1 mixture of 194 and 193 was obtained permitting selective formation of isoswinhoUde ring. Total deprotection of 193 and 194 completed the syntheses of swinholide A (68) and isoswinholide (71), respectively. Fig. (5) shows the retrosynthetic analysis on which is based the OMe Dithiane-cyclic sulphate coupling ^OK Macrolactonization; esterification

Ghosez ! cyclization r ^ o

Ghosez ^'•,^^^ cyclization OH 6 ^ ' OH

MeO,

Wadsworth-Horner Emmons reaction

OH Dithiane-cyclic sulphate coupling

OMe Fig. (5). Retrosynthetic dissection of swinholide A (68) according to Nicolaou

alternative synthetic strategy proposed by Nicolaou. As in Paterson's protocol the final key reactions affording the 44-membered macrodioUde are an esterification and eventually a macrolactonization of the resulting hydroxy acid. Also in this case, both reactions were performed in satisfactory yields by using the Yamaguchi protocol. Diastereoselective formation of the C2-C3 double bond was achieved through extension of the C"3 aldehyde via a Wadsworth-Homer-Emmons olefination reaction. The C3-C32 segment was constructed by coupling of the C3-C17 segment and the C18-C32 segment through the reaction of the C18-C19 cyclic sulphate with the Hthio derivative of the dithiane of the C-17

1231 aldehyde. The C3-C17 fragment was prepared from (5)-dimethyl malate in a synthetic sequence that features a Ghosez cyclization [174] for the assembly of the dihydropyran system. The C18-C32 segment was prepared from L-rhamnose which fiimished three of the nine stereogenic centers present in this fragment. The other stereogenic centers were installed using a combination of reagent-controlled, auxihary-controlled and substrate-controlled reactions. Onnamide A (74) The total synthesis of onnamide A (74) was achieved by Hong and Kishi in 1991 [175] after the completion of the synthesis of mycalamides A and B, reported by the same authors [176] and the synthesis of pederin, reported by Nakata and co-workers [177], Retrosynthetically Fig. (6), onnamide A can be divided in four Onnamide A (74)

MeO ?DMPM

OMe

195 "left hand subunit"

196 "right hand subunit" ?02

TMS-

-CHgCHaLi 197

o

T

AcN

198 Fig. (6). Kishi's retrosynthesis of onnamide A (74)

fragments: the C20-C26 "left hand subunit", 195, corresponding to the pederic acid fragment, the "right hand subunit" C10-C19, 196, containing the interesting acylaminalfimctionalgroup, that is presumed to play a key role in determining the biological activity, the lithium derivative 197, corresponding to the C6-C9 fragment, and the L-arginine-containing side chain 198. The synthesis of the right hand of onnamide (Scheme 12) was modified with respect to that proposed for the construction of the same subunit in mycalamides A and B. The starting material was the ketone

1232 199, prepared according to the literature [178] subjected to reduction and OMe

OMe H/J

P" x^o

1.NaBH(0Ac)3 CeCla/MeOH

H/,

2. Mel/NaH/THF

6.,^.6

OMe

OMe

_ ^ _^

DMe AcO

Ph 200

I.OSO4/ S,S-Corey Ijgand

CH2=CHCH2TMS I

TMSOTf/BF30Et2 MeCN

OBn 201

Q-

2. lm2CO DMe

DMe

DMe 205, X=N3 196,X=NH2 Scheme 12. Synthesis of the C10-C19 fragment of onnamide A (196).

methylation to give the desired methyl ether 200. Several hydride reductions were tested and only sodium triacetoxyborohydride in the presence of CeCls was proved to furnish the desired diastereomer. Protecting group manipulation afforded 201 which was subjected to a Cglycosidation [179] to give exclusively the expected, axially substituted product 202. Corey asymmetric osmylation of 202, followed by carbonate formation afforded 203 which was transformed in the dimethyl acetal 204, identical with the intermediate used in the synthesis of mycalamides A and B. Standard functional groups manipulation allowed for the transformation of 204 into the azide 205, obtained as a 2:1 C-18 diastereomeric mixture favouring the natural configuration. The corresponding amine 196 ("right hand subunit"), obtained by hydrogenation, was configurationaJiy unstable under acidic, basic and

1233 neutral conditions. The "left hand subunit" was prepared in two steps starting ft-om 206, an intermediate in the Nakata's total synthesis of pederin [177] and coupled with the amine 196 giving a separable C-18 diastereomeric mixture (Scheme 13). The unnatural C(18)-p-epimer may be recycled by equilibration in the presence of f-BuOK/THF at reflux. The obtained carbonate 207 was transformed in the corresponding C-

MeO q^

VT^

^OMe

TMS

206

"II

MeO

1^^ TMSCgCCHgCH^Li^^o,"

II

''bMe

'^,^Sn(/hBu)3

+ MeO

OAc r

H AcHN.^ ^N AcN

198 Pd(Ph3)^

I Onnamide A (73)

Scheme 13. Final steps toward onnamide A

10/C-ll epoxide 208, which was subjected to a mixed cuprate addition to give elonged derivative 209. A straightforward fiinctional group manipulation allowed for the transformation of 209 into the vinyltri-nbutyktannane 210, suitable for the synthesis of the C2/C-7 triene of onnamide. Pd(0)-catalyzed coupling of the stannane 210 with 5-iodo amide 198 gave the coupling product as a mixture of geometric isomers. This latter was transformed by using standard procedures to the synthetic onnamide A. The spectroscopic data of the synthetic material was found

1234 to be identical with those of the authentic samples from natural source, thus confirming the relative and absolute stereochemistry, previously assigned by analogy to mycalamides and pederin. CalycuBns (100-102) The calyculins have aroused considerable synthetic interest due not only to their unique polyfunctional structure but also to their intriguing biological activities[180]. However, initial synthetic efforts were addressed to (+)-calyculin (arbitrarily depicted in the 1986 original paper), which is enantiomeric to the natural occurring compound. The Evans group reported the total synthesis of ewf-calyculin A in 1992 [181], which allowed to establish the actual absolute configuration of the natural product. In 1994 Masamune et al published the total synthesis of the natural enantiomer [182]. Shoiri et al have also reported a formal total synthesis of calyculin A [183]. Very recently an alternative synthesis of (+)-calyculin A and (-)-calyculin B was reported by Smith [184] whereas the natural calyculin C was obtained by Armstrong and co-workers [185] Owing to the impressive worlc in this field, rather than being comprehensive, the following discussion will be limited to the description of the general synthetic strategies and of the key synthetic steps. Retrosynthetic analysis of calyculins backbone revealed several possibilities for disconnection involving carbon-carbon bonds. All the synthetic strategies were centred on the initial disconnection at C25-C26 double bond which divided the natural product into two halves of similar functional complexity. Model studies [180d,180m] indicated that Wittig olefination using stabilized ylides derived from C26-C37 phosphonium salt gave the best results in terms of (£^-selectivity and overall yield. The late introduction of the photolabile and easily isomerizable C1-C9 cyanotetraene moiety and of the Cn-phosphate group is another common feature of all synthetic plans. Main disconnections of the Evans' total synthesis of ^n/-calyculin A are depicted in Fig. (7). The key building blocks C1-C25 and C26-C37 were elaborated in homochiral form and eventually coupled in the final stages of the synthesis through a Wittig reaction of the C25 aldehyde with the C26 phosphonium salt. All the synthetic strategy is based on an extensive use of well known author's auxiliary-controlled asymmetric synthesis. 10 of the 15 stereogenic centers (Cio, C12, C13, Cn, C22, C23, C30, C34, C35, €35) were incorporated through auxiliary-based asymmetric aldol, alkylation, hydroxylation, and Michael reactions, whereas the remaining chirality arose from substrate-controlled induction. Truncation of the C1-C25 subunit, Fig. (8), at the three indicated sites revealed four smaller fragments which were coupled according to Eq. (a). Aldol coupling of the C13-C20 methyl ketone (fragment C, 213) with the C21-C25 aldehyde subunit (fragment D, 214) with complete Felkin-Anh stereocontrol of the

1235 ^—ammide coupling OH

MegN

o

/

OH

^Stille

J

OHXOH

OM©

Homer-Emmons ^ ^ Fig. (7). Evans' retrosynthetic analysis of enr-calyculin A O

1CN

213, C 214, D Fig. (8). Evans* retrosynthetic analysis of the C1-C25 subunit of enr-calyculin A

B C + D — • CD

A •

BCD

• ABCD

(a)

newly generated C21 stereocenter was achieved (Scheme 14) using the Mukaiyama-type aldol reaction between the trimethylsUyl enol ether 215 (corresponding to the fragment C) and aldehyde 216. Exposure of the aldol adduct 217 to HF/MeCN/H20 led the desired spiroketal 218, along

1236 with minor amounts of its epimer at the C19 stereocenter. The spiroketal OTES TESO

n 21 : 23

48% HF CH3CN, H2O

Piv0^3xv^i5>o OMe O P M B O T M S BFaOEta

215

MeO

OPMBO

Mukaijama aldol TBSO

O H OR

217

'OTBS

O

O

O

OH OMe OTBS

spiroketalization

O OTICI4 220 ^ aldol

OH 5 H 5Me

221

XP\x<S.X^vi>'^v^^^O* ^ d

OH OAc OMe

^OTBS

H^^^^^x^Nsi3^/\^,x^O 6

OPi OPi OMe

P^=TBS

222 223 Scheme 14. Evans synthesis of the C9-C25 fragment of en/-calyculin A.

218 was then trasformed into the C13 aldehyde 219, the appendage point for the C10-C13 dipropionate subunit (fragment B). Aldol addition of the titanium enolate 220 to the above aldehyde afforded the adduct 221 with wrong stereochemistry at C13. After reduction of the Cn ketone, the C13 hydroxy group was regioselectively inverted under Mitsunobu condition. Conventional elaboration of the acyl group at C9 in 222 afforded the aldehyde 223 to which the tetraene moiety was attached through the sequential application of Homer-Emmons and Stille reactions. Disconnection of the nitrogen rich C26-C37 subunit at the amide junction led the C33-C37 aminoacid and the C26-C32 y-amino azole subunit, Fig. (9). Further disconnection at the C34-C35 bond of the y aminoacid fragment through an anti glycolate aldol reaction fiimished the Dserinale derivative 224. The 2,4 disubstituted oxazole system was obtained by cyclodehydratation and oxidation of the amide alcohol

1237 derivative 225, The stereocenter at C-30 was installed using a chiral OR OH

O U

I

NMeg OR

OR

O

MeO

33^0H NMe2 OR cyclodehydration pxydative aromatization

anti-aldol reaction

MeO*

BocHN

H N. ^COaMe

30^ O

NH2

CH2OH

225 L-serine methyl ester

224

BocHN

OH

Michael addition Curtius rearrangment

Fig. (9). Evans' retrosynthetic approach to the C26-C37 subunit of enr-calyculin A

auxiliary-controlled Michael addition of the enolate derived from propionic acid to tert-butyl acrilate. Even if the retrosynthetic dissection of (-)-calyculin A (100) by Masamune, Fig (10), parallels that by Evans it differs for the timing of coupling of individual fragments (the order was B->BC-^A'BC->AA'BC) and for the approach to the key C9-C25 (228) and C26-C37 fragments (229). The spiroketal subunit (Scheme 15) was constructed starting from 230 through a) Claisen condensation with the litium enolate derived from methyl isobutirrate; b) stereoselective reduction of the Cn ketone, c) standard functional group elaboration to fiimish the silyl enol ether 231 d) Ti^^-mediated Mukaiyama-type aldol reaction with aldehyde 232 e) trasformation into aldehyde 233 f) aldol

1238 reaction of the above aldehyde with the E(0) enolate 234. The OH O

C

=

'^^nr*^K^^°) MegN

OH

OH

O ''/OR

229, C

C3HO

OR

OR

OMe

228, B

O

226, A 227, A' Fig. (10). Masamune's retrosynthetic analysis of calyculin A (100)

17 COgMe

V

f

Methyl Isobutyrate; V ^ ' I / Q ' ^ " 2. MPMBr,

^ 2 3 ^ ^ ^OBn OTrO^O

OTr

OMe-^p,0^^^ 236

21

^ 25 OTES

O ^

OMe g^p^O

238

237

Scheme 15. Masamune synthesis of the C9-C25 subunit of calyculin.

oH

OTES

1239 anthanthanti diastereoisomer 235 was obtained with a 12:1 diastereoselection thanks to the extraordinarily high diastereoeselectivity of the enolate 234 that overrode that (1:4) of the aldehyde 233. With all the carbon in place 235 was trasformed into methyl ketone 236 with a set of standard reactions. The final aldol reaction of cyclohexyl boron enolate of methyl ketone 236 with the aldehyde 237 furnished the acycUc intermediate 238 corresponding to the C9-C25 spiroketal unit. The C33C37 Y-aminoacid was obtained from D-galactose [180f], whereas the 2,4disubstituted oxazole was obtained through condensation of the amide 239 with bromopyruvate, followed by dehydratation (Scheme 16). Conventional functional group elaboration provided the amino-oxazole fragment in excellent overall yield. Smith's retrosynthetic approach, Fig. (11), to en^calyculin A and to ()-calyculin B (the C2 geometrical isomer of calycuUn A) begins with the disconnection at C(2) via a Peterson olefination which would provide both molecules from a common advanced intermediate. Further

^ 239

iX.

2. TFAA ,

'COgEt

S26

OMPM Scheme 16. Masamune synthesis of the C26-C32 subunit of calyculin A.

disconnection as indicated by dashed lines generated five fragments: the phosphonate A (240), the bromoolefin B (241), the spiroketal epoxide C (242), the oxazole D (243) and the lactam E (244). Fig. (12) shows the retrosynthetic approach to the pivotal spiroketal C (242) [180o]. Model synthetic studies [180rl indicated that the stereocontroUed formation of the C(14,15) a epoxide would be achieved via Payne epoxidation of the vinyl spiroketal 245. The corresponding open-chain precursor 246 was envisioned to arise via coupling of epoxide 247 with the sterically hindered ditiane 248. This latter, in turn, was obtained in a three step sequence from alcohol 249, readily available from f5j-dimethyl malate [186]. The epoxide 247 was elaborated (Scheme 17) via Brown asymmetric addition of (Z)-crotyldiisopinocamphenylborane to aldehyde 250, followed by a highly diastereoseiective IBr-induced iodocarbonate cycHzation of the homoallylic carbonate 251 [187]. Treatment of iodocarbonate 252 with K2CO3 (3 eq) in dry MeOH and protection fiimished the desired epoxide 247. The y amino acid segment E 244 was synthetized [180p] (Scheme 18) starting from the known lactone 253 through a) addition of the Weinreb reagent of p-methoxybenzylamine, b)

1240

oxidation to the hemiaminai 255, c) nucleophilic addition of the TMS enol ether of pinacolone to N-acyliminium cation derived from 255, d) conversion of the ketone 256 to a silyi enol ether, ozonolysis with reductive workup affording 257 e) 0-methylation of the primary alcohol =

amide coupling

OH O I

I

MeO* NMe^H WIttig—"HornerEmmons 1CN Peterson

A '

OH^OH\OMe B' w mixed-cuprate coupling

PhosphorusFG

13^ Br O

240, A

•Try

242, C

241, B

Boc I

MeOgC,

N3

OBn

N^.O

\_7

''/

X

243, D

244, E

Fig. (11). Smith's retrosynthesis of enr-calyculin A and (-)-calyculm B

f) N-acylation of the amide to provide the coupling precursor E 244. Recently Armstrong and co-workers reported the total synthesis of calyculin C (101) which differs from the parent compound for the presence of an additional methyl group at C-32. As in other approaches, the initial disconnection at C25-C26 double bond through a Wittig olefination afforded the fragments C1-C25 and C26-C35. The synthesis of the C1-C25 subunit (Scheme 19) involved the initial formation of the C15-C25 spiroketal fragment 259 to which extension of the polypropionate chain was achieved via three subsequent steroselective allylborations. So, the aldehyde 259 was subjected to an allylation reaction via addition of allylmagnesium bromide-ZnCl2 mixture. The addition proceeded with complete Felkin-Anh substrate control affording the wrong stereochemistry at C-15 (compound 260), which was inverted through an oxidation (Swem)-reduction (LiBH^) two-steps procedure. After methylation of the C-15 hydroxyl group, ozonolysis of double bond

1241

PMBO

OTBSOTBS

Fragment C c=:> '^^^^A-cT ^^ '^ = i > < ^ ' y X > J^^^^^^^^OBn 242 Payne spiroketalizatjon HO ^ ^ o>ic " epoxidation 245 246 OTBS aoV^^^^^^'^^OBn = 247 PMR. dithiane coupling / ^ Y I

«

;>

.

s

H^ ^"^^ ^OBn

J>

248

249

Fig. (12). Smith's retrosynthetic approach to the C14-C25 fragment of enr-calyculin A

O

1 \yr-B(lpc)2 250

2-^202 OTBS OBn

iodocarbonate cyclization 252

247

Scheme 17. Smith synthesis of the C20-C25 fragment of enr-calyculin A. PMB

i V

a

H ^ J^

2. AC2O

t \

OTMS PMB

BFaEtgO

/

^

253

254 255 . 256

.^N^ O N ,^ BQN. T H F , ' ' ^ ' v ^ O H N a H , MeJ Vjv/^OMedeprotectlon^ ^^^v/^OMe 2.03, NaBH4 d

|-C 0.^0

e

*^ r \ 0^0

Boc-protection i i ^ 0^0

257 258 Scheme 18. Smith synthesis of the C33-C37 fragment of enr-calyculin A.

in 261 afforded an aldehyde which was subjected to an asymmetric Brown crotylboration reaction which occurred with complete diastereoselectivity, affording, after benzoylation compound 262. More

1242 ^OTBS

^OTBS

MEMO/,

1. Swern 2. LIBH4 3. methylation

MEMO/,^

ZnClp

OBz

^^^ 260

^OTBS

^OTBS

I.O3

BzCI OBz

OBz

2.

OMe

OBz OMe 262

-B(lpc)2

261

derived from (+)-lpc2BOMe, BF3-Et20 .OTBS

1.00

C1-C25 fragment several steps -B(lpc)2

II

OH

derived from (+)-lpc2B0IVIe BF3-Et20

OBz OMe

263

Scheme 19. Annstrong synthesis of the C1-C25 fragment of calyculin C (101).

problematic was the introduction of the ClO-Cll propionate unit. In fact, the subsequent ozonolysis-Brown crotylboration sequence was found to proceed with modest diastereoselectivity to yield a 35:39 mixture of homoallylic alcohols favouring the undesired anti-syn-anti isomer. After ozonolysis of terminal double bond, the introduction of the C1-C8 tetraene moiety in 263 was achieved following the same procedure reported by Evans in its synthesis of ^n^calyculin A. As in other synthetic approaches to calycuHns, the C26-C37 fragment of calyculin C (101) was initially disconnected at C33 amide bond to give the y-aminoacid C33C37 and the C26-C32 aminooxazole units. The first one was obtained in a straightforward manner from Z>-lixose whereas the aminooxazole unit was synthesised starting from L-pyroglutamic acid. Scheme 20 outlines key steps of the synthesis of the aminooxazole moiety. The endomethylation of the bicyclic MO-acetal 264, derived from L-pyroglutamic 1. pTSA .0 264

CH3I

2. MsCI, NEt3 265

^

(Boc)^^^"^^^^^'^^'''^^^^ NH2

267

32%

3. BugSnH, Nal, DME

1,3-Dichloroacetone^

266

(Boc)HN^3N/\j-'0.

K2CO3

N ^ 268

Scheme 20. Armstrong synthesis of the C26-C32fragmentof calyculin C (101).

1243 acid, was obtained with a 60% de using LDA followed by CH3L The C32 methyl group arose from radical deoxygenation of an in i-Z^w-generated iodide obtained from 265. The dimethyl-pirrolidone 266 was converted to the open chain amide 267 via N-Boc-protection followed by Weinreb aluminium-amide opening. The required oxazole fragment 268 was obtained through condensation of amide 267 with 1,3-dichloroacetone under vigorous reflux. Discodermolide (104) Owing to its exceptional pharmacological potential and scarcity of natural material [0.002% (w/w) from frozen marine sponge], discodermolide have stimulated intensive synthetic efforts [188]. The total synthesis of both the unnatural [189] and natural antipodes [190] [(-)- and (+)-104, respectively] by Schreiber and co-workers allowed to establish the correct absolute configuration.

PhS

Me

TBSO

Me

Me

TBSO

OH 272

Me

Me

OH

TBSO

273

n

\

Me

OH 272

^

Me (+)-3-hydroxy-2-mGthylpropionate I^^^COgMe

"^^^'^y' ®s^®''

OH Figure (13)- Schreiber's retrosynthetic analysis of discodermolide (104).

1244 To date, three syntheses of (-)-disco(lermolide [189,191] and two of the natural antipode [190,192] have been described. In this review we will report only the syntheses of the natural product (+)-discodermoUde (104). Very recently an alternative total synthesis of the natural (+)discodermolide has been reported by Paterson [193]. Schreiber and co-workers used an highly convergent approach, depicted in Fig. (13), which allowed the straightforward preparation not only of both enantiomers of discodermolide but also of a radiolabeled variant as well as of several structural variants of the natural product that should be useful in further characterizing the interaction of discodermolide with its receptor. From the retrosynthetic perspective, dissection of the skeleton at indicated sites revealed three fragments (269, 270, 271) of roughly equal complexity The repeating stereochemical triad embedded in the discodermolide backbone suggested that these fragment could arise from the homoallyUc alcohols 272 and 273 that, in turn, originated from a common precursor through asymmetric crotylboration addition to (+)-3hydroxy-methypropionate methyl ester. The homoallyUc alcohol 272 was oxidatively cleaved (Scheme 21) and homologated to the £-enoate 274 via a Wittig olefination. The hemiacetal obtained through reaction with benzaldehyde furnished the Michael adduct 276 with complete Me Me

Me Me

- Dur^ur* M® Me

^^'-^sXX^ I.O3, DMS ^ r^-'^V^^^^'^^COgMe TBSO OH " 2. Ph3P=CHC02Me JBSO OH 272

base ^ |-^'^V^'S;^C02Me 2.HF OR O ^ O

275 OHO

LDess-Martin 3. CSA

^.^

^-ti

Me^S^'/Me^ OH 278

OK/I^

PH^ n^J

Ph 276,R=TBS 277, R=H

Me^V^'^Me OTBS 269

Scheme 21. Schreiber synthesis of the C1-C7 fragment of discodermoUde (269).

Stereoselectivity. After deprotection of the primary alcohol and DessMartin oxidation of compound 277 to the corresponding aldehyde, the acetal 278 was obtained as a mixture of anomers through spontaneous cycUzation of the dimethyl acetal of the C-1 aldehyde with the 5-hydroxy group obtained by cleavage of the benzylidene acetal. The lactone ftinctionality at C-1 of discodermoUde was masked in its reduced form as thiophenyl acetal 269 obtained by treatment of methyl acetal with PhSSiMes, Znl2 and BU4NI. Elaboration of the C8-C15 fragment was achieved (Scheme 22) from the homoallyUc alcohol 273 in a high yielding three steps procedure involving a) silyl protection b) oxidative cleavage of the terminal double bond and subsequent construction of the

1245 cis-trisubstituted olefin by the method of Still and Gennari [194] (condensation of the obtained aldehyde with the potassium salt of Me

TBSO

Me

OH

"

Me

Me

15V.0H

^OPiv

-i^pjvCI

TBSO

273 •*(OMe)2 Scheme 22. Schreiber synthesis of the C8-C15 fragment of discodermolide.

(CF3CH20)POCH(Me)C02Me) and c) reduction of the carbomethoxy group to a hydroxymethyl group, affording compound 279. After protection of the C-15 hydroxyl group and selective deprotection of the C-9 primary alcohol, the acetylene derivative 280 was obtained via Swem oxidation and one carbon homologation using Gilbert's reagent. The third fragment was elaborated (Scheme 23) starting from the /7-methoxybenzyl ether derivative (281) of the homoallyUc alcohol 272. Ozonolysis of the terminal double bond fiimished an intermediate aldehyde which was converted to the diene 283 via Wittig reaction with Me

Me

1.03;NaHMDS

1.Pd{PPh3)3 CH2=CHZnBr

2. Ph3P''CH2l r TBSO

TBSO

HO

OPMB

OPMB

2.TFA

OPMB

283 271 Scheme 23. Schreiber synthesis of the C16-C24 fragment of discodermolide.

(iodomethylene)triphenylphosphorane followed by a palladium-catalyzed coupling of the resulting c/^-vinyl iodide 282 with vinylzinc bromide. Conventional functional group elaboration afforded the methyl ketone 271. The construction of discodermolide backbone, starting from the previously synthesized fragments had required a carefiil setting of proper coupling strategy and of a correct sequence of events (alkylation, reductions, introduction of the C19 carbamoyl functionality that would avoid functional groups' incompatibility and would furnish the best substrate control in order to obtain the desired stereochemical outcome (Scheme 24). The formation of the C7-C8 bond involved the NiCyCrCt mediated coupling of aldehydes and iodoacetylenes developed by Nozaki

1246 and Kishi [195]. The iodoacetylene 284, derived from 280 by treatment with iodine and morpholine, was coupled with aldehyde 269 yielding the propargilic alcohols 285 as a 2:1 mixture of epimers at C-7 favouring the desired one. Conventional functional groups elaboration afforded the allylic bromide 286, which was subjected to an alkylation reaction with the Hthium enolate of the ketone 271, to form the C15-C16 bond. The CMe

Me

^OPiv

.OPIv Me

Me 15^

269, NiCl2/CrCl2

^Me

Scheme 24. Schreiber's coupling strategy toward discodermolide.

16 methyl group was introduced with correct stereochemistry through a second alkylation reaction with methyl iodide, thus affording the full carbon backbone of discodermoUde (104). The final elaboration to discodermolide proceeded by transformation of the thiophenyl acetal at C-1 to the corresponding lactone, DDQ deprotection of the PMB ether, carbamoylation of the C-19 alcohol and reduction of the C-17 ketone. It was observed that reduction occurred with excellent selectivity for the correct R configuration thanks to the neighbouring carbamate group which provides significant chelation control. The highly convergent approach adopted by Marshall and co-workers in the total synthesis of (-i-)-discodermoUde (104) involves elaboration of three stereotriads, Fig. (14), through addition of chiral allenyltin, -indium, and -zinc reagents (of general structure 297) to (5)-2-methyl-3-silyloxypropanal (296). The resulting antUsyn- or ^-yn^^yn-homopropargylic alcohols adducts (294 and 295) were reduced to the corresponding (F)homoallylic alcohols (292 and 293) which, in turn, were subjected to asymmetric Sharpless epoxidation. Addition of Red-Al or methyl cuprate reagents to the resultant epoxy alcohols (290 and 291), respectively, afforded the key precursors aldehyde 287 and alcohol (289), after

1247 conventional functional group elaboration. Alkyne 288 arose from direct addition of the chiral allenylzinc reagent 297 (R=H, M=EtZn) to aldehyde 296 (Ri=TES). Formation of the C7-C8 bond was obtained through addition of the alkyne 288 (as the lithio species) to the aldehyde 287 followed by Lindlar hydrogenation and installation of appropriate protecting groups.

Me

i , ^

Me

Me

OH

Me

OCONH2

discodermolide (104) Me

Me

Me

.OSiEta

OR2

OR2

291 llsharpless Me

OR2

Me

0R2

293

B

reduction

SI ^n

syn

M€\ ( UQ 15^^^

OR2

121

OR2

OPIv

295

294 R=CH20Ac

« < . RiO^ OHO

296

CHaOPiv

Ri=TBS R2=PMP

M

Me/,^

•H 297

Fig. (14). Marshall's synthetic plan for discodermolide (104, Ri= TBS, R2 = PMP)

1248 The 13-hydroxymethyl group in the CI-CI 3 coupled subunit was converted to an aldehyde which was subjected to a Wittig reaction with a-iodoethyUdene triphenylphosphorane to give a (Z)-vinylic iodide (formation of the C13-C14 double bond). A palladium catalyzed Suzuki coupling of this vinylic iodide with a boranate derived from alcohol 289 led to the formation of the C14-C15 bond, thus completing the backbone of discodermolide. Final elaboration to the natural product involves cleavage of cyclic PMP acetal at C-1, transformation of the primary hydroxy group to a methoxycarbonyl, installation of the C-19 carbamate function, cleavage of the remaining alcohol protecting groups and lactonization. ABBREVIATIONS Ac Adda

= =

Bn Boc BOM Bz Cp DCC DDQ DIBAL-H DMAP DMPM DMS HMDS Ipc k-Arg LDA Mdhb MeCtrp METrp MEM MPM MOM NAZA NMO

= = = = = = z=

= = = = = = = = = = = = = = =

1

Acetyl (25, 35, 85, 95)-3-Amino-9-methoxy-2,6,8 trimethyl- 10-phenyl-4,6-decadienoic acid Benzyl f^r^Butyloxycarbonyl Benzyloxymethyl Benzoyl Cyclopentadienyl MA^*-Dicyclohexylcarbodiimide 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone Diisobutylaluminium hydride 4-Dimethylaminopyridine 3,4-Dimethoxybenzyl Dimethyl sulfide bis(trimethylsilyl)Amide Isopinocampheyl a-Ketohomoarginine Lithium diisopropylamide (A^-methylamino)dehydrobutyrate 6-Chloro-A^-methytryptophan Methyltryptophan 2-Methoxyethoxymethyl 4-methoxyphenylmethyl Methoxymethyl Nazumamide A 4-Methylmorpholine-A^-oxide

1249

OTf Piv PMP PPl Red-Al TBAF TBDPS TBS Tee TES TFA TFAA THF TMS TPAP Tr Ts pTSA V-Tyr Xc

z

Trifluoromethanesulfonate Pivaloyl 4-Methoxyphenyl Phosphatase 1 Sodium bis(2-methoxyethoxy)aluminium hydride Tetra-n-butylammonium fluoride ^^rf-Butyldiphenylsilyl ^^rf-Butyldimethylsilyl Trichloroethyl Triethylsilyl Trifluoroacetic acid Trifluoroacetic anhydride Tetrahydrofuran Trimethylsilyl Tetra-n-propylammonium pemithenate Trityl 4-Toluenesulfonyl 4-Toluensulfonic acid Vinylogous tyrosine Chiral auxiliary carbobenzoxy

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Tanimoto, N.; Gerritz, S. W.; Sawabe, A.; Noda, T.; Filla, S. A.; Masamune, S. Angew. Chem., Int. Ed. Engl. 1994, 55, 673-675. Yokokawa, R; Hamada, Y.; Shioiri, T. Chem Commun. 1996, 871-872. Smith, A. B., Ill; Friestad, G. K.; Duan, J. J.-W.; Barbosa, J.; Hull, K. G.; Iwashima, M.; Qiu, Y.; Spoors, P. G.; Bertounesque, E.; Salvatore, B. A. 7. Org. Chem. 1998, 55, 7596-7597. (a) Scarlato, G. R.; DeMattei, J. A.; Chong, L. S.; Ogawa, A. K.; Lin, M. R.; Armstrong, R. W. J. Org. Chem. 1996, 61, 6139-6152. (b) Ogawa, A. K.; DeMattei, J. A.; Scarlato, G. R.; Tellew, J. E.; Chong, L. S.; Armstrong, R. W. J. Org. Chem. 1996,67, 6153-6161. (c) Ogawa, A. K.; Armstrong, R. W J. Am. Chem. Soc. 1998,120, 12435-12442. Lavall6e, P.; Ruel, R.; Grenier, L.; Bissonnette, M. Tetrahedron Lett. 1986, 27, 679-682. Duan, J. J.-W.; Smith, A. B., Ill J. Org. Chem. 1993, 58, 3703-3711. (a) Paterson, I.; Wren, S.P. J. Chem. Soc, Chem. Commun. 1993, 1790-1792. (b) Clark, D. L.; Heathcock, C. H. J. Org. Chem. 1993, 58, 5878-5879. (c) Golec, J. M. C; Jones, S. D. Tetrahedron Lett. 1993,34, 8159-8163. (d) Evans, P. L.; Golec, J. M. C ; Gilliespie, R. J. Tetrahedron Lett. 1993, 34, 8163-8166. (e) Golec, J. M. C; Gilliespie, R. J. Tetrahedron Lett. 1993, 34, 8167-8169. (f) Yang, G.; Myles, D. C. Tetrahedron Lett. 1994, 35, 1313-?. (g) Yang, G. Myles, D. C. Tetrahedron Lett. 1994, 35, 2503-2504. (h) Paterson, I. Schlapbach. A. Synlett 1995, 498-500. (i) Filla, S. A.; Song, J. J.; Chen, L. Masamune, S. Tetrahedron Lett. 1999, 40, 5449-5453. Nerenberg, J. B.; Hung, D. T.; Somers, P. K.; Schreiber, S. L. J. Am. Chem. Soc. 1993, 775, 12621-12622. Hung, D. T.; Nerenberg, J. B.; Schreiber, S. L. J. Am. Chem. Soc. 1996, 118, 11054-11080. (a) Smith, A. B., Ill; Qui, Y.; Jones, D. R.; Kobayashi, K. /. Am. Chem. Soc. 1995, 777, 12011-12012. (b) Harried, S. R ; Yang, G.; Strawn, M. A.; Myles, D. C. J. Org. Chem. 1997, 62, 6098-6099. Marshall, J. A.; Johns, B. A. J. Org. Chem. 1998, 63, 7855-7892. Paterson, I.; Florence, G. J.; Gerlach, K.; Scott, J. P. Angew. Chem. Int. Ed. 2000, 39, 377-380. Still, W. C ; Gennari, C. Tetrahedron Lett. 1983, 24, 4405-4408. (a) Aicher, T. D.; Kishi, Y. Tetrahedron Lett. 1987, 28, 3463-3466. (b) Takai, K.; Kuroda, T.; Nakatsukasa, S.; Oshima, K.; Nozaki, H Tetrahedron Lett. 1985, 26, 5585-5588.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 26 © 2002 Elsevier Science B.V. All rights reserved.

1259

COPPER/TOPA QUINONE-CONTAINING AMINE OXIDASES - RECENT RESEARCH DEVELOPMENTS MAREK SEBELA, IVO FREBORT, MAREK PETRIVALSKY AND PAVEL PEC Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelii 11, 783 71 Olomouc, Czech Republic ABSTRACT: Amine oxidases (EC 1.4.3.6) that contain copper/topa quinone cofactor belong to a new protein group of quinoproteins emerging in recent years. This review brings together information on the general properties of the enzymes and their physiological functions. In plants, these enzymes are involved in processes of development and senescence, they reduce the concentration of toxic amines produced during exposure to stress conditions, provide hydrogen peroxide for wall stiffening and lignification and precursor compounds for biosynthesis of some alkaloids. Major attention is currently being paid to the structure of the active site of the enzymes that contains copper ions and a posttranslationally modified tyrosyl residue, topa quinone. Three-dimensional structures recently obtained for several amine oxidases by X-ray diffraction analysis of the respective crystals provide important structural information about the unique protein folding of the native enzyme and molecular arrangement of the active site. Biogenesis of the quinone cofactor is another issue that is addressed frequently at present. Differences between copper/topa quinone-containing amine oxidases and flavoprotein polyamine oxidases are outlined. Finally, future research directions on amine oxidases and the possibilities of their practical application are discussed.

INTRODUCTION The history of investigation into amine oxidases represents a fascinating interplay of several disciplines using different techniques and approaches. Study on the metabolism of biogenic amines takes its origin from the end of the last century, when organic chemists isolated various substances from biological material, among which were amino acids and a number of diamines and polyamines [1]. For example, the diamines, putrescine and cadaverine were isolated from putrefying animal organs during the years 1885-1887 and named accurately in accordance with their origin. After identifying the polyamine spermine as a phosphate salt in semen, it was isolated and crystallized in 1888. As these findings were then interpreted,

1260 the problem of the metabolic origin of these amines arose. During the period 1890-1910 decarboxylating conversions of the amino acids, lysine, ornithine and arginine were described, resulting in the respective diamines, cadaverine, putrescine and agmatine. The presence of putrescine and cadaverine in putrefied soybean extract was confirmed in 1910. Later, in 1927 the polyamine spermidine was discovered. The metabolism of biogenic amines then became the subject of interest of a newly developing discipline - biochemistry [1]. Currently it has been shown, that there is a new group of enzymes detectable in animal and plant tissues, which decrease the concentration level of amines by the oxidative deamination. These enzymes have been declared as amine oxidases [2]. The first in vitro study on amine oxidases was the demonstration of the presence of tyramine oxidase in liver preparations in 1928. A couple of years later, histaminase, an oxidase sensitive to carbonyl reagents was discovered. During the 30s and 40s, histaminase was reported in lung and later a diamine oxidase that oxidizes putrescine and cadaverine in porcine kidney. The activity of diamine oxidase was also found in plant extracts, especially those prepared from legumes. The enzyme from porcine kidney was partially purified and its identity with histaminase in this tissue was confirmed in 1951. Crude preparation of diamine oxidase from pea seedlings was achieved in 1956. The period 1960-1980 saw an intensive research on the pea and porcine kidney enzymes. Many of their substrates and inhibitors were described. These enzymes were also purified to homogeneity and the kidney enzyme was crystallized. The amine oxidases from bovine and porcine plasma were also characterized at this time. The classification of amine oxidases according the enzyme cofactor subsequently led to distinctions between flavin-containing amine oxidases and copper-containing amine oxidases, the latter including the above mentioned enzymes from pea and porcine kidney. A number of other amine oxidases belonging to these groups were isolated and their properties studied in detail: mitochondrial monoamine oxidase and polyamine oxidase from cereals (both flavin-containing amine oxidases), and a number of plant diamine oxidases (all copper-containing amine oxidases). Various proofs for the presence of a second (organic) cofactor in copper-containing amine oxidases began to appear. The cofactor was primarily thought to be pyridoxal phosphate due to several spectral characteristics. Nevertheless, it was never directly confirmed until the middle of 80s.

1261 In 1984, some indirect evidence was published identifying the organic cofactor of copper-containing amine oxidases with pyrroloquinoline quinone (PQQ). This substance had been known as a cofactor of some microbial oxidoreductases for several years [3]. Its identification as the cofactor PQQ was then indirectly confirmed using instrumental methods in a number of copper amine oxidases, including plant enzymes from pea and lentil. A major breakthrough in the study of amine oxidases as well as in the whole field of protein biochemistry came in 1990 when the existence of a novel cofactor was shown in bovine serum amine oxidase. HPLC isolation of cofactor peptides and their analyses using modem techniques such as amino acid sequencing and mass spectrometry, led in 1990 to the unambiguous disclosure of topa quinone (TPQ) in coppercontaining amine oxidase from bovine serum [4]. Study of amine oxidases then shifted to a new dimension. The occurrence of copper amine oxidase was demonstrated in a number of animal tissues showing high levels of metabolic activity, and also in plants, fungi, yeast and bacteria. The presence of topa quinone was shown in a number of enzymes showing the typical consensus sequence of the active site. Evidence was obtained for the mechanism of the catalytic reaction involving radical intermediate and autocatalytic biogenesis of topa quinone from a tyrosyl precursor, both in the presence of protein bound cupric ions. To date, the complete amino acid sequence is available for several amine oxidases, and some enzymes have been crystallized and analyzed by X-ray diffraction. All these achievements have facilitated better understanding of the protein structure of the enzyme and coordination geometry of the active site, thus providing direct insight into the mechanisms of enzyme action. When other quinonoid cofactors, tryptophan tryptophylquinone (TTQ) and lysyl-tyrosyl quinone (LTQ) were found in bacterial amine dehydrogenase and mammalian lysyl oxidase, respectively [5,6], a completely new class of quinoproteins (or quinoenzymes) in protein classification was declared. At the present time, research on copper amine oxidases revolves more and more around the elucidation of their physiological role. The enzymes of plant origin especially are subjected to intensive study on the interface between biochemistry and plant physiology since they are suspected of playing a key role in essential metabolic pathways in plants [1,7]. At the molecular level, the enzymes affect the concentration of biogenic amines in plant cell. It has been shown that the amines and the products of their catabolism participate in such delicate processes as cell division and

1262 proliferation, apoptosis, senescence, wound healing, and the mechanisms of defense against molecular stress and pathogen infection. Plant copper amine oxidase also takes part in the biosynthesis of several types of alkaloids. During recent years, a lot of work has been done on elucidation of the enzyme action in the lignification process. It has been confirmed that the hydrogen peroxide released by the amine oxidase reaction serves as an input compound for the formation of lignin and suberin. However, research on changes of the activity of copper amine oxidases during development and understanding of the genetic basis of its distribution in different plant organs, e.g. roots, stems and leaves, still represents a big challenge for plant physiologists. Copper amine oxidases Copper amine oxidases (EC 1.4.3.6) [CAOs, amine: O2 oxidoreductase (deaminating)] catalyze the oxidative deamination of biogenic amines to corresponding aldehydes and ammonia, accompanied by a two-electron reduction of molecular oxygen to hydrogen peroxide [7]: RCH2NH3'^ + O2 + H2O -^ RCHO + H2O2 + NH4"^

The enzymatic reaction follows a bi-ter ping-pong mechanism with participation of the quinone cofactor and Cu(II) ions bound in the active site [8]. The enzymes are irreversibly inhibited by carbonyl reagents and reversibly by copper chelating agents (non competitive inhibitors), substrate analogues (competitive inhibitors) and some alkaloids [9,10]. A former classification used the terms monoamine oxidase, diamine oxidase and polyamine oxidase [11]. However, the substrate specificity of the above enzymes is usually quite broad [12], and such a classification was far from accurate. Novel classification of copper amine oxidases comes from both the origin and substrate specificity of the enzymes [13] and includes four groups: (a) mammalian blood plasma amine oxidases showing high affinity for benzylamine and the polyamines spermine and spermidine, (b) mammalian diamine oxidases from kidney and placenta preferentially oxidizing the diamines putrescine and cadaverine, and also histamine, (c) plant diamine oxidases showing very high affinity for putrescine and cadaverine, also oxidizing spermidine at a high rate, (d)

1263 microbial amine oxidases preferentially converting arylalkylamines such as benzylamine, phenethylamine, tyramine and histamine (with the exception of methylamine oxidase). Representatives of all groups were studied in detail [13]. Best known enzymes of the group (a) are the amine oxidases from bovine and porcine blood plasma or serum; The enzymes from rabbit, sheep and equine plasma were also isolated. Mammalian diamine oxidases, the group (b), is represented by the enzymes from porcine kidney, human kidney and placenta, and rat colon. Group (c), the plant diamine oxidases, includes enzymes from legumes (Fabaceae). Several enzymes were purified to homogeneity and characterized in this regard. The starting material for purification is mostly etiolated plant seedlings, less often leaves. Best studied are the enzymes from seedlings of pea Pisum sativum, lentil Lens culinaris [10,12] and from latex of the Mediterranean shrub Euphorbia characias [14]. Finally, the group (d), microbial amine oxidases, includes the enzymes from the filamentous fungus Aspergillus niger, yeasts Hansenula polymorpha and Pichia pastoris. Gram-negative bacteria Escherichia coli, Klebsiella aerogenes and Gram-positive bacteria Arthrobacter globiformis and Arthrobacter strain PI [12,13]. All sources of amine oxidases have been comprehensively reviewed [12,13] and since the enzymes are widely distributed in many organisms and species, it is considered and generally accepted that they are ubiquitous.

Flavin polyamine oxidases Polyamines (namely spermidine) are oxidatively deaminated not only by the action of copper amine oxidases. Flavin-containing polyamine oxidases (PAOs, EC 1.5.3.-) are also involved in their catabolism. An alternative grouping divides both above mentioned catabolic enzymes into those that act on the primary amino group of di- and polyamines (diamine oxidases) and those that act on the secondary amino group of polyamines (polyamine oxidases) [15]. As suggested by Morgan, the latter should be further subdivided according to whether propane-1,3-diamine or 3aminopropanal is the reaction product [16]. The exact nature of the reaction products of PAOs depends on the source of the enzyme [1]. Mammalian PAOs transform spermidine and spermine into putrescine and spermidine, respectively, plus 3aminopropanal. By contrast, plant and bacterial polyamine oxidases

1264 catalyze the conversion of spermidine and spermine to 4-aminobutanal and l-(3-aminopropyl)-4-aminobutanal, respectively, plus propane-1,3diamine. The aminoaldehydes produced in the reaction spontaneously cycle to 1-pyrroline and l-(3-aminopropyl)pyrrolinium, respectively [17], the latter compound occurring mainly in the bicyclic form of 1,5diazabicyclo[4.3.0]nonane in the leaves of various cereals [17]. As typically observed in flavin-dependent oxidases, the overall reaction catalyzed by PAO can be divided into a reductive half reaction, in which the flavin is reduced upon polyamine oxidation, and an oxidative half reaction, in which the reduced flavin is deoxidized by molecular oxygen with the release of hydrogen peroxide [18]. Polyamine oxidation is likely to result in the formation of an imino compound, which is hydrolyzed to produce the final products. In contrast to CAOs [10], the polyamine oxidation by PAOs results in the production of aldehyde and hydrogen peroxide but not in ammonia [15]. In higher plants the wellknown pea seedling copper amine oxidase has been shown to oxidize spermine only very slowly, while spermidine is quickly converted to l-(3aminopropyl)pyrrolinium that undergoes spontaneous cyclization to 1,5diazabicyclo[4.3.0]nonane along with the formation of H2O2 and ammonia [10]. Spermine is oxidized by lentil seedling amine oxidase at the terminal (primary) amino groups producing a dialdehyde [19,20] Physiological aspects of copper amine oxidases Concerning prokaryotes, copper amine oxidases simply allow the organism to grow using the amine as a carbon and energy source. However, the general function of copper amine oxidases in higher organisms has not yet been well understood. The most significant role of the enzymes in plants and animals is considered to be catabolic regulation at the cellular level of biogenic amines, especially the diamine putrescine and poly amines spermidine and spermine. These amines play important roles in fundamental cellular processes such as tissue differentiation, cellular growth and proliferation, transformation in cellular cultures and tumor growth, wound healing, defense activity against parasites and probably also in programmed cell death, apoptosis [7,12,21]. Diamine oxidases found in animal tissues also participate in the metabolism of histamine [12,22]. Primary products of the enzymatic oxidation of mono-.

1265 di- and polyamines, aldehydes and aminoaldehydes, themselves have a regulatory effects on nucleic acids, but are further metabolized to other biologically active compounds [7]. In plants, the polyamines show growth effects, stabilize cellular membranes and delay senescence. Moreover, plant amine oxidases participate in the biosynthesis of some alkaloids and probably also provide hydrogen peroxide for the lignification and stiffening of the cell wall [23,24]. It has been also suggested that amine oxidase may show similarity to or even identity with cytokinin oxidase that cleaves the A^-alkylated adenine derivatives, known as the plant hormones cytokinins, on the secondary amino group [25]. However, CAOs are not known to oxidize secondary amines [12] and our recent results confirm that cytokinins are in fact weak competitive inhibitors of plant amine oxidase [26]. Molecular properties of copper amine oxidases With few exceptions [12,27], copper amine oxidases are homodimers in the native conformation. The molecular mass of the single subunit lies in the range 70-100 kDa. Most of the eukaryotic copper amine oxidases are glycoproteins. Their isoelectric point is usually slightly below pi 7.0, but some enzymes have pi > 7.0 such as pea seedling amine oxidase [12]. The amino acid composition is known for many amine oxidases and in the last decade, the complete amino acid sequence has been determined for a number of them. Some enzymes have been prepared in a crystalline form [12], four of them have been analyzed by X-ray diffraction with complete resolution of their three dimensional structure including detailed spatial conformation of the active site [28-31]. The quinone cofactor of copper amine oxidases Even in the early days of amine oxidase research, it became evident that the organic cofactor showing absorption at 500 nm and conferring a pink color to the enzyme contains a carbonyl functional group [32]. Until the mid eighties, it was believed that the cofactor was pyridoxal phosphate, but nobody succeeded in proving its presence experimentally [33,34]. In 1984, indirect evidence was shown of a covalently bound

1266 pyrroloquinoline quinone (methoxatin, PQQ) in the amine oxidase from bovine blood serum [35,36]. The PQQ had already been known for some time as a dissociable coenzyme of some prokaryotic oxidoreductases [3,37]. Covalently bound PQQ was then shown to be present in a number of other copper amine oxidases, but always indirectly [38-44]. O

O

?

««««—HN—CH—C —NH—CH —C—NH—CH—CCH, CONH,

(CHJ„_-1,2 COO,©

- Asn - TPQ - Asp/GIu -

Fig. (1). Topa quinone as a part of the cofactor consensus amino acid sequence in the active site of copper amine oxidases [4].

The beginning of a new era in the study of copper amine oxidases dates to 1990, when a group of scientists lead by Prof Judith Klinman from the University of California in Berkeley unambiguously eradicated the speculations about PQQ as the cofactor of copper amine oxidases [4]. A ^^c.iabeled phenylhydrazine derivative of bovine plasma amine oxidase was digested with protease thermolysine and the resulting peptides were separated by HPLC. Following the radioactive label, the pentapeptide Leu-Asn-X-Asp-Tyr was isolated in high yield allowing subsequent analysis by mass spectrometry. The unknown amino acid X was identified as a phenylhydrazone of the quinone form of 2,4,5trihydroxyphenylalanine (6-hydroxydopa quinone, named topa quinone, TPQ), Fig. (1). The structure was confirmed by synthesizing a stable model compound 5-(2,4,5-trihydroxybenzyl)hydantoin undergoing rapid autooxidation to the quinone, which was then derivatized with

1267 phenylhydrazine. The unique identity of the spectral properties of the model combined with those of the active site pentapeptide isolated from the amine oxidase from bovine plasma produced firm evidence that topa quinone is in fact the organic cofactor of the enzyme [4]. The results were further confirmed by resonance Raman spectrometry on comparing the spectra of phenylhydrazone and /?nitrophenylhydrazone of bovine plasma amine oxidase with the derivatized pentapeptides of the active site and the model compound. All these spectra showed great similarity in position (wavenumber) and spectral band intensity, while the spectrum of a PQQ model compound differed markedly [45]. Similar experiments confirmed the presence of topa quinone in porcine kidney, pea seedling and Arthrobacter PI amine oxidases. Moreover, the experimental data obtained for intact enzymes excluded the possibility of an artificial topa quinone formation during the proteolysis and peptide isolation [45]. Topa quinone is not a typical oxidoreductase cofactor, since it is an integrated part of the peptide backbone and must be formed in the active site by a posttranslational modification of a tyrosyl precursor. Search for this precursor exploited the data obtained from cloning and sequencing of the gene for peroxisomal copper amine oxidase from methylotrophic yeast Hansenula polymorpha [46]. The yeast is able to form two distinct amine oxidases depending on the inducing amine used in the cultivation medium, methylamine oxidase [46] and benzylamine oxidase [47]. The enzyme derivatized with phenylhydrazine gave, after proteolysis and peptide separation, the cofactor peptide Val-Ala-Asn-X-Glu-Tyr-Val showing the typical resonance Raman spectrum of the topa quinone derivative. Comparing the amino acid sequence obtained by translating the nucleotide sequence of the cloned gene with the peptide sequence obtained, it was found that the precursor of topa is a tyrosyl residue in a typical consensus sequence [47]. Later, the presence of topa quinone was accordingly confirmed in the amine oxidases from porcine serum and kidney and pea seedling by resonance Raman spectrometry of active-site labeled peptides [48]. Comparison of amino acid sequences of these peptides with the sequences of those from bovine plasma and H. polymorpha amine oxidases demonstrated the presence of a consensus sequence Asp-TPQ-Asp/Glu as shown in Fig. (1). Using the pH-dependent shift of the absorption maximum of the enzyme /^-nitrophenylhydrazone, which is considered to be a reliable indirect proof, the presence of topa quinone was also shown

1268 in the amine oxidase from seedlings of chick pea (Cicer arietinum) [48], fenugreek {Thgonella foenum-graecum) [49], sainfoin [50], grass pea (Lathyrus sativus) and sweet pea (Lathyrus odoratus) [51], etc. Recently, the cofactor peptides have also been isolated from semicarbazide-sensitive amine oxidases purified from bovine and porcine aortas [52], sequenced and confirmed to contain the topa quinone. The same topa quinone consensus sequence was also found in the primary structures of amine oxidases from human kidney [53], human retina [54] and rat colon [55], so called "amiloride-binding proteins", and amine oxidase from human placenta [56] that shows 81% identity with bovine plasma amine oxidase [57], bovine lung amine oxidase [58], and amine oxidases from pea and lentil seedlings [59,60], chick pea seedlings [61], and Arabidopsis thaliana [62] obtained by the molecular cloning of respective DNAs. The case of the two amine oxidase forms isolated from the filamentous fungus Aspergillus niger AKU 3302 after induction with w-butylamine is interesting. The dimeric AO-I and monomeric AO-II are encoded by a single gene, which was cloned and sequenced. The primary structure deduced then showed the cofactor consensus sequence Asn-Tyr-Glu, where the tyrosyl Tyr404 is the topa quinone precursor [27]. Recent results show that the AO-II is probably an unfolded inactive precursor of AO-I [63]. The enzymes were derivatized with /^-nitrophenylhydrazine and proteolyzed. Labeled peptides were then purified by HPLC and analyzed by ' H - N M R , MS, and resonance Raman spectrometry, which confirmed the presence of topa quinone in both enzymes. The fact that topa quinone in the derivatized peptides from AO-I was linked by an ester bond to the distant glutamyl residue Glul45 was surprising [64]. However, recent crystallographic data for the amine oxidase from Arthrobacter globiformis [30] and the alignment of its sequence with AOI from Aspergillus niger show that the Glul45 lies in the substrate channel leading to the active site, hence such a link may be created artificially in the derivatized enzyme. Quite recently, model compounds related to the proposed structure of AO-I cofactor have been studied. On the basis of their redox and spectral properties, the authors concluded that the cofactor has been misidentified. The above mentioned carboxylate ester may be considered an unlikely candidate for a biologically functional quino-cofactor [65]. The case of lysyl oxidase from bovine aorta is different. Here the native cofactor is formed by linked lysyl and tyrosyl side-chains (lysyl-tyrosyl quinone, LTQ) [6]. On the other hand.

1269 the lysyl oxidase from the yeast Pichia pastoris contains unmodified topa quinone in the active site and should be classified as a copper amine oxidase [66]. In the amine oxidase from Escherichia coli, the topa quinone was confirmed by a detailed analysis of the cofactor dipeptide X-Asp [67] and the resonance Raman spectrometry of the enzyme and its derivatives[68,69]. The primary structure of the enzyme also contains the cofactor consensus sequence [70]. More bacterial genes were shown to encode proteins containing the topa quinone consensus sequence, such as amine oxidase from Klebsiella aerogenes [71], phenethylamine oxidase and histamine oxidase from Arthrobacter globiformis [72,73], and methylamine oxidase from Arthrobacter strain PI [74]. Amino acid sequences around the position of the cofactor for selected amine oxidases from various sources are given in Table 1. The lengths of the complete amino acid sequences vary from 638 amino acids for A. globiformis phenethylamine oxidase to 762 residues for the enzyme from bovine plasma and the homology is in the broad range of 20-99%, but the important residues determining the enzyme action are conserved throughout all sequences [13]. The homology is high for the amine oxidases having the same substrate specificity obtained from similar organisms or organs, e.g., 92.2% for the amine oxidases from lentil and pea seedlings and 83.0% for the amine oxidases from E. coli and K. aerogenes, but becomes very low when comparing enzymes from bacteria and mammals (around 25%). There are 33 strictly conserved amino acid residues located mostly in the central part of the sequence (in the vicinity of topa quinone) and in the C-terminal region [13,60]. High homology of the primary structure at the C-terminus suggests its structural and functional importance. There are three conserved histidyl residues that are the copper ligands; two of which form the His-X-His motif and one is located near the C-terminus of the protein. Highly conserved asparagine and glutamate (or aspartate) residues in the consensus active site sequences of all known CAOs have been shown to be crucial in maintaining the balance of cofactor mobility versus rigidity expected to be necessary during the dual processes of biogenesis and catalysis, respectively, that all CAOs must accomplish. In addition, a structural linkage between these two highly conserved residues is proposed which spans both subunits of the dimeric CAOs, and may have implications for intersubunit communication [75,76].

1270 Table 1. Alignment of amino acid sequences of several copper amine oxidase around the position of topa quinone. The sequences were obtained by translation the corresponding cDNAs except for the enzymes from porcine kidney and porcine serum and the benzylamine oxidase from Hansenula polymorpha where they were determined by automated Edman degradation of peptides. Homologous consensus sequence around the cofactor is underlined, the tyrosyl precursor of topa quinone is shown as y.

Source of amine oxidase

Amino acid sequence

Reference

Bovine serum (monoamine oxidase)

SVSTMLNyDYVWDMVFYPNGAIE

[57]

Porcine serum (monoamine oxidase)

SVSTMLNxDYVXDMIFHP*

[48]

Porcine kidney (diamine oxidase)

DTSTVYNxDYIXDFIFYYN*

[48]

Porcine aorta (semicarbazide sensitive amine oxidase)

NxDYY*

[52]

Human kidney (diamine oxidase)

TTSTVYNyPYIWDFIFYPNGVME

[53]

Human placenta (diamine oxidase)

TTSTVYNyPYIWDFIFYPNGVME

[56]

Rat colon (diamine oxidase)

TTSTVYNyPYIWDFIFYSNGVME

[55]

Pea seedlings (diamine oxidase)

TIVTVGNyPNVIDWEFKASGSIK

[60]

Lentil seedlings (diamine oxidase)

TVVTVGNyDNVLDWEFKTSGWMK

[59]

Chick pea seedlings (diamine oxidase)

TVVTVGNyPNVLDWEFKTSGWSI

[61]

Arabidopsis thaliana (ataol)

MVATLGNyPYIVDWEFKKSGAIR

[62]

Aspergillus niger (monoamine oxidase, AO-I)

FIITLANyEYIFAYKFPQSGGIT

[27]

Hansenula polymorpha (methylamine oxidase)

QIFTAANyEYCLYWVFMQPGAIR

[46]

Hansenula polymorpha (benzylamine oxidase)

VANxEYV*

[47]

Escherichia coli (monoamine oxidase)

WISTVGNyPYIFDWVFHPNGTIG

[70]

Klebsiella aerogenes (tyramine oxidase)

WISTVGNyPYIFDWVFHPNGTIG

[71]

Arthrobacter globiformis (phenethylamine oxidase)

FFTTIGNyPYGFYWYLYLPGTIE

[72]

Arthrobacter globiformis (histamine oxidase)

FFTTVGNyPYGFYWYLYLPGTIE

[73]

Arthrobacter sixdAU PI (methylamine oxidase)

FIATVANyEYAFYWHLFLDGSIE

[74]

*t[ie unknown amino acid x in the consensus sequence represents the derivatized topa quinone in the sequenced peptide

1

1271 Catalytic mechanism of copper amine oxidases Each subunit of the copper amine oxidase binds one Cu(II) ion. The two cupric ions are indistinguishable in the EPR spectrum. Spectroscopic properties of the copper bound in the enzyme are similar to those of low molecular tetragonal, square-pyramidal or planar complexes of Cu(II) of so called type 2 "nonblue" copper [12,77,78]. Parameters of the EPR spectrum for the enzyme-bound copper correspond to tetragonal complex of Cu(II) with N- and O- ligands [79]. Changes in the EPR spectrum after the reaction of the enzyme with phenylhydrazine or after anaerobic addition of substrate suggest possible interaction of Cu(II) and the organic cofactor [80]. A model of the Cu(II) complex in copper amine oxidases was constructed on the basis of several spectroscopic studies [81-84]. The copper is coordinated in square-pyramidal complex by four equatorial ligands, three nitrogen atoms of histidyl residues, one water molecule, and one axial water ligand. The accuracy of the model was recently confirmed by resolving the crystal structure of the amine oxidase from Escherichia coli [28], as shown in Fig. (2). The distance of the Cu(II) and quinone group of the cofactor favors direct electron transfer between both prosthetic groups [28,29,85]. However recently it has been found that the electron transfer proceeds rather via an integrated water network [31]. The copper can be reversibly removed from the active site by reaction with diethyldithiocarbamate under non-reducing conditions [86-88], or by cyanide after reduction by dithionate to Cu(I) [78]. The catalytic activity of the enzyme can be restored with high yield by addition of free Cu(II) ions to the apoenzyme [78,86]. In the case of pea seedling amine oxidase, addition of other bivalent metal ions does not lead to reactivation [86]. However the activity can be partially restored (from 15%) for the amine oxidase from bovine plasma by adding Co(II) [89]. Addition of Co(II) and Ni(II) can restore the original spectrum of the native enzyme with bovine serum amine oxidase reduced by dithionate [90]. The main role of copper in the active site is to keep the essential amino acid residues in a geometry favorable for substrate binding [78,91] and participate in the reoxidation of the substrate reduced organic cofactor [86,88]. Hypotheses that the copper acts as a Lewis acid [92] or only indirectly participates in the catalysis [78] were based on unsuccessful attempts to detect changes in its redox state by EPR spectroscopy [93].

1272

Fig. (2). Ribbon diagram of the three-dimensional crystal structure of copper amine oxidase from Escherichia coli [28]. Similar structures of amine oxidases from pea seedlings [29], Arthrobacter globiformis [30] and Hansenulapolymorpha [31] lack the domain Dl.

It has been found only recently by EPR spectroscopy that a transition state of Cu(I)-semiquinone radical that might be the looked-for catalytic intermediate directly reacting with oxygen, is generated during anaerobic reduction of the enzyme with a substrate at a laboratory temperature [94]. EPR spectrum of this radical is identical to that of substrate reduced amine oxidases measured in the presence of Cu(I)-stabilizing ligands [84,94,95]. This observation lead to the model of the mechanisms of catalytic reaction as is shown in Fig. (3). The catalytic cycle is composed of two phases [21,96-98]. In the anaerobic phase, the interaction of topa quinone with the primary amino group of a substrate leads to the formation of a quinoketimine. Abstraction of the a-proton of the substrate by a catalytic base (amino acid residue of the active site) then forms a carbanion that is quickly converted to colorless quinoaldimine. The quinoaldimine is then hydrolyzed and releases the product aldehyde, forming a reduced form of the cofactor, aminoresorcinol [99,100]. The Cu(II)-aminoresorcinol formed then exists in equilibrium with the radical

1273

Fig. (3). Mechanism of the substrate oxidation by copper amine oxidases [29]. The scheme shows the roles of copper, topa quinone cofactor and proton abstracting base (Asp) in the catalytic cycle. The oxidized enzyme (a) reacts with an amine substrate giving a Schiff base formation at C-5 of the TPQ (b-c), followed by proton abstraction (d). After hydrolysis and release of the aldehyde, an aminoresorcinol species is formed (e), and the reduced cofactor is reoxidized by molecular oxygen via Cu(I)-semiquinone intermediate (/).

form of Cu(I)-semiquinone, semiquinolamine. Its reaction with molecular oxygen in the aerobic phase leads to release of hydrogen peroxide and ammonia, and reoxidation of the cofactor that completes the catalytic cycle [97-100]. The equilibrium between the intermediates Cu(I)semiquinone and Cu(II)-aminoresorcinol is maintained by rapid electron transfer between copper and the organic cofactor [100,101]. At low temperature, the equilibrium is shifted to the Cu(II)-aminoresorcinol state unless cyanide or other stabilizer is added [84]. Copper-depleted bovine serum amine oxidase has been recently reconstituted with Co(II) ions. The benzylamine oxidase activity of the enzyme was increased to 20% on incorporation of cobalt. Furthermore, Co(II) restored to nearly native level the intensity of the absorption spectrum and the reactions with phenylhydrazine or benzylhydrazine, which had been slowed down or abolished, respectively, in Cu(II)depleted samples. The amine oxidase activity of the Co(II)-derivative, which cannot form a semiquinone radical as an intermediate of the

1274 catalytic reaction, strongly suggests that the Cu(I)-semiquinone is not an obHgatory intermediate in the catalytic cycle of bovine serum amine oxidase [102]. Recently, evidence has been obtained by X-ray absorption spectroscopy that the Cu(I) complex formed after substrate reduction of the enzyme can directly react with dioxygene [103]. This observation is further supported by steady state-kinetics exploring isotopic effects on the oxidative half-reaction of amine oxidase [104]. On the basis of a recently determined X-ray structure for Hansenula polymorpha amine oxidase the authors predicted the existence of a hydrophobic oxygen-binding pocket that is located near the 0-2 position of the reduced cofactor [31], Fig. (1). X-ray crystal structures of three species related to the oxidative half of the reaction of the copper amine oxidase from Escherichia coli have now been determined. Crystals were freeze-trapped either anaerobically or aerobically after exposure to substrate, and then the respective 3-D structures were determined. The oxidation state of the quinone cofactor was investigated by single-crystal spectrophotometry. The structures revealed the site of bound dioxygen and the proton transfer pathways involved in oxygen reduction [105]. The quinone cofactor is regenerated from the iminoquinone intermediate by hydrolysis involving Asp383, the catalytic base in the reductive half-reaction. Product aldehyde inhibits the hydrolysis, making release of product the rate-determining step of the reaction in the crystal. Spectral properties of copper amine oxidases The absorption spectrum of the copper amine oxidases shows a characteristic broad band at around 500 nm (460-510 nm) that confers a typical pink or yellow-pink color to highly purified enzyme preparations [10,12,32]. Absorption in the visible region is caused by the presence of the quinone cofactor and thus is not affected by removal of copper from the enzyme under non-reducing conditions [12,32]. The quinone cofactor shows emission of fluorescence when excited at 280 and 365 nm [41,106]. Electron transition of Cu(II) is seen in circular dichroism spectra at 600-800 nm [12,32,107]. Under aerobic conditions, addition of the substrate leads to a temporary bleaching of the pink color of the enzyme, which is restored after complete consumption of the substrate. However, anaerobic conditions lead to the formation of a stable yellow intermediate

1275 with the absorption maxima at 350, 435 and 465 nm [86,88]. Addition of oxygen then causes rapid reoxidation of the enzyme and restoration of the absorption at 500 nm [99]. The process of reoxidation requires the presence of enzyme bound copper. It does not proceed with a copper depleted and substrate reduced apoenzyme [86,88]. Characteristic absorption spectra of intermediates formed during the catalytic turnover of copper amine oxidases are given in Fig. (4).

O

a cd

o
ostelium discoideum 80 1,2-Didehydroaspidospermidine 1050 re/ro-Diels-Alder reaction 111,138 Diels-Alder reaction 172 Diethyldithiocarbamate 1271 Digenea simplex 41S Digitalis 174 Digitolutein 806 Dihydroangustine 103 6,1037 Dihydroartemisinin 832,833,834 16,17-Dihydrobrachycalyxolide 811,812 Dihydrocubebin 191 Dihydrocyclotheonamide A 1194,1195,1196 5,6-Dihydroflavopereirine 1060 we^o-Dihydroguaiaretic acid 191,240268, 271 Dihydroguaiaretic acid 219,249,473 DihydropenicilHc acid 449 Dihydrophenoxazine 976 1,2-Dihydrosantonin 463 20,21 -Dihydrostrychnobrasiline 1065 Dihydrosweroside 104 8,10-Dihydrosweroside aglucone 141

1314

18,19-Dihydrousambarine 1063 5',6'-Dihydrousambarensine 1063 Dihydroxanthommatin 988,989 3',4'-Dihydroxiflavone 757 2',5'-Dihydroxy-3'-nitrophenylacetic acid 592 Dihydroxybenzoic acid 337 2-Dihydroxymintlactone 456 Dihydroyashabushiketol 883 3,8-Dihyroxy-glucoside-2-hy droxymethy lanthraquinone 657 1,4-Diisothiocyanatobenzene 481 Dimerization ofresveratrol 532 Dimers 544,545,546,547,548,549 ampelopsin D&F 544 anigopreissin A 544 betulifolA&B 545 gnetinA&C 546 gnetinF&G 546,547 leachianol G 547 malibatol A 547 parthenocissin A 548 restrytisol A-C 548,458 tricuspidatol A 549 viniferifuran 549 (+)-e-viniferin 545 l,2-Dimethoxy-4-(£-3'-methyloxiranyl) benzene 362 3,4-dimethoxybenzoic acid 1013 10,13-Dimethyl-9-tetradecen-1 -ol 66 Dimethylallyl-haempferide 802 3',4-O-Dimethylcedrusin 202,269 Dimethylcrocetin 303 (+)-Dimethylisoariciesinol-2-a-xylose 195 7,11-Dimethyloleoside 334,343 9,13-Dimethyltetradecanoic acid 65,66 10,13-Dimethyltetradecanoic acid 65-67 Diminazene (berenil) 786 Dinocophylline B&C 822,823 Dioncopeltine A 822,823 Diosmetin 746,756 Diosmin 752,755-757,759,766 Diospyrin 804 Diospyros montana (Ebeaceae) 804

Dioxapyrrolomycin 488 Dipetalolema viteae 487 Diphylleia 172 Diphyllin 226 Diphyllin acetate 194 Diphyllin acetyl apioside 263 Diphyllin apioside-5-acetate 226 Diphyllin asioside 226 Diphyllin natural products 166 Diphyllinin 194 Diphyllinin crotonate 194 Diphyllinin monoacetate 194 Diphyllin-O-apioside-5-acetate 194 Diphyllin-(9-apisode 194 Diquat 374 Dirca occidentalis 219 genkwanin from 219 (+)-lariciresinol from 219 sitoindoside II from 219 Discobahamins A&B 1202 Discodermia 1175 Discoderm ia calyx 1204 Discodermia dissoluta 1205 Discoderm ia kiiensis 1201,1204 Discodermia sp. 1201,1202 Discodermide 1206 Discodermins A-D 1201,1202 Discodermins E-H 1201,1202 Discodermolide 1205,1206,1243,12451248 Discokiolide A-C 1203 Distichol 562 D ithi ins dipsaci 437 Ditylenchus destructor 460 Ditylenchus dipsaci 493 (-)-3, 4-Divanillyltetrahydroftiran 191 Divarine 1056 L-Djenkolic acid 481 DNA damage effect 984 DNA intercalating activity 1060 DNA repair 925 DNA topoisomerase II 1060 DNA-intercalators 805 Doliocarpus dentatus 229 (-)-liriorinol B from 229

1315 (+)-medioresinol from 229 (+)-pinoresinol from 229 Domoic acid 479 Drechslera rostrata 227 Dreschlera spp. 468 Drosera capemis 1158 Dryopteris filixmas 431 Dysidea herbacea 463 Dysosma 172

a-Ecdysone 700 Ecdysteroid antagonists 555 Ecdysteroids 699,700 Ecdysterone 700 Echimidine 874,876 Echium sp. 872 Echium wildpretti 874 Ectyoplasiaferox 66 Edman degradation 1270 Effective insect growth inhibitors 858 calopeptin 858 dehydrodiisoeugenol 858 galbacin 858 galbegin 858 licarin 858 licarinB 858 sesamin 858 sesamolin 858 veragensin 858 Eflomithine 787,792 Ehrlich ascites carcinoma 303 Ehrlich's reagent 1277 Eichhornia crassipes 359 (5Z,9Z)-5,9-Eicosadienoic acid 80 (11Z, 157)' 11,15-Eicosadienoic acid 89 (1OZ, 15Z)-10,15-Eicosadienoic acid 90 9-Eicosene 361 Eicosyl ferulate 666 Eimeria 804 Elaiophylin 453 Electrophile-Responsive Element 951 Eleocharis microcarpa 361 Ellagic acid 359

Ellipticine 828,829 Emaciation 690 Emanthidine 614 Emetic effect 239 Emetine 144 from Cephaelis ipecacuanha 144 Emodin 637 Encephalartos 1157 Encephalartos friderici-guilielm i 1158 Encephalartos longifolius 1157 Encephalitizoon intestinal is 788 Endlicheria dysodantha 211 dysodanthin A&B from 212 megaphone acetate from 212 Endopolygalacturoase 1133 Endothelin antagonism 237 Endothelin-1 267 Endothelium-Derived Relaxing Factor 951 EnniatinA&B 482 Entamoeba histolyca 836,1063 Enterobacter cloacae 402,1091 Enterodiol 191 Enterofuran 191 Entero lactone 192 Enterolactone lignans 209 Ent-kaur-16a-o]-19-oic acid 811 Ent-kauran-16-en-19-oic acid 811 Enzyme-linked immuosorbent assay 1125 Ephedra spp. (Ephadraceae) 235 ephedradine B from 235 Ephedradine B 235 (-)-Epiasarinin 260 (+)-Epiaschantin 201 (+)Epiashantin 267 10-Epibengazole A 488 Epicalyxin G 902 Epicatechin-3-gallate 359 Epicatechins 748,751 Epicatechins gallates 770 Epicinnzeylanol 850,853 2'-Epicycloisobrachycoumarinone epoxide 798 Epidermophytonfloccosum 227,228 14-Epidihydrocochlioquinone B 460 (-)-Epieudesmin 201

1316

Epigallocatechins 748 Epigallocathechin-3-galIate 755 (+)-Epimagnolin 201 Epiphyas postvittana 231 (+)-Epipinoresinol 201 dimethyl ether 201,241 Epipodophyllotoxin 155,155,210 4-Epipodophylltoxin 193 Epipodorhizol 192 (+)-Epiresinol 268 13-Episclareol 397 (-)-Episesamin 201 (+)-Episesamin 260 Episesmin 246 Episteganangin 196,216 Epithelizing effect 335 (+)-Epiyangambin 201 Epiyangambin 242 Epothilione 811 EpothiloneA 810 10,13-Epoxy-l 1-methyloctadeca-lO, 12dienoic acid 69 Eragrostis curvula 469 pyrocathecol from 469 Erigeron philadelphicus 436 Eriocitrin 744 Eriodyctiol 757 Erylus formosus 80,85 Escherichia coli 330,331,333,402,556, 605,802,1092,1093,1135,1263,1269, 1271,1272, 1274, 1278 Esculetin 314,315,317,318,330-334, 336-340, 342,344 Esculin 317,318,330-342 Espintanol 797,798,807 Estradiol 222 Estrogen agonist/antagonist properties 210 of secoisolariciresinol diglycoside 210 Estrogenic activity 760 3-[a-Ethoxy(a-methyl-thio)]methyl-2thionopyrolidine 1083 Etopophosphate 160 Etoposide 160-163,171,172,212,213 use in combination therapy 171 Etoposide phosphate 160

of lymphoid cancer 171 of myeloid leukemia 171 of refractory testicular cancer 171 of stomach, of ovarian, of brain, of breast, of pancreatic, small and non-small lung cancer 171 Eucalyptus grandis 445 Eucalyptus haemastoma 696 Eucommia ulmoides 235 (±)-pinoresinol diglucoside from 235 (-)-Eudesmin 200 Eudesmin 268 Euglena gracilis 363 Eumelanins 1018 Eunicea Sliceinea 83 Euphorabia characias 1263,1277 Eupomatenoid-5 202 Eupomatenoid-6 232 Europine 874 Eurotium chevalieri 1091,1093,1094, 1097-1099 Evans chiral oxazolidine 1226 Evodia rutaecarpa 487 Exiguaflavanone 801,803 Exoerythrocytic cycle 781 Fagraea racemosa 268 (+)-epiresinol from 268 (+)-isolariciresinol from 268 (+)-pinresinol from 268 (+)-Fargesin 201,267 Fargesin 233 Fargesone A-C 233 Farnesol 331,332 Fenton rection 975,995 Fenugreek (Trigonellafoenum-graecum) 1139,1141,1268 Ferrihemoglobin 985,986,991 mediated oxidation 991 Ficin flavonoids 744 Filospadin flavonoids 744

1317 Fischer ella ambigua 364 Fischerella JAVA 94/20 366 Fischerella muscicola 366,364 F/5c/zere//aNEP95/l 366 Fischerellin 370,374 Fischerellin A 364,368 Fisetin 757,758 Flavin adenine dinucleotide 917,918,951 Flavin mononucleotide 917,918,951 Flavonol quercetin 753 Flavonols 744 Flavonone 743,744 Flexuosol A 569 Fluorenone 655 Fluorescence spectrometry 378 FMN-binding domain 920 Foeniculoside I-IV 556,560 Formononetin 745 l-0-Formyl-4'-Demethoxy-3',4'-methylenedioxymethylrocaglate 203 A^'-Formyl-kynurenine 966,998 8-Formyloct-5-enoate 86 Forsythia 327 Forsythia intermedia 156,174,177, 223 (-)-arctigeninfrom223 Forsythia spp. 156,157,235 pinoresinol monoglucoside from 235 Forsythia suspensa 156,241 Framoside 327,334,343 Fraxetin 314,315,318,331-334,336-340, 342 Fraxidin 315 Fraxin 314,317,318,331-334,336,338-340, 342 Fraxinol 315 Fraxinus 327,333,343 Fraxinus bungeana 317 Fraxinus chinensis 317 Fraxinus formosana 327 Fraxinus insular is 321 insularoside from 321 Fraxinus ornus 313-319,327,328,330, 332,334-340,341,343-345 Fraxinus stylosa 317 cichorin from 314

esculetin from 314 fraxetinfrom314 fraxin from 314 hydroxycoumarin esculin from 314 quercetin from 314 quercetin-3,7-digalactoside from 314 quercetin-3-galactoside from 314 quercetin-3-glucoside from 314 rhamnetin from 314 rutin from 314 Fraxinus uhdei 321 uhoside from 321 Fructus schisandraei 233 Fucus garderi 1152 Fumagillin 458,459 Fumagillol 459 Fumarate dehydrogenase 800 Fungal polysaccharides 1124-1129 Furano flavonoids 744 Furoftiran lignans 200,201 Furanoheliangolides 808 Fusarium solani 404,405 Fusarium spp. 464,482 Futoenone 245 Futoquinol 245 (+)-Galeon 899 Galactomannan 1148 Galacturonorhamnans 1156 Galangin 752 Galanthamine 614 Galanthine 614 Galinum sinaicum 220 bis-glucoside derivatives from 220 Galiosin 657 Galium 631 Gallic acid 359,798 Gallic-/?-coumaric acid 359 Gallic-sinapic acid 359 Gallocatechins 748 Ganoderma lucidum 449 Garbanzol 745 Garcinia cowa 805

1318

Garuga sp. 899 Garuga pinnata 899 Garuganinlll 899 Geduin 815 Geissoschyzine 97 General skeleton 190 oflignans 190 Genistein 750,751,754,757,760,802,803 Genkwanin 219 Genotoxicity assays 675 Geodia 488,1210 Geosmin 353,354 Geotrichum candidum 673 Geotricum louberi 673 GermacreneD 409,413 Germanocrenolide 808 Ghosez cyclization 1230,1231 Giardia intestinalis 1063 Gilbert's reagent 1245 Gingerol 475 Ginko biloba L. 929 GL-311162 used for anticancer treatment 162 Glaucarubin 837 Gleditsia macracantha 1140 Gleditsia thacanthos 1115,1116,1139, 1143 Globodera 429,432,467,471 Globodera pallida 229,474 Globodea rostochiensis 229,430,461, 474,477,486,487 Globodera spp. 428 Glossina spp. 785 Glucantime 828 Glucosinolates 1074 enzymatic breakdown of 1074 Glucotropolin 481 Glucuronomannan 1156 Glunapin 481 Glutamate synthetase (GS) inhibitor 372 y-Glutamylcysteine synthetase 913,951 Glutathinonylspermidine 808 Glutathion reductase inhibition 1036 Glutathione reductase 796 Glycanoxylans 1156

Glyceollin 473 Glyceollin Mil 473 Glyceraldehyde-3-phosphate dehydrogenase 913,951 Glycine max L. 930 Glycinoeclepin A 466 Glycofomis 1131 Glycolysis (GADPH inhibition) 924 Glycoproteins 1129-1137 Glycosidation-induced shift method 8 Glycosphingolipids 1123 Glycyrrhiza glabra 800 GnetinA-C 535,565 GnetinE-I 565 Gnetum parvifolium 530 Gomisin A-C 197,247,256-258,263,265 Gomisin G 223 Gomisin J 197,224,247,263 Gomisin K 197,247 Gomisin L 197,247 Gomisin M 197,240 Gomisin N 197,247,249 Gossypium hirsutum 463 GraminoneB 233,235 Grandisin 198,230,799 Granulocyte phagocytosis 47 of jenisseensosides C&D 47, 55 Gvdiss \iQdL {Lathyrus sativus) 1268 Grevillea robusta 1157 Guaiacylglycerol-p-(9-6'-(2-methoxy) cinnamyl alcohol 211 Guar (Cyamopsis tetragonolobus) 1139 Guayacasin 249 Guiaflavine 1055,1056 Guianensine 1055,1056 Guppy fish (Lebistes retculatus) 689 Gypsogenic acid 29,36 Gypsophila arrostii 38,39,40 Gypsophila bermejoi 44,52 Gypsophila capillar is 42,43 Gypsophila capitata 54 Gypsophila fastigiata 3 8 Gypsophila oldhamiana 41 Gypsophila paniculata 38-40,42,53 Gypsophila paniculata/muralis (baby's

1319 breath) 3 Gypsophila perfoliata 38 Gypsophila saponins 54 Gypsophiloides 3 Gyrocarpine 824,826 >H and ^^C spectra 8,9,22,30,33 of aglycone of dianchinenoside G & H 36 of bisdesmoside41 of dianchinenoside E 33,34 of dianchinenoside F 37 of dianchinenoside G and H 35 ofSAPO30 41 ofSAPO50 41 of saponarioside A 22 ofvaccaroside F30 HA 1002-1004 as a carcinogen 1002-1004 Haber-Weiss reaction 975 (+)-Haedoxan A&B 231,232 (-)-Haedoxan D 231 (+)-Haedoxan D 231 Haemonchus 425 Haemonchus contortus 429,431,432,440, 446, 451,453,457,470,482,484,488,490, 491 Hakea 1157 Haminaea templadoi 90 Hamycin 794,795 Hannoa quassia undulata 466 Hansenula polymorpha 1263,1267,1270, 1272,1274 Hansenula sp. 1087 Hapalindole A 363 Hapalosiphon intricatus 363 Hapalosiphorn fontinalis 363 Haplophyllum hispanicum 263 diphyllin acetyl apioside from 263 tuberculatin from 263 Harmaline 828,829 Harman 828,829 Hattalin 216,269 ouabain receptor 216

Hedera helix 813 Hederagin 814 a-Hederin 814 P-Hederin 814 Helenium 436 Helicoverpa zea 697 Helicteres isora 226 helisorin from 226 helisterculin A&B from 226 Heliothis armigera 850,851 Heliothis virescens 439,485 Heliotropium bovei 874 Heliotr opium floridum 874 Heliotropium megalantum 874 Heliotropium sinuatum 874 Heliotropium sp. 872 Helioxanthin 194,232 Helisorin 226 Helisterculin A&B 226 Helmindiol 451 Helm inthosporium gram ieu 1210 Helycobacter pylori 693 Hemangiomas 693 Hematopoietic cancer cells apoptosis 925 Hemicenttrotus pulcherrimus 1188 Hemoglobin / HA interactions 985-987 Hemolytic activity 41, 42 of Gypsophila paniculata 41 ofSAPO30 41 ofSAPO50 41 (Z)-4-Heneicosenal 81 Hepatic activity 253 Hepatocarcinogenesis 257 Hepatoprotective acitivity 255,257 Hepatospienomegaly 785 Heptamethoxyflavone 752 Heptanoic acid 361 15P-Heptylachaparrinone 815 Herbivorous crustacean zooplankton 355 Hernandia nymphaeifolia 218 (-)-deoxypodophyllotoxin from 218 epiaschantin from 218 epimagnolin from 218 epiyangambin from 218 (-)-yatein from 218

1320

Hernandia nymphaeifolia (Hemandiaceae) 212 (-)-hernone from 212 (-)-6'-hydroxyyatein from 212 (-)-nymphone from 212 Herniaria fontanesii 52,54 Herniaria glabra 50,51 Herniaria hirsuta treatment in arrthritis 50 in infections of urinary & respiratory tracts 50 in kidney disorders 50 in neural catarrh 50 in neuritis 50 in purifying the blood 50 in rheumatism 50 Herniaria saponins A-D 50-52 (-)-Hemone 199 Herpes simplex virus (HSV) 223 Herpetomonas samuelpessoai 1138 Hesperetin 757,762 Hesperidin 744,745,752,759,766,768 HETCOR 11,13,34,37 of bisdesmoside41 of dianchinenoside E&F 34 ofSAPO 30,41,50 Heterakis spumosa 482 Heteroclitin F&G 259 Heterodera 429,432 Heterodera cajani 469 Heterodera glycines 429,454,456,466, 467, 473,477 Heterodera rostochiensis 437 Heterodera schachtii 481 Heterorhabditis UQmditodQS 471 re^ro-Hetero-Diels Alder reaction 113 HeyneanolA 568 Hikizimycin 493 (-)-Hinokinin 192,215,216 Hinokinin 216,231 Hippamine 610,614 Hippeastrine 614 Hippeastrum sp. 614 His2i5 family of inhibitors 368

Histamine 759 Histamine-induced hind paw edema 401 Histiopteris incisa 722 HIV-1 RT inhibition by anolignan A&B 224 HK-mediated neurotoxicity 992 HMBC correlations 9,13,15,21,25,29,34 of bisdesmoside 41 of dianchinenoside E 34 ofSAPO 30,41,50 of saponarioside C 15,25 of saponaside A 21 of vaccaroside D 29 HMQC/HSQC experiment 11,34,37 of bisdesmoside 41 of dianchinenoside E&F 34 ofSAPO 30,41,50 'H and '^C NMR data of 321,324,326,329 of hydroxyframoside A & B 326 of hydroxyomoside 321 of insularoside 321 of isolugrandoside 329 of isolugrandoside acetate 329 iR NMR data 538,539,540,541,542,543 of ampelopsin A 538 of ampelopsin D 538 of ampelopsin F 538 of anigopreissin A 538 of balanocarpol 539 ofbetulifolA539 of betulifol B 539 of cyphostemmin A 539 of cyphostemmin B 540 of gnetin A 540 ofgnetinBV 541 of gnetin C 541 of gnetin F 541 of gnetin G 541 ofleachinoIG 542 of malibatol A 542 ofparthenocissin A 542 of restrytisol A 541 of restrytisol B 543 of restrytisol C 543 of tricuspidatol A 543

1321

of viniferifuran 543 of (+)-^-viniferin 540 of e-viniferin diol 540 HOHAHA spectrum 10,21,29,34 of bisdesmoside 41 of dianchinenosideE 34 of saponariosides A&C 10,21 ofvaccarosideD 29 Holarrhena sp. 837 Holothuria mexicana 72 Holstiine 1048 Homidium 786 Homolycorine 618 (-)-Hopeaphenol 561 (+)-Hopeaphenol 561 Honokiol 217,227,240,249,254 Hopeaphenol 561 Hormosira banksii 440 Homer-Emmons reaction 1221,123 6 Horseradish peroxidase 1133 HRFAB-MS 20,33 of bisdesmoside 41 of dianchinenosideE 33 ofSAPO 30,50,41 of saponarioside A 20 Hugonia 172 Human immunodeficiency virus (HIV) 223 Human papilloma virus (HPV) 223 (-)P-Hydrastine 868 (+)|3-Hydrastine 868 Hydrocholeretic effect 391,399,415 Hydrogenation 66 of 10,13-dimethyl-9-tetradecen-l-oI 66 Hydrolapachol(2-hydroxy-1,4-naphtho quinone) 803 5-Hydroperoxy-eicosatetraenoic acid 265 Hydrothermolysis 40 3-(Hydroxy)methylene-2-thioxopyrrolidine (HMTP) 1073,1077-1079,1083,1084, 1087,1092,1093,1095,1096,1099,1100, 1102,1103 2-Hydroxy-1,4-naphthoquione 804 12-Hydroxy-11 -methoxy-A'^-acetyl-nor-Cfluorocurarimine 1052,1053 12-Hydroxy-11-methoxy-nor-C-

flourocurarine 1052 8-Hydroxy-2'-deoxyguanosine 253 l-Hydroxy-2-hydroxymethylanthraquinone 660 1 -Hydroxy-2-methyl-anthraquinone 660 7-Hydroxy-2-methyl-anthrquinone 676 Hydroxy-2-methylantraquinone 676 2-Hydroxy-3-methoxy strychnine 1030,1052 3-Hydroxyanthranilic acid 965,966,978, 1015 5-Hydroxyanthranilic acid 971 2-Hydroxyanthraquinone 659 5-Hy droxy-benzoquinone-1 -(2' -hydroxy6'-carboxyanil)-4-imide 1003 /?-Hydroxybenzyl isothiocyanate 1076 6-Hydroxycalyxin F 902 Hydroxycoumarin esculin 314 Hydroxycoumarins 315-318 from Fraxinus spp. 315-318 Hydroxyeicosatrienoic acids 759 Hydroxyethylrutosides 756 3-Hydroxyflavone 757,761-764, 768,769 Hydroxyframoside A&B 325,326,333 3-Hydroxykynurenine 965-967,988,997, 999 10-Hydroxyligstroside 334,343 12-Hydroxymalagashanine 1053 2-Hydroxymethlanthraquinone-3-0-^Dglucoside 660 2-Hydroxymethyl-8-hydroxanthraquinone3-0-/?-D-glucoside 660 2-Hydroxymethylanthraquinone 3glucoside 657 2-Hydroxymethylquinizarin 661 12-Hydroxy-myricanone 895 Hydroxy naphthoquinone atovaquone 783 O-Hydroxynitropapuline 593 Hydroxynomuciferine 825 Hydroxyomoside 318,321,322,325,333, 343 0-Hydroxypapuline 592,593 1-Hydroxyphenazine 365 p-Hydroxyphenyl-I-bromoheptadecane 73 (+)-l-Hydroxypinoresinol 200,270 8-Hydroxyquinaldic acid 991

1322

7-Hydroxytectoquinone 661 Hydroxyverniladin 808 11-Hydroxyvittatine 610 (-)-6'-Hydroxyyatein 192 Hymenolepis nana 484 Hyophyllanthin 233 Hypericin 375 Hypericum 371 Hypericum calycinum 797 phloroglucinol from 797 Hyperoside 666 Hypocholesterolemic agent 246 Hypocholesterolemic atherosclerosis 248, 249 Hypocholesterolemic effect 44,54,205 of Gypsophila capitata 44 Hypocholesterolic effects 233 Hypoglycemic 398 Hypoglycin A 476 Hypolepis puncatata 722 Hypolipidemic action 887 Hypolipidemic activity 247 Hypoloside A&B 722 Hypophyllanthin 195,237 Hypoplasia of bone marrow 688 Hypotriglyceridemic effect 246,261 Hypselodory nana 463 Hyp t is verticillata 111 dehydro-p-peltatin methyl ether from 212 5-methoxypodophyllotoxin from 212 Hyriopsis schlegelii 1123 Hyssopus sp. 746 Ibogan 139 Ichthytoxic activity 485 Ictalurus punctatus 353 Idicol 850 Illudane-dienone 716 Illudanes 705 Illudin-M 706

Immunofluorescence detection 935 of the putative NOS 935 Immunofluorescence microscopy 932 Immunohistopathology 792 Immunomodulatory actions 1036 Immunomodulatory effect 50,55 of Acanthophyllum squarrosum 55 of Gypsophila saponins 55 of Vaccaria segetalis 50 Immunopathological reactions 787 Immunosuppressive activity 214 of p-apopicoropodophyllin 214 Immunosuppressive activity 482 Immunosuppressive effects 486 Immunstimulatory activity 797 Imperata cylindrica (Gramineae) 233 graminone Bfrom233 Indanones 700,701,702 by l-acetoxy-(+)-pinoresinol 238 by 1 -acetoxy-(+)-pinoresinol-p-Dglucoside 238 by Anemarrhena asphodeloides (Anthericaceacy) 237 by arctigenin 237 by didemethoxymatairesinol 208 by c/5-hinokiresinol 237 by Forsythia koreana 237 by Forsythia viridissima 237 by Forsythia suspensa 237 by (-)-mataresinol 237 by (+)-pinoresinol 237 by (+)-pinoresinol monomethylether 238 by (+)-pinoresinol-di-j3-D-glucoside 238 by (+)-pinoresinol-p-D-glucoside 237 by podophyllotoxin derivatives 159 by (+)-syringaresinol-di-P-D-glucoside 238 of acetylcholinesterase 472 ofaconitase 912 of cAMP-phosphodiesterase (PDE) 237-240 of cholesterol acyltransferase 247 of cytochrome C oxidase 912

1323

of cytochrome P450s 912 of DNA-topoisomerase II 160-163,166, 167,169,211 of human estrogen sythetase 208 by 3-demethoxy-3-(9-demethymatairesinol 208 of indoleamine-2,3-dioxygenase 912 ofmicrosomalacyl-coenzyme A 247 of photosynthesis 554 of the kinase activity of growth factor 924 of the proto-oncogene 924 of type I DNA- topoisomerase 804,806 of 5-lipoxygenase 887 of cyclooxygenase-1 and - 2 887 Inhibitory activity blastogensis 217 honokiol 217 machilin A 217 matairesinol 217 of mitogen-induced arctigenin 217 Innzeylanine 850 Insecticidal activity 231,232, 426,471, 479,485 Insecticidal effects 852,1136 of ryanodol/isoryanodol-type diterpenes 852 Insularoside 321-323,325,327,333,343 Insulin 222 Inter alia 481 ofentrodiol 208 Interferon-y 951 Interiotherin A 197,223,224 Interiotherin B 197 Interleukin 951 Intramolecular Aldol reaction 111 Intramolecular Claisen-Tishchenko reaction 102 3p-H-Ipecoside 97 Ipomoea cairica 223 Iris japonica 433 Iron regulatory Protein 940 Iron responsive Element 951 Irpex lacteus 439,468 Is at is tinctoria 632

Isoamericanol A 270 Isoampelopsin F 568 Isobrucine 1051,1052 Isobrucine-N-oxide 1051,1052 Isodeoxypodophyllotoxin 193 Isodomoic acid G&H 479 (+)-Isogmelinol 200,241 Isoguattouredigine 825,827 (-)-Isohopeaphenol 561 3a-H-Isoipecoside 97 /raw^-Isokielcorin B 259 Isolariciresinol 195,205 (+)-Isolariciresinol 268 Isolugrandoside 328,329 acetate of 329 Isomagnolin 201,241 Isomagnolol 227 Isonaringin 744 Isophorone 297,298 Isopicropodophyllone 193 Isopicrostegane 196 Isopregnomisin 249 Isoptaquiloside 719,722 Isosakuranetin 744 Isoscopoletin 315,332,333,334,342 Isostrychinine 1051,1052 Isostrychnine-N-oxide 1051 Isoswinholide A 1197,1198 Isothiocyanato-N-(4-nitrophenyl)benzeneamine (amoscanate) 481 Isovelleral 461 Isoxaben 370 Isoxazopodophyllic acid 214,215 Isoyangambin 242 Ivermectin 441,447,453,457,468 Ixodes ricinus 695 Jaspamide 485 J asp is 1210 Jaspis spp. 485,488 Jasplakinolide 485 Jatrogrossidione 809,811 Jatropha grossidentata 809,810 Jatropha isabelli 810

1324

Jatrophone 809 Jeitacin A&B 442 Jenisseensosides A-D 46,47,55 from Silenejenisseensis 46 Jereicopsis 1175 Jereicopsis graphidiophora 1206,1207, 1214 JereisterolB 1206,1207 Jones' oxidation 1209,1225 Juglans 905 Juglans mandshurica 899 Juglone 363,370,470,638,868 Julia olefmation 1219 Juniperus sabina 214 Justicia procmbens var. leucantha 226 diphyllin apioside-5-acetate from 226 diphyllin asioside from 226 diphyllin from 226 justicidin A&B from 226 Justiciaprocumbens 235,226,243,265 justicidin B from 243 justicidin E from 243 neojusticin A from 243 taiwanin E from 243 taiwanin E methyl ether from 243 diphyllin apioside-5-acetate from 226 diphyllin asioside from 226 diphyllin from 226 justicidin A&B from 226 Justicidin A 194,266 Justicidin B 243 Justicidin D (neojusticin A) 194 Justicidin E 243,265 Kadsura heterocollita 240,249,259 burchellin from 249 gomisin M from 240 heteroclitin F from 259 heteroclitin G from 259 kadsurenone from 249 Kadsura interior lll^ gomisin-G from 223 interiotherin A from 223 schisantherin D from 223

Kadsura japonica 255 Kadsuranin 197,224 KadsureninH 240 Kadsurenone 240,241,249 Kaempferol 747,748,750,763,764,769 (-)-a-Kainic acid 478 Kalihinane diterpenes 810 cycloamphilectanes 810 7,20-diisocanoisocycloamphilectane 810 epiamphilectadiene 810 7-isocyano-ll(20),14 7-isocyanocyclo-amphilect-10-ene 810 isocycloamphilectanes 810 KalihinolA 466,810,812 Kappa-carrageenan 1148 Kazmaier's modification 1227 of Ireland-Claisen rearrangement 1227 Keck DCC protocol 1230 Keramamide K 1183 KeramamideL 1183 Keramamides B-J 1181-1183 a-Ketogluturate 638 /m^5-Kielcorin B 259 Kinetin 640 Kishi's retrosynthesis ofonnamide 1231 Klebsiella 1155 Klebsiella aerogenes 1263,1269 Klebsiella pneumoniae 402 Kluyveromyces fragilis 338 Kluyveromyces lactis 1127 Klyne's rule of molecular rotation 8 Kobophenol A&B 535,558,560,561 Kobusin 200,231 Kombamide 1184 Konjac glucomannan 1149 Korundamide A 822,823 Koshikamide A, 1193 Krameria triandra 204 scavenging activity of 204 a-Kratin 667 Kreb's cycle 924,940

1325

Kumusine 1201 Kynurenic acid 965-968,970 Kynurenine 966,968-971,990,998 Lachnella 462 Lachnum papyraceum 446 Lachnumol A 446,447 Lactarius mitissimus 461 Lactuca sativa 187 Lamellomorpha strongylata 1189 Laminaran 1121 from brown algae 1121 Laminar ia 1121 Laminariales 478 Laminine 478 Lamium 392 Lampteromyces japonicus 106 Lapachol methylether 66 Lapdap 783 Lapinone 803 (+)-Lariciresinol 177,188,205,219 Lariciresinol 199 75',8/?,8'7?-(-)-Lariciresmol-4,4'-bis-0-pD-glucopyranoside 199 Lariciresmol-4p-D-glucoside 199 Larix leptolepis 185 Larrea tridentata 187,205,249 Laspeyresia pomonella 231 Lauraceae 849,850 Laurencia implicata 808,809 Laurilia tsugicola 703 Laurus azorica 850 Lawsone 638 Lawsonia alba 837 Leachianol A-G 535,555,563,564 NO-Leghemoglobin complex 937 Leguminosae 637,871,871,931 Leishmania 779,780,783,786,789,790, 793,801,803,805,807,808,810,821,822, 824,828,830 Leishmania amazonensis 229,887 amastigotes of 828 promastigotes of 828 Leishmania decemlineata 851 -859,860

863-866 Leishmania donovani 791,794,797,800, 804,805,806,819,821,828 Leishmania infantum 810 Leishmania major 230,805,784,791,800, 810,811 Leishmania panamensis 821,822 Leishmania tropica 802 Leishmaniasis 781,783,784,785,793 Leishmanicidal activity 793 Lens ciilinaris 1263 Leprae 1155 Leptinotarsa decemlineata 850,854,855, 869-872,874,876 Lethaloxin 451 Leucaena leucocephala 1142,1148 galactomannans from 1148 Leucaena spp. 1139 Leucoanthocyanidins from 747 Leucopaxillus albissimus 493 Leukocyte activity of 510 Leukocytopenia 243 Levamisole 440,454,485 Libocedrus bidwilli 231 Licarin A 247 Licarin B 858 Licarin C 202 Licarin D 247 Licochalcone A 800,801 Licorice 745 Ligistrum 327 Lignans 206,207 as phytoestrogens 206,207 Lignin 1262 Ligstroside 318,333,334,343 Limnocorrals 381 D-seco-Limonoids 815 Linalool 396,397 epoxide of 397 Linoleicacid 361,433,434,461 6-Linolenic acid 361 Linum 172,173 Linum album 173 Linum flavum 173,212 5-methoxypodophyIlotoxin from 212

1326 Linum usitatissimum (Linaceae) 206 Lipid peroxidation activity 253 Lipid-diphosphooligo-saccharide 1131 Lipomannan 1155 Lipopolysaccharide 597,951,1152, Liquiritigenin 745 Liriodendrin 233 Lirioresinol glycoside 268 p-dimethyl ether of 233,268 (-)-Liriorinol B 229 Lister a ovata 1135 Litalin flavonoids 744 Lithistid sponges 1175,1176 Litholytic properties 52,54 of Herniaria fontanesii 52, 54 3,4-Lobatin 808 Lobatin A&B 808,809 Locust bean (Ceratonis siliqud) 1139 A^-Lodosuccinimide 1162 Lolium perenne 696 Lonchocarpus 487 Longicaudatine 1051 Lossen rearrangement 1074 Lucidin 640,657,661,674,675,670 Lucidin glucoside 661 Lucidin primeveroside 642,662,676 Lucidin co-ethylether 661,676 Lucidin co-methyl ether 662,676 Lugrandoside 328 Lupinus Albus 931 Lupus nephritis 269 Luteolin 746,750,757,762,763,769 glycoside of 757 Lutessine 610,612 Lutzomyia 783 Lycoctonine 868 Lycopene 299,300 Lycopsamine 874 Lycorine 581,609-614,617,618 Lycoris radiata 614 Lymphocytic leukemia 925 Lymphokine-activated killer 923,951 Lyngbya majuscula 78 Lyngbya wollei 355 (+)-Lyoniresinol3-a-0-[3-D-glucopyra-

noside 195 Lysine malonate 366 Lysy 1-tyrosy 1 quinone (LTQ)

1261,1268

Macaronesia fortunata 850 Mechilin racemosus 1093 MechilinA 191,217,268 Mechilin G 242,247 Machilus japonica 857 bioactive lignans from 857,858 Machilus thiimbergii 247 Macrocyclic biarylheptanoids (Type III) 894-898 Macrocyclic diaryl ether heptanoids (Type IV) 899-901,905 Macrocyclic trichothecenes 811 Macrocystis alterniflorum 359 Macrocystis spicatum 359 Macrocystis verticilatum 359 Macronesiafortunata 850,851 Macrophage-mediated cytotoxicity 910 Macrozamia communis 3 66 UdiddigdiS^cm Strychnos 1030-1032,1035 Maduramicin 451 Magnolia denudata 247 Magnolia fargesa 233 denudatin B from 233 fargesin from 233 fargesone A-C from 233 lirioresinol-B dimethyl ether from 233 magnolin from 233 pinoresinol dimethyl ether from 233 Magnolia far ges a 245 magnone A&B from 245 schisandrol A from 245 Magnolia far ges a 268 Magnolia obovata 226,227,254 Magnolia officinalis 226 honokiol from 249 magnolol from 249 Magnolia salicifolia 233 Magnolia spp. 240,270 honokiol 240,270 magnolol 240,270

1327

Magnolia virginiana 203,232 4,4'-diallyl-2,3'-dihydroxybiphenyl ether from 232 magnolo from 232 4-methoxyhonokiol from 232 Magnoliae cortex 204 honokiol from 204 magnolol from 204 (+)-Magnolin 200 Magnolin 233,268 Magnolol 227,232,240,249,254 MagnoneA&B 199,245 Magnosalicin 264 Magnosalin 262,263 Magnoshinin 262,263 Major histocompatibility complex 921,951 Malagashanine 1053,1054,1063,1064 Malagashanol 1053,1064,1065 Malate dehydrogenase 800 MALDI mass spectroscopy 11 Malekulatine 824,825,827 MalibatolA&B 562 Manducasexta 439 Mannich reaction 125 Manno-oligomers 1126 Manool 402 Marasmic acid 462 Marasmius 462 MarcfortineA 490,491 MdiVigoXd {Tagetes) 430 Marine fatty acids 63 Matadine 1060 (-)-Matairesinol 156, 157, 192 Matairesinol 177,205,206,217,229,233 (-)-Matairesinol dimethyl ether 192 (-)-Matairesinol-p-D-glucoside 238 Matteson method 1221 for dihalomethylithiation insertion 1221 Matthiola fruticulosa 1075 (-)-Maximowicziol A 899 Mayapple 149,152,153,171,175,176 Measles virus 223 Medicago sativa 696 Medicarpin 473

(+)-Medioresinol 200,229 Mefloquine 782,783,838,839 (+)-Megacerotonic acid 185 Meglumine antimonate (glucantime) 822 Melaconis thelebola 596 Melanin 1019 biosynthesis of 1019 Melanins 1018-1020 Melanogenesis 965 Melanoproteins 1019 Melarsoprol 791,787 Melatonin 965 Melia azedarach 187 Melia cultivar 466 Melinonine F 1060 Mellein 449 Meloidogyne arenaria 456 Meloidogyne incognita 434,435,437,439, 447-50,460,462,463,466,467,469,471, 473,476,483-493,1289 Meloidogyne javanica 429,430,456,464, 466,473,478,482 Meloidogyne spp. 428,430,432,437,481, 487 Menoctone 803 Menoquinones 638 Mentha sp. 746 4-Mepacrine 836 Metastatic secondary tumor 221 in animals 221 Methanolysis 65 of (Z)-9-hexadecenoic acid 70 of 10-hydroxydecanoic acid 65 5-Methdehyropodophyllotoxin 194 Methoxatin 1266 5-Methoxy podophyllotoxin-4-|3-Dglucoside 212 11 -Methoxy-12-hydroxyspermostrychnine 1064-1066 1 -Methoxy-2-methylanthraquinone 662 2-Methoxyanthraquinone 662 7-Methoxyaromadendrin 802 4-Methoxyhonokiol 232 75',8/?,8'/?-(-)-5-Methoxylariciresinol-4,4'bis-O-p-D-glu 199

1328

5-Methoxypodophyllotoxin 193 3 -Methoxy spermostry chnine 1064-1066 Methyl (9-methoxycarbonylnonyl) triphenyl phosphonium bromide 65 Methyl 11-methylpentadecanoic acid 64,65 Methyl 4-(methylthio) butyldithiocarbamate (MBDC) 1081,1082 (Z)-Methyl4-(methylthio)-3-butenyl dithio-carbamate 1081 Methyl 9-deoxy-9-oxo-a-apopicoropo dophyllate 195 TV-Methyl cytisine 487 2-Methylanthraquinone 363,374 Methylated fatty acids 63,64 (-)-De-O-Methyl-centrolobin 902 (-)-N-Methylcytisine 487 (Z)-9-Methyldec-4-enal 83 3-Methyldodec-l-en-3-ol 72 19-Methyleicos-4-en-l-ol 84 9-Methyleicos-4-enal 84 2-(3,4-Methylenedioxyphenyl)-quinoline 820,821 7-Methylesculin 317,333,334,342 7-0-Methylesculin 332-334,342 O'-Methylguanine-DNA-methyl-transferase 913 A^b-Methylharmalane 1060 (4Z,9Z)-24-Methylhexacosa-5,9-dienoic acid 83,84 2-Methylisobomeol 353,365-367 Methyllycaconitine 871 3-Methyloxacylotridecan-2-one 69 4-O-Methylpicropodophyllotoxin 193 Methylpiperbetol 243,244 (-)-Methylpluviatolide 799 Methylpsedolycorine 615 Methylrocaglate 203 (£)-5-Methyltetradec-4-enal 72 4-(Methylthio)-(£,Z)-3-butenyl glucosinolate 1085 4-(Methylthio)-(£,Z)-3-butenyl isothiocyanate (MTBI) 1073,10751079,1082-1084,1090,1092,1093,10981100,1102,1103,1105 3-[4-(Methylthio)-3-buenyl]-5-benzyl-2-

thiohydantion 1089 3-[4-(Methylthio)-3-butenyl]-5-[2-(methyl -thio)-ethyl]-2-thiohydantoin 1089 3-[4-(Methylthio)-3-butenyl]-5-isobutyl-2thiohydantoin 1089 3-(Methylthio)methylene-2-thioxopyrrolidine (MMTP) 1073,1081-1084,1092, 1093,1095,1096,1099,1100,1102,1103 (/?)-17-methyltricos-4-en-l-ol 87 Methylumbeliferyl-phosphate 378 3-[a-Methyoxy(a-methylthio)]methyl-2thioxopyrrolidine(raphantin) 1083 Methyoxy-2-phenylquinoline 820 2,3-Bis(Methyoxycarbonyl)-l-(3,4dimethoxy phenyl)-4-hydroxy-6,7,8 trimethoxynaphthalene 196 Michael addition 1237 re/ro-Michael reaction 110 Miconidin 797,798 Microbacterium lacticum 1122 Microbial transformation ofisophorone 307 of xanthurenic acid 991 of 8-hydroxyquinaldic acid 991 Microbiostatic inhibitory activity 402 Micrococcus lysodeikticus 1122,1124 Micrococcus spp. 1155 Microcystis aeruginosa 359,365,366,367 Microcystis sp. 352,354,258 Microcystis viridis 366 Micromelum tephrocarpum 800 Microschlerodermin A 1215,1216 Microscleroderma sp. 1175,1215,1217, 1218 Microsclerodermins C-E 1216,1217 Microsclerodermin B 1215,1216 Microspectrofluorometry 469 Microsporum canis 227,228 Microsporum gypseum 228 MicrOSproum sp. 1127 Mikania obtusata 810 Milbemycins 425,426,432,453 Mimosa pudica 481 Misakinolide A 1197,1198 Mithramycin 220

1329

Mitochondrial monoamine oxidase 1260 Mitogen-activated protein kinase 753,951 Mitsunobu reaction 1236 MiyabenolA-C 552,556,558,560,561 c/5-Miyabenol C 556,560 possessing o-quinone 164 possessing spiroketal 164 Molinema dessetae 441 Mollugin 666 N(G)-Monomethyl-L-arginine 951 Monomycin 831 Monotropein 666 Morelensin 193,215,216 Morinda lucida 673,805 Mortierella alpina 259 Motuporin 1189,1217-1220, 1222-1227 Mozamides A 1184 Mozamides B 1184 Mucoproteins 1019 Mucuna aterrima 435 Mucuna hassjoo 932 Mukaiyama-type aldol reaction 1235,1237 Munjistin 657,663 methylester of 663 Munnozia maronii 808 Murine cytomegalovirus (MCMV) 223 Murine lymphomas L 1200,1210 Murisolin 441,442 Musa acuminata 474 Musacin C 444 Musca domestica 231 Mutagenic activity 675 of Rubia tinctorum 675 Muzanzagenin 813,814 Myasthenia gravis 486 Mycalamides A & B 1231,1234 Mycelia sterilia 482 Mycobacterium arium 1160 Mycobacterium fallax 69 Mycobacterium luteus 1122 Mycobacterium smegmatis 227,1123,1155 Mycobacterium sp. 1122 Mycobacterium tuberculosis 1122,1155 Mycobacterium malmoense 1122

Mycobacterium tuberculosis var. hominis 673 Mycosphaerella lethalis 451 MycotoxinMT81 805,806 Myocardial ischemia 233 Mvo-inositol 1155 Myrica 905 Myrica gale var. tomentosa 895,899 Myrica rubra 895 Myricanone 895 Myricatomentoside I&II 899 Myricetin 742,746,747,750,757,758, 763,770 Myriophyllum 359 Myriophyllum spicatum 378 Myristica fragrans 226,85 8 dihydrodiisoeugenol from 226 5 '-methoxydehydrodiisoeugenol from 226 Myrothecium hapla 464,414 Myrtoidine 1053,1064,1065 Myzus persicae 850,851,859,861,862 Najas sp. 361 1,2-Naphthoquinone 470 4-Naphthoquinone 470 Naphtoquinones 638 Napththylisoquinoline alkaloids 822 Narciclasine 610,612,613,616 Narciclasine-4-O-D-glucopyranoside 610, 613 Narcissus pseudonarcissus 608 Narcissus tazetta 610,618 Naringenin 745,759,762,768 Natriuretic effect 269 Natsudain 752 Natural algicides in freshwater ecosystems 380,381 Natural phytotoxins 369 Nauclea latifolia 1036 Navelbine 1047,1048,1049 Naviculla pelliculosa 358 Necator americanus 425,481,494 Necrosis 993

1330

NectandrinB 198 Neisseria gonorrhoeae 613 Nemalion vermiculare 1137 Nematocidal activity 439,443,451,454, 461,466, 469,470,474,482,485,887 of fatty acids 434 Nematodes 428,429 Chaenorhabdtis 429 control of 430-432 Nematostatic activity 429,430 Nemerosin 215 Neoglycoconjugates 1159 Neohesperidin 744 Neoipecoside 129 Neoisopecoside 129 Neojusticin A 243 NeojusticinB 194,235 Neolignans 244,551 7-S, 5-5Neolignan 571 7-R, (9-/? Neolignan 571 Neolignans 184 Neoolivil 198 Neoplasic cell lines 752 Neoplastic activity 452 Neosiphonia 1175 Neosiphonia superstes 1207-1210 Neosiphoniamolide A 1210 Neothalibrine 825,827 Nepeta 172 Nerve growth factor 951 Nervous polyarthritis 400 Neuroblastoma 924,927 cell differentiation of 927 Neuroblastoma development 754 Neuroexcitatory activity 479 Neuroleaena lobata 808 2,3-Neurolenin 808 Neurolenin A&B 808,809 Neuroleptic drugs 270 Neurospora crassa 1016,228,1128 Neurotoxicity 965,1004 HK-Neurotoxicity 993 Niacinic acid 968 Nicotiana plumbaginifolia 1146 Nicotine 487

Nicotinic acid 965 Nifurtimox 792 Nigrospora oryzae 227 Nimbinin 815 Nimbolide 815 Nippostrongyliis brasiliensis 465 kalihinol Y from 465 Nirtetralin 195,237 Nitrate reductase-dependent NO production 930 Nitric oxide 909,911-914 chemistry and bioactivity of 909, 911-914 as signal in plant defense response 937-949 Nitroquinoline-A^-oxide 271 Nitrosoglutathion (GSNO) 948,951 S-Nitrosylation 914,924,925,939 Nitzschia pungens 479 'CNMRdata 1052 for monoindoles 1052 NMR studies in structure elucidation 1040 Nitric oxide as mediator of the antitumor immune response 921-923 as plant growth regulator 934,935 cell apoptosis 925,926 cell differenciation 926 classification of 915 in plants 929 synthases 915 synthesis in plants 930 Nobiletin 746,752 Nobilisitine A&B 610,619 Nocardia 451 Nodularia spumigena 367 NO -mediated intramolecular disulfide formation 914 NO-mediated apoptosis 943 in animals 943 NO-modulated transcription factors 946 Non-specific immune response 923 Nonblue 1271 Non-phenolic linear diarylheptanoids (type I) 883,884,905

1331

Non-specific damage on DNA 1004 Non-volatile constituents 297-301 of saffron 297-301 Norambrienolide ether 394 Nordamnacanthal 641,657,663,673 Nordihydroguairaretic acid (NDGA) 191 Normelionine F 1060 Nortrachelogenin 192,233 (+)-Nortrachelogenin 270 NOS-dependent NO production in plant cells 931-934 NOS toe commune 362 Nostoe l^SW95/\0 367 Nostoe sp. 352,366,372 Nothenia anomala 440 Nothenia brasiliensis 446,463-465, 471,483-485,487,488,492,493 Nothenia dubius 432,463,471 Nothogeia fastigiata 1138 Novobiocin 366 Nudiposide 195 (+)-Nyasol 230,231 antiprotozoal agent 230 Nyctanthes arbortristis 807 (-)-Nymphone 199 Obaberine 824,826 Obavatifol 202,219,220 Obovaten 202,219 Obovatinal 219,220 Ocotea foetens Aiton 850 Octadecyl ferulate 666 Octadecanoic acid 361 Odoracin 465 Oleic acid 434 Oleiferin-B 228,230 Oleiferin-F&G 228,230 Oleoside 319 Oleuropein 324,334,343 Oligophagous chrysomelid beetle 871 Oligosporon 449 Olivacine 828,829 from Aspidosper ma olivaceum 144 Ommatins 965,991,992

Ommins 990 Ommidinis 990 Ommochrome 998,1018 Omphalotins A-D 483-486 Omphalotus illudens 706 Omphalotus olearius 483 Onitin 706 Onnamide A 1198,1231,1232,1233,1234 Onychium aiiratum 706 Ophiobolin 468 Ophiobolin K 468 Orbiculamide A 1181 Orchis morio 1144 Oriamide 1184 Origanum sp. 746 Ornithine decarboxylase inhibition 924 Ornoside 318,319,322 Ornosol 319,333 Oroidin 819 Orophea enneandra 204,255 (-)-epieudesmin 204 (-)-eudesmin 204 (-)-phylligenin 204 Oscillatoria 352,358 Oscillatoria agardhii 352,365 Oscillatoria cf chalybea 362,373,379 Oscillatoria latevirens 364 Oscillatoria perornata f attenuata 373, 374,379 Osmanthus 327 Ostertagia circumcinta 453,490 Ostertagia ostertagi 484 Ostreopsis lentcularis 69 Ostrinia nubilalis 186 Otivarin AA\M2 Otoba parvifolia 186 Otoseine 875 Ovalicin 459 Oxandra espinata (Annonaceae) 807 Oxazolidine-2-thiones (goitrogens) 1074 Oxidative coupling of lysine and cysteine 996 2-Oxo-2,3-dihydrotryptamine 117 l-Oxo-4'-Demethoxy-3',4'-methylenedioxyrocaglaol 203

1332

2-Oxo-isocaproate 968 7-Oxoroyleanone 402 Oxotryptamine 118 Oxychlororaphine 365 Oxyhemoglobin 986 Ozonation 355 Ozonolysis 81,86 Paccar di 1215 Paecilomyces lilacinus 432 PAF-induced inhibitors 240 (+)-acetoxypinoresinol dimethyl ether 241 arctigenin 241 arctigenin methyl ether 241 chamigrenal 244 cinnamophilin 240 dehydroschisandrol A 245 denudatin 240 (+)-epipinoresinol dimethyl ether 241 epiyangambin 242 fargesin 241 gomisin M 240 honokiol 240 (+)-isogmelinol 241 isomagnolin 241 isoyangambin 242 justicidin B 243 justicidin E 243 kadsurenin H 240 kadsurenone 240 L-653,150 242 machililin G 242 magnolol 240 magnoneA 245 magnone B 245schisandrin A 244 we^o-dihydroguaiaretic acid 240 methylpiperbetol 243 neojusticin A 243 phillygenin 241 (+)-pinoresinol dimethyl ether 241 piperbetol 243 piperol A 243

piperol B 243 pregomisin 244 puberulin A 243,244 puberulin C 243,244 schisandrol A 245 (-)-syringaresinol 243 taiwanin E 243 taiwanin E methyl ether 243 (+)-yangambin 242 PAF-induced thrombocytopenia 242,243 Palasonin 457 Pallidol 555,560 Palmitic acid 434 Palythoa sp. 69 Panagrellus redivivus 429,432,434,435, 438, 450,455,467,473 Panagrolaimus 488 Pancratium maritimum 610,613,615-617 Pandanus odoratissimus 255 3,4-bis(4-hydroxy-3-methoxy-benzyl) tetrahydrofliran 255 Panek's synthesis 1224,1225 of dipeptide 1224 of p-methylaspartate 1225 Panganensine R 1055,1057 Panganensine X&Y 1055,1057 Panotima angularis 698 Papilio 990 Papillary carcinoma 712 Papillomas 693 Papulin 592,593 Papulinone 592 Paraherquamide A 490,491 Paraherquamides C-G 490 Parasiticidal activity oflignans 229 Paratrichodorus 456 Paratylenchus hamatus 429 Parbendazole 444 Paromomycin 831 Paromomycin (aminosidine) 785 Parthenin 808,809 Parthenocissin A&B 567,568

1333 NO-Participation 936,937 in antioxidant cellular systems 936, 937 Parvaquone 804 Paterson's synthesis 1229 of swinholide A 1229 of iso-swinholide A 1229 Pathogen-induced oxygenase 947 Patulin 449 Pauridianthalyalii 1036 Pear {Pyrus communis) 592 Pederin 1233,1234 Pediastrum simplex 379 Peganum harmala 828 Pelargonidin 748 a-Peltain 193 p-Peltatin 193 methyl ether of 193 Peltatins 222 Penicillic acid 449 Penicillin G 366,374 Penicillium sp. 450,482 Penicillium carneum 490 Penicillium charlesii 490,1128 Penicillium chrysogenum 1281 Penicillium expansum 228 Penicillium nigricans 805 Penicillium paraherquei 490 Penicillium roqueforti 490 Peniophora 462 5-Pentadecen-l-ol 75 Pentamidine (pentacarinat(R)) 791,793 2-«-Pentylquinoline 820 Peppermint 745 Peripheral analgesic properties 400,415 Peripheric antinociceptive effect 416 (f Periplaneta 991 Periplaneta americana 991 Persea indica (lauraceae) 850,851,852 cinnzeylanol from 850 cinnzeylanone from 850 ^/7/-ciniizeylanol from 850 idicol from 850 innzeylanine from 850

perseanol from 850 ryanodanes from 850 ryanodol from 850 ryanodol-14-monoacetatefrom 850 vignaticol from 850 Persea obovatifolia 219 obavatifol from 219 obovatenfrom 219 obovatinal from 219 perseal A&D from 219 Perseal A-D 202,219 Perseanol 850 PerthamideB 1187 (-)-Pessione 823 Peterson olefmation 1239 Phag-inhibitory activity 187 of Melia azedarach 187 Phanerochaete chrysosporium 186,1014 Pharmacological properties 556-559 of resveratrol oligomers 556-559 Phaseolus lunatus All Phaseolus tribe 931 Phaseolus vulgaris 1136 Phase-sensitive NOESY spectrum 12-14, 29,34 of bisdesmoside 41 of dianchinenosideE 34 ofmimusopin 14 ofSAPO30 41 ofSAPO50 41 of saponariosides C 12 of vaccaroside D 29 Phenalenone 474 9-Phenanthrenemethanolhalofantrine chloroqiune 782,820,825,836 Phenolic linear diaryIheptanoids 885-894, 905 Phenoxazinone 965,1014,1009 Phenoxazone 493 Phenylalanin ammonia-lyase 938 p-Phenyllactic acid 592 Phenylpropanoid pathway 154 A^-Phenylquinoneimine 976 vV-Phenyl-a-naphthylamine 359 A^-Phenyl-(3-naphthylamine 359

1334

Pheochromocytoma 927 Pheomelanins 1018 Phillygenin 237,241 Phillyrin 201 Phlebiaradiata 1013 Phlebotomus 783 (-)-Phlligenin 201 9'-(9-(-)-Phlligenin 255 Phllirin 237 Phlorizidin 800,801 Phorn is 172 Phom ops is foeniculi 1128 Phormidium autumnale 358 Phormidium bohneri 365 Phormidium sp. 364 Phosphomannomutase 1140 Phryma leptostachya 231 (+)-haedoxan A from 231 (+)-haedoxan D from 231 (-)-haedoxan D from 231 Phthalimides 1161 Phycobiliproteins 376 PhyllamycinB 196 Phyllamyricin E 194 Phyllamyricoside A 196 Phyllanthin 237 Phyllanthostatin A 196 Phyllanthus acuminatus 211 phyllanthostatin A from 211 Phyllanthus amarus 217 hypophyllanthin from 217 phyllanthin from 217 Phyllanthus myrtifolius 225 phyllamyricin E from 225 phyllamycin B from 225 phyllamyricoside A from 225 retrojusticidin B from 225 Phyllanthus niruri 233,237 hyophyllanthin from 233 hypophy llanthin from 237 nirtetralin from 237 phyllanthin from 237 Phyllantin 191 Physiological effects of phytoestrogens 207

Physostigmine 838 Phytoalexins 468, 472,473,494,745,1289 Phytoecdysteroids 699 Phytoene 3299,300 Phytofluene 299,300 Phytoparasitic nematodes 427,428 Phytopathogenic fungi 418 Botrytis cinerae 418 Fusarium solani 418 Phytophthora nicotianae var. parasitica 418 Rhizoctonia solani 418 Sclerotinia sclerotiorum 418 Sclerotium rolfsii 418 Phytophthora infestans 936 Phytophthora nicotianae var. parasitica 404 Phytotoxic activity 452 Piceid 530,571 Pichia pastoris 1263,1269,1288 Picrocrocin 293,295,296,298-300,303, 306,307 Picropodophyllone 193 Picropodophyllotoxin 166,193 Picrotoxinin 867,868 Pictet-Spengler reaction 128 Pieris brassicae 702 Pier is rapae 439 (+>Pinoresinol 157,177,188, 200, 229,270,474 Pinoresinol 249 (±)-Pinoresinol diglucoside from 235 Pinoresinol dimethyl ether 233 neojusticin B 235 (+)-Pinoresinol dimethyl ether 241 (+)-l-Pinoresinol dimethylether 200 (-)-Pinoresinol di-P-D-glucoside 200 Pinoresinol monoglucoside 235 (+)-Pinoresinol monomethylether 200 (+)-Pinoresinol-p-D-glucoside 200,270 (-)-Pinoresinol-P-D-glucoside 200 Pinosylvin monomethyl ether 472 (±)-Pinresinol 267,268,269 Pinus 905 Pinus canariensis 859

1335 Pinus densiflora 471 Pinus flexilis 887 Pinus massoniana 472,474 Pinus thunbergii 471 Piper betle 243 methylpiperbetol from 243 piperbetol from 243 piperol A&B from 243 Piper betle 469 Piper decurrens 232 Piper futokadsura 240 kadsurenin from 240 kadsurenin H from 240 Piper kadsura 240 denudatin 240 Piper nigrum 819 piperine from 819 Piper puberulum 243 puberulin A&C from 243 Piperbetol 243,244 Piperol A&B 243,244 Piquerol 807 Pirano flavonoids 744 Piricularia oryzae 1210 Pisum sativum L. 832,1263 Plakinastrella sp. 73 Plakortis 492 Planinin 267 Plant growth regulator 585 from phytopathogenic bacteria 585 as 2'-deoxyzeatin riboside 585 as dihydrozeatin 585 as dihydrozeatin riboside 585 as O-hydroxynitropapuIine 585 as indolacetic acid (lAA) 585 as indolaldehyde 585 as isopentenyladenine 585 as methyl ester of IA A 585 as I'-methylzeatin 585 as r'-methylzeatin riboside 585 Plasmodium 779-781,797,798,800,802804,815,825,1030,1035,1036,1063 Plasmodium berghei 1063 Plasmodium chabaudi 789

Plasmodiim7 falciparum 230,231,456, 466,781-783,788,789,797,800,801,804, 805,808,811,813,815,816,819,820, 822-824,828,837-839,1037,1063 Plasmodium lophurae 803 Plasmodium malariae 781,837 Plasmodium ovale 781,789 Plasmodium trophozoite 781 Plasmodium vivax 781,783,789,803,837 Plasmodium yoelii 789,828 Plastoquinone 368 Platelet activating factor (PAF) antagonism and coagulation 240-245 Platelet aggregation 756,758 Platycarya 905 Platycarya strobilacea 899 Platycaryol 899 P38 leukemia Pleurotus ostreatus 227 Plumbagin 470,804 Plumeran 140 Pneumocystis carinii 804,835 Podophyllotoxin 149,154-159,166,168178, 184,193,205,210,220,231,272,799, 858 derivatives of 162,163,164 4-aminoaryl 162 1-o-quinone 162 Podophyllum hexanrum 227 4'-0-demethldehdropodophyllotoxin from 227 picropodophyylone from 227 podophyllum kexandrun 227 picropodophyylone from 227 Podophylum 149,151,153,154,156-158, 169-174,177,178,183,183,188,205 Podophylum bankakri 149 Podophylum emodi 149,153,154,156, 158,171,173,178 Podophylum hexandrum 149,156 Podophylum peltatum L. 149-152,158, 171-178 Podopyllotoxin-based anticancer drugs 160 epipodophylltoxins 160 etopophosphate 160

1336 etoposide 160 etoposide phosphate 160 teniposide 160 Podopyllotoxin-P-D-glucoside 193,210 Podorhizol 192 Polyarthrtis 398 Polydiscamide A 1201 Polygodial 461 Polygyala gazensis 204 eudesmin from 204 kobusin from 204 magnolin from 204 yangambin from 204 Polyphagous lepidopteran 871 Polytheonamides 1193 Ponasterone-A 700 Ponasteroside 700 Populus tremuloides 358 Porlieria chilensis 249 dihydroguaiaretic acid from 249 guayacasin from 249 isopregnomisin from 249 Porson 895 Portiera hornemannii 809 Potam ogeton sp. 361 Pratylenchus 432 Pratylenchus coffeae 436 Pratylenchus curvitus All Pratylenchus neglectus 464 Pratylenchus penetrans 429,436-438,449, 473,477,481 Pratylensus scribneri 472 Precocene II 471 Pregomisin 244,247 (+)-Prennylpiperitol 267 (-)-Prenylpluviatilol 267 Prestegane B 192,249,269 Primaquine 836 (-)-Prinsepiol 200,270 Pristimerin 813,814 Proanthocyanidins 672 Procyanidin 697 Prodelphinidin 697 Proguanil 783 2-/7-Propylquinoline 820,821

Proserpinaca palustris 359 Protease inhibitors 800 Protein S-nitrosylation 913 Proteus m irab His 402,1155 Prunin 745 Prunus sp. 1157 Pseudocolelomic cavity 469 Pseudomanas syringae 583,590- 592, 594,595,598,599,607 Pseiidomonas 969,1134 Pseudomonas aeruginosa 330,331,365, 402,1151 Pseiidomonas alginate 1151 Pseudomonas amygdali 583,587,590, 599 Pseudomonas caryophylli 594 Pseudomonas ciccaronei bacteriocins 595,599 Pseudomonas diminuta 1155 Pseudomonasfluorescens 78,333,334 Pseudomonas sp. {Alteromonas) 69,582, 584,588,597,617,1124 Pseudomonas syringae pv. maculicola 593, 933 Pseudopuropurin 657,663,666,675 Pseudotheonamides A„A„B„C, & D 1194 Pseudoxanthoma elasticum 675 PSIl inhibitors 368,371 Psychodelic drugs 820 cocaine 820 morphine 820 semisynthetic LSD 820 Psychotria campoutans 804 Ptaquiloside 703,705,706,707,709-712, 714,716,719,722-725,727 three dimensional view of 710 Ptaquiloside Z 719,720 Ptaquilosin 723 Pteidanoside's 703,722 toxicity of 703,722 Pteridanoside 722 Pteridioside 704 Pteridium aquilinum var. caudatum 687, 690-692,695,696,698,699,702,703,720, 722,724,725,727

1337

Pteridium arachnoideum 699 Pteridium esculentum 699 Pteridium latiusculum 690,702 Pteris aquilina 691 Pteris cretica 722 Pterocarpanes 745 PterosinA&B 702 PterosinV 700,701,702 Pterosterone 700 papuline 585 papulinone 585 /-zeatin 585 /-zeatin riboside 585 PuberulinA&C 243,244 Puccinia podophylli 176 (/?)-(+)-Pulegone 86,88 Purpurin 640,649,657,663,666,674 Purpuroxanthin 657,666 Putrescine 1259,1260,1263,1276,1277 Pycnamine 824,826 Pycnanthus angolensis 219,473 Pycnoporus 1013 Pycnoporus cinnabarinus 1010-1016 Pycnoporus coccineus 1012 Pycnoporus sanguineus 1012 Pyrimethamine 805 Pyrimidinone 220 Pyrogallic acid 359 L-Pyroglutamic acid 1242 Pyrole-2-carboxylic acid 819 Quadrangularin A 567 Quassin 816,817 Quercetin 314,359,742,746-748,750, 757,758,696,697,714,1008 Quercetin-3,7-digalactoside 314 Quercetin-3-glucoside 314,763 Quercitrin 359 Quilic acid [3-0-P-D-galacttopyranosyl(l->2)[|i-D-xylopyranosyl-(1^3)]-p-Dgiucurono-pyranoside] 21 Quillaia saponaria 54 Quinidine 144,836 from Cinchona ledgeriana 144

Quinimax 836 Quinine 97,780,781,800,835,838,888 Quinizarin-2-carboxylic acid 664 Quinoaldimine 1272 Quinoketimine 1272 Quinolinic acid 965,966,968,970 o-Quinoneimine 976,986,988,992,998, 1000,1002 Quinoneimine 979 Quinonization 1287 Quinoproteins 1259,1270,1273,1277, 1278,1279,1283,1285,1286,1290 Quisqualic acid 476 Quisqualis indica 476 Radicicol (monorden) 452 Radopholus similis 474 Ralstonia solanacearum 933 Raman spectrometry 1267 Raphanus sativus L. 1075 (3R*, 6S*)-Raphanusanins 1084 3R*, 6R*Raphanusanins 1084 Rauwolfia serpentina 1060 Reduction 69,71,123 of 3-methyloxacylotridecan-2-one 69 of methyl 10(rer/-butyldimethyl silyloxy)-2-methyldecanoate 71 relative Van der Waals distances 123, 127 Reidispogia coerulea 1207 Reidispogiolide C 1209 Reidispongia 1175 Reidispongia coerulea 1207,1208 Reidispongiolide A-C 1207,1208 Repandine 824,826 Resda luteola 632 Reserpine 143 from Rauwolfia serpentina 143 Restrytisol A-C 553,558,570,571 Resveratol 753 Resveratrol oligomers 533,534 isolation of 533,534 cw-Resveratrol 555,571 /rara-Resveratrol 560

1338

Resveratrol oligomers 507-511,513-529, 532,556 sources of 513-529 Resveratrol trans-dohydrodlrnQv 555,565 Resveratrolside 530,571 Retrojusticin B 194 Rhabditissp. 432,488 Rhamnaceae 637 Rhamnetin 314 Rheumatoid arthritis 170,486 Rhinacanthin E&F 191,226 Rhinacanthus nasutus 226 rhinacanthin E&F from 226 Rhinoclaviella sp. 673 Rhizobium lipoplysaccharides 931 Rhizoctonia solani 404 Rhizopus oligosporus 1007 Rhodnius prolixus 187,230 Rhodotorula rubra 61?> Rhoiptelea 905 Rhoiptelea chiliantha 895,902 Rhoiptelol A&B 895,902 Rhyncholacis penicillata 471 Riboflavin 1007 Ricinoleate 361 Rishitin 460 Ristocetin 366 Rocaglaol 220 Rodophilia bifida 615 Rollidecin 793 Rollinia emarginta 793 Rolliniastatin-1 793 RoridinE 811,812 Rosmarinus sp. 746 Rotenone 473 Ruberythric acid 629,640,642,657,674 Rubia teep 675 Rubia tinctorum (madder) 629-632, 638,639,641,643,644,647,648,650,653, 656,657,658,666,675-677 anthragallol from 657 lucidin from 657 munjistin from 657 nordamnacanthal from 657 pseudopurpurin from 657

purpurin from 657 rubiadin from 657 xanthopurpurin from 657 Rubiadin 657,664,666,676 Rubiadin 3-(i-primeveroside 657 Rubiadin glucoside 657 Rubiadin-1 -methyl ether 806 Rubianin 664 Rubicunosides A-D 45,46 from Silene rubicunda 45 Russulaceae 461 Rutin 314,666,747,762,763,768,769 Ryanodanes 850 Ryanodine/spiganthine ryanoids 852,853 Ryanodine-type compounds 850,852 Ryanodol 850,856 Ryanodol-14-monoacetate 850 Saccharomyces cerevisiae 227,228,618, 673,1123,1125,1126,1129,1283 Saccharomyces kluyveri 1127 Sacchromyces sp. 1087 Saffron 296,297,299,301-305 Safranal 293,295,296,298,300,303,306, 307 Sainfoin 1268 Sakuranetin 802 Saliva desoleana 407,409,410,411 Composition of 407,409,410,411 Saliva officinalis 391,403,404 Saliva sclarea 404 Composition of 404 Salmonella 1161 Salmonella enteridis 613 Salmonella newington 1160 Salmonella paratyphi A-C 673 Salmonella typhi 673 Salmonella typhimurium 673,675,676, 707,712,722,1002,1093,1099-1101,1104, 1105 Salvia 172 Salvia desoleana 391,406,407,413-419 Salvia miltiorrhiza 235 danshensuan B from 235

1339

Salvia sclarea 391-395,397,398,400-406, 412,413,415-419 Sanguinarine 488 Sanshool 450 Santonin 462,463,466,477,478 SAPO30 41,42 from Gypsophila paniculata 41 SAPO 50 41,42 from Gypsophila paniculata 41 Saponaria officinalis 3,4,10,13,15,18,27, 30,42,54,57 antiinflammatory activity of 27 gypsogenic acid from 5,6,18 gyspogenin from 5,6,18 3(i-hydroxy-oleana-l 1, 13(18)-dien23, 28-dioic acid from 5,6 saponarioside from saponariosides C-M from 10,12, 13,17,18 saponaside A from 17,20-23,42 3, 4-secogypsogenic acid from 5,6 triterpenoid saponins from 17,20 Saponarioside C 26 HMBC correlations of 15, 25 HOHAHA spectrum of 10 MALDI-TOFMSof26 phase-sensitive NOESY spectrum of 12 SAR of podophyllotoxins 167,168 Sarcoma-180 303 Sargassum muticum 1152 SarothalenB 797 Savinin 231 Scenedesmus obliquus 373 Scheriber's synthesis of discodermolide 1244-1246 Schisandra 183 affect on cardiovascular system 183 decrease by heart rate 183 display platelet activating factor antagonist activity 183 for the treatment of hepatitis 183 Schisandra chinensis 203,204,244,247, 249-251,255,258,265

chamigrenal from 244 gomisin N from 249 pregomisin from 244 schisandrin A from 203,244 schisandrol A&B 203 schisandrin C 203 Schisandra rubriflora 205 Schisandrin 238,270 Schisandrin A 197,244 Schisandrin C 224 Schisandrol A 197,245 Schisanhenol 250,251 Schisantherin D 197,223,224,247,256 Schistocerca gregaria 700 Schizandrin 197 5-(-)-Schizandrin C&D 250 Schizonepeta tenuifolia 251, 252 schizotenuin A from 251,252 schizotenuin C, from 251,252 schizotenuin Q from 251,252 Schizonticidal drug 837 Schizosaccharomyces pombe 1093,1097, 1098,1128 Schizotenuin A 251 Schizotenuin C&C, 251 Sclareol 394,402 Sclareolide 394 Sclelerotinia sclerotiorum 405 Scleritoderma sp. 1215 Sclerotium rolfsii 405 Scoparone 315,332-334,342 Scopoletin 315,333,666 Scytonema hofmanni 363 3,4-Secogypsogenic acid 29 Secoilidoids and phenylethanoids 318-329 from Fraxinus ornus 318-329 Secoirridoids 95 (-)-Secoisolariciresinol 156,157,186,188, 191 Secoisolariciresinol 204,205,206 (-)-Secoisolariciresinol diglucoside 191 Secoisolariciresinol diglycoside 248 Secologanin 95-98,100,105,110,111,114118,127,132,137,138,140,143,144

1340 Secoxyloganic acid 100 Segetalic acid 30 Selenastrum capricornutum 373,374, 377-379 Semiquinolamine 1273 Senecio 872 Senecio erraticus 874 Senecio glaber 874 Senecio litura 851,853-855,858-860, 863-866 Senecio microphyllus 874 Senecio palmensis 859 Senecionine 876 Senna occidentalis 1143 Senna s^^. 1139 Ser264 inhibitors 368 Sergolide 815 Serotonin 267 Serotonine 965,965 Serpentine 1060 Serratia marcescens 402 (+)-Sesamin 200,267 Sesamin 231,259,260,264 (-)-Sesamin 268 Sesaminol 249,260,264 Sesamol 249,253 Sesamolin 253,260,231 Sesamolinol 249,253 Sesamum indicumi 249 pinoresinol from 249 sesaminol from 249 sesamol from 249 Sesmin 246,264 Setaria cervi 467 Setaria italica 476 Sharpless asymmetric epoxidation 89 of (E)-2-nondecen-l-ol 89 Shewanella putrefaciens 69 Shigella ambigua 61?> Shigella boidii 673 Shigella flexneri 673,1160 Shigella largei-sachsii 673 Shigella sonnei 673 Shogaol 475 Silandrin 255

Silchristin 255 Silene fortunei 46,47,55 Silenejenisseensis 45,46,47 Silene latifolia 45 Silene species 45 Silene siicculenta 45 Silene villosa 45 Silene vidgaris 47 Silenosides A-C 47,48 from Silene vulgaris 47,48 Silphinene 864,865,866,867,871 Silybin 255 Silybum marianum 255 Silydianin 255 Silymarin 754 Silymonin 255 Simaba guianensis 815 Simalikalactone D 815,816 Simarouba 837 Sinapis alba L. 1074 1 '-0-Sinapoyl-6'-0-galloyl-p-Dglucopyranose 359 Sindbis virus 223 Sinefrmgin 830 Sinensetin 746 Sinigrin 481 Sitoindoside II 219 P-Sitosterol 358 Smith periodate degradation 1156,1157 Snatzkeins A-E 52 from Arenaria fdicaulis 52 Solanaceous steroidal glycoalkaloids 467 a-Solanine (solatriose) 818,868 Solanum tuberosum 936,941 Solarmargine 817 a-Solarmargine (chacotriose) 819 Solavetivone 461 Solricin 135 362 Sophoraflavescens 487 Sophora Japonica 1142 Sorgoleone 370 South American trypanosomiasis 787,788 Spathulenol 402 Spectral data 1078 forHMTP 1078

1341

Spermicidal activity 53 Gypsophila paniculata 53 Spermidine 1263,932 Spermine 1263,932 Spermostrychnine 1064,1065,1066 Sphaerotrichia 1121 (-)-Spinosine 823 (+)-Spinescin 267 Sphinxolides B-G 1207,1208,1209 Spigelia anthelmia 852 Spiro-indolopirrolidine 126 of ^p/ro-indolopirrolidine Spodoptera eridania 871 Sp adopter a litura 850 Spodopterra littoralis 850,852-854,869, 870-872,874-876 Spontaneous apoptosis 926 Sporangium cellulosum 450 Sporotrichum pulverulentum (anamorph of Phanerochaete chrysosporium) 1013 Squamocin 793,794 Squarroside A 31,50 coyncentration dependent immunomodulatory effect of 31 from Vaccaria segetalis 50 Staphylococcus aureus 77,79,83,227, 330-333,402,673 Staphylococcus epidermidis 402,1093, 1095,1097,1098 Staphylococcus spp. 556 Stearic acid 461 Steganacin 196,216 Steganangin 216 Stegangin 196,216 SteganoateB 216,217 SteganolideA 216,217 Steganotaenia araliacea 216 episteganangin from 216 steganangin from 216 stegancin from 216 steganolide A from 216 steganoate B from 216 Stemmadenin 139 Stemonoporol 562 Stenbergine 610,614

Stenophyllol A&B 564 Stephanitis AA3 Stereum purpureum 1133 Stichococcus bacillaris 358 Stille reaction 1236 Stiretrus anchorago 66 Stoichactis helianthus 72 Stomoxys calcitrans 691 Str. chrysomallus 1010 Str lividans 1010 Streblus asper 467 Strenbergia lutea 609-612,614,615 Streptococcus faecal is 71,S3 Streptococcus haemolyticus 613 Streptococcus mutans 64,226,227 Streptococcus pneumoniae 1154 Streptococcus salivarius 402 Streptococcus sanguis 402 Streptomyces ^'mdixmdimsQ 1145 Streptomyces antibioticus 1010 Streptomyces griseolus 830 Streptomyces griseoviridis 444 Streptomyces griseus var autrophicus 454, 1123,1155 Streptomyces incarnatus 830 Streptomyces purpeofuscus 373 Streptomyces sp. 426,442,443,450,453, 488, 1009 Streptomyces toyocaensis 493 Streptosporagium sibiricum 1211 Strictosamide 115,127 Strictosidine 114-116,127,128,130,131, 135 3a-H Strictosidine 97 Strongyloides ratti 486 Strongyloides stercolaris 486 Structure elucidation 535-551 of reseveratrol oligomers 535-551 of ptaquiloside 708 Strychinine 1030,1049,143 from Strychnos nux vomica L.143 Strychnine TV-oxide 1049 Strychnine 868 Strychnobrasiline 1050,1054,1055, 1058,1063

1342

Strychnofendlerine 1064,1065,1063 Strychnos alkaloids 1029-1031, 1040, 1049, 1053,1054,1060,1063-1065,1067 Strychnos atlantica 1050 Strychnos divaricans 1055 Strychnos guianensis 1055 Strychnos monoindolQ 1061 anticancer/protozoal 1061 istonine 1061 cryptolepine 1061 5,6-dihydroflavopereine 1061 harmane 1061 matadine 1061 melinonine 1061 normelinonine 1061 serpentine 1061 strychnoxanthine 1061 Strychnos panganensis 1052,1055 Strychnos sp. 1032-1034 Alkaloid of 1032-1034 Strychnos vom ica 1052 Strychnospermine 1064,1065,1066 Strychnoxanthine 1060 Strychnos alkaloids 1063 chemosensitizing activity of 1063 Strychonos bifurcata 1034,1039 Strychonos camptoneura 1060 Strychonos colubrina 1029 Strychonos decussata 1032,1035,1036 Strychonos diplotricha 1034,1038 Strychonos floribunda 1032 Strychonos grossweilleri 1060 Strychonos henningsii 1032,1039,103 5 Strychonos madagascariensis 1033,1035 Strychonos mitis 1033 Strychonos mostueoides 1034,1035 Strychonos myrtoides 1034,1035,1038, 1039,1050,1053,1065 Strychonos nuxvomica 1029 Strychonos panganensis 103 3 Strychonos pentantha 1034,103 9 Strychonos potatorum 1031,1032,1036 Strychonos spinosa 1033 Strychonos trichoneura 1034 Strychonos usambarensis 1060,1063

Suberin 1262 Succinate dehydrogenase 800 Succinate ubiquinone oxydoreductase inhibitions 924 Suffruticosol A-C 555,566 Sulfinemycin 443 Superoxide dismutase (SOD) 974 Superstolide A&B 1209 Supinine 874 Suramin 342,791 Suzuki coupling 1248 Suzuki-Miyaura cross coupling 74 of 4-bromo-1 -buty 1-9-borabicy clononane 74,75 Sweet pea {Lathyrus odoratus) 1268 Swem oxidation 1245 Sweroside 103,104,105 Swertia chirata (Loganiaceae) 806 Swiholides D-G 1198 Swinholide A&B 1196,1227 Swinhonsterols A-C 1178 Sylvaticin 793 Symphacia obvelata 463,471,478,493 Syndrome type-lA 1130 adipose tissue distribution 1130 cerebellar dy sfunciton 1130 liver insufficiency 1130 peripheral neuropathy 1130 psychomotor retardation 1130 Synechococcus cultures 363 Synechococcus leopoliensis 359,362-364 Synechocystis PCC 6803 366 Synthesis 635-637,642 of alizarin 635-637,642 of mannans and oligomers 1158 Synthetic derivatives of etoposide 162 acetyl salicylic acid 162 4P-aminoaniline 162 2-aminobenzothiazole 162 2-mercaptobenzothiazole 162 Syringa 327 (-)-Syringaresinol 200 (+)-Syringaresinol 200 (±)-Syringaresinol 267,270 (-)-Syringaresinol diacetate 200

1343

Thannilignan 224,225 Theileria 804 Theoezeolide A 1199 Theoneberine 1200 Theonegramide 1186 Tabtoxinine 372 Theonella conica 1189 Tageretin 746,752,758 Theonella cupola 1190 Tagetes sp. 437 Theonella mirabHis 1191 Taiwania cryptomeriodes 232 Theonella sp. 1175,1176,1179,1181, helioxanthin from 232 1184,1188,1193,1198-1200,1212,1217 TaiwanianE 194,243 Theonella swinhowe 1176-1178,1180, methyl ether of 194,243 1186-1191,1194,1196,1197,1200 Takai's homologation 1224 Theonelladins A-D 1200 Tamine 965 Theonellamides A-F 1185 Tamoxifen 222 Theonellapeptolide la-Ie 1187-1189 Taverniera abyssinica 473 Theonellasterol 1176,1177 Taxaceae 185 Theonellasterone 1176,1177 Taxodiaceae 185 Theopalauamide 1186 Taxol 753,811,812,828,830 Theopederins A 1199 Taxus brevifoUa 212 Thermoplasma acidophilum 1155 brevitaxin from 212 Thiarubrine A&C 437 Tazettine 614,618 Thiohydroximate-(9-sulphonate 480 Tecoma stans 1010 2-Thioxopyrrolidines 1096,1098,1099 Tectoquinone 665 Thopsentia sp 464,466 Tellimagrandin II (eugeniin) 359,372 Three dimensional NMR spectroscopy Temuconine 824,825,827 1044-1046 Teniposide 160,163,212,213 Thromboxane A^ 266 Termilignan 224,225,228,231 Thuja plicata 188 Term inalia hellerica 228,231 Thujopsis 172 anolignan Bfrom231 Thymus s^. 172,746 anolignan B from 224 Tigloylgomisin P 197 termilignan from 224, 231 Tingenone 813,814 thannilignan from 224 Toad {Bufo regularis) 689 Terpenic indanones 701 Tobacco {Nicotiana tabacum) 1145 O, V, O \ O -Tetraacetyl-4-(4' '-brom a-Tocopherol 33 7,993,1006 benzyl) strictosidine 127 (±)-a-Tocopherol 251,258,261 a 'aO',0-Tetraacetyl-4-(4"(±)-Y-Tocopherol 261 brombenzyl) vincoside 116 Tolypocladium niveus 486 O, 'O, O', O -Tetraacetylsecologanin 116, Tomatine 818 118, 125 a-Tomatine 467 Tetrahydrobiopterin 917,950 TOP-53 163 Tetrahydrofuran lignans 198,199 used for anticancer treatment 163 3,4,3',4'-Tetramethoxylignan-7-ol 228,230 Topa quinone 1259,1266-1269,1284,1285 2,6,8,10-Tetra-O-methyl NDGA 191 Topoisomerase I inhibitor 825 Teucrium 172 Topoisomerase II expression 924 (+)-Syringaresinol dimethyl ether 200 (-)-Syringaresinol di-P-D-glucoside 200 (+)-Syringaresinol di-P-D-glucoside 200

1344

Total synthesis 66,67,69,71,72,75,77,78, Tribulus terrestris 259 87,90 tribulusamide A from 259 of (1OZ, 15Z)-10,15-eicosadienoic tribulusamide B from 259 acid 90 Tribulusamide A&B 259 Trichinella spiralis 483 of (/?)-22-methy 1-5,9-octacosadienoic Trichoderma spp. 482,1128 acid 87 of 9, 13-dimethyltetradecanoicacid 66 Trichoderma viride cellulase 1145 Trichophyto rubrum 1159 of 10, 13-dimethyltetradecanoic acid Trichophyton mentagrophytes 673 67 Trichophyton spp. 1127,1128 of (5Z,9Z)-5,9-hexacosadienoic 82 Trichormus var. P-9 359 of (Z)-2-methoxy-5-hexadecanoic acid in agar-diffusion assays 359 77 Trichostrongtlus colubriformis 429,440, of (4£,75)-(-)-7-methoxy-4-tetra 443,453,461, 472,484,490,491 decenoic acid 78 of (/?)-2-methoxyhexadecanoic acid 78 Trichothecium 464 of 11-methyl-12-octadecenoic acid 69 Trichphyton mentagrophytes 227 isomagnolol 227 of 7-methyl-6(£)-hexadecenoic acid 71 magnolol 227 of7-methyl-6-octadecenoicacid 72 Triclisia sp/ 824 of methyl (Z)-2-methoxy-6-hexa Tricophton rubrum 228 decenoate 75 A239,\2A(rricophytonmentagro of ent-calyculin A \23A,\235 Tricuspidatol A 568 Toxicity assays Trifluoromethanesulphonic acid 1162 for extracellular promastigote 790 2,2,2-Trifluroethylhydrazonemethyl9Toxiferine-I 143 deoxy-9-oxo-a-apopicropodophyllate 195 from Strychnos toxifera \ 43 Trifolium repens 696 Toxocara canis 470,471,474,887 2,4,5-Trihydroxyphenylalanine 1266 Toxocara mystax 471 Trihydroxystilbene reseratrol 507,511 Toxoplasma 783,804 {-)-trans-2-{2>" ,4 " ,5' '-Trimethoxy Toxoplasma gondii 835 benzyl)-3-(3',4'-ethylenedioxybenzyl) Trachelogenin 192,233 butyrolactone 192 (-)-A^or-Trachelogenin 474 6,7,8-Trimethoxycoumarin 317,332Trachelosperum jasminoides 233 334,342 arctigenin from 233 2-Trimethylsilyloxy-6-hexadecenonitrile76 matairesinol from 233 Triphophyllum peltatum 813,822 nortrachelogenin from 233 Triterpenoid saponins trachelogenin from 233 from Agrostemma githago var. (-)-Trachyloban-19-oic 812 githago 49 Tramesanguin 1012 from Arenaria filicaulis 52 Trametes cinnabarina 1012 from Caryophyllaceae 6,48 Treatment from Dianthus barbatus 32 of Chagas disease 787 from Dianthus superbus var. of cryptosporidiosis 788 longicalycinus 32,33 of malaria 804 from Gypsophila bermejoi 44 from Gypsophila capillaris 43

1345

from Gypsophila perfoliata 38 from Herniaria glabra 51 from Saponaria officinalis 17,20 from Silene fortunei 47 from Silene villosa 45 from Spergularia ramosa 49 from Vaccaria segetalis 27 Trolox 672 Trychostrongylus 425 Trypanocidal activity 800 Trypanosoma berghei brucei 791,797 Trypanosoma berbhei gambiense 785787,791,792,828 Trypanosoma brucei rhodesiense 785787,791, 792,825 Trypanosoma congolense 785 Trypanosoma cruzi 230,673,787,788, 792, 793, 797,799, 801,802, 813,819, 822,828 Trypanosoma spp. 786,779,780,785, 786, 797,800,803,806-808,810,822, 824,828,838 Trypanosoma vivax 785 Trypanosomiasis 781,785,786,787,840 Trypanosomicidal activity 230 of veraguensin 230 of grandisin 230 Trypanothione synthesis 808 Tryptamine 966 Tryptase inhibitor 1129 (-+-)-Tsugacetal 195 Tuberculatin 194,263 Tubulin 753,828 Turbatrix aceti 481 (+)-ar-Turmerone 457 Tylenchulus semipenetrans 456,469 Tyrosin nitrosylation 926 Tyrosine phosphorylation 753 Tyrosol 333 Ubiquinol-10 1006 Ubiquinone oxydoreductase 924 Ugi reaction 1221 Uhdoside 321,322

Ungeremine 610,616,617,618 Ungernia minor 616 Unonpsine 827 Uronicacid 1154,1156 Urospora penicilliformis 1137 Usambarensine 1063 Usambarine 1063 Vaccaria segetalis 27,28,29,50,56 vaccarosides A-H from 27-30,56 VaccarosideD 29,30 enzymatic hydrolysis of 30 COSY of 29 HMBC/HSQC correlations of 29 HOHAHA spectrum of 29 phase-sensitive NOESY spectrum of 29 Vaccaroside E 30 Vaccaroside F 29,30 •H and 'C spectra of 30 acid induced rearrangement of 31 alkaline and enzymatic hydrolysis of 30 FAB- MS of 30 HMBC/HSQC correlations of 29 IR spectrum of 30 Valanimycin 443 Valeriana officinalis (Valerianaceae) 270 (+)-l-hydroxypinoresinol from 270 (+)-pinoresinol-P-D-glucosidefrom 270 (+)-pinoresinol from 270 (-)-prinsepiol from 270 Vanaliculatol 553 Vancomycin 366 Vatdiospyroidol 563 Vaticaffmol 553,562 Vaticaphenol A 563 Veatchine 868 Velbanamine 1047,1048 Velutinal 461 Veraguensin 230,231,799 Verapamil 1063 Veratraldehyde 1013

1346

Veratryl alcohol 1013 Verbascoside 327,328 Verbesina 437 Vemodalin 808,809 Vernonia amygdalina 808 Vernonia brachycalyx 798,811 Verolide 808 Verrucarin A 464 Verrucosin 228 Vertebrate-parasitic nematodes 428 Vesicular stomatitis virus (VSV) 223 Vezical hematuria 689 Vibrio alginolyticus 70 Vibsanol 255 Viburnum awabuki (Caprifoliaceae) 255 9'-0-(-)-phlligenin from 255 vibsanol from 255 Viciafaba 748 Vignaticol 850 Viguirea aspillioides 810 (-)-trachyloban-19-oic- from 810 Vinblastine from catharanthus roseus 828,829, 217,753 Vincristine 144 from Catharanthus roseus 144 Vincamine 144 from Vinca minor 144 Vincosamide 115,116,117,127 Vincoside 127,128,130,135,136 3p-HVincoside 97 Vindoline 1047,1048 (-)-Viniferal 570 Viniferiftiran 570 (-)-a-Viniferin 535,560 2-Viniferin 552,570 (-)-e-Viniferin 530 (+)-£-Viniferin 530,532,551 Viniferin 552,569 £:-Viniferin 553,560 a-Viniferin 553,560 (+)-a-Viniferin 558,560 (-)-^ra^5-e-Viniferin 560 (+)-a-/m«5-e-Viniferin 560 c/^-e-Viniferin 567

£-Viniferin diol 569 Vinorelbine 1048,1049 Virgilia 1157 Virola oleifera 228 3,4, 3' ,4'-tetramethoxylignan-7-ol from 228 oleiferin-B from 228 oleiferin-F from 228 oleiferin-G from 228 Virola seb if era 186 Virola surinamensis 230 grandisin from 230 veraguensin from 230 Vitamin K 638 Vitis vinifera 530 (+)-Vitisftiran A 570 (-)-Vitisfriran B 570 trans-Wimn A 558,568 VitisinA 568 (+)-c/5-Vitisin A 568 l-ytrans-Vitisin B 559,569 (-)-c/5-Vitisin B 569 (+)-Vitisin C-E 559,569 Volatile components 298 of saffron 298 Volatile isothiocyanates 1075 from Raphanus sativus 1075 Wadsworth-Homer-Emmons olefmation 1230 Warabi 691 Wedelia paludosa 810 e/7/-kaura-16-en-l 9-oic acid from 810 Weinerb reaction 1239,1243 Wikstroemia indica 270 (+)-nortrachelogenin from 270 Wikstromol 192 Wittig olefmation 1234 Wittig reaction 65,66,70,72,73,75,81, 83-91 of bromodecanal 83 of 8-formyloct-5-enoate 86 of (iE:)-5-methyltetradec-4-enal 72 of 12-hydroxy-2-methyldodecanal 70

1347

of 6-methyl-2-heptanone 65 of 7-methy 1-2-heptanone 65 of 5-methyl-2-hexanone 66 of 5-pentadecenal 75 Woorenoside-V 268 Wounding-Induced Protein Kinase 946 Wuweizisu B&C 247,251,256,258,263 Xanthan 1155 Xanthommatins 967,988-992,995 Xanthomonas 582,605 Xanthomonas c. pv. armoraciae 596 Xanthomonas c. pv. carotae 596 Xanthomonas c. pv. orizae 596 Xanthomonas c. pv. raphani 596 Xanthomonas campesths 596,1154 Xanthomonas campestris pv. vitians phytotoxins 596 Xanthomonas fragahae 596 Xanthomonas hortorum campestris pv. vitians 596,606 Xanthones 805,806 Xanthopurpurin 657,665,676 Xanthopurpurin 3-methylether 665 Xanthopurpurin dimethylether 665 Xanthoxylol 226 Xanthurenic acid 966 Xenobiotics 755 Xestospongia sp 487 Xiestoaminol A 487 Xiphimena diversicaudatum 460 Xylans 1156 Yamaguchi conditions 1229,1230 (+)-Yangambin 242,243,264 Yatein 157,192 (-)-Yatein 218 YinghaosuA 807 y-Ylidene-y-butyrolactone structure 363 Yohimbine 143 from Corynanthe johimbe K. 143 Zanthoxyllum naranjillo

799

Zanthoxylum ailanthoides 188 Zanthoxylum armatum 267 Zanthoxylum liebmannianum 450 Zeaxnthin 299,300,306 Zefbetaine 610,616,617 /^o-Zefbetaine 616 Zephyranthes flava 616 Zingiber 905 Zingiber officinale 902,474 Zymosan 342

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