Studies in Natural Products Chemistry, Volume 33: Bioactive Natural Products (Part M)

Studies in Natural Products Chemistry

Volume 33 Bioactive Natural Products (Part M) M)

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

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Stereoselective Synthesis (Part A) Structure Elucidation (Part A) Stereoselective Synthesis (Part B) Stereoselective Synthesis (Part C) Structure Elucidation (Part B) Stereoselective Synthesis (Part D) Structure and Chemistry (Part A) Stereoselective Synthesis (Part E) Structure and 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) Bioactive Natural Products (Part H) Bioactive Natural Products (Part I) Bioactive Natural Products (Part J) Bioactive Natural Products (Part K) 1-30 Studies in Natural Products Chemistry: Cumulative Indices Vol. 1-30 Bioactive Natural Products (Part L) Bioactive Natural Products (Part M)

Studies in

natural Products Chemistry Natural Volume 33 Bioactive Natural natural Products (Part M)

Edited by

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

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FOREWORD Natural products present in the plant and animal kingdom offer a huge diversity of chemical structures which are the result of biosynthetic processes that have been modulated over the millennia through genetic effects, including those triggered by environmental stresses. With the rapid developments in spectroscopic techniques and accompanying advances in high-throughput screening techniques, it has become possible to isolate, determine the structures and biological activity of natural products rapidly, thus opening up exciting new opportunities in the field of new drug development to the pharmaceutical industry. Even if the bioactive natural products cannot themselves be used in medicine directly because of the difficulties in obtaining them in sufficient quantities by isolation or synthesis, they may still offer potential new pharmacophores. These pharmacophores may comprise the part of their structures responsible for the activity and they may be more accessible synthetically. The present volume contains 22 articles written by leading experts in natural product chemistry on biologically active natural products. It includes researches on a variety of different classes of natural products including sesquiterpenes, quassinoids, diterpenoids, lignans, oligostilbenes, phenylethanoids, phenylpropanoid glycosides, curcumin analogues, glycosphingolipids etc. Many of these have been found to be active in a number of different disease conditions. I hope that the present volume will provide a large material of interest to natural product chemists, medicinal chemists, and pharmacologists as well as other researchers, particularly those in academia and in the pharmaceutical industry. We would like to express our thanks to Mr. Liaquat Raza and Ms Qurat-ul-Ain Fatima for their assistance in the preparation of the index. We are also grateful to Mr. Wasim Ahmad for composing and typing and to Mr. Mahmood Alam for secretarial assistance.

Prof. Atta-ur-Rahman Federal Minister/Chairman Higher Education Commission/ Director, International Center for Chemical Sciences Karachi, Pakistan

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vii Vll

PREFACE One of the most fertile areas of chemistry is the interface between chemistry and biology. It is there that chemists, armed with modern tools for separation and spectroscopic analysis, bring to light the structural and mechanistic workings of nature at the molecular level. This basic knowledge drives further investigations in diverse areas such as chemical synthesis, protein structure and function, and cell biology, and ultimately leads to advances in the treatment of diseases of humans, animals, and plants, all of which impact upon the quality of our lives. The editors and publishers of "Studies in Natural Products Chemistry" have again done a great service to the natural products community by assembling this 33rd volume in the series, the 13th devoted to Bioactive Natural Products. Here the reader will find timely reviews written by outstanding scientists from around the world on topics ranging from the purely chemical to the very biological. As a contributor to the first volume of "Studies in Natural Products Chemistry" some 18 years ago, it is a special pleasure to thank Professor Atta-ur-Rahman for his leadership and commitment to excellence that in no small part have been responsible for the success of this series.

Eugene A. Mash Department of Chemistry University of Arizona Tucson, Arizona, USA

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IX ix

CONTENTS Foreword

v

Preface

vii

Contributors

xi

Focus on fluorescent proteins GUIDO JACH AND JOCHEN WINTER

3

Structure, function and mode of action of select arthropod neuropeptides GERD GADE AND HEATHER G. MARCO

69

Natural products as modulators of apoptosis and their role in inflammation JOSE LUIS RIOS AND M. CARMEN RECIO

141

Sesquiterpenes classified as phytoalexins A.K. BANERJEE, M.S. LAYA AND P.S. POON

193

Bioactive triterpenes and related compounds from celastraceae NELSON ALVARENGA AND ESTEBAN A. FERRO

239

Structure-activity relationships of sesquiterpene lactones THOMAS J. SCHMIDT

309

Synthetic investigations in the field of drimane sesquiterpenoids PAVEL F. VLAD

393

Quassinoids: Structure diversity, biological activity and synthetic studies IVO J. CURCINO VIEIRA AND RAIMUNDO BRAZ-FILHO

433

The diterpenoids from the genus Sideritis FRANCO PIOZZI, MAURIZIO BRUNO, SERGIO ROSSELLI AND ANTONELLA MAGGIO

493

Recent developments in the asymmetric synthesis of lignans GIUSEPPE DEL SIGNORE AND OTTO MATHIAS BERNER

541

Natural oligostilbenes MAO LIN AND CHUN-SUO YAO

601

Isolation, structure elucidation and bioactivities of phenylethanoid glycosides from Cistanche, Forsythia and Plantago plants T. DEYAMA, H. KOBAYASHI (Late), S. NISHIBE AND P. TU 645 Pharmacological activities of phenylpropanoids glycosides MARINA GALVEZ, CARMEN MARTIN-CORDERO, MARIA JESUS AYUSO 675

x Development of tubulin inhibitors as antimitotic agents for cancer therapy S. MAHBOOBI, A. SELLMER AND T. BECKERS

719

Cholesterol biosynthesis inhibitors of microbial origin HYUN JUNG KIM, IK-SOO LEE AND SAM SIK KANG

751

Structure-activity relationships of curcumin and its analogs with different biological activities LI LIN AND KUO-HSIUNG LEE

785

The Vinca alkaloids: From biosynthesis and accumulation in plant cells, to uptake, activity and metabolism in animal cells MARIANA SOTTOMAYOR AND ALFONSO ROS BARCELO

813

The chemistry of Olea Europaea ARMANDODORIANA BIANCO AND ALESSIA RAMUNNO

859

The chemistry of the genus Cicer L. PHILIP C. STEVENSON AND SHAZIA N. ASLAM

905

New research and development on the Formosan annonaceous plants YANG-CHANG WU

957

Structural and functional aspects of fungal glycosphingolipids ELIANA BARRETO-BERGTER, MARCIA R. PINTO, MARCIO L. RODRIGUES

1025

Phytochemical studies and pharmacological activities of plants in genus Hedyotis/Oldenlandia NORDIN HJ. LAJIS AND ROHAYA AHMAD

1057

Subject Index

1091

xi

XI

CONTRIBUTORS Rohaya Ahmad

Faculty of Applied Science, University Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia

Nelson Alvarenga

Departamento de Fitoquimica, Facultad de Ciencias Quimicas, Universidad Nacional de Asuncion, P.O. Box 1055, San Lorenzo, Paraguay

Shazia M. Aslam

Natural Resources Institute, University of Greenwich, Chatham, Kent ME4 4TB, United Kingdom

Maria Jesus Ayuso

Departmento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain

A.K. Banerjee

Instituto Venezolano de Investigaciones Cientificas, (IVIC) Centra de Quimica, Apartado 21827, Caracas 1020-A, Venezuela

Alfonso Ros Barcelo

Department of Plant Biology (Plant Physiology), University of Murcia, E-30100 Murcia, Spain

Eliana Barreto-Bergter

Instituto de Microbiologia Professor Paulo de Goes, Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, Cidade Universitaria, CCS, bloco I - Ilha do Fundao, Rio de Janeiro, CEP: 21941-590, RJ - Brazil

T. Beckers

Therapeutic Area Oncology, ALTANA Pharma AG, D78467, Konstanz, Germany

Otto Mathias Berner

Kemira Fine Chemicals Oy, P.O. Box 330, FIN-00101 Helsinki, Finland

Armandodoriano Bianco

Scuola di Specializzazione in Chimica e Technologia delle Sostanze Organiche Naturali - Universita di Roma "La Sapienza", Piazzale Aldo Moro 5, Roma, Italy

Raimundo Braz-Filho

Setor de Quimica de Produtos Naturais, Universidade, Estadual do Norte l'luminense Darcy Ribeiro, Avenida Alberto Lamego 2000, 28013-600, Campos dos Goytacazes Rio de Janeiro, Brazil

Maurizio Bruno

Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermor, Italy

T. Deyama

Central Research- Laboratories, Yomeishu Seizo Co., Ltd., Minowa-Machi, Nagano 399-4601, Japan

xii Xll Esteban A. Ferro

Departamento de Fitoquimica, Facultad de Ciencias Quimicas, Universidad Nacional de Asuncion, P.O. Box 1055, San Lorenzo, Paraguay

Gerd Gade

Zoology Department, University of Cape Town, ZA-7701 Rondebosch, South Africa

Marina Galvez

Departmento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain

Guido Jach

Max-Planck-Institut fur Zuchtungsforschung, Carl von Linne-Weg 10, 50829 Cologne, Germany

Sam Sik Kang

College of Pharmacy and Natural Products Research Institute, Seoul National University, Seoul 110-460, South Korea

Hyun Jung Kim

College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, South Korea

H. Kobayashi (Late)

Central Research Laboratories, Yomeishu Seizo Co., Ltd., Minowa-Machi, Nagano 399-4601, Japan

Nordin HJ. Lajis

Laboratory of Natural Products, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selango, Malaysia

M.S. Laya

Institute Venezolano de Investigaciones Cientificas, (IYIC) Centra de Quimica, Apartado 21827, Caracas 1020-A, Venezuela

Ik-Soo Lee

College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, South Korea

Kuo-Hsiung Lee

Natural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC27599-7360, USA

Li Lin

Natural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC27599-7360, USA

Mao Lin

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

xiii Xlll Antonella Maggio

Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermor, Italy

S. Mahboobi

Department of Pharmaceutical Chemistry I, University of Regensburg, D-93040 Regensburg, Germany

Heather G. Marco

Zoology Department, University of Cape Town, ZA-7701 Rondebosch, South Africa

Carmen Martin-Cordero

Departmento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain

S. Nishibe

Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu-Cho, Ishikari-Gun, Hokkaido 061-0293, Japan

Marcia R. Pinto

Instituto de Microbiologia Professor Paulo de Goes, Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, Cidade Universitaria, CCS, bloco I - Ilha do Fundao, Rio de Janeiro, CEP: 21941-590, RJ - Brazil

Franco Piozzi

Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermor, Italy

P.S. Poon

Instituto Venezolano de Investigaciones Cientificas, (IVIC) Centra de Quimica, Apartado 21827, Caracas 1020-A, Venezuela

Alessia Ramunno

Scuola di Specializzazione in Chimica e Technologia delle Sostanze Organiche Naturali - Universita di Roma "La Sapienza", Piazzale Aldo Moro 5, Roma, Italy

M. Carmen Recio

Department de Farmacologia, Facultat de Farmacia, Universitat de Valencia Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Spain

Jose Luis Rios

Department de Farmacologia, Facultat de Farmacia, Universitat de Valencia Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Spain

Marcio L. Rodrigues

Instituto de Microbiologia Professor Paulo de Goes, Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, Cidade Universitaria, CCS, bloco I - Ilha do Fundao, Rio de Janeiro, CEP: 21941-590, RJ - Brazil

Sergio Rosselli

Department of Organic Chemistry, Palermo University, Viale delle Scienze, 90128 Palermor, Italy

xiv XIV Thomas J. Schmidt

Institut filr Pharmazeutische Biologie der Heinrich-HeineUniversitlt Diisseldorf, Universitatsstrasse 1, D-40225 Diisseldorf, Germany

A. Sellmer

Department of Pharmaceutical Chemistry I, University of Regensburg, D-93040 Regensburg, Germany

Giuseppe Del Signore

Institut fur Organische Chemie, Rheinisch-Westfalische Technische Hochschule, Professor-PMet-Str. 1, 52074 Aachen, Germany

Mariana Sottomayor

Department of Botany of Faculty of Sciences and Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alergre, 823,4150-180 Porto, Portugal

Philip C. Stevenson

Natural Resources Institute, University of Greenwich, Chatham, Kent ME4 4TB, UK. and Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey, TW9 3AB, United Kingdom

P. Tu

School of Pharmaceutical Sciences, Peking University, Beijing 100083, P.R. China

Ivo J. Curcino Vieira

Setor de Quimica de Produtos Naturals, Universidade, Estadual do Norte l'luminense Darcy Ribeiro, Avenida Alberto Lamego 2000, 28013-600, Campos dos Goytacazes Rio de Janeiro, Brazil

Pavel F. Vlad

Laboratory of Terpenoid Chemistry, Institute of Chemistry, the Academy of Sciences of Moldova, Academiei Str. 3, Chisinau, MD-2028, the Republic of Moldova

Jochen Winter

Max-Planck-Institut filr Ztlchtungsforschung, Carl von Linne-Weg 10,50829 Cologne, Germany

Yang-Chang Wu

Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan

Chun-Suo Yao

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

Bioactive Natural Products

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Vol. 33 33 Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. © 2006 2006 Elsevier B.V. B.V. All All rights rights reserved. reserved. ©

FOCUS ON FLUORESCENT PROTEINS GUIDO JACH AND JOCHEN WINTER

Max-Planck-Institut fir Ziichtungsforschung Carl von Linne-Weg 10, 50829 Cologne, Germany ABSTRACT: A number of fluorescent proteins have been discovered in marine organisms with the green-fluorescent protein (GFP) from Aequorea victoria representing the first member of this family being isolated and well characterized. These polypeptides show marked differences in their spectral properties. Today, blue-, yellow-, cyan- and red-light emitting proteins are known in addition to GFP. These natural products possess the unique ability to autocatalytically form a cyclized />-hydroxybenzylidene-imidazolidinone structure acting as a light emitting chromophore in the presence of the suitable environment provided by Bcan 3D-structure of the proteins. Fluorescence actually represents the biological activity of these protein. In fact, this intrinsic protein property allows for their non-invasive non-destructive detection in living cells, an ability that has caused enormous interest on all areas of molecular biology employing reporter proteins for gene expression and protein localization studies. Today, GFP is widely used and well accepted as valuable reporter protein. However, during the last decade a number of GFP mutants were described showing altered spectral properties and/or improved solubility upon expression in heterologous systems. In addition, numerous other naturally occurring fluorescent proteins were described such as the red-fluorescent protein from Discosoma sp. (DsRED). A comprehensive description of the available fluorescent proteins is given including the spectral properties, amino-acid sequence alignments, comparisons of the secondary and tertiary structures of the proteins. The mechanisms for this self-catalyzed amino-acid modifications leading to the chromophore formation are best characterized for the GFP although some work was carried out on other proteins as well. This review describes the current knowledge about maturation and (possible) oligomerization of the proteins.

FLUORESCENT OVERVIEW

PROTEINS:

AN

HISTORICAL

Numerous marine invertebrates display an extensive palette of visible fluorescence and coloring. In part, the vibrant coloration is due to a growing family of intrinsically fluorescent proteins. Genes encoding fluorescent proteins have been isolated and described for a variety of coelenterates, both hydrozoa such as Aequorea, Obelia,

4

and Phialidium, and anthozoa such as Anemonia, Discosoma and Renilla. Amongst the fluorescent proteins known today the greenfluorescent protein (GFP) from the jellyfish Aequorea victoria not only represents the first member of this protein family being isolated it also is by far the best understood fluorescent protein. Its history dates back to 1962 when it was first observed as a companion protein to Aequorin (a blue-light emitting chemiluminescent Aequorea protein), exhibiting a bright, greenish fluorescence when excited with ultraviolet light [1]. Soon after that the first emission spectrum of GFP was published by the same group [2]. Interestingly, the light emission of GFP peaked at 508 run a value that is rather close to the green bioluminescence peak of living Aequorea tissue. In addition, the excitation spectrum of GFP showed a (secondary) peak around 470 nm, which overlaps with the blue chemiluminescence peak produced by Aequorin. Based on these data it was assumed that the green glow of intact Aequorea tissues results from the conversion of the blue light emission of Aequorin into green light by GFP. Later on similar color shifts were found in the related coelenterates Obelia (a hydroid) and Renilla (a sea pansy) and it was suggested that the in-vivo excitation mechanism for coelenterate GFPs is based on radiationless energy transfer [3]. The first prove for this assumption was provided by Morise and coworkers in 1974 [4]. They managed to purify and crystallize Aequorea GFP, and to demonstrate the efficient luminescence energy transfer between co-adsorbed Aequorin and GFP. In addition, the absorbance spectrum and fluorescence quantum yield was measured. The next important achievement was the determination of the chemical structure of the GFP chromophore in 1979 [5]. Protein fragments obtained by proteolytic digestion were screened for peptides retaining visible absorbance. Analysis of these chromopeptides finally lead to the (correct) proposal that the chromophore is a 4-(p-hydroxy-benzylidene)imidazolidin-5-one attached to the peptide backbone through the 1- and 2-positions of the ring. Final prove for the chemical structure of the chromophore was provided by Cody et al. [6].

5

Until 1999 Renilla GFP was the only biochemically well characterized fluorescent protein besides the Aequorea GFP. It proved to have a much higher extinction coefficient, resistance to pH-induced conformational changes and denaturation, and tendency to dimerize compared to Aequorea GFP [7]. However, chromophores from Aequorea and Renilla GFPs was shown to be chemically identical [7-9], indicating that the fluorescence properties of the proteins are depending on both, the chromophore and the corresponding environment provided by the amino-acid backbone. The successful cloning of cDNA encoding the Aequorea GFP by Prasher et al. in 1992 and the subsequent demonstration in 1994 that expression of this cDNA in other organisms is sufficient to produce green fluorescence probably are the most important milestones in the history of fluorescent proteins [10-12]. This not only demonstrated that the GFP gene contains all the information necessary for the posttranslational synthesis of the chromophore, and that no jellyfish-specific proteins enzymes are needed. It also caused enormous attention and interest amongst scientists because they instantly noticed the usefulness of GFP with its unique properties to act as a reporter allowing non-invasive and nondestructive detection of gene expression. This ability is a major advantage over commonly used reporter genes such as NPTII, CAT (detection by autoradiography), LUC (detection of light emitted from converted substrate) and GUS (detection of fluorescent or non-fluorescent dyes released from substrate). However, it rapidly turned out that expression of wildtype GFP often results in poor fluorescence yields and that the system is rather insensitive. Further investigations proved that thermosensitivity of GFP protein maturation is one of the major problems leading to the accumulation of improperly folded nonfluorescent, insoluble protein. [13] Several research groups addressed this problem and tried to identify GFP mutants possessing improved properties such as improved light emission properties (higher quantum yield and/or a extinction coefficient) and/or solubility, which both should increase the amount of detectable fluorescence considerably. In. addition, research also focused on the identification of GFP mutants with altered spectral properties (e.g. altered emission peak wavelength) in order to create

6

tools for simultaneous expression monitoring of separate genes or to obtain suitable donor - acceptor pair to investigate FRET (fluorescence resonance energy transfer). In fact, quite a number of different GFP isoforms with an enhanced or altered fluorescence phenotypes were created using various approaches to mutagenize the wildtype GFP coding region [14-21]. Currently, GFP mutants emitting blue, cyan and yellow light are available (BFP, CFP and YFP, respectively). In 1996 the three dimensional structures of crystallized wildtypeGFP and a GFP mutant carrying the chromophore mutation S65T were published almost simultaneously by two independent research groups [22, 23]. Until 1999 the Aequorea GFP remained the only cloned gene encoding a fluorescent protein. All the different GFP isoforms developed and used in molecular biology are derivatives of the GFP 10 cDNA cloned by Prasher and co-workers. Attempts to clone the gene for Renilla-GFV, which is supposed to be several times brighter than the Aequorea-GYV, were not successful. In 1999 Matz et al. reported the cloning of six novel genes from anthozoan species using a PCR approach with degenerate primers derived from conserved amino-acid sequences of known GFPs [24]. Alignments of the deduced amino-acid sequences of these GFP-like proteins with Aequorea GFP showed that the novel proteins share only a low or moderate degree of homology with the Aequorea GFP. The spectral properties of some of these proteins also were markedly different from GFP, with the greatest differences being found between GFP and a protein from reef corals {Discosoma sp.) emitting red light with a peak at 583nm (dsFP583). This protein soon was made commercially available under the name DsRED. About one year after its first description the three dimensional structure of DsRED and chemical structure of the its chromophore was described by two independent groups [25, 26]. The structural data and other experimental evidences proved that the DsRED protein is an obligate tetramer and that the chromophore is GFP-like but possesses an extended n-electron system due to an additional oxidation step [27, 28]. Due to its unique red-fluorescence the DsRED protein gained strong interest amongst researchers, but it soon turned out that the usability of this protein as a reporter is limited by several factors: its

7

tetramerization, its slow maturation rate and its tendency to form aggregates [28]. In order to overcome these limitations several research teams set out to improve the protein properties by means of molecular evolution. In fact, these approaches not only gave rise mutant proteins with faster maturation rates, lowered aggregation and abolished oligomerization, but also to spectral variants emitting far-red light [29-33]. The usability of these DsRED mutants as reporter proteins remains to be seen. hi 2002 Labas and co-workers investigated numerous Anthozoan species and were able to clone 11 new fluorescent proteins with colors ranging from green to red [34]. An additional protein (eqFP611) emitting far-red light was isolated and cloned from Entacmaea quadricolor another anthozoan species [35]. Today 28 members of the family of fluorescent proteins are cloned. Currently research in this field is very active and it is expected that the number of cloned genes encoding fluorescent proteins will grow even further in the near future. NOMENCLATURE During the last two to three years a huge number of new fluorescent proteins were isolated from various organisms and it is now obvious that a unified nomenclature is ultimately needed to distinguish between these proteins and provide them with unique names, hi addition, a unified nomenclature will help to overcome problems arising from the fact, that commonly used abbreviations like BFP, CFP and YFP for blue, cyan and yellow fluorescent proteins are also used in the literature to name completely unrelated proteins such as an E. coli outer membrane lipoprotein related to bundle forming pili (BFPb), a virulence factor (BFP), a cercosporinfacilitator-protein (CFP), culture filtrate proteins (CFPs) from M. tuberculosis , a fluorescent protein from Vibrio fischeri (YFP) [3640], to name only a few examples. Even more confusing is the common practice to provide commercially available FPs with new trivial names resulting in a situation where certain FPs appear under three different names in literature. In 1992, when the first cDNAs encoding the green fluorescent protein (GFP) form Aequorea victoria were cloned names were created by simply adding numbers (probably the number of the

8

clone investigated) to the name of the gene resulting in GFP1, GFP3 and GFP10 [10]. Subsequently, researchers have only worked on the GFP10 cDNA, which therefore represents the ancestor of all Aequorea -GFP known today and is referred to as wildtype - GFP (andnotGFPIO). With green fluorescent proteins becoming available from other organisms scientists started to add the initial letters of the latin names of the organisms as a prefix to the name of the gene. However, this system is not used consequently. Hence, in the literature the wildtype - GFP is also known as AvGFP, but even nowadays in the majority of publications only the term GFP is used for the GFP 1-gene form Aequorea victoria. Furthermore, the system possesses some severe limitations. Since 1994 quite a number of isoforms of the GFP1 protein were created by directed and random mutagenesis in order to improve protein properties. Due to the lack of a defined nomenclature arbitrary names were given to these genes and proteins such as mGFP4, mGFP5, rsGFP4 and smGFP(for modified-GFP, redshifted-GFP and solubility-modified-GFP [13, 41, 42]. Because it is not obvious that these genes are derivatives of the Aequorea victoria GFP1 gene they might be confused with genes from a different organisms named using the above-mentioned system. In addition, problems arise in cases where two organisms share the same initial letters. More recently fluorescent proteins with different colors became available and researchers started to build names for fluorescent proteins and their corresponding genes by using the abbreviation FP (for fluorescent jyotein in contrast to the non-fluorescent colored chromoproteins (CP)) followed by a number indicating the peak wavelength of light-emission [24,43,44]. This nomenclature is also limited since two different fluorescent proteins from different (or even the same) organism can share the same emission peak wavelength resulting in the same name for both of them. Adding the abbreviated latin-name of the source organism as a prefix would help to distinguish between the two proteins, but only if they are originating from different organisms and/or organisms with different names (= abbreviations). To overcome the above-mentioned problems and limitations a novel nomenclature for fluorescent proteins and chromoproteins

9

was proposed in a recent paper [34]. According to this nomenclature the name for a fluorescent protein and its corresponding gene is build by combining a four-letter leader composed of the first letter of the genus name and three initial letters of the species name, followed by definition of color type: BFP, blue, CFP, cyan, GFP, green; RFP, red; YFP, yellow; and CP, chromoprotein (nonfluorescent). Thus a fictitious green fluorescent protein from the fictitious organism "Propose nomenclature " would get the name "pnomGFP". For undefined species the four initial letters of the genus name serve as leader. In the case of multiple unidentified species of the same genus, a number is added to the leader (resulting in "prop 1 GFP", "prop2GFP" etc. for the mentioned fictitious example) In the case of several proteins of the same color type found in the same species, the number is added to the color definition (such as in "pnomGFP 1" and "pnomGFP2"). For A. victoria GFP and drFP583 from Discosoma sp., widely accepted common names, GFP and DsRed, are kept. PROPERTIES OF FLUORESCENT PROTEINS Basks and definitions In this review the term fluorescent protein refers to proteins being able to autocatalytically form a chromophore, thus possessing the intrinsic property to emit light without the need for any substrate, prosthetic group or cofactor. It is important to notice that numerous other light-emitting systems were found in nature. Besides the various classes of marine organisms mentioned above several other bioluminescent species also have emission-shifting accessory proteins, but so far the chromophores all seem to be external cofactors such as lumazines or flavins [40, 45]. Likewise phycobiliproteins and peridininchlorophyll-a protein, which are highly fluorescent and attractively long-wavelength accessory pigments in photosynthesis, use tetrapyrrole cofactors as their pigments [46, 47]. In the literature of fluorescent proteins the terms fluorescence and brightness are often used as synonyms to describe the detected light emission on protein or cell level. Since the usage of these terms is not consistent literature sometimes is misleading especially when it

10

comes to the companion of different GFPs in terms of their "performance" as reporter proteins. In this review the following terms and definitions are used throughout: •

(molar) Fluorescence = (molar) extinction coefficient * quantum yield

•

Apparent fluorescence (brightness) = (molar) extinction coefficient * quantum yield * protein-amount (= fluorescence * protein-amount)

•

Specific fluorescence = (molar) extinction coefficient * quantum yield / amount of protein (= fluorescence/amount of protein)

Protein and chromophore maturation All GFP-like proteins exhibit a p-can motif, formed by 11 ^-sheets, Fig.(l). Several short a-helical segments connect these strands, while one central helix contains the imidazolidinone chromophore. The chromophore is completely encapsulated in this cylinder, thus physicochemically very stable [48]. The a-helical caps at the top and bottom of the p%can support chromophore protection. The neighborhood of the fluorophore contains a number of charged residues and four water molecules to establish hydrogen bonds [49]. The first step in maturation of GFP is the correct folding into the native conformation, with the chromophore being non-fluorescent. Once folding is complete, the tripeptide chromophore motif is deeply buried in the central helix. This 3D-structure presumably promotes maturation of the fluorophore. The fluorescent chromophore is sequentially formed autocatalytically by two distinct chemical processes [50]. Cyclization proceeds through a nucleophilic attack of the amide group of Gly 67 being in close proximity to the carbonyl residue of Ser65, thus leading to a fivemembered imidazolinone ring intermediate. The secondary structure seems to play an important role in the maturation of the chromophore by facilitating attack of the poorly nucleophilic aniido nitrogen of Gly67 on the poorly electrophilic peptidic carbonyl

11

group of Ser65 [48]. Interaction of Arg96 with the carbonyl oxygen of the fluorophore is supposed to activate ring closure and the elimination of a water molecule. In the last step oxidation of the hydroxybenzyl side chain of Tyr66 by molecular oxygen produces the active fluorophore /?-hydroxybenzylideneimidazolinone [49], Fig. (2). The -C=N- double bond of the imidazolidinone moiety is very likely to support the formation of a -C=C- double bond between C2 and C3 of Tyr66 by spontaneous dehydrogenation in the presence of molecular oxygen [48]. This so formed conjugated 71electron resonance system accounts for the fluorescent characteristics [48]. Kinetic studies on chromophore formation suggested a fast protein folding and cyclization process, while oxidation being rate limiting. This model based on elegant experiments comparing the rate of renaturation of

GFP Fig. (1). The B-can-structure of fluorescent proteins

DsRED

12

reduced and non-reduced GFP. Fluorescence develops much faster under non-reduced conditions with the chromophore structure already been formed [50]. The red fluorescent protein (DsRed) from a Discosoma coral forms a GFP-like chromophore structure but with an extended conjugated 71-electron system, thus leading to a red-shifted spectrum. The fluorophore-containing tripeptide consists of amino acids Gln66-Tyr67-Gly68, Fig. (3). Interestingly, DsRED chromophore maturation proceeds via a green-light emitting intermediate state. Therefore, a mechanism similar to GFP was suggested for the first steps of chromophore maturation, with the imidazolinone moiety being derived from Gln66 and Gly68. The final dehydrogenation step then introduces an additional double bond thus extending the 7C-electron system of the chromophore. Two extraordinary features support such extended rc-system: 1. The Ca of Gln66 is observed to be sp2 hybridized, thus creating a double bond with the peptide N-atom of Gln66. The Ca at the identical amino acid residue in GFP exhibits a normal tetrahedral configuration. 2. Phe65 is connected with Gln66 by a unique cis peptide bond which positions the sp2 Ca of Gln66 in the same plane as the chromophore. The analogous peptide bond in GFP is trans configurated [25]. Dehydrogenation of Gln66 requires molecular oxygen, while possible H2O2, presumably generated by the first oxidation step, is ineffective in forming the double bond [27]. Spectral tuning (Effects of mutations)

With regard to spectral tuning of the chromophore by its protein environment, two classes of mechanisms seem to operate in FPs. First, covalent modification of the chromophore through extension of the system of conjugated electrons or through introduction of charge can affect spectra by fundamentally altering the chromophore resonance properties. For example, deprotonation of the phenolic oxygen of the GFP chromophore causes a dramatic red-shifting of chromophore absorbance nearly 100 nm), and is the mechanism of the fluorescein-like 489 nm absorbance peak of GFP. Second, manipulation of electrostatic interactions between the chromophore and its surrounding protein environment

13

folding lOmin)

cyclization

~N

V

dehydration

oxidation (t 1/2 ~20-83min)

H2O2?

Fig. (2). Proposed mechanism for the GFP chromophore formation

t,/2~3min

14 14

folding cyclization dehydration oxidation

dehydration

GREEN

RED Fig. (3). Proposed mechanism for the DsRED chromophore formation

changes the spectra due to selective interaction with photoexcited or ground states. In GFP substitutions of the second position of the chromophore (Tyr66) directly affect spectral properties of the protein, since the aromatic 7i-electron ring system of the hydroxybenzylidene moiety has an important influence on the fluorescence. Replacement of Tyr66 by Phe (Y66F), which lacks an electron donor group on its aromatic side chain, produces blue-shifted excitation and emission spectra (excitation: 358nm, emission: 442nm). In the mutant Y66H (today known as BFP, see below) the imidazole feature of His with

15 15

its stronger ability to withdraw electrons shows a hyperchromatic effect (excitation: 382nm, emission: 448nm), Fig. (4). Presence of the indole side chain of tryptophane in the mutant Y66W (CFP, see below) shifts the spectrum to 433nm and 457nm for excitation and emission, respectively, Fig. (4). The most dramatic effect on fluorescence is derived from the charged phenolate of Tyr66. Up to now pointmutations substituting amino-acid residues of the chromophore leading to altered spectral properties have only be described for Aequorea GFP. However, mutation located outside the chromophore can also change the spectra of fluorescent protein, indicating that the chromophore environment provided by the fi-can structure of the protein is of equal importance, Fig. (5). It is obvious that mutations replacing Ser65 of wtGFP are not directly involved in the conjugated 7t-electron system of the fluorophore. Nevertheless, introduction with aliphatic residues such threonine, alanine, or glycine at this position leads to mutant proteins with red-shifted excitation spectrum and higher molar extinction. These substitutions are thought to prevent Glu222 from deprotonation due to a change in the hydrogen-bonding network around the chromophore. This uncharged Glu leads to a complete ionization of Tyr66 with a phenolate moiety [49]. In the T203Y mutant of GFP, stacking the it-electron system of the tyrosine side chain on the rc-electron system of the chromophore causes red-shifting due to relative stabilization of the photoexcited state to produce yellow fluorescent protein Fig. (4). Not only for GFP, but also for the red fluorescent protein (DsRED) non-chromophore mutations were described altering its spectral properties. The underlying mechanisms, however, are less well understood. Replacing Lys83 by Arg, Glu, Asn, Pro, Phe, Trp (W) and Met results in proteins emitting green light (peaking at 499nm) instead or in addition to the red emission peak at 582nm [28]. Amongst these exchanges the ration of red to green light emission varies considerably. In the mutant K83R the red emission peak is completely abolished. Obviously the second dehydrogenation step required to form the complete DsRED chromophore either is blocked or very inefficient in these proteins. Thus the chromophore formation stops after the first maturation step resulting in a green-light emitting GFP-like chromophore [28].

16 16

GFP

O

GFP-Y66H (BFP)

HN

11

=\

O

V - NH HO —f

N j»

O

GFP-Y66W (CFP)

GFP-S65G/T203Y (YFP)

DsRED NH,

x.

Fig. (4). Known chromophore structures of fluorescent proteins

17

v

T203

S205

E222

Fig. (5). GFP-chromophore and its environment Environment of the GFP ehromophore according to [22] (modified). Side-chains are marked with the one-letter code for the amino-acid and the residue number, whereas groups of the main chain are labeled with the residue number alone (in italics). Hydrogen bonds are shown as dotted lines.

Introduction of the mutations K70R, K70M,Y120H and S197T into the wildtype DsRED protein shows similar results. The "fluorescent timer" is another DsRED mutant with the unique ability to change its light emission peaks from green to red over time. This property results from the pointmutations VI05A and S197T present in "fluorescent timer" [32]. Green fluorescent proteins The proteins of this class and especially its first member, the Aequorea GFP are by far the best understood fluorescent proteins,

18

often serving as model to understand or explain the properties of other fluorescent proteins. Therefore these proteins were chosen the first to be discussed in this review. For a long time the GFPs from Aequorea victoria and Renilla reniformis were the only well characterized proteins, with Aequorea GFP gaining more importance for molecular biology due to the availability of the corresponding cDNA. Meanwhile also genes encoding Renilla GFP became available. Recently a series of other naturally occuring GFP proteins were isolated and spectroscopically analyzed [34]. Table 1 provides an overview over these proteins. It is important to note that in the original publication three additional GFPs (amajGFP, dstrGFP and clavGFP) were described. With emission maxima around 486nm, however, their spectral properties are much more related to those of the cyan fluorescent proteins (CFPs). Consequently, in this review these proteins will be referred to as amajCFP, dstrCFP and clavCFP and will be discussed in the corresponding chapter. Over the last decade quite a number of different recombinant isoforms of Aequorea GFP with improved fluorescence and folding properties were generated and described in literature [14-21]. Commonly used GFP isoform are listed in table 2 together with their spectral properties and the described improvements. Unfortunately, a closer look reveals that some of these published data are inconsistent, best visible upon comparison of the experimental data describing the relative fluorescence of the proteins between each other (on protein and cellular level) as well as with the data describing their extinction co-efficients and quantum yields. When reading the original publications, it becomes obvious that quite different experimental setups were used by the different researchers to measure protein concentrations and fluorescent properties of the recombinant GFPs, some of which appear to be errorprone. This might explain the observed inconsistencies. Unfortunately, a comprehensive, comparative study investigating these proteins under similar experimental conditions is lacking.

19 Spectra

For the majority of GFPs knowledge about the mechanisms and processes responsible for the fluorescence of the proteins is lacking. However, in this respect the Aequorea GFP and its derivatives were investigated intensively and certainly can serve as model for the other proteins [48, 49]. Depending on the chromophore-structure three different types of spectra can be distinguished for wildtype and mutant Aequorea GFP, Fig. (7). Based on these findings Tsien defined three classes of Aequorea GFPs [51]. Class-1 proteins carry the wildtype chromophore and display the typical type-1-spectra. These spectra are complex in that there is are two excitation peaks (usual a major and a minor one) and a single emission peak. In class-2 proteins the chromophore contains a phenolate anion and the excitation-spectra is "red-shifted": the major excitation peak at 399nm disappeared and the amplitude of former minor peak is increased several fold resulting in a type-2- spectrum. Class-3 proteins contain chromophores with neutral phenol. In the resulting spectra (type-3) the minor excitation peak disappeared leaving the major peak at 399nm alone. These proteins still emit green light. Labas and co-workers analyzed the spectral properties of various GFPs isolated from non-bioluminescent anthozoan species with emission peaks between 499 and 516 nm [34]. Interestingly, all anthozoan GFPs displayed spectral properties similar to the type-1 and -2 spectra of Aequorea GFP, Fig. (7). Apparently, these types of spectra can be used in general as a simplified system to group the proteins. However, whether or not the chromophore structures of the type-1 and -2 anthozoan GFPs are related to the corresponding chromophores of Aequorea GPF remains to be determined. As already mentioned wildtype Aequorea GFP possesses a class1 chromophore resulting in a complex type-1-excitation/emission spectrum with a major excitation peak at 395 nm and a three times smaller minor excitation peak at 475 nm. Interestingly, the wavelength of emission peak varies slightly with the excitation wavelength used: excitation at 395 nm results in a single emission peak at 508 nm, whereas excitation at 475 nm gives maximum light emission at 503 nm [52]. In addition, it was found that at alkaline pH values (pH 10-11) the amplitude of the

498 500 500

2 2 2

Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined * Recombinant isoforms of Aequorea GFP are described in table 2.

506 497

2

-

Scolymia cubensis

scubGFPl

518

508 2

-

Ricordea florida

516 506

2

rfloGFP

508

494 2

-

Dendronephtya sp. Montastraea cavernosa

dendGFP

512

503

2

-

Discosoma sp.3

dis3GFP

mcavGFP

499 (403)480

2

-

nd

nd

506

506

506

500

Ammonia sulcata

nd

nd

nd

nd

496

510

500

2

2

508

405(481)

1

508 496

395(471) 399 (482)

1 1

Peak wai elength's (nm) Em. Ex.

asulGFP (asFP499)

K5E, K10E, N66M

Zoanthus sp.

zoanGFP (zFP506) (zsGreenl)

zoanGFP-0 (ZFP506-N66M-NA)

-

Renilla reniformis

rrenGFP2 (hrGFP)

N66M

-

zoanGFP-I (ZFP506-N66M)

-

Renilla muelleri

-

Ptilosarcus sp.

ptilGFP Renilla reniformis

-

Heteractis crispa

hcriGFP

rmueGFP

-

Aequorea victoria Condylactis gigantea

Type of Spectra

Mutation

GFP*

Organism

cgigGFP

Protein

Cloned green fluorescent proteins

rrenGFPl

Table l.

nd

nd

nd

nd

nd

nd

nd

nd

35600

nd

nd

nd

nd

nd

-

nd

-

-

nd

-

-

1,8

1,8

1

-

nd

nd

nd

nd

nd

nd

0,63

nd

-

nd nd

-

-

-

-

rel. spec. FL

nd

nd

nd

0,80

27600 nd

QY

EC

[34]

[34]

[34]

[34]

[34]

[43]

[55]

[55]

[24]

[54]

[53]

[53]

[34]

[34]

[34]

[10]

Ref

20

21 Table 2. Protein

Properti es of ccimmon isoform s ofAe quorea (IVY Mutation

GFP(gfplO)

Pe ik wavele ngth's (nrn) Em. Ex. 395 (471)

EC

508

25000 30000 25000 30000

GfplO.l

Q80R

395 (471)

508

S65C

S65C

479

507

QY

EC*C

rel.

rel. spec. FL** Prot/Cell

Ref.

0,79

1975023700

1

1/1

[10]

0,79

1975023700

1

1 /I

[11]

6/nd

[17]

0,64

33280 37120

1,6

6/nd

[17]

S65T

S65T

489

511

52000 58000

GFPmutl (EGFP)

F64L S65T

488

508

56000

0,60

33600

1,5

35/39

[18]

GFPmut2

S65A V68L S72A

484

508

n.d.

n.d.

-

-

19/85

[18]

GFPmut3

S65G S72A

504

512

n.d.

n.d.

-

-

21 /77

[18]

Cycle3GFP (smGFP)

F99S M153T VI63 A

398 (480)

508

30000

0,79

23700

1,1

nd/42

[14]

mGFP5

VI63 A I167T S175G

395 473

510

n.d.

n.d.

-

-

nd/20

[13]

mGFP5(S65T)

S65T VI63 A I167T S175G

475

510

n.d.

n.d.

-

-

nd/33

[13]

GFPS65T/S147P

S65T S147P

496

512

n.d.

n.d.

-

-

(6*S65T)

[15]

smRS-GFP

S65T F99S VI63 A I167T

n.d.

n.d.

n.d.

n.d.

-

-

1,68 /nd

[42]

Emerald

S65T S72A N149K M153T I167T

487

509

57500

0,68

39100

1,8

nd/ (8*S65T; 5*EGFP)

[51]

18/

Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined * for bacteria grown at 37 °C, ** as measured for protein-samples (Prot.) or whole cells (cell)

475-nm excitation peak increases at the expense of the 395-nm peak [9]. It was concluded that purified GFP protein samples are actually protein populations consisting of molecules containing either

22

deprotonated/ anionic chromophores giving rise to the 475-nm peak or protonated/ neutral chromophores responsible for the 395-nm peak [19, 52]. Since phenols almost always become much more acidic in their excited states it has to expected that the protonated/neutral chromophore will be deprotonated in the excited state. This light-induced ionization of the chromophore to the anion was proven experimentally [56] and explains why excitation of the neutral chromophores gives emission at greater than 500 nm, similar to but not quite identical to the direct excitation of anionic chromophores. Class-2 GFPs with phenolate anions in the chromophore are commonly used as reporters in molecular biology because they combine high brightness with simple excitation and emission spectra peaking at wavelengths very similar to fluorescein, a popular small-molecule fluorophore. The ionization of the phenol group of the chromophore is caused by a replacement of Ser65 by Thr, Gly, Ala, Cys, or Leu, with S65T being the most commonly used mutation [13, 17, 18, 41]. The GFP mutant RSGFP4 carrying the triple mutation F64M, S65G, Q69L, shares the same fluorescence phenotype. In both S65T and RSGFP4, the wildtype 395-nm excitation peak is suppressed due to the neutral phenol, whereas the 470nm peak is enhanced five- to sixfold in amplitude and shifted to 489^90 nm due to the anion [17, 41, 57]. The probable mechanism by which replacement of Ser65 promotes chromophore ionization [58, 59] is that only Ser65 can donate a hydrogen bond to the buried side chain of Glu222 to allow ionization of that carboxylate, which is within 3.7 °A of the chromophore. Gly, Ala, and Leu cannot donate hydrogen bonds, and Thr and Cys are too large to adopt the correct conformation in the crowded interior of the protein. Such residues at position 65 force the carboxyl of Glu222 to remain neutral. The other polar groups solvating the chromophore are then sufficient to promote its ionization to an anion, whereas if Glu222 is an anion, electrostatic repulsion forbids the chromophore from becoming an anion as well. The chromophore of class-3 proteins contains a neutral phenol group due to repression of chromophore ionization caused by the mutation Thr203 to He [52, 60]. Presumably a chromophore anion

DTLVNRIELK--'GIDFKEDGNILGHK VQCF SRYPDHMKQHDFFKSAMPEG-YVQE- RTIFFKDDGNYKTRAEVKFEG Q. . .K. . . .G - . 1 . .--.M I. . .A. . .E. . .MN NRAYTG..EEI--S.Y.LQSF...-FTY.- . N. RYQ. G. TAIVKSDISL. D GKFIVNVDF. - •AK. LRRM. PVMQQD NRT.TK..EDI—S...IQSF.A.-F.Y.- ..LRYE.G.LVEI.SDINLIE EMF.Y.V.Y.-- .RN.PN..PVMKKT NRT.TK..NDI--S.Y.IQSF.A.-FMY.- ..LRYE.G.LVEI.SDINLIE .KF.Y.V.Y.-- .SN.PD..PVMQKT NRT.TK. . .DI--A.Y.VQSF.A. -FFY. .NLR.E.GAIVDI.SDISL.D .KFHYKV.YR-- . NG. PSN. PVMQKA IKV.AK..KEI--P....QSL.G.-FSW.- . VSTYE . G. VLSATQ. TSLQ. .CIICKVKVL--. TN. PAN. PVMQK. NKV.AK..—KDHP....QSL...-FTW.- .VSNYE.G.VLTVKQ.TSL.. .CIICK.KAH-- . TN. PA. . PVMQKR NKV.TD...DI--P....QSLSD.-FTWR- .VS*Y**G.VLTVTQDTSLK. .CIICN.KVH-- .TN.P.N.PVMQN. NRV.AK..EDI—A.Y..QTF...-.FW.- . SMTYE. Q. ICIATNDITMMEGVD. CFAYK. RFD- -.VN.PAN.PVMQR. NRA.VN. .KDIPDIFKQTCSG.D.-GFSWQ ..MTYE.G.VCTASNHISVD. ..FYYV.RFN-- . EN. PPN. PVMQKR NRV.TE..ADI--T.Y..QSF...-.SW.- ..MTYE.K.ICTI.SDISL.. .CFFQN.RFN-- .MN.PPN.PVMQK. NRA.TE. .TEIADYFKQSFEFG..-FSW.- .SFT.E.GAICVATNDITMV. GEFQYD.RFD-- .LN. * *. .PVMQK. NRA.TE..KEISDYFKQSFEFG..-FTW.- .SFT.E.GAICVATNDI.MV. .EFQYN.RFD-- .VN.P.. . * *MQK. NRA.VN..EDIPDIFKQTCSG.N.G.SWQ- . .MTYE.G.VCTATSNISW. . .FNYD.HFM—.AN.PL. . PVMQKR NRV.TE..QDI--V.Y..NSC. A. - . TWD-.SFL.E.GAVCICN.DITVSV EENCMYH.S.FY.VN.PA..PVM-K.

GFP amacGFPl rrenGFPl rrenGFP2 rmueGFP ptilGFP asulGFP hcriGFP cgigGFP mcavGFP rfloGFP dendGFP scubGFPl scubGFP2 dis3GFP zoanGFP

Fig. (6). Alignment of the amino-acid sequences of cloned naturally occurring GFPs

M—SKGEELFTGWPIL VE LDGDVNGHKFSVSGEGEGDATYGKLTLKF-ICT-TGK-:LPVPWPTLVT-T-FSYG .-I. .V. 1. H R D. . . .El ..-...-.-LG. . .--DLAKLGLKE.M.TK IN .E.L.GD.A..ME.V...NILE.TQEV.I-SV.-K.A-:PLPFAFDI.SVA-.... .V-..QILKN..LQE.MSFK.N .E.V..N.V.TME.C.K.NILF.NQLVQI-RV.-K.A- PLPFAFDILSPA-.Q.. .--..QILKN.CLQEVMSYK.N .E.I..N.V.TME.C.K.NILF.NQLVQI-RV.-K.A- PLPFAFDI.SPA-.Q.. PLPFAFDI.SIA-.Q.. .--NRNVLKN. .LKE.M SAKASVE . I. .N.V. .ME . F .K.NVLF .NQLMQI-RV. -K.GPLPFAFDILSHA-.Q.. . YPSIKETMRVQ LS ME.S. .Y.A.KCT.K. . . KPYE .TQS .NI-TI. -E .GPLPFAFDILSHA-.R.. . CSYIKETMQSK .Y ME.K. .D.N.KCTA. .K . EPYK. SQS . TI-TV. -E .GPLPFAFDILSHA-.Q.. . YPWIKETMRSK .Y ME. . . .N.A.KCTAV. . .KPYK.SQD.TI-TV. -E.GPLPFAYDIL.-.V.D.. ME.A V. E . D.K. KPFD.TQ.MDL-TVI-E . A.--TSVAQEKGVIK.DMKMKLR .--.ALK. EMK.K LK MV.V. . .QS.QID. . . K . KPYE . SQK. T L - E W - E PLLFSYDIL.-.I.Q.. . G..FSYDI.T.-A-LH.. . N.IKEDMRVK .H ME.N. . . .A.VIE. . .K.RPYE.TQ. .NL-TVK-E.AP .—QRAGMKVKEHMK. K LR MG.T. . .KH. A.N. T . D. YPYQ. .QI. .L-.VEGSEP-..FAFDI.SA-A-.Q.. ..FAFDI.SA-A-.Q.. . QSAGKKNWKDFMK . T LR M. .A. . . KP . A. N. T . D .NPYG . IQS . .L-TVD-GN.P LT ME.V. . . LP . KIR. D.K.KPYQ . SQE . T L T W K - G..FSYDI.T.-M-.Q.. . P.--.ALK. EMK.N .—AQSKHGL . KEMTMK YR M E . C D . . . .VIT. . . I. YPFK . . Q A I N L C W E - ..FAEDI.SA-A-.N.. G . P-

GFP amacGFPl rrenGFPl rrenGFP2 rmueGFP ptilGFP asulGFP hcriGFP cgigGFP mcavGFP rfloGFP dendGFP scubGFPl SCubGFP2 dis3GFP zoanGFP

23

Alignment of the amino-acid sequences of cloned naturally occurring GFPs

VLLEFVTA-AGITH--GMDELYK-* .F. . .FS.-C.H. .--. QHETAIA.-HSTIK--KIEGSLP-. TAIAQL.S-L.KPL—.SLHEWV-. TAIAQM.S-I.KPL--.SLHEWV-. TAIAQL.T-I.KPL--.SLHEWV-. EQH. S . V. -SYSQV--PSKLGHN- . EQH.N.R.-S YFNDSG . - . KQH.Y.V.-SYSKV--PSKIGRQ-. .K.HEHAE-.--R.--.LSRKA.-. LSEDA.AH-NSPLE--KKSQAKA-. ARYSPLPK-S.LVEVQ.KAIMTA-. K.R.HAK.RSSLSP--TSAKER.A. K.Q.-YAK-.RSGL—HLP..Q.-. E.T.VAE.-RYSSL--EKIGKS.A. NQKWHL.E-HA.AS--.--SALP-.

GFP amacGFPl rrenGFPl rrenGFP2 rmueGFP ptilGFP asulGFP hcriGFP cgigGFP mcavGFP rfloGFP dendGFP scubGFPl scubGFP2 dis3GFP zoanGFP

Fig. (6). (cont.)

LEYNYNSHNVYIMADKQKNG-IKVNFKIRHNIEDGSVQLADHYQQN-TPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHM . . . .F P..AN..-L G.G T.-V.L I.I L.T.I...R..T.... IVGMQP.YESMYTNVTSVI.-ECIIAFKLQTGKHFTYHMRTV.KSK-K.VETM.LYHFIQ. R. VKTNVDTASGYW ITGLQP . FE . VY. N. GVLV. -QVILVYRLNSGKFY. C H M R T L M K S K - G W K . F . EYHFIQ. R. --EKTYVE . GGFVEQ. E ILGIEP . FEAMY. NNGVLV. -EVILVYKLNSGKYY. C H M K T L M K S K - G W K E F . SYHFIQ. R. --EKTYVE. GGFVEQ. E ILGMEP.FE.VY.NSGVLV.-EVDLVYKLESGNYY.CHMKTF.RSK-GGVKEF.EYHFIH.R.--EKTYVEEGSFVEQ.E G.-LLLRDTPALMLA. . --GHLSCFMET- . YKSKKE .K. . EL .FHHLRMEKLNISDDWK-TV TCGWEP.TETV.PR. TNGWEP. -TETVIPRGGGIL-MRDVPALKLLGNK.HLLCVMETTYK-SKKKGE.AKPHFH.LRMEKDSV. DDEKTI TDGWEP . STETVI--P . DG. - . VAARSPALRLR. KGHLICHMETTY-K. N K E — . K. . EL. FHHLRMEKLSVSDDGK-TI TLKWEP.TEIMYAR.---GV-L.GDVNMALLL.G.GHYRC.--FKT-.YKAKKV.R...Y.FV--DHRIEIVSHD.DYNK TVKWEP.--TE..FER-DGL-LRGDIAMSLLLKG.GHYRC.FKTIY-..—KRK.NM.GY.FVDHCIEIQ.HDKDYNMAV TLKWEP. TEKLHVR. GLLV. N. NMALLLEGGGHYLCDFKTTYKAKK-WQLPDYHFVDHRIEILSNDSDYNKVKLYEHGV TVKWEP . TEIMY. .NGV-L . GEVNMALLL* . K. HYRC . LKTTY-KAKNNV. -HP. GY. .VDHCIEI LE.RK. .V . GGV-L. GEVNMALLLK. K.HYRC . FKTTYKAKNPVP .TA. . *Y. .VDHCIEIT E.N. . YV TVKWEP.TEIMRV TMKWEP.—TE..FER-DGM-LRGDIAMSLLLKG.GHYRC.FETIY-K.--NKV.KM..Y.FVDHCIEIT-SQQDYYNW MTD.WEPSCEK.IPVPKQGI-L.GDVSMYLLLK..-GR.RCQFDTV-YKAKSV.RKM..W.FI--.HK.TRE—DRS.AK

GFP amacGFPl rrenGFPl rrenGFP2 rmueGFP ptilGFP asulGFP hcriGFP cgigGFP mcavGFP rfloGFP dendGFP scubGFPl scubGFP2 dis3GFP zoanGFP

24

25 wavelength (nm) 350

400

450

500

550

600 650 I' ' '

1

• • • I i

1,0-

••• Excitation ~~ Emission

0,8type-1-GFP

0,6 0,40,2 0,0 1,08

0,8

•£

0,4-

I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' type-2-GFP

0,2 0,0

' ' • • I

1,00,8 0,6 0,4 0,2 0,0

Fig. (7). Reference spectra for type 1,2 and 3 - GFPs

type-3-GFP

26

cannot be adequately solvated once the hydroxyl-group of Thr203 is gone, so the chromophore is neutral in almost all the ground-state molecules. However, the emission is still at 511 nm because the excited state remains acidic enough to eject a proton. Factors affecting protein folding and maturation

When expressed at temperature ranging from 20 to 23 °C protein maturation of wildtype GFP is quite efficient, whereas at higher temperatures accumulation of improperly folded non-fluorescent, insoluble protein increases dramatically [13]. This effect was called thermosensitivity. Furthermore it was found that only the folding process of the protein is sensitive to higher temperatures. Once matured GFP is stable, soluble and fluorescent at temperatures up to 65 °C. GFP isoforms only containing chromophore mutations such as S65T, S65C etc. show a similar behavior, indicating that these amino-acid exchanges do alter the spectral properties of the proteins but do not significantly alter the initial protein folding required to form the typical B-can-structure (Jach, unpublished). Nevertheless, chromophore mutations do lead to faster maturation of the chromophore itself (about 4 times), which is supposed to happen once the initial folding is finished [52]. Since Aequorea will probably never encounter warm water the thermosensitivity of GFP most likely is of no consequence under natural conditions. Some researchers used the poor maturation efficiency of GFP at higher temperatures in pulse-chase experiments following the fate of fluorescent protein matured at low temperatures after restoration of normal warmth and simultaneous suppression of new fluorescence [61, 62]. So far nothing is known about the occurrence of thermosensitivity effects in case of GFPs from other marine organisms, table 1. However, these proteins have only recently been described and knowledge about their biochemical properties is rather limited. This point will certainly be addressed in future research on these proteins. The demonstration that the expression of GFP cDNA is sufficient to produce green fluorescence in heterologous organisms has caused enormous interest in the scientific community to use the protein as a reporter for non-destructive monitoring of gene expression. However, it quickly turned out that thermosensitivity of

27

GFP protein maturation clearly limits the sensitivity of this system. This problem was addressed in several independent approaches employing various protocols such as DNA shuffling and errorprone-PCR to create and screen randomly mutagenized GFP proteins for improved properties. These approaches gave rise to the GFP isoforms cycle3GFP, GFPmutl (=EGFP), GFPmut2, GFPmut3, mGFP5, and GFP-S65T/S147P [13-15, 18]. The individual point-mutations present in these GFP derivatives are given in table 2. It appears that in all cases two to three amino-acid exchanges are required to improve the protein folding behavior. Interestingly, some point-mutations (M153T and especially VI63 A) were found in independent experimental approaches by several groups, indicating that they may play a major role in GFP folding [63]. As a matter of fact folding mutations do not increase the intrinsic light emission properties of GFP molecules given by the product of extinction coefficient and fluorescence quantum yield. They rather increase the percentage of properly matured molecules under adverse conditions, such as elevated temperatures (e.g. 37°C) and protein concentrations that promote aggregation [64-66]. However, under conditions not severely hampering the GFP folding (e.g. low temperatures) the presence of known folding mutations (as single mutations or in various combinations) has little or no beneficial effect due to the obvious fact that folding efficiency cannot exceed 100%. Additional research proved that the combinatorial use of folding and chromophore mutations is possible leading to protein isoforms with markedly improved apparent fluorescence as for example in the variants mGFP5(S65T), smRS-GFP and Emerald (see table 2) [51]. The beneficial effects of both sets of mutations and their apparent additive effect, suggests that they may play separate roles in the folding or maturation process. Knowledge about the recently described Anthozoan GFPs is still rather limited and chromophore or folding mutations have not yet been described for the these proteins. Factors affecting the mature proteins Photobleaching and -isomerization

Irradiation of FPs causes light emission according to the spectra of the individual protein, but can also induce two distinct changes of

28

the spectral properties itself referred to as photobleaching and photoisomerization [19]. The term photobleaching describes the (general) loss of fluorescence light emission due to exposure of the sample to light of the excitation wavelength for a prolonged time and/or at high intensities. In this respect fluorescent proteins behave just like any other fluorescent dye (the only difference probably is the energy input needed to achieve bleaching). In contrast, photoisomerization describes the light-induced transition between two different forms of a molecular structure (as for example the switch of double-bond conformation from trans to cis, e.g. all-trans retinal to 11-cis retinal). In wildtype-GFP photoisomerization progressively descreases the excitation/absorption peak and 395nm and increases the secondary peak at 475nm thus causing a shift of the spectra rather than a decline of the peaks (as caused by photobleaching) [19]. Clearly, bleaching sets the ultimate limit on the amount of light emission obtainable from a fluorescent protein. Maximum fluorescence can only be achieved by minimizing photobleaching via limiting one or both of the following: (1) the time of exposure to, or (2) the intensity of the exciting light [51]. However, either of these strategies may compromise the quality of the results or limit the types of analyses that can be performed because the signal to noise ratio is unavoidably decreased. Furthermore, kinetics-based assays performed over an extended period of time may not be possible. Fluorescent proteins with improved resistance towards photobleaching could help to overcome these kinds of problems. In fact, all "second generation" protein isoforms derived from Aequorea GFP (cycle3GFP, GFPmutl (EGFP), mGFP5, Emerald etc., see table 2) are known to be less susceptible to photobleaching. Renilla GFP (rrenGFPl) is also known to be relatively resistant towards photobleaching, whereas the remainder of proteins has not yet been investigated with respect to this property. Photoisomerization of wildtype-GFP can be explained as a lightinduced shift from the neutral chromophore to its anionic form [49, 56, 67] caused by the occasional loss of protons during the reversible proton transfers happening during the light absorption/emission cycles. Probably the reversible proton transfer occurs via the hydrogen bonds of a buried water and Ser205 to Glu222 [59, 64], while the phenolate oxyanion is solvated and

29

stabilized by the rotated side chain of Thr203. In the crystal structure of monomeric wildtype GFP, Thr203 exists in two conformations: approximately 85% with the hydroxyl-group facing away from the phenol oxygen, and 15% with the hydroxyl rotated toward it [59]. This proportion agrees well with the spectroscopic estimate for the ratio of neutral to anionic chromophores at equilibrium [56]. Photo-isomerization is of importance for GFPs showing complex type-1 spectra such as GFP, cgigGFP and hcriGFP. However, for the latter two proteins data concerning their photoisomerization have not yet been published. GFPs with type-2 spectra can be regarded at permanently and stably isomerized proteins no longer suffering from this phenomenon. Oligomerization and Aggregation

The Aequorea GFP possesses a weak tendency to form homodimers. This dimerization usually occurs only at high protein concentrations and is most prominent for the wildtype GFP protein. Interestingly, depending on the conditions wildtype GFP can be crystallized either as a monomer or a dimer indicating that oligomerization of GFP is not obligatory [23, 59]. Yang and coworkers determined the three-dimensional structure of dimeric wildtype GFP and found that the protein interface is formed by the hydrophobic residues Ala206, Leu221, and Phe223 as well as the hydrophilic residues Tyr39, Glul42, Asnl44, Serl47, Asnl49, Tyrl51, Argl68, Asnl70, Glul72, Tyr200, Ser202, Gln204, and Ser208. Although there is hardly any report about artifacts or dysfunctions caused by GFP dimerization [68] this tendency to dimerize can be further reduced or eliminated by introducing mutation such as Phe223Arg, Leu221Lys or Ala206Lys, thus replacing hydrophobic residues of the protein-protein interface by charged amino-acids. For rrenGFPl, rrenGFP2 (hrGFP) and asulGFP it is known that they form stable dimers [69]; Jach, unpublished). The high stability of these dimers is reflected by the fact that these proteins only dissociate under denaturing conditions. The zoanGFP (zsGreenl) appears to form dimers and/or tetramers [55], whereas for the other GFPs (see table 1) these data have not been published yet. Mature Aequorea GFP as well as its recombinant derivatives are known to be highly soluble [51]. Formation of protein aggregates has only been seen for the wildtype Aequorea GFP [19]. In GFP

30

isoforms carrying folding/solubility mutations this aggregation tendency is abolished. Not very much is known about the recently described GFPs from Anthozoan species, but it was stated that almost all of them form protein aggregates (at least in vitro). For a number of anthozoan FPs it has been demonstrated that the Nterminal stretch of amino-acids plays a major role in this respect and that substitution of amino-acid residues responsible for the high positive net-charge of the N-terminus by negatively charged residues results in non-aggregating protein isoforms [55]. For zoanGFP such a mutant was generated by introducing the exchanges LysSGlu and LyslOGlu (for more details see the section about red fluorescent proteins). pH and salt

It was found that the excitation and emission spectra of green fluorescent protein (GFP) and its mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells [51]. Typically, GFP and its mutant derivatives are fully fluorescent at pH values ranging from about pH 6.5 to 10. Above pHIO fluorescence strongly declines. At pH values of 4 and below GFP is non-fluorescent, Fig. (8). Renilla GFP (rrenGFP2) shows a quite similar behavior. 100 rrenGFP2 (taGFP)

mGFPS

asulGFP (FP499)

Fig. (8). pH-optima of some green fluorescent proteins

31

In contrast, the pH optimum of asulGFP is shifted towards more acidic conditions and ranges from pH 5 and 8, Fig. (8). Even under relatively strong acidic conditions (pH 3) this particular proteins shows 50% fluorescence (Jach, unpublished). For EGFP and GFP-S65T detailed pH titrations ranging from pH 5 to 8 indicated 10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (EGFP), 5.9 (GFP-S65T) and apparent Hill coefficients of 1 [70]. Under these conditions the fluorescence spectral shape, lifetime, and circular dichroic spectra were found to be pH independent, whereas at pH values below 5, the fluorescence response was slowed and not completely reversible. Due to this it was concluded that GFP pH sensitivity involves simple protonation events at a pH of 5 and above, but both protonation and conformational changes at lower pH values. The pH sensitivity of GFP in living cells was found to be similar to that of purified protein. Furthermore fluorescence responded very rapidly to a pH changes thereby demonstrating the usefulness of GFP as a non-invasive intracellular pH indictor [70, 71]. Salt concentrations, especially inorganic anions (chloride), can vary widely without impacting the fluorescence of GFP and its mutant isoforms. Further work is required to characterize the other members of the green fluorescent protein family (e.g. anthozoan GFPs) with respect to pH and salt dependence of fluorescence. Temperature

Once matured GFP remains fully fluorescent up to 65°C [51]. At temperatures higher than 65 °C the light emission declines (slowly) probably due to unfolding/denaturing of the protein, so that the chromophore is no longer completely shielded by the surrounding fl-can. At 78°C the fluorescence loss of GFP is 50 % [9]. As yet, for the majority of other GFP proteins temperature stability has not been investigated. Blue fluorescent proteins All blue fluorescent proteins known today are derivatives of Aequorea GFP. The first member of these class of proteins was obtained by intentional introduction of a single point mutation

32 32

replacing the second amino-acid of the chromophore (Tyr66) by Histidine [63]. Since the gained protein displayed rather poor fluorescence compared to GFP some work was carried out to identify improved randomly mutagenized isoforms of this very first BFP, but with limited success only. Even the best performing BFP still suffers from a relatively low fluorescence quantum yield and relatively easy bleaching [72, 73]. A list of the most commonly used BFPs in given in table 3. Not very surprisingly, BFPs were found to benefit considerably from the presence of folding mutations originally described for GFP [42]. Crystal structures for several BFPs have been solved [64, 74]. The BFPs should not be confused with other blue-light emitting chemiluminescent proteins (e.g. spent aequorin and lumazinecontaining proteins from Photobacterium phosphoreum) sharing the same acronym [45]. Table 3. Name

GFP derived BFP and its isoforms Mutation

Pe ak wavelc ngth's (nin) Ex. Em.

EC

QY

EC*(

rel. spec. FL

Ref.

abs.

rel.

Prot. / Cell

0,24

5040

1

Nd/1,0

[52]

22300

0,30

6690

1,3

Nd / 2,9

[63]

440 (447)

26300 (31000)

0,17 (0,26)

4471 (8060)

0,9 (1,6)

Nd/5,6

[65]

448

n.d.

n.d.

-

-

0,15*/nd

[42]

BFP (GFP-Y66H)

Y66H

384

448

21000

P4-3

Y66H Y145F

382

446

EBFP

F64L Y66H Y145F

380 (383)

smBFP

Y66H F99S M153T VI63 A

385

Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined * compared to the fluorescence of cycle3-GFP (=smGFP, Davis 1998)

Spectra

As mentioned above BFPs are mutant isoforms of the Aequorea GFP carrying a histidine residue at position 66. In consequence, an imidazole is placed in the chromophore [52] causing a shift of the excitation and emission peaks towards shorter wavelengths. Further work led to the isolation of improved BFP isoforms with slightly higher molar extinction coefficient and quantum yield (increasing

33 33

total light-emission by about 30 %) by introducing the pointmutation Tyrl45Phe in addition to Tyr66His [63]. All BFPs share rather simple excitation and emission spectra consisting of a single excitation and emission peaks at 383nm and 447 nm, respectively, Fig. (9). Factors affecting protein folding and maturation

The first described BFP (GFP-Y66H, see table 3) also suffers from the thermosensitivity effect described for GFP. In fact, folding of this protein appears to be even more sensitive to elevated temperatures, probably due to the altered steric properties of the mutated chromo- phore. In contrast, the latest recombinant protein wavelength (nm) 350 1,08 c a u £

400

450

500

550 -f-

600

650

••• Excitation — Emission

0,8-

BFP

0,6-

O

_:

0,4

0,20,0 Fig. (9). Typical excitation and emission spectra of BFPs

isoforms (EBFP, smBFP) hardly show any thermosensitivity (Jach, unpublished observation). In the variant P4-3 introduction of the mutation Tyrl45Phe leads to a 30% increase of fluorescence and a three-fold increase of apparent fluorescence, indicating that the major effect of the pointmutation is on protein folding [63]. Later it was shown that addition of the point-mutation Phe64Leu, which was already known to improve GFP folding, further improves the folding of BFP. The resulting clone is commercially available as EBFP. Davis and Viestra combined the Tyr66His chromophore mutation with the folding mutations originally found in cycle3-GFP (Phe99Ser,

34

Metl53Thr and Vail63Ala) giving rise to smBFP [42]. Although neither the extinction coefficient, nor the quantum yield or the relative apparent fluorescence with respect to the original BFP were determined for this protein it is likely that the presence of these mutation will improve the protein folding considerably resulting in increased apparent fluorescence. However, comparative experimental data are missing, so it is impossible to judge the relative "performance" of EBFP and smBFP. Factors affecting the mature protein Photobleaching and -isomerization

All BFPs currently known are very sensitive to prolonged and/or intense excitation light and bleach very fast. For BFP and EBFP the rate of bleaching was estimated to be 40 - 60 times higher with respect to EGFP [75]. Data about the photobleaching of smBFP are lacking, but it is likely that this protein will show similar results, since the only difference to BFP lies in the addition of folding mutations (Phe99Ser, Metl53Thr, Vail63Ala) not altering the spectral properties of the protein. Clearly, the usability of the blue-fluorescent proteins as reporters is limited by their high bleaching rates. Whether further improvement of the properties of theses proteins by means of molecular evolution is possible remains to be seen. Oligomerization and Aggregation

Due to their extremely close relationship to GFP it is highly likely that these proteins share the weak dimerization tendency with its ancestor Aequorea GFP [19]. Although experimental evidence is lacking it has to be assumed that the introduction of mutations blocking the dimerization of GFP (Phe223Arg, Leu221Lys or Ala206Lys) will also be beneficial for GFP-derived BFPs. The known BFPs are highly soluble and do not show significant protein aggregation. pH, salt and temperature

In the published literature there are hardly any data about the impact of these parameters on fluorescence and protein stability of BFPs. The pKi values for the BFP-chromophore were found to be quite similar to those of the achestral GFP-chromophore, although steepness of the slope of the curve describing the pH-dependence of BFP fluorescence is somewhat lower, Fig. (10).

35 35

In term of temperature stability BFPs showed a behavior comparable to that of GFP (Jach, unpublished). No significant losses of fluorescence were seen for mature protein heated to about 65°C. 100%

20%

Fig. (10). pH-dependence of the fluorescence of blue fluorescent proteins

Cyan fluorescent proteins The first described member of the cyan fluorescent proteins (CFPs) resulted from a rationally designed chromophore mutation of Aequorea GFP. Heim and co-worker replaced Tyr66 with Tip and found the peak wavelength for excitation and emission of this GFP derivative (GFP-Y66W) to be shifted to 436 and 476 nm, respectively [52]. Because of this blue-green/cyan light emission the protein was called cyan fluorescent protein or CFP. Since the fluorescence of this protein was several-fold lower compared to GFP researcher attempted to further improve protein properties by random mutagenesis and succeeded to generate improved CFP isoforms. However, only minor improvements were achieved and the best performing GFP-derived CFP known today, ECFP, still is rather dim (see table 4). Recently, a small set of additional cyan fluorescent proteins were described: amajCFP, clavCFP and dstrCFP [34], Table 5, Fig. (11). Unfortunately, besides their spectra not very much is known about these proteins and, in particular, quantitative data allowing to compare the strength of their light emission to ECFP are lacking.

36 36

Nevertheless, one of these proteins has already been made available commercially under the name amCyanl (Clontech). (Note that the latter three proteins were originally classified and published by Labas et al. as the green fluorescent proteins amajGFP, clavGFP and dstrGFP. As mentioned in the chapter about GFPs the renaming and re-classification of these proteins is justified by the fact that their spectral properties are much more related to those of the cyan fluorescent proteins). Spectra

Due to the substitution of Tyr66 by Tip the chromophores of the GFP derived CFPs contain an indole instead of a phenol or phenolate [52]. In consequence, the spectra of these proteins show excitation peaks at 434nm and emission peaks at 476nm to 486nm, which is intermediate between those of GFPs with neutral phenol and anionic phenolate chromophores, Fig. (12). In fact, these proteins possess double-humped rather than conventional single excitation and emission peaks. The origin of the doubled Table 4. Clone

GFP-derived cyan fluorescent proteins (CFPs) Mutation

Pe ak wavelf ngths (mn) Ex. Em.

EC

QY

EC* QY

rel. spec. FL

Ref.

rel.

Prot. / Cell

-

-

n.d. / n.d.

[52]

0,42

10038

1

n.d. / 1

[63]

32500

0,40

13000

1,3

n.d./1,3

[63]

21200

0,39

8268

0,8

n.d./l,6

[63]

CFP (GFP-Y66W)

Y66W

436

485

nd

nd

W7

Y66W N146I M153T VI63 A

434 (452)

476 (505)

23900

WIB(ECFP)

F64L S65T Y66W N146I M153T VI63 A

434 (452)

476 (505)

W1C

S65A Y66W S72A N146I M153T VI63 A

435

495

Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined

37 Naturally occurring CFPs and mutant isoforms

Table 5. Protein

Organism

amajCFP (amFP486) (amCyanl)

Anetnonia mqjano

amajCFP-II (amFP486-K68M)

Anemonia majano

amajCFP-lH (amFP486-K68M-NA)

Fe iik waveU njjths (mn) Ex. Em.

EC

QY

rel, spec. FL

Ret

458

486

n.d.

n.d.

1

[24]

K68M

458

486

n.d.

n.d.

1,5

[55]

Anemtmia majano

K6E K68M

4S8

486

n.d.

n.d.

1,5

[55]

dslrCFP (dsFP483)

Dacosoma striata

-

456

484

n.d.

n.d.

n.d.

[24]

clavCFP (CFP484)

Clavularia sp.

-

443

483

n.d.

n.d.

n.d.

[24]

Mutation

Ex: excitation, Em; emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined

emission peaks must be vibrational levels or other quantum states that equilibrate within the lifetime of the excited state, because their shapes and relative amplitudes are the same regardless of the excitation wavelength [51]. Clearly, the amount of light emitted by these molecules is drastically reduced only reaching levels of 3050% compared to their achestor GFP. In contrast to the GFP derived CFPs the naturally occurring anthozoan CFPs (amajCFP, dstrCFP and clavCFP) show simple spectra with single excitation and emission peaks [34], Fig. (12). The excitation maxima found clearly differ from the GFP-derived CFPs. They are shifted about 10-24nm towards longer wavelength (see table 5), whereas emission maxima of these proteins are in the range of 483nm to 486nm, which is quite comparable to the other CFPs. Interestingly, according to their deduced amino-acid sequences anthozoan CFPs possess GFP-like chromophores

38 38 ECFP amajCFP dstrCFP clavCFP

MGKGEELFTGWP .ALSNKFIGDDMK .SCSKSVIKEEML . KCKFVFCLSFLVLAITNANIFLRNEADLEEKTLRIPKALTTMGVIKPDM

ECFP amajCFP dstrCFP ClavCFP

-ILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT GK-LPVPWP -MTYHM. .C....Y.T.K.. .N.KPYE.TQ. ST.KV.MANG. P-. AFSFD -.DLH.E.TF...Y.EIK.K.K.QPNE.TN.VTLEV.K .GP..FG.H K.KLKME.N. . . .A.VIE KPYD.TH. .NLEVKE .AP. .FSYD

ECFP amajCFP dstrCFP ClavCFP

TLWTLTWGVQCFSRYPDHMKQHDFFKSAMPEGWQERTIFFKDDGNYKT I.S.VFKY.NR. .TA. .TS.—P.Y. .Q.F.D.MSY. . .FTYE.G.VATA I.CPQFQY.NKA.VHH. . — N I . .YL.LSF. . . .TW. .SMH.E.G.LCCI SW. . .MT.E.K.IV.V I.SNAFQY.NRALTK. . .—DIA.Y. -QSF

ECFP amajCFP ds trCFP C lavCFP

RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQK SW. ISLK.NCFEHKSTFH.VN.PA. . PVMAK.TTGWDP. FEKMTVC.GIL TNDISLT . NCFYYD. KFT. LN. PPN. P W Q K . TTGWEP. TERLYPR . KSDISM. E . SFIYE . RFD . MN. PPN . PVMQK. TLKWEP. TEIMYVR. GVL

ECFP amajCFP dstrCFP ClavCFP

NGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALS KTKK. .TM.P. . W E H R I . — K. DVTA. LMLQGGGNYRC. FHTS . - . VLIGDIHHALTVE. GGHY. CDIKTVYRAKKAALKM. GY. . VD. KLVIW V. D.S.SLLLEGGGHYRCDFKSIYKAKKV.K. . .Y. FVDHRI

ECFP ama j CFP dstrCFP clavCFP

KDPNEKRDHMVLLEFVTAAGITHGMDELYK RTDLD. GGNS . Q. TEHAV. H. . --SWPF* NNDK.FMKVEEHEIA.ARHHPFYEPKKDK* EIL.HDK.YNKVTLYEN.VARYSLLPSQA*

Fig. (11). Alignment of the amino-acid sequences of cloned CFPs

and do not contain an indole, Fig. (11). This emphasizes the importance of the interaction between the chromophore and its environment for the spectral properties of the individual protein. Unfortunately, quantitative data excitation coefficients and the quantum yields of these proteins are lacking, so their brightness can not be compared to the other CFPs. For amajCFP a mutant displaying a 1,5-fold increase in brightness upon expression in E. coli was described and found to contain the point-mutation Lys68Met [55]. However, whether or not this mutation affects the

39 wavelength (nm)

350 1

1,0-

8 c

0,8-

| a

0,6-

400 ' ' ' I•' ECFP

450

500 1

600

550 ' ' I '

650

••• Excitation — Emission

0,20,0

-"-+•

1,0

8

0.8

|

0,6

I

amajCFP (amCyani)

0,2 0,0

Fig. (12). Typical spectra of cyan fluorescent proteins

light emission of the protein remains unclear. It might as well be that the increased brightness results from improved folding behavior of the protein. Factors affecting protein folding and maturation

Inspired by the successful optimization of GFP using molecular evolution techniques similar strategies were applied to the GFP derived CFP (GFP-Y66W) resulting several improved protein isoforms. Interestingly, although generated independently these proteins proved to contain two beneficial point-mutations previously described for improved GFP isoforms, Metl53Thr and

40

Vail63Ala, helping them to overcome thermosensitivity by positively influencing the protein folding. Hence these mutations are expected to exert the same effects on the CFP proteins. Other helpful pointmutations found in these proteins include Phe64Leu, Ser65Thr, Ser72Ala and Asnl46Ile [51]. Knowledge about the behavior of the anthozoan CFPs is rather limited. As already mentioned (see above) the amajCFP-II protein (harboring the mutation Lys68Met) was found to show improved brightness [55], which might either be attributed to higher light emission (spectral mutation) or improved protein maturation (folding mutation). More work is required to clarify this point. Factors affecting the mature protein Photobleaching and -isomerization

The cyan-fluorescent proteins were found to be almost as photostable as GFP indicated by the fact that the rate-constant for bleaching only was about 30% higher than the rate of EGFP [75]. In practical terms this slight difference is not limiting the usability of GFP derived CFPs as reporter proteins in molecular biology. Photoisomerization has not been reported for GFP derived CFPs. Data about the photobleaching and -isomerization of the recently described amajCFP and derived isoforms are not yet available in published literature. Oligomerization and Aggregation

All CFPs being descendants of Aequorea GFP are expected to share the (rather weak) dimerization tendency with its achestor. Due to the close relationship between GFP and CFP introduction of mutations blocking the dimerization of GFP (Phe223Arg, Leu221Lys or Ala206Lys) should also be beneficial for CFPs. However there is no further evidence for this assumption and its truth remains to be proven. The anthozoan protein amajCFP as well as its mutant amajCFPII (see table 5) form tetramers as judged by semi-native gelelectrophoresis [55]. For the anthozoan CFPs protein oligomerization and aggregation needs to be investigated. When it comes to protein aggregation the known GFP derived CFPs should behave just like Aequorea GFP with the mature proteins being highly soluble not giving rise to significant protein

41 41

aggregation. In contrast, amajCFP forms protein aggregates and the other anthozoan CFPs are likely to behave in the same manner. Introduction of a negatively charged residue (Lys6Glu) into the amajCFP protein resulted in the non-aggregating protein version (amajCFP-III), indicating that the high positive net-charge at Nterminus of the protein is responsible for protein aggregation as already described for other anthozoan FPs [55]. pH, salt and Temperature

For the GFP derived CFPs, amajCFP and its mutant isoforms as well as dstrCFP and clavCFP no data were found in published literature describing the pH-dependence, salt-sensitivity and temperature-stability of these proteins. As close relatives of GFP the mature GFP-derived CFPs are supposed to be able to tolerate relatively high temperature (up to 65 °C) before denaturing starts. However, this can only be speculated since experimental data are missing. Yellow fluorescent proteins Together with the protein classes BFP and CFP, also the first yellow fluorescent proteins (YFPs) were originally derived form Aequorea GFP by introduction of amino-acid exchanges at positions 65 (the first amino-acid of the chromophore) and 203 [51]. In fact, these mutants were rationally designed based on crystal structure of GFP in order to create a fluorescent protein sufficiently different in its fluorescence properties to allow for double-labeling experiments, FRET and other experimental applications. Very recently the first naturally occurring yellow fluorescent protein (zoanYFP) was cloned and spectroscopically analyzed [34], Fig. (13). However, further work is required to characterize this protein in more detail. In this review we refer to the GFP derived and naturally occurring YFPs and their isoforms as given in tables 6 and 7. Today GFP derived YFPs are widely used as reporters in molecular biology [76].

42 42 Spectra

As mentioned above the point-mutations at positions 65 and 203 were introduced in order to gain additional interaction of 7Uelectrons and to enhance polarizability around the chromophore, thus reducing excited state energy and increasing both the excitation and emission wavelengths [51]. In the GFP derived YFPs residue 65 is Gly (or Thr) instead of Ser to promote ionization of the chromophore, whereas Thr203 is replaced by an aromatic residues (His, Trp, Phe, and Tyr). In consequence, the aromatic ring of the amino-acid at position 203 is stacked next to the phenolate anion of the chromophore resulting in excitation and emission wavelengths increased by up to 20 nm (30), Fig. (14). When mutation Gln69 to Lys (Q69K) is added in addition to the mentioned pointmutation of positions 65 and 203 the maximum light emission is shifted by 1-2 nm, resulting in an emission peak around 529 nm [51]. The threedimensional structure of zoanYFP and the chemical nature of its chromophore remains to be determined. Consequently, knowledge about the mechanisms underlying the fluorescence properties, Fig. (14), of this protein is lacking. Factors affecting protein folding and maturation

For YFPs derived from the Aequorea GFP (table 6) it has to be expected that they also do suffer from the thermosensitivity effect described for GFP. Indirect evidence is provided by the fact that the point-mutations described to improve the folding of GFP are also helpful for the GFP derived YFPs (see below). However, no direct evidence is present in published literature.

43 Table 6. isoforms Name

GFP-derived yellow fluorescent protein (YFP) and its Mut.

Pe ik wavele ngths (ntn) Em. Ex.

EC

QY

EC* QY

rel. spec. FL

Ref.

rel.

Prot. / Cell

0,70 45850

1

n.d./1

[51]

48500

0,78 37830

0,8

n.d./ 2

[51]

529

62000

0,71 44020

0,9

n.d./ 8,3

[51]

514

527

83400

0,61

50874

1,1

n.d./9,7

[51]

S65G S72A K79R T203Y

514

527

94500

0,60 56700

1,2

n.d./16,7

[51]

Citrine

S65G V68L Q69M S72A T2O3Y

514

524

77000

0,76 58520

1,3

n.d.

[77]

EYFP-F46L

F46L S65G V68L S72A T203Y

515

528

78700

0,61

48007

1

n.d./ 194

[78]

SEYFP

S65G V68L S72A M153T VI63 A S175G T203Y

515

528

101000

0,56

56560

1,2

n.d./29,1

[78]

Venus (SEYFP-F46L)

F46L S65G V68L S72A M153T VI63 A S175G T203Y

515

528

92200

0,57

52554

U

n.d./291

[78]

abs.

GFPS65G/S72A/T203F

S65G S72A T203F

512

522

65500

GFPS65G/S72A/T203H

S65G S72A T203Y

508

518

10C-Q69K.

S65G V68L Q69K S72A T203Y

516

lOC(EYFP)

S65G V68L S72A T203Y

Topaz

Ex: excitation, Em: emission, EC: extinction coefficient, QY: quantum yield, n.d.: not determined

44 Table 7.

Naturally occurring YFPs and mutant isoforms EC

QY

rel. spec. FL

Ref.

538

nd

nd

nd

[24]

(494) 528

538

nd

nd

nd

[55]

(494) 528

538

nd

nd

nd

[55]

Name

Organism

Mutation

zoanYFP (zFP538) (zsYellow)

Zoanthus sp.

-

(494) 528

zoanYFP-II (ZFP538-M129V)

Zoanthus sp.

Ml 29V

zoanYFP-III (zFP538-M129V-NA

Zoanthus sp.

K5E K9T Ml 29 V

Pe ak wavelt :ngths (n & £ Apoptosome

Caspase 9

Effector caspase

Fig. (2). Mechanism of apoptosis

U

dATP

148 148

Bcl-2 family proteins Bcl-2 was first discovered as a proto-oncogene in follicular B-cell lymphoma. It has since been identified as a mammalian homologue to the apoptosis repressor Ced-9. The mitochondrial-dependent pathway in the human Bel-family includes both pro-apoptotic (Bax, Bak, and Bok) and anti-apoptotic proteins (Bcl-2, BCI-XL, Bcl-w, etc.) [10]. Although the precise mechanisms of the mammalian Bcl-2 family of proteins are still a matter of debate, it has been established that their main function is to regulate the release of cytochrome c and other proteins from the mitochondria [24]. The relative ratios of anti- and pro-apoptotic Bcl-2-family proteins dictate the ultimate sensitivity to or resistance of cells to various apoptotic stimuli, including hypoxia, radiation, anticancer drugs, oxidants, Ca2+ overload, ceramide, and growth-factor/neurotrophin deprivation [25]. During apoptosis, the pro-apoptotic Bcl-2 family members are activated through several mechanisms such as dephosphorylation or proteolytic cleavage brought about by caspases. They are then translocated to the mitochondria. The translocation of Bax, Bid, or Bad can induce the release of the proteins contained in the intermembrane space, including cytochrome c, which, only after subsequent coupling with a heme group, functions to induce caspase activation [25,26]. The anti-apoptotic proteins Bcl-2 and BCI-XL prevent cytochrome c release, thus preserving cell survival. Cytochrome c release is thus an early event during apoptosis, occurring hours before phosphatidylserine exposure and loss of plasma membrane integrity. As mentioned above, it is only after cytochrome c release that caspases areactivated and the cell undergoes apoptosis. The actual apoptotic process occurs through the formation of an "apoptosome" (comprised of cytochrome c, apoptosis protease activating factor 1 (Apaf-1) and procaspase-9). This apoptosome then recruits procaspase-3, which is cleaved and activated by the active caspase-9 and is subsequently released to mediate apoptosis (Fig. 2) [27]. In some cellular systems, cytochrome c is necessary, but not sufficient, for cell death. In these systems, a second mitochondrial activation of caspases is promoted by a protein with the dual name of Smac/DIABLO [28,29], which is released from the mitochondria with

149 149

cytochrome c in a coordinated fashion during apoptosis. While cytochrome c activates Apaf-1, Smac/DIABLO relieves the inhibition of caspases by binding to the IAPs, thereby allowing caspase-9 to activate caspase-3 [30]. Alterations in the amounts of Bcl-2 proteins have been associated with diseases in which too much or too little cell death occurs (this is referred to as cell loss and cell accumulation, respectively). These diseases include cancer, autoimmune disorders such as lupus, immunodeficiency associated with human immunodeficiency virus (HIV) infection, and ischemia-reperfusion injury during stroke and myocardial infarction, among others [31]. Death ligands and death receptors The "death receptors" of the TNFR family include TNFR1, Fas (CD95), DR3/WSL, and the TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) receptors. When these receptors are bound by a ligand (TNF, Fasligand (FasL), etc.), apoptosis may occur [25]. Fas is a glycosylated cell surface molecule that can also be found in soluble form. It is expressed on several different cell types (mainly in macrophages, activated T and B lymphocytes, and in organs such as the thymus, liver, spleen, lungs, testes, brain, intestines, heart, and ovaries), and its expression can be augmented both by cytokines (interferon-y (IFN-y) and TNF) and by lymphocyte activation. In contrast, expression of FasL is more tightly regulated, often being induced only under very specific conditions. Moreover, FasL expression is restricted to immune cells including T and B lymphocytes, macrophages and natural killer cells, and to non-immune sites such as the testes, kidneys, lungs, intestines, and eyes [25]. A fuller understanding of the Fas-FasL interaction is necessary to comprehend better the signaling pathway involved in death receptorinduced apoptosis. Inappropriate expression of Fas and FasL on lymphocytes and other immune cells has previously been documented in patients with HIV infection; it has also been implicated in the loss of lymphocytes that characterizes this immunodeficiency syndrome [32]. Conversely, hereditary mutations in the death domain of the Fas gene are known to cause an autoimmune lymphoproliferative syndrome in humans [33].

150 150

Researchers have discovered that Fas-mediated apoptosis is blocked by a molecule that has been assigned various names, but is usually referred to as c-FLIP (cellular FLICE-inhibitory protein) (other names include FLAME, I-FLICE, Casper, etc.) [34,35]. The expression of c-FLIP is down-regulated by IL-2 [36,37], a fact which explains why IL-2 can sensitize activated T-cells to Fas. Ligation of a death receptor does not necessarily lead to caspase-8 activation and death. TNFR1 can also activate nuclear factor-KB ( N F - K B ) in cells that are resistant to TNFR1-mediated apoptosis. Inhibition of this transcription factor can thus sensitize the cells to this particular form of cell death [38]. In summary, these are only some of the major molecular pathways for the induction and control of caspase activation and apoptosis following a variety of stimuli for cell death. It should be noted, however, that apoptosis induced by ultraviolet (UV) light and other stimuli has been shown to occur in animals lacking caspase-9 [39], thus suggesting at least one alternative to the apoptosome. APOPTOSIS AND INFLAMMATION Defects in the physiological pathways of apoptosis have been shown to play a role in many diseases (Table 2). Consequently, there has a been a great surge of interest in devising therapeutic strategies for modulating the key molecules that make life-or-death decisions in cells. In addition to targeting the core components of the cell-death machinery (caspases, IAPs, and Bcl-2 family proteins) directly, opportunities exist to affect apoptosis indirectly by modulating the input into cell-death pathways through protein kinases, protein phosphatases, and transcription factors, as well as by affecting the cell-surface receptors for cytokines, neurotrophins, cardiotrophins, and growth factors [19]. For example, the inhibitor of the N F - K B (IKB) kinase (IKK) complex has emerged as a potential target for promoting the apoptosis of cancer cells. N F - K B has been found to suppress cell death in a variety of contexts, and has also been seen to induce the transcription of several anti-apoptotic genes, including members of the Bcl-2 family and some IAPs. Moreover, N F KB hyperactivity has been observed in several types of cancer. Certain non-steroidal anti-inflammatory (NSAIDs) drugs, as well as synthetic triterpenoids and other compounds have been reported to inhibit the

151

catalytic activity of IKK. These agents should be examined not only for their ability to sensitize tumors to apoptosis, but also for their potential in treating inflammatory diseases in which N F - K B has been implicated [19, 40].

Table 2.

Diseases associated with defective apoptosis regulation

Insufficient apoptosis

Cancer, autoimmunity, restenosis, persistent infections, atherosclerosis, metabolic disorders.

Excessive apoptosis

Neurodegenerative disorders (Alzheimers's disease, Parkinson's disease), hematological disorders, autoimmune disorders (graft versus host disease, type I diabetes, rheumatoid arthritis), ischemia, heart failure, inflammation, osteoarthritis, human immunodeficiency virus, bacterial infections, allograft rejection, trauma.

There are several areas in which the modulation of apoptotic death could advance medical treatment of disease in the future: 1) Control of malignant diseases, 2) Delay of premature senescence/neurodegenerative disorders, 3) Treatment of transplant rejections, 4) Regulation of tissue regeneration/repair, and 5) Regulation of inflammatory diseases through the induction of apoptosis and phagocytosis, which would aid in suppressing and/or resolving the inflammatory response [41,42]. The rest of this section will deal with how new findings concerning the apoptotic process may influence the treatment of inflammatory diseases. To date, one of the main methods of controlling inflammations has been the use of glucocorticoids, which have proven highly effective in attenuating the inflammatory response. These hormones are able to induce apoptosis in the monocytes, macrophages, and T lymphocytes involved in the inflammation reaction. However, in fibroblasts and glandular cells such as hepatocytes and ovarian follicular cells, glucocorticoids actually protect against apoptosis induced by cytokines, cyclic adenosine monophosphate (cAMP), tumor suppressors and death genes. This phenomenon is probably due to composite regulatory crosstalk among multiple nuclear coactivators or corepressors, which mediate the transcription regulation of the genes by interacting with the glucocorticoid receptor [43]. Another line of research in this area includes the study of the apoptotic processes of other cells, which play major roles in the immune system. Recent work has thus focused on the apoptosis of a key component of

152

this system, namely the polymorphonuclear (PMN) leukocytes known as neutrophils. Aged neutrophils have been found to undergo spontaneous apoptosis in the absence of cytokines or other proinflammatory agents prior to their removal by macrophages. In acute inflammation, however, the number of neutrophils within tissues may reach excessive levels due to recruitment from circulation and also because their constitutive apoptotic pathway is delayed by the action of local inflammatory mediators [44]. Because the potential for inflammatory neutrophil tissue damage via the release of toxic reactive oxygen species and granule enzymes such as proteases is very high, death by apoptosis and safe removal by phagocytic cells helps limit tissue damage during the resolution of inflammation. It has been demonstrated that neutrophil apoptosis can be promoted by a wide variety of agents, including granulocyte-macrophage colony stimulating factor (GM-CSF), P2 integrins, and TNF-a. In the regulation and execution of neutrophil apoptosis, members of the Bcl-2 family and caspases (caspases-1, -3, -4 and -8) are also involved. Cell surface receptors and protein kinases, particularly mitogen-activated protein kinases (MAPK), also play critical roles. These and other aspects related to the regulation of neutrophil apoptosis have recently been reviewed [45]; from the research to date, it is clear that promoting the safe clearance of apoptotic cells may be a new avenue for therapy in inflammatory responses which persist rather than resolve [46]. Controlling apoptotic processes may also be useful in devising new treatments for numerous inflammations of the skin. One of the principal cell groups found in the skin is that of the keratinocytes, the epithelial cells which comprise the epidermis of the skin and the epithelium of the oral mucous membranes. The main function of these cells is to provide an intact epithelial covering for the body to serve as an impermeable barrier. While keratinocyte stem cells are found in the deepest tips of the dermal papillae, proliferating keratinocytes are found in the basal and the immediately suprabasal epidermis. There are other cellular elements in the epidermis, which influence epidermal keratinocyte function, notably melanocytes. These cells produce melanin, a complex heteropolymer which protects against the toxic effects of light. Once synthesized, melanin is transferred to keratinocytes to form supranuclear shields against ultraviolet radiation effects. In addition, keratinocyte function is influenced by the cytokine-releasing epidermal Langerhans cells. These

153 153

are specialized dendritic antigen-presenting cells that reside in the upper epidermis, but which emigrate to regional lymphatic when presented with exogenous antigens. In many inflammatory skin diseases, recirculating lymphocytes can enter the epidermis in response to cytokine and chemoMne release. Keratinocyte apoptosis has been observed not only during a number of biological processes, such as epidermal differentiation, but also in certain skin diseases in which the epidermis is the target of immunological cytotoxicity [47,48]. The skin, and especially the epidermis, is constantly at risk for induction of cytotoxicity by ultraviolet radiation, oxidant stress, cytokines, chemokines, and neuropeptides, as well as cytotoxic lymphocytes and macrophages. Modulation of the members of the Bcl-2 family is thus a potential mechanism for the defense of the epidermis against apoptosis. Since immunohistochemical analyses have demonstrated that Bcl-2 proteins are localized in the basal keratinocyte layer, it has been proposed that these proteins may be a major inhibitor of apoptosis in keratinocytes. Indeed, transgenic mice which overexpress Bcl-2 in the epidermis showed decreased susceptibility to apoptosis induced by ultraviolet radiation or 12-0-tetradecanoylphorbol 13-acetate (TPA) [49]. However, despite the intrinsic defenses of the basal layer of the epidermis against the induction of apoptosis, keratinocyte apoptosis is largely involved in a number of immunological skin diseases, including lichen planus, graft versus host disease, photosensitive lupus, erythema multiforme, and other forms of dermatitis. By the same token, it has been demonstrated that keratinocyte apoptosis represents the major mechanism in the pathogenesis of atopic dermatitis as well as other eczematous disorders. The process begins as T cells infiltrate the skin and upregulate the Fas receptor on the keratinocytes, thus rendering them susceptible to apoptosis by IFN-y. Apoptosis is simultaneously induced by the FasL expressed on the surface of the T cells themselves [50]. Chondrocytes, the only cell type found in normal mature articular cartilage, are responsible for the maintenance and repair of this tissue, which happens to be the site of pathologic matrix remodeling and degradation in arthritis [51]. Indeed, chondrocyte apoptosis is a feature of osteoarthritic cartilage and is closely associated with extracellular matrix degradation. Fibrillated cartilage from osteoarthritic joints has been found to contain apoptotic cells in both the superficial and mid zones. In

154

contrast, very low numbers of apoptotic cells are detected in normal cartilage. This is due to the fact that cartilage does not contain mononuclear phagocytes; therefore, apoptotic bodies are more likely to exert pathogenic effects on this tissue. Chondrocyte apoptosis in osteoarthritis may be the consequence of aberrant hypertrophic chondrocyte differentiation, but it may also be induced by extracellular stimuli such as FasL and other cytokines. In addition, nitric oxide (NO) production may lead to chondrocyte apoptosis. Various factors thus contribute to the pathogenesis of cartilage degradation. Inhibitors of NO synthesis and chondrocyte apoptosis may therefore be of therapeutic value after cartilage injury and in osteoarthritis [52]. In contrast to osteoarthritis, rheumatoid arthritis is a chronic inflammatory synovitis dominated by the presence of macrophages, lymphocytes, and synovial fibroblasts and which ultimately leads to the destruction of bone and cartilage. The effectiveness of therapies that are directed against TNF-a and IL-1 lends credence to the notion that macrophages are a crucial target for therapeutic intervention in this disease [53]. Because inadequate or insufficient apoptosis appears to play a significant role in the increase in cellularity of rheumatoid synovial tissue, lack of apoptosis is seen as contributing to the perpetuation of the disease. The mechanisms that prevent apoptosis of inflammatory cells in rheumatoid arthritis have recently been reviewed by Pope [54], whose findings support the idea that interventions aimed at enhancing apoptosis in the synovium are potentially effective forms of treatment in patients suffering from this disease. ANTI-INFLAMMATORY NATURAL PRODUCTS As mentioned above, the resolution of inflammation involves clearing away the excess of inflammatory cells by apoptosis and the subsequent recognition and removal of apoptotic cells by phagocytes. One hallmark of inflammation, especially in the skin, is macrophage infiltration, which quite often can mediate chronic inflammations such as psoriasis, atopic dermatitis, and chronic contact dermatitis [55]. In both atopic dermatitis and allergic contact dermatitis, a disruption of the epidermal barrier occurs, leading to spongiosis, which in turn can rupture the intercellular attachments of the keratinocytes, thus generating vesicles. Secretion of IFN-y by T lymphocytes then promotes Fas upregulation in

155 155

keratinocytes, which undergo apoptosis as part of the normal inflammatory reaction. In the case of keratinocytes, however, the inflammatory infiltrate is the cause rather than the consequence of their apoptosis [56]. Finally, apoptosis is implicated in the resolution of T cellmediated cutaneous inflammation, such as that associated with delayedtype hypersensitivity (DTH) reactions [57]. There are thus several different mechanisms by which apoptosis can be involved in the inflammatory response. The fact that the apoptosis of inflammatory cells can be regulated pharmacologically makes this phenomenon extremely interesting, as it can therefore be exploited to develop new drug therapies [44]. In fact, as discussed above, the effect of many anti-inflammatory drugs, such as glucocorticoids, NSAIDs, and anti-oxidant agents, is based on the role these drugs play in this particular physiological process. Glucocorticoids and their implication in anti-inflammatory effects Glucocorticoids, which play a major role in attenuating the inflammatory response, do so via a mechanism in which a specific receptor is implicated. As mentioned above, the anti-inflammatory action of glucocorticoids is exerted by two complementary, but opposite pharmacological effects: a) by an apoptotic mechanism that induces the death of the inflammation-provoking cells such as monocytes, macrophages, and T lymphocytes; and b) by an anti-apoptotic mechanism that protects the resident cells of the inflamed tissue by arresting the apoptotic signals evoked by cytokines, cAMP, tumor suppressors, and death genes on glandular cells and fibroblasts. This latter mechanism involves the modulation of several survival genes such as Bcl-2, BCI-XL, and N F - K B in a cell-specific manner [43,58]. Modification of transcription signals and apoptotic effects

The glucocorticoids have been found not only to increase apoptosis, but also to modulate the expression of apoptosis-related markers in both unstimulated and IL-2-stimulated T lymphocytes. In one study, this class of drugs induced apoptosis while reducing Bcl-2, Fas, and CD25 expression. Only negligible effects were detected on Bax expression, a fact which points towards a potential mechanism by which some corticoids exert their anti-inflammatory effects [59].

156 156

In another study on the effects of glucocorticoids on proliferation and apoptosis, as well as on the activity and expression of N F - K B in intestinal epithelial cells, glucocorticoids were seen to modulate the repair mechanisms of intestinal epithelial cells in vitro while profoundly modulating the inflammatory regulator N F - K B , a known regulator of apoptosis and inflammation [60]. On the other hand, glucocorticoids decreased the expression of cyclooxygenase-2 (COX-2). This is of interest because upregulated COX-2 expression plays a relevant role in pathological processes characterized by increased local prostaglandin (PG) production and consequently in the process of inflammation itself [61]. Role of cells in inflammation and their modification by apoptosis

While the role of neutrophils in asthma remains relatively obscure, eosinophils have been shown to play a major part in the onset and maintenance of the bronchial inflammation and tissue injury in this condition [62]. Like other leukocytes, eosinophils present in excessive numbers in inflamed tissues are removed by apoptosis, which allows for the elimination of dangerous cells [63,64,65]. Conversely, a defect in eosinophil apoptosis leads to the development and persistence of allergic airway inflammation in asthma. It is also thought that a defect in apoptosis might contribute to the chronic tissue eosinophilia associated with the malady. It has been determined that this delay of eosinophil apoptosis in asthma occurs in part due to the production of GM-CSF. Traditional treatments for asthma are based almost exclusively on the use of inhaled glucocorticoids, which totally reverse the delayed eosinophil apoptosis in this condition [66]. It is clear that a better understanding of the mechanisms underlying eosinophil apoptosis would help delimit the molecular events involved in eosinophil accumulation in the blood and tissues, which in turn would uncover potential new targets for the treatment of allergic diseases in general, and asthma in particular [67]. One line of research that has proven of interest is that concerning the role of caspases in eosinophil apoptosis. Thus, although it has been established that treatment with dexamethasone induces eosinophil apoptosis via a mechanism mediated through the glucocorticoid receptor, as evidenced by the fact that the effect was nullified by the glucocorticoid receptor antagonist mifepristone [68], it has also been shown that this dexamethasone-induced apoptosis and activation of c-jun

157 157

NH2-terminal kinase (JNK) and p38 MAPK activity in eosinophils is regulated by caspases. Interestingly, the caspases involved are not the common apoptosis-related caspase-3 or -8, as is the case in other cells. The elucidation of the role of caspases in eosinophil apoptosis may thus facilitate the development of more specific and effective treatments for this type of allergic inflammation [69]. Neutrophils, for their part, have been implicated in mediating the tissue damage associated with chronic inflammatory diseases. Glucocorticoids have been found to exert significant inhibitory effects on both neutrophil activation and neutrophil functions such as chemotaxis, adhesion, transmigration, apoptosis, oxidative burst, and phagocytosis [70]. Since phagocyte recognition, uptake, and nonphlogistic degradation of neutrophils and other leukocytes undergoing apoptosis all promote the resolution of inflammation, this hitherto unrecognized ability of glucocorticoids to potentiate the nonphlogistic clearance of apoptotic leukocytes by phagocytes has potential implications for therapies aimed at promoting the resolution of inflammatory diseases [71]. Basophils comprise another group of cells actively involved in allergic inflammations. The apoptogenic effects of glucocorticoids on these cells might have implications for the mechanism of action of these drugs in allergic inflammation [72]. Mast cells, positioned in the asthmatic airways, also play a major role in the maintenance of the condition. During the active disease, these cells are primed to secrete preformed and newly generated inflammatory mediators, neutral proteases, cysteinyl leukotrienes, cytokines, and chemokines [73]. The local delivery of glucocorticoids to the affected tissues has been found to reduce significantly the number of mast cells present. In one study in which glucocorticoids were directly applied to mouse dermis, the number of mast cells decreased. No direct effect of the glucocorticoids on the mast cells themselves was observed; thus, the decrease was probably the result of increased apoptosis [74]. T-cells are involved in various inflammatory pathologies such as asthma, multiple sclerosis, and human idiopathic polymyositis. The modification of these cells may thus be an important mechanism to help avoid the damage caused by these afflictions. In the natural disease course of multiple sclerosis, for example, apoptosis contributes to the elimination of T-cells from the inflamed central nervous system. Using corticoids to induce apoptosis could contribute to the down-regulation of

158 158

T-cell activity, thereby terminating the inflammation of the central nervous system [75]. Moreover, corticoids may reduce allergen specific T-cells through apoptosis, which is one of the mechanisms of effectiveness of corticoids in asthmatics [76], as well as induce apoptosis of endomysial T-cells in human idiopathic polymyositis, thus modifying the inflammation [77]. Interestingly, the expression of Bcl-2 may be an important factor in protecting the lymphocytes in inflamed synovia! membranes from glucocorticoid-induced apoptosis [78]. Inflammatory cytokines and apoptosis

Phagocytosis of apoptotic cells by macrophages leads to the production of anti-inflammatory cytokines as a way of preventing inflammation. Kurosaka et al. [79] have demonstrated that human serum potentiates the production of two anti-inflammatory cytokines, IL-10 and TGF-p, by both TPA-treated THP-1 cells and human monocyte-derived macrophages. This enhanced response to the presence of apoptotic cells also results in the suppression of the production of the pro-inflammatory cytokine IL-8. In addition, human IgG and FcyRI appear to be critical in triggering the production of anti-inflammatory cytokines by macrophages in response to apoptotic cells. Glucocorticoids repress the expression and release of numerous cytokines in macrophages, thymocytes, and CD4+ splenocytes; in addition, a protein-protein interaction with transcription factors such as N F - K B is involved in their anti-inflammatory activity. The only defect of immune suppression brought about by this class of drugs detected so far concerns the induced apoptosis of thymocytes and T lymphocytes [80]. Non steroidal anti-inflammatory drugs (NSAIDs) The anti-inflammatory mechanism of the NSAIDs is principally related to any or all of the following three factors: the inhibition of different enzymes implicated in the aracMdonic acid metabolism, the modification of the effects of relevant mediators, and the inhibition of the tissue damage produced by free radicals. It is thought that the phenomenon of overexpression of the enzymes implicated in inflammation may be avoided by the effect of apoptotic mechanisms on inflammatory cells. The role of eicosanoids and NSAIDs, with either a positive or negative

159

effect, thus provides exciting possibilities for new anti-inflammatory agents. Arachidonic acid metabolites and apoptosis

The arachidonate cascade includes the COX pathway to form prostanoids and the lipoxygenase (LOX) pathway to generate several oxygenated fatty acids, collectively called eicosanoids. While the exact mechanism remains unknown, it has been shown that eicosanoids play a dual role in regulating both cell survival and apoptosis in various types of cells [81]. One recent study, for example, demonstrated that arachidonic acid metabolites are involved in the regulation of apoptosis in human polymorphonuclear neutrophil granulocytes [82]. NSAIDs such as the selective COX-2 and LOX inhibitors were initially developed to suppress inflammation and pain by inhibiting the production of PGE2 and its metabolites. Since then, however, the arachidonic acid metabolites, particularly 12-hydroxy-6,8,11,14eicosatetraenoic acid (12-HETE), 5-HETE, and PGE2, have been found to play a pivotal role in prostate cancer. In a recent study with human prostate cancer cell lines, for instance, the addition of a 5-LOX inhibitor induced apoptosis and decreased cell life duration. In contrast, 5-HETE prevented cell death and led to the overexpression of COX-2 as well as a clear increase in PGE2 production. Inhibiting COX-2 may thus be a possible key to the treatment of prostate cancer since selective inhibitors of COX-2 actually reduce PGE2 production in this cancer, and this, in turn, leads to cell apoptosis [83]. Moreover, while overexpression of 12LOX and 15-LOX-1 in prostate cancer cells stimulates prostate tumor angiogenesis and growth, the expression of 15-LOX-2 is reduced during the initiation and progression of prostate tumors. It has been found, however, that 15(5)-HETE, the product of 15-LOX-2, inhibits proliferation and causes apoptosis in human prostate cancer cells. This fact suggests an inhibitory role for 15-LOX-2 in the progression of the prostate tumor [84]. The relationship between 5-LOX activity and apoptosis has been reported in several different studies. Anderson et al. [85] studied the effect of the compound MK886, which at nM concentrations is a selective in vivo inhibitor of 5-LOX, while at uM concentrations it inhibits the proliferation of monoblastoid cells by means of an apoptotic mechanism. In human pancreatic cancer tissues, which show a marked

160 160

expression of 5-LOX and the leucotriene B4 (LTB4) receptor, it has been demonstrated that the inhibition of 5-LOX not only blocks the proliferation of cancerous cells, but also that it induces their apoptosis [82]. Moreover, the 5-LOX branch of the arachidonate cascade is also responsible for membrane peroxidation, oxidative stress, and apoptosis of peripheral blood mononuclear cells; thus, the administration of substances such as vitamin E, which inhibits 5-LOX activity, may be helpful in controlling oxidative stress-related diseases [86]. Lipoxins (LXs) are LOX-derived eicosanoids generated during inflammation that inhibit chemotaxis and adhesion of PMN neutrophils. They also function as putative braking signals for PMN neutrophilmediated tissue injury. Lipoxin A4 (LXA4), for example, promotes the phagocytosis of apoptotic PMN neutrophils by monocyte-derived macrophages, thus acting as an endogenous stimulus for PMN neutrophil clearance during inflammation [87]. Moreover, LXA4 has been shown to inhibit TNF-a-stimulated neutrophil adherence to epithelial monolayers at nM concentrations. This is noteworthy as TNF-a not only induces disruption of mucosa architecture, but also enhances colonocyte apoptosis via a caspase-3-independent mechanism [88]. Nuclear factor-KB (NF-KB) and apoptosis

comprises a family of inducible transcription factors that serve as relevant mediators of the inflammatory response. This factor is also involved in protecting cells from undergoing apoptosis in response to DNA damage or treatment with cytokine [89]. Normally, N F - K B is kept inactive by a cytoplasmic inhibitor of KB (IKB) proteins, which are phosphorylated by a cellular kinase complex known as IKK, made up of two kinases, IKK-a and IKK-p. The phosphorylation of IKB by these kinases leads to the degradation of the proteins and to the translocation of N F - K B to the nucleus. Once in the nucleus, N F - K B activates gene expression of cells exposed to growth factors and cytokines [90,91]. Activation of the N F - K B pathway is thus involved in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and asthma [92], while an altered N F - K B regulation is in part responsible for the inflammatory response of other pathologies such as Alzheimer's disease [93]. Taking this into consideration, then, it is not surprising that the mechanisms of actuation of various pharmacological agents are based on

NF-KB

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how these anti-inflammatory drugs modulate N F - K B effects. In the case of glucocorticoids, the repression of the entire N F - K B pathway is implicated in their mechanism of action, while different NS AIDs inhibit this pathway at various stages, including that of IKK inhibition [89]. Despite evidence that N F - K B is generally an antiapoptotic factor, there are cases in which N F - K B acts as a proapoptotic [94]. For this reason, the modification of N F - K B could be an interesting target for different antiinflammatory drugs. Inhibition of N F - K B activation by NSAIDs has been described in many cells. These drugs, however, failed to impair IKB kinase activity, the processing of N F - K B , or the expression of N F - K B dependent genes such as iNOS in hepatic cells. Moreover, selective COX-2 inhibitors did not promote apoptosis in hepatocytes under inflammatory conditions, a fact which suggests that PGs are not required to maintain cell viability [95]. Nitric oxide (NO) and apoptosis

NO, derived from L-arginine (L-Arg) by the enzyme nitric oxide synthase (NOS), is involved in the regulation of relevant physiological and pathophysiological functions. The mechanisms by which NO exerts its effects include activation of guanylate cyclase, formation of peroxynitrite, apoptosis, and COX regulation [96]. Apoptosis induction mediated by NO involves mitochondrial depolarization and is blocked by Bcl-2 overexpression [97]. NO is generated under inflammatory conditions and may serve as a cytotoxic molecule to produce cell death along either an apoptotic or a necrotic pathway. NO formation is established to initiate apoptosis, characterized by upregulation of the tumor suppressor p53, changes in the expression of pro- and anti-apoptotic Bcl-2 family members, activation of caspases, and DNA fragmentation. Prestimulation of macrophages with cytokines or low-level NO has been shown to activate the transcription factor N F - K B and to promote immediate early gene expression of COX-2 [98]. In a recent study, the NO radical quenching activity of NSAIDs and steroidal drugs demonstrated that NSAIDs directly scavenged generated NO and prevented the reduction of cell viability and apoptotic nuclear changes in neuronal cells without affecting the induction of iNOS. In contrast, corticoids, which had no scavenging effects in vitro, showed

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almost no protective effects. These data suggest that the protective effects against apoptosis of the NSAIDs studied might be due mainly to their direct NO radical scavenging activities in neuronal cells [99]. Antioxidant compounds and apoptosis

While apoptosis is related to the production of reactive oxygen intermediates, the modulation of apoptosis by antioxidants correlates to the modification of cell proliferation. There is evidence that oxidative stress acts as a major determinant of apoptotic cell death [100]. For this reason, the ability to differentiate and modulate apoptosis and necrosis by antioxidants opens up a wide range of possibilities in anti-inflammatory therapies [101]. It has been well established that fatty acid metabolites of LOX and cytochrome P450 are implicated in essential aspects of cellular signaling, including the induction of apoptosis. The enzymatic and non-enzymatic products of polyunsaturated fatty acids thus control cell growth and apoptosis, and the spontaneous oxidation of polyunsaturated fatty acids gives rise to reactive aldehydes and other products of lipid peroxidation that are potentially cytotoxic and which may also signal apoptosis [102]. MODULATION OF APOPTOSIS BY NATURAL PRODUCTS Anti-inflammatory properties have been attributed to many natural products, some of which are antioxidants while others inhibit the arachidonic acid metabolism. Still others act through a mechanism related to the modification of transcription signals. The apoptotic effects of natural products have received little attention to date, although more in-depth studies of these effects would help determine the precise mechanisms through which these compounds act as anti-inflammatory agents. The majority of studies in this area have focused on the apoptotic effects of phytochemicals in cancer research with the goal of identifying anti-cancer agents, but much of this research could also be applied to the study of the anti-inflammatory properties of natural products. Such is the case of alkaloids or phenolics such as flavonoids, both of which are widely studied in phytochemical and pharmacological research. For this review, we have selected a few representative natural products, many of which have previously been described as anti-

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inflammatory agents. We have, however, also included several compounds whose mechanisms and known properties may be related to the inflammatory process. We thus classify this review into two main groups, namely apoptosis-inducing compounds and those compounds with anti-apoptotic effects. Natural products as activators of apoptosis and their implications in inflammatory diseases Alkaloids

Alkaloids comprise the most relevant group of phytochemical compounds with pharmacological and biological activities. Indeed, a wide range of alkaloids has been described as being analgesics, antiinflammatories, or anticancer agents, with various mechanisms of action depending on their pharmacological effects. As for their role in the resolution of the inflammatory process, recent data have implicated the apoptosis of specific cells, a fact which makes the investigation of apoptosis an interesting new approach to studying different compounds, both known and new. The xanthine theophylline has been used for several decades in the treatment of asthma. This compound produces different effects at the cellular level, including phosphodiesterase isoenzyme inhibition, adenosine antagonism, catecholamine secretion enhancement, and the modulation of calcium fluxes. Recently, theophylline was found to have both immunomodulatory and anti-inflammatory properties; therefore, interest in its use in patients with asthma has been renewed [103]. Recent studies have thus discovered that at low doses, theophylline is able to decrease airway inflammation, accelerate eosinophil apoptosis, and decrease recruitment of lymphocytes and neutrophils to the lungs. Although it is classified as a phosphodiesterase inhibitor, its exact therapeutic mechanism of action remains undetermined [104]. Of the new mechanisms that have been included in the potential mode of action of theophylline, one is the apoptosis of inflammatory cells. In eosinophils and lymphocytes, for example, this effect is due to the compound's ability to inhibit phosphodiesterase, which leads to an even more pronounced increase in intracellular cAMP levels than that which occurs when adenylate cyclase, the enzyme that synthesizes cAMP, is activated. This inhibition and the resulting cAMP level increase thus lead to

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apoptosis in the aforementioned cells. Since inducing apoptosis is generally beneficial in allergic inflammations, the use theophylline in combination with corticoids may also be appropriate to induce apoptosis in eosinophils and lymphocytes [105]. Inflammatory cytokines such as GM-CSF and IL-5 are upregulated in bronchial asthma, and because they inhibit granulocyte apoptosis, they cause neutrophil and eosinophil infiltration into the airways. The administration of theophylline counteracts this affect by accelerating granulocyte apoptosis, which is important not only in combating the inflammation, but also in controlling granulocyte longevity regardless of the elevation of intracellular cAMP levels. Studies have shown that after theophylline administration, the percentages of GM-CSF-induced delayed apoptosis increased in both neutrophils and eosinophils, and that the percentage of IL-5-induced delayed eosinophil apoptosis was also higher. Moreover, cAMP-increasing agents inhibited granulocyte apoptosis both in the control and in anti-Fas antibody-treated cells, with the expression of Bcl-2 protein also decreasing after incubation of eosinophils with theophylline [106]. Additionally, theophylline was shown to induce apoptosis of leukemia cells in humans [107].

Sanguinarine, isolated from the root of Sanguinaria canadensis, possesses both anti-inflammatory and antioxidant properties. In addition, this alkaloid has displayed anti-proliferative and apoptotic effects against human epidermoid carcinoma cells and normal human epidermal keratinocytes. While treatment with sanguinarine has been shown to decrease the viability of both kinds of cells, this loss of viability occurred at lower doses of the compound and was much more pronounced in the carcinoma cells than in the normal keratinocytes [108].

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In a study on the mechanisms of apoptosis induced by Chinese and Western anti-rheumatic drugs in human T-cells, Lai et al. [109] demonstrated that Tripterygium wilfordii and its alkaloid tetrandrine can cause T-cell death. It is noteworthy that the apoptotic effect of tetrandrine was selective toward especially activated T-cells. Although the cytotoxicity of this compound was mediated through apoptotic mechanisms, Fas/FasL interaction was not required. Moreover, tetrandrine-induced T-cell DNA damage required caspase-3 activity. The induction of apoptosis brought about by tetrandrine was much faster than that caused by treatment with glucocorticoids, and did not require de novo protein synthesis. These results suggest that the anti-inflammatory and irnmunosuppressive properties of tetrandrine are mediated by novel mechanisms that have yet to be determined [40]. Nevertheless, cepharanthine, an alkaloid analogous to tetrandrine, has been shown to restore the aberrant in vitro morphogenesis of apoptotic cells treated with both TNFoc and plasmin. This alkaloid suppressed the TNFa-stimulated N F - K B activity by partially preventing the degradation of IKBO: protein in NS-SV-AC cells. In addition, cells which were pretreated with cepharanthine and which then received subsequent treatment with both TNFa and cepharanthine exhibited suppressed production of matrix metalloproteinase 9 [111]. Other alkaloids with potential interest as proapoptotic agents are acutiaporberine from Thalictrum acutifolium, which induces apoptosis by down-regulating the Bcl-2 gene while simultaneously up-regulating the Bax and c-myc genes [112]; acrocynine, a cytotoxic alkaloid isolated from Achronychia baueri used to synthesize active proapoptotic agents [113]; sinococuline from Stephania sutchuenensis [114]; solamargine from Solarium incanum [115]; and cryptolepine and neocryptolepine, both isolated from Cryptolepis sanguinolenta [116].

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OCH 3

OCH,

OCH 3 Cepharanthine

Tetrandrine

CH3O

OCH3 OCH3 OCH,

CH3O

OCH3

OCH3 Acutiaporberine

Phenolics

Phenolics and their functional derivatives are widely found throughout the plant kingdom. One defining characteristic of these compounds is that their aromatic ring usually contains at least one hydroxyl substituent. In a broad sense, phenolics, which are classified according to their structural skeleton, are basically derivatives from simple phenols and phenolic acids, phenylpropanoids including coumarins and lignans, flavonoids and related compounds, and stilbenes. Some of these compounds which show anti-inflammatory activity are reviewed in this section.

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Tiram'-resveratrol, a natural product obtained from grapes and grape products such as red wine, has recently been shown to have antiinflammatory properties. Indeed, resveratrol is a potent inhibitor of both N F - K B activation and NF-KB-dependent gene expression due to its ability to inhibit IKK activity. By this mechanism, resveratrol blocks the expression of genes that promote inflammation and protect against apoptosis [117]. Moreover, resveratrol induces apoptosis through activation of p53 activity in those cells that express wild-type p53 protein, but not in p53-deficient cells. This mechanism may thus be responsible for resveratrol's proven anti-carcinogenic activity [118].

/raws-Resveratrol

Capsaicin is a natural compound that has been described as both antigenotoxic and anti-carcinogenic. In addition, it is surmised to have a potential chemopreventive activity [119]. The compound's antiinflammatory properties have been demonstrated in different in vivo pharmacological tests, which have shown that it inhibits, among others, carrageenan-induced inflammation in rats and croton oil-induced mouse ear edema. These effects are associated with its interference of phospholipase A2 (PLA2), the enzyme that produces arachidonic acid from the membrane phospholipids. Moreover, the proapoptotic effects of capsaicin are widely documented in the literature [120]. Although the exact mechanism responsible for the proapoptotic effects of capsaicin remains unknown, several different mechanisms have been proposed, including the inhibition of plasma membrane nicotinamide adenine dinucleotide reduced (NADH) oxidase activity [121], regulation by Bcl-2 and calcineurin [122], and the overexpression of the p53 tumor suppressor gene and/or c-myc proto-oncogene [123]. Since tumor promotion is related to inflammation, the anti-inflammatory and antitumoral effects of capsaicin are most likely directly related to each other and are thus both of interest. In addition, the activation of N F - K B by

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external signals provoked the migration of this factor to the nucleus, where it binds to a specific segment of DNA. This triggers the expression of a variety of rapid-response genes involved in important physiopathological reactions, including inflammation [120]. Vanilloids such as capsaicin are recognized at the cell surface by vanilloid receptor type 1 (VR1), which mediates the effects of capsaicin in VR1-expressing cells. There are, however, pathways which are not mediated by VR1 through which vanilloids are able to induce apoptosis. [124]. Macho et al. [125], for example, demonstrated that capsaicin induces apoptosis in transformed cells and produces a rapid increase of reactive oxygen species (ROS). Interestingly, this latter effect is not a consequence of calcium signaling; thus, the apoptotic pathway may be separated from that which mobilizes calcium. Moreover, the authors evoke the implication of a possible vanilloid receptor in calcium mobilization, but not in ROS generation.

CH 3 O

Capsaicin

Curcumin, the major component of the spice turmeric {Curcuma longa), exhibits anti-inflammatory and antioxidant activities, and inhibits both the generation of ROS and the JNK pathway. This compound reduces or inhibits not only the effects of PLA2 and phospholipase Cyl (PLCyl), which are involved in arachidonic acid release; but also the protein kinase C (PKC) activity induced by treatment with TPA. It has also been shown to reduce the inhibition of tyrosine protein kinase activity and to inhibit oxidative DNA damage both in the epidermis of mice as well as in cultured fibroblast cells of mice. The compound also inhibits the generation of ROS, including superoxide and hydrogen peroxide in peritoneal macrophages. Finally, it has been shown to inhibit the expression of proto-oncogenes such as c-fos, c-jun and c-myc [126,127,120]. Different researchers see a relationship between these effects and the ability to induce apoptosis. Thus, Kuo et al. [128] demonstrated that while protein synthesis inhibitors did not affect the apoptosis-inducing activity of curcumin, antioxidant agents prevented it,

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suggesting that curcumin-induced cell apoptosis is mediated by ROS, a proposal that gives Bcl-2 an important role in the early stages of curcumin-triggered apoptotic cell death. Recently, Piwocka et al. [129] demonstrated that curcumin induces caspase-3-independent apoptosis in human multidrug-resistant cells. In addition, Deeb et al. [130] demonstrated that a mixture of curcumin and TNF-related apoptosisinducing ligand (TRAIL), a member of the TNF family of cell deathinducing ligands, induced the cleavage of procaspase-3, procaspase-8, and procaspase-9, as well as the truncation of Bid and the release of cytochrome c from the mitochondria. These effects indicate that, at least in prostatic cancer cells, treatment with the aforementioned mixture triggers both the extrinsic and intrinsic pathways of apoptosis, thus defining a potential use of curcumin to sensitize cancer cells via TRAILmediated immunotherapy. In another study, however, experimental dietary supplementation of curcumin was accompanied by decreases not only in the activation of apoptosis by cyclophosphamide, but also in that of JNK. These results thus demonstrate that curcumin can inhibit chemotherapy-induced apoptosis through inhibition of ROS generation and blockade of JNK function, suggesting a possible negative effect on breast cancer patients undergoing chemotherapy [131].

Curcumin

Studies of the methanolic extract of Alpinia oxyphylla have shown that this extract suppresses the promotion of skin tumors in mice and that it induces apoptosis in cultured human promyelocytic leukemia cells. In addition, two phenolic diarylheptanoids isolated from the active extract, yakuchinone A and yakuchinone B, were found to ameliorate tumor promotion as well as inhibit both TPA-induced epidermal ornithine decarboxylase (ODC) activity and ODC RNA expression. Moreover, yakuchinones A and B reduced production of the TNF-a in the TPAstimulated skin of mice. Furthermore, both compounds inhibited the TPA-induced expression of COX-2 at both transcriptional and

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translational levels. Doubtless, the anti-inflammatory properties attributed to these compounds are related to these inhibitory effects [132].

CH 3 O

Yakuchinone A

o CH3O

Yakuchinone B

[6]-Gingerol, which is the major pungent ingredient of the ginger rhizome (Zingiber ojjicinalis), has also been shown to exhibit strong antiinflammatory activity while [6]-paradol, a closely related compound from the same species, possesses chemopreventive potential. Both compounds showed similar effects when studied as potential inducers of apoptotic cell death [133]. H

OH

CH3O

CH3O

CH3

[6]-Paradol

Various phenolics have been found to induce apoptosis in human cancer cells, with some of them serving as potential anti-inflammatory

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agents as well. It is thought that the mechanism of action in both cases is related. Of the compounds studied, honokiol, from Magnolia officinalis, induced apoptotic cell death in different types of human cancer cells using a three-pronged attack: modulation of Bcl-XL and Bad proteins, induction of the release of mitochondrial cytochrome c, and activation of caspase-3 [134]. Another compound, humulone, isolated from hops extract (Humulus lupulus), induced apoptosis in the promyelocytic leukemia cell line HL-60 by means of a mechanism which implicated the compound's antioxidative activity [135].

OH

OH O Honokiol

O

Humulone

Flavonoids are natural products with a wide range of pharmacological effects, including anti-inflammatory and antioxidative properties [89]. Some of them, e.g. naringin, naringenin, quercetin, and myricetin, inhibit the enhanced expression of iNOS through down-regulation of N F - K B binding activity [89,136]. Because N F - K B is involved in both inflammatory diseases and apoptosis, the modulation of N F - K B activity may be a suitable target for altering the inflammatory process. The LOX inhibitors nordihydroguaiaretic acid (NDGA) and baicalein were found to induce apoptosis and inhibit proliferation of different breast cancer cells in vitro. In contrast, the LOX products 5-HETE and 12-HETE had mitogenic effects, stimulating the proliferation of the same cell lines. Blocking both 5-LOX and 12-LOX pathways led to several different effects, including apoptosis in breast cancer cells. This effect occurred through cytochrome c release and caspase-9 activation, as well poly-(ADP-ribose) polymerase (PARP) cleavage. Blocking these pathways also reduced the levels of the anti-apoptotic proteins Bcl-2 and Mcl-1 while increasing the levels of the pro-apoptotic protein Bax [137]. The 12-LOX inhibitors might be of interest for the treatment of

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Alzheimer's disease, first because of the chronic inflammation which occurs in this illness, and second because the neurodegeneration associated with Alzheimer's is related to the accumulation of amyloid p peptide. Thus, studies with the 12-LOX inhibitor baicalein found that this compound attenuated both neuronal apoptosis as well as the c-jun protein over-expression induced by amyloid P peptide (25-35) on rat cortical cells. In contrast, neither the broad spectrum LOX inhibitor NDGA nor the 5-LOX inhibitor caffeic acid exhibited cell protecting effects [138]. As for baicalein's mechanism of action, Chang et al. [139] demonstrated that the apoptotic effect of this compound on human Hep G2 cells was induced by mitochondrial dysfunction and Bcl-2 regulation. Quercetin and its 3-rhamnoglucoside rutin were studied as possible modifiers of tumor formation, but neither showed any effect in this respect [140]. In contrast, the aglycone quercetin was found to induce apoptosis in HeLa cells by means of a mechanism which reduces the level of expression of Hsp27 and Hsp72 [141]. Moreover, the same authors [142] demonstrated that quercetin can induce apoptosis and necrosis in vitro in the kidney cells of monkeys, although the percentage of affected cells was very low. On the other hand, quercetin, apigenin, myricetin, and kaempferol are all able to induce apoptosis in human leukemia cells. When administered separately, each of these compounds caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of PARP. They also induced loss of mitochondrial transmembrane potential, elevation of ROS production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of procaspase-9 processing [143]. The authors correlate the apoptogenic potency of these compounds with the presence of hydroxyls in ring B, along with the absence of a 3-hydroxyl in ring C. OH

H< OH

O Baicalein

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Tangeretin is a citrus flavone that inhibits the release of both ROS by human neutrophils and histamine by human basophils. In addition, it inhibits the proliferation of malignant tumor cells in vitro. Hirano et al. [144] demonstrated that the compound's anticancer activity is mediated in part through induction of apoptosis, but that this occurs without affecting the immune cells. OCH 3 H3C0.

Studies with tectorigenin and its 7-glucoside, tectoridin, both isolated from the Korean plant Belamcanda chinensis, long used as an antiinflammatory, found that these compounds suppressed PGE2 production by rat peritoneal macrophages that had been stimulated by either TPA or thapsigargin. This effect arose from the inhibition of COX-2 induction in the inflammatory cells; interestingly, the compounds do not directly affect the activity of either COX-1 or COX-2 [145]. Among other effects, tectorigenin has been shown to induce the transformation of human promyelocytic leukemia cells into granulocytes and monocytes/ macrophages, thus causing apoptotic changes of DNA in cells. Tectorigenin also inhibits autophosphorylation of epidermal growth factor (EGF) receptors by EGF itself, and decreases the expression of Bcl-2 protein [146]. The study of this compound with other related isoflavones demonstrated that the cytotoxic properties of tectorigenin depend on the isoflavone configuration and the presence of the 5hydroxyl group. Genistein, or 6-demethoxy tectorigenin, was shown to exert inhibitory effects on IL-3, IL-5, IL-6, and GM-CSF bioactivities, a fact which sheds light on its possible mechanism of action as an anti-inflammatory and immunosuppressive agent [147]. Moreover, genistein induces apoptosis in prostate cancer cells [148] and in non-small-cell lung cancer cell lines

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[149]. Studies of this compound demonstrated that genistein causes a typical DNA laddering, which is a hallmark of apoptosis. HO.

Finally, Kuntz et al. [150] have established that the capability of flavonoids for inhibiting growth and inducing apoptosis cannot be predicted on the basis of their chemical structures or substituents. A great number of pharmacological effects for licorice {Glycyrrhiza glabrd) have been documented, including its anti-inflammatory properties. Glycyrrhizic acid has been found to inhibit both LOX and COX; it also inhibits PKC and downregulates the EGF receptor. Although research on other phenolics has shed light on their various applications, it is the polyphenols that are thought to be mainly responsible for inducing apoptosis in cancer cells [151]. For instance, the major polyphenol isolated from green tea {Camellia sinensis), epigallocatechin gallate, has been found to exhibit multiple biological effects, including antioxidative, anti-inflammatory, and antiproliferative properties. The pharmacological effects of this compound are myriad and include reduction of the inhibitory effect of peroxinitrite on COX, attenuation of NO generation, blocking of iNOS expression and activity, inhibition of N F - K B binding activity [152], and inhibition of DNA synthesis [153]. This compound also protects against cancer by causing cell cycle arrest. Finally, it has been found to induce apoptosis in different cell lines by forming internucleosomal DNA fragments [154,155,156]. With respect to the activity of tannins, Momose et al. [157] have demonstrated that gallic acid exhibits higher activity than the tannins derived from it. Gallic acid was found to induce apoptosis in different cell lines; this induced cell death was mediated by ROS such as hydrogen peroxide and superoxide anion, as well as by Ca2+. However, the induction of apoptosis depends on the acid's distinctive structural features rather than on its antioxidative activity [158]. In fact, the

175

apoptotic effect of gallic acid seems to require the presence of Ca2+ since the depletion of this ion from the culture medium reduced the acid's apoptosis-inducing activity. In contrast, lack of Ca2+ did not affect the activity of either tannic or caffeic acid [157].

OH

Epigallocatechin gallate

Caffeic acid phenethyl ester, isolated from the propolis found in bee hives, has also been shown to induce apoptosis in different cell lines, probably by modulating the redox state of cells. This ester was studied under different experimental conditions and in the presence of various agents, including Bcl-2, which protects cells from oxidative stress. Indeed, this agent had a protective effect against the apoptosis induced by caffeic acid phenethyl ester [159,160]. OH

In a study on different anthraquinones from Rheum palmatum, Chen et al. [161] found that emodin, but not physion or chrysophanol, induced apoptosis in HL-60 cells though activation of the caspase 3 cascade. The

176 176

authors further demonstrated that this effect was independent of ROS production, and they hypothesized that the presence of a hydroxy substituent on C-6 was essential for the apoptotic activity of the compound. Terpenoids

A great number of triterpenes have been studied as anti-inflammatory agents, both in vivo and in vitro. The mechanism of action has been found to depend on the type of terpene as well as its structural group [162,163,164]. In Hata et a/.'s [165] study of the possible pro-apoptotic effect of a number of triterpenes on a melanoma cell line in mice, many of the compounds tested showed anti-inflammatory activity. These tests, together with similar studies on the apoptotic effects of the same compounds, indicate that the ability to induce apoptosis in proinflammatory cells is strongly correlated to anti-inflammatory activity. Of the 21 triterpenes assayed, betulinic acid and its methyl ester, along with lup-28-al-20(29)ene-2p-ol and lup-28-al-20(29)-en-3-one all inhibited cell proliferation by inducing apoptosis. The authors suggest that the carbonyl group at C-27 may be essential for the apoptotic effects of these compounds. While previous papers [163,164] had already described the relevance of this carbonyl group for the anti-inflammatory activity of triterpenes, more research on the apoptotic effects on proinflammatory cells is necessary to better understand these compounds.

Ri

Betulinic acid Betulinic acid methyl ester Lup-28-al-20(29)ene-2p-ol Lup-28-al-20(29)-en-3-one

a-H, P-OH a-H, P-OH a-H, p-OH =O

R2 COOH COOCH3 CHO CHO

177

Ginsenoside Rg3 from Panax ginseng not only induced classic apoptotic morphology, but it also interfered with the expression of Bcl-2, caspase-3, and apoptosis-related genes in human prostate carcinoma LNCaP cells. By the same token, ginsenoside Rg3 activated the expression of the cyclin-kinase inhibitors p21 and p27, arrested LNCaP cells at Gl phase, and subsequently inhibited cell growth through a caspase-3-mediated apoptosis mechanism [166]. Moreover, ginsenosides Rbl, Rb2, and Re were shown to be metabolized by intestinal bacteria to yield ginsenoside Ml, which in turn inhibited cell proliferation and induced cell death by regulating apoptosis-related proteins [167].

Ginsenoside Rg3

Parthenolide, isolated from Tanacetum parthenium and other species, is a sesquiterpene lactone widely investigated for its anti-inflammatory activity [168,169,170]. Recent in vitro studies have shown that this compound inhibits the N F - K B pathway. A study on the effect of parthenolide in endotoxic shock in rodents showed that treatment with this compound stopped nitrotyrosine formation, PARP synthetase expression, and apoptosis. It also reduced iNOS mRNA content in the tissues studied. All these effects are brought about by the compound's inhibition of N F - K B [171]. In addition, parthenolide mimicked the effects of IKBOC in that it inhibited both N F - K B DNA binding activity as well as Mn-SOD expression, while simultaneously increasing paclitaxel-induced apoptosis of breast cancer cells [90].

178 178

Triptolide from Tripterygium wilfordii was also shown to exhibit antitumoral and anti-inflammatory effects by inhibiting cell proliferation, inducing apoptosis, and inhibiting both N F - K B and AP-1 transeriptional activity. However, since this compound neither inhibited growth nor induced apoptosis in cells with mutant p53, it clearly requires a functional p53 to exhibit any proapoptotic, anti-inflammatory, or antitumoral effect. [172].

Parthenolide

Jolkinolide B from Euphorbia fischeriana was found to induce the apoptosis of human prostate cancer cells, but its exact mechanism has yet to be determined [173].

Jolkinolide B

Zerumbone, from Zingiber zerumbet, was shown to effectively suppress free radical generation, pro-inflammatory protein production, and cancer cell proliferation while additionally inducing the apoptosis of these same cells. An analysis of the relationship between the compound's structure and its activity indicated that the a,p-unsaturated carbonyl group in the sesquiterpene structure is a requisite for the compound's

179

effects since ot-humulene, an analogous compound without the carbonyl, is ineffective [174].

Zerumbone

a-Humulene

Other terpenoids with potential proapoptotic effects are farnesol and geranylgeraniol [175], y-tocotrienol and P-lonone [176], perillyl alcohol [177], limonene [178], and paclitaxel [179], all of which induce apoptotic cell death via a signaling pathway that is independent of G2/M arrest and microtubules [180]. Lectins

In general, lectins are divalent or multivalent carbohydrate-binding proteins with the ability to agglutinate cells. They have been used in biochemistry, immunology, and molecular physiology, but the interest in this phytochemical group has increased greatly since recent studies have established their utility as potential pharmacological agents. One of the potential uses of lectins is as a modifier of cell evolution. Thepen et al. [181], for example, have reported the resolution of cutaneous inflammation after local elimination of macrophages by an apoptotic mechanism. For this study, an immunotoxin composed of an antibody directed against the high-affinity IgG receptor CD64 and the lectin ricinA was synthesized. This immunotoxin was found to induce apoptosis in cultivated macrophages while leaving the low CD64- expressing macrophages unaffected. This activity was corroborated in vivo on transgenic mice expressing human CD64; the cutaneous inflammation was resolved in 24 h, and both the skin temperature and vasodilatation decreased. Apart from their postulated immunostimulatory properties, lectincontaining extracts from mistletoe (Viscum album) have also been found to induce apoptosis [182]. The activity of the extracts depends on the

180 180

manufacturing process, host tree, and time of harvest [183]. Although the lectin content of these extracts is strongly correlated with their apoptosis-inducing properties on cultured lymphocytes, its presence does not totally account for their biological activity; thus, other compounds are probably involved in the modulation of the mistletoe lectin activity [184,185]. In the case of the lectin isolated from Korean mistletoe (V. album coloratum), for example, the activity against tumor cells occurs by means of an apoptotic process that is mediated by Ca2+/Mg2+-dependent endonucleases [186]. Three additional lectins, obtained from Canavalia brasiliensis (ConBr), Dioclea violacea (DVioL), and Dioclea grandiflora (DGL), have all been found to stimulate T-cell activation and apoptosis in vivo, but they also produce important side effects, including inflammation associated with high endothelial venule necrosis [187]. Other natural products

Oh et al. [188] isolated the principle (3i?,6i?)-4-methyl-6-(lmethylethyl)-3-phenylmethylperhydro-l,4-oxazine-2,5-dione from the fruiting bodies of Isaria japonica to study its effects as an apoptosisinducing agent on human leukemia HL-60 cells.

\

^s

T

(3^,6«)-4-Methyl-6-(l-methylethyl)-3-phenylmethylperhydro-l,4-oxazine-2,5-dione

Inhibitors of apoptosis and their implications in inflammatory diseases Alkaloids

The alkaloid boldine has been found to decrease dopamine-induced cell death, including apoptosis, in PC 12 cells through a scavenging action on ROS and inhibition of both melanin formation and thiol oxidation [189].

181 181

CH3O

CH3O OH Boldine

Phenolics

As mentioned above, experimental dietary supplementation of curcumin was accompanied by a decrease in the activation of apoptosis by cyclophosphamide, as well as a decrease in JNK activation. These results demonstrate that curcumin can inhibit chemotherapy-induced apoptosis by inhibiting ROS generation and blocking JNK function, effects which would be counterproductive for breast cancer patients undergoing chemotherapy [131]. Agastinol and agastenol, two lignans from Agastache rugosa, have been found to inhibit etoposide-induced caspase-3 induction in U937 cells. Both compounds have a similar potency range and thus seem to comprise a new type of anti-apoptotic agent [190]. Other natural products

Cat's claw (Uncaria tomentosa and Uncaria guianensis), an herbal medicine from the Amazon, is widely used to treat inflammatory disorders. Both species of cat's claw provide effective antioxidant and anti-inflammatory activities, but Uncaria guianensis is the more potent of the two. Non-alkaloid fractions from both species have been found to decrease lipopolysaccharide (LPS)-induced TNF-a and nitrite production in RAW 264.7 cells, and oral pretreatment for 3 days with Uncaria tomentosa actually prevented TNFa mRNA expression and apoptosis. Interestingly, the pharmacological properties of these species do not seem to be dependent on the presence of oxindole or pentacyclic alkaloids [191].

182

OH

OH OCH 3

CHjO

OGH3

CH3O Agastinol

Agastenol

CONCLUSIONS The study of natural products as potential anti-inflammatory agents is an exciting topic for future research. Many known compounds have been described as anti-inflammatory agents because of their ability to affect the arachidonic acid metabolism and/or the induction of new proinfiammatory agents and proteins. However, a new approach to the investigation of natural products and their part in inflammations has arisen from the recent revisiting of the role of glucocorticoids and theophylline in inflammatory processes. Thus, while glucocorticoids have been found to promote safe clearance of apoptotic cells through phagocytes, theophylline has been shown to induce apoptosis in eosinophils and lymphocytes. Both drugs produce beneficial effects in allergic inflammation, especially in the treatment of asthma if used in combination with corticoids. Moreover the application of apoptotic agents could be highly effective in the resolution of skin diseases such as eczematous dermatitis. Recent research seems to single out phenolics as being those natural products with the most potential for use as anti-inflammatory and proapoptotic agents. Many of these compounds are proven antioxidants that have been found to inhibit the enzymes implicated in the inflammatory process as well. Some of the mechanisms in question may be related to their pro-apoptotic effects and as such may provide ways to potentiate the compound's actual pharmacological effects. Alkaloids and terpenoids may also be of interest, but the research on them to date has focused more on their use as anticancer agents.

183 183

ABBREVIATIONS = adenosine diphosphate = acquired immunodeficiency syndrome = apoptosis inducing factor AIF AP-1 = activator protein-1 Apaf-1 = apoptosis protease activating factor-1 Bcl-2 = B-cell leukemia oncogen-2 cAMP = cyclic adenosine monophosphate = cluster of differentiation CD CD64 = FcyRI (immunoglobulin G receptor I) CD95 = Fas c-FLIP = cellular FLICE-inhibitory protein c-fos = cellular family of genes c-jun = cellular family of genes c-myc = cellular family of genes COX, COX-1, COX-2 = cyclooxygenase, eyclooxygenase-1, -2 DNA = deoxyribonucleic acid = delayed-type hypersensitivity DTH = epidermal growth factor EGF FADD = Fas-associated death domain FasL = Fas ligand FcyRI (CD64) = immunoglobulin G receptor I FLICE = FADD-like interleukin-ip converting enzyme GM-CSF = granucolyte-macrophage colony stimulating factor 5-HETE(12-,15-) = 5-hydroxy-6,8,l 1,14-eicosatetraenoic acid (12-, 15-) = human immunodeficiency virus HIV Hsp, Hsp 27,Hsp 72 = heat shock protein = inhibitor of apoptosis protein LAP = interleukin-ip converting enzyme (caspase-1) ICE = ICE and Ced-3 homologue ICH = C-FLIP, FLAME, Casper I-FLICE = immunoglobulin G IgG IKB = family of inhibitory proteins of NF-KB = inhibitor of the NF-KB kinases, IKB kinase IKK IL,-lp, -2,-3,-5,-6,-8,-10 = interleukin, interleukin-2,-3,-5,-6,-8,-10 INF-y = interferon-y = c-jun NHrterminal kinase JNK = lipoxygenase, lipoxygenase-5, -12, -15 LOX, -5,-12,-15 = lipopolysaccharide LPS = leukotriene, leukotriene B 4 LT, LTB4 LX,LXA4 = lipoxin, lipoxin A4 MAPK = mitogen-activated protein kinase = nordihydroguaiaretic acid NADG NADH = nicotinamide adenine dinucleotide reduced

ADP

AIDS

184 NF-KB

NO NOS, iNOS NSAID ODC PARP PG, PGE2 PKC PLA2 PLC PMN mRNA ROS Smac/DIABLO SOD TGF, TGF-(3 TNF, TNFa TNFR1 TPA TRAIL UV VR,

:

nuclear factor-KB = nitric oxide ; nitric oxide synthase, inducible nitric oxide synthase = non-steroidal anti-inflammatory drug = ornithine decarboxylase = poly-(ADP-ribose) polymerase = prostaglandin, prostaglandin E2 = protein kinase C = phospholipase A2 = phospholipase C = polymorphonuclear = messenger ribonucleic acid = reactive oxygen species = second mitochondria-derived activator of caspases/direct IAP binding protein = superoxide dismutase = tumoral growth factor, tumor growth factor-(3 = tumor necrosis factor, tumor necrosis factor-a = TNF receptor-1 = 12-0-teradecanoylphorbol 13-acetate = TNF-related apoptosis-inducing ligand = ultraviolet light = vanilloid receptor type 1

Cell lines cited in text A431 B16 2F2 Caco-2 C3H 10T1/2 CH27 CREF DU145 GMK H460 HaCaT HeLa Hep3B HepG2 HL-20 HL-60 L5178Y LLC-PK1 LNCaP MCF-7 Molt4B

:

human epidermoid carcinoma cells mouse melanoma derived subclone cells : human colon cancer cells = mouse embryonic fibroblasts = human squamous lung cancer cells = non-tumorigenic rat embryo fibroblasts = human prostate carcinoma cells = monkey kidney cells = non-small lung cancer cells = human carcinoma keratinocytes = human negroid cervix epitheloid carcinoma cells = human hepatocytes = human hepatome cells = human Caucasian promyelocytic leukaemia cells = human promyelocytic leukaemia cells = mouse lymphoma cells = renal tubular cells = androgen-sensitive human prostate cancer cells = human breast adenocarcinoma cells = human lymphoid leukaemia cells :

185 NHEKs NSCLC NS-SV-AC PCa PC 12 PLA-801 PBMCs RAW 264.7 SK-N-SH THP-1

= normal human epidermal keratinocytes = human non-small cell lung cancer cells = normal human salivary gland cells = prostate adenocarcinoma cells = undifferentiated cells = cultured NSCLC cells = human peripheral blood mononuclear cells = mouse monocyte macrophages = human neuroblastoma cells = human monocytic cells

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pIC50(exp)

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33 Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 33 © 2006 2006 Elsevier B.V. All Allrights reserved. ©

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SYNTHETIC INVESTIGATIONS IN THE FIELD OF DRIMANE SESQUITERPENOIDS PAVEL F. VLAD Laboratory of Terpenoid Chemistry, Institute of Chemistry, the Academy of Sciences of Moldova, Academiei Str. 3, Chisinau, MD-2028, the Republic of Moldova ABSTRACT: In this article, the results of the author's investigations in the field of the synthesis of drimane sesquiterpenoids, a group of terpenic compounds which possess a wide variety of biological activities (antifeedant, antibacterial, antifungal, anticomplemental, cytotoxic, antiallergic, piscicidal, molluscicidal, plant growth regulatory, insecticidal and others) have been reviewed. The original structural- and stereoselective methods for obtaining natural compounds in the optically-active form, and also of important intermediates on the pathway to them, including alcohols, poliols, ketones, lactones and acids, have been elaborated. Most of the compounds obtained are polyfunctional. Much attention has been focussed on the synthesis of drimenol, a drimanic sesquiterpene key compound, which is widely used as a starting substance in syntheses of many biologically active drimanes. Much effort has been devoted to elaborate the syntheses of drim-8-en-7-one and drim-5,8-diene-7-one, compounds possessing a great synthetic potential. Indeed, using those above a large number of natural compounds, such as isodrimenin, 7-oxoisodrimenin, 7-oxo-5,6dehydroisodrimenin, /raras-tetrahydroactinidiolide and intermediates for the syntheses of polygodial and warburganal, compounds with various and high biological activities, were prepared.

INTRODUCTION Drimanes are sesquiterpenes with a carbon skeleton of drimane whose structure and stereochemistry are depicted in formula (1) [1]. The name "drimane" comes from the name of a South American tree Drimys winteri Forst., from the bark of which the alcohol (-)-drimenol (2), the first representative of this group of sesquiterpenoids, has been isolated [2]. The chemistry of drimanes had been particularly intensively developed within the last 15-20 years, when many members of this group of terpenoids were isolated and investigated. It was shown that they are sufficiently widespread in the nature. Drimanes have been isolated from higher plants, fungi, microorganisms, and marine organisms [3-4]. Drimanes have been of interest first of all owing to their high and various biological activities, considered in detail in excellent reviews [3-4]. Among them are

394

substances with diverse activities: antifeedant, antibacterial, antiviral, antifungal, cytotoxic, phytotoxic, anticomplemental, plant growth regulatory, piscicidal, molluscicidal, and others [3,4]. The practical value of many drimanes and their often small content in natural sources stimulated their synthetic preparation [5, 6]. Both total and partial syntheses of drimanes have been described in literature. The former have two drawbacks: they are, as a rule, multistep procedures and consequently the yields of final products are low. In addition, these compounds are obtained in racemic form [6]. As it is known, usually only one of the enantiomers of such compounds possesses biological activity and hence the yield of the desired stereoisomer a priori can not exceed 50%, but actually it is lower. Moreover, in most cases the racemates resolution is not a simple, empiric process. The latter approach to drimane syntheses is based on their structural relationships with higher terpenoids: di-, sesterand triterpenoids. With their cleavage, drimanic compounds can be obtained. Such partial syntheses of drimanes are attractive since the target compounds are obtained in natural, optically-active form and besides most of these syntheses involve a few steps [6, 7].

OH

(5)

(6)

(7)

Most of the known partial syntheses of drimanes were carried out starting with bi- and tricyclic diterpenoids. However, bicyclic labdane diterpenoids mostly are structurally similar to drimanes. Their only

395

difference from drimanes is the presence of an extra isoprene unit in their side chain. Elimination of that unit yields the desired drimanes. Labdane diterpenoids have one more important advantage since most of them, for example, sclareol (3), manool (4), larixol (5), labdanolic acid (6), the mixture of AX3-cis- and trans-neoabienols (7) and others, are easily available and can be obtained in large amounts [8]. In this review, the results of our investigations in the field of the synthesis of a number of optically-active, drimanic compounds from norlabdanic derivatives, the cleavage products of many available labdanoids, and also from a mixture of neoabienols (7) are summarized. The respective literature data are also discussed in the present article. A number of the prepared drimanes are naturally occurring biologically active substances. Others have been used as intermediates in the syntheses of important natural bioactive polyfunctional drimane sesquiterpenoids. Below the syntheses of the representatives of the drimane (1) group are described. 1 Synthesis and Use of (-)-Drimenol (2) Besides the Drimys winteri, drimenol (2) was also found in many other natural sources [3,4]. For example, it has been isolated from some species of the plants of the Warburgia [9] and Porella [10] families, from Polygonum hydropiper [11], some liverworts [12], Ferula ceratophylla [13] and fungi [14]. According to [3], drimenol (2) has the plant growth regulatory activity comparable with that of heteroauxin (indole-3-acetic acid). However, more important is the fact that drimenol (2) has been used as a starting compound for the synthesis of a series of natural biologically active drimanes and nordrimanes. So, two syntheses of the drimane dialdehyde polygodial (8) were carried out starting with the drimenol (2) (Scheme 1). In one of them [15], drimenol (2) was oxidised into drimenal (9), which then transformed into acetal (10). The latter was oxidised into aldehyde (11) with selenium reagents. Treatment of aldehyde (11) with ptoluenesulphonic acid gave the target polygodial (8) (a 30% overall yield), isolated earlier from some plants and marine organisms [3, 4]. In the second synthesis [16], drimenol (2) was acetylated and its acetate (12) was oxidised with selenium reagents into a mixture of

396

CHO (2)

CHO

a) PCC, CH2C12, 75.4%; b) CH(CH 2 OH) 2 , H 1 , 88.5%; c) SeO2 (cat), (p-MeOC 6 H 4 ) 2 SeO, 45%; d) TsOH, acetone, 100%; e) Ac 2 O, Py, 98%; f) K 2 CO 3 , MeOH, 100%; g) (COC1)2, DMSO, 98%.

Scheme 1 hydroxy acetates (13) and (14). Saponification of the latter gave diol (15), which was oxidised by the Swern reagent into polygodial (8) in a high yield. The particular interest of researchers to polygodial can be explained by its various biological activities: antifeedant [17, 18], antibacterial [10, 19], cytotoxic [10, 20], allergenic [10], piscicidal, molluscicidal, anticomplemental, and plant growth regulatory [3, 4]. Polygodial is a powerful analgetic agent (15 times more powerful than aspirin) [10,21]. Some substances (anetol, saphrol, methylevgenol) have the synergistic effect on polygodial (8), intensifying its antifungal activity 32-128 times [22]. It is necessary to note that polygodial has bitter taste [17]. Drimenol (2) has also been used as a starting compound for the synthesis of another drimanic dialdehyde, warburganal (16), which is

397

similar to polygodial (8) in the variety and level of biological activities [23] (Scheme 2). _OAc

CHO

(17)

(16)

a) SeO 2 , dioxane, A, 45%; b) KOH, MeOH, 100%; c) DMSO, (CF 3 CO) 2 O, 64%.

Scheme 2 Drimenyl acetate (12) was oxidised with selenium dioxide into the acetoxy diol (13). This compound was saponified into triol (17) which on oxidation with DMSO and (CF3CO)2O gave the final product (16). Afterwards, the yield at the step of the triol (17) oxidation was improved using the Swern reagent [24]. Warburganal possesses antifungal, antibacterial, antimicrobial, cytotoxic, antiallergic, antifeedant, insecticidal, plant growth regulatory, helicoccidic activities, and a bitter taste [3, 4]. Scheme 3 shows the synthesis of (-)-cinnamodial (ugandensidial) (18), the warburganal-related dialdehyde, from the drimenol (2) [25]. Cinnamodial also has a bitter taste [3], and possesses antimicrobial, antifeedant, piscicidal and antihelmintic activities [3, 4]. Cinnamodial (18) has been simultaneously isolated from Cinnamosma fragrans [26] and from Warburgia ugandensis [27], and then from other plants [4]. Cinnamodial (18) was obtained from drimenol (2) in 12 steps in an overall 10% yield. The diol (22) was synthesised from drimenol (2) according to the method [28], and further synthesis was continued as described in [25] (Scheme 3). Drimenol (2) has also been used as a starting material for the preparation of uvidin C (26), a metabolite of fungus Lactarius uvidus Fries [29]. The eight-step synthesis was carried out according to Scheme 3 and includes oxidation of the diol (22) with m-chloroperbenzoic acid. The overall yield of (26) was 20% [28].

398 OH

OH

'lOH (2)

\z

(19)R=O (20) R=NNHTs

OH

CHO

OH (26)

OAc (18)

a)Ac2O/Py;OsO4,NMO;NBS,CH2Cl2;KOH,MeOH; b) TsNHNH2, BF3 Et2O, QH^; c)BuLi,THF; Py; PCC; D1BAL, THF; e) Ai^O, Py, DMAP; f) SeO2 (cat), (p-MeOCyi^SeO, dioxane; g) K2CO3, MeOH; h) (COCl^, DMSO; i) m-CPBA, CH2C12.

Scheme 3 Swedish chemists [30], starting with drimenol (2), accomplished the synthesis of drim-8-en-7-one (27), thus confirming its structure and determining its stereochemistry (Scheme 4). Drimenyl acetate (12) was epoxidised with m-CPBA and the product was saponified into (28). Its oxidation with the Brown reagent, the base isomerisation of the resulting epoxy aldehyde, and the sodium borohydride reduction of the isomerisation product afforded the diol (29). Its monoacetate (30) was oxidised with the Jones reagent into the keto acetate (31) whose hydrogenolysis gave the target drim-8-en-7-one (27). It should be mentioned that this synthesis has no preparative importance since it involves many steps and at some stages (for example "c") mixtures of products were formed. Furthermore, some intermediates have not been characterised.

399 ,OH

(12) OH (28)

I

(29)R=H (30) R=Ac

,OAc

(31) (27) a) m-CPBA, CHjCl2; KOH, MeOH; b) CrO3, EfcO; KOH, MeOH; NaBH,, MeOH; c) Ac2O, Py; d) Jones reagent; e) Zn, AcOH.

Scheme 4

CHO

CHO

'V,r

OH

(1) 8aH

CHO

OH

(1) 8PH

(38)

Drimenol (2) has also been used in the syntheses of a series of natural nordrimanic compounds: the fragrant hydroxy ketone (32) [31] isolated from tobacco [32]; isonordrimenone (33) isolated also from tobacco [33];

400

and its unnatural isomer (34) [34], polygonal (35), isopolygonal (36) and polygonone (37) [35] isolated from Polygonum hydropiper L [21, 37]. Finally, drimenol (2) was transformed into the saturated 8aH- and 8(3Hdrimanes (1) found in benzenes [37]. From the data mentioned above it follows that drimenol (2) is a valuable synthon for the synthesis of drimanic compounds, including bioactive ones. This fact stimulated the elaboration of the methods for its preparation from available natural compounds, first of all, from labdanoids. One of the first syntheses of natural drimenol (2) was accomplished by Wenkert and Strike [38] from dehydroabietic and podocarpic derivatives of the type (38), obtained from resin acids. Because of its complexity due to a multistep path (ca 25 steps) and also of no preparative importance, these syntheses are not discussed here. Pelletier et al. [39] carried out the drimenol (2) synthesis from the ambreinolide (39), the product obtained from a series of available labdanoids [7, 8] (Scheme 5). Dehydrogenation of the ambreinolide (39) OR HOH

I

(41)R=H

I „ (42)R=Ac

OH

(2)

(43)

a) DDQ, dioxane, A; b) O,, CH2C17, -70°C; Red-Al, C 6 H 6 ; c) Ac 2 O, Py; d) POC13, Py; KOH, MeOH; e) BF 3 Et 2 0, CH2C12.

Scheme 5 with DDQ led to A -dehydroambreinolide (40), whose ozonolysis followed by reduction of the resulting products with Red-Al gave (+)drimane-8a,l 1-diol (41). Its monoacetate (42) was dehydrated with POCI3 into a mixture of unsaturated acetates followed by their saponification to a lz 112

401

mixture of alcohols (2) and (43) separated by column chromatography on silica gel. The total yield of the drimenol (2) in this seven-step synthesis was ca 13%. It is necessary to mention that authors [40] showed that the albicanol (43) isomerises into the drimenol (2) in high yields (93%) on treatment with boron trifluoride etherate.

CO 2 Me

CO,Me

(2)

a) SOC12, Py; b) Na 2 S 2 O 4 , PTC; O3,CH2C12, MeOH; H 2 O 2 , NaOH; c) NaBH,, MeOH; d) (CH 3 ) 2 C(NH 2 )CH 2 OH, H 3 BO 3 ; e) PhSeOH, H 2 O 2 ; f) O 3 , CH2C12, MeOH; CrO 3 , H 2 SO 4 ; g) NaBH,, MeOH; CH 2 N 2 ; h) HMPA, A; i) LiAlH 4 , THF.

Scheme 6 The drimenol (2) was also synthesised from the available labdanoid gispanolone (44) [41] (Scheme 6). The latter was dehydrated into the unsaturated ketone (45), the conjugated double bond of which was reduced, and the resulting product was ozonolysed, giving the keto acid

402

(46). Its reduction with NaBELt led to a mixture of epimeric hydroxy acids (47), which on interaction with 2-amino-2-methylpropanol-l afforded a mixture of 4,5-dihydrooxazols (48). This mixture was dehydrogenated with phenylseleninic acid into a mixture of compounds (49), which was converted into the keto acid (50) by subsequent ozonisation and the Jones oxidation. Reduction of this keto acid with NaBH4 followed by methylation with CH2N2 afforded the hydroxy ester (51). On its heating with HMPA, the drimic acid ester (52) has been obtained. Its reduction with LiAltLt led to the drimenol (2). This synthesis of the drimenol (2) includes 11 steps, the overall yield being 8%. A shorter synthesis of the drimenyl acetate (12) was elaborated by Barrero et al. [42] from the sclareol (3) (Scheme 7). Oxidative cleavage of

(3)

(12)

(42) a) OsO 4 , NaIO 4 , 73%; b) tBuMezSiCl, NaH, 99%; c) 0,, CH2C12, MeOH, -78°C; NaBH 4 , MeOH, 95%; d) SnCL,, CH 2 C1 2 ,25%.

Scheme 7 the sclareol (3) side chain by osmic acid and sodium periodate led to the acetoxy aldehyde (53), whose enol silylation gave a mixture of the acetoxy silyl esters (54). On its ozonolysis, followed by reduction of the ozonolysis products with NaBH4, the 11-monoacetate of drimanediol (42) has been formed. On its interaction with SnCU, the drimenyl acetate (12) was obtained in low yield. The total yield of the desired product in this four-step synthesis was ca 17%. The final stage of this synthesis turned out to be less efficient. Synthesis of the drimenol (2) from the readily available larixol (5) has been recently described in [43] (Scheme 8). The exocyclic double bond of

403

larixol has been selectively epoxidised with oxone. The obtained epoxy diol (55) was then reduced into the triol (56), whose C-6 hydroxy group OH

(59)

OH

(60)

(2)

a) oxone. CH2C12, acetone, H 2 O, NaHCO 3 . 18-crown-6, 81%; b) LiAlH 4 , THF, 94%; c) Ac 2 O, Py, 99%; OsO 4 , NaIO 4 , THF, H 2 O, 84%; d) tBuMe 2 SiCl, NaH, THF, 96%; e) O 3 , CH2C12, MeOH, -78°C, IVfeS, 78%; f) collidine, 200°C, 77%; g) NaBH,, EtOH, 0°C, 89%.

Scheme 8 was selectively acetylated and the resulting compound was cleaved with OsO4-NaIO4 into the diacetoxy aldehyde (57). This compound was converted into the mixture of the silyl enol esters (58), ozonolysis of which led to the diacetoxy aldehyde (59). On subsequent heating of compound (59) eliminated the acetic acid, affording the diene aldehyde (60). Reduction of (60) with NaBELt gave only drimenol (2) as a result of selective 1,4-addition. Selectivity of this method and sufficiently high yields of products at all stages of this synthesis are its main advantages. The overall yield of this eight-step synthesis was 32.5%.

404

We accomplished the drimenol (2) synthesis from the sclareol (3) [44] (Scheme 9). This was the first synthesis of a drimanic sesquiterpenoid

(64) A' (65) A8

(67) A s

8 14

(66)A < > (68)A8(12) a) KMnO 4 , AcOH, H 2 O; KOH, MEOH, A; H 2 SO 4 ; b) CH 3 Li, Et 2 O; c) H 2 O 2 , BF 3 Et 2 O; d) KOH, EtOH, A.

Scheme 9 from a labdanic diterpenoid. This synthesis, which correlated these two groups of terpenic compounds, confirmed the stereochemistry of the drimenol (2). The sclareol (3) was oxidised according to the method [45], and the obtained hydroxy acid (61) was introduced in reaction with methyl, lithium. The reaction product was a mixture of the ditertial diol (62) (18% yield) and of the hydroxy ketone (63) (80%). The latter on interaction with a mixture of concentrated (93.6%) hydrogen peroxide and boron trifluoride etherate afforded a mixture of the unsaturated ketones (64)-(66) and of the isomeric acetoxy drimenes (12), (67) and (68). All these substances were separated and isolated in individual form by column chromatography with SiC^'AgNOs and characterised. On saponification of the acetate (12), drimenol (2) was obtained. Later on in [46] it was established that, instead of the unstable, easily lactonised hydroxy acid (61), in the reaction with methyl lithium it is possible to use the commercially available norambreinolide (69), a common cleavage product of many labdane diterpenoids [7, 8]. In this case the yield of the hydroxy ketone (63) is a little lower (65%), but it is more convenient to carry out

405

this reaction. The total yield of the drimenol (2) in this synthesis is small (6.5%), therefore this method is of no preparative interest. It is necessary to note that later on we elaborated an alternative, shorter route for preparation of the hydroxy ketone (63) from the sclareol (3) [47] (Scheme 10). On the ozonolysis of the sclareol (3) and subsequent treatment of the ozonolysis products with ammonium chloride, the dimeric product (70) is formed [48]. Its further ozonolysis gave the (3-diketone (71).

~CH,

(3)

OAc

'OH

(72)

a) O 3 , MeOH, 5-10°C; NH4C1, 80%; b) O 3 , hexane, -65...-70°C; H 2 O, A, 100%; c) NaOH, EtOH, A; d) H 2 O 2 , CH2CI2, (CF 3 CO) 2 O/NaHCO 3 (l:l), 100%; e) H 2 SO 4 , EtOH, r.t, 56%; f) KOH, MeOH, 98%; g) 1 mmole FSO3H, C 3 H 7 NO 2 , -8O...-85°C, 1 h, 7 1 % ; h) 10 mmoleFSO3H, -8O...-85°C, 5 min, 77%.

Scheme 10 Alkaline cleavage of the latter produced a mixture (1:1) of the hydroxy acid (61) and the hydroxy ketone (63). As a result, this three-step synthesis led to the hydroxy ketone (63) in an overall 37% yield. Furthermore, the hydroxy acid (61) can be converted into (63), as is indicated in Scheme 9.

406

Recently, we have elaborated an alternative method of the drimenol (2) synthesis from the hydroxy ketone (63) of preparative value [49]. In the article [46] it was shown that on oxidation of the hydroxy ketone (63) with trifluoroperacetic acid under certain conditions, the 11-monoacetate of drimane-8a,ll-diol (42) is obtained in the quantitative yield. On treatment of the compound (42) at room temperature with 30% solution of concentrated sulphuric acid in ethanol by using 10 ml of this solution per 1 g of the compound (42), the crystalline drimenol (2) was obtained in 56% yield, which could be purified by recrystallisation from n-hexane [49] (Scheme 10). In such a way, at stage e) selective dehydration and transesterification of the hydroxy acetate (42) took place. Finally, we elaborated a highly efficient, structure- and chemoselective, stereospecific one-step method for the synthesis of the racemic drimenol (2) and hydroxy acetate (42) by the superacidic, low temperature cyclisation of E,E-farnesol (72) and its acetate (73), respectively [50] (Scheme 10). 2 Synthesis and Use of Drimane-8a,ll-diol and Its 11-Monoacetate Drimane-8a,ll-diol (41) and its 11-monoacetate (42) are suitable starting compounds for the synthesis of a series of drimanes and not only of them. Only the diol (41) was found in natural sources and was isolated from tobacco [51] and from a special gland of African elephant [52]. Data about the synthesis of these compounds from the ambreinolide [39] and of the hydroxy acetate (42) from the sclareol (3) have been already reported [42]. Barrero et al. [42] showed also that if the reduction of the ozonolysis product of the mixture of esters (54) is done with L1AIH4 instead of NaBH4, the diol (41) is obtained in a 95% yield (Scheme 11). Ohloff and Giersch [53] accomplished the synthesis of the drimanediol (41) from the norambreinolide (69). The latter was reduced into the semiacetal (74), whose acetate (75) on pyrolysis gave the dihydrofuran (76). Its ozonolysis and subsequent reduction of the ozonolysis products with NaBH4 afforded the diol (41). Unfortunately, the yields of the products in [53] are not given. As it was described above, the hydroxy acetate (42) was transformed into the drimenol (2) [39]. It should be mentioned that the reverse conversion of the drimenol (2) into the diol (41) [54] also takes place. For this purpose, the drimenyl acetate (12) was

407

epoxidised into the epoxy acetate (77) which was further reduced to the diol (41) (Scheme 11).

(54)

a) O 3 , MeOH, CH2C12, -78°C; LiAlH,, THF, 95%; b) iBujAlH, toluene; c) Ac 2 O, Py; d) 350°C; e) O,, EtOH; NaBH 4 ; f) m-CPBA, CH2C12; g) LiAlH,, THF.

Scheme 11 Earlier it was indicated (Schemes 5, 7, 10) that the hydroxy acetate (42) was transformed into the drimenol (2) [49], its acetate (12) [42] or a mixture of the drimenol (2) and the albicanol (43) [39]. Authors [55] accomplished the targeted synthesis of the albicanol (43) and its acetate (68) starting with the hydroxy acetate (42) (Scheme 12). The hydroxy acetate (42) was dehydrated with thionyl chloride into the mixture of acetates (12), (67) and (68), which was subjected to oxidation with mCPBA. On that treatment the acetates (12) and (67) are selectively oxidised, but the acetate (68) remained unreacted. The latter was than isolated by chromatography and saponified into the albicanol (43). The overall yield of the albicanyl acetate (68) and the albicanol (43) was 61% (at the saponification step the yield of (43) was quantitative). Barrero et al. [42] synthesised a mixture (1:1) of the albicanyl acetate (68) and its isomer (67) from the hydroxy acetate (42) by its successive acetylation into the diacetate (79) and pyrolysis of the latter (Scheme 12). It should be mentioned that the albicanyl acetate (68) is a biologically active substance, being a powerful antifeedant for fish [56]. The albicanol (43) itself is inactive, but it is easily acetylated with acetic anhydride in

408 ,OAc

OAc

,-OAc

(67)+(68)

(41)

'"OH

a) SOC12, DMAP, Py; b) m-CPBA, CH2C12, NaHCO 3 ; c) KOH, MeOH; d) Ac 2 O, Et 3 N, DMAP, THF, A, 92%; e) collidme, A,; f) TsCl, DMAP. Py; g) Nal, Zn, (CH 2 OMe) 2 , A, 59%.

Scheme 12 pyridine to give the active compound (68) [57]. The albicanol (43) was isolated from liverworts [12, 58], where it was found for the first time, and also from marine molluscs [56], where it is present together with its acetate (68), the predominant metabolite. It is necessary also to note that the diol (41) has been used as a starting compound for the synthesis of drim-9(ll)-en-8a-ol (80), one of the two C-8 epimeric metabolites of the fungus Aspergillus oryzae [59], used in Japan in baking and in manufacturing of some beverages (sake and others). On tosylation, the diol (41) afforded the monotosylate (81) which was transformed to the unsaturated alcohol (80) on treatment with Nal and Zn [60] (Scheme 12).

409

We succeeded in the elaboration of several syntheses of the drimanediol (41). One of them [49] was already discussed above. On peracidic oxidation, the hydroxy ketone (63) led to 11-monoacetate of drimane8a,ll-diol (42), which on alkaline saponification gave the diol (41) in almost quantitative yields (Scheme 10). In two syntheses of the diol (41), the sclareol (3) has been used as a starting compound. The sclareol oxidation product, the sclareol oxide (82) [61], on bromination in methanol afforded the dibromomethoxy derivative (83) which on interaction with potassium hydroxide eliminated hydrogen bromide, giving the unsaturated oxide (84). Its successive ozonolysis and reduction of the ozonolysis products with LiAlELi led to drimane-8a,lldiol (41). The overall yield of the diol (41) from the sclareol (3) was ca 32% [62] (Scheme 13).

(83)

(84)

a) KMnO 4 , acetone, 80%; b) 2 mol Br2, MeOH, 76%; c) KOH, toluene, PEG-600, A, 76%; d) O 3 , CH2C12> 65...-70°C; LiAlH,, EtjO, 70%.

Scheme 13 In the other synthesis of the diol (41) from the sclareol (3) [63], the latter was ozonised according to [64]. Under these conditions, a mixture of C-12 epimeric bisnorlabdanic oxidoketones (85) has been formed in a high yield. Its oxidation with monoperphtalic acid afforded a mixture of acetoxy oxides (75), from which acetic acid has been eliminated to give the unsaturated oxide (76). The latter was ozonised and the ozonolysis products were reduced with NaBtLj into the diol (41). This four-step synthesis led to the diol (41) in an overall 44% yield (Scheme 14).

410

However, the preferable method for the diol (41) synthesis is its preparation from a mixture of the isomeric neoabienols (7) [65], which has been isolated from the highly boiling fraction on distillation of oleoresin of silver-firs, for example, Siberian silver-fir [66]. On heating of the oleoresin, the cis-abienol (86), presented in it, suffers the sigmatropic shift giving a mixture of stereoisomeric neoabienols (7) [67]. On the exhaustive ozonisation of this mixture of neoabienols in methanol, followed by reduction of the resulting peroxidic products with NaBH4, drimane-8ot,l 1-diol (41) was obtained in a high yield (74%) (Scheme 14). H_

V-n

0Ac

(3)

"'OH

a) Oj, NaIO4, MeOH, H2O, -78°C, 91%; b) MPPA, Et2O, 90%; c) DMSO, NaHCO3, 150°C, 80%; d) O3, MeOH, -70°C; NaBH4 to a mixture of C-8 epimeric 14,15-bisnorlabdan-8-ol-13-ones (93) and (94). The latter mixture was subjected to Norrish II photolytic cleavage to afford compounds (80) and (89) (Scheme 15). On the basis of polarimetric data, authors [68] concluded that the alcohols, isolated from the fungi, belong to the series of ent-drimanes. The results of the work [60] (Scheme 12) are also in conformity with this conclusion. OH

(87)R,=OH,R2=CH3 (88)R,=CH3,R2=OH

(3) R,=CH3, R2=OH (92) R!=OH, R2=CH3

(90) 8R (91) 8S

"'OH i_H (93) RrCH 3 , R2=OH (94)R!=OH,R2=CH3

(80)

a) rn-CPBA, CH2C12, NaHCO.,, 78%; b) LiAlH,, THF, 75%; c) KMnO4, MgSO4, acetone, 100%; d) hv, petroleum ether, Ar.

Scheme 15 Afterwards, G. Dominguez et al. [69] accomplished the synthesis of alcohols (80) and (89) from the methyl ether of the natural quinone royleanone (95) (Scheme 16). They investigated three different approaches for compounds (80) and (89) preparation from the quinone (95). It was found that the following ones were optimal. The quinone (95) was ozonised, and the ozonolysis products were oxidised with periodic

412 OH

OMe

(80) + (89)

(101)

(102)

acid and methylated. The obtained keto diesther (96) was reduced to a mixture of C-ll epimeric triols (97), which were transformed to acetonides (98). The free hydroxylic group of (98) was reduced via the sulphoester, and the mixture of compounds (99) was obtained. Hydrolysis of the acetonide group of (99) gave the diols mixture (100), whose glycolic group was cleaved by oxidation and the resulting product was then reduced to the alcohol (101). The latter was transformed to the selenophtalimide (102) which on oxidation with H2O2 afforded the corresponding selenoxide spontaneously rearranged to give a mixture of the alcohols (80) and (89). a) O3, CH2C12, -80°C; H5IOfo EtOH, H2O; CH2N2; b) LiAlH,, THF; c) acetone, CuSO4; d) PySO3, THF; LiAlH,; e) AcOH, H2O; f) Pb(OAc)4. O f t , MeOH; LiAlH,, Et2O; g) N-(phenylseleno) phtalimide; h) H2O2, CH2C12.

Scheme 16 Although manool (4) and royleanone (95) belong to the same stereochemical series, the results of polarimetric measuring, carried out in [69], turned out to be opposite to what Brazilian chemists concluded [68]. Authors [69] drew the conclusion that the natural drimenols, isolated by authors [59], belong to the normal drimane row.

413

To clarify this confusing situation, we [70] carried out the synthesis of the alcohol (80) from sclareol (3) which had been earlier used by authors [68] as one of the intermediates in their synthesis of the same substance (80) (Scheme 17). Taking into consideration that the hydroxy ketone (90) is unstable and is easily transformed to sclareoloxide (82) [8], sclareol (3) was first acetylated according to the method [71] to the diacetate (103), which was cleaved to the acetoxy ketone (104) by ozonolysis. Norrish II photolytic cleavage of this ketone yielded the acetate (105). Under these conditions a part of the starting material remained unreacted. The acetate (105) is an extremely unstable substance. As a result, it decomposes on chromatographic purification to give the hydrocarbon (106). Apparently, this was the reason why authors [72] obtained only the hydrocarbon (106) on photolysis of the acetoxy ketone (104).

(3)

(105)R=Ac (106) (80) R=H a) AcCl, DMA, 93%; b) O3, MeOH, CH2C12> -60T; Cu(OAc)2H2O, MeOH, toluene, 70°C, 52%; c) hv, hexane.99%; d) SiO2, r.t, 100%; e) KOH,EtOH,A.

Scheme 17 Taking into account this fact we saponified the crude photolysis product and obtained the alcohol (80) which is more stable and can be purified by chromatography. Positive optical rotation of the alcohol (80) obtained by us demonstrated that metabolites of Aspergillus oryzae [59] belong to entdrimane series, and their absolute configurations should be depicted by formulas (87) and (88). The overall yield of the alcohol (80) synthesised from sclareol (3) in four steps was 45% (taken into consideration the unreacted acetoxy ketone (102) at the photolysis stage).

414

4 Synthesis of Drim-8-en-7-one and Drim-5,8-dien-7-one To date, drim-8-en-7-one (27) has been found only in one natural source, tobacco [30]. Authors [30] accomplished also its first multistep and

•(27)

-co2

(110) (HI) (112) a) H2SO4, MeOH, A, 100%; b) K2Cr2O7, AcOH, A, 41.5%; o) KOH, EtOH, A; HC1, 98%; d) SeO2, dioxane, A, 93%; e) NaOH, EtOH; HC1, 100%; f) 200°C, 80%.

Scheme 18 low-efficient synthesis from drimenol (2) (Scheme 4). Another known synthesis of the racemic ketone (27) [73] also involves many steps and is not considered here. Drim-8-en-7-one (27) has been found among the neutral products of the sclareol (3) oxidation with chromic mixture [74]. We have found that this substance possesses a strong ambergris odour, and may be of interest for perfumery [75] and tobacco industry [76]. The discovery of these important properties stimulated the search for a new, shorter, simpler and more efficient synthesis of the ketone (27) from available starting materials. We could accomplish such a synthesis from commercially available norambreinolide (69) [77]. The latter was converted according to the known procedure [78] to a mixture of esters (107) whose oxidation with K2Cr2O7 in acetic acid led to the keto ester (108). On its saponification, the corresponding keto acid (109) was obtained, which spontaneously decarboxylated to give the drimenone (27) in an overall 40.5% yield. A disadvantage of this method is the low yield of the keto ester (108). All attempts to improve it by variation of chemical oxidants and reaction conditions were unsuccessful [77]. We could obtain

415

an acceptable yield (65%) of the keto ester (108) only on electrooxidation of the mixture of esters (107) [79] (Scheme 18). This method allowed the simplicity of drimenone (27) isolation; the electrooxidation product was saponified and separated into the acidic and neutral parts. The latter consisted of the drimenone (27) which was purified by crystallisation. Thus, this ketone became a relatively available compound. This fact and also the specific chemical structure of drim-8-en-7-one make it attractive as a starting compound for the syntheses of the natural polyfunctional drimanic sesquiterpenoid, including the biologically active ones. Indeed, its functional groups, double bond at C-8-C-9 and the carbonyl group at C7, activate the carbon atoms C-6, C-ll and C-12, providing a certain synthetic niche for the ketone (27). Below are the data concerning the syntheses of poly functional drimanes from drim-8-en-7-one (27). The keto ester (108) is easily dehydrogenated with SeCh to the dienone ester (110). On its saponification, the corresponding acid (111) is obtained. Unlike the keto acid (109), the keto acid (111) is sufficiently stable and decomposes only on heating to give drim-5,8-dien-7-one (112) [79] (Scheme 18). Like the drim-8-en-7-one (27), the ketone (112) is also of interest as a starting compound in the syntheses of polyfunctional drimanes. 4.1 Synthesis of 1 l-Acetoxydrim-8-en-7-one from Drim-8-en-7-one

ll-Acetoxydrim-8-en-7-one (31) is of interest as an intermediate in the syntheses of biologically active drimanes, particularly, of warburganal (16). For the first time, compound (31) was obtained by authors [30] as an intermediate in the synthesis of drim-8-en-7-one (27) from drimenol (2) (Scheme 4). Later on this substance was prepared by Chilean chemists [80] (Scheme 19). On the interaction of drimenyl acetate (12) with selenium oxidants a mixture of compounds (13), (14) and (113) was obtained, in which the latter predominates (65% yield). Oxidation of the hydroxy acetate (113) with pyridinium dichromate and tert-butyl hydroperoxide led also to a mixture of three compounds (114), (115) and (31). The yield of the keto acetate (31) reached only 20% at this stage, and the overall yield of (31) from (12) was 13%. Barrero et al. [81] carried out a more efficient synthesis of the keto acetate (31) from the unsaturated acetate (67), which they obtained earlier

416

(12)

(113)

OH

(16)

a) SeO 2 (cat), (p-MeO-C6H4)2SeO, dioxane, A; b) 2Py Cr2O7> tBuO 2 H, O f t ; c) Na 2 Cr0 4> AcjO, AcOH, AcONa, 70°C, 9 1 % ; d) H 2 O 2 , NaOH, MeOH, 88%; e) N2H4, AcOH, A, 95%; f) Ac 2 O, Py, 92%; g)NaBH4, EtOH, 96%; h) MsCl, Et 3 N, DMAP, THF, A, 8 1 % ; i) SeO 2 , dioxane, A; KOH,MeOH, 7 1 % .

Scheme 19 [42] from sclareol (3) via the 11-monoacetate of drimane-8a,l 1-diol (42). The acetate (67) was converted into the keto acetate (31) by oxidation of sodium chromate in a very high yield (91%) (Scheme 19). The same authors [81] performed the transformation of the keto acetate (31) into biologically active warburganal (16) by two convergent pathways, (Scheme 19). According to one of them, compound (31) was epoxidised, and the epoxy ketone (116) was subjected to a WhartonBollen rearrangement. The resulting diol (117) was acetylated to give the

417

hydroxy acetate (113). According to the second, a shorter route, the keto acetate (31) was reduced to the hydroxy acetate (118), which was isomerised to the hydroxy acetate (113). Its subsequent oxidation with selenium dioxide and saponification led to the triol (17), whose conversion to warburganal (16) was described above. We carried out a three-step synthesis of the keto acetate (31) from drim8-en-7-one (27) in the overall yield of 66.5% [82] (Scheme 20). The ketone (27) was enolacetylated to the diene acetate (119), whose oxidation with MPPA gave the keto alcohol (120), acetylated to the keto acetate (31). It should be noted that the structure of the keto alcohol (120) was determined not only on the basis of spectral data, but also was confirmed by X-ray [83].

(31) O

> f £ ^

^OAc

°

(27) (119) (120) a) CH3C(OAc)=CH2, TsOH, A, 84%; b) MPPA, Et2O, 80%; c) Ac2O, Py, 99%.

Scheme 20 We proposed also an alternative method to pass from the keto acetate (31) to the triol (17) (see below). 4.2 Synthesis of 11,12-Diacetoxydrim-8-en-7-onefrom

Drim-8-en-'/'-one

For the first time, the 1 l,12-diacetoxydrim-8-en-7-one (121) was prepared in the racemic form by Japanese chemists [84] and was then converted in two stages via the epoxide (122) into the racemic triol (17). This triol was further transformed to biologically active (l)-warburganal (16) by the same authors in a very complicated way of eight steps. Afterwards, warburganal (16) was obtained from (17) in one step by the Swern oxidation [24] (Scheme 21). The keto diacetate (121) was obtained in optically active form from royleanone (95) [69]. The ozonisation of the latter, and subsequent treatment of the resulting products by hydrogen peroxide in alkaline medium and then with Pb(OAc)4, gave the dicarboxylic acid (123). This diacid was converted in two steps into drim8-en-11,12-diol (124) followed by the acetylation to the diacetate (125). Chromic anhydride oxidation of diacetate (125) afforded the (+)-keto diacetate (121) (Scheme 21).

418

OH

(16)

(95)

^ (125)R=Ac

*•

(127)

(124)

(128)

Later on the (+)-keto diacetate (121) was synthesised by Nacano et al. a) H 2 O 2 , NaOH, MeOH, H 2 O, 82%; b) N 2 H,H 2 O, 23%; c) (COC1)2, DMSO, 96%; d) O3, CH2C12, -78°C; H 2 O 2 , NaOH, 90%; Pb(OAc) 4 , C f t , MeOH, 80%; e) CH 2N2, 100%; LiAlH,, Et 2 O, 90%; f) Ac 2 O, Py, 100%; g) CrO 3 , AcOH, 69%; h) KMnO 4 , acetone, 60%; i) hv, pentane, 73%; j) hv, O 2 , maso-tetraphenylporphine, CC14, 72%; k) LiAlH4, THF, 100%.

Scheme 21 [85] also by oxidation of the diacetate (125) with CrC>3. However, the diacetate (125) was obtained from manool (4) oxidised to the ketone (126) followed by photolysis to give the diene (127) [86]. Subsequent photooxidation of this diene to the endoperoxide (128), its lithium aluminium hydride reduction to the diol (124) [88] and acetylation led to the diacetate (125) [85] (Scheme 21).

419

We elaborated two pathways for obtaining the keto acetate (121) from drim-8-en-7-one (27) [82] (Scheme 22). According to one of them, the keto acetate (31) was brominated to ll-acetoxy-12-bromodrim-8-en-7-one (129), in which the acetoxy group was substituted for the bromine atom to give the keto acetate (121) in an overall 28% yield from drim-8-en-7-one (27) (5 steps). According to the second pathway, compound (27) was

(31)-

(121) OH (132) l l a OH (133) lip OH a) NBS, CaCO3, CC14, A, 60%; b) AcOK, DMSO, 84%; c) AcOK, DMF, 65%; d) K2CO3, MeOH, 72%; e) KOH, O2, MeOH, 50%; f) SOC12, Py, 78%; NaBHj, EtOH, 94%; g) O2, eosin, hv, tBuOH, 2,6-lutidine, 40%.

Scheme 22 brominated with NBS to afford the dibromoketone (130) in a high yield. Its structure was confirmed by X-ray data [79]. The keto diacetate (121) was then obtained from (130) by substitution of both bromine atoms by acetoxy groups. In this two-step synthesis of the product (121) from the ketone (27) the overall yield was reached to 59%. It is necessary to note that in [69], the keto diacetate (121) has been described as a crystalline substance, whereas in [85] as an oil. We did not succeed in crystallising this compound either. However, on saponification of (121), we obtained the keto diol (131) in the crystalline form. The structures of (121) and (131) were confirmed by spectroscopic and X-ray data [88]. It should be noted that the keto diacetate (121) served as a starting compound for the synthesis of one more natural drimane (+)-fuegin (135) [85], isolated from Drimys winteri Forst [89]. On saponification of

420

compound (121) in the presence of oxygen a mixture of epimeric semiacetals, (132) and (133), was formed, whose further dehydration and subsequent reduction gave hydroxyeuryfuran (134), photooxidated into fuegin (135). 4.3 Synthesis of 7-Oxoisodrimenin and Isodrimenin from Drim-8-en-7-one

7-Oxoisodrimenin (136) has been isolated from Porella cordeana [90], though much earlier it had been synthesised on the Beckmann mixture OH.

OH j

(16)

•i.H

(141)a-epoxi(68%) (142) |3-epoxi(29%)

I

(17)

a) Beckmann mixture; b) CrO 3 , Py, 38%; c) PCC, CH2C12, 92%; d) MnO 2 , CH2C12; e) (CH 2 OH) 2 , TsOHHjO, C6H6, A, 94%; f) LiAlH4, Et 2 O; 10% HC1, 95.5%; g) LiAlH 4 , Et 2 O, 88.5%; h) m-CPBA, CH2C12; i) Et 2 NLi, THF.A, 54%; j ) (COC1)2, DMSO, 70%.

Scheme 23 oxidation of drimenin (137) and isodrimenin (138), isolated for the first time from Drimys winteri [91]. 7-Oxoisodrimenin (136) was also obtained on oxidation of natural isodrimeninol (139) with the Collins reagent [10]. Nacano et al. [85] have prepared 7-oxoisodrimenin (136) on oxidation of the mixture of oxosemiacetals, (132) and (133).

421

We obtained 7-oxoisodrimenin by oxidation of the keto diol (131) with an excess of PCC [92]. On oxidation of (131) with MnO 2 or PCC (2 mol. equivalents), a mixture of the semiacetals (132) and (133) and keto lactone (136) was obtained. One of these semiacetals, (133) was isolated by crystallization, and its structure and stereochemistry were confirmed by Xray data [93]. The mixture of semiacetals (132) and (133) was further oxidised to the keto lactone (136). This lactone has no biological activity itself, but it has been used as an intermediate in the total synthesis of bioactive (±)-warburganal (16) [84]. The ethylene ketal (140) of the keto lactone (136) was converted into the keto diol (131), whose transformation to warburganal (16) was discussed above (Scheme 23). There are many syntheses of isodrimenin (138), both total in the racemic form, and partial in the optically active form from available bi- and tricyclic diterpenoids or other drimanes. These syntheses will not be discussed here since most of them have already been reviewed [6,7]. Isodrimenin (138) possesses antifeedant activity against larva of kolorado beetle [4]. Besides, compound (138) can be transformed to biologically active warburganal (16) via the following series of transformations: (138) -^ (136) - • (140) -> (131) -> (122) - • (17) -» (16). There is also known another transition from isodrimenin (138) to warburganal (16) via the diol (124), the isodrimenin reduction product [91] (Scheme 23). On the m-chloroperbenzoic acid oxidation, the diol (124) gives a mixture of the epoxy diols, (141) and (142). The predominant one (141) (68% yield) reacts with lithium diethyl amide to afford the triol (17), oxidised to warburganal (16) [87, 94]. We prepared isodrimenin (138) from drim-8-en-7-one (27) according to the following sequence of transformations: (27) —> (130) —» (121) —» (131) -» (136) -> (143) - • (138) (schemes 22-24) [92].

422

4.4 Synthesis of5,6-Dehydro-7-oxoisodrimeninfrom Drim-8-en-7-one

5,6-Dehydro-7-oxoisodrimenin (144) was isolated from Porella cordeana [90], though earlier this compound (144) had been synthesised

(136)

OAc

(112)

(145)

\Z

(146) (147)

(144)

a) (CH 2 SH) 2 , SnCl 2 2H 2 O, THF, A, 92%; b) MC1 2 6H 2 O, NaBtt,, DMF, 85%; c) SeO 2 , AcOH, A, 72.5%, d) NBS, CCU, A, 76%; e) AcOK, DMSO, 95%; f) K 2 CO 3 , MeOH, 60%; g) PCC, Me 2 CO, 98%; h) CH 3 C(OAcj=CH 2 , TsOH, A, 78%; i) O 2 , hv, tetraphenylporphine, CC14, 69%.

Scheme 24 by dehydrogenation of 7-oxoisodrimenin (136) with selenium dioxide [91]. However, the keto lactone (144) prepared in such a way is contaminated with selenium, which is difficult to get rid of. We elaborated two pathways for the preparation of compound (144). In one of them, the ketone (112) has been brominated to the dibromide (145), by replecing the bromine atoms with the acetoxy groups. The resulting product (146) has been saponified to the keto diol (147), which was oxidated to the target lactone (144). The overall yield of compound (144) was 42%. It is necessary to note that the dibromide (145) is formed also on bromination of drim-8-en-7-one (27) [79]. According to the second route, the keto lactone (136) has been enolacetylated to the enol acetate (148) photooxidated into the keto lactone (144). The overall yield of (144) from drim-8-en-7-one (27) (six steps) was 22.4%.

423

c (99%)

4.5 Synthesis ofDrim-7fi8/3,9a-triolfrom Drim-8-en-7-one

Polyhydroxylated drimanes are of interest as potentially biologically a)H2O2, NaOH, MeOH, 96%; b) KBH4, MeOH, 100%; c) Ac^O, Py, 97%; d)HClO4, THF; e)KOH,MeOH, 93%; f) (CH3)2CO, CuSO4, HC1O4, 74%; g) SOC12, Py, 79%.

Scheme 25 active substances and as starting compounds in the syntheses of bioactive labdanic diterpenoids (forscolin, manoyloxides). We carried out the synthesis of drim-7|3,8(3,9a-triol (149) starting with drim-8-en-7-one (27) [95]. The latter was epoxidised to the epoxy ketone (150), which was reduced to the epoxy alcohol (151), whose acetate (152) was transformed, on interaction with HCIO4, to a mixture of the triol (149) (41% yield) and its monoacetate (153) (54%). Compound (153) gives the triol (149) on saponification, and vice versa, the triol (149) is quantitatively acetylated to the monoacetate (153). The triol (149) forms the acetonide (154). That conversion proves its absolute configuration at C-8 and C-9, confirmed by spectral data. It is interesting to note that on phosphorus oxychloride treatment the acetoxy diol (153) is transformed into the epoxy acetate (152), but the dehydration products were not formed (Scheme 25).

424

5 Synthesis of (-)(3aS,7aS)-ft"a«s-tetrahydroactinidiolide from Drim-8en-7-one 7?ans-tetrahydroactinidiolide (155) has been isolated from tobacco [96]. This compound is of interest since it can easily be transformed to dihydroactinidiolide (156) [97, 98], an important odorous component of the fragrances of tea [99], tobacco [100], tomato [101], essential oils from Actinidia polygama [102] and Acacia famesiana [103], and also the one of the components of the red ants pheromone [98]. Dihydroactinidiolide (156) was patented as an analeptic for the respiratory depression [104]. We carried out a short and efficient synthesis of transtetrahydroactinidiolide (155) from drim-8-en-7-one (27) [105]. On exhaustive ozonolysis of drim-8-en-7-one (27) and subsequent treatment of resulting products with hydrogen peroxide, the keto acid (157) was obtained in a quantitative yield. The Baeyer-Villiger oxidation of this acid leads directly to (-)(3aS, 7aS)fra«s-tetrahydroactinidiolide (155) (Scheme 26).

(157)

(155)

(156)

a) O 3 , AcOEt; H 2 O 2 , 100%; b) m-CPBA, conc.H 2 SO 4 , CH2C12, 67%; c) LDA, THF; (PheSe) 2 , HMPA, 59%; H 2 O 2 , THF, AcOH, 37% [97].

Scheme 26 Thus, starting with drim-8-en-7-one (27), we succeeded in performing the syntheses of a series of drimanic compounds. 6 Synthesis of (+)-drim-8-en-ll-oic Acid Drim-8-en-l 1-oic acid (158) is a convenient synthon for the preparation of polyfunctional drimanes. This acid has been used as an intermediate in the total synthesis of 1,6,7-tridesoxyforskolin (159), a related compound to forskolin (160), possessing ionotropic, antihypotensive and other activities [106]. Optically active compound (159) was isolated together with forskolin from the plant Coleus forskohlii [107]. The synthesis of the acid (158) was accomplished by us starting with the mixture of esters (107) [108], obtained from norambreinolide (69)

425

[78]. The mixture (107) reacts with methyl lithium to give a mixture of alcohols, (161) and (162), separated by chromatography. Alcohol (162) was dehydrated into the diene (163) cleaved by ozonolysis to a mixture of acids (158) and (164), separated by chromatography. It was found that on acid treatment, the unsaturated alcohol (161) is easily cyclised to the oxide (165), which is formed together with a mixture of hydrocarbons (Scheme 27). The overall yield of the unsaturated acid (158) was 28.8%. OH

_CO-,Me

OH

OAc OH (160) a) CH3Li, Et2O, 90%; b) TsOH, O f t , A, 90%; c) O3, AcOEt, Py, -50°C.

Scheme 27 7 Synthesis of 8a-Acetoxydrimane-ll-oic Acid 8a-Acetoxydrimane-ll-oic acid (166) is a convenient synthon for the preparation of biologically active drimanes [72]. The synthesis of this compound was accomplished by us [109] from commercially available

426

sclarodiol (167) [8]. The latter was exhaustively acetylated to the diacetate (168) followed by selective saponification to give the hydroxy acetate (169) which was then oxidised to the acetoxy aldehyde (53). Its enolacetylation and recrystallisation of the reaction product led to the trans-enol acetate (170), which was ozonised to afford the acetoxy acid (166) in a good yield. The overall yield of (166) from sclarodiol (167) was 16% (Scheme 28). It should be mentioned that authors [72] synthesised the acetoxy aldehyde (53) from labdanolic acid (6) in 5 steps in an overall 50 %

lOAc

(171)R=CH2OH (172) R=CHO

yield. a) Ac2O, Et3N, DMAP, 80%; b) NaHCO3, MeOH, H2O, 58%, c) CrO32Py, CH2C12, 63%; d) O3, AcOH, Py, -70°C, 88%; e) O3, NaBU, 45%; f) (COC1)2, DMSO, 25%; g) NaClO2, 90%.

Scheme 28 They obtained the enol acetate (170) in 92% yield, probably, as a mixture of cis- and tram- isomers. Compound (170) was ozonised, and the resulting product was reduced with NaBELi to the hydroxy acetate (171) which, on the Swern oxidation, afforded the acetoxy aldehyde (172). The

427

latter was then oxidised to the acetoxy acid (166). The overall yield of (166) from labdanolic acid (6) was 4.6% (Scheme 28). Thus, the present review summarises the results of our investigations, concerning the synthesis of a series of drimanic sesquiterpenoids with biological activity or being valuable intermediates on the way to important, biologically active natural compounds. The literature data concerning the presence of these compounds in natural sources, their biological activity and methods of preparation are also discussed. Starting from available labdanoids, sclareol and neoabienols, a large number of drimanic sesquiterpenoids have been prepared. In many of these syntheses, the commercially available norambreinolide served as an intermediate on passing from labdanoids to drimanes. It is well known that norambreinolide is a cleavage product of a large number of labdanoids. In particular, using norambreinolide as a convenient method for preparing the drim-8-en-7-one, a derivative with a high synthetic potential, was worked out. Using this compound, a series of drimanic substances has been prepared. At present, the investigations in the synthesis of drimanic polyfunctional sesquiterpenoids are in progress in our laboratory. ABBREVIATIONS

Ac

AcOH (Ac2O)2O

NBS BuLi m-CPBA DMAP DMA DIBAL DDQ DMF DMSO HMPA NMO MPPA

PTC Py

Acetyl Acetic acid Acetic anhydride N-Bromosuccinimide n-Butyllithium m-Chloroperbenzoic acid 4-Dimethylamino pyridine N,N-Dimethylaniline Diisobutylaluminium hydride 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone N,N-Dimethylformamide Dimethyl sulphoxide Hexamethylphosphoric triamide N-Methylmorpholine-N-oxide Monoperphtalic acid Phase transfer catalyst Pyridine

428

PCC Red-Al Tert t-Bu THF TsOH TSNHNH2 TsCl

= = = = = = = =

Pyridinium chlorochromate Sodium bis(2-methoxy)-aluminium dihydride Tertiary Tertiary butyl Tetrahydrofuran p-Toluenesulphonic acid p-Toluenesulphonic acid hydrazide p-Toluenesulphonyl chloride (tosyl chloride)

ACKNOWLEDGEMENTS The author Kuchkova, preparation contributed

is sincerely grateful to Doctors Aculina N. Aricu, Kaleria I. and Olga C. Iliashenco for skillful assistance in the of the manuscript. Thanks are also due to co-workers who to these investigations, whose names are in references.

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[70] Vlad P.F., Aricu A.N. and Ciocirlan A.G.; Russ. Chem. Bull. Int. Ed, 2004, 53, 453-455. [71] Ohloff G.;Helv. Chirn. Acta, 1958, 41, 845-850. [72] Lithgow A.M., Marcos I.S., Basabe P., Sexmero J., Diez D., Gomez A., Estrella A and Urones J.G.; Natur. Prod. Lett, 1995, 6, 291-294. [73] Banerjee A.K., Correa J.A. and Laya-Mino M.; J. Chem. Soc, Synop., 1998, 710711. [74] Vlad P.F., Coltsa M.N., Sibirtseva V.E and Kustova S.D.; Zh. Obshch. Khim. (J. Gen. Chem. USSR), 1980, 50, 213-217. [75] Vlad P.F., Belfer A.G., Coltsa M.N. and Sibirtseva V.E., USSR Patent 777055 (1980). [76] Iastrebova A., Panfil G., Rotundu F., Cojocaru D., Diaconu G., Ciubara M., Vlad P., Coltsa M., Mironov G., Ceban P., Botsan V. and Muntean G.; Patent of the Republic of Moldova 235 (1995). [77] Vlad P.F. and Vorobieva EA.; Khim. Prirod. Soedin. (Chem. Nat. Compd.), 1983, 146-149; USSR Patent, 767083 (1980). [78] Stoll M. and Hinder M.; Helv. Chim. Acta, 1954, 37, 1859-1865. [79] Koltsa M.N., Mironov G.N., Malinovskii S.T. and Vlad P.F.; Russ. Chem. Bull. (Engl. Transl), 1996, 45, 208-214. [80] Razmilic I., Lopez J. and Cortes M,; Synth. Commun., 1993, 23, 1155-1173. [81] Barrero A.F., Cortes M., Manzanenda E.A., Cabrera E., Chahboun R., Lara M. and Rivas A.R.; J. Nat. Prod., 1999, 62, 1488-1491. [82] Vlad P.F., Popa D.P., Gorincioi E.C., Coltsa M.N. and Mironov G.N.; Russ. Chem. Bull. Int. Ed, 2000, 49, 98-101. [83] Chumakov Yu.M., Simonov Yu.A., Mazus M.D., Popa D.P. and Vlad P.F.; Crystallography Reports (Engl. Transl), 2000, 45, 244-247. [84] Nakata T, Akita H, Naito T. and Oishi T.; Chem. Pharm. Bull, 1980, 28, 21722177. [85] Nacano T., Villamizar J.E. and Maillo M.A.; Tetrahedron, 1999, 55, 1561-1568. [86] Nacano T. and Maillo M.A.; Synth. Commun., 1981,11, 463-473. [87] Nacano T. and Martin A.; J. Chem Research (S), 1989, 52-53. [88] Kravtsov V.N., Gorincioi E.C., Mironov G.N., Coltsa M.N., Simonov Yu.A. and VladP.F. Crystallography Reports (Engl. Transl.), 2000, 45, 289-291. [89] Appel H.H., Bond R.P. M and Overton K.H; Tetrahedron, 1963,19, 635-641. [90] Harrijan G.G., Ahmad A., Baj N., Glass T.E., Gunatilaka A.A. L. and Kingston D.G.I.; J. Nat. Prod, 1993,56, 921-925. [91] Appel H.H, Connolly J.D, Overton K.H. and Bond R.P.M.; J. Chem. Soc, 1960, 4685-4692. [92] Vlad P.F., Gorincioi E.C., Coltsa M.N. and Deleanu C; Russ. Chem. Bull. Int. Ed, 2000, 49, 546-548. [93] Kravtsov V.N., Simonov Yu.A. Gorincioi E.C., Coltsa M.N. and Vlad P.F. Crystallography Reports (Engl. Transl), 2000, 45, 789-791. [94] Razmilic I., Sierra J., Lopez J. and Cortes M,; Chem. Lett., 1985, 1113-1114. [95] Gorincioi E., Popa D., Mironov G., Coltsa M. and Vlad P.; Abstracts of XXVI National Sympozium on Chemistry, Calimanesti-Caciulata, Valcea, Roumania, 2000, 141.

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[96] Kaneko H. and Hoshino K.; Agric. Biol. Chem., 1969, 33, 969-970.; Schumacher J.N. and Heckman R.A.; Phytochemistry, 1971,10, 421-423. [97] Hoye T.R. and Kurth M.J.; J. Org. Chem., 1978, 43, 3693-3697; Strekowski L, Visnick M. and Battiste M.A.; J. Org. Chem., 1986, 51, 4836-4839. [98] Mori K. and Nacazono Y.; Tetrahedron, 1986, 42, 283-290. [99] Bricout J., Viani R., Muggler-Chavan F., Marion J.P., Reymond D and Egli R.H.; Helv Chim. Acta., 1967, 50, 1517-1522; Ina K., Sacato Y. and Fukami FL; Tetrahedron Lett, 1968, 2777-2780. [100] Kaneko H. and Ijichi K.; Agric. Biol. Chem., 1968, 32, 1337-1340. [101] Viani R., Bricout J., Marion J.P., Muggler-Chavan F., Reymond D. and Egli R.H.; Helv Chim. Ada, 1969, 52, 887-891. [102] Sacan T., Isoe S. andHueon S.B.; Tetrahedron Lett., 1967,1263-1627. [103] Demole E., Enggist P. and Stall M.; Helv Chim. Acta., 1969, 52, 24-32. [104] Schumacher J.N.; US Patent 3.576.008. [105] Vlad P.F., Gorincioi E.C., Aricu A.N. and Coltsa M.N.; Nat. Prod. Lett, 1999,13, 1-4. [106] Hashimoto S., Sonegawa M., Sakata S. and Ikegami S.; J. Chem. Soc, Chem Commun., 1987,24-25. [107] Gabetta B., Zini G. and Danieli B.; Phytochemistry, 1989,28, 859-862. [108] Coltsa M.N., Mironov G.N. and Vlad P.F.; Khim. Prirod. Soedin. (Chem. Nat. Comp.), 1991, 214-220. [109] Coltsa M.N., Mironov G.N. and Vlad P.F.; Khim. Prirod. Soedin. (Chem. Nat. Comp.), 1991,499-502.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©

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QUASSINOIDS: STRUCTURAL DIVERSITY, BIOLOGICAL ACTIVITY AND SYNTHETIC STUDIES IVO J. CURCINO VIEIRA* AND RAIMUNDO BRAZ-FILHO Setor de Quimica de Prodntos Naturals, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego 2000, 28013-600, Campos dos Goytacazes, Rio de Janeiro, Brazil. (*Email: curcino@uenf. br) ABSTRACT: Quassinoids are bitter constituents of Simaroubaceae and the secondary metabolites characteristic of this family. The generic term quassinoids arises from quassin, the name of the first structurally identified member of this class isolated from specimen Quassia amara. Quassinoids can be divided into distinct groups according to their basic skeletons C!8, C19, C20, C22 and C2s. The chemistry and biogenesis of quassinoids have been reviewed several times. They remain exclusively of Simaroubaceous origin and biogenetically can be regarded as degraded triterpenoids and are almost certainly derived from tetracyclic triterpenes. Several quassinoids have been isolated and structurally elucidated and the majority of them have been biologically tested, including antifeedant, inseticidal, herbicidal, antLparasitic, antimalarial and anticancer activities. The interest in the chemistry of quassinoids has accelerated rapidly with the American National Cancer Institute finding in early 1970s, showing that these compounds display marked antileukemic activity (e.g. bruceantin). Chemical modifications of biologically inactive quassinoids have been performed, attempting to yield active ones, either by esterification or conversion of glycosides to the corresponding aglycones. Many studies on semisynthesis of rings member, intermediates and total synthesis of the molecular backbones or same leads such as bruceantin have been published. This review covers the structural variations, biological activity and some quassinoids synthetics studies.

1. STRUCTURAL DIVERSITY OF QUASSINOIDS /. / Introduction In elapsing the scientific development, the phytochemical has been using growing efforts to search new secondary metabolites of vegetable origin, not only for its economic value for the human society but also for what they represent in aiding the amplification of

434

the knowledge on the natura phenomena. The tracks that motivate and guide that type of investigations are based most of the times on the popular use of several plants. With that approach researchers could isolate and identify the quassinoids, micromolecules by proceeding that, what was verified so far, i.e. they are produced exclusively by plants of Simaroubaceae family. There are lot several of those plants, it tending to be of popular use in the amebiasis treatment, disinters, arthritis and intestinal parasites, in the combat the fever and, also as insecticides. Several pharmacological studies led to the conclusion that quassinoids are the main ones responsible for the healing properties of those plants. For such review the collected data are from 1985 up to 2004 focusing on all the isolated and identified quassinoids, as well as, the main biological activities and some synthetic processes. 1.2 General Features of Quassinoids The review containing structural data, biological activities and chemical modifications of quassinoids was published last decade with references up to September 1984 by Polonsky [1]. According to Simao et al. [2] more than two hundred quassinoids were isolated and identified until December 1984.

cm,. Quassinoids are degraded triterpenes. They present five basic skeletons: a) skeleton of 25 carbon atoms (C25), denominated simarolidane skeleton, b) skeleton of 22 carbon atoms (C22),

435

denominated here of picrolemmane skeleton; c) skeleton of 20 carbon atoms (C20), denominated quassotidane skeleton; d) skeleton of 19 carbon atoms (Cw), denominated cedralidane skeleton; and e) skeleton of 18 carbon atoms (Cig), denominated of lauricolactme skeleton [2]. The quassolidane skeleton (C20) is of larger occurrence. Quassinoids are heavily oxygenated lactones (5-lactone in the quassolidane skeleton and yfoctone in the cedrotidane and lauricolactane skeletons, and 8-lactone and y-lactone in the other skeletons), with variable of groups hydroxyl amount, hydroxyl esterified, carbonyt, methoxyl and carbomethoxyl. In the quassolidane skeleton, these oxygenated functions can be found in most of carbon atoms, except carbons C-5, C-9, C-19 and C-29. Carbons C-l, C-2, C-7, C-ll and C-12 almost meet that obligatorily oxygenated, except in the quassinoids of the simarolidane type, once they do not happen oxygenated in C-12 position [1]. Ring D of quassolidane skeleton can present one group hydroxyl in carbon C-l 5, group that is generally esterified with various small fatty acids. In ring B, the carbon C-6 can present group hydroxyl, esterified or not. Ring A introduces one or two double bond in all skeletons. All isolated quassinoids he has only a group methyl in the carbon C-4 so far. Quassinoids O-glycosylated were isolated from species of genera Bntcea and Picrasma. 1.3 Biogenesis of Quassinoids Biogenetically quassinoids can be regarded as degraded triterpenoids and are almost certainly derived from tetracyclic triterpenes. The conversion of the tetracyclic triterpene into quassinoid has been experimentally verified by using labeled mevalonate precursors [3-5] and follows the route originally proposed by Arigoni et al. [6] but extended by further degradation to quassinoids as shown in Scheme 1. Support for this biogenetic picture has also come from the isolation of compounds representing the various stages and structural variations among quassinoids.

436 H

HO

Q5 Skeletons

Ct8 Skeleton

Scheme 1. Biogenesis of Quassinoids The postulated precursor, A7 - euphol (1) is proposed to undergo skeletal rearrangement to the hypothetical 7oc-hydroxy apo compound (2). Ring D is then oxidatively expanded to the 5-lactone (3) [6]. During these transformations one of the methyl groups at C-4 and four terminal carbons at the side chain must be lost. Opening of the 8-lactone (3) and relactonisation to the 7a-hydroxyl would then

437

give the intermediate 5-lactone (4) from which the basic skeletons observed amongst quassinoids can be derived. Thus, the lactonisation through the C-17 hydroxyl leads first to C25 skeleton quassinoid, while lactonisation through the hydroxyl at C-21 and subsequent oxidation of the residual C-17 hydroxyl may result in second C25 skeleton quassinoid. The C22 and C20 skeletons quassinoids may then be formed by cleavage of the C-13 - C-17 bond while the C19 skeleton compounds require the additional loss of the carbon atom C-16. The C18 skeleton results from the C19 skeleton quassinoid by loss of one carbon atom in ring A, presumably due to a benzylic rearrangement resulting in a ring contraction. 1.4 Structures of Quassinoids To follow what has been described, all quassinoids (divided by basic skeletons) were isolated and identified since 1985 to June 2004. 1.4.1 (

Quassinoids

xI

.,o

..,-"

HO,

o \ It J 5

c ^O

O

H

ft

6

o R-

O \\

iX i

A

^o

•o

/

7R=H 8R=C1

Until 1985 only three quassinoids with C18 skeleton had been isolated from Simaroubaceae species, Samaderine A (5) isolated from Samadera indica [7], and laurycolactone A (6) and B (7) isolated from Eurycoma longifolia Jack [8].

438

Then, two decades passed and only three more quassinoids with Ci8 skeleton were isolated from Eurycoma longifolia, eurycolactone B (8), C (9) [9] and D (10) [10], in a total of six quassinoids with Ci8 skeleton, isolated and identified so far. Compound (8) is the first halogenated quassinoid separated from plant sources. 1.4.2 Ci9 Quassinoids o

11. Ri= O; R2= O 12. Ri= OH-a; R2= O 13. 5,6-dehydro; Ri= OH-a; R2= O 14. 3,4-dihydro; Ri= O; R2= O 15. R]= O; R2= OH-a

o

18 17. 5,6-dihydro

Only nine quassinoids possessing the C19 basic skeleton were isolated until 1985: samaderins B (11), C (12), D (13), an 3,4dihydrosamaderin B (14) from the stem and leaves of Samadera indica, cedronin (15) and cedronolin (16) isolated from the fruits of Simaba cedron, eurycomalactone (17) and 5,6dehydroeurycomalactone (18) from Eurycoma longifolia Jack, and shinjulactone B (19) isolated from Ailanthus altissima Swingle [1]. Two decades passed and twenty-three more quassinoids were isolated from Simaroubaceae species in a total of thirty-two quassinoids with C19 skeleton identified so far. Eurycoma longifolia Jack is one of the most well known folk medicines for intermittent fever (malaria) in Southeast Asia [11]. This plant possesses the largest number of quassinoids with C19 skeleton identified so far. 7a-hydroxyeurycomalactone (20) [12], 6ahydroxyeurycomalactone (21) [13], eurycolactone E (22) [10], eurycomalide B (23) [14], and quassinoids (24) and (25) [15] isolated from the E. longifolia Jack and cymosanine (26) [16] isolated from

439

Simaba subcymosa possessing an y-lactone linked to carbons C-12 and C-13 in ring C in basic skeleton. Quassinoid (24) showed the presence of an exomethylene group in ring A. Cymosanine (26) showed an oxymethylene group (CH2-30), involving the formation of a tetrahydrofuran ring linked to carbon C-13. The p-configuration of the methyl group in position C-4 in ring A in cymosanine (26) and quassinoid (24) has been the unedited factor in all quassinoids.

OH

20. Ri= O; R2= OH-a; R3= H2 21. R,= O; R2= O; R3= OH-a 22. R,= OH-a; R2= O; R3= H2 23. 5,6-dehydro; Ri= OH-a; R2= O; R3= H 25. 3.4-dihvdro: Ri= OH-a: R2= O: R3= OH-a

26

The eight new quassinoids C19 type shinjulactone B (19) with a presence of a l,2-seco-l-nor-(5->10)-aZ>eo-picrasan-2,5-olide skeleton have been isolated from Simaroubaceae species. The structures of polyandrol (27) and 15-(9-acetyl-5(<S)polyandrol (28) isolated from Castela polyandra were established by a combination of spectroscopic and X-ray analysis [17,18]. Cedronolactone B (29) and cedronolactone C (30), isolated from wood of Simaba cedron were shown, exhibited in vitro cytotoxicity (IC50 6.5 and 49 u,g/mL) against P-388 lymphocytic leukemia cells [19]. Brucea javanica Merr. is a shrub, which is distributed from Southeast Asia to northern Australia having its seeds been used in the treatment of dysentery, malaria and cancer [20]. Javanicolide A (31) and Yadanziolide D (32) were isolated from seeds of B. javanica Merr., and their structures were elucidated by the analysis of spectral data and chemical evidence [20].

440

Ailanthm malabarica is a large indigenous tree from India and Southeast Asia and is used in the treatment of dyspepsia, dysentery, bronchitis, opthalmia and snake bites [21]. Ailanquassin A (33) was isolated from the wood of Ailanthus malabarica, and its structure was established by a combination of spectroscopic and X-ray analysis [21]. Two new quassinoids (34) and (35) with a presence of a 1,2seco-l-nor-(5—»10)-«&eo-picrasan-2,5-olide skeleton were isolated from wood Ewycoma longifolia Jack [15]. OH

OH ..-•""

R,

o

H ,

Four quassinoids Cw skeleton type with ring D contracted have been isolated from Eurycoma longifolia Jack., longilactone (36) [22], eurycolactone F (37) [10], 6-dehydroxylongilactone (38) [12], and eurycomaoside (39) [23]. The eurycomaoside (39) represents the first entry of the series of quassinoids with C19 skeleton possessing a glycosyl moiety at C-l. The 6-dehydroxylongilactone (38) showed potent activity (IC50 0.66 ng/mL) against P-388 leukemia cells. OH

OH OH 39

441

Quassinoid (40) isolated from the roots from Eurycoma longifolia Jack. [14] showed lactonization rarely-occurred C ring with hydroxyl group at C-ll with the carbonyl group at C-13, and eurycolactone A (41) showed a novel carbon framework with ring A contracted [9].

42

41

Cedronolactone E (42) isolated from Simaba cedron possesses a unique pentacyclic structure [24]. The structural similarity between cedronolactone C (30) may suggest a biogenetic relation between the former and the latter (Scheme 2). The cleavage of hemiketal at C-ll produces the cyclic ketone intermediate (30a), which via Michael addition of the ketone oxygen atom to C-4 from the si face produces OH

O H Michael addition „

30

Scheme 2. Proposed Biogenetic pathway from Cedronolactone C (30) to Cedronolactone E (42)

Ailantinol G (43) a quassinoid isolated from Aikmthus altissima was evaluated for its antitumor effects promotion against Epstein-Barr virus early antigen activation introduced by 12-0tetradecanoylphorbol-13-acetate in Raji cells [25], OH

HO, OH

442

1.4.3 C20 Quassinoids Despite their diversity in structure in quassinoids with C20 skeleton certain general features have been rationalized as follows [1]: i) The number and the positions of the methyl groups are the same of all skeletons; ii) Quassinoids are heavily oxygenated 8-lactones in the C20 skeleton, with the exception of C-5 and C-9 and the methyl groups at C-4 and C-10 oxygenated functions have been found on all the other carbon atoms; iii) Ring A may have the structures (a), (b), (c), (d), and (e); iv) Ring C may possess at position C-8 either a methyl group or a hydroxymethyl group which forms a hemiketal bridge to C-11 or an oxide bridge to C-13; v) Ring D and ring B may have at C-15 and/or C-6 hydroxyl groups which are generally esterified with a small fatty acid e.g. acetic, 2-methylbutiric, isovaleric, senecioic, 2-hydroxy-2methylbutyric, 3,4-dimethyl-4-hydroxyvaleric acids. OH

OH

o

o

H

°

I H (e)

To proceed, they are striped, all quassinoids identified since the year of 1985 up to 2003. The identified quassinoids are divided by Simaroubaceae species. 1.4.3.1 Quassinoids from Ailanthus altissima Swingle Ailanthus altissima Swingle, the tree-of-heaven, is native of China and was introduced in Europe around the end of the 18th century. Ailanthus altissima is used in Chinese traditional medicine as a bitter aromatic drug and in the treatment of colds and gastric diseases [26]. Seven quassinoids were isolated from Ailanthus altissima Swingle, ailantinol A-F (44-49) [26-28], and their structures were elucidated from spectral data. The ailantinol E (47) and F (48) were evaluated for its antitumor promoting effects against Epstein-Barr virus through early antigen activation introduced by 12-0-

443

tetradecanoylphorbol-13-acetate in Raji cells. Quassinoids were found to show potent activity without showing any cytotoxicity [25].

Shinjulactones L (50), M (51), N (52) [29-30], and shinjuglycosides E (53), and F (54) [31] were isolated from the root bark of Ailanthus altissima. OAc

OH HQOl OH

HO. OH

CH;OH

9H

,

HO,

GbO4l

1.4.3.2 Quassinoids from Ailanthus excelsa Chemical examination of Ailanthus excelsa has been carried out by several workers resulting in the isolation of quassinoids, alkaloids and

444

terpenoids [32], Three quassinoids (55), (56) and 3,4dihydro excelsin (57) were isolated from the stem bark of Ailcmthus excelsa, and structural elucidation is based on the analysis of spectroscopic data [32]. Quassinoids (55) and (56) showed the presence an A ring aromatized. OH HO O I

1.4.3.2 Quassinoids from Ailanthus vilmoriniana Six quassinoids, named vilmorinine A-F (57-62) were isolated from the cortex of Ailanthus vilmoriniana. The structures were elucidated by various spectroscopic methods, X-ray analysis, and computational chemical methods. Vilmorinines did not show cytotoxic activity (IC50 > 100 ug/mL) [33-34]. OH

O, , O H V

COzH "OR?

R2 Ri

SapOH 59. POH 60.POH 61. POH 62. pOH

H R4

R2

R3

aH aH aH aH aH

Me PMe H PMe H PMe H PMe H aMe

445

1.4.3.4 Quassinoids from Brucea antidysenterica Milt, Various quassinoids with antileukemic activity have been isolated from the Ethiopian tree Brucea antidysenterica Mill, including the antileukemic compound bruceantin (234) by kupchan and associates [35]. Eight quassinoids named bruceanols A-H (64-71) were isolated from Brucea antidysenterica, and all of these compounds exhibited cytotoxicity against murine lymophocytic leukemia (P-388) [35-38], Bruceanols G (70) and H (71) were evaluated against three cancer cell lines: SK-MEL-5 (melanoma), COLO-205 (colon cancer), and KB (nasopharynx carcinoma). These compounds were only marginally cytotoxic in the melanoma cell line with ED50 values of 4.08 and 6.37 uM, respectively. However, bruceanol G (70) showed activity against the COLO-205 and KB cell lines with ED50 values of 0.44 and 0.55 uM, respectively [38]. Quassinoid glycosides named bruceantinoside (72) and the new bruceanic acids B (73), C (74), and D (75) were isolated from wood Brucea antidysenterica [39-40]. Bruceanic acid D (75) was cytotoxic against P-388 (ED50 0.77 ug/mL).

446 OH

OAc

1.4.3.5 Quassinoidsfrom Bntceajavanica (L.) Merr. Brucea javanica Merr, is a shrub, which is distributed from Southeast Asia to northern Australia having its seeds been used for the treatment of dysentery, malaria and cancer [20]. Seeds ofBrucea javanica are known as "Ya-dan-zi" in Chinese folklore and have been used as a Chinese medicine for cancer, and the main active compounds of the plant has been extensively studied and thirty-four quassinoids have thus far isolated the last two decades. Quassinoids glycosides, yadanziosides A (76), B (77), C (78), D (79), E (80), G (81), H (82) and dehydrobrasatol (83) and dehydrobraceantinol (84) [41], and yadanziosides K (85), M (86), N (87), O (88) [42], and P (89) [43] were isolated from seeds Brucea javanica Merr..

447 447 OH HO.

io

GlcO.

GlcO1

GlcO,

Three quassinoids named yadanziolides A (90), B (91), and C (92), and four quassinoids glycosides named yadanziosides F (93), I (94), J (95), and L (96) were isolated from water-soluble fraction methanol extract of seeds of Brucea javanica Merr., and their structures were determined by spectral and chemical means [44]. Yadanziosides F (93), I (94), J (95), and L (96) were demonstrated to have in vivo antileukemic activity against the murine P-388 lymphocytic leukemia.

448 OH

OH

Bruceosides C (97) isolated from the fruits of B. javanica [45] demonstrated potent cytotoxicities against human epidermoid carcinoma of the nasopharynx (KB) (ED50 < 0.1 u,g/mL), human lung carcinoma (A-549) (ED50 < 0.44 ug/mL), colon carcinoma (HCT-8) (ED50 < 4.51 ug/mL), melanoma (RPMI) (ED50 < 0.1 ug/mL), and CNS carcinoma (TE-671) (ED50 < 0.29 ug/mL), as well as murine lymphocytic leukemia (P-388) (ED50 < 5.11 |J.g/mL) [45]. Bruceosides D (98), E (99), and F (100) show selective cytotoxicity in the leukemia and non-small cell lung, colon, CNS, melanoma, and ovarian cancer cell lines with log GI50 values in the range of-4.14 to -5.72 [46]. HO, GfcO. GfcO

449

Javanicin (101) an unusual quassinoid with seco ring A had its structure and relative configuration established unequivocally by single crystal X-ray analysis [47]. Javanicolides B (102) [20], C (103), and D (104) [48] isolated from the seeds of Brucea javanica showed weak cytotoxicity against P-388 murine leukemia cells with an IC50 values of 8, 10 and 18 Hg/mL, respectively, whereas javanicosides A (105), B (106), C (107), D (108), E (109), and F (110) had no activity [20, 48]. OAc

OH

HO,

I H

H 103

HO_

OH J.R .COjCH,

YY OH f O

HO. OH

HO.. J.R

rr

GkO.

,CO2CH3

GfcO,

107

106 OH

I H

H

1.4.3.6 Quassinoids from Castela peninsularis The structure of a new bitter-tasting quassinoid, named peninsularinone (111) isolated from the roots of Castela peninsularis

450

were determined by NMR spectroscopic and single X-ray analysis [49].

9H

Ill

1.4.3.7 Quassinoids from Castela texana A new quassinoid, ll-0-/ra«s-p-coumaroyl amarolide (112) isolated from Castela texana and the structure was elucidated by spectroscopic analysis. Compound (112) is the first coumaroyl quassinoid derivative to be isolated from nature [50]. Testing in the antimalarial bioassay showed that (112) possessed moderate antimalarial activity without potent cytotoxicity.

o o

112

1.4.3.8 Quassinoids from Castela tortuosa Castela tortuosa Liemb, a medicinal plant known as "chaparro amargo" in Mexico, was administered by the ancient Mexican people to treat liver diseases and is currently used to heal stomach aches and spasmodic pain [51], Quassinoids, castelalin (113) [52] and chaparramarin (114) [51] have been isolated and identified from the bark of C tortuosa.

451

Chaparramarin (113) exhibited moderate insect inhibitory activity against the lepidopteran pest insect, Heliothis virescem (tobacco budworm) £51]. Three quassinoids glycosides, casteloside A (115), casteloside B (116), and casteloside C (117) were also from the bark of Castela tortuosa [53-54]. OH

116. R= OH

1.4.3,9 Quassinoidsfrom Castela polyandra The structures of six new quassinoids, l-e/w-holacanthone (118), 15O-acetyl-glaucarubol (119), 15-O-aeetyl-A4>5-glaucarubol(120), l-epi5-wo-glaucarubolone (121), 1-e/w-glaucarubolone (122), and A4'5glaucarubol (123) all isolated from the twigs and thorns of Castela polyandra, were established by a combination of spectroscopic and single-crystal X-ray analysis [18].

452

1.4.3.10 Quassinoids from Eurycoma harmandiana Eurycoma harmandiana Pierre is a small Simaroubaceae plant (Thai name: Ian-don) distributed in the border regions between Thailand and Laos. Three new unusual 15a-0H quassinoids named iandonosides A (124), B (125), and iandonone (126) were isolated from the roots of Eurycoma harmandiana [55]. OH

^,OH

126

1.4.3.11 Quassinoids from Eurycoma longifolia Jack. Eurycoma longifolia Jack is one of the most well known folk medicines for intermittent fever (malaria) in Southeast Asia [11]. This plant possesses the largest number quassinoids with C19 skeleton identified so far. Seven novel highly oxygenated quassinoids were isolated from the leaves of Eurycoma longifolia, 13a(21)-epoxyeurycomanone (127), 15-acetyl-13oc(21)-epoxyeurycomanone (128), 12,15-diacetyl13a(21)-epoxyeurycomanone (129), 12-acetyl-13,21dihydroeurycomanone (130), 153-acetyl-14-hydroxyklaineanone (131), 6a-acetoxy-14, 15f3-dihydroxyklaineanone (132), 6a-acetoxy, 15(3-hydroxyklaineanone (133) [12]. The quassinoids (127-133) showed moderate antileukemic activity against P-388 cell lines, with IC50 14.0, 6.6, 7.2, 0.94, 7.8, 12.0, and 15.0 (ig/mL, respectively. The new 12-ep;-ll-dehydroklaineanone (134) isolated from the leaves Eurycoma longifolia showed moderate activity (plant growth inhibitor) against cucumber seedling [56]. Two highly oxygenated quassinoids, (135) and 13(3,18dihydroeurycomanol (136) were also isolated from the roots of Eurycoma longifolia [57-58].

453

Eurycomanol-2-O-P-D-glycopyranoside (137) isolated from the «-butanol extract roots of E. longifolia showed moderate activity against Plasmodium falciparum with IC50 of 1.590±0.169 |ig/mL, less potent than chloroquine (225) and quinine (222) [59]. OAo

OAo

OR2 O.

127. R,=R2= H 128. R,= H; R2= Ac 129.R1=R2=Ao OH OH

OAc 132. R= OH 133. R= H

1.4.3.12 Quassinoids from Hannoa chlorantha Hannoa chlorantha Planch, is a shrub used in Angolese traditional medicine, and the last two decades only a quassinoid was isolated of Hannoa chlorantha, 14-hydroxychaparrinone (138) [60]. HO,

454

1.4.3.13 Quassinoids from Harmoa klaineana Hannoa klaineana Pirre et Engler decoctions are used in African traditional medicine against fever and intestinal diseases [61]. Two quassinoids glycosides 15-0-P-D-glycopyranosyl-21hydroxyglaucarubolone (139) and 15-0-a-D-xyloruranosyl(l-»6)-pD-glycopyranosyl-21-hydroxyglaucarabolne (140) were isolated from the roots of Hannoa klaineana [62].

1.4.3.14 Quassinoids from Picrasma ailanthoides Planchon Quassinoids of the Japanese Picrasma ailanthoides Planchon (= P. quassioides Bennett) have been investigated in detail and more than twenty quassinoids have been obtained until 1984. However, very few reports for quassinoid glycosides have been found [63]. Eight quassinoids glycosides, picrasinoside A (141), B (142), C (143), D (144), E (145), F (146), G (147), H (148), and quassinoids hemiacetals, picrasinol A (149), B (ISO), C (1S1), and D (152) were isolated from Picrasma ailanthoides the last two decades [63-66].

455 OMe

OMe

GlcO,

MeO, OGk

MeO, ""OH

MeO,

I H 151

152

OH

150

1.4.3.15 Quassinoids from Picrasma crenata Picrasma crenata (Veil.) Engler is a Brazilian tree, which is used in traditional medicine to treat Diabetes mellitus, gastric disturbance and hypertension [67]. Three quassinoids quassin type, Pdihydronorneoquassin (153), 16-P-O-methylneoquassin (154), and 16p-0-ethylneoquassin (155) were isolated from Picrasma crenata [6768].

456 OMe

OMe HO,

MeO,

MeO,

MeO, OH

OMe

. i \ H 155

154

153

1.4.3.16 Quassinoids from Picrasma javanica Picrasma javanica is a medium-sized tree found in New Guinea, Southeast Asia and Indonesia. Decoctions of its bark are used in folk medicine as a febrifuge and as substitute for quinine [69]. OMe

OMe HO,, MeO,

MeO,

157. H l«0. H 164. H 166. OH 170. H

H OH H H H

H aOMe H O H aOH H O OH O

159. Me 162. Ac 163. PhCO 167. H 169. Ac

MeO. OMe

OMe

HO, MeO,

MeO, OMe

O MeO,

172. H Ac OH O 177. OH Me H O 179. OH Me H aOH

OMe HO,

175. R!= Me R2= H 176. R,= H R2= OH

MeO,

R2 Me Me Ac OH Me H Me H Me H

457

Twenty-four new picrasane quassinoids have been isolated from the leaves, roots and bark of Picrasma javanica, and they were named of javanicin B (156) [69], E (157), F (158), G (159) [70], H (160), I (161), J (162) [71], K (163), L (164), O (165), R (166), S (167), T (168) [72], N (169), P (170), Q (171) [73], U (172), V (173), W (174), X (175), Y (176) [74], Z (177), dihydrojavanicin Z (178), and hemiacetaljavanicin Z (179) [75]. Quassinoids glycosides has also been isolated of Picrasma javanica, and were denominated of javanicinoside B (180), C (181) [69], D (182), F (183), G (184), H (185) [76], I (186), J (187), K (188), L (189), and A (190) [77].

MeO,

"OGfc

180. Ac 183. Ac 184. Me 185. H

Ac Me Me Ac

OH H H OH

MeO,

187. Me OAo 188. H OAo 189. Me OH 190. H OMe

OH OH OH H

J. 4.3.17 Quassinoids from Quassia amara Quassia amara Wood is still widely used in traditional medicine and some quassinoids and quassinoid glycosides isolated from Quassia have received renewed attention due to their biological activity as potential antitumor agents [78].

458

HO

MeO. OMe

MeO.

= H 194

Six new quassinoids were isolated from Quassia amara Wood, dihydronoraeoquassin (191) [79], ll-a-0-(P-D-glycopyranosyl)-16a-O-methylneoquassin (192), l-a-0-methylquassin (193), 12-cthydroxy-13,18-dehydroparain (194), and 16-a-O-methylneoquassin (195), and 11-acetylparain (196) [78]. 1.4.3.18 Quassinoids from Quassia indica Indaquassin C (197), D (198), and E (199) were isolated from bark of Quassia indica. Their structures were determined by spectroscopic and chemical evidence [80].

OH

1.4.3.19 Quassinoids from Simaba multiflora

459

Two new quassinoids, 13,18-dehydro-6a-senecioyloxychaparrin (200) and 12-dehydro-6o>senecioyloxychaparrin (201), have been isolated from the fruits of Simaba multiflora [81]. HO, HO,.

200

1.4.3.20 Quassinoids from Simaba guianensis Simaba guianensis is a small tree that occurs in the flooded areas of the Amazon basin and is locally known as "cajurana". Its red fruits that ripen during the time of flooding are highly appreciated by fish and its bark is very bitter and is used by native populations against fevers [82], The new antimalarial (IC50 3.9-4.1 ng/mL) quassinoid named gutolactone (202) was isolated from the bark of Simaba guianensis [82]. OH I O .,

1.4.3.21 Quassinoids from Simaba orinocencis on

460

Simaba orinocencis Hunth (= S. multiflora) is a native tree found in the Amazonian riversides and seasonally inundated areas of South America [83]. A new antimalarial quassinoid, namely, orinocinolide (203), was isolated from the root bark of Simaba orinocencis [83]. The orinocinolide (203) was potent against Plasmodium falciparum clones D6 and W2 (IC5o 3.27 and 8.83 ng/mL). Orinocinolide (203) also inhibited growth of human cancer cells SKMEL, KB, BT-549, and SK-OV-3 [83], 1.4.3.22 Quassinoids from Soulamea amara Soulamea amara Lam., is a Simaroubaceae indigenous to Vanuatu (New-Hebrides). The new quassinoid 15-0-benzoylbrucein (204) was isolated from the aerial parts, and the structure has been established from spectral data and by single-crystal X-ray [84].

1.4.3.23 Quassinoids from Soulamea fraxinifolia Soulamea raxinifolia is a small tree New Caledonia that occurs in forest gallery of low altitude. It is characterized by the composed, barefaced leaves.

205

The studied sample comes from the region of Dumbea and the present survey has been led on peels of stems and leaves [85], From

461

the stem bark and leaves, a new quassinoid 6a-acetoxypicrasine B (205) was isolated [85]. 1.4.4 C22 Quassinoids Until 1985, only two quassinoids possessing the C22 basic skeleton were known: sergeolide (206) [86] and 15-deacetylsergeolide (207) [87] isolated from the roots of Picrolemma pseudocoffea. Sergeolide (206) is the first quassinoid to possess a butenolide function [1]. Two decades passed and only a new quassinoid with C22 skeleton was isolated so far. The new quassinoid (208) contained the y-lactone function bonded to ring A, in angular fashion, instead of linear for sergeolide (206) [88-89]. OH

O

o 208

1.4.5 C25 Quassinoids Until 1985, only six quassinoids possessing the C25 basic skeleton were known: simarolide (209) from the bark of Simarouba amara, picrasin A (210) isolated from Picrasma quassioides, soulameolide (211) from Soulamea tomentosa, simarinolide (212) and guanepolide (213) from the root bark of the Simaba cf. orinocencis, and deacetylsimarolide (214) isolated from the fruits of Simaba moretii [1].

462

Since then, seven more C25 quassinoids have been discovered: Odyendane (215) and odyendene (216) from the Odyendea gabonensis [90], klaineanolide A (217) and B (218) from the Hannoa klaineana [61], indaquassin F (219) from the Quassia indica [80], javanicinoside E (220) isolated from the Picrasma javanica [76] and the new epimer from simarolide (221) [91].

MeO,

R,0

H 209. Rj= H; R2= Ac; R3= pH 210. R!= Me; R2= Ac; R3= 0H; 2,3-dehydro 214. R!=R2= H; R = PH 221. Ri= H; R2= Ac; R3= ocH

212

213

2. BIOLOGICAL ACTIVITY OF QUASSINOIDS 2.1 Antimalarial Activity One estimates that throughout the world more than 300 million clinical cases of malaria occur every year, and over 1 million people

463

die of malaria [92], The vast majority of deaths occurs among young children in Africa, especially in remote rural areas with poor access to health services. In malaria-endemic regions and countries this disease is not only a health problem, but it also causes delay in economic development [92]. Malaria is a disease caused by Plasmodium protozoa and transmitted by of mosquito vectors (Anopheles spp.), bites [93] to men, monkeys, rodents, birds, and reptiles [94]. The main agents involved in human malaria fever are four species of Plasmodium protozoa (single-celled parasites): P. fakiparum, P. vivax, P. ovale, and P, malarie. Among these, P. falciparum accounts for the majority of infections, and it is the most lethal one. The control of disease is, among other factors, hampered by the ongoing spread of multidrug-resistant of Plasmodium falciparum [95] and the resistance of vector (Anopheles spp.) to insecticides [96]. Although malaria is a curable disorder if promptly diagnosed and adequately treated, a limited number of drugs for its treatment are available today [97]. In fact, the two most effective drags for malaria, quinine (222) and artemisinin (223), originate from plants; it is probable that other plants still contain undiscovered antimalarial substances. Many researches have focussed on trying to isolate and purify antimalarials from plants. The first studies about the antimalarial activity of Simaroubaceae's extracts date back to 1947 [98]. Cedronin (224) was isolated from Simaha cedron, a species popularly believed in South America to have antimalarial properties. It was examined for in vitro and in vivo antimalarial activities and for cytotoxicity against KB cells. Experimental results showed that cedronin was active against chloroquine-sensitive and resistant strain, with an ICso of 0.25 u,g/mL (0.65 ujnol/mL). It was also found to be active in vivo against P. vinkei with an IC50 of 1.8 mg/Kg (4.7 nM/Kg) in the classic 4-day test. Cedronin (224) show a rather low cytotoxicity against KB cells (IC50- 4 jig/mL, 10.4 uM); however its toxic/therapeutic ratio (10/1.8) remains lower than chloroquine (225) (10/0.5) [99].

464

222

225

223

Hannoa chlorantha and Hannoa klaineana are used in traditional medicine of Central African countries against fevers and malaria. Four stem bark extracts from H. klaineana and for quassinoids from H. chlorantha were examined in vitro against P. falciparum NF 54 [100]. OH

O H I H Otiglate 226.Ri=R 2 =R 3 =O 227. Rt= O-tiglale; R2= H; R3= OH 22&R,=R 3 =H;R 2 -OH

O

229

The extracts displayed good activities, while the quassinoids were highly active, with IC50 values very below 1 (xg/mL, those chaparrinone (226) and 15-desacetylundulatone (227) being much lower than 0,1 |ig/mL (0.037 and 0.047 M-g/mL, respectively). Chaparrinone (226) is five times more active than 14hydroxychaparrinone (228) against P. falciparum, indicating that the hydroxyl function at C-14 is unfavourable for antiplasmodial activity [100]. As 14-hydroxychaparrinone (228) has a seven-time higher cytotoxic activity against P-388 cells than chaparrinone (226), the latter compound has the better antiplasmodial therapeutic index. All four quassinoids were also evaluated in vivo in a standard 4-day test. 15-desacetylundulatone (227) was proven to be the most active compound, almost totally suppressing the parasitaemias of OF1 mice for at least 7 days, while both chaparrinnone (226) and 14hydroxychaparrinone (228) were active for at least 4 days.

465

Quassinoids have ED50 values much lower than 50 mg/Kg body weight/day and none of them caused obvious side effects. The keto function at C-2 in 15-desacetylundulatone (227) is apparently of crucial importance for its high activity. 6-a-Tigloyloxyglaucarubol (229) was not active at all. Chaparrinone (226) is considered the most interesting of the investigated quassinoids and its in vivo antimalarial potential will be examined further [100]. In recent literature [101] the synthetic compound 3,15-di-Oacetylbruceolide (230), a derivative from bruceoside showed a potent in vitro antimalarial activity against Plasmodium falciparum equivalent to that of chloroquine (225) [102] and also a potent in vivo activity against Plasmodium berghei in mice, with low cytotoxicity against mouse mammary tumor, representing a model in the same host.

OCOCH,

o

o 230

The results obtained showed that 0.46 + 0.06 mg/Kg per day of 3,15-di-O-acetylbruceolide (230) caused 50% suppression of P. berghei in mice, while the ED50 values of chloroquine (225) and artemisinin (223) were 0.2 and 5.6 mg/kg per day [101]. 2.2 Anti-EW Activity In order to combat the Human immunodeficiency virus (HIV), the causative agent of the debilitating disease acquired immune deficiency syndrome (AIDS), colossal amount of money, manpower time and energy have been dedicated to research on compounds which can be developed as therapeutic agents. Research groups have devoted their efforts to hunt for compounds in different plant species, which inhibit HIV replication

466 466

and/or activities of HIV enzymes. A number of terpenoids and their derivatives, such as quassinoids have been reported to be anti-HIV agents [103]. Eighteen quassinoids glycosides and nine known quassinoids isolated from Brucea javanica, Brucea antidysenterica and Ailanthus altissima were tested for inhibitory activity against HIV replication in H9 lymphocytic cells [103]. Bruceoside-B (231), as well as yadanzioside-B (77) and -L (96), showed good EC50 values of 3,5, and 5 uM, respectively. However, their therapeutic indexes (TI) of 1, 0.8, and 2, respectively, indicate that they are toxic as anti-HIV agents. Likewise, quassinoid shinjulactone A (232) had a good EC50 value of ca. 5 uM but was cytotoxic at this concentration. Shinjulactone B (19) and ailantinol A (43) showed no cytotoxicity and were marginally active with EC50 values of 28 and 30 [oM, respectively. The most promising compound was shinjulactone C (233); this compound had a TI of >25 and showed significant anti-HIV activity with an EC50 value of 10.6 uM. Further studies on analogs and related compounds to increase the pharmacological profiles of (233) are in progress [103]. OH

OH

OH

GlcO

2.3 Anticancer Activity

O

HO

234

467 467

Since the isolation and characterization of braceantin (234) by Kupchan and associates over 30 years ago [104], several quassinoids have been tested against several types of tumors [1]. Several quassinoids isolated from Brucea antidysenterica in the series of antitumor agents of the National Cancer Institute, including: Bruceanol-A (64) and Bruceanol-B (65) [35] showed significant (T/C >120%) antileukemic activity in vivo against P-388 lymphocytic leukemia (3-day dosing) at T/C= 130% (0.5 mg/Kg), 129% (1 mg/Kg) and 134% (2 mg/Kg), and 123% (0.5 mg/Kg), respectively. The control, 5-fluorouracil, had T/C= 135% (200 mg/Kg, 1-day dosing). Braceantinoside C (72), Yadanzioside G (81) and Yadanzioside N (87) showed significant (EDso< 4,0 ug/mL) cytotoxicity in vitro against P-388 and L-1210 lymphocytic leukemia tissue culture cells at ED50= 2.12 and 3.50, 1.25 and 2.58, and 4.56 ug/mL, respectively [39]. The control drag, 5-fluorouracil, used in this assay showed EDso= 3.72 and 1.94 ug/mL, respectively. Bruceanol C (66) demonstrated potent cytotoxicity against human KB, A-549 lung carcinoma, and HCT-8 colon tumor as well as murine P-388 lymphocytic leukemia with ED50 values of < 0.04, 0.48, AK^J.,,

X 80-90% A K ^ . O

142

OH 80-90%

'

O

143

Ar = piperonyl, veratryl or 4-metoxyphenyl

Ar

145

de = 85:15-87:13

a) LDA, THF, TiCI4, -78°C-rt, 24h; b) THF, LiOH, H2O2, rt 12-24 h, c) i. AC2O, MeOH -78°C ii. NaBH4, 1 h; d) LiAIH4, THF, 12 h

Ar Ar

45-63% 148

Ar = piperonyl, veratryl or 4-metoxyphenyl

Y

X b Ar X 70-80%

o 149

Y

OH

as above

o 144

de = 88:12-92:8 a) LDA, hexane/THF, CuCI 2 , DMPU, -78°C-rt, 12 h; b) LiOH, THF/H 2 O 2 , reflux 2 4 ^ 8 h.

Scheme (26). Asymmetric synthesis of dibenzylbutyrolactones and dibenzylbutandiols by Kise et al

572

Gordon and co-workers used iV-4-pentenoyloxazolidinone 150 as chiral starting material in the asymmetric synthesis of (-)-sesaminone (159), Scheme (27) [90]. 9. o

0

O

OH

-OH

159 a) Bu2BOTf, CH2CI2, 0°C, Et3N, 0 to -78°C, piperonal; b) LiBH4, H2O, THF, 0°C; c) TBDPSCI, imidazole, THF; d) MOMCI, Et3N, CH2CI2; e) OsO4, NMO, (-BuOH, H2O, THF; f) NalO4, H2O, THF, 0°C; g) 5-lithio-1,3-benzodioxole, THF, -78°C to 0"C; h) MnO2, CH2CI2; i) TBDMSOTf, Et3N, CH2CI2; I) TiCI4, CH2CI2, -78°C; m) 48% HF, C5H5N, MeCN, 0°C.

Scheme (27). Asymmetric synthesis of (-)-sesaminone by Gordon et al.

Asymmetric aldol reaction according to the Evans procedure between 150 and piperonal produced aldol adduct 151 with excellent

573

diastereoselectivity (19:1) which could be further increased via purification to give the product free of isomers in 82% yield. Reduction of the aldol adduct 151 with lithium borohydride in the presence of water afforded diol 152 again in 82% yield. Both alcohols of diol 152 were consecutively protected first with TBDPSC1 and then with M0MC1 to give the diprotected 153. At this point, dihydroxylation of the double bond and subsequent cleveage of the diol 153 provided aldehyde 154. 1,2Addition of 5-lithio-l,3-benzodioxole to 154 proceeded smoothly to give after oxidation of the secondary alcohol the aryl ketone 155 in 76% yield. Aryl ketone 155 was first transformed into a mixture of silyl enol ethers 156 (7:1) and then cyclized to tetrahydrofuran 157 using titanium tetrachloride. Tetrahydrofuran 157 was produced in good yield together with a small amount of a-tetralone 158. Finally, deprotection of 157 with fluoric acid afforded (-)-sesaminone 159 in 84% yield. Yamauchi et al. produced the chiral monoprotected diol 161 analogous to the procedure of Gordon in 4 steps and 56% overall yield, Scheme (28) [91]. Dihydroxylation of the double bond with osmium tetroxide followed by cleavage of the obtained diol and oxidation of the transient lactol with silver carbonate-celite gave lactone 162 in excellent yield. Reaction with methyl chloro formate proceeded stereoselectively to afford 163 as single isomer. Treatment of 163 with lithium aluminium hydride gave the desired triol 165 in 63% yield together with the corresponding hemiacetal 164 (22%). The hemiacetal 164 was converted into 165 via sodium borohydride reduction. The total yield of triol from lactone 163 was 76 %. Cyclization of the triol to the tetrahydrofuran ring-system was achieved by an intramolecular SNI reaction using 10-camphoric acid as catalyst. The desired 166 was obtained as a mixture of diastereoisomers in a ratio of 1:1. Pyridinium chlorochromate oxidation furnished aldehyde 167 which partially epimerized resulting in a 2:3 ratio. Stereoselective ochydroxylation of 167 was performed with triisopropylsilyltrifluoromethane in the presence of DBU and DMAP. The unstable silyl enol ether was oxidated using osmium tetroxide after which the crude product was treated with A^^'-dimethylethylenediamine and finally chromatographed successively on silica gel. The resulting unstable ahydroxyaldehyde 168 was immediately reacted with tetrabutylammonium fluoride to cleave the silyl group affording ( l S ^ S ^ (169) as a single isomer in 78% yield.

574

OH .0

TBDPSO

U ^ 0 > 92%

r

Bn

a.b.c

v

TBDPSO 162

161

160

A ii

47%

HO, HO HO—

OTBDPS

HO—-

X

-OTBDPS

H3CO2C—-

22% 164

63% 165

^—OTBDPS 163

54% 83%

\ HO — ^

/ ^-OTBDMS 166 1:1

CX 0 >

h 72%

0=^N-

i,k 71%

HO 0 = =*

^-OTBDMS sole isomer

OTBDMS

167 2:3

I /

/ 78%

168

^O

) 0

HO")— 10:1 a) THF, n-BuLi, CICOAr, -78°C, 3 h; b) Mg, ArBr, CuBr(CH3)2S, THF/(CH3)2S (3:1), -48 to 0°C. 3 h; c) NaHMDS, (-BuO2CCH2l, THF, -78°C to -48°C, 6 h; d) LiAIH4, THF, 20 h; e) i. LiOH/H2O2, THF, 0°C, 5h; ii. BH 3 , THF -10°C, 14 h iii. PTSA, benzene, reflux, 2 h; f) NaHMDS, THF, Mel, -78°C, 3 h.

Scheme (29). Asymmetric synthesis of peperomins by Sibi et al.

576

In a similar way, compound 176 was used as starting material in the synthesis of several lignans, Schemes (30) and (31) [93]. A radical addition was used to introduce the remote aryl group: 176 was treated with 3-methoxybenzyl bromide in presence of an equimolar amount of samarium triflate as lewis acid and BU3S11H and Et3B as radical promoters. Only one diastereomer was detected in the reaction. However, a side product was formed due to the addition of the ethyl group to the double bond which could be removed after purification to give 177 in very good yield. Treatment of 177 with NaHMDS and quenching the resulting anion with 3-methoxybenzyl iodide afforded only the syn product 178. Removal of the chiral auxiliary was then performed utilizing LiOH and H2O2. Reduction of the acid moiety with borane and subsequent cyclization gave the desired dibenzobutyrolactone ring system 180. Finally, treatment with boron tribromide provided (-)-enterolactone 21.

H3CO

COOEt

21

180

a) SmiOTfh, 3-methoxybsnzyl bromide, CHzCI/rHF (4:1), Bu 3 SnH, EtaB/Oj, -78°C; b) NaHMDS, THF, S-MeOCjH^CHal, -78"C to -54"C; 0) LiOH, H2O2; d) BH3/THF, -15"C, 18 h; e) PTSA, benzene, reflux 4 h; f) BBr 3 , CH 2 CI Z , 0'C

Scheme (30). Asymmetric synthesis of (-)-enterolactone by Sibi et al.

577

Using the same approach, the easily accessible lactones 175 and 176 were converted in good chemical yields into (-)-isoaretigenin 177 and (-)arctigenin 178 after removal of protecting groups via hydrogenolysis, Scheme (31). O

0 COOEt

1

V" Ph 176

0

II

0

0 Ph 176

f) H2, Pd/C, EtOAc, AcOH, 1.5 h. Scheme (31). Asymmetric synthesis of (-)-isoarctigenin and (-)-arctigenin by Sibi et al.

Asymmetric cycloaddition reactions Asymmetric Diels-Alder reactions have found widespread use in the total synthesis of complex natural products. However, only few examples have been reported on their application towards the asymmetric synthesis of lignans. Charlton et al. used an asymmetric Diels-Alder reaction as a key step in the total synthesis of (—)-a-dimethylretrodendrin (193), Scheme (32), [94]. The aldehyde 185 was prepared in three steps and in good overall yield starting from 3,4-dimethoxybenzaldehyde. Irradiation of 185 in a benzene solution containing dissolved an excess of SO2 afforded the key intermediate 186. Thermolysis of 186 produced the diene 187 which could be trapped with chiral fumarate 188 yielding a mixture of diastereomers in a ratio of 9:1. After purification, the cycloadduct 189 was isolated in 44% yield. Lactonization of the secondary hydroxy group with y-ester followed by transesteriflcation and opening of the lactone were achieved in an one-pot fashion utilizing sodium methoxide in

578

methanol to give acid ester 191 in excellent yield. Catalytic hydrogenation followed by reduction of the ester group and refluxing the crude product in a benzene/p-toluenesulfonic acid mixture afforded the lactone 192 as sole product in 72% yield. Finally, epimerization of the C2 carbon gave optically pure (-)-a-dimethylretrodendrin (193). CO2R RO ,C

CO2Me "Ph

188

MeO. MeO

OMe OMe 193 a) benzene, pyridine, SO2, hv, 6 h, rt; b) 188, ZnO, toluene, reflux,1 h; c) NaOMe/MeOH, rt, 24 h; d) H2, Pd/C, EtOAc/AcOH, rt, 24 h; e) THF, LiEt3BH, rt 19 h; f) benzene, p-TsOH, reflux, 14 h; g) (-BuONa/f-BuOH, reflux 24 h.

Scheme (32). Asymmetric synthesis of (-)-a-dimethylretrodendrin by Charlton

579

hi addition, a modified version of this methodology was applied some years later to the synthesis of (-)-deoxypodophyllotoxin (202) [95] as well as other aryltetralines [96]. Ketone 194 was synthesized in 9 steps starting from piperonal, Scheme (33).

OCH 3

H3C0 194

197 58%

H3CO"

OCH 3

H3C0

y

0CH3

OCH3

H3CO

OCH3 199

^CH20H '.,., 'CO2H

H3CO

g 30 % overall from 192

198

Q. v. 0

0GH H3CCf ^ f 3 OCH3 202

a) n-BuLi, THF, -78°C; b) toluene, reflux, 196, 44 h; c) BF3Et2O, CH2CI2, -20°C then LiAIH4, -55°C-rt; d) Pd/C, H 2 , MeOH/AcOH, rt, 89 h; e) (CF3CO)2O, reflux, 2h; f) NaBHj, /-PrOH, 15 h, rt; g) benzene, p-TsOH, reflux, 17.5 h.

Scheme (33). Asymmetric synthesis of (-)-deoxypodophyllotoxin by Charlton et at

Treatment of 194 with n-BuLi afforded the benzocyclobutenol 195 in 71% yield. Thermolysis of 195 in refluxing toluene and treatment with methyl (5)-mandelate (196) gave a mixture of cycloadducts via the ahydroxy-a-aryl-o-quinodimethane 197 intermediate. The major

580

cycloadduct 198 was isolated in 58% yield. Reduction of the hydroxy group was acieved using BF3'Et2O followed by LiAlHj treatment. A mixture of three isomers were obtained in a ratio of 15:1:2. Isolation of the major isomer 199 was very difficult due to the similar polarity of the three products. Therefore, the crude was used directly in the next steps without purification. The mandelate group was cleaved by catalytic hydrogenolysis after which refluxing the crude product in trifluoroacetic anhydride gave a product, presumed to be the anhydride 200. Reduction of 200 with NaBH4 resulted in a mixture of y-hydroxy acids in a ratio of 3.3:1 providing the desired y-hydroxy acid 201 as the major product. Finally, lactonization via /?-toluenesulphonic acid concluded the total synthesis of (-)-deoxypodophyllotoxin (202) in 30% overall yield starting from 198. Pelter et al. used (51?)-menmyloxy-2(5/^-furan°ne (100) as chiral dienophile in the asymmetric synthesis of (—)-isopodophyllotoxin (209), Scheme (34) [97]. Treatment of starting material 203 with the chiral dienophile 100 in refluxing toluene gave, via the arylisobenzofuran 204 generated in situ, a complex mixture of 5 products. After purification by silica gel chromatography the major product 205 could be isolated in 37.5% chemical yield. Raney-nickel reduction of the cycloadduct 205 afforded isopicropodophyllotoxin (206), along with a small amount of the C-10 epimer. After cleavage of the menthyloxy group by using a mixture of sodium borohydride and potassium hydroxide in EtOH, products 207 and 208 were obtained in 52% and 44% yields, respectively. Finally, ZnCl2 mediated lactonization of 207 completed the asymmetric synthesis of (-)-isopodophyllotoxin (209),

581

37.5%

MeO

y OMe OMe 205

OMenth

OMe

MeO

y

OMe

OMe 206 + epimer

MeO

y OMe OMe

209 a) toluene, 100, reflux, 9 h; b) Ra-Ni, EtOAc, H2, 60 psi, rt, 15 h; c) NaBH4, EtOH, 20 h; rt; d) THF, ZnCI2, MS 4 A, reflux, 6 h.

Scheme (34). Asymmetric synthesis of (-)-isopodophyllotoxin by Pelter et al.

Jones and co-workers [98] concluded a very efficient synthesis of (-)podophyllotoxin (1) based on an asymmetric Diels-Alder addition to 1aryl-2-benzopyran-3-one [99]. The o-quinonoid pyrone 210 reacted smoothly when (5i?)-menthyloxy-2(5//)-furanone (100) was used as dienophile, Scheme (35). The cycloaddition proceeded with high facial selectivity as well as very high regioselectivity affording 211 as sole isomer in 79% chemical yield. The cycloadduct 211 underwent ring opening with acetic acid to give the acid 212 in 87% yield. Hydrogenation followed by oxidation with lead tetraacetate converted 212 into the

582

acetate 213. Hydrolysis of 213 under carefully controlled acidic conditions gave two epimeric lactols 214 and 215 in a ratio of 1:1 which were separable via chromatography. Brief treatment of the individual lactols with diazomethane followed by facile reduction with LiEtaBH furnished the two methyl podophyllates 216 and 217. The former was readily converted into methyl podophyllate 217 by treatment with hydrochloric acid. Final lactonization using ZnCb completed the total synthesis of (-)-podophyllotoxin (1) in 15% overall starting from 210.

9°2 H p-menth

CH 3 O'

64% a) MeCN, 100, 50°C, 29 b) HOAc, 49°C, 13 h; c) EtOAc, Pd/C, H2, 40 h; d) HOAc, THF, rt, Pb(OAc)4, 3 h; e) dioxane/HCI (3:1). 41°C; f) CH 2 N 2 , Et2O/MeOH (24:1), 0°C; g) THF, LiEt3BH, -78°C, 1h; h)THF/HCI (1:1.5), rt, 3.5 h i) THF, ZnCI2, molecular sieves, reflux, 2.5 h.

Scheme (35). Asymmetric synthesis of (—)-podophyllotoxm by Jones and co-workers

583

Miscellaneous Wirth and co-workers reported on the application of the easily accessible chiral diselenide 218 to the asymmetric synthesis of furofuran lignans, Scheme (36) [100]. Cleaveage of the diselenide group in situ with bromine followed by treatment with silver triflate afforded the electrophilic selenium species 219. Allowing 219 to react with alkene 220 for 15 min followed by addition of 2,3-butadien-l-ol (221) gave 222 in 56% chemical yield and excellent selectivity (16:1). Compound 222 was then subjected to radical cyclization with triphenyltin hydride in the presence of AIBN to give tetrahydrofuran 223 as a mixture of two diastereoisomers in a ratio of 1:1 and 64% yield. Dihydroxylation of the double bond and subsequent oxidation gave the two corresponding aldehydes 224 and 225. Finally, after removal of the protecting group with tetrabutylammonium fluoride, cyclization of the mixture containing both isomers to the hemiacetal occurred spontaneously affording (+)samin (226) in 67% yield.

Et '''OH Se) 2 218

226

a ) i . Br2, Et 2 O,-78°C, 15 min ii. AgOTf iii.-100°C, 220 15 min iv. 221, 3 h; b) Ph 3 SnH, AIBN, toluene, 90°C, 1 h; c) N-morpholine-N-oxide, acetone, t-BuOH, OsO 4 , H 2 O, 4 h; d) NalO 4 , THF, 8 h; e) n-Bu 4 NF, THF, 0°C, 4 h.

Scheme (36). Asymmetric synthesis of (+)-samin by Wirth and co-workers

584

In addition, the strategy was extended to the asymmetric synthesis of diaryl furofurans, Scheme (37) [101]. Compound 227 was synthetized using the same route as above in 23% overall yield. Oxidation of the vinylic double bond with osmium tetroxide and subsequent treatment with periodic acid afforded directly furofuran 228. It is noteworthy that cleavage of the diol to give the aldehyde, removal of the protecting group, isomerization at C-4 and cyclization could be obtained in one pot, albeit in moderate yield. Finally, Grignard addition gave the furofuran lignan (+)-mesembrine (229) in 45% yield. OMe TBDMSO

a

J

-'OH

SeOTf

219

^ 2 3 %

M e O

OMe

227

228

229

a) OsO 4 , N-morpholine-N-Oxide, acetone, f-BuOH, H2O, 10 h; b) H5IO6, THF, H2O, rt, 13 h; c) 4-methoxyphenyl magnesium bromide, THF, 70°C, 3h.

Scheme (37). Asymmetric synthesis of (+)-mesembrine by Wirth

Yamauchi et al. completed an ex-chiral pool synthesis of a samin-type lignan, Scheme (38) [102]. The diprotected tetraol 230 was obtained from Z-glutammic acid by a 15 step procedure in 7-8% overall yield [103]. The diprotected tetraol 230 was treated with boron trifluoride diethyl etherate in dichloromethane to give the tetrahydrofuran 231 in 84%-87% yield. After deprotection with tetrabutylammonium fluoride, the resulting diol was oxidized by dihydridotetrakis(triphenylphospine)ruthenium(II) to provide two lactones 232 and 233 in a ratio of 2:1. Lactone 232 was transformed in samin-type lignan 234 by diisobutylaluminium hydride reduction in 70% yield.

585 MeO.

84-87% H " 4 — H H TBDPSO-"

^"OH

230

TBDPSO"^

231

234

a) BF 3 Et 2 0, CH2CI2, 0°C, 30 min,; b) n-Bu4MF, THF, rt, 1 h; c) RuH2(PPh3}4, acetone, toluene, reflux, 1.5 h; d) DIBAL, toluene, -75°C, 1 h. Scheme (38). Asymmetric synthesis of samin-type Hgnan by Yamauchi

Ohmizu and co-workers explored the use of (S)- and (i?)-3-(2,2-dimethyll,3-dioxolan-4yl)-cw-2 propenoate (235 and 236, respectively) as chiral building blocks in the asymmetric synthesis of lignans, Scheme (39) [104,105]. Michael addition of cyanohydrin 237 to the ester 235 proceeded smoothly in the presence of 2 equivalents of HMPA. The Michael adduct 238 was obtained in 94% chemical yield and very high diastereoselectivity (93%). The 1,4-adduct 238 was then converted into lactone 239 by treatment with sodium metaperiodate in methanol. Aldol reaction of 239 with veratraldehyde was carried out in THF at -78°C using LDA as base. After removal of the silyl group, the ketone 240 was recovered as sole product in 84% yield. Reduction of the ketone 240 with sodium borohydride afforded the diol 241 together with its C-4 stereoisomer in 86% and 8% yields, respectively. Diol 241 was further reduced with LiAlH4 to tetraol 242 and finally cyclized to (+)-fargesin (243). hi addition, synthon 244, accessible by the same procedure in 67% overall yield and 93% de, was used in the asymmetric synthesis of (-)picropodophyllone (247). Reduction of 244 with sodium borohydride in hot THF-methanol followed by selective oxidation of the resulting diol with Fetizon's reagent gave the y-lactone 245 in 67% yield. Aldol reaction of 245 with 3,4,5-trimethoxybenzaldehyde followed by intramolecular Friedel-Crafts like cyclization promoted by trifluoroacetic acid afforded the product 246. Without any purification, 246 was treated

586

with tetrabutylammonium fluoride completing the synthesis of (-)picropodophyllone (247) in 83% overall yield starting from adduct 245.

MeO2C

1 0

MeO2C

Xx

235

T XT 0

236 H

JPMe

243 a) LDA, THF, 2 eq. HMPA, 235, -100°C, 30 min.; b) NalO4, H2SO4, MeOH, 40°C, 24 h; c) LDA, THF, -78°C veratraldehyde, 30 min; d) TBAF, CH2CI2, AcOH, rt, 2 h; e) NaBH4, MeOH, 0°C, 30 min; f) LiAIH4, THF, 60°C, 1 h; g) pyridine, MsCI, 0°C, 6 h. TBDMSO CN

TBDMSO .CN

OMe

TBDMSO CN

67%

>/

A 244

245

\\ O

*OCH3 OCH3 246



'

U

H 278

98%

H

H A

H 279

_ u Ar^S^OAc c,d , r ^ ^ o 80% Ar^/j^OH 48% Ar^^W H Ho 280 281 ee > 98%

a) LiAIH4, CH2CI2:Et20 (3:2), rt; b) CAL, vinyl acetate, benzene, rt, 74%; c) PDC, DMF, rt; d) p-TsOH, HCI 5N, toluene reflux; e) Acetic anhydride, DMAP, pyridine, rt; f) CAL, EtOH, isopropyl etherbenzene (5:2) Scheme (43). Asymmetric synthesis of cw-dibenzylbutyrolactones by Chenevert et ah

Diester 275 was prepared according to the method of Ward [111]. Reduction of the diester with lithium aluminium hydride furnished the desired meso diol 276. Diol 276 was subjected to the enzymatic esterification reaction by treatment with Candida antartica lipase (CAL)

591 591

in benzene using vinyl acetate as acyl donor to give monoester 277 in 74% yield and with an enantiomeric excess greater than 98%. Oxidation of the primary alcohol group and subsequent acidic cyclization concluded the total synthesis of 278. To synthesize the enantiomeric form a modified procedure was used: diol 276 was acylated with acetic anhydride in pyridme in the presence of DMAP to give the meso-diacetate 279. At this point transesterification of 279 by utilizing ethanol and CAL gave 280 as sole isomer in 80% yield. The conversion into the natural product 281 was then achieved as described above. Berkowitz and co-workers disclosed a new synthesis of (-)podophyllotoxin via enzymatic desymmetrization of a meso diacetate, Scheme (44) [112,113]. The bromo acetal 282 was synthesized from piperonal in 80% yield by using a two step procedure. Hydroxymethylation of 282 proceeded smoothly using s-BuLi in the presence of solid paraformaldehyde affording 283 in 82% yield. Treatment of 283 with neat acetylenedicarboxylate under acid catalysis gave the cycloadduct 285 via isobenzofuran 284 in high yield. It is noteworthy that the reaction could be performed in 20 gram scale. Catalytic hydrogenation of 285 occurred exclusively from the less hindered exo-face providing after reduction with LiAlELt and acetylation the meso diacetate 286 in an excellent 87% yield. Desymmetrization of the meso diacetate 286 was conducted under carefully monitored conditions by treating 286 with porcine pancreatic lipase (PPL) in DMSO at pH 8. The reaction could be performed in several gram scale affording monoacetate 287 in 66% chemical yield and 95% enantiomeric excess. The chiral monoacetate 287 was then converted into the silyl-protected aldehyde through a three step procedure commencing with silylation of the primary alcohol followed by deacetylation and finally Swern oxidation to afford the product 288 in 97% yield. Treatment of 288 with base lead to a retro-Michael ring opening giving 289 in 90% yield. The secondary alcohol was protected as 2-(trimethylsilyl)ethoxymethylether and the aldehyde oxidized to acid 290. The acyl oxazolidinone functionality was then introduced by treatment of 290 with carbonyl diimidazole followed by reaction with 2-lithium-oxazolidinone. Compound 291 was recoverd in 60% yield. Michael addition on 291 using ArMgBr and CuCN cleanly produced the 1,4-adduct 292 as sole isomer. Desilylation of 292 with TBAF, followed by spontaneous lactonization provided lactone 293 in 62% yield. Finally, epimerization to

592

the termodinamically less stable trans-lactom under Kende's conditions [114], separation of the two isomers by chromatography and removal of the protecting group completed the total synthesis of (-)-podophyllotoxin

287

288

90% I

OH OTIPS

r

CHO

SEMO

SEMO

OTIPS

OTIPS

m,n 93%

289

290

291 85%

SEMO

H3CO

y

SEMO

OCH3

H3C0

OCH3 293

OTIPS

y 0CH3 0CH3 292

a) n-BuLi, THF, (CH2O)n; b) HOAc, DMAD, 80°C; c) H2, Pd/C; d) LiAIH4, Et2O, reflux; e) Ac2 O, pyr, DMAP, -5°C; f) PPL, 10% DMSO, 50mM KPO4, buffer, pH 8; g) TIPSCI, imidazole, DMF, rt; h) K2CO3, MeOH; i) (COCI)2, DMSO, CH2CI2, Et3N, -78°C; I) NaOMe, MeOH, rt; m) SEMCI, /-Pr2NEt, CH2CI2, rt; n) NaCIO2, NaH2PO4, (-BuOH, 2-methyl-2-butene; o) i. carbonyl diimidazole, THF ii. n-BuLi, oxazolidinone -78°C; p) 3,4,5-trimethoxyphenylmagnesium bromide, CuCN, THF, 10°C, 2.5 h; q) TBAF, THF, 50°C; r) i. LDA, -78°C ii. pyr-HCI; s) MgBr2, EtSH, Et2O-Benzene 4-1, 0°C to rt.

Scheme (44). Asymmetric synthesis of (-)-podophyllotoxin by Berkowitz and co-workers

Uemura et al. investigated the use of (arene)chromium complex in a formal total synthesis of (-)-steganone (7), Scheme (45) [115]. Treatment

593

of 294 with (-)-l,2,4-butanetriol followed by methylation afforded the 1,3-diequatorially substituted dioxane derivative 295 in 73% yield. Chromium complexation proceeded smoothly giving the corresponding arenium complex 296 in 67% yield. A bromine atom was then stereoselectively introduced by lithiation of 296 and subsequent bromination with l,2-dibromo-l,l,2,2-tetrafluoroethane providing 297 in 47% yield and 90% enantiomeric excess. Furthermore, after one fractional crystallization from ether-hexane, the enantioselectivity could be increased to greater than 99%. Acidic hydrolysis of the acetal followed by reduction of the obtained aldehyde furnished the alcohol 298 in 77% overall yield. Suzuki cross-coupling between 298 and 2-formyl-4,5methylenedioxyphenylboronic acid (299) in the presence of Pd(PPli3)4 gave the desired coupled biaryl 300 in 67% yield without formation of the corresponding atropisomer. Protection of the hydroxy group as silyl ether followed by reaction with methyl lithium at -78°C afforded a 5:1 mixture of secondary alcohols 301. Allylation of the secondary alcohol 301 was then performed by reaction with NaH and allyl bromide. After demetallation by air, 302 was recovered in 57% overall yield. Finally, deprotection with tetrabutylammonium fluoride followed by bromination of the primary alcohol and reaction with sodium dimethyl malonate afforded 303 in 45% yield. This concluded the formal total synthesis of (— )-steganone (7) as Meyers et al. had earlier converted 303 into the natural product [116].

594

OMe

H3CO

OCH3 OCH3 *Cr(CO)3 297

ee = 90%

(CO)3Cr

H3CO. H3CO

a) (S)-1,2,4-butanetriol, TsOH, MeOH, reflux, 6 h; b) Mel, NaH, THF, DMF; c) Cr(CO)6, butyl ether, heptane, THF, 130°C, 24 h; d) n-BuLi, toluene, -78°C, 1,2-dibromo-1,1,2,2-tetrafluoroethane; e) 6N HCI, THF, 40°C, 1h; f) MeOH, NaBH4, 5 min, rt; g) 299, Pd(PPh3)4, aq. Na2CO3, MeOH, reflux, 1 h; h) NBuMe2SiCI, imidazole, CH2CI2, i) MeLi, Et2O, -78°C; I) allyl bromide, NaH, THF, DMF; m) hv, O2, Et2O; n) n-Bu4NF, THF; 0) CBr4, PPh3, CH2CI2, 0°C; p) NaCH(CO2Me)2, MeOH.

Scheme (45). Formal total synthesis of (-)-steganone by Uemura

In addition, intermediate 300, produced using Uemura's methodology, was used in another very elegant synthesis of (-)-steganone (7) by Molander and co-workers, Scheme (46) [117]. The primary alcohol of 300 was converted into the corresponding bromide 304 by utilizing

595

methanesulphonic anhydride in the presence of triethylammonium bromide. Stille coupling between 304 and 305 was high yielding using the weakly coordinating AsPli3 as ligand to give the chromium complexed product 306 in almost quantitative yield. At this point, Smk promoted 8endo radical cyclization was achieved using 3.5 equivalents of Smk in fert-butanol in the presence of HMPA. The cyclized product 307 was obtained as a single isomer in 73% yield. PCC-Treatment of 307 buffered with NaOAc in CH2CI2 accomplished the oxidation of the secondary alcohol as well as decomplexation of the chromium complex in one pot. The resulting ketone 308 was obtained in 85% yield as a mixture of two atropisomers in which the absolute stereochemical information of alcohol 307 was retained only at the lactone ring juncture stereocenters. Equilibration of the stereocenters was achieved by treating ketone 308 with DBU in THF at reflux affording (-)-steganone (7) in 82% yield.

r-q 91% "H3CO,

(CO)3Cr 300

OCH3 304

H 3 crj / (CO)3Cr

0 73%

H3CO, H3CQ

OCH3

(CO)3Cr

306

82% H3CO'

a) Ms2O, Et3NHBr, Et 3 N, CH2CI2, 0°C, 1 h; b) 298, Pd2(dba)3, AsPh3, THF, reflux; c) 3.5 eq Sml 2 , 2 eq f-BuOH, THF/HMPA, 0°C; d) PCC, NaOAc, CH2CI2; e) DBU, THF, reflux.

Scheme (46). Asymmetric synthesis of (-)-steganone by Moiander et at

596

Conclusion In the last ten years significant progress has been made in the asymmetric synthesis of lignans demonstrated by the multitude of elegant total syntheses reported in this review. As new lignans with interesting biological activies are isolated at a frequent rate, it is very likely that in the future we will see more exiting total synthesis of this extraordinary class of compounds. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] II1] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]

Harworth, R.D. /. Chem. Soc, 1942,448-456. Whiting, D.A. Nat. Prod. Rep., 1985,2,191-211. See for example: Ayres, D.C.; Loike, J.D. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press: Cambridge 1990 Rao, C.B.S. In Chemistry of Lignans, Andhra University Press, Andhra, 1978. Ward, R.S. Nat. Prod. Rep., 1999,16,75-96. Ward, R.S. Nat. Prod. Rep., 1997,14,43-74. Raffaelli, B.; Hoikkala, A.; Leppala, E.; Wahala", K. J. Chromatogr. B, 2002, 777, 29-43. Wang L.-Q. J. Chromatogr. B, 2002, 777,289-309. Canel, C ; Moraes, M.R.; Dayan, F.E., Ferreira, D. Phytochemistry, 2000, 54, 115-120. See for example: Ward, R.S. Tetrahedron, 1990, 46,5029-5041. Morimoto, T.; Chiba, M.; Achiwa, K. Heterocycles, 1992, 33,435-462. Meragelman, K.M.; McKee, T.C.; Boyd, M.R. /. Nat. Prod, 2001, 64, 14801482. Lee, C ; Kim, H.; Kho, Y. J. Nat. Prod, 2002, 65, 414-416. Kuo, Y.-H.; Huang, H.-C; Kuo, L.-M.Y.; Chen, C.-F. J. Org. Chem., 1999, 64, 7023-7027. Valcic, S.; Montenegro, G.; Timmermann, B.N. J. Nat. Prod, 1998, 61, 771-775. Sung, S.H.; Kim, Y.C. J. Nat. Prod, 2000, 63,1019-1021. Wang, B.-G.; Ebel, R.; Bambang, W.N.; Prijono, D.; Frank, W.; Steube, K.G.; Hao, X.-J.; Proksch, P. J. Nat. Prod, 2001, 64,1512-1526. Cavin, A.; Potterat, O.; Wolfender, J.-L.; Hostettmann, K.; Dyatmyko, W. J. Nat. Prod, 1998, 61, 1497-1501. Kraus, G.; Spiteller, G. Phytochemistry, 1997, 44, 59-67. Bartlova, M.; Opletal, L.; Chobot, V.; Sovova, H. J. Chromatogr. B, 2002, 770, 283-289 and references cited therein. Suto, K.; Ito, Y.; Sagara, K.; Itokawa, H. J. Chromatogr. A, 1997, 786, 366-370. Kominami, G.; Ueda, A.; Sakai, K.; Misaki, A. /. Chromatogr. B, 1997, 704, 243-250. Kuo, Y.-H.; Li, S.-Y.; Huang, R.L.; Wu, M.-D.; Huang, H.-C; Lee, K.-H. /. Nat Prod., 2001, 64,487-490. Liu, S.; Tian, X.; Chen, X.; Hu, Z. /. Chromatogr. A, 2001, 928,109-115.

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598 [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78]

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©

601 601

NATURAL OLIGOSTILBENES MAO LIN, CHUN-SUO YAO Institute ofMateria Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China ABSTRACT: More than 200 naturally occurring oligostilbenes isolated from plant kingdom are grouped into I-V structural types. They are a special kind of polyphenolic natural products with multi-faceted bioactivities. This article will review their classification, distribution, spectral characteristics, biological activities (anti-fungal, antibacterial, antioxidant, anti-inflammatory and anticarcinogenic activities, etc.) and mimetic biosynthesis.

INTRODUCTION

The naturally occurring oligostilbenes are a special group of polyphenolic compounds polymerized from resveratrol or other stilbene units (such as isorhapontigenin, oxyresveratrol, etc.). They usually possessed novel and complex structures and are widely distributed in plant families. A number of structures of stilbene oligomers have been elucidated. A conservative estimate suggests that the number of naturally occurring oligostilbene has increased to more than 200 in recent years with the development of modern spectroscopic techniques, especially various kinds of 2D-NMR and mass spectral techniques. Their multi-faceted biological activities including anti-inflammatory, anti-viral, anti-tumor, antibacterial and anti-fungal are attracting the worldwide medicinal chemists. These compounds are considered to play an important role in the protective effects of plants against fungal and bacterial invasion. They also appear to have considerable potential for pharmaceutical uses as chemopreventive and anti-inflammatory agents against neoplastic changes and inflammatory effects in human body. The biomimetic synthesis of oligostilbenes is based on their biogenetic pathway using different stilbene monomers or £-viniferin as precursor in the synthesis of new types of compounds with strong activity for drug development

602

In 1951, the first resveratrol (1) polymer, hopeaphenol (2), was isolated from Hopea odorata and Balanocarpus heimti by King and his co-workers [1]. But its structure was not established as a resveratrol tetramer until 1966, when a single crystal, a dibromide derivative of deca-O-methyl ether of 2 had been prepared and analyzed by X-ray method [2]. Langcake and Pryce reported that 1 existed as a phytoalexin in infected grapevine leaves, since then (+)-s-vinferin (3) and a-vinferin (4) have been isolated from the infected grapevine leaves [3,4]. Thereafter, about 20 oligostilbenes have been isolated from five plant families. In 1993, Sotheeswaran and Pasupathy [5] divided naturally occurring resveratrol

2 hopeaphenol

3 (+)-&-viniferin

4 a-vinferin

oligomers into two major groups: Group A contained at least one oxa-cyclic ring, usually the fra«s-2,3-diaryl-2,3-dihydrobenzofuran moiety, while group B did not contain any oxygen heterocyclic rings. All the oligostilbenes of group A appeared to be formed from resveratrol via the dimer ^-viniferin. However, oligostilbenes of group B were polymerized directly from various kinds of stilbene monomers without s-viniferin being an intermediate. Most naturally occurring oligostilbenes belong to group A, only a few of them belong to group B. With the increase of naturally occurring oligostilbenes, a variety of other oligomers containing different stilbene monomers, especially isorhapontigenin (S), oxyresveratrol (€), as well as their glycosides were obtained from plant resources. Thus, we proposed a new classification method as follows: all naturally occurring oligostilbenes hitherto known could be classified into the following five major groups (I-V) according to the different stilbene monomers present in their structures. Each basic type was subdivided into two groups (A and B) depending upon whether they

603

contained oxygen heterocycles or not. Group I was further divided into group I-A, I-B, I-C and I-D respectively. 1. Classification of Oligostilbene The stilbene monomers that polymerized to oligostilbenes are depicted in the following: Ri

R2

R3

R4

1

resveratrol

H

H

OH

H

5

isorhapontigenin

H

OCH3

OH

H

6

oxyresveratrol

OH

H

OH

H

7

piceatanol

H

OH

OH

H

8

rhapontigenin

H

OH

OCH3

H

9

gnetol

OH

H

H

OH

The numbering of oligostilbenes complies with the following rule: Each

OH

Fig. (1). Numbering of resveratrol (A) and (-)-s-viniferin (B)

stilbene monomer is numbered respectively and the different units are distinguished by a, b, c, etc; as an example, the numbering of 1 and 3 are shown in Fig. (1). I. Oligostilbenes polymerized from compound 1. Most naturally occurring oligostilbenes belong to this type and are further divided into four groups (I-A, I-B, I-C and I-D) according to whether they contain oxa-cyclic rings and the type of polymerization. I-A group: this group contains at least one five-membered, oxa-cyclic ring, usually the 2-aryl-2, 3-dihydrobenzofuran ring. About 60 oligomers

604

10 (-)-s-viniferin

11 ampelopsin A, R=OH 12 ampelopsin B, R=H

14 amuresins C

16 amuresins H

13 miyabenol C

15 amuresins D

17 amuresins"L

18 anigopreissin

belonging to this group have been isolated from nature. (+)-s-Viniferin (3) and (-)-£"-viniferin (10) are the basic units, which form various structures of oligostilbene with a straight chain including dimers, trimers, tetramers and pentamers. For example, 3 and miyabenol C (13) belong to I-A group. But the oligomers containing a moiety of bicyclo [5,3,0] decane or 10-oxabicyclo [6,3,0] undecane ring system, such as ampelopsin A (11) and ampelopsin (12), are not included in this groups. Some oligostilbenes with benzofuran ring also belong to this group. Anigopreissin A (18) was the first oligostilbene containing an unsaturated benzofuran ring [8J. Recently, amuresins C (14), D (15), E, F, H (16) and L (17) have been isolated from Vitis amurermsis [6,7], In the structures of amuresins D, E and F, the locations of 4-hydroxybenzene moiety and 3,5-dihydroxybenzene of those compounds were interchanged compared with those of 14, which are the new type of oligostilbenes.

605

Some oligostilbenes with cis olefinic protons, such as maximol B (19) [9] and gnemonol L (20) [10], have been found in plants. I-B group: Oligostilbenes are polymerized directly from resveratrol monomers but do not contain any oxygen heterocyclic ring in their structures, such as ampelopsis D (21) [11]. Until now, 20 oligomers of this group have been found in plants.

OH OH

20 gnemonol L

19 maximol B

Caraphenols B (22) and C (23) are a pair of isomers with different connectivity recently isolated from Caragana sinica [12].

21 ampelopsin D

22 caraphenol B

23 caraphenol C

I-C group: Oligostilbenes containing at least a bicyclo [5,3,0] decane or bicyclo [6,3,0] undecane ring system in their structures. Compound 11 or 12 is the basic moiety of I-C group. In the past few years, more than 40 oligomers, including trimer (+)-vinferol D (24) [13], tetramer (+)-viniferol A (25) [14], hexamer vaticanols D (26), H, I and heptamer vaticanol J have been successfully isolated from the genera of Vitis and Vateria [15,16]. Recently, the only octamer, vateriaphenol (27), has been isolated from Vateria indica [17].

606

24 (+) viniferol D

25 (+)-viniferol A

27 vateriaphenol A

26 vaticanol D

28 hemsleyanoside F

29 gnemonoside B

30 gnemonoside K

I-D group: this group includes all oligostilbene glycosides. The structures are usually formed by an aglycone of I-A and I-C groups and connected with 1-3 glucoses composing O- glycoside or C-glycoside. Since 2001, about 28 glycosides belonging to this group have been found as natural products. Hemsleyanoside F (28) is an I-C type of stilbene dimer-C-glucoside from Shorea hemsleyama and Gnetum gnemonoides, respectively.

31 gnemonoside H

32 gnemonoside F

607

Two glycosides of resveratrol polymer, gnemonoside B (29) and K (30) have cis olefinic bonds in their aglycones, The aglycone moieties of gnemonoside H (31) and F (32) are dimer and trimer of resveratrol, and each of them is connected with 3 glucoses, respectively [19,20]. Recently, two glycosides of stilbene dimer named compound 1 (33) and compound 2 (34) were obtained from an aqueous extract of the roots of Polygonum cuspidatum. Among them, 34 was a new type of resveratrol dimer possessing a four-membered ring [21].

33 compound 1

34 compound 2

35 shegansu B

II. Oligostilbenes polymerized from compound 5. More than 20 polyphenols of this type have been isolated from plants. II-A group: shegansu B (35) was obtained from Belamcanda chinensis in 1997 [22], which later was also obtained from most of the Gnutum species. Recently, gnetupendin D (36) was isolated as the first isorhapontigenin glycoside from Gnetum species having 35 as aglycone moiety [23]. A pair of isorhapontigenin isomer (gnetuhainin N (37) and O (38)) polymerized from gnetulin (41), and an isorhapontigenin unit (with a dihydrobenzofuran moiety at C-7c,8c,5b and 4b-O positions and different stereochemistry at H-7c and H-8c) were isolated from Gnetum hainanense [24]. II-B group: gnetifolins C (39) and D (40) were obtained by our research group in 1992 [25]. At first, gnetifolin C was assigned a structure with

OCH,

36 gnetupendin D

37 gnetuhainin N

38 gnetuhainin O

608

an eleven-membered ring (39) according to the 2D-NMR spectroscopic methods. For further confirmation of the structure, an oxidative reaction was carried out. Oxidation of acetylated gnetifolin C (39a) with Lemieux-Johnson's method (OsO4/NaIO4 in dioxan-HbO) produced two products, a ketone (a) and an aldehyde (b) Fig (2). The reaction indicated that the structure of gnetifolin C (39) was identical with a known compound,

MeO-

HO

OH

HO

39 gnetufolin C

OCH,

40 gnetufolins D

OH

OCH, •Ms

41 gnetulin

42 gnetuhainin R

gnetulin (41)[27], which has an exo-double bond with a five-membered ring in the molecule. Gnetuhainin R (42), the first isorhapontigenin tetramer isolated from Gnetum hainanense, was attributed to II-B group [28]. Up to now, most of the isorhapontigenin oligomers have been isolated from Gnetum species in China. It may be a characteristic of Gnetum species in China. III. Oligostilbenes polymerized from piceatanol. Only eight piceatanol dimers have been reported up till now.

609 OCH 3

OAc

OCH 3

OAo

OCH

OAc

OCH,

39a Fig. (2) The oxidation reaction of gnetufolin C

III-A group: this group of oligomers is polymerized from two units of 7, with a five-membered oxygen heterocyclic ring or a benzo-dioxane ring. For example, cassigarol D (43) belongs to the former, and cassigarol E (44) belongs to the latter [29a, 29b].

OH

42 gnetuhainin R

43 cassigarol D

OH

44 cassigarol E

III-B group: piceatanol oligostilbenes without any oxygen heterocyclic ring in their molecules. This type of polyphenol was formed directly from two units of 7 through carbon-carbon bond. Cassigarol A (45) is an example for this group. [30]. IV. Oligostilbenes coupling from 1 and 6 units or two units of 6. IV-A group: oligostilbenes containing a benzo-oxygen heterocyclic ring. 17 compounds have been found involving dimers and trimers from plants such as gnetumontanin A (46) [31]. An oxyresveratrol dimmer, gnetuhainin S (47) [28] and parvifol A [32] were isolated from Gnetum hainanesis and Gnetum pravifolium, respectively. Both compounds are identical and have been reported almost simultaneously. Gnemonol A (48) and gnemonol C (49) from Gnetum gnemon are polymerized from 1 and 6, respectively [33].

610

HO

^ ^

OH

45 cassigarol A

46 gnetumontanin A

48 gnemonol A

47 gnetuhainin S

49 gnemonol C

IV-B group: oligostilbenes from 1 and 6 units connected by carbon-carbon bond forming cyclic or chain oligomers. For example, andalasin A (50) coupled with two units of 6 has been isolated from Morus macroua [34]. Gnetuhainin D (51) polymerized from 1 and 6 was obtained from Gnetum hainanense [35].

OH HO

50 andalasin A

51 gnetuhainin D

OH

52 gnetuhainin K

V. Oligostilbenes polymerized by other monostilbene units besides I-IV group. V-A group: gnetuhainin K (52) is a polymer of 5 and 9 isolated from Gnetum hainanense [36]. Recently two oligostilbenes, gneafricanin A (53) and B (54) have been isolated from Gnetum africanum: 53 was polymerized from 5 and 6 units, whereas 54 from 5 and 7 units [37]. V-B group: oligomerstilbenes connected by different stilbenes units with carbon-carbon bonds such as gnetuhainin J (55), which was polymerized from 5 and 6 units [38].

611

53 gneafricanin A

54 gneaf ricanin B

55 gnetuhainin J

2. Distribution of Oligostilbenes Resveratrol oligophenols have been found mainly from five plant families in the past [5]. Owing to the rapid increase in oligostilbenes in the recent years, the distribution in plants have so far been extended to nine plant families, namely Dipterocarpaceae, Vitaceae, Cyperaceae, Leguminosae, Gnetaceae, Iridaceae, Celastraceae, Paeoniaceae and Moraceae. More than 200 naturally occurring oligomerstilbenes have been isolated from these plants. Most compounds are resveratrol dimers, trimers, and tetramers. A few compounds belong to pentamer, hexame,r heptamer and octamer. 3. Biogenesis and Conformations of Oligostilbene Initially it was proposed that all naturally occurring oligostilbenes of I-A with dihydrobenzofuran moieties were formed from 1 via the dimer e-viniferin, such as the proposed biosynthetic route of 13 [38]. This biosynthetic pathway may be the main biogenetic route for oligostilbenes, but other biogenetic precursors seem to exist during biogenesis of oligostilbenes of group A, because many oligostilbenes with various novel skeletons have been separated as in the cases of amurensins B-F and J [39,6,40]. Amurensins B, 14 and J, each with a cis dihydrobenzofuran

56a 7a,8a-cfe-£-viniferin 56b iso-e-viniferin Blg.(3) Plausible intermediate of biogenetic pathways of oligostilbenes

612

12 ampelopsin B 21 (-)-ampelopsis D 57 (+)-ampelopsin F Fig. (4) Plausible biogenetic pathways of 12,21 and 57 from 3 moiety, might be formed from 1 via 7a,8a-cis-ff-viniferin (56a ), while amurensins D-F might be formed via iso-a-viniferin (56b) [6] Fig, (3). Unfortunately, 56a and 56b were not isolated from plants till now. It was concluded that the biogenesis of oligostilbenes is very complex and implies multiplicity.

[A]

613

[AJ + [B]

25

*=a 2 *=0 61 Fig.(5) Plausible biogenetic pathways of stilbenetetramers from 10

In the last few years, Niwa M. et al. reported that oligostilbenes have mainly been isolated from Vitaceae Dipterocarpaceae, Cyperaceae, Leguminosea and Gnetaceae;oligostilbenes isolated from Vitaceaeous plants are chemically different from those from other families as shown in the cases of e-viniferin and hopeaphenol. 3 has only been isolated from Vitaceaeous plants, but 10 has been isolated from plants of other families. Therefore, Vitaceaeous plants have a specific biogenetic pathway distinguished from other plants such as Dipterocarpaceae, Cyperaceae, Leguminosae. 3 seems to be a biogenetically important precursor for many

614

oligostilbenes isolated from Vitaceaeous plants. Niwa M. et al. also designed a transformation of 3 in different acids by the biomimetic pathway leading to obtain a variety of oligostilbenes. Isomerization and/or rearrangement of 3 yielded compounds 12, (+)-ampelopsin F (57) and 21. The difference of products is apparently due to the difference of the position of protonation at the initial stage of the reaction as shown in Fig. (4) [41]. The oxidative coupling along with isomerization may also transform 3 to 2, (+)-vitisin A (58), (-)-vitisin B (59), (+)-vitisin C (60), (-)-isohopeaphenol (61) and 25 as shown in Fig. (5)[42]. All the mentioned transformation and oxidative coupling reactions provide us information of the reactivity based on the biogenetic pathway and further confirmation of absolute structures of the resveratrol oligomers.

4. Spectral Characters of Oligostilbenes The structural elucidation of naturally occurring oligostilbenes depends on a modern spectroscopic evidence, such as MS, NMR, IR and UV. All kinds of 2D-NMR techniques (including COSY, HMQC, HMBC, NOESY) play important roles in the structural elucidation of oligostilbenes. We will mainly summarize their 1 H NMR and B C NMR characteristics in this section.

4-1 tHNMR and13CNMR Characteristics In *H NMR and 13C NMR spectra of oligostilbenes, the signals of various monostilbenes are useful to determine their polymerization degrees. The different monostilbenes could be distinguished by XH NMR signals of ring A Fig. (6). Signals of ring A appear as A2B2 system for resveratrol, ABX system for 5, 6, 7 and 8. There is one methyloxy signal more in 5, 8 than in 6 and 7. Following is an example of the NMR features of resveratrol oligomers. The trans double bonds (H-7, H-8) of resveratrol units appear in three forms after polymerization. 1. Still in trans double bond, the proton signals appear as two doublets at 8 6.4~7.2 with coupling constant of

615

15.0~17.0Hz. The carbon signals appear at 5 128-135 (C-7) and 5 120~125(C-8) respectively. A few of them in cis double bond, the proton signals appear at 8 5.5-6.1(J = 12 Hz). 2. As part of a dihydrobenzofuran moiety Fig. (6), H-7a and H-8a are in trans relationship and C-7a is generally linked to the oxygen atom. The signals of H-7a and C-7a in NMR spectra appear at lower field of 8 5.2-6.0 and 8 85-95, respectively, due to the deshielding effect of the oxygen atom. H-8a and C-8a appear at 8 4.2-4.8 and 8 45-60 respectively. The coupling constant between H-7a and H-8a is within 3.5~8.6 Hz when they are in trans relationship. 3. As members of aliphatic ring, the signals of H-7 and H-8 appear at 8 2.5-4.5 and 8 35-65 respectively.

Fig (6) Polymerization of resveratrol in a dihydrobenzofuran moiety

Most of the 4-hydroxybenzene groups (ring A) in 1 display signals of A2B2 system in aH NMR spectra after polymerization, presenting two coupled doublets ( J = 7.5-9.0 Hz) at 8 7.0-7.5 for H-2 and H-6, and at 8 6.4-6.9 for H-3 and H-5. In 13C NMR, the signals of C-2 and C-6 appear at 8 127-130, and of C-3 and C-5 at 8 114-117. Only a few of them showed signals of ABX system in tetramers and pentamers. The 3,5-dihydroxybenzene group (ring B) in 1 showed signals of AB2 system at 8 5.8-7.0 after polymerization, such as H-lOa, 12a, 14a in Fig. (6). The 1HNMR proton signals of H-12a appear as a triplet with coupling constant between 1.0-2.5Hz and proton signals of H-10a,14a appear as symmetrical doublets with the same chemical shift and coupling constant. In 13C NMR, C-lOa and 14a have the same chemical shift at 8 105-108, and the chemical shift of C-12a appears at 8 100-103. When the 3,5-dihydroxybenzene group was involved in a dihydrobenzofuran moiety, two meta coupled protons such as H-12b and H-14b showed two doublets at 8 6.0-6.6. In 13C NMR, the signals of C-12b appeared at 8 95-100, and of C-14b at 8 105-110.

616

The quaternary carbons of C-l and C-9 (not attached to a double bond) in resveratrol exhibited signals at 8 130~136 and 5 141~149, respectively, after polymerization. The quaternary carbons attached to a hydroxy group in resveratrol oligomers showed signals at 8 155~162. The stereochemistry of H-7a, H-8a and H-7b, H-8b of oligostilbene with bicyclo (5,3,0) or (6,3,0) decan or undecane ring system could be determined by 1 H-NMR spectrum. The coupling constant ( J = 11.0-12.8 Hz) of H-7a and H-8a suggested the stereochemistry between H-7a and H-8a to be trans, such as 11 (J=11.7Hz) and 2 (/=12.5Hz). The chemical

62 (+)-viniferol C.

63 (+) vaticanol B

64 vaticaphenol A

shift value of H-8a at 8 4.25-4.44 indicated the stereochemistry between H-8a and H-7b to be and as shown in the case of 2, 63 and vaticaphenol A (64). This upfield shift is caused by a shielding effect of the aromatic group B at C-7b. The chemical shift value of H-8a at 8 5.0-5.4 indicated the stereochemistry between H-8a and H-7b to be syn as shown in the case of 62. The coupling constant value of H-7b and H-8b (J=11.7 Hz) indicated the stereochemistry between H-8b and H-7b to be trans as shown in the case of 62. A coupling constant of J=3.0-3.9Hz in 63 and 64, which have the same ring system as 62, suggested a cis stereochemistry between H-7b and H-8b. This consideration was further supported by NOE difference experiments and molecular mechanics calculation as well as biogenetical synthesis [14,43]. 4-2. MS Characteristics Since the oligostilbenes have high molecular weights and mutiplet hydroxyl groups, it is generally difficult to obtain their molecular ion peaks in EI-MS except for some dimers or those without a hydroxyl group in the structures. Modern MS techniques, such as FAB-MS, FD-MS, ESI-MS and LSI-MS were used to obtain their molecular ion peaks.

617

4-3. UV Characteristics

The maximum absorption bands of oligostilbenes depend on the conjugation system in their structures. The maximum absorption bands appear at nearly 320 nm for oligostilbenes having trans double bonds, and at about 280 nm for oligostilbenes without trans double bonds in their structures. Oligostilbenes with a benzofuran ring have maximum absorption band at about 340 nm. 4-4. IR Characteristics The IR spectra of oligostilbenes exhibit absorption bands for hydroxyl groups (3200-3500 cm"1), benzene group (1450-1600 cm"1) and double bonds (1610-1670 cm"1). There is a strong absorption band at 965 cm"1 if trans double bonds exist in the structure. An ambiguous absorption band at 730-675 cm"1 will appear in certain cases, if there are cis double bonds in the molecule. 4-5. Absolute Configuration of Oligostilbene In 1990, two chiral carbons of 10 had been established to be 7a/? and 8ai? respectively, by Kurihara et al. [44]. After that, in 1996, 7aS and 8aS had been established for 3. Their CD spectra showed negative and positive Cotton effects at 237nm, respectively. The absolute configuration of a large number of oligostilbenes belonging to I-C group has been established by Ito et al. [46]. The absolute configuration of 11 was

lla

65

66

618

3

V"S

CHO

67

68

established by the following method: methylation by methyl iodide to give a penta-methyl ether (lla), which lead to oxidation giving the corresponding ketone (65). The observation of NOE between H-8a and H-2b (6b) suggested the configuration of the 4-methoxyphenyl group at C-7b to be pseudo-axial against the adjacent carbonyl group in 65. The structures of 65 and 66 show positive and negative Cotton effects respectively. On the basis of octant rule and by comparison of CD spectra, the ketone of 65 showed a positive Cotton effect at 357nm indicating that the absolute configuration of 11 should be represented as in structure 65. The absolute configuration of 12 has been established by comparison of its CD with that of 11. The three positive absorption maxima at 208-211, 233-236 and 287-288nm showed strong resemblance to that of 11. Therefore, the absolute configuration of 12 is identical to 11 as shown in 12. For some tetramer oligostilbenes such as 58, the absolute configuration has been established by the same method. Two parts of degradation products 67 and 68, with aldehyde groups, have been obtained after oxidation. One degradation product, 67 should have the same absolute configuration as 3. The other degradation product 68 was a seven-membered ring aldehyde resembling 65 in wavelengths and the CD spectrum. Thus the entire absolute configuration of 58 could be established by the combination of chemical reactions and NOE as well as C D spectrum [47]. 4-6. X-ray Crystallographic Analysis Most naturally occurring oligostilbenes have been isolated as amorphous powder, therefore it was very difficult to determine their structures by

619

X-ray crystallographic analysis. Hitherto, only the structure of 2 was established by X-ray crystallographic analysis of its bromide. Recently, the structure of 47 isolated from Gnetum hainanense has been verified by X-ray crystallographic analysis [28], 5. Biological Activities Oligostilberies are a large group of phenolic substances isolated from 5 principle plant families. About 70 of more than 200 individual known compounds have been tested for various biological activities. Many studies suggested that this group of constituents exhibited potent biological activities, including antimicrobial, antioxidant, anti-inflammatory, antihepatotoxic, antitumor and other activities. The various in vitro biological activities of oligostilbenes have been tested while their in vivo pharmacological efficacies were less documented in the past studies. 5-1. Antifungal and Antibacterial Activities Since compounds 3 and 4 were first isolated from infected grapevin leaves together with 1, so antifungal activity studies on 1 and its oligomers have been conducted. In the preceding studies, 1 showed moderate antifungicidal and antibacterial activities, while its polymers possessed greater or broader bioactivities [5], Almost 10 compounds have been screened by filter paper disc method (FPDM) in the Mueller Hinton Agar medium using Staphylococcus, Esherichia coli and Staphylococcus aureus etc. in the past years [38, 48, 49, 50, 51, 52], In 2002, Tomoko N. reported the antibacterial activity of the extract prepared from 181 species (75 families) of tropical and subtropical plants, which were screened against various types of pathogenic bacteria. Among the 505 extracts, 53 of them from barks of Shorea hemsleyana and the roots of Cyphostemma bainesii. Some of them showed significant activity in reducing the viable cell number of methicillin-resistant Staphylococcus aureus. The active compounds were all identified as stilbene derivatives. Hemsleyanol D (69), a stilbene tetramer, isolated from S. hemsleyana was the most effective [53].

620

5-2. Antioxidant Activity

Various reactive oxygen species (ROS) are generated from enzymatic and nonenzymatic processes during the normal metabolism in cells. If excessive ROS are not eliminated promptly, the accumulated ROS will induce cytotoxicity, which plays important role in pathophysiology of

69 hemsleyanol D

70 gnemonol B

many diseases, such as inflammation, atherosclerosis, reperfusion injury and cancer. Antioxidants can terminate ROS chain reaction by disturbing initiation and propagation steps of the reaction and play important protective role against the damage caused by various diseases. Thus the studies on antioxidant activities have attracted many pharmacologists. Lliya I. et al. [54] found that many stilbenes showed potential antioxidant activities by comparing the antioxidant activities of nine compounds including monomers 2, 5, 6, dimmer 10, trimmers gnemonol B (70), I (71), K (72) and 20 with Vitamin E for lipid peroxide inhibition and super oxide scavenging activity [10]. Oligostilbene dimers 53, 54, gneafricanins D (73), and E (74), isolated from Gnetum africanum, showed inhibition of lipid peroxide and super oxide scavenging activities as shown in Table 1 [37,55].

71 gnemonol I

72 gnemonol K

621

73 gneafricanin D

74 gneafricanin E

Ten natural polyphenolic products, i.e. longusone A (75), longusols A (76 ), B ( 77) and C (78), were isolated as the active constituents together with 10 phenolic compounds from the methanolic extract of the whole plant of cyperus longus by a bioassay-guided separation. The antioxidative action for l,l-diphenyl-2- picrythydrazyl (DPPH) radical showed that frans-scirpusin B (79) was the most potent (SCso=2.8|j,M), and the scavenging activity of stilbene dimers (75, 76, 77, 78, 79 and 80) was stronger than those of monomers (1,7) except for pallidol Table 1. [56]. Liu G. T et al. investigated the antioxidation activities of nine compounds including oligostilbenes 16 and 81, monostilbenes and isoflavones by several models in vitro, among them 1, 5, 16 and 81 have potent antioxidation activities in vitro. Furthermore, 16 showed significant protective effects on neurotoxins induced oxidative cell injuries and apoptosis in primary cultured cerebellar granule cells and bovine aorta endothelial cells through inhibiting release of cytochrome c from mitochondria and activation of Caspase-3 [57a, 57b].

OH

75 longusone A

78 longusol C

76 longusol A

79 frans-scirpusin A

77 longusol B

80 rrans-scirpusin B

622

Compounds 25, 26 and 27 belonging to I-C group, isolated from Vatica rassak and V. indica, showed antioxidation and SOD like activities, indicating that this group of oligomers also has antioxidation activity and superoxide scavenging activity [58,59, 60]. Two glycosides of stilbene dimer, 33 and 34 were isolated from Polygonum cuspidatum. Pharmacological studies showed that 34 which has a special skeleton showed moderate inhibition (inhibition rate 36%) in the formation of MDAat a concentration of 2 pM [21]. The results of recent research on antioxidation activities have revealed that the radical scavenging activity depends on the structure and multiple phenolic hydroxyls of the oligostilbenes. Table 1 Natural oligostilbenes with antioxidant activity* ^ompoiixiua

Super oxide scavenging activityriCsoTuM)!

lipid peroxide inhibition activity[ICsn(nM)l

16

15

69 59 79 57 20 20 10 33 30 26 29

19 7 50 25 33 34 13 50 32 29 45 81 36

LangusoneA Longusol A Longusol B Longusol C Treres-seirpusin A 7>*aras-scirpusin B Cassigatol E Cassigarol G Pallidol Gnemonol K Gnemonol L Gnemonol B Gnemonol I (-)-*-viniferin Gneafricanin A Gneafricanin C Gneafricanin D Gneafticanin E Gnetin F Bisisorhapontigenin B Compound 1 Compound 2

DPPH radical [SCsoOiM)] 4.6 9.3 4.3 5.0 8.2 2.8 3.2 4.5 29

* DPPH=l,l-diphenyl-2-picrylhydrazyl

Studies on the structure-activity relationship of oligostilbenes drew the following conclusion: (i) the presence of conjugated system with double bonds and para-hydroxy groups, which have better electron-donating properties and are responsible for electron delocalization. This is a radical target; (ii) multiple hydroxy groups on different positions of the molecule, for example, ortte-dihydroxystilbene forming metal chelate complex can

623

increase the radical scavenging capability; (iii) cyclic sizes of oligomer and different attachment of rings have different contributions to radical scavenging capability. Thus, the estimation of oligostilbene as antioxidants has a pharmacological prospect. 5-3. Anti- Inflammation and Anti-Carcinogenic Activities Leukotrienes (LTA4, B4, C4, D4, E4, F4) and prostaglandins (PGS) are a series of important metabolites of arachidonic acid (AA) via 5-lipoxygenase (5-LO) and cyclooxygenase (COX). They were found to play a major role in the pathogenesis, including cell proliferation, inflammation and immune response, platelet aggregation, smooth muscle contraction and maintenance of fluid and electrolyte balance. Therefore, inhibitors of 5-LO and COX are known to have anti-inflammatory and anti-carcinogenic activities. Many oligostilbenes from Gnetum species showed a variety of biological activities on LTs. A stilbene dimer (Gn-3) separated from Gnetum parvifolium exhibited potent inhibitory activities on LTC4 and D4 enzymes and their receptors [61, 62]. Some oligostilbenes polymerized by 1 and 5 obtained from Gnetum or Vitis species were observed to exhibit potent inhibition action on LTB4. Compounds 81,2, 61, 58, (+)-vitisinfuran A (82), amurensin F (83) and 35 showed inhibitory activity on LTB4 at concentrations of 10"5 -10"4 mol'L"1

81 heyneanol A

82vitisinfuranA

with an inhibitory ratio of 76%, 56%, 60%, 63%5 72%, 67% and 98% respectively [22, 40]. 15 and 35 also showed slight activity on biosynthesis

624

of LTD4 [6, 22]. In the research for anti-inflammatory and/or cancer chemopreventive activities of natural products, Lee SH et al estimated the inhibitory effects on the COX activities of over 600 species of wild plants in Korea. 4 was isolated as a main active constituent in the dose-dependent inhibition of COX from Carex humilis (originally from Caragana chamlagu), with an IC50 of 7|J.M and 3-4 fold stronger than 1 [64]. Therefore, 4 exhibited anti-inflammatory and cancer chemopreventive activities.

83 amurensin F

84 kobophenol A

A series of oligostilbenes has been tested for various inflammatory models. Among them, compounds 16, 81 and 2 showed strong inhibitory effects both in vitro and in vivo. Their IC50 of inhibition of PMN chemotaxis induced by fMLP were respectively 4.8xlO"13, 4xlO"10 and 1.6xlO"10 mol-L"1 in vitro. They showed significantly the inhibition of inflammatory response with multiple drug administration pathways. In the inflammatory model of croton, oil-induced ear edema and DNFB induced the delayed-type hypersensitivity. The results suggested that 16, 81 and 2 are worthwhile for further studies [65]. 5-3-1. Protein Kinase C (PKC), a family of serine/threonine kinases, is one of the major regulatory enzymes, which involves multiple cellular responses. Since the activation of PKC has been implicated in both inflammatory and proliferative processes, the inhibitors of PKC may be of potential therapeutic value. Compounds 4, 13 and kobophenol A (84) were separated from Caragana sinica by bioassay-guided fractionation, which were evaluated

625

for their PKC activity in vitro. 4 and 13 showed inhibition activity at a concentration of 8-6 l^M range and were more potent than the tetramer, 84. While not showing appreciable isoenzyme selectivity, the three compounds were selective in inhibiting PKC over the cAMP-dependent kinase (PKA). 4 showed modest activity (IC50 47)LIM) in zymosan activated leukocytes in whole blood and also potently inhibited the proliferation of NHEK cells (IC50 0.4 ^M) and MCF-7 mammary tumor cells (IC50 3.6(j.M). The 50-100-fold decrease in activity of 4 after a short-term exposure (8h) in the MTT assay suggested that its anti-proliferative activity is not the result of direct acute cytotoxicity. 4 also showed anti-inflammatory activity in the carrageenin induced mouse hind paw edema model. The research indicated that 4 may be useful in treating hyperproliferative or inflammatory skin diseases [66]. 5-3-2. Tumor Necrosis Factor (TNFa) is one of the pro-inflammatory cytokines, which seems to be involved in the initiation and amplification of the inflammatory process. Thus, searching for an inhibitor or modulator of TNFa is one of the main tasks in the quest for anti-inflammatory leading compounds and drugs. HO

OH

85 gnetupendin C

87gnetumontanin B

86 gnetin D

88 gnetumontanin A

626

Two tetramers (59, 60), and gnetupendin C (85), gnetin D (86), gnetumontanin B (87) (dimer) and gnetumontanin A (88) (trimer) were isolated from Vitaceae and Gnetaceae respectively. Pharmacological test revealed that 85, 86 and 87 showed potent inhibitory activity on TNFa with an inhibitory ratio of 59.50, 67.23 and 58.1% respectively at concentration of 10"5 mol-L"1. The IC 50 value of 89 was 1.49xlO"6 mol-L"1, for the inhibition of TNFa production by murine peritoneal macrophages [23, 31].

5-4. Cytotoxic A nti- Tumor A ctivity

Much bioassay-guided research has been conducted to find cytotoxic anti-tumor agents in plants especially those known to be used in folk medicine for this purpose. The choice and number of cell lines used in these bioassays were very variable. OH

HO OH

OH

89 resveratrol trans-dehydrodimer

OH

90 pallidol

Resveratrol frans-dehydrodimer (89) and pallidol (90), constituents of grapes (Vitis spp.), which are formed in response to the microbial attack by the fungal grapevine pathogen Botrytis cinerea, showed modest cytotoxicity against human lymphoblastoid cell (CEM) with IC50 values of 49 |iM, and 32 pM respectively [63]. Malibatol A (91) and B (92) only inhibit cytotoxicity to the host cell (CEMSS), with IC50 values of 13 and 21 i^g-ml"1 respectively [68]. Vatdiospyrodol C (93) from Vatica diospyroides displayed the most potent activity against oral epidermoid carcinoma (KB, EC50 1.0 \ig-m\~1), colon cancer (CoI2, EC50 1.9 M-gml"1), and breast cancer (BC1, EC 50 3.8 \xg-ra\~1) cell lines. This is the first example in which significant cytotoxic

627

91 malibatol A

92 malibatol B

93 vatdiospyrodol C

activity against human cancer cell line has been reported for a tetramer. As the decamethyl derivative of 93 did not show significant activities in the cell lines tested in this study, therefore the hydroxyl groups in 93 play an important role in the cytotoxic activity of 93 [69]. Recently, a patent for cancer remedy and preventive medicines of 93 and 4 has been applied by Linuma, M. et al. [60]. 5-5. Inhibition of Tyrosinase Activity

Melanin biosynthesis inhibitory compounds are useful not only as skin whitening agents but also used in cosmetics and as a remedy for disturbances in pigmentation. Tyrosinase is one of the key enzymes for melanin biosynthesis in plants, microorganisms and mammalian cell [70]. Therefore, many tyrosinase inhibitors have been tested in cosmetics and Pharmaceuticals for preventing overproduction of melanin in epidermal layers. Tyrosinase is also one of the most important key enzymes in the insect molting process. Thus the investigation of inhibitors of this enzyme may provide important clues for developing new insect control agents or whitening agents in cosmetic products and inhibition of the mammalian cancer cell. Artogomezianol (94) and 50 isolated from Artocarpus gomezianus are two oligostilbenes with straight chain. They showed moderate tyrosinase inhibitory activity with IC50 values of 68 and 39 fxM, respectively. The inhibition of 50 is about two times stronger than 94, which possesses only one 4-substituted resorcinol structure (ring C). The relationship of 4-substituted resorcinol skeleton and tyrosinase inhibitory activity has been discussed in reference [71].

628

5-6. Antihepatotoxic Activity

There are a variety of crude drugs in China and Japan, which are effective in liver diseases. Ohizumi Y et ah found that some Vvtaceaeous plants such as Ampelopsis brevipedunculata Trautv, A. brevipedunculata Trautv.var. hancei Render and Vitis coignetiae Pulliat et Planch exhibited significant antihepatotoxic activity at a concentration of 1 mg-ml"1 in carbon tetrachloride (CCU), and D-galactosamine-induced cytotoxicity of primary cultured rat hepatocytes [72]. Further studies showed that 3 was a main active principle from the extract of Vitis coignetiae which showed strong hepatoprotective activity against injuries of primary cultured rat hepatocytes induced by CCU and D-galactosamine (D-GalN). 3 also reduced ALT values significantly in CCU-treated mice at a dose of 30 mg-kg"1. Besides 3, 11, ampelopsin C (95), 57, 58 and ds-vitisin A (96) were also present in the extract. Among them, 95 and a mixture of 58/96 increased ALT values approximately 4.1 and 5.7 times respectively, compared to the control value, when given at 30 mg-kg"1 to CCU-treated or non-treated mice, also showing strong hepatotoxins [73]. Surprisingly, the extract of Vitis coignetiae, an Oriental crude drug used in treatment of liver disease, contained two contradictory constituents [72].

94 artogomezianol

95 ampelopsin C

96as-vitisinA

Four oligostilbenes, 21, ampelopsins E (97), H (98) and cis ampelopsin E (99), were obtained from Ampelopsis brevipedunculata var. hancei. Among them, 97 and 99 showed antihepatotoxic activity at a dose of 0.1 mg-ml'1 in the culture medium, reducing the elevation of GPT levels by 64 and 73% respectively [11].

629

97 ampelopsins E

98 ampelopsins H

99 cis ampelopsins E

A stilbene polymer (Gn-3) isolated from Gnetum parvifolium inhibited the development of liver injury in mice caused by CCU, N-acetyl-P-aminophenol (APAP) and Bacillus Calmette-Guerin (BCG) plus bacterial lipopolysaccharide (LPS) at a dose of 50 mg-kg^-d"1 sc administered for 3 d; thus Gn-3 was found to have liver protective effects [74] 5-7. Other Biological Activities

Kim YC et al. found that 4 and 84 have inhibitory activity on acetylcholinesterase (AChE). The cholinergic system is in relation with Alzheimer's disease. A promising therapeutic strategy for re-activating the central cholinergic function has been used for inhibitors of AchE, which is responsible for the metabolic hydrolysis of ACh. 4 and 84 from methanolic fraction of Caragana chamlague inhibited AchE activity in a dose-dependent manner and the IC50 values were 2.0 and 115.8 |uM respectively. However, AChE inhibition of 1 or a glycoside of 8 was not significant. Therefore, 4 might be a valuable AChE inhibitor because it has an appropriate bulky structure that masks AChE and prevents ASCh from binding to AChE in a noncompetitive manner. In contrast, 84, which is a tetramer with a bulky structure, was less active due to the difficulty of accessibility to AChE [67]. Ecdysteroids are the steroid hormones of insects, where they regulate moulding and metamorphosis. As ecdysteroids are essential to the normal development of insects, it was presumed that some plant secondary metabolites might have the ecdysteroid antagonistic activity and be capable of affecting insect development. Such compounds would be useful

630

tools for the elucidation of ecdysteroid gene regulation and as potential lead compounds for the development of new classes of insecticides [75]. Dinan L. reported that three resveratrol trimers, suffruticosols A (100), B (101), C (102), and one monomer cis resveratrol from Paeonia suffruticosa are active as ecdysteroids (antagonists (EDso) 10-50 pM vs. 5xlO"8|iM of 20-hydroxyeedyson), but inactive as agonists in the Drosophila melanogaster BII cell bioassay for ecdysteroids agonists/ antagonists [75]. It was reported that 12 and 4 from the methanolic extract of the seeds of Iris clarkei, antagonized the action of 20-hydroxyecdysone with ED50 of l.OxlO'5 and 3.3xlO"5 M respectively. [76]. The potencies of 12 and 4 are similar to those of 100-102 (ED50,1.4xl0~5-5.3xl0"5M) and eis-resveratrol (EDso at 1.2xlO"5M) [76] as well as three oligostilbenes from the seeds of Carex pendula (EC50 at 31, 19 and 37^M M) [77] indicating that resveratrol oligomers are potentially significant constituents in reducing insect predation on plants.

100 suffrutieosol A

101 suffruticosol B

102 suffruticosol C

6. Biomimetic Pathways The biological effects of some naturally occurring oligostilbenes were usually greater and/or broader than their monostilbenes, but because of the low content, strong polarity as well as similar chromatographic properties, it was quite difficult to isolate the pure compounds for further pharmacological studies. Recently, some biomimetic syntheses have been reported which may be a way to obtain this kind of bioactive oligostilbenes by semi-synthesis and total synthesis. Now, we will submit for discussion in following four sections.

631

6-1. Catalysis by Oxidase Langcage P. et al. proposed that the viniferin like compounds could be obtained from resveratrol when resveratrol was treated with horseradish peroxide and H2O2. The major products (103-106) of this reaction were

103

103-106

Rl

R2

R3

R4

R5

H

H

H

H

H

H

OH

OH

H

OH

R6

104

OH

H

105

H

OH

H

H

OH

H

106

H

H

OH

H

H

OH

obtained in 40% yield [78]. Coupling reaction of resveratrol or s-viniferin, with an additional unit of resveratrol could take place by the same process. Similar coupling reactions have been achieved with other 4-hydroxylated stilbenes. Ito and Niwa isolated 3, 57 and 59 successfully from the same source, and synthesized those viniferin oligostilbenes by the same process [79]. The methods were special for the synthesis of oligostilbenes. 6-2. Catalysis by Metal A. Coupling reaction using ferric chloride (FeC^) as oxidant 1, The polymerization from homo-monomer: Compound 16 isolated from Vitis amurensis showed strong biological activity [7, 65] and its biomimetic synthesis was achieved as shown in Fig. (7). Oxidative coupling reaction of 1 with FeCl3 as oxidant produced an intermediate, (±)-£-viniferin (107), by silica gel column chromatography. After acetylation, it was dehydrogenated by treatment with 2,3-dichloro-5,6- dicyano-1, 4benzoquinone (DDQ) to afford an intermediate(108) and the desired compound 16 in 20% yield [7].

107

108 Fig. (7) The mimetic synthesize of 16

16

632

MaO

109 shegansuB

110 111 112 bisisorhapontigeninA bisisorhapontigenin B triisorhapontigeninA

HO

113 bisisorhapontigenin C

^ ^

OH

114 bisisorhapontigenin D

115 tetraisorhapontigenin A

Shegansu B, the isorhapontigenin dimer from Belamcanda chinensis, was synthesized from 5 with FeQa as oxidant [22]. In the reaction procedure, seven compounds (109-115) of dimeric, trimeric and tetrameric polymers were obtained. Their possible formation mechanisms were also deduced respectively [80, 81]. For instance, the mechanism of 115 was described as shown in Fig. (8). 2, The polymerization from hetero-monomer: A group of oligostilbenes polymerized from 1 and 5, such as gnetuhainin J, K, L and Q, isolated from Gnetum hainanesis [36, 35a], represented a type of hetero-monomer stilbenes. In order to study the biogenetic pathway of hetero-monomer oligostilbenes, a probe work was carried out. One molecule of 1 and two molecules of 5 (1:2 mol) were dissolved in acetone and stirred with FeCl3-6H2O (0.12 mol) at room temperature for 24 hrs. The suspension was filtered. The filtrate was evaporated to remove off acetone

633

.OH

-115

Fig. (8) The mechanism of coupling reaction of 115 in vacuo and extracted with EtOAC, which was concentrated to dryness. The residue was chromatographed on silica gel and ODS Rp-18 to obtain nine compounds, which were identified as 107,116,117,118,119,120,

116 Fe-3

117 Fe-4

119 Fe-7

118 Fe-5

120 Fe-8

634

121 Fe-9

122 Fe-10

121, 122 and 114 respectively, by spectra analysis, especially 2D NMR spectra. The desired stilbene dimers, such as gnetuhainin L and Q, were not obtained. But compounds 118-121 were identified as hetero-monomer stilbene oligomers of Compound 119, which has a novel skeleton with one unit of 1 and two units of 5. The skeletons of 120, 121 and 122 were similar to those of the naturally occurring compounds 97 and 15 [11, 6, 821.

Fig. (9) Condensation of 44 and 123 by Ag2O

B. Coupling reaction using silver oxide (Ag2O) as oxidants. Two oligostilbenes of 44 and 123 (III-A group) from Cassia garrettiana were synthesized by Baba K. et al by 7 in acetone with Ag2O as oxidant [29] Fig.(9). C. Coupling reaction using vanadium oxytrichloride (VOCI3) as oxidant

124 miyabenol A

125 miyabenol B

Fig. (10) The oxidation of miyabenol A and B by vanadium oxytrichloride

635

Kawabata J et al. isolated two compounds, miyabenol A (124) and miyabenol B (125), from Carex species. The former belongs to I-A group and the latter to I-C group. In determining the stereochemistries of 124 and 125, oxidative coupling of 124 with VOCI3 as oxidant gave compound 125. Their spectral data were in agreement with those of the naturally occurring compounds as depicted in Fig. (10) [83]. 6-3. Photooxidative Coupling Reaction Using UVas Photooxidant

In the course of polymerization reaction, UV was always used as photooxidant to prepare oligostilbenes. For example, 58 was converted to its cis isomer 96 by photochemical transformation as shown in Fig. (11) [47]. The structure-activity relationship analysis indicated that cis configuration of the stilbene unit is the most important factor in combretastatin group for inhibition of cancer cell growth. In order to obtain oligostilbenes with cis olefinic protons, our research group designed and achieved two oxidative coupling reactions using FeCl; and UV as oxidants respectively. With FeCb as an oxidant, the coupling reaction of 1 at room temperature produced an intermediate (±)-£-viniferin after silica gel chromatography. Irradiation of (±)-e-viniferin in anhydrous

58(+)-vitisinA 96 cis- vitisin A Fig. (11) Photochemical transformation of 58 and 96

alcohol with UV light (254 nm, 200W) as photooxidant for 2.5 hrs afforded two object products in 25% and 20.0% yield after column chromatography, as shown in Fig. (12). Among them, 126 is a

636

czs-£-vinferin with a 51.4% inhibition rate on TNFa at concentration of 10"5 mol-L"1, and 127 is a phenanthrene derivative with a 58.1% inhibition rate on TNFa [84].

OH +

HO

127 resveratrol

7b,8b-cw-£-viniferin

2b,14b-dehydrobisresveratrol

Fig. (12) The synthesis of 126 and 127 by photochemical transformation

6-4. Coupling Reaction with Acid The natural products 2, 12, 21, 57, 58, 59 and 60 have been isolated from Vitaceaeous plants. Niwa M. et al reported the regiospecific and stereospecific transformations of e-viniferin to above oligostilbenes using various acids as polymerizing agents, such as H2SO4, HCl and CF3SO3H, according to the biogenetic pathways of oligostilbenes, see Figs. (3) and (4) [41, 42].

128 y-viniferin

129 y-2- viniferin

Fig. (13) The structure of 128 and 129 verified by HCl

Korhammer S et al. isolated two stilbene tetramers, y-viniferin (128) and y-2- viniferin (129), form Vitis roots. Treatment of 128 with 0.1% HCl in MeOH afforded 129, Fig.(13); their structures were verified as unambiguous [85]. Sotheeswaran S. et al. reported that the formic acid could be used as a cyclization agent [86]. Both resveratrol trimers of stemonoporol (130) and

637

copalliferol A (131) were isolated from Dipterocarpaceae plant. 130 was treated in formic acid to afford 131, which confirmed that the former was a precursor of the latter [Fig.(14)].

HCO2H or toluene-p-sulDhonic

130 stemonoporol 131 copalliferol A Fig. (14) Chemical conversion of 130 and 131 into formic acid

In order to get various cyclo-oligostilbenes, our research group designed a dimerization reaction between natural compounds 1 and 5 using 80% formic acid as catalyst. A series of tetralins, isorhafomicols A, B, C and D (132a-135) and resformicols A, B and C (132b-134b) were obtained this way. All structural assignments were made on the basis of various spectral evidences, including 2-DNMR techniques. To our surprise, during ?L

,OH

OH OH

132aD132bD

133a (133b)

OCH3

134a (134b)

1, R=H 5,R=OCH3

135

Fig. (15) Tetralins of isorhapontigenin and resveratrol by formic acid

638

dimerization both the structures of 134a and 134b were found to lose a substituted phenyl group, i.e, 3-methoxy-p-hydroxy-phenyl and j?-hydroxy- phenyl, respectively. Moreover, 135 successively lost two substituted benzene rings and one methoxy group during the course of trimerization. This type of reaction has not been reported before. The probable mechanisms both cyclodimerization and cyclotrimerization are discussed respectively below [87]. The cases mentioned above indicated that strong, electron-donor substituents lead to the formation of a 6-membered ring. In the investigation of the Diels-Alder reaction from 1 and 5, a side reaction, involving loss of a substituted benzene ring during dimerization or trimerization, driven by ejection of a methoxy group was found. It is a very special reaction we have ever seen. It may be formed through some transition states as shown in Figs. (15) and (16).

134a

CH S O,

639

HO

^"^

OH

135 Fig. (16) The proposed mechanism of 135

CONCLUSION The natural oligostilbene is a type of the important polyphenolic compounds distributed widely in the plant kingdom. A large number of pharmacological tests indicated that these compounds possess strong biological activities, especially for a variety of cytokines. Recently, some cytokine modulators, such as selective blockers of IL-1(3 and TNFot receptors, have been employed clinically. The use of these drugs has some disadvantages regarding their high cost, important side effects and route of administration (subcutaneous injections). The fact that oligostilbenes are widely distributed in plants and plenty of them have important anti-cytokine activities, studies on the development of therapeutic agents would be beneficial. The natural oligostilbenes or its derived agents could be used alone or in association with other available effective drugs, allowing a reduction in costs and /or side effects and possibly leading to an increase in effectiveness. Large-scale studies on biological activity, phytochemistry and biomimetic synthesis reveal that their constituents would have considerable potential for pharmaceutical uses as chemopreventive agents against neoplastic change and inflammation in human body. The review of oligostilbene may assist relevant fields to seek for new, effective and low side effects drugs. ACKNOWLEDGEMENTS The National Natural Science Foundation of China financially supported some studies of relevant oligostilbenes in this review by a grant No. 30,

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ISOLATION, STRUCTURE ELUCIDATION AND BiOAcnvrnES OF PHENYLETHANOID GLYCOSIDES FROM CISTANCHE, FORSYTHM AND PLANTAGO PLANTS T. DEYAMA,1 H. KOBAYASHICLAIE),1 S. NISHIBE,2 and P. TU3 1 Central Research Laboratories, Yomeishu Seizo Co., Ltd, Mnawa-Machi, Nagano 399-4601, Japan 2 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tohetsu-Cho, IsMkari-Gun, Hokkaido 061-0293, Japan 3 School of Pharmaceutical Sciences, Peking University, Beijing 100083, PR Chirm ABSTRACT: Many Phenylethanoid glycosides were isolated from Cistanche Herb, produced from Cistanche deserticola, C. salsa, and other Cistanche plants, C tubulosa, C sinensis and C. phelypaea (Grobanehaceae). Cistanoside A-I and tubuloside A-E were isolated from C. deserticola and C. tubulosa, respectively, together with known phenylethanoid glycosides; echinacoside, acteoside and isoacteoside. They possessed rhamnosyl (1—»3)glucosyl group. Suspensaside and forsythiaside, possessed rhamnosyl (1—»6)glueosyl group, were isolated as main components from Forsythia suspensa (Oleaceae), not from F. viridissima. On the other hand, acteoside and J3-hydroxyacteoside were isolated from F. viridissima as main components, not from F suspensa. Plantamajoside, isoplantamajoside and hellicoside, possessed glucosyl (1—»3)glucosyl group, were isolated from Plantago asiatica (Plantaginaceae) together with acteoside, P-oxoacteoside, P-hydroxyacteoside, and campenoside. Several phenylethanoid glycosides, forsythiaside, suspensaside, acteoside, |3hydroxyacteoside and plantamajoside, showed an antibiotic activity against Staphylococcus aureus. Forsythiaside, suspensa- side, plantamajoside and isoplantamajoside showed a strong inhibitory activity against cAMP phosphodiesterase, whereas acteoside and P-hydroxyacteoside showed a weak activity. Plantamajo- side, hellicoside exhibited a strong inhibitory activity on arachidonic acid-induced mouse ear edema. Acteoside and isoacteoside showed a weak activity. Suspensaside showed a continuous hypotensive activity on spontaneously hypertensive rat (SHR).

646

INTRODUCTION Cistanche (Orobanchaceae) plants parasite on the root of Chenopodiaceae and Tamaricaceae plants and are distributed in Middle Asia from China to Turkey, Arab and North Africa Cistanchis Herba (Roucongrong in Chinese) has specified as the fleshy stem of Cistanche deserticola Y. C. Ma and used for staminal tonic, treatment of male impotentz, female sterility, and cold sensation in the loins and knees in Chinese Pharmacopeia 2000 [1]. Other Cistanche plants, such as C. salsa (C. A. Mey) G Beck, C. sinensis G Beck and C. tubulosa (Schrenk) Wight have been used for the similar purposes [2-4]. We reported the pharmacognostical studies on Cistanche plants from the morphological, phylogenetic and chemical viewpoints [5-8]. The dried Forsythia fruit has been used since ancient times as an antidote, an anti-inflammatory agent and a diuretic in Japan, Korea and China [9]. In Japan, the dried fruit originating from F suspensa Vahl or F viridissima Lindley (Oleaceae) is listed in the Japanese Pharmacopeia XTV as crude drugs "Forsythiae Fructus". The dried fruit from F. koreana Nakai is also used as crude drugs in Korea On the other hand, the Forsythia leaves are used as health tea for cold in China. The Plantago aerial parts have been used since ancient times as a diuretic, an anti-inflammatory and an anti-asthmatic drug "Plantaginis Herba" in Europe and Asia [10,11]. Plantaginis Herba from Plantago asiatica L. (Plantaginaceae) is listed in the Japanese Pharmacopeia XTV as an important crude drug and an aqueous extract is also used as a medicine. The aerial parts of P. depressa Wild, P. major L. and P. lanceolata L. are also used as herbal medicines in Europe and Asia [10,11]. The seeds of P. asiatica have been used since ancient times as a diuretic, an antitussive, an expectorant and an anti-inflammatory drug in Japan and China [12]. In China, the seeds of P. depressa have been also used for the similar purposes. P. psyllium L. is cultivated in Spain and France for the seeds. The dried ripe seeds from this plant (Psyllium) are used in Europe as domulcents and in the treatment of chronic constipation, while the seeds from P. ovata Forskal (Ispaghula husk) are used for similar purposes in India and Pakistan [13]. We shall describe the isolation and structure elucidation and HPLC analysis of the phenylethanoid glycosides from several Cistanche plants growing in China, Mongolia, Kazakhstan, Pakistan, Turkey, Bahrain and Qatar, and from Forsythia plants and Plantago plants used as important herbal medicines.

647

PHENYLETHANOID GLYCOSIDES FROM CISTANCHE PLANTS Isolation of phenylethanoid glycosides from Cistanche plants Many reports on the phenylethanoid glycosides from Cistanche plants have been published in this twenty years. They are acteoside (verbascoside;l), 2'-acetylacteoside (2), isoacteoside (3), echinacoside (4), cistanoside A-E (5-9), cistanoside Q H (10,11), salidroside (rhodioloside; 12), decaffeoylacteoside (13), osmanthuside B (14), syringalide A 3'-a-rhamnoside (15), isosyringalide A 3'-a-rhamnoside (16), crenatoside (17), tubuloside A-E (18-22), jionoside D (23) and poliumoside (24), and tentatively named sinenside A (25), sinenside B (26) and are listed in Table 1. 1. Phenylethanoid glycosides from Cistanche salsa (C. A. Mey) Bunge Fourteen phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), isoacteoside (3), echinacoside (4), cistanoside A-E (5-9), cistanoside G, H (10,11), salidroside (12), decaffeoylacteoside (13) and osmanthuside B (14) were isolated from the whole body of C. salsa in China[14]. Recently, tubuloside B (19) has been isolated from 75% ethanol extract of C. salsa stem[15]. 2. Phenylethanoid glycosides from Cistanche deserticola Y. C. Ma Seven phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), echinacoside (4), cistanoside A-C (5-7) and cistanoside H (11) were isolated from 50% ethanol extract of C. deserticola [16]. 3. Phenylethanoid glycosides from C. tubulosa (Schrenk) G Wight Seven phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), isoacteoside (3), rhodioloside (12), decaffeoylacteoside(13), syringalide A 3'-a-L- rhamnoside (15), and crenatoside (17) were isolated from 95% ethanol extract of C. tubulosa in China [17]. Five new phenylethanoid glycosides, tubuloside A-E (18-22) were isolated and their structures were elucidated, together with six known glycosides, acteoside (1), 2'-acetylacteoside (2), isoacteoside (3), echinacoside (4), syringalide A 3'-a-Lrhamn oside (15), isosyringalide A 3'-a-L-rhamnoside (16), from C. tubulosa in Pakistan [18,19]. 4. Phenylethanoid glycosides from Cistanchephelypaea (L.) Cout. Five phenylethanoid glycosides, acteoside (1), 2'-acetyacteoside (2), echinacoside (4), tubuloside A (18) and tubuloside E (22) were isolated from C. phelypaea in Qatar [20]. 5. Phenylethanoid glycosides from Cistanche sinensis G Beck in China Two new phenylethanoid glycosides, tentatively named sinenside A (25) and sinen-

648

OR,

coutn: R=H

HO

caff: R=OH fer: R=OCH3

Table 1. Phenylethanoid Clyeosides from Cistanche plants No.

Name

R3

R4

R5

R«

H

R2 rham(OH)3

caff

H

OH

OH

Ri

1

acteoside (verbascoside)

2

2-acetylacteoside

COCH3

rham(OH)3

caff

H

OH

OH

3

acteoside isomer(isoacteoside)

H

riiam(0H)3

H

caff

OH

4

echinacoside

H

rham(OH)3

caff

5

cistanoside A

H H H H

rham(OH)3

caff

glc glc

OH OH OCH3

OH

rham(OH)3

fa-

glc

OCH3

OH

rham(OH)3

caff

H

OCH3

OH

rham(OH)3

fer

H

OCH3

OH

H

rham(OH)3

H

OCHj

OH

H

H H H

6

cistanoside B

7

cistanoside C

8 cistanoside D 9 cistanoside E 10

cistanoside G

H

rham(OH)3

11

cistanoside H

COCH3

rham(OH)3

12

salidroside(rhodioloside)

H

H

13

decaffeoylacteoside

rham(OH)3

H H H

14

osmaiithuside B

rham(OH)3

OH

H

OH

OH

OH

H

H

OH

H

OH

OH

count

H

H

OH

rham(OH)3

15

syringalide A 3' o-rhamnoside

H H H

caff

H

H

OH

16

isosyringalide A 3' a-rhamnoside

H

rham(OH)3

coum

H

OH

OH

18

tubuloside A

COCH,

rham(OH)3

caff

glc

OH

OH

19

tubuloside B

COCH3

rham(OH)3

H

caff

OH

OH

20

tubuloside C

COCH3

rham(Ac)3

caff

glc

OH

OH

21

tubuloside D

COCH3

rham(Ac)3

coum

glc

OH

OH

22

tubuloside £

COCH3

rham(OH)3

eoum

OH

OH

H H

rham(OH)3

caff

H H

OH

OCHj

rham(OH)3

caff

rham(OH)3

OH

OH

caff

glc glc

H H

OH

caff

23 jionoside D 24

poliumoside

25

sinenside A

H

rham(OH)3

26

sinenside B

COCH3

rham(OH)3

H 2 OH

B HO 17

crenatoside{orobanchoside)

OH

OH

649

side B (26), have been elucidated by LC/MS, as major components together with acteoside (1), 2'-acetylacteoside (2), isoaeteoside (3), echinacoside (4), eistanoside A (5), eistanoside C (7), tubuloside A (18) and tubuloside B (19), jionoside D (23) and poliumoside (24), from Cistanche sinensis G Beck in China [21,22]. HPLC analysis of phenylethanoid glycosides from Cistanche plants and Cistanchis Herba We reported HPLC analysis of the phenylethanoid glycosides of crude drug Cistanchis Herba, and C. deserticola, C. salsa, C. salsa vat. albiflora, C. tubulosa and C, sinensis in China. They were similar to one another except C. sinensis, which was different from the others; the major phenylethanoid glycosides were sinenside A (25) and sinenside B (26) [8]. We measured the content of acteoside (1), 2'-acetylacteoside (2), isoaeteoside (3), echinacoside (4), eistanoside A (5), cistanoside C (7) and tubuloside A (18) of C. deserticola in China, C. salsa in China and Turkey, C. tubulosa in China, Pakistan and in Bahrain, and C. phelypaea in Qatar [6]. Further our study on Cistanche plants by HPLC showed that most Cistanche plants have 1-7, osmanthuside B (14) and tubuloside E (22) [23]. HPLC chromatogram of Cistanche plants were shown in Fig. 1. HPLC analysis of phenylethanoid glycosides from callus of Cistanche tubulosa, C deserticola and C. phelypaea Contents of acteoside (1), 2-aceiylacteoside (2), isoaeteoside (3), echinacoside (4), eistanoside A (5), eistanoside C (7) and tubuloside A (18) in callus and regenerated plants of C tubulosa and C. phelypaea were analysed by HPLC [6\. It is very interesting thai all callus, regenerated plants of C. tubulosa and C phelypaea showed no peak of eistanoside A (5) nor eistanoside C (7), as well as their normal plants. It seemed that 5 and 7 are able to be used as the marker compounds in chemotaxonomy of Cistanche plants. Callus of C desrticola is abundant in echinacoside (4) and poor in eistanoside C (7) and tubuloside A (18). Structure elucidation of phenylethanoid glycosides from Ciatanche plants Structure elucidation of phenylethanoid glycosides were carried out on the bask of spectral analysis and chemical evidences. Assignment of the 13C-NMR chemical shifts of phenylethanoid glycosides were le-examined and shown in Table 2. [24] Two dimensional NMR spectra, such as ^ - ^ ( C O S Y ) , *H-

650 13

C(HMQC and HMBC) directly afforded the importnat structural informatioa Acteoside (verbascoside: 1) was isolated as an amorphous powder. The sonic spray ion mass (SSI-MS) showed the negative ion at m/z 623 (M-H)", 461(M-163)' and 161 in Table 3. By acetylation with acetic anhydride-pyridine, 1 gave the amorphous nonaacetate, suggesting the presence of five alcoholic acetoxyl groups at 8 All O.OIOr

"i"335'W|

la

14 19 18

i 3 ,61

30

40

] I 50

AU 0.100j

TTSl

0.050:

18

J3 10

20

30

2 40

50

UV 335 m

0.100

25

26

0.050

aiC.ckserttoh fiom China b.Csalsa fxm China

19

, 18 10

20

30

LUj 40

50

Fig.1. HPLC Chromatogram of Cistanche plants

c.C.tuhdasa from China ACtubulosa from Pakistan e.Csinensis from China

651 651

1.87,1.94,2.02, 2.08 and 2.10 (3H, each), and four phenolic acetoxyl groups at 5 2.27, 2.28 (3H, each) and 2.30 (6H). By methanolysis[25] with acetyl chloride in methanol, 1 afforded methyl caffeate and 3,4-dihydroxy- phenethyl alcohol, and by acid-hydrolysis with 10% sulfuric acid, 1 gave glucose and rhamnose in a ratio of 1 to 1. The 13C-NMR chemical shifts of 1 were shown in Table 2. Compound 1 was identified as acteoside [26,27] (verbascoside [28,29]) by comparison with authentic sample. 2'-Acetylacteoside (2) was isolated as an amorphous powder. The SSI-MS showed the negative ion at m/z 665 (M-H)", 503 (M-163)", 461 (M-205)" and 161 in Table 3. The spectral data of 2 suggested the structural resemblance to 1 except for the glucose moiety in 13C-NMR spectrum (Table 2) and for the presence of an alcoholic acetoxyl group at 5 1.99 (3H) in ^ - N M R spectrum. Acetylation of 2 gave the octaacetate, which was identified as the nonaacetate of 1. The location of the acetoxyl group in the glucose moiety was determined by its acetylation shift value in the 13C-NMR chemical shifts. The 13C-NMR spectrum of 1 showed the signals at 104.1, 75.9 and 81.6 ppm due to C-l', C-2' and C-3' carbons of glucose moiety, respectively, whereas those of 2 showed the signals of corresponding carbons at 101.6,75.1 and 80.3 ppm, respectively, in Table 2. The differences in the chemical shifts of the corresponding carbon atoms of 2 and 1 were -2.5 (C-l1), -0.8 (C-21) and -1.3 ppm (C-31) in Table 4. The differences showed that an acetoxyl group was linked to the C-2' hydroxyl group of the glucose moiety [30]. Compound 2 was identified as 2'-acetylacteoside [31] by comparison with an authentic sample. Isoacteoside (3) was isolated as an amorphous powder, whose 13C-NMR chemical shifts were shown in Table 2. The SSI-MS showed the negative ions at m/z 623 (M-H)", 461 (M-163)" and 161 in Table 3. Compound 3 was identified as isoacteoside [25] by comparison with authentic sample. Echinacoside (4) was isolated as an amorphous powder. The SSI-MS showed the negative ions at m/z785 (M-H)", 623 (M-163)"and 161 in Table 3. The 13CNMR spectrum of 4 was silimar to that of 1 except for six carbon signals due to a glucose moiety (in Table 2). The respective signals at 74.5 and 69.2 ppm, due to C-5' and C-6' carbons of glucose, showed upfield shift by 1.6 ppm and downfield shift by 6.9 ppm, respectively, indicating that a glucose moiety is linked to C-6' hydroxy group of inner glucose (in Table 5). Compound 4 was identified as echinacoside [32] by comparison with authentic sample. Cistanoside A (5) was isolated as an amorphous powder. The SSI-MS showed negative ions at m/z 799(M-H)", 637(M-163)" and 161 in Table 3 and the field desorption mass (FD-MS) exhibited positive ion at m/z 823 (M+ Na)+. The 'HNMR spectrum of 5 showed the signal at 8 3.84 due to an aromatic methoxyl

652

group. By aeetylation, 5 afforded the undeeaaeetate, whose 'H-ISIMR spectrum exhibited the presence of eight alcoholic acetecy groups at 8 1.87,1.94,2.00,2.10 (3H each), 1.98 and 2.03 (6H each), and three phenolic acetoxyl groups at 5 2.29 Table 2. 13 C-NMR Chemical 1 2 3 Aglycone 1 131.6 131.9 131.4 2 117.2 117.2 117.1 3 146.0 145.9 146.0 4 144.4 144.4 144.6 116.6 116.6 116.3 5 121.4 121.4 121.3 6 72.3 a 72.3 72.5 36.5 36.2 36.6 P Ester 1 127.7 127.7 127.7 2 115.5 115.5 115.1 146.6 146.6 146.7 3 4 149.5 149.6 149.5 116.4 116.4 116.5 5 123.2 123.2 123.1 6 148.0 148.1 147.2 7 114.8 114.7 114.9 8 9 168.3 168.1 169.1 Glucose 104.1 101.6 104.3 75.4 2' 75.1 75.9 3' 80.3 84.0 81.6 70,0 4' 70.3 70.7 75.6 76.1 5' 76.0 6' 62.3 62.2 64.6 Rhamnose V 102.8 103.1 102.7 2" 72.0 72.3 72.1 3' 71.7 72.3 72.1 4' 73.6 74.0 73.8 5' 70.7 70.4 70.7 17.8 6' 18.4 18.4 Glucose

r

1" 2» 3" 4" 5" 6" OCHj CH 3 CO

20.9 171.5

Shifts of Phenylethanoid Glycosides (in Methanol-rfj) 4 7 11 6 8 5 9 10 131.4 131.5 131.6 131.6 131.6 131.6 117.1 113.9 113.9 113.9 114.0 113.9 145.7 148.7 148.7 148.8 148.8 148.8 144.3 145.7 145.9 145.9 145.9 145.9 116.4 116.1 116.2 116.6 116.6 116.2 121.3 122.4 122.4 122.4 122.4 122.4 72.1 72,1 72.2 72.3 72.3 72.3 36.4 36.6 36.6 36.6 36.7 36.7

12

130.7 130.8 116.1 156.7 116.1 130.8 72.3 36.3

131.8 116.2 144,5 145.9 117.1 121,2 72.4 36.3

130.8 130.8 116.2 156.7 116.2 130.8 72.0 36.4

101.8 74.8 83.1 70.3 77.9 62.5

104.4 75.1 78.1 71.8 77.9 62.8

127.5 115.3 146.4 149.5 116.4 123.1 148.1 114.7 168.3

127.6 115.4 146.6 149.5 116.5 123.1 148.1 114.7 168.3

127.6 112.1 150.7 149.3 116.5 124.2 148.0 115.1 168.3

127.7 115.5 146.7 149.6 116.2 123.2 147.9 114.8 168.3

127.2 112.1 150.7 149.3 116.2 124.2 147.8 115.2 168.2

103.9 75.9 81.5 70.2 74.5 69.2

104.1 75.9 81.5 70.3 74.6 69.3

104.1 76.1 81.4 70.3 74.7 69.4

104.1 76.0 81.6 70.3 76.1 62.4

104.2 76.0 81.4 70.3 76.0 62.5

104.2 75.5 84.8 70.1 77.7 62.8

104.1 75.5 84.6 70.1 77.7 62.7

102.7 72.1 72.1 73.7 70.5 18.3

102.8 72.1 72.1 73.7 70.6 18.4

102.8 72.2 72.2 73.7 70.7 18.4

102.8 72.1 72.0 73.8 70.7 18.4

102.8 72.2 72.1 73.8 70.8 18.4

102.7 71.9 72.3 74.0 70.3 17.9

102.7 102.9 72.3 71.6 72.3 72.1 74.0 73.7 70.2 70.3 17.9 17.8

104.4 74.9 77.6 71.3 77.6 62.5

104.5 74.9 77.6 71.4 77.6 62.6 56.6

104.6 75.0 77.7 71.5 77.7 62.6 56.6 56.6

56.6

56.6 56.6

56.6 20.9 171.6

(3H) and 2.31(6H). The 13C-NMR spectrum of 5 showed almost the same chemical shifts as those of 4 except for the signals due to aglycone moiety (in Table

653 653 Table 2. Continued 14 13 Aglycone 1 131.6 130.7 2 116.3 131.2 3 144.6 116.1 4 146.0 156.6 5 117.1 116.1 6 121.3 131.2 72.1 72.3 a 36.2 36.5 P Ester 1 127.1 130.8 2 3 116.8 4 161.1 5 116.8 6 130.8 7 147.5 g 114.8 9 168.2 Glucose 1' 104.1 104.1 2' 75.5 76.0 3' 84.6 81.5 4' 70.2 70.1 5' 77.7 75.9 6' 62.7 62.3 Rhamnose 1' 102.7 102.7 72.2 2' 72.1 3' 72.1 72.3 4' 74.0 73.7 5' 70.2 70.7 6' 18.4 17.9 Glucose 1" 2" 3" 4" 5" 6" Rhamnose 1" 2" 3" 4" 5" 6" OCH3 CH3 CO

15

16

17

18

19

20

21

22

23

24

130.6 130.8 116.1 156.6 116.1 130.8 72.1 36.2

131.4 117.0 146.0 144.5 116.3 121.2 72.2 36.5

129.1 113.8 145.7 145.7 115.5 118.2 77.8 72.3

131.8 117.2 145.9 144.5 116.3 121.4 72.5 36.2

131.7 117.1 145.9 144.4 116.3 121.3 72.4 36.3

131.8 117.1 145.9 144.4 116.3 121.4 71.8 36.2

131.7 117.1 145.9 144.4 116.3 121.3 71.9 36.2

131.7 117.1 145.9 144.4 116.2 121.3 72.5 36.2

132.9 117.1 147.4 144.5 112.9 121.2 72.1 36.5

131.5 117.1 146.1 144.7 116.5 121.3 72.1 36.6

127.6 115.2 146.6 149.6 116.4 123.1 147.0 114.6 168.2

127.1 131.2 116.8 161.2 116.8 131.2 147.5 114.7 168.2

126.9 114.6 146.2 149.2 115.8 122.6 167.3 147.6 113.8

127.6 115.4 146.7 149.8 116.6 123.3 148.3 114.6 168.2

127.7 115.2 146.7 149.5 116.5 123.1 147.2 114.9 169.0

127.4 115.3 146.8 149.9 116.6 123.2 148.4 114.3 168.0

126.8 116.9 131.4 161.5 131.4 116.9 148.0 114.4 168.1

126.9 131.3 116.8 161.3 116.8 131.3 147.7 114.6 168.0

127.7 115.3 146.8 149.8 116.5 123.2 148.0 114.7 168.3

127.7 115.3 146.8 149.8 116.4 123.2 148.0 114.8 168.0

104.1 76.0 81.5 70.3 75.9 62.3

104.1 76.1 81.5 70.2 75.9 62.3

98.4 81.3 76.7 69.7 77.2 61.4

101.6 75.0 80.5 70.7 74.8 69.2

101.8 74.8 82.6 70.3 75.4 64.4

101.6 75.5 80.1 70.5 74.6 69.1

101.6 75.0 80.3 70.7 74.6 69.1

101.6 75.0 80.4 70.7 76.0 62.1

104.2 76.2 81.7 70.4 76.0 62.4

104.4 76.2 81.6 70.5 74.7 67.6

102.8 72.1 72.1 73.7 70.5 18.3

102.8 72.1 72.1 73.7 70.6 18.4

101.5 71.4 71.3 72.9 69.5 17.7

103.9 71.9 71.4 73.6 70.7 18.5

102.8 72.1 71.8 73.7 70.5 17.8

99.5 70.0 71.2 71.4 68.2 18.0

99.6 70.1 71.2 71.4 68.2 18.0

103.1 71.8 71.8 73.5 70.7 18.4

103.0 72.4 72.1 73.8 70.6 18.5

103.0 72.3 72.0 73.8 70.4 18.4

104.6 74.6 77.7 71.8 77.7 62.6

104.6 74.6 77.7 71.9 77.7 62.6

104.6 75.0 77.8 71.9 77.8 62.6

102.3 72.3 72.0 74.0 69.9 18.0 56.5 20.9

20.9

171.5

171.6

20.4 20.9 171.2 171.5

20.4 20.9 171.2 171.5

20.9 171.4

654

2). Compound 5 gave methyl cafifeate and 3-methoxy-4-hydroxyphenethyl alcohol by methanolysis, and afforded glucose and rhamnose in a ratio of 2 to 1, on acid hydrolysis. Consequently, the structure of cistanoside A (5) was determined to be 2-(4-hydroxy-3-methoxyphenyl) ethyl-(>a-L 6)]-(4-O-caffeoyl)-2-O-acetylTff-D-glucopyranoside. Tubuloside D (21) was isolated as an amorphous powder. The SSI-MS showed negative ion at m/z 937 (M-H)~. The 'H-NMR spectrum showed the presence of four alcoholic acetoxyl groups at 81.81, 1.93, 1.96 and 2.09 (3H each, s). Acetylation of 21 gave the heptaacetate, of which ' H - N M R exhibited the prsence of eight alcoholic acetoxyl groups at 8 1.87,1.94,1.96,1.99,2.10 (3H each, s), 2.02 (9H, s) and three phenolic acetoxyl groups at 8 2.27,2.30 and 2.32 (3H each, s). The 13CNMR spectrum of 21 was similar to that of 20, except for the signals due top- coumaric acid moiety in Table 2. Methanolysis of 21 with acetyl chloride in methanol showed the presence of methyl />coumarate and 3,4-dihydroxyphenethyl alcohol. Acid hydrolysis of 21 with sulfuric acid gave glucose and rhamnose. Consequently, tubuloside D (21) was determined to be 2-(3,4-dihydroxyphenyl) ethyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl (1^3)-0-|$-D-glucopyranosyl(l—• 6)]-(4-O-j9-coumaroyl)-2-O-acetyl-y?-D-glucopyranoside. Tubuloside E (22) was isolated as an amorphous powder. The SSI-MS showed negative ion at m/z 649 (M-H)~. The ^ - N M R spectrum of 22 exhibited the signals due to a methyl group of rhamnose at 8 1.07 (3H, d, J=6Hz), an acetoxyl group at 8 1.98 (3H, s), benzylic methylene protons at 8 2.72 (2H, t, J=7Hz), a glucose anomeric proton at 84.54 (1H, d, J=8Hz), a rhamnose anomeric proton at 8 5.00 (1H, brs), two olefinic protons at 8 6.34 and 7.66 (1H each d, J=\6Hz) and aromatic protons at 8 6.50-7.46 (7H). Acetylation of 22 gave the octaacetate, of which the

659

'H-NMR spectrum exhibited the presence of five alcoholic acetoxyl groups at 5 1.87,1.95,2.02 (3H each, s) and 2.10 (6H s), and three phenolic acetoxyl groups at 5 2.27,2.28,2.31 (3H each, s). The 13C-NMR spectrum of 22 was almost identical with that of 2, except for the the signals due to the j>cournaric acid moiety (in Table 2). Tubuloside E (22) was determined to be 2-(3,4-dihydroxyphenyl) ethyl-0-a-Lrhamnopyranosyl( 1 —>3)-2-(9-acetyl-4-O-j!>coumaroyl-P-D-glucopyranoside. Jionoside D (23) was isolated as an amorphous powder and showed negative ion at m/z 637 (M-H)" in FABMS. The 'H-NMR spectrum of 23 exhibited an anomeric proton of glucoe at 8 4.37 (1H, d, ^ 8 . 1 Hz) and that of rhamnose at 8 5.18 (1H, d, J=\2 Hz). The 13C-NMR spectrum was also identical with that of jionoside D [36]. Poliumoside (24) was isolated as an amorphous powder and showed negative ion at m/z 769 (M-H)" in FABMS. The 'H-NMR spectrum of 24 exhibited an anomeric proton of glucoe at 8 4.27 (1H, d, ^ 8 . 0 Hz) and two that of rhamnose at 8 4.52 (1H, d, J=2.Q Hz) and 8 5.08 (1H, d, J=\ .5 Hz) and sujested phenylethanoid glycoside. The 13C-NMR spectrum was also identical with that of poliumoside [37]. Compound 24 was identified as poliumoside [37], by comparison with authentic sample. Sinenside A (25) showed negative ions at m/z 110 (M) ", 769 (M-l)", 607 (M-163)" and 161 in SSI-MS. The UV spectra showed absorption maxima at 233.6 and 329.1 ran. Known phenylethanoid glycosides, compound 1,3-5, and 7 also showed the SSI-MS ions at m/z (M-l)', (M-163)", and 161 (Table 3). Fragment ion at m/z (M-163) " was determined as the cleavage of caffeoyl group (M-C9H7O3)". Sinenside A (25) was tentatively determined to be 2-(4-hydroxyphenyl) ethyl-O-a-L-rhamnopyranosyl(l—»3)-O-|j5-D-glucopyranosyl (1—>6)]4(9-caffeoyl-/?-D-glucopyranoside. Sinenside B (26) showed SSI-MS ions at m/z 811 (M-l)", 649 (M-163)", 607 (M-163-42)" and 161 (Table 3). Fragment ion at m/z 607 was determined as the cleavage of caffeoyl and acetyl groups (M-C9H7O3- COCH2)". Known acetylated phenylethanoid glycosides, 2 and 18-20 also showed the ions at m/z (M-l)", (M-163)", (M-163-42)" and 161(Table 3). Sinenside B (26) was tentatively determined to be 2-(4-hydroxyphenyl) ethyl-O-a-L-rhamnopyranosyl (1—>3)-0-|/?-Dglucopyranosyl (1 —>6)]-(4-O-caffeoyl)-2-O-acetyl-y5-D-glucopyranoside. SPECTROSCOPIC ANALYSIS OF PHENYLETHANOID GLYCOSIDES Phenylethanoid glycosides consist of phenyethyl glucosyl ether, acyl ester (coumaroyl, feruroyl and caffeoyl) and glycosyl groups. The structure of isolated phenylethanoid glycosides was determined or identified by comparison with UV, IR, MS

660

and NMR spectra and chemical evidence. 13 C-NMR chemical shifts of isolated phenylethanoid glycosides were shown in Table 2. At present, two dimensional NMR spectra, such as ^HfCOSY), 1H13 C(HMQC and HMBC) directly afford the importnat structural information. Chemical shifts by methylation, glycosyMon and acylation suggested the information of substituted carbon positions. 13

C-NMR chemical shifts of phenylethanoid gtycosides

Studies on the structure elucidation of phenylethanoid glycosides of 13C-NMR, Hie change of chemical shift value were observed. Tab4e4Acetylat»n Shifts ofCarbon Signals in Methanol-di

104.1

101.6

101.6

-2.5

-Z5

1035

101.6

101.6

8 (184) -23

2

75.9

75.1

75.0

-0.8

-05

755

75.0

75.5

-0.9

-0.4

3'

81.6

803

80.4

-13

-12

81.5

80.5

80.1

-1.0

-1.4

1

2

22

8(2-1)

8(22-1)

4

18

20

8 CSM) -23

TaHe 5. Glycosylatwn Shifts of Carbon Signals inMethanoM,

Glucose

1

4

5

6

8(4-1)

8(5-1)

8(6-1)

2

19

5"

76.1

74.5

74.6

74.7

-1.6

-1.5

-1.4

76.0

74.8

8 (18-2) -12

6"

623

692

693

69.4

65

7.0

7.1

622

692

7.0

Table 6. Methylation Shifts of Carbon Signals in Methanol-rf4

1 Aglycone 2 117.2 4 144.4 5 116.6 6 121.4

7

8

9

113.9 145.9 116.6 122.4

114.0 145.9 116.6 122.4

113.9 145.9 116.2 122.4

5

6

113.9 145.7 116.1 122.4

113.9 145.9 116.2 122.4

8(5-4) -3.2

8(7-1) -3.3

8(8-1) -3.2

8(9-1) -3.3

1.5 0.0 1.0

1.5 0.0 1.0

-0.4

1.5 1.0

Table 6. Continued

4 Aglycone 2 117.1 4 144.3 5 116.4 6 121.3

8(6-4) -3.2

1.4

1.6

-0.3

-0.2

1.1

1.1

8(8-1) -3.4 4.1 1.0

8(8-7) -3.4 4.0 1.0

Table 6. Continued Ester

2 3 6

1

7

g

115.5 146.6 123.2

115.5 146.7 123.2

112.1 150.7 124.2

4 115.3 146.5 123.1

5

6

8(6-4)

8(6-5)

115.4 146.6 123.1

112.1 150.7 124.2

-3.2 4.2 1.1

-3.3 4.1 1.1

661

Acetylation of C-21 position at inner glucose caused the acetyMon shifts of C-l', 2' and 3' carbons of glucose. Their shifts values were about (-2,4) ppm, from (-0.4) to (-0.9) ppm and firni (-1.0) to (-1.4) ppm, respectively (in Table 4). GlycosyMon of C-6' position at glucose caused the glycosylation strife of carbon signals of C-51 and C-6' position, the former changed from (-1.2) to (-1.6) ppm and the latter shifted about (+7.0) ppm, respectively, as shown in Table 5. MethyMon shifts of phenol groups were shown in Table 6. In Table 6, C-2,4, 5 and 6 carbons in aglycone changed in the range from (-2.5) to (-2.7) ppm, about (+4.4) ppm, from (-0.6) to (-1.0) ppm and about (+1.0) ppm, respectively, by methylation of hydroxyl group at C-3 position of 3,4-dihydrojq?phenylethyl group. A similar change of chemical shifts value was observed by methylation of hydroxyl group at C-3 position of eafleoyl group in Table 6. The chemical shifts value of C-2,3 and 6 carbon signals changed about (-3.3), (+4.1) and (+1.0) ppm, respectively.

MS spectra of phenylethanoid glycosides MS spectra of phenylethanoid glycosides from Cistanche plants were measured by the SSI method. Compounds 1, 3-5, 7,14 and 25 showed negative ions at m/z (M-H), (M-163) and 161 in Table 3. Fragment ion at m/z (M-163) was deter mined as the cleavage of caffeoyl group (M-C9H7O3). Compounds 2, 18-20 and 26 showed negative ions at m/z (M-H) ", (M-163)", (M-205) "and 161 in Table 3. Fragment ion at m/z (M-205)" was determined as the cleavage of caffeoyl and acetyl groups (M - C9H7O3 - COCH2).

PHENYLETHANOID GLYCOSIDES FROM FORSYTHM PLANTS Isolation of phenylethanoid glycosides from Forsyihia plants Four phenylethanoid glycosides, forsythiaside (27), suspensaside (28), acteoside (1) and P-hydroxyacteoside (29), were isolated from the Forsyihia fruit and leaves [39-44]. They are listed in Table 7. 1. Phenylethanoid glycosides from Forsythkt suspensa Forsythiaside (27) and suspensaside (28) were isolated from the methanol extract of fruit [39,40] and forsythiaside (27) from the methanol extract of leaves [43,44], respectively. 2. Phenylethanoid glycosides from Forsythia viridissima Acteoside (1) and P-hydroxyacteoside (29) were isolated from the methanol

662

extract of Suit [41] and aeteoside (1) from the methanol extract of leaves [43,44], respectively. 3. Phenylethanoid glyeosides fiom ForsytMa komana Forsythiaside (27), suspensaside (28), acteoade (1) and pMrydroxyacteoside (29) were isolated from the methanol extract of fruit [42], andfcrsythiaside(27) and aeteoside (1) from the the methanol extract of leaves [43,44], respectively. Structure elucidation of phenylethanoid glyeosides from Forsythia plants The structure elucidations of the novel compounds, fbrsythiaside (27), suspensaside (28) and (3-hydroxyacteoside (29) were carried out on the basis of the analysis of 'H- and I3C-NMR spectra and chemical evidences [39,40]. They are listed in Table 7. Forsythiaside (27) was obtained as pale yellowish powder, C29H36O15, mpl44150 °C, whose molecular weight was confirmed by the observation of m/z 647 (M+Na) +on field desorption mass spectrometry (FD-MS). The spectral data of 27 suggested that 27 bears a marked structural resemblance to aeteoside (1). Alkaline treatment of 27 followed by acid hydrolysis gave caffeie acid, 3,4-dihydroxyphenethyl alcohol, D-glucose and L-rhamnose. The 13C-NMR of 27 indicated the attachment of the cafleate moiety at the C-4' carbon of glucose and of the rhamnose moiety at the C-61 carbon of glucose (Table 8). The chemical shift of the C-a carbon of 27 at 72.2 ppm suggested the linkage of the 3,4-dihydroxyphenethyl moiety to the C-l1 carbon of glucose. Consequently, the structure of fbrsythiaside (27) was established as 3,4Klihydroxyphenethyl-«-L-rhamnopyranosyl-(l—*6)4^£>«arreoyl-P-D-glucopyranoside. Suspensaside (28) was obtained as colorless powder, C29H36O16, mpl77-182°C, whose molecular weight was confirmed by the observation of m/z 640 (M4) on FD-MS. The molecular formula of 28 differs in composition by an increment of one unit of oxygen atom relative to that of 27. The lH-NMR spectrum of 28 resembled that of 27 except for the disappearance of the signal (8 2.80,2H, t, J=7 Hz) assigned to two benzyl protons of the phenethyl moiety. The spectral data of 28 suggested that it bears a marked structural resemblance to 27. The chemical reaction and ^C-NMR spectral data suggested that the phenylethanol moiety of 28 consists of |3,3,4-trihydroxyphenethyl alcohol (Table 8). The 13C-NMR of 28 indicated the attachment of the caffeate moiety at the C-41 carbon of glucose and of rhamnose moiety at the C-61 carbon of glucose. The chemical shift of the C-a carbon of 28 at 76.8 ppm suggested the linkage of the P,3,4-trihydroxyphene1hyl moiety to the C-l' carbon of glucose. With regard to the problem of the absolute configuration at the C-P position in 28, the molecular optical rotation differences

663

between 27 and 28 (A[M]D -3.6°) and between deacylforsythiaside dimethyl ether and deacylsuspensaside dimethyl ether ( A | M ] D +3.0°) were compared with the molecular optical rotation of (+)-phenylethane-l,2-diol [(+)-P-hydroxyphenethyl alcohol] (|M|D +83.2°). It was expected that the molecular optical rotation value attributable to the P-hydroxyphenethyl moiety of 28 having the ^-configuration at

caff

No. 1 27 28 29 30 31 32 33 35 36 37 38

Table 7. Phenylethanoid Glycosides from Forsythia and Plantago plants R, R, R2 Name R4 Ri H rham caff H OH acteoside (verbascoside) forsythiaside H rham OH H caff suspensaside H rham H caff OH P-hydroxyacteoside rham H H caff OH plantamajoside glc H H caff OH hellicoside H OH H caff glc glc isoplantamajoside H caff H OH H 3,4-DPCG H caff H OH campenoside rham caff H H OH p-oxoacteoside rham caff H H OH lavandulifolioside rham-arab caff H H OH forsythoside B rham apio caff H OH

Rfi

R?

H H OH OH H OH H H H

H H H H H H H H OCOCH3 =O

H H

H H

CH 2 OR 3

Table 7. continued No. Name 34 plantasioside 17 orobanchoside(crenatoside)

R, H rham

R2 H caff

R3

caff H

C-P position would probably be nearly equal to that of (+)-phenylethane-l,2-diol. The value of the molecular optical rotation differences suggested that P,3,4-trihydroxyphenethyl moiety is a racemate. Consequently, the structure of suspensaside (28) was established as DL-P3,4-trihydroxyphenethyl-(9-a-L-rhamnopyranosyl-(l->6)-4-O-caffeoyl-P-D-glucopyranoside. P-Hydroxyacteoside (29) was obtained as an amorphous powder, C29H36O16, mpl77-183 °C, whose molecular weight was confirmed by the observation of. m/z 663 (M+Na) + on FD-MS. The 'H-NMR spectrum of 29 resembled that of acteoside (1) except for the disappearance of the signal assigned to the two benzyl protons of the phenethyl moiety. The chemical reaction and 13C-NMR spectral data

664

of 29 clearly suggested that 29, like 28, consists of P,3,4-trihydroxy-phenethyl moiety and a rhamno-glucose moiety containing a caffeoyl group (Table 8). The 13 C-NMR spectrum of 29 indicated the attachment of the caffeate moiety at the C-4'carbon of glucose and of the rhamnose moiety at the C-31 carbon of glucose. The chemical shifts of the C-a carbon of 29 at 76.3 ppm and of the C-l' carbon of Table 8. "C-NMR Chemical Shifts of Phenylethanoid Glycosides in (Metlianol-3)-4-O-cafFeoyl-P-D-glucopyranoside. With regard to the problem of the absolute configuration at the C-P position of the P-hydroxyphenethyl moiety, the molecular optical rotation difference between deacylacteoside dimethyl ether and deacyl-P-hydroxyacteoside dimethyl ether (A [M]D +14.1°) was compared with that between deacylforsythiaside dimethyl ether and deacylsuspensaside dimethyl ether (A[M]D +3.0°), and a related compound, (+)-phenylethane-l,2 -diol [(+)-P-hydroxyphenethyl alcohol]([M]D +83.2°). The value of the molecular optical rotation difference suggested that the P-hydroxyphenethyl moiety has both S- and i?-configuration in a ratio of approximately 7:5. PHENYLETHANOID GLYCOSEDES FROM PIANTAGO PLANTS Isolation of phenylethanoid glycosides from Plantago plants Thirteen phenylethanoid glycosides, plantamajoside (30), hellicoside (31), acteoside (1), isoplantamjoside (32), 3,4-dihydroxyphenethyl alcohol-6-O-caffeoyl-PD-glucoside (3,4-DPCG) (33), plantasioside (34), P-hydroxyacteoside (29), campenoside (35), P-oxoacteoside (36), orobanchoside (oraposide, crenatoside;17), lavandulifolioside (37) and isoacteoside (3), were isolated from the Plantago herbs [4549]. Two phenylethanoid glycosides, acteoside (1) and forsythoside B (38), were isolated from the Plantago seeds [50,51]. 1. Phenylethanoid glycosides from P. asiatica Plantamajoside (30), hellicoside (31), acteoside (1), isoplantamjoside (32), 3,4dihydroxyphenethyl alcohol-6-O-caffeoyl-P-D-glucoside (3,4-DPCG) (33) and plantasioside (34) were isolated from the methanol extract of herb [45,46] and acteoside (1) from the methanol extract of seed [50], respectively. 2. Phenylethanoid glycosides from P. depressa Acteoside (1), P-hydroxyacteoside (29), campenoside (35), P-oxoacteoside (36) and orobanchoside (17) were isolated from the methanol extract of herb [47] and acteoside (1) from the methanol extract of seed [50], respectively. 3. Phenylethanoid glycosides fromi? major Plantamajoside (30), acteoside (1) and isoplantamajoside (32) were isolated from the methanol extract of herb [48]. 4. Phenylethanoid glycosides from P. lanceolata Acteoside (1), plantamajoside (30), lavandulifolioside (37) and isoacteoside (3) were isolated from the methanol extract of herb [49]. 5. Phenylethanoid glycosides from P. ovata Acteoside (1) and forsythoside B (38) were isolated from the methanol extract

666

of seed [51]. 6. Phenylethanoid glycosides from P. psyllium Acteoside (1) and forsythoside B (38) were isolated from the methanol extract of seed [51]. Structure elucidation of phenylethanoid glycosides from Plantago plants The structure elucidation of the novel compounds, plantamajoside (30), hellicoside (31), plantasioside (34), P-oxoacteoside (36), orobanchoside (17) was carried out on the basis of the analysis of 1 H- and 13C-NMR spectra and chemical evidences [4549]. Another known compounds were identified by comparison with respective authentic samples or comparison of their spectral data with those reported in the literatures. Plantamajoside (30) was obtained as an amorphous powder, whose structure was elucidated as 3,4-dihydroxyphenethyl-O-P-D-glucopyranosyl-(l^'3)-4-Ocaffeoyl-P-D-glucopyranoside on the basis of the analysis of 1H- and 13C-NMR spectra (Table 8) and chemical evidences by Ravn et al [48]. Hellicoside (31) was obtained as an amorphous yellow light powder, whose molecular formula was confirmed by the observation of m/z 679 [M (C29H36O17) + Na] + by positive ion FAB mass spectrometry. The 'H-NMR spectrum resembled that of 30 except for the disappearance of the signal assigned to two benzyl protons of the phenethyl moiety. The 'H-NMR spectrum of acetate of 31 showed the presence of seven alcoholic acetoxy and four phenolic acetoxy groups, and a proton at the benzyl position bearing an acetoxy group. These data suggested that 31 bears a marked structural resemblance in the sugar moiety to 30 and in the phenethyl moiety to 29. Partial hydrolysis of 31 gave desrhamnosyl P-hydroxyacteoside and 4-caffeoylglucose. Total hydrolysis of 31 yielded only glucose. The results clearly indicated that 31 consists of a P,3,4-trihydroxy-phenethyl moiety and a glucose-glucose moiety containing a caffeoyl group. The 13C-NMR of 31 supported the attachment of the caffeate moity at C-4' position of the inner glucose, the glucose moiety at C-3' position of the inner glucose and the linkage of the inner glucose moiety to the C-a position of P,3,4-trihydroxy-phenethyl alcohol (Table 8). Consequently, the structure of hellicoside (31) has been established as P,3,4-trihydroxyphenethyl-O-P-D-glucopyranosyl-( 1 ^3)-4-0-caffeoyl-P-D-glucopyranoside. For the absolute configuration at C-P position of the P-hydroxyphenethyl moiety, the molecular optical rotation difference between31 and 30 was compared with that of (+)-phenylethane-l,2-diol [(+)-P-hydroxyphenethyl alcohol]. The value as expected ( A [ M | D + 9 8 . 1 O ) suggests that the P-hydroxyphenethyl moiety of 31 has the S-configuratioa

667

Plantasioside (34) was obtained as an amorphous powder, whose molecular formula (C23H24O11) was confirmed by the observation of ions at m/z 499 (M + Na) + and m/z 477(M + H) + by positive ion FAB-mass spectrometry. The 'H-NMR spectrum of 34 resembled that of 3,4-dihydroxyphenethyl dcohol-6-O-caffeoylp-D-glucoside (3,4-DPCG) except for displaying signals at 5 4.53 (1H, dd, J= 3, 10 Hz) assignable to a C-p proton and 5 3.93 (1H, dd, J= 3,13 Hz), 5 3.65 (1H, m) assignable to C-<x protons instead of 8 2.77 (2H, t, J= 7 Hz), 5 3.56 (1H, m) and 8 3.73 (1H, m) in the phenethyl moiety. The ^ - N M R spectrum of the acetate showed the presence of two alcoholic acetoxy and four phenolic acetoxy groups but no presence of a proton at the benzyl position bearing an acetoxy group as that of acetate of 29. Hydrolysis of 34 with acid afforded only glucose. These data suggested that 34 bore a marked structural resemblance in the linkage between a glucose and a phenethyl moiety to that of oraposide from Orobanche spp which contains, besides the glycosidic linkage, an ether linkage between a glucose and a phenethyl moiety [36,52]. The 13C-NMR spectrum of 34 was correlated with those of 3,4-DPCG (33) and oraposide (17) (Table 8). The spectrum of 34 supported the attachment of the caffeate moiety at the C-6' position of the glucose moiety and the presence of an ether linkage between a glucose moiety and phenethyl moiety, besides the glucosidic linkage. Consequently, the structure of plantasioside (34) has been established as l',2'-[P (3,4-dihydroxyphenyi)-a, P-dioxoethanol]-6'-O-caffeoyl O-P-D-gJucopyranoside. P-Oxoacteoside (36) was obtained as an amorphous powder, whose molecular formula was confirmed by the observation of m/z 639 \M (C29H34O16) + H] + and m/z 661 [M(C2S)H34Oi6) + Na] + by positive ion FAB mass spectrometry. The ^ - N M R spectrum showed signals due to methyl protons, anomeric protons of a sugar moiety, aromatic protons of a phenethyl moiety bearing a carbonyl group at the P-position and protons of a caffeate moiety. The reaction of 36 in methanol with excess diazomethane gave deacyl-P-oxoacteoside dimethyl ether. Acid hydrolysis of 36 gave P-oxo-P-(3,4-dimethoxyphenyl)-ethanol, caffeic acid , D-glucose and L-rhamnose. P-Oxo-P-(3,4-dimethoxyphenyl)-ethanol was identical with the compound which was synthesized by the reaction of 3,4-dimethoxy-acetophenone with iodobenzene. These results clearly suggested that 36 consists of a P-oxo-P-(3,4dihydroxyphenyi)-ethyl moiety and a rhamno-glucose moiety containing a caffeoyl group. The 13C-NMR spectrum of 36 (Table 8) supported the attachment of the caffeate moiety at C ^ ' position of the inner glucose, the rhamnose moiety at C-3' position of the inner glucose and linkage of the inner glucose moiety to the exposition of P-oxo-P-(3,4-dihydroxyphenyl)-ethanol. Tthe structure of P-oxo-acteoside (36) has been established as P-oxo-P-(3,4-dihydroxyphenyl)-ethyl-O-a-Lrhamnopyranosyl-(l^'3)-P-D-(4-O-caffeoyl)-glucopyranoside.

668

Orobanchoside (oraposide, crenatoside:17) One of phenylethanoid glycosides from P. depressa was identical with orobanchoside from Orobanche rapumgenistae, whose structure was elucidated as P,3,4-trihydroxyphenethyl-O-a-L-rhamnopyranosyl-(1^2)4-0-caffeoyl-P-D-glucopyranoside by C. Andary et al [28]. In the process of structural elucidation of 30, it was found that the chemical shifts at the C-a and P positions of the phenethyl moity in the 13C-NMR spectrum of orobanchoside isolated from Plantago were consistent with those of 34 and oraposide [52], but 2different from that of 29. In addition , the positive ion FAB-mass spectrum of orobanchoside gave only the ion at m/z 645 as (M + Na)+, which was consistent with that of oraposide (molecular formula C29H32O14) [52]. Thus, the structure of orobanchoside should be replaced by that of oraposide. Furthermore, the 13C-NMR spectra of oraposide, and crenatoside (17) which was isolated from Orobanche crenata by Afifi et al. [36], were compared. As a result, the 13C-NMR spectra of orobanchoside, oraposide and crenatoside (17) in methanol-^ were completely superimposed (Table 8). The spectral data of orobanchoside were also in agreement with those of oraposide and crenatoside (17), and the X-ray analysis of orobancho- side supported these spectral data [52]. It was concluded that orobanchoside, orapo- side and crenatoside (17) are the same compound, that is, l',2'-[p(3,4-dihydroxyphenyl)-a,P-dioxoethanol]-4'-0-caffeoyl-0-a-L-rhamnopyranosyl-(1^3)-(9-P-Dglucopyranoside. BIOLOGICAL ACTIVITIES Antibacterial activity It has been known for many years that the Forsythia fruit is effective in the treatment of skin diseases. The antibacterial activities of the phenyletanoid glycosides from Forsythia fruit were examined [40,42]. A higher antibacterial activity against Staphylococcus aureus was observed in phenylethanoid glycosides. The minimum inhibitory concentration (MIC) of phenylethanoid glycosides against Staphylococcus aureus Terashima was determined by a broth dilution method. Forsythiaside (27), suspensaside (28), acteoside (1) and P-hydroxyacteoside (29) exhibited MIC activities of 3.2 mM (2.0 mg/ml), 4.1 mM (2.6 mg/ml), 3.2 mM (2.0 mg/ml) and 2.0 mM (1.3 mg/ml), respectively. This suggests that the antibacterial activity of Forsythia fruit is attributable to the phenylethanoid glycosides present in them. Plantamajoside (30) from Plantago herb also exhibited MIC activity of 4.1 mM (2.7 mg/ml) [45].

669

Inhibitory effect on cyclic AMP phosphodiesterase It is known that cyclic AMP inhibits the release of a chemical mediator fiom the mast cell. So when cyclic AMP phosphodiesterase is inhibited by some inhibitor, the concentration of cyclic AMP is increased to inhibit the release of the chemical mediator from the mast celL Inhibitors against cyclic AMP phosphodiesterase may be useful in the therapy for allergic diseases. In addition, by the presence of 5-lipoxygenase, free arachidonic acid is converted to leukotrienes, which is one of the chemical mediators known as a slow reacting substance of anaphylaxis. Therefore, specific inhibitors of 5-lipoxygenase may be useful in the therapy for allergic diseases. Phenylethanoid glycosides were assayed for their inhibitory effect on beef heart cyclic AMP phosphodiesterase [53]. Forsythiaside (27) and suspensaside (28) from F sumensa showed a high inhibitory effect with IC50 of 11.0 x 10'5 mol/1 and 18.3 x 10 mol/1, respectively. On the other hand, the IC50 of both acteoside (1) and fJhydroxyacteoside (29) from F. viridissima was over 50 x 10"5 mol/1. Plantamajoside (30) and hellicoside (31) from P. asiatica showed a high inhibitory effect with IC50 of 16.0 x 10"5 moM and 16.9 x 10"5 mol/1, respectively [45]. Inhibitory effect on 5-lipoxygenase Phenylethanoid glycosides fiom ForsytMa fiuits were assayed for their inhibitory effect on 5-lipoxygenase from rat peritoneal cells [54]. Forsythiaside (27), suspensaside (28), acteoside (1) and P-hydroxyacteoside (29) showed a high inhibitory effect with IC50 of 2.50 x 10"* M, 7.97 x 10 4 M, 5.27 x 10* M and 19.3 x 10"6 M, respectively. Phenylethanoid glycosides from Pktntago herbs were assayed for their inhibitory effect on 5-lipoxygenase from RBL-1 cells [45] Plantamajoside (30), isoplantamajoside (32), hellicoside (31), acteoside (1) and P-hydroxyacteoside (29) showed a high inhibitory effect with ICJO of 3.73 x 10"7M, 0.42 x 10"7 M, 3.16 x 10'7 M, 13.6 x 10"7 M and 49.8 x 10"7 M, respectively. The anti-inflammetory and anti-asthmatic action of Forsythia fruits and Plantago herbs may be ascribed to the inhibition of cyclic AMP phosphodiesterase and 5-lipoxygenase by the phenylethanoid glycosides contained in these herbal medicines. Antihypertensive activity The antihypertensive activity in the aqueous extracts of F smpensa has been

670

clinically reported [9]. The blood pressure in anesthetized spontaneously hypertensive rats (SHR) was therefore directly measured by carotid cannuration [54]. In fact, the aqueous extracts of F. suspensa showed antihypertensive activity (35 mm Hg, 10 mg/kg, i. v.). The most potent activity occurred in suspensaside (28), which showed high inhibitory activity on cyclic AMP phosphodiesterase. These facts suggested the possibility that there is some correlation between the therapeutic effect of herbal medicines and biological activities of phenylethanoid glycosides.

Antiallergic activity Plantamajoside (30) from P. asiatica and acteoside from P. lanceolata were tested for inhibitory effect on arachidonic acid-induced mouse ear edema [49]. Plantamajoside (30) showed a high inhibitory effect with inhibitions of 12 % at 1 mg / ear and of 25 % at 3 mg / ear. On the other hand, acteoside (1) showed a weak inhibitory effect with inhibitions of 6 % at 1 mg / ear and of 14 % at 3 mg / ear. This result showed good correlation with the inhibition of cyclic AMP phosphodiesterase and 5-lipoxygenase. Analgesic activity Acteoside (1) was isolated as an analgesic principle by activity-guided separation, from the leaves and stems of Lippia triphylla (L'Her) O. Kuntze (Verbenaceae), called Cedron and has been used as a calmative and carminative for stomachache in Peru. Acteoside (1) showed analgesia on acetic acid-induced writhing mice by 300mg/kg, p.o. and 2mg/kg,z.v and exhibited a weak sedative activity on the prolongation of pentbarbital-induced anesthesia and on the depression of locomotion enhanced by methanphetamine [55]. Antistress effect Cistanchis Herba has been used for staminal tonic and treatment of male impotentz [1]. The effect on sex (licking, mounting and intromission) and learning behaviours were studied in the chronic hanging stressed-adult male mice. The phenylethanoid glycosides fraction of Cistanchis Herba (20mg/kg, p.o.) showed the marked protective effect against decrease of sex and learning behaviours. Acteoside (1), cistanoside A (5) and cistanoside B (6) also exhibited the same effect by the administration of 2Qmg/kg,/>.0. Echinacoside (4,10mg/kg,/>.o.) showed the effect against decrease of sex behaviour, but little effect on learning behaviour [56].

671 671

Inhibitoiy effect on lipid peroxidation in rat liver microsomes Seven phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), isoacteoside (3), echinacoside (4), cistanoside A (5), cistanoside C (7) and tubuloside A (18) showed a strong inhibitorey effect on lipid peroxidation in phospolipid-ascorbic acid system. Their IC50 values were 1.82,7.10,1.73,4.63,1.92,4.50 and 2.20 / M , respectively [57]. Kadota et al reported that acteoside (1), 2-acetylacteoside (2), isoacteoside (3) and tubuloside B (18) significantly suppressed NADPH/CCU-induced lipid peroxidation in rat liver microsomes and prevented cell damage induced by exposure to CCU or D-galactosamine [58]. Neuroprotective effect Tubuloside B (19) significantly attenuated MPP+-induced cytotoxicity, DNA fragmentation and intracellular accumulation of reactive oxygen species (ROS). These results indicated that tubuloside B (19) prevent MPP+-induced apoptosis and oxidative stress and may be applied as an anti-Parkinsonian agent [59].

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Chinese Pharmacopeia Committee of Ministry of Public Health of the People's Republic of China. The Chinese Pharmacopeia. Beijing: Chemical Industry Publishing House; 2000, ppl03. Editorial Committee of Flora of China, Chinese Academy of Sciences. The Flora of China; vol. 69. Science Press; 1990, pp82. Institute of Materia Medica, Chinese Academy of Medicinal Sciences, Manual of Chinese Medicines; vol 4. Bejing: The People's Health Publishing House; 1961, pp347. Jiangsu New Medical Colledge. Chinese Materia Medica Dictionary. Shanghai: Shanghai Science and Technology Publishing House; 1978, pp895. Moriya, A.; Tu, P.; Karasawa, D.; Arima, H.; Deyama, T; Kegasawa, K.; Natural Medicines, 1995,49,383-393. Moriya, A.; Tu, P.; Karasawa, D.; Arima, H.; Deyama, T; Hayashi, K.; Ibe, N.; Kegasawa, K.; Natural Medicines, 1995,49,394-400. Tomari, N.; Ishizuka, Y; Moriya, A; Kojima, S.; Deyama, T; Mizukami,

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H.; Tu, P.; Chem. Pharm. Bull., 2002,25,218-222. Tu, P.; Wang, B.; Deyama, X; Zhang, Z.; Lou, Z.; Acta Pharmaeutica Smica, 1997,32,294-300. Jiangsu New Medical College. Chinese Materia Medica Dictionaiy. Shanghai: Shanghai Sd. and Tech. Publishing House; 1978, pp. 111-1113. Hansel, R.; Keller, K.; Rimpler, H.; Schneider, G Hagers Handbuch der Pharmazeutischen Praxis 5. Heidelberg, New York; Springer Verlag; 1994, pp. 221-239. Jiangsu New Medical College. Chinese Materia Medica Dictionary. Shanghai: Shanghai Sd. and Tech. Publishing House; 1978, pp. 401-403. Jiangsu New Medical College. Chinese Materia Medica Dictionaiy. Shanghai: Shanghai Sd. and Tech. Publishing House; 1978, pp. 403-405. Stuart, M. The Encyclopedia of Herbs and Herbalism. London: Qrbis Publishing; 1979, p. 242. Following our pharmacognostical studies on Cistanchis Herba and research for Citanche plants in China, comercial Cistanchis Herba is mainly produced from C. deserticola, and so Kobayashi's work of Cistanchis in our laboratory should be done witht C. deserticola: a) Kobayashi, H.; Karasawa, H.; Miyase, T; Fukushima, S.; Chem. Pharm. Bull, 1984,32, 3009-3014; b) Idem ibid., 1984, 32, 3880-3885.; c) Idem ibid., 1985, 33, 1452-1457.; d) Karasawa, H.; Kobayashi, H; Takizawa, N ; Miyase, T.; Fukushima, S.; Yakugaku Zasshi, 1986,106,562-566.; e) Idem ibid., 1986, 106,721-724. Lei, L.; Tu, P.; Wu, L.; Proceeding of The Second Symposium on Herba Cistanchis and Desert Medicinal Plants, Xinjiang Hetian, China, May, 2002,p76-78. Xu, W.; Qui, S.; Zhao, J; Xu,Y; Wan, K.; Yan, Q.; et d; Zkmgcaqyao (Chinese Thaditional and Herbal Drugs), 1994,25,509-513. Song, Z.; Tu, P.; Zhao, Y; Zheng, J.; Zhongpaoyao ((Chinese Traditional and Herbal Drugs), 2000,31,808-810. Yoshizawa, F.; Deyama, T; Takizawa, N ; Usn^nghani, K; Ahmad, M.; Chem. Pharm. Bull., 1990,38,1927-1930. Kobayashi, H; Oguchi, H.; Takizawa, N.; Miyase, T; Ueno, A.; Usmanghani, K.; Ahme4 M.; Chem Pharm Bull, 1987,35,3309-3314. Deyama, T; Yahikoaawa, K.; Al-Easa, H.; Rizk, A.; Qatar Univ. Sci J. 1995,15,51-55.

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Kitagawa, S.; Tsukamoto, H.; Hisada, S.; Nishibe, S.; Chem. Pharm. Bull, 1984,32,1209-1213. Kitagawa, S.; Nishibe, S.; Baba, H.; Yakugaku Zasshi, 1987,107,274-278. Kitagawa, S.; Hisada, S.; Nishibe, S.; Phytochemistry, 1984, 23, 1635-1636. Kitagawa, S.; Nishibe, S.; Benecke, R.; Thieme, H.; Chem. Pharm. Bull, 1988,36,3667-3670. Ravn,H.; Nishibe, S.; Sasahara, M.; Li, X.; Phytochemistry, 1990, 29, 3627-3631. Nishibe, S.; Tamayama, Y; Sasahara, M.; Andary, C ; Phytochemistry, 1995,38,741-743. Nishibe, S.; Sasahara, M.; Jiao, Y; Yuan, C ; Tanaka, T.; Phytochemistry, 1993,32,975-977. Ravn, H.; Brimer, L.; Phytochemistry, 1988,27,3433-3437. Murai, M.; Tamayama, Y; Nishibe, S.; Planta Med., 1995,61,479-480. Kawamura, T.; Hisata, Y; Okuda, K.; Hoshino, S.; Noro, Y; Tanaka, T; Kodama, A.; Nishibe, S.; Natural Medicines, 1998,52,5-9. Nishibe, S.; Kodama, A.; Noguchi, Y; Han, Y; Natural Medicines, 2001, 55,258-261. Andary, G; Wylde, R.; Maury, L.; Heitz, A.; Dubourg, A.; Nishibe, S.; Phytochemistry, 1994,37,855-857. Nishibe, S.; Kitagawa, S.; Hisada, S.; Baba, H.; Yasui, S.; Narita, T; Yoshioka, K.; J. Pharmacobio-Dyn., 1987,10, s48. Kimura, Y; Okuda, H.; Nishibe, S.; Arichi, S.; Planta Med., 1987, 2, 148-153. Nakamura, X; Okuyama, E.; Tsukada, A.; Yamazaki, M.; Satake, M.; Nishibe, S.; Deyama,T; Moriya, A.; Maruno, M.; Nishimura, H.; Chem. Pharm. Bull, 1997,45,499-504. Sato, X; Kojima, S.; Kobayashi, K.; Kobayashi, H.; Yakugaku Zasshi, 1985,105,1131-1144. Moriya, A.; Theses of Graduate School of Agricuture, Gifu University, Gifu, Japan, 1995. Xiong, Q.; Hase, K.; Tezuka, Y; Tani, T; Namba, X; Kadota, S.; Planta Med, 1998,64,120-125. Sheng, G; Pu, X.; Lei, L.; Tu, P.; Li, C ; Planta Med, 2002,68,966-970.

(Ed.) Studies Studies in in Natural Products Chemistry, Chemistry, Vol. Vol. 33 33 Atta-ur-Rahman (Ed.) © 2006 2006 Elsevier Elsevier B.V. B.V. All All rights rights reserved. reserved. ©

675 675

PHARMACOLOGICAL ACTIVITIES OF PHENYLPROPANOIDS GLYCOSIDES MARINA GALVEZ, CARMEN MARTIN-CORDERO, MARIA JESUS AYUSO. Departamento de Farmacologia, Facultad de Farmacia, Universidad de Sevilla, Spain ABSTRACT: The pharmacological assays and activities of natural phenylpropanoid glycosides, extracted from a variety of plants are summarized in this review, such as antioxidant, anti-inflammatory, healing, antimicrobial and antitumoral-chemopreventive. Structureactivity relationships are also discussed.

INTRODUCTION The phenylpropanoid glycosides (PPGs) are a group of derivatives of phenylpropane, distributed on Gamopetalas (Lamiales, Oleales, Asterales...) and located in most of the vegetable tissues and on the pollen, [1-4]. This group is characterised by having a caffeoyl and hydroxyphenylethyl moieties, both of which are linked to (3-glucose by ester (C-4) and glucosidic (C-l) linkages. Other sugars such as rhamnose, xylose or arabinose may be attached to C-3, C-4 or C-6 of the glucosyl residue [2]. This group of compounds has also been defined as phenethyl glycosides (PhGs), phenyletanoid glycosides or caffeoyl phenylethanoid glycosides (CPGs). We are going to use the term phenylpropanoid glycosides (PPGs) Fig- (1). Various plants used in traditional medicine contain significant amounts of PPG: For example, different species of Scrophularia genus, have been traditionally used for several skin inflammatory ailments. The leaves of species of Buddlej'a have been applied topically as a poultice or lotion for the healing of wounds and ulcers. Ballota nigra is commonly used for their neurosedative activity and Pedicularis sp. are a Chinese folk medicinal h erb, found e specially i n T ibet, u sed to t reat m alignant s ores, collapse, exhaustion, and relieves uneasiness of body and mind.

676 Fig. (1). Chemical structures of PPG (phenylpropanoids glycosides) mentioned. .OR,

R1 H P-D-api ara ara ara ara P-D-xyl P-D-api a-L-rha H caf H H H P-D-glu a-L-rha p-D-api fer caf H fer Ac caf fer H H a-L-rha P-D-api P-D-api H H H a-L-rha H H H H p-D-xyl P-D-glu caf H

Name 2' -Acetilverb ascos ide Alyssonoside Angoroside A Angoroside B Angoroside C Angoroside D Arenarioside Ballotetroside Brandioside Calceolarioside A Calceolarioside B Cistanoside C Cistanoside D Cistanoside F Echinacoside Forsythiaside Forsythoside B Isomartynoside Isoverbascoside Lavandulifolioside Leucosceptoside A Luteoside A Luteoside B Luteoside C Martynoside Myricoside Pedicularioside A Pedicularioside A Pedicularioside M Persicoside Phlinoside A Phlinoside C Poliumoside Rossicaside A Samioside Teucrioside Trichosanthoside A Trichosanthoside B Tubuloside A Tubuloside B Verbascoside

R2 caf fer caf fer fer fer caf caf caf caf H caf fer fer caf caf caf H H caf H caf H H fer caf caf fer fer caf caf caf caf caf caf caf caf caf H H caf

0 H

Caffeoyl

3OO^Y Feruloyl

R3 a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-arab (1-2) a-L-rha a-L-rha H H a-L-rha a-L-rha a-L-rha a-L-rha H a-L-rha a-L-rha a-L-rha a-L-ara (1-2) a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha a-L-rha (1-3) P-D-api P-D-api a-L-rha a-L-rha P-D-glu P-D-glu (1-2) a-L.rha a-L-rha (1-2) a-L.rha a-L-rha P-D-glu (l-4)a-L-rha P-D-api (1-4) a-L-rha a-L-lyx (1-2) a-L-rha P-D-xyl (l-4)a-L-rha P-D-xyl (l-4)a-L-rha a-L-rha a-L-rha a-L-rha 0 ||

OH

Ho'SZ^Sj ^ O H Rhamnose

R4 H H H H H H H H H H H H H H H H H H H H H H H H CH 5 H H CH , H H H H H H H H H H H H H

R5 H H H H CH, H H H H H H CH, CH, CH, H H H H H H H H H H H

H H H

H H H H H H H H H H H H H

OH H0

R6 Ac H H H H H H H H H H H H H H H H H H H H P-D-api P-D-api p-D-api H H H H H P-D-glu H H H H H H H H Ac Ac H OH

k5~° A V^H -0

iOH HC .HO Arab mose

H

Apiose

677

Roles in Plant Physiology It has been detected that the plants increase the phenylpropanoids synthesis in different situations, like, defense against herbivores; protection of microorganism attack or invasion by other species. Besides, some external factors that can increase the free radicals levels, such as stress, high light (more UV incidence), low temperatures, pathogen infections, nutrient deficiency [5], drought or ozone exposition [6], can induce a higher production of PPG by the plants. On the other hand, the PPG are precursors for other complex molecules, also useful to the plants [7, 8]. From these phenylpropanoids, other phenols have been synthesized such as flavonoids, coumarins, lignans, stilbenes, psoralenes, pterocarpanes, tannins, which also act against the oxidative stress [9]. All these functions that have the phenylpropanoids, have conduced to the genetic engineer to modify the biosynthetic pathways, hi that way, it has induced illness resistance, or foraged plants digestibility increase [10].

METHODOLOGY The data has been obtained after a revision of the literature available by different data basis, such as MedLine or Chemical Abstracts, and by the references in the literature. The order of the tables is based on the alphabetical order of the family, following "The International Plant Name Index", by The Royal Botanic Gardens, Kew, The Harvard University Herbaria and Australian National Herbarium (www.ipni.org/index.html), for the nomenclature. RESULTS The data is presented in table 1 divided in five columns that include the family of the plant specie where the phenylpropanoid glycoside (PPG) has been isolated, the specie, some of the popular uses of the plant, the trivial name of the PPG, the assayed activity with the result, also including the negative results, and the reference.

Globulariaceae

Globularia trichosantha

Mussatia sp.

Diuretic, laxative, carminative, tonic.

Euphoric effect

Table 1. Pharmacological activities of PPG. Species Popular use Family Acanthaceae Asthma, Barleria prioniits Fever, Cold Bignoniaceae Markhamia lutea Diarrhea, Asthenia, Infections

Crenatoside (=oraposide) Rossicaside A Trichosanthoside A Trichosanthoside B Verbascoside

Mixture of 4cinnamoylmussatioside + 4dimethylcaffeoylmussatioside + 4-pmethoxycinnamoylmussatioside

Luteoside C Verbascoside

Luteoside A Luteoside B

Isoverbascoside

Verbascoside

PPG

• • • •

Active Active Active Active

Antioxidant: Scavenging activity (DPPH) (TLC) • Active

• Inactive as inhibitor of prostaglandin biosynthesis. Antimicrobial: • No effect in the range 0,05-5 mg/strip. (microorganisms not shown on the paper) Cardiovascular effects 1 No effect on blood pressure and heart rate. Platelet agregation • Inhibition of rat ADP-induced platelet aggregation. It might be through cAMP-phosphodiesterase inhibition.

Antiviral: • Active against RSV. • Inactive against VZV, mCMV, HSV 1 and 2. • Active against RSV. • Active against RSV. • Inactive against HSV-1 and 2. • Active against RSV. • Active against RSV. • Inactive against HSV-2 and CMV. Anti-inflammatory:

Antiviral: • Active against RSV.

Assayed activity

[14]

[13]

[13]

[13]

[13]

[12]

[11]

References

00

OS

678

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Lamiaceae Ballota nigra Flu, cold, nervousness.

(+)-E-caffeoyl-L-malic acid Arenarioside Ballotetroside ForsythosideB Verbascoside

(+)-E-caffeoyl-L-malic acid Arenarioside Ballotetroside ForsythosideB Verbascoside

(+)-(E)-caffeoyl-Lmalic acid Alyssonoside Angoroside A

(+)-(E)-caffeoyl-Lmalic acid Alyssonoside Angoroside A Arenarioside Ballotetroside Forsythoside B Lavandulifolioside Verbascoside

PPG

• • • •

Inactive Inactive Inactive Inactive

• Active • Active • Active • Active -Methal chelation (Cu 2+) • Inactive

• Inactive • Inactive Antioxidant: -Inhibition of in vitro Cu (2+) - induced LDL peroxidation • Active

Inactive Inactive Active Inactive Active Inactive Active E. faecalis; P aeruginosa; E. coli; E. aeroenes; K. pneumoniae; K. oxytoca • Inactive

• • • • " • • •

Antibacterial: • S. aureus; S. aureus (methicillin- resistant); P. miral • Inactive

Assayed activity

[16]

[16]

[15]

[15]

References

679

Leonurus cardica

Leonotis nepetaefolia

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Lamiaceae (cont.) B. nigra (cont.)

Lavandulifolioside

Lavandulifolioside

Lavandulifolioside Martynoside Verbascoside

(+)-E-caffeoyl-Lmalic acid Alyssonoside Angoroside A Arenarioside Ballotetroside Forsythoside B Lavandulifolioside Verbascoside

(+)-E-caffeoyl-L-nialic acid Alyssonoside Angoroside A Arenarioside Ballotetroside Forsythoside B Lavandulifolioside

PPG

• Active • Active • Active • Active • Active • Active • Active Antioxidant: Scavenging activity {DPPH) • Active • Active " Active Chronotropic • Negative Hypotensive; • Active

• Active • Active • Active • Active • Active • Active Sedative: BZD; D;n receptor agonists, • Active

Antioxidant (cont.): -Scavenging activity in vitro and in vivo against Oa"" OH • Active

Assayed activity

, 0HC1,

[19]

[19]

[18]

[17]

[17]

References

680

Phlomis armeniaca

Nepeta uaraintca

Table 1. Pharmacological activities of PPG, (cont.) Popular use Family Species Lamiaceae (cont.) Marruhium vulgare

Verbascoside

Verbascoside

Leucosceptoside A Martynoside Phlinoside B

Forsythoside B

Verbascoside

(+)-E-eaffeoyl-Linalic acid Arenarioside Ballotetroside Forsythoside B Verbascoside

(+)-E-caffeoyl-Lmalic acid Arenarioside Ballotetroside Forsythoside B Verbascoside

PPG

• COX-2 inhibitor • COX-1 inhibitor • COX-2 inhibitor • Selective COX-2 inhibitor Immunomodulatory: • Increases chemotactic activity. • Positive effect on respiratory burst of neutrophils. Antitu moral: - Cytotoxic activity against RLh-84; S-I80; P-388; HeLa and hepatocytes • Cytotoxic on dRLh-84, S-180 and P-388/dl. • No cytotoxic on hepatocyte • No cytotoxic • No cytotoxic • Cytotoxic on dRLh-84, S-180. • No cytotoxic on hepatocyte • Cytotoxic on dRLh-84, S-180 and P-388/dl. • No cytotoxic on hepatocyte - Apoptosis induction • Active on HL-60

• Active • Active • Active • Active Enzymatic inhibition (anti-inflammatory): • Inactive.

Antioxidant: Inhibition of Cu (2+)- and AAPH induced LDL oxidation • Active

Assayed activity

[24]

[23]

[22]

[21]

[20]

References

681

Sideritis licia

P. samia

P. pungens

P. physicalyx

Table 1. Pharmacological activitieg of PPG, (cont.) Family Species Popular use Lamiaceae (cont.) P. monocephala

Lavandulifolioside Leucosceptoside Martynoside Verbascoside

Samioside Verbascoside

Samioside

Samioside Verbascoside

Forsythoside B Alyssonoside

Forsythoside B Leucosceptoside A Martynoside Physocalycoside Verbascoside Wiedemannioside C

Alyssonoside Forsythoside B Verbascoside

PPG [25]

Antioxidant: Scavenging activity (DPPH) • Active • Active • Active Antioxidant: Scavenging activity (DPPH) • Active • Active • Active • Active • Active • Active Vasocontracting: against free radical-induced impairment of endothelium-dependent relaxation. • Active • Active Antibacterial: S. aureus; S. epidermidis; E. cloacae; E. coli; K. pneumonia: P. aeruginosa. • Active • Active Antifungal-.Candida albicans, C. glabrata, C, tropicalis • Active Antioxidant: Scavenging activity (DPPH) " Active • Active Anti-inflammatory: against carrageenan-induced paw edema • Active • Active • Active • Active

[29]

[28]

[28]

[28]

[27]

[26]

References

Assayed activity

682

Loganiaceae

Inflammations, wounds and ulcers

Conjunctive congestion, clustered nebulae.

B. globose

B. officinalis

Buddleja cordata

Teucrium polium

Table 1. Pharmacological activities of PPG, (cont.) Species Popular use Family Lamiaceae (cont.) Stachys sieboldii

AngorosideA Calceolarioside Campneoside Echinacoside Forsythoside B Verbascoside

Verbascoside

Verbascoside

Echinacoside Verbascoside

Verbascoside

Verbascoside

Poliumoside

Verbascoside

Stachysoside C Verbascoside

PPG

Active

Healing: Fibroblast-protector against ROS. • Active • Active Antibacterial • Active Antitu moral •Active Enzymatic inhibition: In vivo COX and 5-LOX inhibition. -Inactive "Inactive "Inactive "Inactive -Inactive -Inactive

•

Antibacterial: • Active against S aureus. • Mechanism: Affecting protein synthesis Antibacterial:

Anti-anoxia: Inhibition of the KCN-induced anoxia in mice • Active • Active Anti-nephritic: • Preventive of nephritis and glomerulonephritis An titu moral: Cytotoxic activity against dRLh-84, S-180, p-388/Dl, HeLa • Cytotoxic on dRLh-84, S-180, p-388/Dl.

Assayed activity

[36]

[37]

[37]

[35,36] [35]

[34]

[33]

[32]

[31]

[30]

References

00

683

Oleaceae

Myoporaceae

Forsylhia sp.

Fraxinus sieboldiana

Allergic and inflammatory diseases

Poliumoside Verbascoside

Cold, headache, sores chest pain

E. gilesii

Verbascoside

p-hydroxyverbascoside Suspensaaside

Forsythiaside

Calceolarioside B Calceolarioside A Verbascoside

Verbascoside

Verbascoside

AntivirahHIV • Active • Inactive • Inactive Antiinflamatory: Kffect on arachidonic acid metabolism •Active as inhibitor of 5-HETE and LTB4 in rat peritoneal cells and human peripheral PMN-L •Active as inhibitor of 5-HETE and LTB4 in rat peritoneal cells and human peripheral PMN-L •Active as inhibitor of 5-HETE and LTB4 in rat peritoneal cells and human peripheral PMN-L "Active as inhibitor of 5-HETE and LTB4 in rat peritoneal cells and human peripheral PMN-L

• Active Cardiotonic • Chronotropic positive • Inotropic positive. • Increase of coronary perfusion rate by increase of c AMP Hypo-Hypertension: •No effect on arterial pressure Inhibition of platelet aggregation: • Active • Active Inhibition of serotonin release: • Active

[44]

[43]

[42]

[39-41]

[39-41]

Verbascoside Antimicrobial

[38]

Neuroprotective: Against MMP-induced apoptosis and oxidative stress in PC12 neuronal cells. •Active. Parkinson prevention.

Fever, headache, pain, conjuntivitis, inflammation.

References

Assayed activity

PPG

Eremohyla ahernifolia

Table 1. Pharmacological activities of PPG, (cont.) Popular use Family Species Loganiaceae (cont.) B. officinalis (cont.)

2

684

Oleaeeae (cont.)

Sores, headache, pain, hypertension

Inflammations

Ligustrum robustum

L. vulgare

Forsythia sp. (cont.)

Table 1. Pharmacological activities of PPG, (cont.) Species Popular use Family Antioxidant: DPPH scavenging activity •Active •Active

Assayed activity

Verbascoside

Osmanthuside B Verbascoside

Q O

• Inactive • Active Vasocontracting • Active (Inhibition of NO production)

•Active •Active Antioxidant: Protection against hemolysis of red blood cells induced by AAPH Ligupurpuroside A • Active Ligupurpuroside B • Active LigurobustosideM • Active LigurobustosideN • Active LigurobustosideO • Active Osmanthuside B • Active Osmanthuside B6 • Active Verbascoside • Active Antioxidant: - Inhibition of in vitro Cu (2+)- induced LDL peroxidation Isoverbascoside • Active Ligupurpuroside A • Active Verbascoside • Active - Inhibition of in vitro peroxyl radical-induced LDL peroxidation Isoverbascoside • Active Ligupurpuroside A • Active a's-Ligupurpuroside B • Inactive ftww-Ligupurpuroside • Inactive

Forsythiaside P-hydroxyverbascoside Suspensaaside Verbascoside

PPG

[48]

[47]

[46]

[45]

[44]

References

00

en

685

Orobanchaceae

Calceolaria kypericina Cistanche deserticala

Syringa vulgaris

Malaria

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Oleaceae (cont.J_ Olea europaea

2*-acetyl-verbaseoside Cistanoside A Cistanoside F Echinacoside Isoverbascoside Syringalide A 3*-arhamnopiranoside Tubuloside A Tubuloside B Verbascoside

Verbascoside

2'-acetyl-verbascoside Isoverbascoside Tubuloside B Verbascoside

Calceolarioside A

Verbascoside

Verbascoside

PPG

• Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH, superoxide anion and NO scavenger.

Platelet aggregation: • Active Anti-hepatotoxic: In vitro protection against CCU and D-GalN • Active • Active • Active • Active Inhibition of LPS and D-GalN-induced apoptosis on hepatocytes. • Active Antioxidant: -DPPH and superoxide anion scavenging activity • Active as DPPH,s uperoxide anion and NO scavenger. " Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH and superoxide anion scavenger. • Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH, superoxide anion and NO scavenger. • Active as DPPH and superoxide anion scavenger.

Antioxidant :Scavenging activity (ATBS) • Active Antiviral: * Inactive against-HI¥ Heart rate effect: • Decrease of heart rate. Antihypertensive effect: • Active

Assayed activity

[55,56]

[54]

[53]

[52]

[51]

[51]

[50]

[49]

References

686

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Orobanchaceae(cont.) C. deserticola (cont.) 153]

Antioxidant: -Inhibition of NADPH-CCU -induced lipid peroxidation • Active • Active • Active • Active -Inhibition of ascorbic acid and Fe(2+)-induced lipid peroxidation • Active " Active • Active • Active • Active • Active

[55]

156]

[55]

References

Assayed activity

• Active " Active • Active Enzymatic inhibition: inducible Nitric Oxide synthase (iNOS) 2 '-acetyl-verbascoside • Inactive Cistanoside A • Inactive Echinacoside • Inactive Isoverbascoside • Inactive Tubuloside A • Inactive Tubuloside B • Inactive Verbascoside • Inactive. Xanthine oxidase (XOD) 2'-acetyl-verbascoside • Inactive Cistanoside A • Inactive Cistanoside F • Inactive Echinacoside • Inactive Isoverbascoside • Active

2 '-acetyl-verbascoside Cistanoside A Cistanoside F Echinacoside Isoverbascoside Syringalide A 3'-ccrhamnopiranoside Tubuloside Tubuloside A Verbascoside

2'-acetyl-verbascoside Isoverbascoside Tubuloside B Verbascoside

PPG

687

Pedaliaceae Harpagophyium procumbens

O. hypericina

O. hederae

Orobanche caerulescens

C. salsa

Inflammatory degenerative diseases,skin lesions, fever, tonic.

Kidney deficiency and neurasthenia Tonic for impotence, inflammation, cystitis, feces softener

Table 1. Pharmacological activities of PPG, (cont.) Species Popular use Family Orobanchaceae (cont.) C. deserticolci (cont.)

6'-0-acetylVerbascoside Isoverbascoside Verbascoside

Calceolarioside A Calceolarioside B Calceolarioside C

Verbascoside Orobanchoside

Caerulescenoside 3'-methyl crenatoside Isoverbascoside Verbascoside Campneoside II Crenatoside Derhamnosyl verbascoside

Verbascoside

Syringalide A 3'-arhamnopiranoside Tubuloside A Verbascoside Tubuloside B

PPG

• Active • Inactive

Enzymatic inhibition: Elastase • Strong activity

Platelet aggregation inhibition: • Active • Active Platelet aggregation inhibition: • Active • No effect • No effect

Antioxidant: Inhibition of LDL oxidation • Active • Active " Active • Active • Active • Active • Active

• Inactive • Inactive • Active. Neuro-protecting effect against MMP • Active by inhibition of caspases activation

Enzymatic inhibition: Xanthine oxidase (XOD) (cont.) • Inactive

Assayed activity

[59]

[58]

[58]

[58]

[57]

[55]

References

688

Schrophulariaceae

Polygonaceae

Brandisia hancei

Polygonum lapatifotium

Necrotic osteitis, rheumatoid arthritis, hepatitis, hyperlipemias

Dysentery, articular pain, inflammations

Same as P. lanceolata

P. major P. media

Wound healing, respiratory problems, cancer

P. lanceolata

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Plantaginaceae Plantago cynops

2' -acetyl verbascoside Brandioside Poliumoside Verbascoside

Vanicoside B

Lapathoside A Vanicoside B

Lapathoside A Vanicoside B

Homoplantaginine Verbascoside

Plantamajoside

Cistanoside F Isoverbascoside Lavandulifolioside Plantamajoside Verbascoside

Verbascoside

PPG

two-stage

two-stage

skin

skin

Antioxidant:Inhibition of free radical-induced hemolysis of red blood cells and superoxide radical generation. • Active • Active • Active • Active

Antitumoral-chemopreventive Inhibition of EBV-EA induction by TPA " Active • Active Anti-tumor-promoting effects on mouse carcinogenesis induced by DMBA and TPA • Active • Active Anti-tumor-promoting effects on mouse carcinogenesis induced by NO donor • Active

[60]

Antibacterial • Active Anti-inflammatory:-Inhibition of arachidonic acid-induced edema • Inactive " Inactive • Inactive • Active " Active Antibacterial: against S. aureus; E. coli • Active Antitumoral-chemopreventive: EGFR, TK and tumor growth inhibition • Active • Active

[65]

[64]

[64]

[64]

[63]

[62]

[61]

References

Assayed activity

689

MonochAsthma savatierii

Digitalis purpurea

Castilleja linariaefolia

Table 1 • Pharmacological activities of PPG, (cont.) Family Species Popular use Schrophulariaceae (cont.) B. hancei (cont.)

Dehydroverbascoside Verbascoside

Calceolarioside A Calceolarioside B Forsythoside Plantainoside

Isoverbascoside Verbascoside

2'-acetylverbascoside Brandioside Poliumoside Verbascoside

2'-acetylverbascoside Arenarioside Brandioside Isoverbascoside Verbascoside

2'-acetylverbascoside Brandioside Poliumoside Verbascoside

PPG Antitumoral: Antiproliferation on A7r5 cells • Active • Active • Active • Active Enzymatic inhibition: Xanthine oxidase • Inactive • Inactive " Inactive • Competititve inhibitor • Inactive Prevention against arteriosclerosis • Active " Active • Active • Active Antitumoral: Cytotoxic activity against P-388 • Active • Active Enzymatic inhibition:PKCa • Active • Active • Active • Active Enzymatic inhibition: Aldose reductase in vivo. • Inactive • Strong activity

Assayed activity

[70]

[69]

[68]

[66]

[67]

[66]

References

690

P. plicata

P. lasiophyris

P. alashanica

Schrophulariaceae (cont,) Pedicularis sp. Tonic for treatment of debility, collaptse, exhaustion, swating, seminal emission, senility

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular

Verbascoside Martynoside

Martynoside

Cistanoside D

Leucosceptoside A Martynoside

Cistanoside C Pedicularioside A Verbascoside

Cistanoside D Echinacoside Isoverbascoside Pedicularioside A Verbascoside

Cistanoside Echinacoside Isoverbascoside Pedicularioside A Verbascoside

PPG

Antioxidant: -Inhibition of autoxidation of Hnoleic acid in CTAB • Active • Active • Active • Active • Active -Protection against oxidative hemolysis • Active • Active • Active • Active • Active Antitumor-chemopreventive: DNA adducts-reparation: dAMP+; dAMP-OH; dAMP(NH) dGMP+; dGMP-OH; PoliG-OH; TMP -Active • Active • Active Antioxidant: Scavenging activity against Ch~ and OH • Active • Active Antioxidant: Protection against oxidative hemolysis • Active Chemopreventive: DNA adducts-reparation: T Active Motor level: Retardation of skeletal muscle fatigue. • Active • Active

Assayed activity

[77, 78]

[76]

[72]

[75]

[73,74]

[72]

[71]

References

691

P. striata

Asthenia

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Schrophulariaceae (cont.) P. spicata

Pedicularioside A Verbascoside

Verbascoside

Isoverbascoside

Isoverbascoside Verbascoside

Pedicularioside A Pedicularioside M Pedicularioside N Verbascoside

Echinacoside Pedicularioside A Verbascoside

Isoverbascoside PermethylVerbascoside Verbascoside

Cistanoside C

PPG

• Active Protection against oxidative hemolysis • Active • Active • Active Scavenging activity (OH" and O2~) • Active • Active • Active • Active Antitumoral-chemopreventive: Induction of cellular differentiation on: • SMMC-7721 and MGc803 . MKN45 and MGc803 Antiproliferation on MGc803 cell line • Active Telomerase inhibition on MKN45 cell line • Active DNA adducts-reparation: T-OH • Active • Active

Chemopreventive: DNA adducts-reparation: T' • Active Antioxidant: Inhibition of FeSO,t-induced lipid peroxidation • Active • Inactive

Assayed activity

[87]

[85]

[83,84] [85,86] [84]

[75, 82]

[81]

[72]

[80]

[79]

References

692

Scrophularia albida

Rehmannnia glutinosa

Penslemon linarioides

Tonic, antianemic, antipyretic

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Schrophulariaceae (cont.) P. striata (cont.) Antitumoral-chemopreventive (cont.): DNA adducts-reparation: T" • Active • Active • Active • Active DNA adducts-reparation: dGMP-OH • Active Repair effect on DNA damage Enzymatic inhibition: "Inactive as xanthine oxidase inhibitor. "Inactive as xanthine oxidase inhibitor. Metal chelating effect: (ferric ion) • Active • Inactive

Assayed activity

• Active Enzymatic inhibition: PKCa Leucosceptoside A • Active Poliumoside " Active Verbascoside • Active Immunosuppressive: Study of hemolytic plaque forming cells in mice. Cistanoside F •Active Isoverbascoside •Active Jionoside A-l •Active Jionoside B-l •Active Purpureaside C •Ative Verbascoside •Active Antitu moral: Cytotoxic activity against dRLh-84; S-180; P-388/D1; HeLa (+) syringaresinol-o-p• Active D-glucopyranoside

Isoverbascoside Permethyl Verbascoside Verbascoside

Pedicularioside A Verbascoside

Verbascoside Verbascoside

Leucosceptoside A Pedicularioside M Pedicularioside N Verbascoside

PPG

[32]

[90]

[69]

[80]

[72,75]

[88]

[76]

References

693

S. ningpoensis

S. scorodonia Skin inflammatory ailments

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Schrophulariaceae (cont.) S. scopolii

Angoroside C Verbascoside

Verbascoside

Verbascoside

Isoverbascoside

Angoroside D

Angoroside C

Angoroside A

Verbascoside

Angaroside A Angaroside B Angaroside C

PPG [32]

Antitumoral: Cytotoxic activity against dRLh-84; S-180; P-388/D1; HeLa •Active •Active "Active Antiviral: against HSV and VSV "Inactive Anti-inflammatory: Inhibition of PGE2, LTC4 TXB2 and NO release in calcium ionophore-stimulated mouse peritoneal macrophages and human platelets. •Active as inhibitor of PGE2,TXB2, LPS-induced TNF-a and LPS-induced NO release. •Active as inhibitor of PGE2 (COX-2) accumulation. •Active as inhibitor of PGE2,TXB2 and LPS-induced NO release. •Active as inhibitor of PGE2 (COX-2) accumulation. "Active as inhibitor of PGE2, LPS-induced TNF-a and LPSinduced NO release. •Active as inhibitor of PGE2 (COX-2) accumulation. "Active as inhibitor of PGE2, LPS-induced TNF-a and LPSinduced NO release •Active as inhibitor of PGE2 (COX-2) accumulation. •Active as inhibitor of TXB2, LPS-induced TNF-a and LPSinduced NO release •Active as inhibitor of PGE2 (COX-2) accumulation. Antitumoral: Cytotoxic activity against: HeLa, Vero and BHK-21 •Inactive Antitumoral-chemoproventive DNA adducts-reparation: d-AMP-OH and d-GMP •Active •Active

[93]

[91]

[92]

[91]

References

Assayed activity

694

Verbenaceae

Clerodendron bungei and C. tnchotomum

Caryopteris incana

Veronica persica

Verbascum macrurum

Malignan lung cancer, rheumatism, rheumatic articular pain

Table 1. Pharmacological activities of PPG, (cont) Family Species Popular use Schrophulariaceae (cont,) Scutellaria armeniaca Assayed activity

Deshamnosylisoverbascoside Deshamnosylverbascoside Isoverbascoside Verbascoside

Incanoside Isoverbascoside Phlinoside A Verbascoside

• Cytotoxic on B16F10 • Cytotoxic on B16F10

• Cytotoxic on B16F10

Antioxidant: Scavenging activity against DPPH, OH' and O2". • Active • Active • Active • Active Antitumoral: Cytotoxic activity againstB16F10, MK.-1 and HeLa • Cytotoxic on B16F10

Antitumoral: Cytotoxic activity against dRLh-84, S-180, P-388/dl HeLa and hepatocytes. Teucrioside • Cytotoxic on all cell lines.. Verbascoside • Cytotoxic on dRLh-84; S-180 and P-388, Leucosceptoside A • No cytotoxic. Martynoside • No cytotoxic. Antioxidant: DPPH scavenging activity and preserving activity 6'-O-a-Lagainst sunflower oil oxidative rancidity. arabinopyranasyl•Active martynoside + 6'-O-PD-xylopyranosylmartynoside Martynoside •Active Verbascoside •Active Antioxidant: -Scavenging activity against DPPH Persicoside • Active Verbascoside • Active

PPG

[97]

[96]

[95]

[94]

[23]

References

695

Premna subscandens

L. multiflom

Lippia dulcis L. canescens

Lantana camara

Lantana camara

Malaria

Cough and bronchitis

Table 1. Pharmacological activities of PPG, (rout.) Popular use Family Species Verbenaceae (cont.) C. trichatamum Hypertension

Verbascoside

Verbascoside

Arenarioside Diacetyl- martynoside Isoverbascoside Leucosceptoside A Martynoside Verbascoside

Verbascoside

Verbascoside

Isomartynoside Isoverbascoside Leucosceptoside Martynoside Verbascoside

Isomartynoside Isoverbascoside Jionoside D Leucosceptoside A Martynoside Plantainoside C Verbaseoside

PPG Antiviral: Inhibition of HTV-1 integrase • Inactive • Active • Inactive • Inactive • Inactive • Inactive • Active Enzymatic inhibition: ACE • Active • Active • Active • Active • Active Antitumoral: Cytototic activity on L-1210 • Active Enzymatic inhibition: PK.C • Active, by catalytic domain Antitu moral: Antiproliferative effect against B16F10, MK-1 and HeLa • Active on B16F10. • Inactive • Active on B16F10 • Inactive • Inactive • Active on B16F10 Anti-inflammatory: TXA3 synthesis inhibition • Inactive Healing: Promotion of collagen network formation in vitro • Active

Assayed activity

[104]

[103]

[101,102]

[100]

[100]

[99]

[98]

References

696

Not given on the paper

Verbena oficinalis

Table 1. Pharmacological activities of PPG, (cont.) Family Species Popular use Verbenaceae (cont.) Stachytarpheta Liver cayennensis diseases, flues, cough, arthritis Verbena litoralis

Verbascoside

Purpureaside C

Purpureaside B

Purpureaside A

Isoverbascoside

2' -acetyl verbascoside Desrhamnosil Verbascoside

Isoverbascoside Jionoside Verbascoside

Verbascoside

PPG

Antiviral against virus Aujeszky Antitumoral-chemopreventive: •Induction of HL-60 differentiation Antimicrobial against a E. coli Antiviral against virus Aujeszky Antimicrobial against a E. coli Not antiviral against virus Aujeszky Antimicrobial against a E. coli Not antiviral against virus Aujeszky Chemopreventive: "Scavenging activity against O2" and lipid peroxidation •Antimetastatic effect on lung metastasis with B16 melanoma. •Inhibition of tumor-induced angiogenesis through downregulation of MMP expression in vitro. "Active against nephritis, through the inhibition of cellular proliferation.

Chemopreventive No increase of NGF • Active • Active • Active Anti-inflammatory • Active against carrageenan-induced mouse paw edema Antimicrobial against a E. coli

Anti-inflammatory: • Active against carrageenan-induced mouse paw edema • Inhibition of bradykinin and histamine effects.

Assayed activity

D'3]

[111] [112]

[HO]

[108]

[108] [109]

[107] [108]

[106]

[105]

References

697

698

DISCUSSION Before starting to discuss the pharmacological activities shown by PPGs, it is interesting to take into account that it is difficult to compare all the results due to the different experimental models used. Moreover, in each assay, different kinds of PPGs with more than one structural difference have been compared, therefore being hard to understand, which moieties or structure modifications could contribute to each activity. Nonetheless, it has been found that some structure-activity relationships, which are discussed below, and based on this, some suggestions for further analysis of PPGs are given. Antioxidant activity An antioxidant can exert its activity through different mechanisms of action such as: Radical scavenging or free radicals (ROS), metal chelating, inhibition of lipid peroxidation or inhibition of endogenous enzymes that generate ROS [72]. PPGs can act as antioxidants through different mechanisms. They possess in vitro scavenging activity against superoxide, hydroxyl, hydrogen peroxide hippochlorite and nitric oxide radicals, tested out of and within cell systems tests [17, 55, 56, 65, 75, 82, 96, 110]. Curiously, some of them (pedicularioside M, N and martynoside) have shown an antioxidantprooxidant dual effect, being hydroxyl scavengers at high concentrations but becoming radical generators at low doses. This dual effect could be due, in part, to the capacity of binding to iron III, reducing it to iron II. However, others P PGs s uch as v erbascoside, h ave o nly s hown s cavenging p roperties [75]. Even so, it has been concluded that there is a structure-activity relationship between the different PPGs. There is a direct relationship between the antioxidant activity and the number of hydroxyl groups, and an inverse relationship with the increasing of higher monosaccharides moieties. Detailing what was mentioned above, in both, enzymatic and cellular models, it has been proven that verbascoside is the compound with the highest scavenging power. It has published different potency orders, such as:

699

Verbascoside > Forsythoside > Arenarioside > Ballolletroside or, Verbascoside > Pedicularioside A > Pedicularioside M > Leucosceptoside A > Martynoside N > permethylverbascoside [75]. There was only one assay where two PPGs, brandioside and poliumoside, were better superoxide scavengers than verbascoside. This fact could be due to the rhamnosyl moieties, that might be more potent against superoxide radical [65]. All these scavenging capacities are based on one fact: The polyphenol compounds are attacked by superoxide or hydroxyl radicals predominantly at the o-dihydroxy site. The semiquinone radicals formed from the reaction of the o-dihydroxy structures with the radicals are quite stable, probably because of the presence of hydrogen bonding. However, if the PPG has a methoxy radical at the ortho position, the aroxyl radical derived from its reaction with superoxide or hydroxyl radical is less stable owing to the absence of intramolecular hydrogen bonding [114]. Despite this being known, it might be interesting to try to develop some of the assays described in bibliography, under the same conditions, with all the possible variations, and including isoverbascoside and derivatives. There is some controversy in the bibliography about the chelating properties of the PPGs. Some authors found that some are iron chelants [80], whereas others have proven the opposite for ferric [75] and cupric [16] irons. Another antioxidant mechanism of action is the inhibition of the lipid peroxidation. It can be detected by different methods, but, in most of them, the peroxidation is induced by F enton reaction, in which hydroxyl radicals are generated. Therefore, it would be logical to think that those PPGs that have shown hydroxyl radical scavenging, will be active as lipid peroxidation inhibitors. But it is necessary to develop these kind of assays, because, the absorption properties of these compounds into cells or the ways in which they may offer protection to the exterior of the cell wall, still remain unknown [35]. All the PPGs tested showed activity [53, 55, 80], being isoverbascoside, an isomer of verbascoside, a slightly higher than verbacoside. On the other hand, it has been studied that some PPGs affects the expression or activation of endogen enzymes that produce free radicals, like xanthine oxidase [72, 82] or inducible nitric oxide synthase [56]. But, the PPGs assayed (2-aeetylverbascoside, cistanoside, echinacoside, forsythoside

700

isoverbasocoside, pedicularioside, tubuloside, and verbascoside) do not affect those enzymes, except isoverbascoside, which is a competitive xanthine oxidase inhibitor [55, 67]. Therefore, most of the PPGs only reduce the ROS levels acting as radical scavengers. This antioxidant activity, and to be more exact, the scavenging activity, is one of the responsible m echanisms of other pharmacological activities that have been described for PPGs, as anti-inflammatory, healing and antitumoral-chemopreventive activities. Anti-inflammatory activity The inflammatory response involves many types of tissues and cells. These cells produce some common modulators like: eicosanoids, cytokines, ROS, and nitrogen intermediates. The eicosanoids are classified into three big groups, prostaglandins and prostacyclins, leukotrienes, and thromboxanes. The prostaglandins are lipid mediators implicated, not only in inflammation, but also, in other pathological processes, such as edema, fever, hyperalgia, cancer or Alzheimer's disease. The cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of PGE2, TXB2 and prostacyclines from arachidonic acid. Leukotrienes are involved in immune-regulation, asthma, inflammation, and various allergic conditions. In the presence of 5-lipooxygenase (5-LOX), free arachidonic acid is converted to 5-HPTE, which is then reduced to 5HETE or dehydrated to a non-stable intermediate LTA4 [115]. LTA4 is further converted enzymatically to leukotrienes, LTB4 and LTC4 [116, 117]. The LTC4 is a leukocyte chemotaxic that participates in cell adhesion, superoxide production, calcium translocation and the release of different enzymes. Therefore, there are different targets to attack the inflammatory process. Due to this, the study of the anti-inflammatory activity by the PPGs has been studied following different experimental models, trying to justify their mechanism of action as anti-inflammatory agents. Verbascoside has shown carrageenan-induced rat paw edema inhibition [105].

701

Liao and cols.,[36] published that different PPGs, as, angoroside A, calceolarioside, capnoepside, echinaeoside, forsythoside, and verbascoside, do not inhibit to COX or 5-LOX on rat peritoneal leukocytes at 50 [ig/mL. However, has recently been studied the effect of PPG on COX enzymes in two different publications. On one hand, Sahpaz and cols. [21] found that arenarioside, forsythoside, and verbascoside, were the strongest COX-2inhibitors at 100 uM. Moreover, these compounds did not exhibit any significant inhibition on COX-1 at the same concentration. The authors defended the existence of a structure-activity relationship: the possession of two o r t hree s ugar u nits i n t heir s tructure c ould c ontribute t o t he se lective inhibition on COX-2. On the other hand, ballotetroside, with four sugar units, exhibited a weaker activity, and, interestingly is more active over COX-1 than COX-2. They think that the addition of a sugar unit (in this case arabinose on position C-2 of rhamnose) increases the steric hindrance, which prevents the molecule easily getting to the active site of the enzyme. The study of forsythoside and arenarioside is important because they have the same PPG structure, with the only difference in the kind of third sugar moiety joined to C-6 of glucose. Comparing the activity showed by each active compound, we also would hypothesize even more that apiose moiety joined to C-6 of glucose seems to contribute to COX-2 inhibition more than xylose moiety. Nevertheless, the presence of xylose on this position exerts the same effect on COX-2 inhibition than verbascoside, a diglycoside PPG. Diaz and cols. [92] studied more PPGs (angoroside A, C and D, isoverbascoside and verbascoside), as anti-inflammatory compounds, and their effect not only over COX-1 and 2, but also over TX-synthase, and NO generation. Angoroside A, C and D and isoverbascoside exhibited inhibitory activity on COX-1 in A23187-stimulated macrophages. The inhibition was more evident with angoroside C and A. Of all tested compounds, only angoroside A, C and verbascoside showed a significant effect on TXB2release. All compounds strongly inhibited LPS-induced NO production, being more active angoroside A, D and isoverbascoside. All compounds except angoroside C also inhibited the accumulation of PGE2 that means that they are active inhibiting COX-2. All compounds, except angoroside C,

702

strongly inhibited LPS-induced TNF-a production, being more potent in the case of angoroside D and isoverbascoside. These authors concluded that verbascoside is the most active compound on TX-synthase inhibition; therefore, caffeoyl moiety is an important function for this activity. The replacement by a feruloyl radical, leads to a complete loss of this activity. The attachment of a caffeate moiety at C-6 of glucose (case of isoverbascoside) appears to be favorable for COX-1 activity and TNF-a release inhibition, although it is detrimental for TX-synthase inhibition activity. On the other hand, the attachment of arabinose is favorable for NO activity and detrimental for TNF-a activity, but, if in this case, the caffeoyl is replaced by a feruloyl moiety, it is favorable for NO and TNF-a activity. Comparing these results with those showed by Sahpaz and cols. [21], we also can conclude that, it should be interesting to develop the assays of Diaz and cols [92], on forsythoside and arenarioside as well, and study the effect of the methoxylation (feruloyl radicals) on diglycosides like leucosceptoside and martynoside, not only on triglyeosides as angoroside. Also it might compare the activity of forsythoside with poliumoside and angoroside A, all of them with three sugars moieties, but, differing on the last one, apiose, rhamnose and arabinose, respectively, joined at the same position of the second sugar moiety, rhamnose. Other authors have been studying other aspects of the inflammatory process. Kimura and cols. [44] assayed different PPGs as forsythiaside, suspensaside, verbascoside and P-hydroxyverbascoside, on the 5-HETE and LTB4 inhibition. They conclude that two adjacent phenolic hydroxyl groups (caffeoyl radicals) are essential for potent inhibition of the formation of 5HETE and LTB4. The inhibition of 5-LOX enzyme is reversible and noncompetitive. With these results, we also suggest that a hydroxylation in position (3 could decrease this activity. Besides, it seems that there is no influence of the position of the second sugar moiety (rhamnose on position C-3 or C-6 of the glucose) or the metoxilation of the hydroxyl groups. But, surprisingly, despite the compounds inhibited the formation of 5-HETE at concentrations 10" -10"3 M, at concentrations between lO^-lO"4 M they stimulated the formations of TXB2 and 6-keto-PGFa.

703

Xiong and cols.,[56] also studied the influence on the inhibition of nitric oxide (NO) by PPGs in activated macrophages, because during inflammatory reactions NO is also produced by iNOS in different cells, such as macrophages, hepatocytes and renal cells. NO acts as a defense and regulatory m olecule with h omeostatic a ctivities [118]. H owever, i t i s a lso pathogenic when it is excessively produced. NO, per se, is a reactive radical, damaging directly to functional normal tissue [119-123]. The PPGs assayed 2'-O-acetylverbascoside, cistanoside A, echinacoside, isoverbascoside, and tubuloside A and B, and verbascoside, could specifically scavenge NO radical at high concentration (200 JJM) without attenuation of iNOS RNA-expression or iNOS protein levels or iNOS activity. The compounds that had a disaccharide moiety (2'-0acetylverbascoside, isoverbascoside, tubuloside B, verbascoside) showed a better inhibitory potency than those with a trisaccharide (cistanoside A, echinacoside, and tubuloside A). This result suggests that an increase in the number of monosaccharide units in glycosylated sugar attenuates the scavenging activity of phenylethanoids for maerophage-generated NO radical [56]. Healing activity Another interesting activity to take into account is the healing properties exerted by some PPGs. The process of wound healing involves a variety of processes such as inflammation, cell proliferation and contraction of the collagen lattice formed [124]. After wounding, several types of cells are recruited into the site of the injury to carry out the processes of repair. Following the neutrophils and monocytes, fibroblasts are attracted into the site to initiate the repair proliferating phase. They are the cells that secrete the collagen fibers and the glycosaminoglycans of the new granulation tissue, and subsequently effect the remodeling of the granulation tissue into mature dermis. They also secrete different growth factors that stimulate proliferation, differentiation and migration of other cells involved in the wound healing [125]. Mensah and cols.[35] showed the effect of two PPGs, echinacoside and verbascoside, both differentiated on the number of sugar moieties, as

704

protectors on human dermal fibroblasts against hydrogen p eroxide-induced oxidant injury. This activity could be attributed to the antioxidant property that has this kind of compounds. Therefore, this antioxidant effect may be one of the mechanisms that facilitate the wound healing. Furthermore, Sudo and cols. [104] studied the effect on collagen by different PPGs. The collagen protein plays diverse important roles, forming connective tissues, basement membranes and core proteins for bone formation. However, the production of an excess amount of collagen in the liver and lung causes flbrosis and eventually, liver cirrhosis. For wounding, the rapid formation of collagen is required in the early phase and effective resorption of excess collagen in the later phase. In each case, a proper turnover of the collagen protein is necessary for the maintenance of homeostasis. They found that verbascoside, at a concentration of 20 |ig/mL formed a more complex network of collagen fibers. These authors compared the activity shown by verbascoside with martynoside, which loose the collagen fibers formation, concluding that the caffeoyl moieties must be required for the expression of the biological activity. Moreover, verbascoside made thinner and densely distributed collagen fibers. This activity could be another mechanism of action in the wound healing activity, since production of minute networks with more slender collagen fibers is favorable for rapid granulations and for avoidance of formation of ugly scars which result from thick collagen fibers. Antimicrobial activity Some PPGs have been tested against Gram-positive and Gram-negative bacteria. Didry and cols. [15] performed the assay over five strains Staphylococcus aureus including one methicillin-resistant strain, five strains of Enterococus faecalis, three strains of Pseudomonas aeruginosa, five strains of Escherichia coli, three strains of Proteus mirabilis and one of each strain of Enterohacter aerogenes, Klebsiella pneumoniae, K. oxytoca with, , arenarioside, ballotetroside, forsythoside and verbascoside. These compounds do not possess antimicrobial activity up to 128 |a,g/mL. Verbascoside, forsythoside, arenarioside possessed a moderate inhibitory

705

effect against S. aureus and P. mirabilis. Arenarioside showed the most significant results from all of them. But, in this publication, the authors did not show the effect that the tested PPGs have on the rest of the bacteria that we have mentioned above. Kyriakpoulou and cols,[28] discovered that samioside, is more active than verbascoside against S. aureus, S. epidermidis, Enterobacter cloaceae, E. coli, K. pneumoniae and Pseudomonas aeruginosa. This result could make to think that an additional sugar moiety, in this case, apiose, at C-4 of rhamnosa could contribute to the antibacterial activity. To confirm this, it would be interesting to study some other PPG that have modifications on the sugar moieties, or feruloyl radicals in stead of caffeoyl ones. Despite the PPG showing a weak activity, it has developed further antimicrobial assays. Pardo and cols, reported [34] the MIO= 400 p.g/mL and MBC= 800 jxg/mL of verbascoside. Moreover, Avila and cols. [33] studied its mode of action in vitro. There is no evidence of inclusion of [3H]-leucine into the cell, whereas [3H]-thymidine and [3H]-uridine were not observed. That resulted in the conclusion that the mode of action of verbascoside is through the inhibition of protein production, since leucine is an important metabolite in protein synthesis. Antitumoral-chemopreventive activities The PPGs exert a cancer chemopreventive activity through different mechanisms: Repair ofDNA Adducts:

DNA damage can be caused by the environmental agents, such as ionizing radiation, UV light, and a variety of chemicals as well as normal metabolism in which reactive oxygen species are formed as side-products. With regard to ionizing radiation, there are direct and indirect ways to damage DNA. With direct effect, ionization occurs within the DNA itself and generates base radical cations and base radical anions. Indirect damage is caused by

706

hydrated electrons and hydroxyl radicals produced by ionization taken place in close vicinity to DNA reacting with DNA. DNA gives up base electron adducts and base radical anions [126-128]. Considering the scale of electron affinity of the different nucleosides, pyrimidine is found to be a much better electron acceptor than purine. On other hand, the purine bases, with the electron-rich imidazole, react with hydroxyl radical (OH*) faster than the electron-deficient pyrimidine [127]. This fact reflected the electrophilic nature of the hydroxyl radical. Concerning cellular DNA, a two-component hypothesis has been developed. According to this hypothesis, the e lectron loss c enters (radical cations) end up with the purines, particularly with the guanine moiety, whereas the final site of deposition of the ejected electron is with the pyrimidines, particularly with thymine [127]. The two-components hypothesis implies that in DNA there are mechanism of electron and positive hole transfer by which the initially generated and randomly distributed electron gain and loss centers are tunneled into the T and G "traps" respectively. The PPGs tested have been active both in purine and pyrimidine adducts, reducting or oxidizing radicals [73,76, 87, 88, 93,126, 129]. In table (2) it is summarized the different PPG that have been tested against some DNA base adducts, explaining the mechanism of action.

707 Table (2): PPG repairing DNA adducts activity and mechanism of action. Base adduct Mechanism Repair potency Ref. dAMP is rapidly protonated to Cis> Ver > Ped for [74] NH2 NH2 NH 2 give N-protonated radicals, dAMP*" [73] strong reductants. PPGs can Verb > Ped> Cis react with hydrated electron for dAMP(-H)' due to the electrophilic phenyl-substituted unsaturated dAMP- and dAMP(-H)' carboxylic group It may undergo a dehydration Cis > Ped [129] reaction by which a very weak oxidizing radical is converted into a strong oxidizing radical, which leads to a neutral Ncentered radical, that is dAMP-4-OH oxidizing. This can be repair by PPGs NH2 There is a ring-opening Cis > Ped [129] reaction, to give a strong ,OH reducing radical, which is converted into FAP. Can be repaired if it goes dAMP-g-OH under dehydration. This adduct mainly exists in Cis > Ped [129] the oxidizing stats and can be reverted to dGMP or hydrated 'dGMPbyPPGs.

-u:->

i,JL>

dGMP-4-OH ^H 1

1

\

^

HN

%

>

HjiN

N

•

N H

R

N *

N

R

dGMP-5-OH

i

K,

I

R

y-R

OH

It is a reducing radical because there is a little unpaired spin density on the nitrogen. But, if it goes under dehydration, becomes an oxidizing radical. PPGs can repair it by donating electron. Predominantly it is a reducing radical. The repaired proportion by PPG is very little.

Cis > Ped

[129]

Cis > Ped

[129]

R

dGMP-8-OH

oi

IT R

TMP radical anion

PPGs react through the electrophilic phenyl substituted unsaturated carboxylic group.

Cis > Ped > Verb [126]

708

The PPGs are mainly nucleophilic, (electron donors) that react easily with the oxidizing adducts (electrophilics), and thanks to the non-saturated carbonyl- phenyl moiety, which is electrophilic, they can act as electron acceptors too. In a wide sense, we can conclude that the PPGs are able to repair the DNA due to their phenol groups and to the electron transfer process by the formation of a complex in minor groove of the double helix [89]. Due to the two-components hypothesis mentioned above, it is reasonable to say that by repair TMP radical anion, PPGs can repair indirectly other base radical anions produced in cellular DNA by radiation. In case of dAMP and dGMP-8 OH, there is redox ambivalence. This is a general property of radicals since they are in-between two stable oxidation states; Therefore, they can be oxidized or reduced depending on their reaction partner. The proportion repaired, depends not only on the reducing activities of PPGs but also on their concentration in the repaired system. Finally, there has been found a structure-activity relationship, the repair activities of PPGs toward oxidizing hydroxyl adducts of dGMP and dAMP are also positively related to the number of phenolic hydroxyl groups.[129]. Prevention against tumor formation induced by carcinogens

The PPGs were able to inhibit the tumor formation induced by different carcinogenic agents, such as galactosamine, [63, 64] lipopolysaccharide [54] due in part to the increase of their elimination. Induction of cell differentiation:

Verbascoside and isoverbascoside are able to induce differentiation on different cell lines. In case of verbascoside, on MKN45 [85] and human gastric adenocarcinoma (MGc803) [86] cell lines and for isoverbascoside, human hepatocellular carcinoma (SMMC-7721) [83], MGc803 [84], and human promyelocytic leukemia (HL-60) [109]. Despite the mechanism by which these two compounds induce differentiation in these cell lines remains to be investigated, some of the observed effects are the accumulation of cells

709

in GQ/GI phase, Mid the decrease in S phase. This arrest is a common phenomenon in the cells undergoing induction of differentiation [130]. Besides, isoverbacoside induces the upregulation of protein expression of p53, p21/WAF, and pl6/INK4, as well as the suppression of C-myc expression [84]. The cyclin-dependent kinase inhibitors such as p21 and pi6 proteins play central roles in this process. These proteins exert their functions by combining with cyclins/CDKs complexes, conducting finally to the blockage of the cell cycle at Gl-S checkpoint [131,132]. p53 is a tumor suppression gene, that can exert its cell cycle arresting function by up-regulating the expression of p21/WAFl protein [131, 132]. On the other hand c-myc is an oncoprotein, that acts as a nuclear transcription factor, and regulates the cell cycle by promoting the transcription of DNA synthesis related genes and therefore, impelling cell cycle into S phase [131,132]. Another important fact related to the induction of the cell differentiation is the antioxidant activity that these compounds have. The tumoral cells have lower antioxidant levels; therefore, ROS scavengers might induce this differentiation. Another mechanism closely connected to the antitumoral activity is the inhibition of oncologic signals and enzymes implicated on proliferation, differentiation and transformation processes. Verbascoside and homoplantaginin inhibit EGFR [63] and tirosin-kinase (TK). Calceolarioside A and B, forsythoside, leucosceptoside A, plantainoside, poliumoside, and verbascoside inhibit a-isoform of protein-kinase C (PKC) [69, 100]. The potency of this activity might be related to the number of sugar moieties in an inverse way, but it required more assays with other PPG to find the relationship. It has been found that verbascoside interacts with the catalytic domain of this enzyme, but a structure-activity relationship was not discussed. Apoptosis activation:

Verbascoside is also able to induce apoptosis [24]. The apoptosis is the programmed cell death functioning to conserve tissue homeostasis. It is

710

induced by the activation of cysteine proteases (caspases), ceramide formation by sphingomyelinase, activation of MAPK cascade and generation ofROS. Their mechanism of action still remains unknown, but it has been observed that those PPGs that also have pro-oxidant activity, can exert apoptosis activation as well, probably due to a generation of DNA damage through the hydrogen peroxide generation. This DNA damage might lead to the apoptosis process because of the accumulation of DNA fragments. Surprisingly, better results have been obtained with in vivo than with in vitro assays. Telomerase inhibition:

Verbascoside is able to inhibit another enzyme related to tumor cells, the telomerase [85]. The tumoral cells express this enzyme that elongates the 3' ends of telomere [133]. Telomere shortening and telomerase activity have been detected in almost all human tumors but not in normal somatic tissues [133, 134]. Zhang and cols, [85] showed that the telomerase inhibition by verbascoside may involve telomere-lenght regulation. Although verbascoside can arrest tumor cell growth, repair DNA oxidative damage and induce cell apoptosis and differentiation, whether telomerase inhibition by verbascoside may lead to tumor cell apoptosis, and whether all the cell cycle changes exist as a common mechanism, is not yet known. Anti-angiogenic and anti-metastasis

Finally, the effect of verbascoside on others carcinogenic steps, angiogenesis and metastasis, have been studied [111, 112], where ROS may also be important. This activity has been tested in vivo. Cytotoxicity:

Surprisingly, of all the cell lines studied, verbascoside only showed a cytotoxic effect on the cells with a murine origin [23, 32, 91]. Other PPGs

711

have been studied and it seems to be a structure-activity relationship, where the possession of feruloyl radicals, decreases the activity. We can conclude suggesting more deep assays that contribute to the knowledge of the wide antitumoral activity that verbascoside and isoverbascoside have, because they seem to be promising antitumoralchemopreventive agents. Moreover, it is thought that more than one mechanism of action is implicated. Besides, it should be interesting to develop all these assays with other PPGs, to compare their activity and be able to find more exact structure-activity relationship. ABBREVIATIONS A7r5 AAPH B16F10 BZD cAMP

CCUCOX CTAB dAMP dAMP(NH) dAMP+ dAMP-OH D-Gal dGMP dGMP+ dGMP-OH DMBA DPPH dRLh-84 EBV-EA EGFR H2O2 HeLa

=

= =

=

Rat aortic smooth muscle cells. 2,2'-azo-bis(2-amidinopropane)dihydrochloride Murine melanoma. Benzodiazepine cyclic 3',5'-adenosine monophosphate Carbon tetrachloride Cyclooxygenase Cetyl trimethylarnmonium bromide 2' -deoxyadenosine 5' -monophosphate dAMP N-protonated radical adduct dAMP radical cation dAMP hydroxyl radical adduct D-Galactosamine 2'-deoxyguanosine 5'-monophosphate dGMP radical cation dGMP hydroxyl radical adduct 7,12-dimethylbenz(a)anthracene 1,1 ,-diphenyl-2-picrylhydrazyl radical Rat hepatoma Epstein-Barr virus early antigen Epithelial growth factor receptor Hydrogen peroxide Human epithelial carcinoma

712

HeLa HIV HL-60 HSV-1 HSV-2 L1210 5-LOX LPS mCMV MDA MGc803 MK-1 MMP MPP NO Of OH P-388 P-388-D1 RSV S-180 SMMC-7721 T" TK TLC TMP TNF-a T-OH TPA

Human uterus carcinoma. Human immunodeficiency virus Human promyelocytic leukemia Herpes simplex vims type 1 Herpes simplex virus type 2 Lymphocytic mouse leukemia 5-lipooxygenase Lipopolysaceharide Murine Cytomegalovirus Malondialdehyde Human gastric adenocarcinoma Human gastric adenocarcinoma Metaloproteinases l-methyl-4-phnylpyridinium ion Nitric oxide. Superoxide Hydroxyl radical Murine lymphocytic leukemia. Mouse lymphoid neoplasma Respiratory syncytial virus Sarcoma Human hepatocellular carcinoma TMP radical anion Tirosin kinase Thin layer chromatography. Thymidine-5' -phosphate Tumor necrosis factor Thymine-hydroxyl radical adduct 12-0-tetradeeanoylphorbol-13-acetate

713

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©

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DEVELOPMENT OF TUBULIN INHIBITORS AS ANTIMITOTIC AGENTS FOR CANCER THERAPY S. MAHBOOBI1, A. SELLMER1 and T. BECKERS2 Department of Pharmaceutical Chemistry I, University ofRegensburg, D-93040 Regensburg, Germany, Germany; phone: (+49) 941-9434824, Fax: (+49) 941-9431737, E-mail: [email protected] 2

Therapeutic Area Oncology, ALTANA Pharma AG, D-78467 Konstanz, Germany, Phone:(+49) 7531 842974; Fax:(+49) 7531 8492974; E-mail: Thomas. Beckers@altanapharma. com ABSTRACT: Cancer represents one of the most threatening diseases of mankind. Within the last decade, our understanding of malignant cell growth and regulation of the cell cycle machinery has offered several new molecular targets with the promise of higher selectivity in human cancer therapy. Within mitosis, aP-tubulin heterodimers, building up the mitotic spindle, are still an attractive target in the development of anticancer drugs. In the following article, we review the recent advances in the development of tubulin interfering agents. These agents are divided according to their mode of action into colchicine site binder, vinca-alkaloid related drugs and those interacting with the Taxol binding site and functioning as stabilising agents. Since clinically used compounds such as Paclitaxel or Vincristine are facing severe disadvantages, namely a small therapeutic window, restrictions in bioavailability and solubility, a complex synthesis and most importantly development of drug resistance in patients, special emphasis is laid on the development of synthetic small molecule tubulin inhibitors (SMTIs). These new agents offer promise for the rational design of new chemotherapeutic drugs by their simple structures and potential broad applicability in 2nd and 3 rd line standard chemotherapy regimens towards resistant tumours. In this regard, most SMTIs are not P-glycoprotein substrates. Most tumours can only grow beyond a critical size by inducing the formation of new blood vessels, a process called neovascularisation. SMTIs as well as natural tubulin inhibitors have been described to interfere with this angiogenic process and some, like combretastatin A4 phosphate, are even described as selectively damaging tumour vasculatures. Finally, the present review emphasises the preclinical and clinical status of tubulin inhibitors in cancer therapy.

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INTRODUCTION Cancer represents one of the most threatening diseases of mankind. In spite of great efforts in basic research, cancer is still the second leading cause of death in industrialised nations and this picture will get even worse in near future. Within the last decade a better understanding of the cell cycle machinery offered several new targets with the promise of higher selectivity in cancer therapy. Uncontrolled proliferation is a hallmark of cancer cells, thus selective interference with the M-phase of the cell division cycle is most important. Within mitosis, after condensation of nuclear chromatin and disruption of the nuclear envelope, the mitotic spindle is formed which segregates the daughter chromatids. Cyclindependent protein kinases (CDKs) and their inhibitors (CKIs) [1] are key regulators of the cell cycle machinery and microtubules [2] and microtubule associated proteins (MAPs), building up the mitotic spindle, are of high interest in anticancer drug development. In the present review, we report about recent development of compounds that interfere with the dynamics of tubulin polymerisation and depolymerisation by direct binding to tubulin (for earlier review see [3,4, 5,6,7]. Tubulin, a heterodimer of closely related and tightly linked globular a- and p-tubulin proteins, is the essential structural element of the mitotic spindle, a- and P-tubulin heterodimers are polymerised parallel to cylindrical axis-building, helical, hollow tubes called microtubules, forming the mitotic spindle. The mitotic spindle is a bipolar, self-organising machine that gathers energy from nucleotide hydrolysis to segregate sister chromatids accurately into daughter cells [8,9]. The rapid switch between growing and shortening states of microtubules in the process of dynamic instability and driven by P-tubulin dependent GTP hydrolysis is essential for the movement of chromosomes [10]. A modification of microtubule properties is induced by binding of microtubule associated proteins (MAPs) [11,12]. Besides their function in mitosis, microtubules are also associated with important cellular processes, namely axonal transport and cell movement [2]. Tubulin binding agents interfere with the dynamic instability of microtubules and thereby arrest mitotic cells in the M-phase of the cell division cycle, finally leading to induction of apoptosis. Most, if not all, tubulin inhibitors bind within B-tubulin to distinct epitopes. They are

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categorised according to their tubulin binding sites or to the first compound described (e.g. colchicines or vinca alkaloid like tubulin binding agents). Colchicine itself and colchicine analogues predominantly bind to a high affinity site, the so-called colchicine binding site located at the intradimer interface between a - and p-tubulin, facing the lumen of the microtubule. Colchicine (1) Fig. (1) inhibits microtubule formation and disrupts microtubules as a tubulin destabilising agent. HjCO.

/^O

H 3 CO'

0

OCH3 1 Colchicine

3

Taxol INN Paditaxe!

Fig. 1: Chemical structures of Colchicine (1), Vinblastine (2), Vincristine (2a) and Taxol (3).

Vinblastine (2) and several vinca-related drugs bind to a different site, as do a number of other drugs that bind competitively with each other, but do not compete with the colchicinoids. Like the colchinoids, vinca alkaloids destabilise microtubules. The Taxol (3) binding site is located in a pocket that is lined by several hydrophobic residues and is well defined from crystal structures of ap*-tubulin. It represents the putative binding site for other microtubule stabilising drugs like epothilones [2,11]. Other binding sites have been postulated according to tubulin interfering agents with a binding behaviour distinct to that of taxanes, vinca alkaloids and colchinoids.

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From a therapeutic point of view, compounds of low molecular weight with oral bioavailability and high therapeutic index are very attractive as lead structures. By inhibition of tumour cell proliferation and neovascularisation, tubulin inhibitors are potent anticancer drugs. Nevertheless, clinically available compounds such as Paclitaxel or Vinchristine are facing severe disadvantages. [13]. hi the following we therefore focus on different structures with low molecular weight, socalled "small molecules tubulin inhibitors" or SMTIs. Most of these SMTIs can be placed in the category of colchicine-site binders. THE FAMILY OF COLCHICINE-SITE BINDING TUBULIN INHIBITORS The family of colchicines-site binders includes compounds of diverse structure unified by interference with the colchicine binding epitope. The respective compounds can be clustered according to their chemical structure mainly into I) colchicines and compounds with colchicine like substructures, II) combretastatins and phenstatins, III) compounds having an indole core structure, IV) quinolones, V) sulphonamides and VI) naturally occurring as well as synthetic compounds. Colchicinoides Colchicine (1), the first known tubulin binding agent isolated from the plant colchicum autumnale, is too toxic to be used as an anticancer agent. Nevertheless it has been used in the treatment of gout and other inflammatory diseases since the 6th century a.c. [14]. Binding of colchicine induces an alteration in tubulin dimer structure and hinders micro tubule assembly. The drug (1) additionally can bind to tubulin at a second, lower affinity site in a reversible manner [15]. Another highly active and naturally occurring colchicine analogue is cornigerine (la) Fig. (2), derived from colchicum cornigerum [16]. In order to reduce the toxicity, a lot of efforts have been made to provide new analogue as potential anticancer drugs. Previous experimental observations as well as structure evaluation programmes (SAR) suggested that the colchicine binding site of B-tubulin has very stringent structural requirements. The three methoxy groups in the A-ring

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seem to be essential for high binding affinity. The seven-membered ring and the C (7) side chain do not seem to be essential for binding to tubulin. Furthermore, the seven-membered C-ring may also be exchanged by a benzene ring, as shown by the high biological activity of allocolchicine (lb) (IC50 = 1 . 4 |jM in inhibition of tubulin polymerisation) and (lc), which was shown to inhibit the tubulin polymerisation reversibly and much more potently than colchicine itself without enhanced toxicity (for review see [17,18] ). Further allocolchicinoids, exhibiting high tubulin binding affinity and potent inhibitory activities against solid human tumour cell lines, have been reported [19]. ZD 6126 Ongoing research has resulted in ZD 6126 (Id) [20], the water-soluble phosphate prodrug of N-acetylcolchinol, a novel tubulin binding agent directly targeting tumour vasculature. It is currently developed by AstraZeneca in phase II clinical trials in solid tumour patients and was inlicensed from Angiogene Pharmaceuticals Inc. in 1999. In preclinical studies, single doses of ZD 6126 (200mg/kg i.p.) induced haemorrhage and necrosis in the PC14PE6 NSCLC nude mice metastasis tumour model with some selectivity towards tumour endothelial cells [20]. In a 2nd preclinical study, ZD6126 showed significant effect on tumour vasculature in different nude mice xenograft models at well-tolerated doses up to 16fold below the MTD of « 400mg/kg (i.p. or i.v.). In the Calu-6 NSCLC model, lOOmg/kg ZD 6126 (i.p. for 5d) in combination with 4mg/kg Cisplatin had more than additive effects on tumour growth delay [21]. Phase I clinical trials for definition of a MTD, DLT and circulating endothelial cells (CECs) as a surrogate marker has been finished recently, showing reasonable tolerability, a rapid clearance of Nacetylcolchinol with t\a = 2-3h and a 2 fold increase in CECs 4-6h after ZD6126 infusion [22,23].

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H3C OPO 3 H 2 H3C 1a

Cornigerine

1b R = CO 2 CH 3 Allocolchidne 1c R = COCH3

1e ZD6126

EtO. H3C

4 2-Methoxyestradiol (NSC-659853)

Ring expanded homologes of 4 exhibit structural similarity to colchicine

Fig. 2: Colchicinoide derivatives and synthetic steroids exhibiting antiproliferative activities

2-Methoxyestradiol / NSC-659853 2-Methoxyestradiol / NSC-659853 (4), a cytotoxic human metabolite, binds to the colchicine site of tubulin with an affinity of IC50 = 4.7 \iM. On the basis of a hypothetical relationship to the colchicine structure, a series of B-ring expanded 2-ethoxyestradiol analogues were synthesised, in which the B-ring of the steroid was replaced by the B-ring of colchicines. While the resulting analogues showed significant affinity to the colchicine binding site consistent with the proposed structural resemblance, derivatives having a ketone at C-6 surprisingly behaved like Paclitaxel [24]. Further investigations on 2-methoxyestradiol have been performed and shown anti-proliferative effects, both on hormone dependent and independent breast cancer cells [25] as well as on antiangiogenetic activity [26]. The pharmacological profile has furthermore been shown to be especially dependent on steric and electronic influences of the substituent in position 2 [27]. Phase II trials with solid tumours, sponsored by EntreMed, are ongoing (National Cancer Institute, Maryland, USA, web site: [email protected]).

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Combretastatins, Heterocombretastatins and Phenstatins Combretastatin A-4 A second group of tubulin inhibitors is represented by compounds exhibiting the combretastatin structure. Combretastatin (5) itself, as well as combretastatins A-l (5a), A-4 (5b) and A-2 (5d), have first been extracted from a South African tree Combretum caffrum by Pettit et al., depicting affinity to the colchicine binding site [28]. Syntheses of various analogues of this simple molecule, thereafter, have been reported [16,29, 30] (see also Scheme 1).

CBr4, PPh3

Bu3SnH Pd(PPh3)4 H3C

H3C

B(OH)2 Pd(PPh3)4 Na 2 CO 3

H 3 C . '' ° CH 3 H3C

(Suzuki Cross-Coupling) Combretastatin -A4

Scheme 1: Synthesis of Combretastatin-A4 according to Gaukroger [36]

Combretastatin-A4 (5b) is one of the most potent antimitotic agents [31] also active towards multi-drug-resistant (MDR) cell lines [18]. Research on combretastatin A-4 (5b), which has so far reached phase II in clinical development, has focussed on the structure dependency of antimitotic combretastatin agents [32], improvement of water solubility and in vivo activity [16]. One of the most important findings has been the introduction of an amino group instead of the phenolic hydroxy group as shown by the amino stilbene AC-7728 (5e), increasing water solubility and efficacy [31]. Based on the structural features of (5e), different heterocombretastatins (e.g. 5f-5h) have been prepared retaining IC50 values for inhibition of tubulin polymerisation in the low nanomolar range [33].

726

The water-soluble prodrug of combretastatin A-4, Combretastatin-A43-O-phosphate (CA4P) (5c) was licensed by Oxigen Inc. from Arizona State University in 1997. Like ZD6126 (le), CA4P acts as a tumour vascular targeting agent leading to tumour necrosis by shut-down of blood flow [34] (for review see [35]). CA4P was investigated in three phase I clinical trials and the results from the Ireland Cancer Centre Trial have been published recently [36]. CA4P currently is evaluated in phase II clinical trials [37], monitoring tumour blood flow as the critical parameter for optimal dosing of tumour vasculature targeting drugs. A-289099 and A-l05972 The recently developed oxadiazoline A-105972 (5i) displayed reasonable cytotoxic activity towards a panel of human cancer cell lines in vitro [38], but its utility in vivo was limited by a short half-life. Further efforts led to the identification of the indolyloxozoline A-259745 (5k) [38] that demonstrated a better pharmacokinetic profile and three times increased survival of tumour bearing nude mice upon oral dosing. The mechanism of tubulin interference and the enantio selective synthesis of A-289099 (5k, S-enantiomer) have been investigated, confirming the competition with colchicine [39]. After the preclinical development no further investigation has been reported. Another group of closely related compounds is the phenstatins. Phenstatin (6a) and hydroxyphenstatin (6b) resulted from a SAR-study on combretastatin A-4 and exhibited potent inhibition of cancer cell growth [29]. Further modifications have been performed, resulting in highly potent agents, namely 243869 (6c) with in vitro toxicity of IC50 = 0.58 nM towards HeLa cervical carcinoma cells [40,41]. SAR information about a series of 3-aminobenzophenone compounds (e.g. 6d and 6e), based on the mimic of the aminocombretastatin molecular skeleton, revealed that by the introduction of an amino group instead of a hydroxy group inhibition of tubulin polymerisation through binding to the colchicine binding site could fully be preserved or even improved [42].

727 727 H3C O H3C

NH2

H3C

H3C H3C

5a Ri = R2 = OH, R3 = R4 = CH 3 Combretastatin A-1 5b R-i = H, R2 = OH, R3 = R 4 = C H 3 Combretastatin A-4 5c R, = H, R2 = OP(O)(ONa)2, R3 = R4 = CH 3 Combretastatin A-4-3-O-phospate 5d R-) =H, R2 = OH, R 3 and R4 briged byCH 2 Combretastatin A-2 5e Ri = H, R2 = NH 2 , R3 = R4 = CH 3 AC-7739/AVE-8063

5f

'

x

5g

"

x

_

N=N

NH 2

5i A-105972

-CH 3

5k S-enantiome: A-289099 Racemate: A-259745

51 Heterocombrestatines e.g. 2-benzoylindoles

6 6a 6c 6d 6e

R-i = H , R2 = OH, R 3 = O C H 3 Phenstatin R, = R2 = OH, R3 = OCH3 H ydroxyphenstati n R1 = H; R2 and R3 = methylendioxy R1 = H, R2 = NH 2 , R3 = OCH 3 R1 = H, R2 = NH 2 , R3 = OC 2 H 5

Fig. 3: Chemical structures of different combretastatins and phenstatins

Incloles Several compounds possessing an indole moiety as a core structure have been developed during recent years Fig. (4).

728 728

8 7

Dlarylindoles

6-Methoxy-2-(4-methaxyphenyl)3-(3,4,5-trimetoxybenzoyl)benzo[b]thiophene

9

X = S, Benzthiaphene-derivatives X = NH, R = H, OCH 3 , F e.g. 6-Methaxy-2-(4-methoxyphenyl) indole

C4H9 10

12-Formyl-5,6-dihydro-indolones

N-Pyndin-4-yIH1-(t-chIorbenzyl)lndol-3-yl]glyoxyl-am(d (D-24851)

12 R = H, D-84131 12a R = OCH 3 , D-64144 12b R = F, D-8118?

13 Nocodazole

Fig. 4: Tubulin polymerisation inhibitors possessing an indole moiety as a core structure

Diarylindoles, e.g. (7), classified as heterocombretastatins with respect to the ethylene bridged diaryl structure and the 3,4,5-trimethoxy substitution pattern, displayed cytotoxicity especially towards leukaemia, non-small cell lung and CNS cancers [33, 43]. A modification of the tubulin inhibitor (8) [44] led to the thiophene and indole analogue (9) with remaining high cytotoxicity (IC50-values in the range of 10 to 100 nM) [45]. The structure of (9) also represents a fragment of the tetracyclic tubulin inhibitor (10) [46] that showed similar activity.

729 729

D-24851 (INN: Indibulin) A structurally related member of this group is presented by N-(pyridin4yl)-[l-(4chlorbenzyl)-indol-3yl]glyoxylamide / D-24851 or Indibulin (11), a compound currently in phase I clinical development by Baxter Oncology. D-24851 destabilises microtubules and blocks cell cycle transition specifically at the G2-M phase by binding to B-tubulin, competing with colchicine for binding [47]. D-24851 is highly cytotoxic in vitro towards a panel of established human tumour cell lines including SKOV3 ovarian cancer, U87 glioblastoma, ASPC-1 pancreatic cancer cells as well as towards tumour cell lines with various resistance phenotypes. hi vivo, a complete tumour regression in rats bearing Yoshida AH13 sarcomas was observed. D-24851 displayed no neurotoxic effects at efficacious doses in rats as studied by a deficit in motor function and reduction of nerve conductance velocity. No data on D-24851 analogue, developed by Zentaris GmbH, have been published so far. 2-Aroylindoles Another recently published class of potent antimitotic agents is 2-aroylindoles (12-12b), exhibiting ICso-values in a low nanomolar range (IC50 = 20 to 75 nM) towards various carcinoma cell lines. 2-Aroylindoles bind to the colchicine site of (3-tubulin, however, and in contrast to colchicine (1), vincristine (2a), nocodazole (13) or taxol (3), they showed no significant influence on the GTPase activity up to 10 fiM [48,49]. In addition, angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay was inhibited by D-64131 and analogue. Since 2-aroylindoles are easy to synthesise (Scheme 2), the role of the indole substitution pattern was studied in more detail. hi terms of SAR information, substituting the indole structure (azaindoles; X or Y = N) yielded inactive or compounds of low cytotoxicity. The same holds true for N-alkylation or substitution of the methoxy-group in the indole core structure of (12a) vs. a methyl group. In summary, the results from the cellular cytotoxicity screening suggested the benefit of the 5-methoxy-indolyl-group for potent antitumour activity. The 3-methoxyphenyl- (12a) (IC50 < 3.2 pM) and the 3-fluorophenyl(12b) derivatives showed even higher cytotoxic activity than the unsubstituted compound (12). An insignificant electronic effect of the substituents in the aromatic ring is observed. The respective aroylindoles

730

have furthermore been shown to be no MDR/MRP substrates with no cross resistance with various resistance phenotypes [50]. In xenograft studies, no signs of systemic toxicity were observed after p.o. dosages of up to 400 mg/kg of D-64131 [13].

X and Y = C or N

Scheme 2: Synthesis of 2-aroylindole derivatives and the respective precursors. Conditions: i: THF, - 78 °C; ii: Pyridiniumchlorocromate (PDC), CH2C12, 20 °C; iii: THF, -78 °C; iv: NaOH, EtOH, A; v: Tetrabutylammoniumfluoride (TBAF), THF, A.

Quinolones Quinolones and related structures represent another class of compounds affecting the colchicine-binding site of tubulin. The synthetic 2-phenyl-4-quinolones (14) Fig. (5) structurally related to naturally occurring anti-mitotic flavonoids (15) [17] displayed promising activity and impressive differential cytotoxicity towards human tumour cell lines with IC50 values in the low micromolar to nanomolar range, comparable to that of colchicine. The most active compounds from this series were obtained by introducing of functional groups with non-bonding electrons e.g. -NRR', -OCH3, Cl or F at the 6'position of the A-ring and the 3'position of the C-ring. The structurally related 2-phenyl-l,8naphthylpyridin-4-ones (16), containing an additional nitrogen position 8 of the aromatic system, also exhibited potent cytotoxicity in particular towards the ovarian cancer 1A9 and the P-gp-expressing, vincristine resistant HeLa/KB-VIN cell lines.

731

14

2-Phenylquinolones e.g.R = N-pyrrolidine

14a

Fluorinated 2-Phenylquino!ones e.g. NSC 656158

15

Flavones

16

related quinazolones

14 b

Fig. 5: Chemical structure of quinolones, flavones and quinazolones

Further investigations based on SAR-studies resulted in fluorinated analogue (e.g. 14a and 14b), exhibiting high cytotoxicity against renal and melanoma tumour cell lines with ICso-values in the in vitro tubulin polymerisation assay of 0.46 |j,M (14b). The biaryl system, composed of rings A and C, being probably analogous to the biaryl-system occurring in many antimitotic agents, as well as the ketone functional moiety have been shown to be essential for a strong interaction with tubulin [51,52,53, 54]. Sulphonamides E7010 / ABT-751 Sulphonamides as E 7010 (17) inhibit tubulin polymerisation by binding to the colchicine site, exhibiting potent anti-proliferative activity in vitro (IC50 = 0,2 - 40ng/ml), currently investigated in clinical phase II studies [55,56] by Eisai/Abbott in patients with solid tumours. The pharmacophore structure responsible for tubulin binding seems to be the 3-pyridinyl-4-methoxybenzenesulfonamide substructure [57] Fig. (6). New analogue have been published recently by Abbott having a higher cytotoxicity but are disadvantageous because of short half-lives of t V2 < lh [58]. Closely related structures being active against multidrugresistant-cancer cells are an illustrative example of the different modes of

732 732

anticancer-drug-action that may result from small pharmacophore modifications. T138067 (Batabulin) Tl38067 (18), currently in phase III clinical trials in therapy of liver cancer, inhibits tubulin polymerisation by irreversible binding to cysteine239 on (3-tubulin isoforms 1, 2, and 4. The covalent modification of Ptubulin inhibits the polymerisation of the a(3-tubulin heterodimer into microtubules, leading to cell arrest followed by apoptosis induction [57,59]. A recently described analogue of T138067, exhibiting an amide structure instead of fluorine substituent in the B-ring and less lipophilicity, retained the same mechanism of action and an increase in potency towards HeLa and MDR expressing and non-expressing human mammary carcinoma cells [60]. E7070 (INN: Indisulam) hi contrast, the antimitotic compound E7070 (19), synthesised by Eisai Co. Ltd (Tsukuba Research Laboratories, Ibaraki, Japan) disturbs P388 murine leukaemia cells by disrupting the cell cycle progression and not by inhibition of tubulin polymerisation. This compound, structurally closely related to E7010 and Tl 38067, caused accumulation of cells in the Gl but not in the M-phase, as typically observed for tubulin inhibitors and can therefore be classified as cell cycle inhibitor. Human tumour xenograft models demonstrated that E7070 could cause a tumour regression in three of five colorectal and two out of two lung cancer models [57,61]. Phase II clinical trials are ongoing.

H2NSO:

17

E7010

18 T138067

Fig. 6: Structures of antimitotic sulphonamides. Dependent on the pharmacophore core structure the respective compounds can be classified as reversible (17) or irreversible (18) tubulin inhibitors respectively as cell cycle inhibitor (19).

733 733

Miscellaneous Compounds Affecting the Cokhicine Binding Site: Indanones, RPR112378 andRPR115781 (E)-2-Benzylidene-l-indanones (20) e.g. indanocine/NSC698666 [62] and the water-soluble prodrug SDX103 are in preclinical development by Salmedix. Indanocine (20) is a synthetic indanone that has been identified by the NCI Developmental Therapeutics Programme. Indanocine interacts with the colchicine binding-site of B-tubulin, potently inhibiting tubulin polymerisation in vitro and inducing apoptosis in cancer cells at concentrations that do not impair the viability of normal non-proliferating cells [63]. RPR112378 (21) and RPR115781 (21a) extracted from the Indian plant Ottelia alismoides were identified in a screening programme for new anti-mitotic drugs by a research group at Rhone-Poulenc Rorer / Aventis Pharma. The more potent destabilising tubulin inhibitor (21) (IC50 =1.2 \iM, tubulin polymerisation in vitro) competes with Colchicine for binding and reacts with the sulfhydryl groups of tubulin. RPR-112378 furthermore was shown to be cytotoxic with IC50 = 20nM towards the HeLa / KB human cervical carcinoma cell line [64]. No further preclinical data, or development, have been published on this new class of compounds until now. OH HO' CH3

20 Indanones, e.g.

21

RPR112378

21a

RPR115781

Indanocine NSC 698668

Fig. 7: Chemical structures of indanocine, RPR112378 and RPR 115782, compounds affecting the colchicine binding site

SYNTHETIC SMALL MOLECULES OF DIFFERENT STRUCTURE TARGETING BINDING SITES DISTINCT FROM THAT OF COLCHICINOIDS COBRA-0 and COBRA-1 Based on the structure of ap-tubulin dimer, resolved by electron crystallography of zinc-induced tubulin sheets, a previously unrecognised

734 734

hydrophobic binding pocket on the surface of a-tubulin was identified. A novel synthetic drug targeting this unique binding cavity in a-tubulin was synthesised, designated Cobra-0 because of its mono-THF head portion attached to a long Cn-chain, resembling the shape of a cobra. COBRA-0 (22) and the analogue COBRA-1 (22a), the first enantiomerically pure prototype compounds targeting this cavity, bind to a-tubulin and exhibit weak cytotoxicity in concentrations > 100 fiM. Treatment of human breast cancer and glioblastoma cells with COBRA-1 caused destruction of microtubule organisation and induction of apoptosis [65]. SPIKET-Compounds SPIKET-P (23), a novel synthetic spiroketal Pyran [66], represents another pharmacophore identified by docking simulations [67] with the marine product Spongistatin 1 (24), a macrolide polyether [68]. SPIKET compounds target the spongistatin binding site of [3-tubulin and exhibit potent cytotoxicity with IC50 values against the NCI panel of human cancer cell lines in the sub-nanomolar range (for review see [5]). The preclinical and clinical status of the compounds as mentioned in the sections before is summarized in table 1. H25C1:

OH

22

WHI-261 /Cobra 0

22a

OH

Cobra 1 OH

13 H

23 SPIKET-P1 AcO'

CH, H3C

OAc

OH

24 Spongistatin 1

Fig. 8: Synthetic Small Molecules of Different Structures Targeting Various Binding Sites

735

Table 1: SMTIs currently investigjated in preclinical / clinical studies Compound Name (Nr.) ZD-6126 N-Acetylcolchinolphosphate (le) 2-Methoxyestradiol NSC-659853 (4) CA4P (Prodrug) (5c)

Status in Company ic 50 development values in vitro Phase II n.p. Astra Zeneca ongoing

Phase II ongoing Phase II

A-289099 (5k)

Ceased

D-24851 (11)

Phase I

D-64131 (12) E-7010/ ABT-751 (17) T-13 8067/ Batabulin(18)

4.7 uM

0.9-3 nM Oxigene INC (drug) 7nM

36-285 nM Preclinical 24 - 144 nM Phase II 0.2-40 ng/ml Phase III 11-165 (liver cancer) nM

Indanocine NSC-698666 (20)

Preclinical

EntreMed

< 20 nM

Abbott Laboratories Baxter Oncology Baxter Oncology Abbott Laboratories Tularik Inc.

Salmedix

Remarks

Ref.

vascular targeting agent

[22,23]

angiogenesis inhibitor vascular targeting agent

[24,26, NCI website] [30,34,36 37] [39] [47]

angiogenesis inhibitor

[50] [56]

Irreversible P-tubulin binder

[59]

[63]

VINCA SITE BINDING AGENTS Vinca Alkaloids Vinblastine (2) and Vincristine (2a) are the leading compounds of the widely recognised antimitotic Vinca alkaloids, which represent a chemical class of major interest in cancer chemotherapy. The natural compounds are only present in minute quantities in the leaves of the Madagascan periwinkle, Catharanthus roseus. Several hundred analogues have been synthesised and evaluated for their pharmacological profile. The recent success of vinorelbine in human chemotherapy has encouraged the search for analogues with a new activity and tolerability profile. The clinical trial on Vinflunine has been reported recently (25). Vinflunine is the bifluorinated analogue of Vinorelbine (25a), which has used in the treatment of NSCLC and advanced breast cancer since 1992 [69].

736

Vinflunine was also successfully investigated in a phase-I trial by Pierre Fabre for use in the therapy of metastatic breast cancer in combination with Doxetaxel [70]. In comparison with Vinorelbine (25a), by the use of Vinflunine (25), MDR related drug resistance is developed less readily and the overall response rate in vivo with 64% is superior [71, 72, 73]. Phase III studies in NSCLC compared to standard 2nd line therapy with Doxetaxel and treatment to resistant bladder cancer are ongoing. Cryptophydns The Cryptophycins are a unique family of 16-membered macrolide antimitotic agents originally identified in blue-green algae (cyanobacteria) belonging to Nostocaceae (for review see [74]). The parent compounds of the series, Cryptophycin-1 or Cryptophyem-A (26) were found to block cells at mitosis in the low picomolar concentration range [75]. CH3

H3C

.. . CH 3

COOCH3 25

Vinflunine

25a

H

OCOCH 3 COOCH3

Vinorelbrine

26

R ~H Cryptophycin-1 (Cryptophycln-A) 2Sa R = CH 3 Cryptophyein 52 (LY 355703)

Fig. 9; Chemical structures of different Vinca site binding agents

Cryptophycin-52 (26a), a member of the cryptophyein family developed by Eli Lilly and produced by total chemical synthesis [76], was selected from diverse synthetic analogues displaying superior potency, stability and amenability of clinical formulation [77,76]. Cryptophycin-52 in vitro binds non-covalently to tubulin at a single high affinity site, which presumably overlaps with the Vinblastine binding site [75]. Proliferation of diverse tumour cell lines was inhibited with IC50 values of 13 pM to 232 pM, minimally affected by P-gp or MRP overexpression [78]. Data from phase I and II trials showed severe toxicities as long

737

lasting neuroconstipation and neurosensory toxicity without objective tumour responses leading to trial suspension [79,80]. Dolastatms The Dolastatins are cytotoxic cyclic pentapeptides isolated from the marine shell-less mollusk Dolabella auricularia [81].

27

27a

27b

Dolastatin 10 (NSC-376128)

Dolastatin 15

Cematodin (LU-103793)

27c

Synthadotin (LU-223651 / ILX -651 / LU223851)

Fig. 10: Dolastatins as potent inhibitors of tubulin polymerisation.

They were first described in 1990 as inhibitors of tubulin polymerisation (IC50 = 1,2 joM for Dolastatin 10) mediated through high affinity binding to the vinca binding site [82]. The Dolastatins include Dolastatin-10 / NSC-376128 (27), the analogue Auristatin / TZT-1027, as well as Dolastatin-15 (27a) and the analogues Cematodin / LU-103793 (27b) and ILX-651/ LU-223651 or synthadotin (27c). In a phase I study on Dolastatin 10, patients with advanced solid tumours developed peripheral sensory neuropathy, which was not dose limiting [83]. hi phase II studies with patients having metastatic prostate and colorectal carcinoma, Dolastatin 10 was very well tolerated but lacked significant clinical efficacy [84, 85]. Cematodin, the synthetic and water soluble

738 738

analogue of dolastatin 15 does not inhibit the binding of vinblastine to tubulin and is taken into account in this context due to similarity in structure. Scatchard analysis of Cematodin binding to tubulin indicates that there are two affinity classes and spindle dynamics are effected through a distinct molecular mechanism by binding to this novel site in tubulin [86,87]. Table 2: Preclinical / clinical status of Vinca site binding agents Compound Name (Nr.) Vinflunine (25)

Cryptophycin (LY-355703) (26a) Dolastatin 10 NSC-376128 (27) Cematodin LU103793 NSC D-669356 (27b) Synthadotin ILX-651 LU223651 (27c)

Status in development Phase III (NSCLC, bladder) Phase II suspended

Ref.

ICso-values in vitro 18nM

Company Pierre Fabre

[73]

13-232pM

Eli Lilly

[79,80]

Phase II ongoing

0.5 nM (L1210 cells)

NCI

[83]

Phase II finished, further investigation recommended Phase II

0.1 nM

Abbott

Abbott Ilex Oncology

Remarks

No vinca-site binding

[87,90]

[88,91, Company Info]

Investigations in a phase I trial determined the MTD with 2.5 mg/m and neutropenia, peripheral oedema as well as liver function test abnormalities as dose-limiting toxicities [89]. Phase II trails in patients with malignant melanoma noted only a small percentage of responders, but significant duration of response in patients with liver metastases [90]. Only a few data are published on ILX-651 developed by Ilex Oncology. Data from a phase I study have been reported showing reasonable tolerability but short plasma half-life [91]. ILX 651 is currently in multicentric phase II studies in advanced melanoma or NSCLC patients with additional trials scheduled.

739

TAXOL, SEMISYNTHETIC ANALOGUES AND TAXOL RESEMBLING COMPOUNDS Paclitaxel (Taxol) (3) is one of the most broadly used as anti-cancer agents with therapeutic value in the treatment of breast, ovarian carcinoma and NSCLC. However, despite high initial response rates to Paclitaxel, many patients relapse and develop drug resistance. Another limitation of Paclitaxel in the clinical use is the poor solubility and the toxicity exerted by its vehicle, i.e. Cremopher EL containing polyoxygenated castor oil [92]. Doxetaxel (Taxotere) (28), a semisynthetic analogue displays considerable activity against several types of solid tumours including those of the breast, lung, head and neck as well as ovary. Compared to Paclitaxel, Doxetaxel has broader activity [93,94]. Having impact on the clinical setting, analogues with a broader efficacy, higher tolerability and no cross-resistance are urgently needed [95]. BMS-184476 and BMS-188797 Data on two of these new taxanes, namely BMS-184476 (29) and BMS188797 (29a), were published by Bristol-Myers-Squibb (BMS), which also developed Paclitaxel [96]. In a comparative preclinical study, both analogues were found to have cytotoxic potency similar to Paclitaxel but overcome two different forms of Paclitaxel resistance. BMS-184476 was found to be clearly superior to Paclitaxel especially on A2780 ovarian carcinoma, HCT/pk, a moderately Paclitaxel-resistant colon carcinoma and L2987 lung carcinoma. BMS 184476 is currently in phase II clinical trials in breast, NSCLC, oesophageal and gastrointestinal cancers [97].

740

X2H2O

OH O

OCOCH

30a RPR 109881A 29 R = CH2SCH3, BMS-184476 29a R = H.BMS-188797

O

31

O

QH

IDN 5109

Fig. 11: Semisynthetic Taxol analogues

TXD-258 and RPR-109881A Two taxane analogues being in clinical development by Aventis Pharma are TXD-258 (30) (phase II) and RPR-109881A (30a) in phase III. RPR109881 was prepared as a single diastereomer by partial synthesis from 10-deacetylbaccatin III, the major natural taxoid extracted from the needles of taxus species. It has been shown to be active against tumours sensitive to Doxetaxel. In tumour models being poorly sensitive to Doxetaxel, the activity of RPR-109881 was similar. Nevertheless, RPR109881 had substantially lower affinity as Doxetaxel for P-gp in highly resistant cell lines and was found to be active in several cell lines moderately resistant to taxoids and Vincristine. In a phase I dose finding study, the recommended dose of RPR-109881A given as a one hour infusion on days 1 and 8 of a 21-day cycle [98] was determined to be 45mg/m2. Currently, phase III studies in metastatic breast Cancer are ongoing (Company info 2004). TXD-258, after i.v. and p.o. administration, has been reported to be effective towards various human tumour xenografts including MDR

741 741

positive and taxane resistant models, and to cross the blood-brain-barrier [99]. TXD-258 is currently in clinical phase II trials, but so far no data have been published. IDN-5109 Substitutions in the 14fl-hydroxy-10-deacetylbaccatin III (14-OHDAB) synthon, a diterpene present in the needles of Taxus wallichiana, led to a new class of taxanes from which IDN-5109 [13-(N-boc-Bisobutylisoserinyl)-14hydroxybaccatin-l,14-carbonate] (31) was selected because of its enhanced antiproliferative activity and lack of cross resistance in tumour cell lines expressing the MDR phenotype [92]. IDN5109 is a poor substrate of P-gp, hence is highly active towards MDR positive cancer cell lines and has oral bioavailability of about 50% [100,101]. IDN-5109 given p.o. was highly active towards the human ovarian carcinoma xenograft 1A9 and HOC 18 in vivo (90-100 % tumour regressions) and showed activity towards the Paclitaxel-resistant MNBPTX1 xenograft (10 % tumour regression) [92]. The oral efficacy of this taxane, likely related to the inability to be a substrate of the P-gp, allowed an adequate intestinal absorption and is a unique feature among the taxanes and presumably a benefit for clinical use [101]. IDN-5109 was first described by Indena, licensed to Bayer AG in March 2000 and is currently in phase II clinical trials in patients with aggressive refractory non-Hodgkin's lymphoma. Recently clinical data on the toxicity and efficacy profile have been published [102] (for review see also [103]). Paclitaxel Prodrugs Further efforts in reducing the dose-limiting side effects of Paclitaxel are reflected by the synthesis of Paclitaxel prodrugs, e.g. (32), designed for bioreductive activation. Blocking the C2'-OH group, which is important for activity, has recently been employed to generate several Paclitaxel prodrugs exhibiting diminished cytotoxicity [104].

NATURAL COMPOUNDS OF DIVERSE STRUCTURE WITH TAXANE-LIKE Several natural compounds of diverse structure, shown in Fig. (12), affecting the taxane-binding site on tubulin are currently developed in clinical trials, namely (+)-Discodermolide, Epothilone B, BMS-247550 (Azaepothilone B, NSC-710428) and Eleutherobin.

742

(+)-Discodermolide (XAA-296) The lactone-bearing polyhydroxylated alkatetraene (+)Discodermolide (33), isolated from the sponge Discodermia dissoluta, was described as a stabilising tubulin inhibitor with up to 100 fold higher activity as Taxol [105]. (+)-Discodermolide competitively inhibits the binding of [3H] Paclitaxel (3) to tubulin, is a poor P-gp substrate and effective towards Paclitaxel resistant ovarian carcinoma cells with mutated B-tubulin isotypes [106,107]. In a study using the taxane-resistant NSCLC cell line A549-T12, requiring taxol for normal cell division, (+)Discodermolide could not be substituted for taxol [108]. Further studies showed a strong synergism between Taxol and (+)-Discodermolide, which was a surprise and could not be observed with Epothilones or Eleutherobin. Thus, new combination regimens with (+)-Discodermolide might be feasible for clinical use [106]. (+)-Discodermolide is currently developed in a phase II clinical trial in solid tumour patients by Novartis. CH 3

CH 3

5H 3

CH 3

^

C H z

R^

HO,,,

'''CH 3 33

OH

(+)-Discodermolide

OH

= H,X = O Epothllone A 34a R = CH 3 , X = O Epothilone B 34b R = CH 3 , X = NH BMS-247550

34

H3C

O-Ri H3«

CH

3

"2

O _"A

R, - CH 3 , R2 = o K3 - un Eleutherobin

s ^%

H

° V " TJ^CH

35 (yH 2

,0

36a R-, = H, R2 = COOCH3, R3 = H Sarcodictyin A 16b R-I = H, R2 = COOCH2CH3, R3 = H Sarcodictyin B 36c R1 = H, R2 = COOCH3, R3 = OH Sarcodtatyin A 36d R, = H, Rj = COOCH3, R3 = H Sarcodictyin D

n

• Jrocanoyl 3

!

E E E E Z

Fig. 12: Natural compounds of diverse structure affecting the taxoid site.

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Epothilone B (Patupilone, EpoB) Epothilones A (34) and B (34a) (CGP-47906, EpoB, EPO-906), developed by Novartis, are 16-membered macrolides isolated from the mycobacterium Sorangium cellulosum. Epothilones were originally isolated and structurally resolved by G. Hoefle and colleagues in 1996 [109]. Epothilones A and B act by stabilising microtubules, competing with Taxol for binding to B-tubulin. These agents showed slightly higher in vitro cytotoxicity towards taxane resistant tumour cell lines [107,110]. In contrast to Taxol, Epothilones A and B have no endotoxin like properties [111]. Initial data on a phase I and II studies with EpoB have been published showing alopecia, neuropathy, nausea and diarrhoea as most prominent toxicities [112,113]. In preliminary results from a phase II study, EpoB was well tolerated at 2.5 mg/m2 i.v. [114], improving diarrhoea control which was observed being the DLT [113]. Currently Phase II clinical trials in patients with breast, colorectal, ovarian, NSCLC, renal and ovarian cancer are on-going. BMS- 247550 (Azaepothilone B, NSC-710428, INN: Ixabepilone) BMS-247550 (INN: Ixabepilone) (34c) is a semisynthetic analogue of Epothilone B developed by Bristol-Myers Squibb currently in phase II/III clinical trials. Like Epothilone B, BMS-247550 is a stabilising tubulin antagonist with broad anti-tumour activity [115].The drug is active towards Taxol sensitive and insensitive cell lines, including A2780Tax ovarian carcinoma cells with defined B-tubulin mutations (F270 -» V or A364 -> T; [116]. BMS-247550 has a potent tubulin polymerisation capacity (2,5 fold more potent as Taxol), a broad cytotoxicity (mean IC50 of 3.9 nM) and is efficacious in vivo after i.v. and p.o application [115]. A MTD with neutropenia as DLT (without G-CSF co-treatment) was determined and objective responses in breast and cervical cancer patients refractory to prior taxane therapy were observed [117]. In a clinical trial, the formation of microtubule bundles in peripheral blood mononuclear cells (PBMCs) was monitored and cell death occurred after peak microtubule bundle formation [118]. Eleutherobin The anti-mitotic diterpene Eleutherobin (35) and Sarcodictyins A - D (36a-d) are structurally related, natural compounds isolated from the marine soft coral Eleutherohia spec, and Sarcodictyon roseum,

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respectively [119], whose chemical synthesis has been published [120]. Eleutherobin is a competitive inhibitor of Taxol binding to tubulin, thereby enhancing tubulin assembly and stability. The in vitro cytotoxicity towards human tumour cell lines (IC50 10 nM - 60 nM) is comparable to that of Epothilone A with Sarcodictyins A and B being at least 10 times less potent [119]. Eleutherobin is a substrate of P-gp, whereas there are conflicting results concerning the cross resistance towards Taxol resistant ovarian carcinoma cell lines with the mutated B tubulin isotype M40 [116,119,121]. Eleutherobin, identified by researchers at the Scripps Institution of Oceanography in La JoUa, was licensed to Bristol-Myers Squibb and entered clinical trials but clinical development was not performed. Table 3: Status of several natural / semisynthetic compounds of diverse structure, affecting the taxoid site currently in clinical development Compound Name (Nr.) BMS-188797 (29a) TXD-258 (30) RPR-109881A (30a) IDN-5109

Status in development

IC50-values in vitro

Company

Phase II

Bristol-Myers partial Taxol Squibb resistance Aventis Pharma

Phase III

Aventis Pharma

Phase II

Bayer / Indena

Phase II

(31)

(+) Discodermolide (33) Epothilone B CGP-47906 EpoB / EPO 906 (34a) BMS-247550 Azaepothilone B NSC-710428 (34c) Eleutherobin (35)

Remarks

Phase II

8 - 36 nM

Novartis

Phase II ongoing

0.2 - 0.8 uM

Novartis

Phase II/III

Ceased

Bristol-Myers Squibb

[97] [99] [98, Company info 2004] [92,102, 103]

No P-gp substrate, no Taxol resistance [106,108, Synergism with Taxol, no Taxol Company web page] cross resistance No Taxol cross [107,110-114] resistance

3.9 uM (mean) Bristol-Myers No Taxol cross Squibb resistance

10-60nM

References

No P-gp substrate

[115,118]

[119]

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The abbreviations used are: cdk, cyclin dependent kinase; cki - cdk inhibitor; CEC, circulating endothelial cells; DLT, dose limiting toxicity; GTP, guanosinetrisphosphate; MAP - microtubule associated protein; MDR, multidrug resistance; MRP, multidrug resistance protein; MTD - maximal tolerated dose; NSCLC - non-small cell lung cancer; PBMCs, peripheral blood mononuclear cells; P-gp - P-glycoprotein; SAR, structure-activityrelation; SCLC, small cell lung cancer; SMTI, small molecule tubulin inhibitor; REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]

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Atta-ur-Rahman (Ed.) (Ed.) Studies Studies in in Natural Products Products Chemistry, Chemistry, Vol. Vol. 33 33 Atta-ur-Rahman ©2006 2006 Elsevier Elsevier B.V. B.V. All All rights rights reserved. reserved. ©

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CHOLESTEROL BIOSYNTHESIS INHIBITORS OF MICROBIAL ORIGIN HYUN JUNG KIM1, IK-SOO LEE1* and SAM SIK KANG2* 'College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, South Korea 2 College of Pharmacy and Natural Products Research Institute, Seoul National University, Seoul 110-460, South Korea ABSTRACT: Coronary heart disease is the leading cause of morbidity and mortality in the developed countries, which is due to abnormal deposition of lipids in the inner walls of coronary arteries. Higher levels of low-density lipoprotein (LDL) cholesterol are believed to be a major risk factor of coronary heart disease. Thus, inhibition of de novo cholesterol biosynthesis has been known to be one of the most efficient approaches in the regulation of LDL cholesterol levels. Many attempts have been made to find cholesterol biosynthesis inhibitors for development as hypocholesterolemic agents. Microbial secondary metabolites have been used as valuable natural sources in the development of novel cholesterol biosynthesis inhibitors. Mevastatin and lovastatin were isolated from the fungi, Penicillium citrinum and Aspergillus terreus, respectively, as potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which is involved in the rate-limiting step of cholesterol synthesis in mammals. These findings have led to the development of 'statins', which are drugs of choice in the treatment of hypercholesterolemia. HMG-CoA reductase inhibitors have also been shown to decrease the synthesis of other biologically important isoprenoid compounds derived from mevalonate, including ubiquinone and dolichol. And there has been continued interest in developing hypolipidemic agents that inhibit the enzymes involved specifically in the later stages of cholesterol biosynthesis. Cultured animal cells and intact animals have generally been used for the screening of cholesterol biosynthesis inhibitors. Coverage of the review includes chemical and biological aspects of the cholesterol biosynthesis inhibitors originated from microorganisms and their semisynthetic and biotransformed analogues reported to the present.

INTRODUCTION Cholesterol (C27H46O) (1) is the most widely occurring sterol in all the animal tissues as a constituent of animal membranes. Since it was first isolated from human gallstone, this compound was named "cholesterol" from the Greek word for "bile solids". Cholesterol is synthesized from

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five-carbon isoprene units in the liver [1,2]. The structure of cholesterol is shown in Fig. (1). It constitutes a major part of the membranes of the central and peripheral nervous systems. In addition, it is an important precursor of specific biological products including bile acids, steroid hormones and vitamin D [1,2].

Fig. (1). Structure of cholesterol

The lipids including cholesterol and triglycerides are transported in the blood as part of large molecules called lipoproteins. There are four major families of blood (plasma) lipoproteins. Chylomicrons transport exogenous triglycerides and cholesterol from the gastrointestinal tract to the tissues. They are degraded by lipoprotein lipase and free fatty acids are absorbed in the peripheral tissues. The remnants of chylomicrons are taken up in the liver cells, where cholesterol is stored, or metabolized to the bile acids, or else released into very low-density lipoproteins (VLDLs). VLDLs transport cholesterol and de novo synthesized triglycerides from the liver to the peripheral tissues. VLDL remnants, namely, low-density lipoproteins (LDLs) still contain much cholesterol, some of which are absorbed into the peripheral tissues or taken up in the liver on endocytosis via LDL receptors. High-density lipoproteins (HDLs) absorb cholesterol derived from the peripheral tissues including arteries, and then, transfer it to VLDLs and LDLs. HDLs also are transported to hepatocytes via HDL receptor, SR-B1 [2,3]. Although cholesterol is one of the most important physiological constituents in mammals, higher total cholesterol levels, more specifically, increased low-density lipoprotein (LDL) cholesterol levels are known to be a major risk factor of coronary heart disease [4-7]. Elevated levels of circulating cholesterol, specifically LDL cholesterol, result in the migration and penetration of LDL into the arterial walls, and lead to lipid

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accumulation in smooth muscles and in macrophages (forming foam cells). Accumulated LDLs are oxidized, which also increases proliferation of smooth muscles and deposition of connective tissue components in response to growth factors and cytokines. These deposits result in a disease process called atherosclerosis, which can cause blood clots to form that will ultimately totally stop blood flow. If this happens in the coronary arteries, a heart attack will occur. Coronary heart disease has been the leading cause of death in the developed countries for the past half century [3,8]. The clinical studies have indicated that the lowering of total and LDL cholesterol levels reduces the risk factor of coronary heart disease [4-7]. Endogenous and exogenous pathways determine plasma levels of cholesterol and lipoproteins. Plasma cholesterol levels and coronary heart disease risk can be reduced by decreasing cholesterol biosynthesis in hepatocytes, increasing its catabolism to bile acids, increasing excretion with bile acids, and by reducing its absorption from the intestine [3,6,7,9,10]. The more profound knowledge about cholesterol absorption, biosynthesis and metabolism has allowed the development of several cholesterol-lowering drugs with different mechanisms of action, with the purpose of reducing both morbidity and mortality associated with coronary heart disease [3]. CHOLESTEROL BIOSYNTHETIC PATHWAY Cholesterol is formed biosynthetically from isopentenyl pyrophosphate (active isoprene). The majority of cholesterol in the body derives from de novo biosynthesis in the liver [1,2]. Cholesterol synthetic pathway has been assumed to occur primarily in the cytoplasm and endoplasmic reticulum (ER). However, more recent evidences have suggested that the enzymes, except squalene synthase, squalene epoxidase and oxidosqualene cyclase, are partly localized in the peroxisomes, which are essential for normal cholesterol synthesis [11]. Acetoacetyl-CoA thiolase catalyzes the initial step in cholesterol biosynthesis, the condensation of two molecules of acetyl-CoA into forming acetoacetyl-CoA, which is then followed b y its condensatio n with a third molecule of acetyl-CoA to produce the six-carbon compound HMG-CoA via HMG-CoA synthase [12-15]. HMG-CoA is converted to mevalonate by HMG-CoA reductase. HMG-CoA reductase requires two

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molecules of NADPH as a donor of two electrons during the conversion of HMG-CoA to mevalonate. This reaction catalyzed by HMG-CoA reductase is the rate-limiting step, which is the major point of regulation on the cholesterol biosynthetic pathway [1,2]. In the next step, C-3 and C5 hydroxyl groups of mevalonate are reactivated by successive phosphorylations with three ATP molecules, to yield 3-phospho-5pyrophosphomevalonate. Then, the phosphate group at C-3 and the carboxyl group at C-l are removed, yielding isopentenyl pyrophosphate (IPP), a five-carbon activated isoprene molecule. By isomerization, isopentenyl pyrophosphate is converted into dimethylallyl pyrophosphate (DAPP), another activated isoprene unit. A head-to-tail condensation of IPP and DAPP generates the ten-carbon isoprene, geranyl pyrophosphate (GPP). GPP further undergoes another head-to-tail condensation with IPP, yielding farnesyl pyrophosphate (FPP), the fifteen-carbon isoprenoid. The NADPH-requiring enzyme, squalene synthase, catalyzes the tail-to-tail condensation of two molecules of FPP, forming 30-carbon isoprenoid, squalene. Squalene undergoes a two-step reaction to yield lanosterol. The first step is catalyzed by squalene monooxygenase, which requires NADPH as a cofactor to add one oxygen atom at the end (between C-2 and C-3 positions) of squalene to yield an epoxide. In the next step, squalene-2,3-oxide is cyclized by squalene-2,3-oxide cyclase to lanosterol [1,2]. Lanosterol is converted into cholesterol in a series of nineteen enzyme reactions [18]. The production of cholesterol from lanosterol involves the reduction of the double bond at C-24, demethylations of ge/w-dimethyl at C-4 and a tertiary methyl at C-l4, and isomerization of the double bond from C-8 to C-l. Two major pathways involving the same enzymes have been proposed [16-18]. Cholesterol biosynthetic pathway is outlined in Fig. (2).

755

acetyl CoA

acetaacetylCoA thiolase

acetoacetyl CoA HMG-CoA synthase

3-hydroxy3-methylglutaryI CoA (HMG-CoA)

mevalonate

HMG-CoA reductase

phosphorylation decarboxylatlon

isopentenyl pyrophosphate

isopentenyl adenosine

isomerase dimethylallyl ' pyrophosphate (DAPP)

geranyl pyrophosphate (GPP)

famesyl pyrophosphate (FPP) X2 dolichol, ubiquinone

squalene squalene monooxygenase

squalene-2,3-oxide squalene-2,3-oxide cyclase

lanosterol

756

lanosterol

A24-sterol reductase^

lanosterol 14ademethylase

4,4,14-trimethylcholest8-en-3B-ol lanosterol 14ademethylase

4,4-dimethylcholesta8,14-dien-3p-ol

4,4-dimethylcholesta8,14,24-trien-3p-ol

A14-sterol reductase

4,4-dimethylcholest8-en-3B-o! I t

4,4-dimethylcholesta8,24-dien-3B-ol

C4-methyl sterol oxidase decarboxylase 3-ketosterol reductase

I » I

cholesta-8,24-dien-3p-ol (zymosterol)

cholest-8-en-3B-ol

sterol A8-A7 isomerase

cholest-7-en-3p-ol (lathosterol)

cholesta-7,24-dien-3p-ol A5-desaturase

7-dehydrocholesterol

7-dehydrodesmosterol

A -sterol reductase

desmosterol A24-sterol reductase

cholesterol

Fig. (2). Cholesterol biosynthetic pathway [1,2,16,17]

757

INHIBITORS OF CHOLESTEROL BIOSYNTHESIS Inhibition of endogenous cholesterol biosynthesis has been known to be one of the most efficient approaches in the regulation of LDL cholesterol levels. Based on the several screening assay systems on enzymes, cell lines and laboratory animals, a number of cholesterol biosynthesis inhibitors have been isolated and identified from microorganisms as the lead compounds, some of which have been developed as therapeutic agents to regulate cholesterol metabolism. Hydroxymethylglutaryl-CoA (HMG-CoA) Reductase Inhibitors An important class of active agents that potently inhibit HMG-CoA reductase has evolved from extensive studies for microbial secondary metabolites. Since Brown and Goldstein have reported that the rate of cholesterol biosynthesis is determined by the activity of HMG-CoA reductase [19,20,21], this enzyme has been known to be a prime target for discovery of novel therapeutics against hypercholesterolemia. Several fungal secondary metabolites were isolated as useful inhibitors of endogenous cholesterol biosynthesis and developed as commercially available hypolipidemics. Endo and Hasumi have extensively reviewed natural, semisynthetic and synthetic HMG-CoA reductase inhibitors in 1993 [22].

758

In 1976, mevastatin (ML-236B, 6-demethylmevinolin) (2) was first reported as a potent competitive inhibitor of HMG-CoA reductase from the culture of Penicillium citrinum [23,24], which is identical with compactin, an antifungal compound isolated from P. brevicompactum [25,26]. Lovastatin (mevinolin, monacolin K) (3) has been isolated from the cultures of Aspergillus terreus [27] and Monascus ruber [28,29], separately.

R= H mevastatin (2) R= CH 3 lovastatin (3)

Fig. (3). Structures of mevastatin and lovastatin

In addition to mevastatin and lovastatin, several other related derivatives showing inhibitory activities against HMG-CoA reductase were isolated from cultures of the fungi mentioned above. ML-236A (4), ML-236C (5) and dihydrocompactin (6) have been isolated from P. citrinum [23,30]. o0H

ML-236A (4)

ML-236C (5)

dihydrocompactin (6)

Fig. (4). ML-236A, ML-236C and dihydrocompactin isolated from Penicillium citrinum

759

Monacolins J (7), L (8), M (9) and X (10), dihydromonacolin L (11) and 3a-hydroxy-3,5-dihydromonacolin L acid (12) were isolated from cultures of M. ruber [31-34]. Mevinolinic acid (13) and dihydromevinolin (14) were produced by A. terreus [27,35]. Of them, HMG-CoA reductase inhibitory activities of dihydrocompactin and dihydromevinolin were comparable to those of mevastatin and lovastatin [36].

monacolin L (8)

monacolin J (7)

monacolin M (9)

,,OH

'"OH monacolin X (10)

dihydromonacolin L (11)

3a-hydroxy-3,5-dihydromonacolin L acid (12)

Fig. (5). Monacolins J, L, M, X, dihydromonacolin and 3oc-hydroxy-3,5-dihydromonacolin L acid from Monascus ruber

760

mevinolinic acid (13)

dihydromevinolin (14)

Fig. (6). Mevinolinic acid and dihydromevinolin isolated from Aspergillus terreus

The structural feature of these compounds is the hexahydronaphthalene ring system which is functionalized with a-methylbutyric acid ester and a P-hydroxy-8-lactone linked by an ethylene bridge. The p-hydroxy-8lactone portion of these compounds can be easily opened, and it is converted to the 3',5'-dihydroxyheptanoic acid [22]. This hydroxy acid portion of their structures, which resembles the HMG portion of the HMG-CoA, is responsible for the activity, and it is known to interact competitively with the HMG binding domain of the enzyme active site [37]. A systematic investigation of structure-activity relationship (SAR) has shown that the introduction of an additional aliphatic group on the carbon a to the carbonyl group in a-methylbutyric acid ester linkage of lovastatin increases its potency. This result has led to the synthesis of simvastatin (15) and this modification increased the intrinsic inhibitory activity of lovastatin by 2.5 times [38]. o 0H

simvastatin (15) Fig. (7). Structure of simvastatin, a semisynthetic analogue of lovastatin

761 761

Microbial transformation method along with SAR studies has provided some novel metabolites that inhibit cholesterol biosynthesis. The phosphorylated derivatives (16 and 17) were produced by the conversion using several fungi [39]. Also, L-669262 (18) has been produced by microbial transformation of simvastatin, which is more active than simvastatin, its parent compound [40].

R= H (16) R=CH3(17)

Fig. (8). Microbial transformation products of statins

Pravastatin (CS-514) (19) has been produced by microbial transformation of mevastatin (compactin, ML-236B) [41,42]. Cytochrome P-450sca from Streptomyces carbophilus was described in detail as being responsible for this conversion [43]. It differs from lovastatin in that it contains a P-hydroxyl group at C-6 position of the hexahydronaphthalene ring. This modification makes it more hydrophilic than lovastatin. In

762 762

addition, the p-hydroxy-8-lactone ring has been opened to afford 3',5'dihydroheptanoic acid, the active form. Lovastatin, simvastatin and pravastatin are all widely used in the treatment of hypercholesterolemia and are known to be the first generation statins.

pravastatin (19)

Fig. (9). Structure of pravastatin

Lovastatin (Mevacor™) (3), simvastatin (Zocor™) (15), pravastatin (Pravachol™) (19), atorvastatin (Lipitor™) (20), cerivastatin (Baycol™, withdrawn on August 1, 2003) (21), and fluvastatin (Lescol™) (22) were introduced to lower total cholesterol levels, and especially LDLcholesterol levels to prevent coronary heart disease. These HMG-CoA inhibitors inhibit de novo synthesis of cholesterol in the liver. The ratelimiting enzyme in cholesterol synthesis is HMG-CoA reductase, which catalyzes the conversion of HMG-CoA to mevalonate. The resulting decrease in hepatic cholesterol synthesis leads to increased synthesis of LDL receptors and thus increased clearance of LDL-cholesterol in plasma [44,45]. Recently, two new and more potent statins (also called "superstates") have been studied for their LDL cholesterol-lowering ability, toxicity and drug-drug interaction [46,47]. Rosuvastatin (23) is a highly potent, selective and relatively hydrophilic statin with a low propensity for muscle toxicity and drug interaction. Pitavastatin (24) is known to be another statin with a high oral bioavailability. Since its catabolism is not mediated by cytochrome-P450, it reduces the potential of drug-drug interaction [46,47]. Rosuvastatin was approved for marketing in the U.S.A. in August, 2003 [48].

763 HOOC

,OCH3

COOH OH

atorvastaiin (20)

OH

cerivastatin (21)

OH

OH

COOH

fluvastatin (22)

COOH

COOH

msuvastatin (23)

pitavastatin (24)

Fig. (10). Structures of the synthetic statins

764

Squalene synthase inhibitors The findings of HMG-CoA reductase inhibitors resulted in the development of clinically effective hypocholesterolemics. However, HMG-CoA reductase inhibitors have also been shown to decrease the synthesis of other biologically important isoprenoid compounds derived from mevalonate including, ubiquinone, dolichol and isopentenyl f-RNA. Thus, there has been continued interest in developing hypolipidemic agents that inhibit the enzymes involved specifically in the later stages of cholesterol biosynthesis. Squalene synthase is one of those enzymes involved in the later stages of cholesterol biosynthesis, which has been targeted recently for the development of new therapeutic agents [49]. Screening of a number of fermentation cultures of microorgamsms afforded potent inhibitors of squalene synthase. Squalestatins 1 (25), 2 (26) and 3 (27) were isolated from cultures of Phoma sp. C2932 [50-52]. All of these three compounds possess the hydrophilic core unit, [15(la,3a,4p,5a,6a,7P)]-4,6,7-trihydroxy-2,8-dioxabicyclo-[3.2.1]octane3,4,5-tricarboxylic acid. These compounds showed potent inhibitory activities against rat liver squalene synthase (IC50 = 15.2,15.1 and 5.9 nM, respectively) [52].

OAc

4

X

COOH

OH squalestatin 1 (zaragozic acid A) (25)

OH

OH squalestatin 2 (26)

765 OAc

HOOC HOOC OH squalestatin 3 (27)

Fig. (11). Structures of squalestatins 1-3

Zaragozic acids A (25), B (28) and C (29) were also isolated from the cultures of MF5453 (ATCC 20986), Sporormilla intermedia and Leptodontium elatius, respectively [53-56], Among them, absolute stereostructure of zaragozic acid A is identical to that of squalestatin 1 [53]. These compounds exhibited dose-dependent inhibition of cholesterol biosynthesis as potent competitive squalene synthase inhibitors. Moreover, zaragozic acid A (squalestatin 1) showed inhibitory activity against cholesterol biosynthesis in freshly isolated rat hepatocytes (IC50 = 39 nM), and reduced the serum cholesterol level in mammals including marmosets, mice and rats in vivo [56,57]. It was postulated that zaragozic acids mimic effectively the intermediate presqualene diphosphate in the enzyme reaction catalyzed by squalene synthase [56].

zaragozic acid B (28)

766

OAc

zaragozic acid C (29)

Fig. (12). Sturctures of zaragozic acids B and C

Extensive studies on the structure-activity relationships (SAR) in a series of compounds derived from squalestatins 1 (zaragozic acid A) (25) and 3 (27) have been performed, focusing on the role of the carboxylic acids at C-3, C-4 and C-5 positions of [lS-(la,3a,4p,5a,6oc,7p)]-4,6,7trihydroxy-2,8-dioxabicyclo-[3.2.1]-octane-3,4,5-tricarboxylic acid [5861]. Consequently, the modification of the carboxylic acid at C-5 in squalestatin 1 analogues was not tolerated, but the carboxylic acids at C-3 and C-4 were not absolutely required for the retention of squalene synthase inhibitory activity [58]. In further SAR study for C-3 heterocycle-substituted derivatives of squalestatins 1 and 3 [61], the inhibitory activities of squalestatin 3 analogs showed a greater dependence on the nature of the C-3 substituent, which is different from those of squalestatin 1 analogues. Potent squalene synthase inhibitory activities equivalent to those of squalestatins 1 and 3 were retained in C-3 analogues substituted with a tetrazol-5-yl functionality which closely mimics the parent carboxylic acid (see Table 1). Also, electrostatic potential maps studies showed that squalene synthase inhibitory activity similar to that of the methyl ester (IC50 = 220 nM) was retained only in those C-3 heterocycle-substituted squalestatin 3 analogues for which electrostatic potential maps of the C-3 substituent were closely similar to that of a methyl ester [61]. Squalene synthase inhibitory activities of several analogues substituted with a heterocyclic moiety at C-3 are shown in Table 1.

767 767 Table 1. In vitro inhibitory activities of the synthetic analogues of squalestatin 1 (R! = COOH, R 2 = 4,6dimethyl-2-octenoyl) and squalestatin 3 (Ri = COOH, R 2 = H), substituted with a heterocyclic moiety at C-3, against rat squalene synthase |61]

R2O,

OAc

HOOC HOOC synthetic analogues of squalestatins 1 and 3 ICso (nM)

R2 Ri

R2

4,6-dimethyl2-octenoyl

H

COOH

12

26

COOMe

7

220

R.

A

4,6-dimethyl2-octenoyl

H

92

505

63

-

SyN

Am ri NyN

4

25

mA

-

785

72

310

SyN

146

1181

357

2875

172

1940

57

442

NH 2

Me

A NyO

NHMe

10

147

NHMe

Me 43

156

Me

H N N

N'NH Me

d Aa SH

-

312

768

7"-Hydroxylated analogue (30) of zaragozic acid A was produced from zaragozic acid A by microbial transformation using Streptomyces cyanus ATCC 55214, which inhibited the human squalene synthase in a dosedependent manner with an IC50 of 0.091 nM in HepG2 cells [62].

OAc

Fig. (13). Microbial transformation product of zaragozic acid A

Zaragozic acids D (31) and D2 (32) were further isolated from cultures of the keratinophilic fungus Amauroascus niger as potent inhibitors of rat liver squalene synthase with IC50 values of 6 and 2 nM, respectively. Structures of zaragozic acids D and D2 are related to those of the previously described zaragozic acids A, B and C, which contain 4,6,7trihydroxy-2,8-dioxabicyclo~[3.2. l]octane-3,4,5-tricarboxylic acid core unit and hydrophobic alkyl and acyl side chains [63].

OH zaragozic acid D (31)

769

OH zaragozic acid D 2 (32)

Fig. (14). Zaragozic acids D and D2 isolated from Amauroascus niger

Four zaragozic acid derivatives, F-10863 A (33), B (34), C (35) and D (36), were recently isolated from culture broths of the fungus Mollisia sp. SANK 10294. These compounds also contained a 4,6,7-trihydroxy-2,8dioxabicyclo-[3.2.1]octane-3,4,5-tricarboxylic acid core like previously reported zaragozic acid compounds. However, they contained two hydrophobic acyl side chains. It was found that F-10863 A is identical to zaragozic acid D3 (33) which was produced by Libertella sp. [54]. All the four compounds exhibited potent inhibitory activities against rat liver microsomal squalene synthase (IC50 values of 0.7, 1.3, 1.6, 2.0 nM, respectively) in a dose-dependent mode. Also, the compounds F-10863A and B showed inhibitory activities against sterol biosynthesis in freshly isolated rat hepatocytes (IC5o= 6.7 and 11 nM). Furthermore, F-10863A exhibited in vivo serum cholesterol-lowering activity in marmosets and hamsters [64].

F-10863A(33) (zaragozic acid D3)

770

F-10863B(34)

F-10863C(35)

Fig. (15). Structures of zaragozic acids F-10863A through D isolated from Mollisia sp.

771

Isolation and structure elucidation of a novel family of cholesterollowering constituents, viridiofungins A (37), B (38) and C (39), from Trichoderma viride, were reported and they were potent in vitro inhibitors of squalene synthase (IC50 values = 41.6, 19.3 and 0.29 |a.g/mL) in HepG2 cells. These compounds also showed broad-spectrum and potent antifungal activities [65,66].

HO

viridiofungin A (37)

H3CO

O H3CO

viridiofungin B (38)

772

viridiofungin C (39) Fig. (16). Structures of viridiofungins A through C from Trichoderma viride

Bisabosqual A (40) was isolated from the culture broth of Stachybotrys sp. RF-7260. Its structurally related compounds, bisabosquals B (41), C (42) and D (43) were also isolated from Stachybotrys ruwenzoriensis RF6853. All the bisabosqual compounds inhibited squalene synthases of microbial and mammalian origins. Bisabosqual A showed significant inhibitory activity against the microsomal squalene synthases from HepG2 cells and rat liver. Its IC50 values were 0.95 and 2.5 JJ. g/mL, respectively. Bisabosqual A also showed broad-spectrum antifungal activity in vitro. Bisabosqual C exhibited inhibitory activities similar to those of bisabosqual A [67,68].

....OH O

OHC CHO bisabosquat A (40)

CHO bisabosqual B (41)

773

H

t •

H1

OHI 0

OHC

OHC

CHO

CHO

Disabosqual C(42)

bisabosqual D (43)

Fig. (17). Structures of bisabosquals A-D from Stachybotrys sp.

CJ-15,183 (44) has been isolated from the fermentation culture of the fungus, Aspergillns aculeatus CL38916 as a squalene synthase inhibitor. The compound potently inhibited rat liver and human squalene synthases. In addition, it showed antifungal activities against filamentous fungi and yeast. The structure was elucidated to be an aliphatic tetracarboxylic acid compound consisting of an alkyl y-lactone, malic acid and isocitric acid moieties by spectroscopic analyses [69].

o COOH HOOC'

Y

O

COOH

CJ-15,183 (44)

Fig. (18). Structure of CJ-15,183 from Aspergillus aculeatus

774

CJ-13,981 (45) and CJ-13,982 (46) were isolated from the fermentation broth of CL15036, an unidentified fungus, as new squalene synthase inhibitors. They inhibited human liver microsomal squalene synthase with IC50 values of 2.8 and 1.1 uM, respectively. Based on the spectroscopic analyses, the structures of CJ-13,981 and CJ-13,982 were elucidated to be highly carboxylated fatty acids, 3-hydroxy-3,4dicarboxy-15-hexadecenoic acid (45) and 3-hydroxy-3,4-dicarboxyhexadecanoic acid (46), respectively [70],

COOH

CJ-13,981 (45)

COOH

CJ-13,982 (46)

Fig. (19). Structures of CJ-13,981 and CJ-13,982 isolated from CL15036

Recently, a Streptomyces species microorganism that produces squalene synthase inhibitors was screened from soils, and two active inhibitors were isolated from its culture broth. Structures of these inhibitors were identified as macrolactins A (47) and F (48) on the basis of spectral analyses. The IC50 values for macrolactin A were 0.11, 1.66, and 1.08 uM for the squalene synthases of pig liver, rat liver and Hep G2 cells, respectively. Macrolactin F also showed significant inhibitory activities with IC50 values of 0.14, 1.53, and 0.99 uM, respectively. Kinetic results for macrolactins A and F showed that they appear to be

775

non-competitive inhibitors of rat liver squalene synthase. However, both macrolactins A and F exhibited no inhibitory activity against the squalene synthase of the fugus Candida albicans [71],

macrolactin A (47)

macrolactin F (48)

Fig. (20). Structures of macrolactins A and F

776

Cholesterol Biosynthesis Inhibitors Screened by Using Cell Lines A family of 10-membered lactones was detected by chemical screening for inhibition of cholesterol biosynthesis. Taxonomic studies and fermentation conditions of the screened microorganisms have demonstrated that they belong to the species Penicillium simplicissimum and Penicillium corylophilum [73]. Decarestrictines A (49), B (50), C (51) and D (52) were isolated as active constituents. In vitro tests using the HepG2 cell assay showed the decarestrictines to be inhibitors of cholesterol biosynthesis [72,73], which exhibited inhibition effects of about 40%, 20%, 30% and 50% in the concentration of 100 nM, respectively. Decarestrictines were highly selective in that they exhibit no significant antibacterial, antifungal, antiprotozoal and antiviral activities [74]. Hypolipidemic activity of decarestrictine D was also confirmed in rats in vivo [72,73]. Of them, decarestrictine D (tuckolide) was selected for further study. Toxicity studies revealed that decarestrictine D displays good tolerability, showing a lack of change in a standard set of defined safety parameters [75]. Synthetic studies of this compound have been performed to provide a clue for the design of new cholesterol biosynthesis inhibitors [73-75].

OH decarestrictine A (49)

OH

o

decarestrictine B (50)

HO1" OH decarestrictine C (51)

OH decarestrictine D (52) (tuckolide)

Fig. (21). Decarestrictines A-D

777 777

The purification, structure elucidation, and antihyperlipidemic activities of nine compounds of the decarestrictine family, descarestrictines E to M (53-61), of P. simplicissimum were also reported. All of the compounds exhibited inhibitory effects on cholesterol biosynthesis in HepG2 liver cells in vitro [76].

O

H 3 CO

0

decarestrictine E (53)

OH decarestrictine H (56)

OH decarestrictine K (59)

0

HO decarestrictine F (54)

OH decarestrictine I (57)

decarestrictine G (55)

OH decarestrictine J (58)

OH decarestrictine L (60)

Fig. (22). Decarestrictines E-M

decarestrictine M (61)

778

Enzyme inhibition assays in vitro led to the isolation and synthesis of a variety of therapeutic agents and biologically active constituents as potent inhibitors of cholesterol biosynthesis [22,49]. Also, the cultured human liver cell lines including HepG2 and Chang liver cells have been traditionally used as an effective model for screening of cholesterol biosynthesis inhibitors from natural sources including plant materials, foods and microorganisms [77-81]. The methods using human liver cells have been shown recently to provide valuable profiles for the evaluation of inhibition mode against cholesterol biosynthesis based on the analysis of incorporation and distribution of radio-labeled precursor into nonsaponifiable lipids [80,81]. In a more recent search for new cholesterol biosynthesis inhibitors from microorganisms, a modified assay system was established in vitro to screen cholesterol biosynthesis inhibitors using Chang liver cell line [82,83]. Of a total of 83 microbial EtOAc extracts screened, the EtOAc extract of the fungus Hormoconis resinae showed significant inhibitory activity in the in vitro assay system. Bioactivity-guided fractionation of the extract led to the isolation of a cholesterol biosynthesis inhibitor, and the structure of this compound was elucidated as a new ergostane-type steroidal analogue, 3 P-hydroxy-1,11 -dioxo-ergosta-8,24(28)-diene-4aearboxylic acid (62) [84].

27

(62)

Fig. (23), Structure of 3p-hydroxy-l,l l-dioxo-ergosta-8,24(28)-diene-4a-carboxylic acid isolated from Hormoconis resinae

779 779

Details of its structure elucidation and biological activity are presented herein. The [M+H]+ peak at mlz 471,3118 (calcd. mlz 471.3110) shown in HR FABMS of 62 was assigned the molecular formula. The TH and 13C NMR spectra showed an exomethylene signal [8H 4.67 (brs), 4.73 (d, J=1.0 Hz); 8 C 107.0,157.1], two tertiary methyl signals [8H 0.71, 1.51; 8C 12.3, 19.8], a secondary methyl [S H 0.96 (d, J=6.5 Hz); 8C 18.9], two isopropyl methyl signals [8H 1.02 (d, J=7.0 Hz), 1.03 (d, J=7.0 Hz); 8C 22.3, 22.4], a hydroxymethme [SH 3.95 (ddd, J=6.3, 10.3, 11.0 Hz); 8C 75.3] and a tetrasubstituted double bond [8C 136.1,160.7,201.0], a ketone [Sc 211.8], and a carboxyl group [8c 180.8]. These findings suggested that this compound is an oxygenated tetracyclic steroid analogue. The structure of 62 was established by analysis of HMBC and " H - ' H COSY spectral data. The proton signals of isopropyl group at H-25, H-26 and H-27, and the methylene protons at H-23 showed correlations with the exomethylene signals at C-24 and C-28. The methylene protons at H-2 were correlated to the carbons at C-l, C-3 and C-10. The methine protons at H-3 and H-4 showed long-range couplings with the carboxyl group at C-29. The proton signals at H-7p\ H-14 and H-12 were respectively correlated to the carbons at C-8 and C-9 and C-ll in a conjugated enone. Also, H-12, H-14 and H-17 protons showed correlations with C-18. Another tertiary methyl, H-19 was correlated to the C-l, C-5, C-9 and C10 carbons. The secondary methyl protons at H-21 also showed longrange couplings with C-l7, C-20 and C-22 carbons. The connectivities of C-14, C-15, C-16 and C-17 were clarified by the 'H-'H COSY correlations. The stereochemistry of 62 was determined by NOESY and selective NOE correlations, and unambiguous structure of this compound was assigned to be 3p-hydroxy-l,ll-dioxo-ergosta-8,24(28)-diene-4acarboxylic acid as shown in Fig (23). The compound exhibited moderate to significant cholesterol biosynthesis inhibitory activity with an IC50 value of 8 ng/mL when compared with that of mevastatin (8 ug/mL), a commercially available HMG-CoA reductase inhibitor. However, this compound showed an inhibitory profile different from that of mevastatin. Analysis of the extracts of cells treated with 62, revealed the presence of accumulated lanosterol in the sterol fractions as evidenced by TLC, whereas no labeled sterol intermediate was detected in case of mevastatin under the same condition.

780

CONCLUSIONS Inhibition of cholesterol biosynthesis constitutes an important strategy to the lowering of higher blood total and LDL cholesterol levels. Several in vitro assay systems have been used as screening methods for developing novel leads for cholesterol biosynthesis inhibitors. Two major enzyme inhibition assays, targeting for HMG-CoA reductase and squalene synthase, respectively, have led to the isolation and synthesis of a variety of therapeutic agents and biologically active constituents as potent inhibitors of cholesterol biosynthesis. The findings of HMG-CoA reductase inhibitors resulted in the development of anti-hypercholesterolemic agents that could effectively lower the serum LDL cholesterol level. Microorganisms have been used as valuable sources for developing therapeutic agents to regulate the cholesterol metabolism. An important class of active agents that potently inhibit HMG-CoA reductase has evolved from extensive studies for microbial secondary metabolites. Lovastatin, simvastatin and pravastatin are all widely used in the treatment of hypercholesterolemia and are known to be the first generation statins. On the other hand, HMG-CoA reductase inhibitors have also been shown to decrease the biosynthesis of other biologically important isoprenoid compounds derived from mevalonate. Thus, there has been continued interest in developing hypolipidemic agents that inhibit the enzymes involved specifically in the later stages of cholesterol biosynthesis. Zaragozic acid derivatives were isolated from several microbial species and they exhibited dose-dependent inhibition of cholesterol biosynthesis as potent squalene synthase inhibitors. Viridiofungins, bisabosquals, macrolactins, CJ-15,183, CJ-13,981 and CJ-13,982 were also isolated as mammalian squalene synthase inhibitors of microbial origin. In addition, 10-membered lactone compounds, namely, decarestrictines, were identified by chemical screening for inhibition of cholesterol biosynthesis using in vitro and in vivo assay systems. 3(3-Hydroxy-l,lldioxo-ergosta-8,24(28)-diene-4a-carboxylic acid was isolated as an inhibitor of cholesterol biosynthesis using human Chang liver cell system.

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Atta-ur-Rahman (Ed.) (Ed.) Studies Studies in Natural Products Products Chemistry, Chemistry, Vol. 33 33 Atta-ur-Rahman © 2006 2006 Elsevier Elsevier B.V. B.V. All All rights rights reserved. reserved. ©

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STRUCTURE-ACTIVITY RELATIONSHIPS OF CURCUMIN AND ITS ANALOGS WITH DIFFERENT BIOLOGICAL ACTIVITIES1 LI LIN and KUO-HSIUNG LEE* Natural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill Chapel Hill, NC27599-7360, U. S. A. ABSTRACT: Curcumin is a constituent of the yellow pigments isolated from Curcuma longa, which is commonly named turmeric and is the major ingredient of the spice curry. Curcumin possesses many bioactivities, such as anti-oxidant, anti-inflammatory, antiviral, antifungal, cancer chemopreventive, and cancer chemotherapeutic properties. This review paper is prefaced by a general introduction to the origin and bioactivity of curcumin, followed by general synthetic routes to curcumin and its analogs. The major part of this paper examines the current structure-activity relationship studies of curcumin and various curcumin analogs relating to different bioactivities and distinct biological targets of interest to scientists worldwide. Finally, the advantages of curcumin and related compounds, which have multiple biological targets in cancer treatment, are discussed.

INTRODUCTION Plants of the Zingiberaceae family have been used as spices and indigenous medicine in Asia for thousands of years. The rhizome of Curcuma longa (Zingiberaceae), which is commonly named turmeric, is used as a spice (e.g., curry), flavoring agent, food preservative, coloring agent, and medicine for treatment of inflammation and sprain in India, China, and other Asian countries. Curcumin (1) [diferuloyl methane; 1,7bis-(4-hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5-dione] is the major constituent of the yellow pigments isolated from Curcuma longa (Zingiberaceae) and other Curcuma species. The main components of turmeric include curcumin, demethoxycurcumin (2), and bisdemethoxycurcumin (3), together referred to as curcuminoids, Fig. (1). Curcumin was first isolated in 1870. Its chemical structure was determined • To whom correspondence should be addressed. Tel: (919) 962-0066. E-mail: [email protected] f Antitumor Agents 241.

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in 1910 [1] and subsequently confirmed by synthesis. Curcumin has a unique conjugated structure including two methoxylated phenols and the enol form of a heptadiene-3, 5-diketone linking the two phenols, giving a bright yellow color. OH

o OCH,

2 Demethoxycurcumin OH

OH

O

3 Bis-demethoxycurcumin

Fig. (1). Structures of curcuminoids in Curcuma longa

Over the last few decades, curcumin has received increasing interest from researchers worldwide. In addition to its well-known antiinflammatory application, curcumin has been found to possess multiple therapeutic effects, such as reducing blood cholesterol, preventing LDA oxidation, inhibiting platelet aggregation, suppressing thrombosis and myocardial infarction, suppressing symptoms associated with type II diabetes, rheumatoid arthritis, multiple sclerosis and Alzheimer's disease, inhibiting HIV replication, enhancing wound healing, increasing bile secretion, protecting from liver injury, cataract formation, and pulmonary toxicity and fibrosis, showing antileishmaniasis and anti-atherosclerotic properties, as well as preventing and treating cancer [2]. Besides its numerous reported therapeutic effects, curcumin is non-toxic even at high dosages. It has been classified as "generally recognized as safe" (GRAS) by the National Cancer Institute [3]. Many curcumin analogs have been developed from the lead compound curcumin based on structure-activity relationship (SAR) studies and optimization of compounds as drug candidates.

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Curcumin has been reviewed from the aspects of its biology and mechanisms of action [2, 4]. In this paper, a comprehensive review will be presented with respect to SAR of curcumin and its analogs regarding different bioactivities.

GENERAL SYNTHETIC ROUTES TO CURCUMIN AND ITS ANALOGS Certain curcumin analogs differ structurally from curcumin only in their phenyl ring substitutions. The general synthetic strategy for this analog series is the condensation of substituted benzaldehydes with 2,4pentanedione [5], Fig. (2). Boron oxide is used in this condensation reaction to form a boron complex with 2,4-pentanedione and protect C-3 from Knoevenagel condensation. Other analogs are l,5-diphenyl-l,4diene-3-ones, which are synthesized by coupling aromatic aldehydes with acetone or cyclic ketones under acidic conditions [6].

o

o

0,7 eq, B 2 O 3

sV 0

Vp

o

o

OH

1.2eq. ArCHO, 2 eq. 2- i. 5 eq.BuNH 2 3 . HC1

O

R'

0 5 eq

HC1

R

(n= 0,1,2)

O

R' ^

HC1

R' ^

v

R

Fig. (2). General synthetic routes to curcumin and selected analogs [5]

STRUCTURE-ACTIVITY RELATIONSHIPS DIFFERENT BIOLOGICAL ACTIVITIES

(SARs)

FOR

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Curcumin exhibits diverse biological activities and has numerous biological targets [2]. Various research groups have studied the SAR of curcumin and its analogs with respect to different activities, hi this part of the review, the relationships between the structures of curcumin and its analogs and their targeted activities will be discussed and summarized. Anti-inflammatory activity Turmeric has been used for the treatment of inflammation for thousands of years in Asian countries. The active constituents were found to be curcuminoids, including curcumin as well as its two natural analogs, demethoxycurcumin and bisdemethoxycurcumin [7]. Demethoxycurcumin showed the most potent anti-inflammatory activity among these three natural curcuminoids [8]. According to present studies, curcumin acts by diverse anti-inflammatory mechanisms and at several sites along the inflammation pathway, which are summarized in Fig. (3) [9]. Stimulus

J J-

Cell membrane disruption Phospholipase )

COX II | —

|—

NO | —

Lipo-oxygenasi — | LOXI

Leukotnene

T"^

Prostaglandins

T

| Thromboxane

T

Prostacylin

T

Collagenase, elastase, hyaluronidas MCP1, Interferon-inducible protein, TNF, IL-12 I—

: Indicates inhibition by/ (curcumin

Fig. (3). Sites of action of curcumin along the inflammation pathway [9] MCP-1, monocyte chemoattractant protein-1; COX-II, cyclo-oxygenase-II; TNF, tumor necrosis factor; IL-12, interleukin-12.

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Sodium curcuminate, tetrahydrocurcumin, and curcumin were reported to have greater anti-inflammatory activity than phenylbutazone at low doses using carrageenin-induced rat paw edema and cotton pellet granuloma assays [10]. Various semi-synthetic analogs of curcumin, Fig. (4), were screened for anti-inflammatory activity using the same animal assay [11, 12]. Diacetylcurcumin (5), diacetoxycurcumin (10), and tetrabromocurcumin (4) were the most potent analogs among those studied. The study concluded that the presence of the j3-diketone moiety is important for the anti-inflammatory activity and addition of alkyl groups at ortho positions of the phenyl rings decreased potency. OH

O

Br

O

O

Br

H,C

H,CO AcO

HO

Fig. (4). Curcumin and semi-synthetic curcumin analogs tested for anti-inflammatory activity [11, 12]

Nurfina et al. designed and synthesized symmetrical curcumin analogs [13]. Anti-inflammatory activity was evaluated by inhibition of the carrageenin-induced swelling of rat paw. The structures of these synthetic analogs and their inhibitory potencies are shown in Table 1. The rank order of their potencies was 14 > 15 > 24 > 1 (curcumin) > 21 > 16 > 3 > 20 > 19. Other analogs showed no anti-inflammatory activity in this assay. Based on comparison of the structures and potencies, the following SAR conclusions were made: 1) The unsubstituted compound 11 showed little inhibitory activity. Therefore, appropriate substitutions on the phenyl rings are necessary for the anti-inflammatory activity. 2) Proper substitution at para positions of the phenyl rings is also crucial. Analogs without para substitution (11, 12,and 17) did not show anti-inflammatory activity. A para phenolic hydroxy group is essential for potent anti-inflammatory

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activity, as analogs without the para phenolic hydroxy group exhibited weak or no anti-inflammatory activity. 3) Likewise, substitution at positions ortho to the phenolic hydroxy group is important for the activity. Di-methyl substitution (14) enhanced the activity most, followed by diethyl (15), di-methoxy (24), and then di-isopropyl (16). However, ditetrabutyl substitution (22) diminished the anti-inflammatory activity, probably due to its bulkiness. Table 1. Curcumin derivatives tested in an anti-inflammatory bioassay [13] R2

_Comgmind___ 1 (curcumin) 3 11 12 13 14 15 16 17 IS 19 20 21 22 23 24

R2 H H H H H

H H H OCH3 H H H H H H

H

R3 OCH3 H H OCH, OCH3 CH3 CH 2 CH 3 i-C 3 H 7 H H

H H OCH3 t-C 4 H 9 E OCH3

OH

O

R4 OH OH H H OCH3 OH OH OH H Cl OCH3 CH3 OCH3 OH E OH

R2

RS

H H H H OCH3 CH3 CH2CH3 i-C3 H7 H H H H H t-G,H 9 H OCH3

__EDsoJimg/^}

38±4 73 ±5 — — --

13+2 22 ±6 58 ±21 — — 82 ±7 80 ±18 50 ±22 —

28±5

Cyclovalone (25), which differs from curcumin in the linker between the two phenyl rings, and three analogs showed anti-inflammatory activity based on inhibition of the enzyme cyclooxygenase [14], Fig. (5). Curcumin was used as a reference, and compounds 26, 27, and 28 were more potent than curcumin. Compounds 26 and 28 showed greater inhibition than compounds 25 and 27, respectively. Therefore, in this series, di-methyl substitution enhanced the ability to inhibit cyclooxygenase more than mono-methoxy substitution. Compound 25 showed greater inhibitory activity than compound 27; however compounds 26 and 28 had similar activity. Hence, the authors concluded that modification of the cyclic ketone from cyclohexanone to

791

cyclopentanone increased the inhibitory activity if the phenyl substitution was methoxy, but had no significant effect on analogs with di-methyl substitution.

H,CO OH

HO CH,

Fig. (5). Cyclovalone and its three analogs [14]

hi addition to curcumin, many other constituents in the Zingiberaceae family possess anti-inflammatory activity [15]. Some of these constituents, such as yakuchinones A and B isolated from Alpinia oxyphylla Miquel [16], Fig. (6), are diarylheptanoids and structurally similar to curcumin. H,CO

29 Yakuchinone A

30 Yakuchinone B

Fig. (6). Structures of yakuchinoine A and yakuchinone B [16]

Anti-oxidant activity Many of the therapeutic effects of curcumin are attributed to its strong antioxidant property. Most natural antioxidative compounds can be classified into two types: phenolic compounds and /S-diketone compounds [17]. Sesaminol from sesame belongs to the former type, and ntritriacontan-16,18-dione from the leaf wax of Eucalyptus belongs to the latter type. However, few antioxidative substances possess both phenolic hydroxy and /3-diketone groups in one molecule, and curcumin has both features.

792

Curcumin has both antioxidant and pro-oxidant effects in oxygen radical reactions, acting as a scavenger of hydroxy radicals or a catalyst in the formation of hydroxy radicals, depending on the experimental conditions [18-20]. The antioxidant effect of curcumin presumably arises from scavenging of biological free radicals. The antioxidative activity of the three natural curcuminoids and their hydrogenated analogs, Fig. (7), were examined in three antioxidative bioassay models, the linoleic acid auto-oxidation model, rabbit erythrocyte membrane ghost system, and rat liver microsome system. The results obtained from the three models were consistent, and revealed that curcumin was the strongest antioxidant among the natural curcuminoids and tetrahydrocurcumin had the strongest antioxidative activity among the hydrogenated curcuminoids [17]. Among all six compounds, tetrahydrocurcumin showed the highest potency, implying that hydrogenation of curcumm and demethoxycurcumin increased their antioxidative ability. Absence of one or both methoxy groups resulted in decreased antioxidant activity in both natural curcuminoids and tetrahydrocurcummoids. A second paper also reported that the presence of methoxy groups in the phenyl rings of curcumm enhanced the antioxidant activity [21]. OH

O OCH3 HjCO

HjCO.

HO"

1 Curcumm OH

OH

OCH,

HO

OH

31 Tetrahydroureumin O

O

O

H 3 CO.

HO'

2 Demethoxycurcumin OH

HO

32 Tetrahydro-demethoxycurcumin O

O

3 Bis-demethoxycurcumin

OH

HO

v

O

v

"OH

33 Tetrahydro-bis-demethaxyeurcumin

Fig. (7). Curcuminoids and tetrahydrocurcuminoids

Venkantesan et al. [22] used three models to investigate the importance of the phenolic hydroxy groups, as well as other substituents in the phenyl rings of curcumin and curcumin analogs, to antioxidant

793

activity. The curcumin analogs used in the bioassays are shown in Table 2. The three antioxidant bioassay models were inhibition of lipid peroxidation, free radical scavenging activity by the DPPH method, and free radical scavenging activity by the ABTS+* method. Table 2. Chemical structures of curcumin analogs [22] OH

O

QO OCH,

31 Tetrahydrocurcumin

Cmpd

Ri

R2

R3

Lipid peroxidation inhibition IC50 (jiM)

DPPH scavenging IC50 0«M)

ABTS*'scavenging TEAC 3 min

1 3 5 11 13 14 19 20 21 22 31 34

IS min

20.02 1.30 3.37 2.61 3.09 32.08 3.04 2.19 4.96 4.31 1.33 2.01 NA 1.85 2.33 >250 NA 3.36 1.57 2.78 NA 1.90 2.98 15.32 3.43 0.89 21.75 1.28 1.13 0.63 2.14 2.04 NA >250 2.05 0.67 1.52 NA >250 1.95 >250 2.67 1.86 2.49 NA 23.72 t-C 4 H 9 1.07 0.81 0.98 6.48 t-C 4 H 9 18.22 2.52 2.08 2.37 1.83 3.32 2.36 3.07 30.32 OH OEt H 1.11 NA till 90 35 H 1.09 NA SCH 3 H --IC50 is the concentration required for 50% inhibition of lipid peroxidation or scavenging of DPPH radical. TEAC is the trolox equivalent antioxidant capacity, which is defined as the mM concentration of a trolox solution having the antioxidant capacity equivalent to a 1.0 mM solution of the substance under investigation. OCH3 H OCH3 H OCH3 CH3 H H OCH3

OH OH OCOCH3 H OCH3 OH OCH3 CH3 OCH3 OH

9 min

H H H H OCH3 CH3 H H H

In all the three models, the phenolic curcumin analogs were more potent than the non-phenolic analogs. This result indicates that the phenolic groups are important to the antioxidant activity. The presence of methoxy groups in curcumin increased antioxidant activity in all the three models, which was in agreement with previous reports. Ethoxy substitution also enhanced the activity to some extent. Compound 14, which has two methyl groups at ortho positions relative to the phenolic hydroxy groups, showed the highest inhibitory activity in the lipid peroxidation model. However, compound 22, with bulkier ^-butyl groups flanking the phenolic group, was ten-fold less active than compound 14.

794

The decreased potency of 14 probably resulted from the offsetting influences of the electron donating and steric effects. In the three models, tetrahydrocurcumin showed comparable activity with curcumin, which implies that the enhanced electron delocalization of the double bonds may not be essential in terms of curcumin's antioxidant activity in these three bioassay models. l,5-Diphenyl-l,4-pentadiene-3-ones and cyclic analogs were synthesized by Sardjiman et al. [23]. The structures and antioxidant activity (inhibition of lipid peroxidation) of these analogs are shown in Table 3. Table 3. 1, 5-Diphenyl-l,4-pentadiene-3-ones and cyclic analogs and antioxidant activities [23]

Compound 25 26 36 37 38 39 40 41

OCH3 CH3 H

R2 H CH3 H

C2H5 i-C 3 H 7 t-C 4 H 9 OCH3

C2H5 i-C 3 H 7 t-C 4 H 9 OCH3

Cl

Cl

Ri

Lipidjjcroxidation inhibition ICso^yM)^ >4 2.8 ±0.0 » 4

2.0 ±0.3 4.4 ± 1.1 » 4

1.6 ±0.4 Inactive 11.0 ± 1.3

Curcumin

Compound 27 28 42 43 44 45 46 47

R, OCHj CH3 H

R2 H CH3 H

C2H5 i-C 3 H 7 t-C 4 H 9 OCH3

C2H5 i-C 3 H 7 t-C 4 H, OCH3

2.2 ±0.2

Cl

Cl

» 4

Anti-oxidant activity IC 5 0 (|JM)

>4 2.5 ±0.2 » 4 >4

» 4 0.9 ±0.2

795

Compound 48 49 50 51 52

•

Ri

Rj

H OCH3 CH3 OCH3 Cl

H H CH3 OCH3 Cl

Anti-oxidant activity IC50((J.M) »4 >4 •

1.3 ±0.4 2.9 ±0.4 Inactive

Compared with the structure of curcumin, these synthesized analogs retain para hydroxy groups in the phenyl rings but lack one carbonyl group and methylene group in the linker between the phenyl rings. The bioassay results revealed the following SAR information. 1) Electron withdrawing substitutions in the phenyl rings (41, 47, and 52) resulted in loss of antioxidative activity. 2) Bulky alkyl substituents (e.g. isopropyl, fe/t-butyl) at ortho positions relative to the phenol groups (38, 39, 44, and 45) retarded the antioxidative activity. 3) Small alkyl groups (e.g. methyl, ethyl) and electron donating groups (e.g. methoxy) at ortho positions (26, 37, 40, 28, 43, 46, 50, and 51) enhanced the activity. Dimethoxy substitution potentiated the antioxidative activity more than monomethoxy substitution. Several analogs were more potent than curcumin. Therefore, the truncation of the linker did not result in decreased antioxidative activity, but rather, enhanced the activity. Recently, Youssef et al. [24] reported the synthesis of curcumin analogs as potential antioxidant and cancer chemopreventive agents. The general structures of the synthesized analogs are shown in Fig. (8). These compounds were tested for scavenging ability of DPPH free radicals and in an ATP chemiluminescence assay. The SAR conclusions from the results were mainly consistent with the prior conclusions drawn by other researchers. In addition, they found that di-substitution of the central methylene group resulted in decreased antioxidative activity.

796

R'

X R R' R"

XO R"

= = = =

H, CH 3 CH 3 , C 2 H 5 , C3H7 H, CH 3 , OCH3 H, CH 3 , OCH 3 , OC 2 H 5

R"

Fig. (8). General structures of curcumin analogs synthesized by Youssef et al. [24]

The antioxidant mechanisms of curcumin have been under extensive investigation. The predominant conclusion is that curcumin is a classical phenolic chain-breaking antioxidant, which donates H atoms from the phenolic groups [25-30]. However, some conflicting results suggest that H atom donation takes place at the active methylene group in the diketone moiety [31, 32]. Ligeret et al. published the latest report [33]. They evaluated the effects of curcumin derivatives (Table 4) as well as curcumin on the mitochondrial permeability transition pore (PTP), which can release apoptogenic factors from mitochondria to induce apoptosis. The authors postulated that PTP opening is closely related to the antioxidant property of curcumin. From the data on mitochondria swelling, O2* and HO* production, thiol oxidation, and DPPH* reduction, the authors concluded that the phenolic hydroxy groups are essential for activity and are more effective at the para position than at the ortho position, hi addition, an electron-donating group at the ortho position relative to the phenolic group is also required for activity, but bulky substituents, such as f-butyl, are not favorable. In contrast, electron-withdrawing substitution, such as NO2, reduced activity. Based on the observations that antioxidant activity was attenuated when the P-diketone moiety was replaced by a cyclohexanone ring, but was not found with ferulic acid, they concluded that the p-diketone could not induce, but did contribute to the activity of curcumin derivatives. Their conclusions agree with the prevailing SAR for antioxidant activity.

797

Table 4. Structures of curcumin derivatives [33]

o

Compound 1 (Curcumin) 3 11 13 22 24 53 54 55 56 57

o

o

R2 OH OH

Rl OCH3 H H OCH3 t-C4H, OCH, OH

H

OCH3 OH OH OCH3 OCH2O OC4H, OH

OCH3 NO2

R3 H H H OCH3 t-C 4 H 9

OCH3 H H H H

NH

HN

Compound 25 36 39 40 58 59 60 61 62 63 64

o

R2 OH OH OH OH H OCH3

R3 H H t-C4H, OCH3 H

OCH3

OCH3

OCH3

OCH3 NO2

OC4H, OH

H H

Rl OCH3 H t-C4H, OCH3 H OH OCH2O

H H

However, a curcumin analog without phenolic and methoxy groups was reported to be as active as curcumin in terms of scavenging hydroxy radicals and other redox properties [34]. Considering the variety of test free radicals, solvents, and pH ranges used in the literature, Wright employed theoretical chemistry to interpret the controversy [35]. First, he explored the stable conformer of curcumin,

798

pointing out that the enol form is the most stable, followed by the transdiketo form, and then the cw-diketo form, Fig. (9). Calculations showed that the phenolic O-H is the weakest bond in the curcuminoids. This theoretical result favors the necessity of a phenolic OH group for the antioxidant activity of curcumin and its analogs. However, the C-H bond of the methylene group becomes active when radicals with very high bond dissociation enthalpy, such as methyl and ?-butoxyl radicals, are used. Thus, the variety of the attacking radicals in the bioassay system is likely responsible for differing experimental results. OH

o

0CH3 OH cu-diketone form

trans-diketone form

Fig. (9). Three forms of curcumin

Anti-HIV activity In addition to reverse transcriptase and protease, HIV-1 integrase has become a new target being explored in order to find more effective treatments for AIDS. HIV-1 integrase is the enzyme catalyzing integration of the double-stranded DNA of HIV into the host chromosome. Curcumin was reported to have inhibitory activity against HIV-1 integrase [36]. Other classes of compounds have also shown inhibited HIV-1 integrase in enzyme assays, but few have shown specificity against HIV-1 integrase, and even fewer were active in cell-based assays [37]. However, curcumin was reported to have moderate activity in cell-based assays, in addition to its activity in enzyme assays [38]. Therefore, modified curcumin analogs were developed for anti-HIV potency evaluation, as well as mechanism of action study [37, 39]. Mazumder et al. [39] synthesized curcumin analogs (Table 5) as probes for an anti-HIV-1 integrase mechanism study. Evidence suggested that curcumin does not bind to the DNA-binding domain of HIV-1 integrase [40] or the same binding site of another HIV-1 integrase

799

inhibitor, NSC 158393 [41]. Compounds 11 and 19, which have no hydroxy group in the phenyl ring, did not inhibit HIV-1 integrase. Therefore, hydroxy groups in the phenyl rings are apparently essential for the inhibitory activity. Compounds 65 and 66 exhibited greater activity than compound 1 (curcumin). Hence, replacing one or two methoxy groups of curcumin with hydroxy groups increased the activity. Compound 31 did not show inhibitory activity in this bioassay, suggesting that the unsaturated linker also contributes to the inhibitory activity. Three compounds (65, 66, and 67) showed very high potency. They have at least one catechol (3,4-dihydroxybenzyl) substructure as a common feature. More conclusions about this SAR study were obtained from further investigation of curcumin analogs as inhibitors of HIV-1 integrase. In addition to the catechol unit and an unsaturated linker, the syn disposition of the C=C-C=O moiety in the linker and the coplanarity of the structures are important to the integrase inhibitory activity of curcumin analogs [37]. The SAR conclusions drawn from experimental data are consistent with QSAR studies performed with MOE and Cerius2 programs [42]. Fig. (10) summarizes the anti-HIV-1 integrase SAR of curcumin analogs. However, no therapeutic index of these tested compounds was reported. Table 5. Structures of curcumin analogs and anti-HIV-1 integrase ability [39] OH

o

H3CO

Compound

Rl

R2

R3

R4

1 2 3 11 19 31 65 66 67

OCH3 H H H H

OH OH OH H OCH3

OCH3 OCH3 H H H

OH OH OH H OCH3

OH OCH3

OH OH

OH OH

OH OH

3-Processing

Strand transfer

IC 50 0*M) 150 140 120

IC 50 (jiM) 140 120

>300 >300 >300 6.0± 1.5 18.0+9.0 9±7

80±20 >300 >300 >300 3.1±0.1 9.0+ 3.0 4.0± 1.5

800 Coplanarity

syn disposition of enone

,,----.N

unsaturated linked 1

catechol unit

J

>

catechol unit

Fig. (10). Schematic picture of structural features favoring anti-HIV-1 integrase activity

Chemopreventive activity Chemoprevention is a relatively new concept. It attempts to use natural and synthetic compounds to intervene at early stages of cancer before invasive disease begins [43]. Nontoxic agents are administered to otherwise healthy individuals who may be at increased risk for cancer. Some potential diet-derived chemopreventive agents include epigallocatechin gallate in green tea, curcumin in curry, and genistein in soya. Curcumin has demonstrated wide-ranging chemopreventive activity in preclinical carcinogenic models of colon, duodenum, forestomach, mammary, oral cavity, and sebaceous gland and skin cancers. It is under Phase I clinical trial as a chemopreventive agent of colon cancer by the National Cancer Institute. The mechanisms of the chemoprevention induced by curcumin are pleiotropic. It enhances the activities of Phase 2 detoxification enzymes of xenobotic metabolism, such as glutathione transferases [44] and NAD(P)H: quinone reductase [45]. It also inhibits procarcinogen activating Phase 1 enzymes, such as cytochrome P450 1A1 [46]. With regard to the mode of chemopreventive action in colon cancer, curcumin exhibits a diverse array of metabolic, cellular, and molecular activities, including inhibition of arachidonic acid formation and its further metabolism to eicosanoids [47]. In order to search for new chemopreventive enzyme inducers and to elucidate the structural features responsible for the ability of curcumin to induce Phase 2 detoxification enzymes, Dinkova-Kostova et al.[48, 49] examined some natural as well as synthetic curcumin analogs for ability to induce NAD(P)H: quinone reductase (a prototype for phase 2 detoxification enzymes) in murine hepatoma cells. Compounds

801 801

containing a Michael reaction acceptor, such as phenyl-C=C-C=O, were expected to have phase 2 enzyme inducer potency [49]. Fig. (11) shows the structures of the curcumin analogs and their potencies in the QR assay in murine hepatoma cells. OH

o

H3CO

o

OH

OCH,

OCH3

HO

1 Curcumin CD = 7.3

OH HO

2 Demethoxyeurcumin CD = 9.5 OH

OH

O

3 Bisdemethoxycureumin CD =11.5 OH

OH

O OCH, OH

31 Tetrahydrocurcumin CD = 35.7

72 2,4-pentadione Inactive

75 Inactive

Fig. (11). Structures of curcumin analogs and their inducer potencies (CD//iM) in the QR assay in Hepa lc lc.7 murine hepatoma cells [48] (CD value is the concentration required to double the quinone reductase specific activity.)

Dibenzoylpropane (71) was inactive as an inducer probably due to its lack of an enone moiety. Dibenzoylmethane (69) was quite active with a CD value of 0.8 fiM; the keto-enol tautomerization of the /3-diketone can provide the Michael reaction acceptor feature. The three natural curcuminoids showed similar CD values; therefore, the methoxy group in the phenyl ring does not greatly affect the potency. However, the CD

802

values of compounds 11 and 68 indicated that the introduction of orthohydroxy groups on the phenyl ring dramatically enhanced the chemopreventive potency. Tetrahydrocurcumin (31), which as previously described is a potent anti-oxidant, possessed moderate potency (CD=35.7 juM) in the QR assay. Its potency may be due to the contribution of the keto-enol tautomerization of the /3-diketone moiety. 2,4-Pentadione (72) and l,l,l,5,5,5-hexafluoro-2,4-butanedione (73), which do not possess a phenyl ring, were inactive. Therefore, although the presence of a j8diketone moiety in the structure could enhance the activity, it is not sufficient to induce activity. Anti-prostate cancer Prostate cancer is the most common cancer in males living in developed countries and the second leading cause of cancer death. Curcumin has been reported to have anti-prostate cancer activity in vitro and in vivo [5052]. Although mechanisms of action have been postulated [53], they are not yet fully understood. The androgen receptor (AR) plays an important role in prostate carcinogenesis; therefore, anti-AR reagents such as hydroxyflutamide are widely used for the treatment of prostate cancer. Sixty-one curcumin analogs were synthesized or isolated from natural sources in our laboratory (Table 6) [54-58] and evaluated for AR inhibitory activity in prostate cancer cell lines. Among these analogs, compounds 2 and 9 exhibited the greatest inhibitory activity against the transcription of AR in three prostate cancer cell lines LNCaP, PC-3, and DU-145. Other analogs exhibited lower activity, and the rest showed no inhibition [55, 56]. Based on the bioassay results, the present SAR of curcumin analogs as anti-AR reagents is as follows. 1) A conjugated (3diketone moiety is crucial for the activity. Converting the /3-diketone moiety to pyrazole (105-108) resulted in decreased activity, and saturating (31, 109-115) or eliminating (116-121) the C=C bonds resulted in decrease or loss of activity. 2) When the methylene group in the linker was not substituted, replacing the phenolic hydroxy groups with methoxy (2) or methoxycarbonylmethoxy (4) groups resulted in significant increase in the inhibitory activity. 3) When the phenyl ring substitution of curcumin was retained, adding an ethoxycarbonylethyl group to the central methylene group (9) greatly increased the anti-AR activity. 4) All electron-withdrawing substitutions in the phenyl rings resulted in the loss of anti-AR activity. 5) The cyclic diarylheptanoids did not show

803 803

significant anti-AR potency; thus, the acyclic structure may be required for this activity. It is noteworthy that curcumin (1), which was reported to have anti-prostate cancer activity, was found to be inactive in the anti-AR bioassay. Therefore, curcumin does not inhibit the growth of prostate cancer cell through an anti-AR mechanism. The mechanism for how the synthetic curcumin analogs inhibit the transcription of AR remains unknown. Further modifications are ongoing in our laboratory to expand the SAR and optimize anti-AR activity. Table 6. Curcumin analogs synthesized for anti-AR assay [55, 56] A) Symmetric aryl rings with unsaturated diketone linker OH

O OCH, N

OCHj

H 3 C(H 2 C) 2 O

Compound 1 (Curcumin) 3 13 21 76 77 78 79 80 81 82 83 84 83 86 87 88 89 90 91 92 93 94 95 96 97

Rl H H H H H CH3 (CH2 ) 2 COOH (CH 2 ) 2 COOH (CH2 ) 2 COOH (CH2 ) 2 COOEt (CH 2 ) 2 COOEt (CH 2 ) 2 COOEt (CH2 ) 2 COOEt (CH2 ) 2 COOEt (CH 2 ) 2 CH3 H H H H H H H H H H H

R2 H H H H H H H H H H H H H H H H H F H CF3 H F F F NO2 H

R3 OCH3 H OCH3 OCH3 OCH3 OCH3 H OCH3 OCHj OCH2 OCH3 OCH3 OCH3 N(CH3)2 OCH3 H F H F F H H H H H NO2

R4 OH OH OCH3 OCH3 OCH2 COOCH3 OCH3 H OH H OH H OCH3 OCH3 OCH3 O(CH2 ) 2 CH3 F H H OCH3 H OCF3 F OCH3 H OCH3 OH

R5 H H OCH3 H H H H H H H H H OCH3 H H H H H H H H H H H OCH3 OCH3

MS H H H H H H H H H H H H H H H H H H H H H H H OCH3 H H

804 98 99 100 101 102

H H H

H H H

NO2

N(CH3)2

OCHj OCH3

OEt

OEt

OCH3

H H

B) Asymmetric aryl rings with unsaturated diketone linker OH

O

R,,

Compound 2 103 104

Rl H OCHj OCH3

Rl'

R2

R2'

OCH3

OH OCH3 OH

OH OH OCH2COOCH3

OCH3

C) Symmetric/asymmetric aryl rings with unsaturated pyrazole linker HN

_Comgound______ 105 106 107 10g

Rl OCH3 OCH3

N

Rl' OCHj

H

H H

OCH3

OCHj

R2' OH OH OH OCH3

R2 OH OH OH OCH3

D) Symmetric aryl rings with saturated linker

H3CO

Compound 31 109 110 111 112 113 114 115

OCH,

Rl OH OH OH OCHj OCHj OCHj OCH3 OCHj

R2

R3

R4

=0 =0 -OH =0 =O -OH =0 -OH

=0 -OH -OH =O -OH -OH -OH -OH

H H H H H H CH3 CH3

H H H

805 805 D) Symmetric phenyl rings with diketone linker R,

0

O

R2

Compound

R2 OCH3 H H H H H

Rl

H OCH3 H H

116 117

118 119 120 121

H

H

R3 OCH3 OCH3 OCH3 NO2 NO2 H

R3' OCH3 OCH3 NO2 NO2 NO2

H

H

CH2 COPh

E) Cyclic diarylheptanoids R,0

Compound 122 123 124

Rl

R2 H H OH

H H CH3

JR3_ C=O C=N-OH C=O

H3CO

_jCojnpjound_

Rl H CH 3 CH3 Ac

R2

R3

125 126 127 128

H H CH3 Ac

c=o c=o c=o

129

H

H

130

C=O

0

o

R4

H H Br Br

806

Anti-angiogenesis In order to maintain growth, a tumor must develop new blood vessels to obtain a constant supply of oxygen and nutrients and remove waste. Angiogenesis is the growth of new blood vessels from preexisting vasculature and is stimulated by biochemical signals. Inhibiting angiogenesis is a therapy to starve the cancer cells, Curcumin has demonstrated anti-angiogenic activity in vitro and in vivo [45, 59, 60]. The molecular mechanisms of anti-angiogenesis of curcumin were postulated to be the down-regulation of the expression of proangiogenic genes [61] and the down-regulation of matrix metalloproteinase [62]. The design of curcumin analogs as potential anti-angiogenic agents has been explored [63-66]. Hydrazinocurcuminoids modified from eurcurninoids by Shim et al. [64] showed greater anti-proliferative potency than curcuminoids in bovine aortic endothelial cells (BAECs) (Table 7). In contrast to curcumin, which inhibited the proliferation of several epithelial and fibroblast cells non-selectively, hydrazinocurcuminoids showed specificity against endothelial cells. Thus, proper modification on the linker between the two phenyl rings cannot only enhance the anti-angiogenic activity but also the specificity. The rank order of the potencies of the curcuminoids was 1 > 2 > 3. The hydrazinocurcuminoids showed the same trend. Therefore, the elimination of methoxy groups from the phenyl rings resulted in decreased anti-angiogenic potency. Robinson et al. [66] designed and synthesized curcumin analogs as anti-angiogenic agents. They postulated that the two aromatic rings of curcumin might be crucial for its binding to the potential receptor; therefore, they retained these rings and replaced the unsaturated j3diketone linker of curcumin with an enone or dienone linker. The impact of substitutions in the aromatic rings was also explored by preparation of chlorinated analogs. The structures of the analogs and their percent inhibition of endothelial cell proliferation are shown in Table 8. (Because curcumin was not tested in parallel, no conclusion could be drawn regarding to the relationship between structure and activity of curcumin.) The introduction of /?ara-methoxy groups on the phenyl rings increased the anti-angiogenic potency (cf. compounds 134 and 135). The introduction of two ortho-chloto groups on one phenyl ring also enhanced the inhibitory activity against the proliferation of endothelial cells (cf. compounds 135 and 136). Replacing one phenyl ring with a naphthyl ring

807

had little effect. In the dienone series, the most potent analog was compound 48, which has no substitution in the phenyl rings. Replacing the dienone moiety with a diene-cyclohexanone moiety, replacing the phenyl rings with pyridyl rings, or substituting the phenyl rings with 4-hydroxy-3methoxy moieties or two ortho-chlorines, all resulted in a decrease in the anti-angiogenic activity. Table 7. Structures of curcuminoids and hydrazinocurcuminoids and their inhibitory potencies against BAECs [64] Compound

Rl

R2

Inhibition of BAECs ICSOQtM)

1 2 3

OCH3 OCH3

OCH3

15±3 22 ±5 53 ±6

105 106 107

OCH3 OCH3

131

OCH3 OCH3

132

H

H

133

JU

H H OCH3

H H OCH3

H

0.52 ±0.04 1.8 ±0.3 5.8 ±0.2 0.93 ±0.04 2.4 ±0.1 8.7 ±0.2

lAnH

Table 8. Percent inhibition of in vitro endothelial cell proliferation by curcumin analogs [66] Structure

3 fig/ml

25 H 3 CO.

.OCH,

90.1

96.0

96.6

97.7

94.4

97.7

98.2

98.1

48

134

808 808 135

92.9

97.5

92.8

94.4

92.2

94.7

89.1

96.9

92.9

96.7

87.1

90.4

136

137

138

139

140

a

cr

CONCLUSION Curcumin is a natural product that possesses multiple biological activities, has various biological targets, and modulates numerous signal transduction pathways. Most cancer biologists suggest that, because tumor cells always have multiple pathways to escape the host defense mechanisms, a drug that is specific for modulation of one signal transduction pathway in the tumor cells may not be adequate [2]. Curcumin is an ideal anti-cancer drug candidate that affects multiple pathways and yet is still pharmacologically safe. However, due to its natural occurrence and long history of dietary use, curcumin cannot be patented. Hence, curcumin is a good lead compound for the development of better analogs that are patentable and more potent in the targeted activities [58, 67-70]. ACKNOWLEDGEMENTS This work was supported by National Cancer Institute Grant CA-17625 awarded to K. H. Lee.

809

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©

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THE VINCA ALKALOIDS: FROM BIOSYNTHESIS AND ACCUMULATION IN PLANT CELLS, TO UPTAKE, ACTIVITY AND METABOLISM IN ANIMAL CELLS MARIANA SOTTOMAYOR1 AND ALFONSO ROS BARCELO2 Department of Botany of Faculty of Sciences and Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre, 823, 4150180 Porto, Portugal, 2 Department of Plant Biology (Plant Physiology), University ofMurcia, E-30100 Murcia, Spain. ABSTRACT: The leaves of Catharanthw roseus (L.) G. Don (formerly Vinca rosea L.) were used in traditional medicine as an oral hypoglycemic agent and investigation of this activity ultimately led to the serendipitous discovery of the cytostatic terpenoid indole alkaloids vinblastine and vincristine. These compounds were the first natural anticancer agents to be clinically used and, together with a number of semisynthetic derivatives, are universally known as the Vinca alkaloids. Due to its important pharmaceutical alkaloids, C. roseus has now become one of the most extensively studied medicinal plants and much has been discovered about the biosynthetic pathway of terpenoid indole alkaloids, the regulation and compartmentation of the pathway, and the mechanisms of accumulation of those compounds inside the plant cell. The biosynthesis of vinblastine involves more than twenty enzymatic steps, nine of which are now well characterized at the enzyme and gene level and, recently, regulatory genes of the initial part of the pathway (ORCAs) have been cloned, in what consists a highly promising strategy for the manipulation of the pathway. On the other hand, the activity of vinblastine and vincristine in human cells has been thoroughly studied. The cytostatic activity has been shown to result from interference with tubulin, but the precise mechanism of action is still not perfectly understood. Uptake and extrusion in human cells has been characterized, specially the extrusion mechanism responsible for resistance to the drugs, and their metabolism in the human body has also been studied. Together, the above mentioned studies enable to establish some interesting evolutionary links between the enzymes involved in plant biosynthesis of the anticancer alkaloids and the enzymes involved in animal metabolism of the drugs, and also, possibly, between their vacuolar transport in plant cells and multidrug resistance in human cancer cells.

INTRODUCTION The so-called Vinca alkaloids are dimeric terpenoid indole alkaloids well known by their antimitotic activity, which has made them extremely

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useful drags in cancer therapy for more than thirty years. This designation includes the natural products vinblastine and vincristine, Fig. (1), extracted from the leaves of the plant Catharanthus roseus (L.) G. Don (previously Vinca rosea L.)» and a number of semi-synthetic derivatives like vindesine, vinorelbine and the recently developed vinflunine, Fig. (1). OH

CH31

Vinblastiue

, ,,, OOOCH3 I H J 'COOOJ3 0H CII3

CHO

*

ocoai3 'OOOCH3 "

Fig. (1). Structure of natural and semi synthetic Vfnca alkaloids. Shaded areas indicate the structural differences from vinblastine.

Catharanthus roseus, known as the Madagascar periwinkle, was used in traditional medicine as an oral hypoglycemic agent in the treatment of diabetes mellirus, and investigation on this activity ultimately led to the discovery of the anticancer alkaloids, almost simultaneously, by two totally independent groups: the group of Noble and collaborators in Canada [1,2] and the group of Svoboda and collaborators from the EM

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Lilly Company, Indianapolis, in the United States [3]. The chain of events leading to the discovery of vinblastine by the group of Noble is particularly interesting and represents a paradigmatic example of the role serendipity often plays in scientific discoveries. A full description of the discovery, including the casual details that started the whole process, was published by Robert Noble in 1990 [4]. After their discovery, the Vinca alkaloids became the first natural anticancer agents to be clinically used, and they are still an indispensable part of most curative regimens used in cancer chemotherapy nowadays. On the other hand, the plant producing these alkaloids, C. roseus, has become one of the most extensively studied medicinal plants. The levels of vincristine and vinblastine in the plant revealed to be extremely low and, for pharmaceutical production, approximately hah0 a ton of dry leaves is needed to obtain 1 g of vinblastine [4]. This fact stimulated intense investigation in alternative methods for the production of vinblastine and vincristine, namely chemical synthesis and plant cell cultures. However, chemical synthesis showed not to be viable due to the high number of transformations involved, and the anticancer alkaloids were never detected in cell cultures, which express alkaloid metabolism very poorly [5, 6]. The biosynthetic pathway of terpenoid indole alkaloids in C. roseus has also been intensively studied with the objective of developing a manipulation strategy to improve the levels of the anticancer alkaloids in the leaves of the plant [5, 7-10]. This review intends to put together what is known about the biosynthesis and accumulation of Vinca alkaloids in the plant Catharanthus roseus, with what is known about their uptake, mechanism of action and metabolism in animal cells. This will enable to highlight the curious matching of some of the enzymes involved in alkaloid biosynthesis in plant cells with some of the enzymes involved in alkaloid metabolism in animal cells, as well as to highlight the likely relation between the putative alkaloid accumulation mechanism in the vacuole of plant cells, and the transport mechanism responsible by multidrug resistance in animal cells. These similarities suggest that, during plant/herbivore co-evolution, plants have developed toxic chemical defenses against herbivores, like the Vinca alkaloids in C. roseus, by recruiting the same type of enzymes and transport mechanism that animal herbivores have, on their side, recruited for the defense against the very same compounds.

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THE VINCA ALKALOIDS AND THEIR CLINICAL USES The first Vinca alkaloid to be discovered was isolated in 1957 by Noble and collaborators from the Western University of Ontario, London, Canada, who named the alkaloid vincaleukoblastine, in view of its origin and its effect on immature white cells - leukoblasts [1, 2]. Later, the name was shortened to the less cumbersome vinblastine. Almost at the same time, the group of Svoboda and collaborators, at the Eli Lilly Research Division in the United States, detected two compounds with antitumour activity in C. roseus [3]. One of them was the already identified vinblastine, the other one was named leurosine. The two groups came in touch in a conference held by the New York Academy of Sciences in 1958, and worked in close collaboration thereafter. Clinical trials confirmed the usefulness of vinblastine in the treatment of Hodgkin's disease, lymphoma and other cancers, and the drug was introduced in the clinic shortly after. Leurosine was proved to be unsuitable for cancer therapy due to its toxicity, but Svoboda later isolated another alkaloid, which was also cytostatic and suitable for therapy [11]. This compound was first named leurocristine, then vincaleukocristine and finally vincristine. Vinblastine and vincristine have now earned a place among the most valuable agents used in cancer chemotherapy. They are dimeric terpenoid indole alkaloids differing only in that vincristine has a formyl group at a position where vinblastine has a methyl group, Fig. (1), but, although their chemical structure is very similar, they differ markedly in the type of tumors they affect and in their toxicity. The basic structure of terpenoid indole alkaloids includes an indole nucleus derived from tryptophan, via tryptamine, and a versatile C9 or CIO unit arising from the monoterpenoid secologanin (see biosynthesis below). The anticancer alkaloids are built from two different terpenoid indole units derived from the precursors vindoline and catharanthine, this later suffering a rearrangement during the dimerization reaction to give rise to the so called velbenamine or cleavamine part of the dimeric molecule, Fig. (2). The direct product of the dimerization reaction is the dimer oc-3',4'anhydrovinblastine whose potential for cancer therapy is also being investigated [12]. However, apart from the reference cited, no report about the anticancer action of anhydrovinblastine was found in indexed

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scientific publications.

CH3 CH 3

Vindolme

Vinblastine Fig. (2). Biosynthesis of vinblastine from the monomeric precursors catharanthine and vindoline. Anhydrovinblastine is the direct product of the dimerization reaction and the precursor of the anticancer drugs. Shaded areas indicate the structural differences between the precursor catharanthine and the deavamine part of anhydrovinblastine.

Vinblastine, with the commercial names Velbe®, Velban® and Vinblastine®, is used alone and as a component of combined regimens with other anticancer drugs in the treatment of Hodgkin's disease and other lymphomas, in advanced carcinoma of the testis, in Kaposi's sarcoma and histiocytosis X. It can also be used in the treatment of breast carcinoma and choriocarcinoma. The use of vinblastine is mainly limited by its hematological toxicity due to destruction of the bone marrow [1315]. Vincristine, with the commercial names Oncovin , Vincasar , Vincrisul®, Pericristine®, and Kyocristine®, is used as a component of combination therapy in the treatment of Hodgkin's disease and lymphomas, and also in acute leukemias, sarcomas and carcinomas. Because of its relative lack of hematologic toxicity it is widely used as a component of many chemotherapeutic regimens. Combined with prednisone it produces complete remission in up to 90% of children with

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acute lymphocytic leukemia. The major and dose-limiting adverse effect of vincristine is neurotoxicity, specially to the peripheral nervous system [13-15]. Soon after the introduction of vinblastine and vincristine in clinical usage, during the 1970's, intensive chemical research was undertaken in order to try to obtain semi-synthetic derivatives of Vinca alkaloids showing higher activity, lower toxicity, and a wider spectrum of anticancer efficacy (for reviews see [16, 17]). The Eli Lilly company developed several series of derivatives with modifications in the vindoline part of the dimeric structure, culminating with the approval of vindesine, Fig. (1), for clinical treatments. Vindesine, with the commercial name Eldisine® and Enisone®, has a vincristine-like spectrum of activity, and is used mainly in the treatment of melanoma, acute lymphoblastic leukaemia and advanced non-small cell lung cancer [13, 14, 16]. Vindesine is approved in Europe and other areas but, in the United States, vindesine is approved only for investigational use [15]. In 1975, Potier and collaborators proposed that, inplanta, the dimeric vinblastine type alkaloids resulted from the coupling of catharanthine and vindoline and, in light of this hypothesis, they reported for the first time the chemical synthesis of a dimer with the natural configuration through a modified Polonovski reaction [18, 19]. This reaction resulted in the formation of an iminium dimer which, after reduction with NaBKj, yielded a-3',4'-anhydrovinblastine, Fig. (2), later proved to be the first dimeric biosynthetic precursor of vinblastine in the plant. The group of Potier investigated possible modifications of anhydrovinblastine and produced vinorelbine, Fig. (1), which was the first active derivative with an altered cleavamine (catharanthine) moiety [20, 21]. Vinorelbine demonstrated important antitumour properties associated with reduced toxic side effects and its application was developed during the 1980's by the French pharmaceutical company Pierre Fabre Medicaments, under the commercial name Navelbine . Vinorelbine is now widely used in the treatment of non-small cell lung cancer and breast cancer, and several other potential indications are under clinical investigation, like lymphoma, esophageal cancer and prostatic carcinoma [16, 22, 23]. Furthermore, it has been proved that vinorelbine is well absorbed orally with no unpredictable toxic effects and an oral formulation of the drug was registered in France in 2001 [13, 16]. The main side effect of vinorelbine is hematological toxicity.

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Pursuing the effort to obtain new useful Vinca alkaloids, the research divisions of Pierre Fabre produced a new family of derivatives using superacidic chemistry, from which vinflunine, a difluorinated derivative of vinorelbine, was selected for detailed preclinical investigations. Results showed that vinflunine is more active than vinorelbine, vinblastine or vincristine against a number of murine tumours and human tumour xenografts, and it entered phase I clinical trials in 1998, phase II in 2000, and is entering phase III in 2003 [16, 23, 24] . For a review on preclinical anticancer properties of vinflunine see [25]. The vinflunine case demonstrated that the Vinca alkaloids remain a drug family where it is still possible to identify new members with unprecedented and promising pharmacological properties. When the ongoing research on the mechanisms of action of Vinca alkaloids unravels the precise relation structure/function of the dimeric molecules, it should be possible to rationally design a new generation of Vinca alkaloids with new therapeutic properties. BIOSYNTHESIS OF VINCA ALKALOIDS IN CATHARANTHUS ROSEUS After the discovery of the anticancer properties of vinblastine and vincristine, the elucidation of their structure, Fig. (1), was a natural step achieved in the early 60s [26, 27], and it was shown that they were dimeric terpenoid indole alkaloids - as already stated above. Simultaneously, further studies of the plant C. roseus revealed that this plant is an amazing chemical factory, producing more than 100 different terpenoid indole alkaloids, including two other with important pharmacological activity: ajmalicine, used as an antihypertensive, and serpentine, used as sedative [6, 28]. Terpenoid indole alkaloids (TIAs) comprise a large family of secondary metabolites, with around 3000 members identified, including several with important biological activity, like the Vinca alkaloids, the rat poison strychnine, and the antimalarial drug quinine [7], [29, 30]. They are almost restricted to four plant families of dicotyledones: Apocynaceae, Loganiaceae, Rubiaceae and Nyssaceae [9, 31]. In the plant, alkaloids are thought to play a defense role, mainly as deterrent factors against herbivorous pests, and some have been shown to be toxic against certain fungi and bacteria [32, 33]. A number of reports about the

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antibiotic or antifeedant activity of the TIAs present in C. roseus has been published [34-40]. (7YTOPI.ASM

.tryplamine

— strictosidinc RER

16-hydroxv. ' ™ tabereomne

... strict™dmc aglvcome Vincristine

tabersonine "

cat haranthine

Anhydrovinblastinc deacety] vindol i ne

vindoline

Fig. (3). Compartmentalization of the biosynthetic pathway of terpenoid indole alkaloids in plant cells. G10H: geraniol 16-hydroxylase; SLS: secologanin synthase; TDC: tryptophan decarboxylase; STR: strictosidine synthase; SGD: strictosidine (3-D-glucosidade; T16H: tabersonine 16-hydroxylase; OMT: S-adenosyl - Lmethionine : 16-hydroxytabersonine - 16-O-methyltransferase; NMT: 5-adenosyl - L-methionine : 16-methoxy - 2,3-dihydro-3-hydroxytabersonine - A'-methyltransferase; D4H: desacetoxy vindoline 4-hydroxylase; DAT: acetylcoenzyme A : 4-O-deacelylvindoline 4-O-acetyltransfcrase; PRX: peroxidase.

The great pharmacological importance of the dimeric alkaloids, allied with its low availability, stimulated intense research in the biosynthesis of

821 821

TIAs in C. roseus and in the regulation of the pathway, with the aim of eventually manipulating plant metabolism in order to obtain higher levels of the anticancer alkaloids. The biosynthesis of vinblastine has shown to be highly complex, involving more than twenty enzymatic steps, and a great deal is already known about the pathway, the enzymes and genes involved, and about their regulation. However, considerable parts of the pathway remain relatively hypothetical, and enzymatic characterization is still lacking for many steps. The biosynthetic pathway of vinblastine revealed to be highly compartmentalized inside the cell, since a number of enzymes were shown to be localized in different cellular compartments, either experimentally, or by inference from the presence of targeting signal peptides in their aminoacid sequences, Fig. (3) [8, 41]. Enzymes and genes involved in branching points of the early stages of biosynthesis, like geraniol hydroxylase, tryptophan decarboxylase and strictosidine synthase, Fig. (4), have been thoroughly characterized, and the last 6 steps in the path leading to vindoline, Fig. (6), one of the monomeric precursors of vinblastine, Fig. (2), have received much attention as well, with several enzymes/genes of these late steps being characterized. This part of the pathway is not expressed in cell suspension cultures, what possibly accounts for the absence of dimerics in this system. The dimerization step itself has received considerable attention and is thought to be mediated by a class III plant peroxidase [42-44]. In spite of all this, the pathway from primary metabolism to vinblastine and vincristine still includes many transformations that remain to be characterized, even at the level of the biosynthetic intermediates. Previously, the biosynthesis of TIAs has been reviewed in [7, 8, 10, 45]. Biosynthetic pathways What is known about the biogenetic routes leading to the biosynthesis of the dimeric akaloids vinblastine and vincristine in C. roseus is represented in Fig. (4) to (6). Enzymes and genes that have been characterized are indicated, and the subcellular compartmentalization of the pathway is presented in Fig. (3). The basic structure of TIAs includes an indole nucleus derived from tryptamine, the decarboxylation product of the aminoacid tryptophan, and a versatile C9 or CIO terpenoid unit arising from the iridoid glucoside secologanin, Fig. (4).

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Tryptophan is a product of the shikimate pathway and is converted into tryptamine by tryptophan decarboxylase (TDC), Fig. (4). TDC is a cytosolic soluble enzyme that occurs as a dimeric protein, and it was shown to exhibit a high substrate specificity and to be under posttranslational control [46-52]. A cDNA clone encoding TDC was isolated by DeLuca et al. [53] and the foil gene was characterized by Gooddijn et al. [54, 55] who found that TDC is encoded by a single copy gene without introns. The Tdc promoter has also been cloned and its regulation characterized [56, 57]. Glyceraldehyde / pyruvate pathway Geraniol

OH

Shikimate pathway

I

COOH

10-Hydroxygeraniol OH

L-Tryptophan

lOGlu NH

Tryptamine

Secologanin

Fig. (4). Early steps of the biosynthesis of terpenoid indole alkaloids in Catharanthus roseus. Triple arrowheads indicate multiple steps. G10H: geraniol 16-hydroxylase; TDC: tryptophan decarboxylase; STR: strictosidine synthase.

The terpenoid portion of TIAs is derived from secologanin, whose monoterpene precursor geraniol, is produced by the recently discovered Rommer or triose phosphate / pyruvate pathway, responsible for the synthesis of isoprenes like geraniol in the plastids [58-60].

823

The first committed step in the biosynthesis of secologanin is the hydroxylation of the C-10 position of geraniol by geraniol 10-hydroxylase (G10H), Fig. (4), which was one of the first cytochrome P-450 monooxygenases to be characterized in plants [61-63]. The enzyme and the associated NADPHxytochrome P-450 reductase were purified to homogeneity from cell suspension cultures of C. roseus and characterized [64, 65]. G10H was shown to be able to hydroxylate both geraniol and its cis isomer nerol. The end product alkaloid catharanthine was proved to be a reversible, linear, noncompetitive inhibitor of G10H, while vindoline and vinblastine were less inhibitory but still interfered with activity [66]. G10H was found to be localized in provacuolar membranes [67], although the same authors state later that what they had characterized as vacuolar membranes could in fact represent a differentiated form of endoplasmic reticulum [61]. In spite of this ambiguity, G10H is considered by most reviewers to be localized in the vacuolar membrane [5, 8, 41]. The NADPHxytochrome P-450 reductase (CPR) was found to be similar to the mammalian enzyme and both G10H and CPR were cloned and the genes characterized [68-70]. The pathway leading from 10hydroxygeraniol to secologanin has been relatively well characterized [8, 10, 29, 71] and the enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, secologanin synthase (SLS), Fig. (4), was also shown to be a cytochrome P450 [72, 73]. The stereospecific condensation of tryptamine and secologanin under the action of strictosidine synthase (STR), Fig. (4), is the first committed step in TIAs biosynthesis, and it yields the glucoalkaloid 3-a(S)strictosidine, which is the central biogenetic precursor of all TIAs [7477]. STR was first purified by Treimer and Zenk [78, 79] and Mizukami et al. [80] from cell cultures of C. roseus. The enzyme was found to have a high substrate specificity, and to suffer no inhibition by end-product alkaloids, such as vindoline and catharanthine [78-80]. It was observed that STR occurred as different isoenzymes [81, 82], and the subcellular localization was determined to be the vacuole [83]. The complete mRNA sequence of Str was determined by Pasquali et al. [84], who also showed that STR is encoded by a single-copy gene, indicating that the above mentioned isoenzymes are formed post-translationally from a single precursor. Comparison of the primary structure of the STR protein with the amino acid sequence deduced from the Str mRNA showed the

824

presence of a signal peptide of 31 amino acids in the amino-terminal sequence. This signal peptide appears to be essential for vacuolar targeting of STR, according to results obtained with transgenic N. tabacum [83]. The promotor of Str has been studied in great detail and has enabled the identification of transcriptional factors involved in the regulation of TIAs biosynthesis [9, 85, 86] (see section "Regulation..." below). Strictosidine is the general precursor of several divergent pathways leading to the multitude of TIAs accumulated by C. roseus. Somewhere, downstream of strictosidine formation, the pathway of TIAs suffers several ramifications mostly uncharacterized. The less ill characterized branches are the ones leading to catharanthine and vindoline, the monomeric precursors of the Vinca alkaloids, Fig. (2). Those branches will be the ones discussed here.

21

" lH ,,oau

OH

4,21-Dehydrogeissoschizine

Strictosidine aglycone

CH3OOC

CH 2 OH

Stemmadenine

CH 3

COOCH3

Tabersonine Fig. (5). Biosynthesis of catharanthine and tabersonine from strictosidine, the central precursor of all terpenoid indole alkaloids. SGD: strictosidine p-D-glucosidade.

825 825

The first step following strictosidine synthesis is the removal of its glucose moiety by strictosidine p-D-glucosidade (SGD) with formation of an unstable aglycone, Fig. (5) [87]. SGD is encoded by a single copy gene in C. roseus and is most likely associated with the ER, as suggested by in vivo staining and by the presence of a putative ER signal sequence in the protein [88]. Deglucosylated strictosidine is converted via several unstable intermediates into 4,21-dehydrogeissoschizine from which catharanthine and vindoline are believed to derive, Fig. (5). This part of the pathway has been scarcely characterized - it includes an undetermined number of steps, seems to involve the intermediate stemmadenine, and the branching point for the 2 paths giving rise to catharanthine and vindoline has been proposed to be dehydrosecodine by Blasko and Cordell [71], and to be stemmadenine by Verpoorte et al. [89]. The 6 last biosynthetic steps leading to the production of vindoline from the intermediate tabersonine have been thoroughly characterized and are represented in Fig. (6) [45, 90]. The first step in the conversion of tabersonine to vindoline is hydroxylation of the C-16, which is catalyzed by the enzyme tabersonine 16-hydroxylase (T16H), Fig. (6). Characterization of T16H indicated the enzyme is a cytochrome P-450 monooxygenase [91], what was confirmed by the molecular analysis of the isolated cDNA [92]. Southern analysis suggests the presence of at least two T16H genes in C. roseus. The following step in the biosynthesis of vindoline is the Omethylation of 16-hydroxytabersonine to yield 16-methoxytabersonine by the enzyme S-adenosyl-L-methionine: 16-hydroxytabersonine-16-0methyltransferase (OMT), Fig. (6) [45, 93]. Only a preliminary identification of OMT has been carried out in crude desalted extracts from C. roseus leaves [91, 94]. O-methylation of 16-hydroxytabersonine is followed by an uncharacterized hydration step, and then by iV-methylation of the Nindole by the enzyme S-adenosyl - L-methionine : 16-methoxy - 2,3dihydro - 3-hydroxytabersonine - iV-methyltransferase (NMT), originating desacetoxyvindoline, Fig. (6). NMT has been roughly characterized by DeLuca et al. [95], and partially purified by Dethier and DeLuca [96] from young leaves of 6 months old C. roseus plants. Subcellular localization studies indicated that NMT is specifically associated with the membranes of thylakoids [46].

826

COOCH3

Tabersonine

16-Hydroxytabersonine

NMT €H3

„

H

COOCH3

16-Methoxvtabersonine

D4H

1 uM). Among vinblastine, vincristine and vmorelbine, vincristine demonstrates the highest overall affinity for tubulin and vinorelbine the lowest [177]. Vinflunine has been shown to bind to tubulin with even lower affinity than vinorelbine [178]. Affinity is thus ranked vincristine > vinblastine > vinorelbine > vinflunine, parallel to drug toxicity, but not to therapeutic potential. These values correlate inversely with drug doses used in clinical treatments, since vincristine is used at the lowest dose and vinorelbine at the highest. Binding of Vinca alkaloids to tubulin is reversible, and the precise location and number of binding sites in tubulin dimers and microtubule ends is not clear. Rai and Wolff [176], using a fluorescent vinblastine derivative, detected and characterized a single high affinity binding site in P-tubulin and also detected the presence of several low affinity sites that were not possible to characterize. More recently, nuclear magnetic resonance analyses revealed the presence of three binding sites in the oc/ptubulin dimer for vinorelbine and vinflunine [179]. At 30° C, binding of

839 839

vinflunine to tubulin was hardly detected, in agreement with the significant lower affinity of this drag to tubulin. In reconstituted microtubule systems and in cells, the interaction of Vinca alkaloids with microtubules results in different effects depending on drug concentration: i) at low concentrations (< 1 uM = nanomolar range), the drugs diminish microtubule dynamics increasing the time spent in the "resting state"; ii) at intermediate concentrations (1-2 uM), they depolymerize microtubules and inihibit assembly; and Hi) at high concentrations (> 10 uM), they induce self-aggregation of tubulin with formation of large paracrystals and other aggregates [172, 176]. In all cases, normal microtubule function is compromised. In spite of all that is known about the interaction between Vinca alkaloids and tubulin, the precise molecular location and chemical bonds established during that interaction are not known what prevents a rationale design of new Vinca alkaloids. The different therapeutic profiles and toxicities of the Vinca alkaloids are thought to be partially due to the different affinities they show towards tubulin [172, 177], namely towards different tubulin isotypes that may have tissue specific expression [180]. It is interesting to remark that, although vinorelbine and vinflunine show significant lower affinity to tubulin than vincristine and vinblastine, a fact correlated with their lower toxicity, they actually present a higher anticancer therapeutic action, a feet correlated with their higher intracellular accumulation (see section "Uptake..." below) [23, 25, 181]. This means that vinorelbine and vinflunine present a differential effect between normal cells, where lower affinity to tubulin prevents toxic effects in spite of Mgh intracellular concentrations, and cancer cells, where the weak interaction of the high concentrated drugs is sufficient to induce a strong effect. There is no explanation for this feet but Ngan et al. [181] consider that nontumor cells, with normal checkpoint proteins, may tolerate better the relatively less powerful inhibitory effects of vinflunine and vinorelbine on microtubule dynamics, than cancer cells, with abnormal cell cycle regulation. Mechanism of inhibition of cell proliferation The mechanism of action of the Vinca alkaloids was initially thought to involve the depolymerization of spindle microtubules and induction of

840

paracrystalline tubulin-F/«ca alkaloid aggregates, the effects observed at intermediate and high concentrations of the drugs [167-169]. However, the more recent observation that, at low concentrations, the Vinca alkaloids inhibit the microtubule dynamic behavior raised two questions: i) whether this effect could also block mitosis and ii) which of the two situations is actually involved in anticancer therapy with this group of drugs. Investigating this question, Jordan and collaborators [182] observed that, at the lowest effective concentrations of five Vinca alkaloids, inhibition of cell proliferation and blockage of HeLa cells at metaphase occurred with little or no microtubule depolymerization and no spindle disorganization. With increasing drug concentrations, the authors observed that the organization of microtubules and chromosomes started to deteriorate. Dhamodharan et al. [183] also observed that low vinblastine concentrations (nM levels) block mitosis in BS-C-1 cells, in association with suppression of microtubule dynamics but in the absence of appreciable changes in microtubule mass. Recently, it has been shown that the precise parameters of microtubule dynamics that are inhibited at low concentrations by vinorelbine and vinflunine differ significantly from vinblastine [181]. In spite of those differences, further investigations showed that all the three drugs produced remarkably similar effects on spindle organization. In all cases, proliferation inhibition seemed to be induced my mitotic block at the metaphase/anaphase transition with formation of aberrant spindles, consistent with induction of block by suppression of microtubule dynamics [23]. As a whole, the results presented above indicate that low concentrations of Vinca alkaloids, probably similar to therapeutic concentrations, have an antiproliferative activity that is due to inhibition of mitotic spindle function by changing the dynamics of microtubules rather than by depolymerizing them. A growing body of evidence seems to indicate that, specially in cancer cells, where mitosis regulation is already disrupted, the suppression of microtubule dynamics with mitosis arrest induces a signaling cascade leading to cell death by apoptosis (a type of programmed cell death) [172-174]. Other cellular targets of Vinca alkaloids Vinca alkaloids are toxic molecules that easily cross cellular membranes due to their lipophilicity and interfere with a multitude of cell targets.

841 841

Although tubulin and microtubules are undoubtedly the main target responsible by their antieancer action, other targets may also contribute to this action, while some others may be associated with their toxic side effects. Recently, it has been shown that proteasomes, the proteolytic machinery of the ubkjuitin/ATP-dependent proteolysis pathway, can also be considered a target of vinblastine. Proteasomes have a crucial role in the regulation of the cell cycle, and proteasome inhibitors can block cell cycle progression and induce apoptosis in certain cell lines. Vinblastine seems to have an inhibitory effect on proteasomes and could thus interfere with mitosis also through this path [165]. Another target of Vinca alkaloids seems to be DNA. Tiburi et al. [184] showed that vinblastine, vincristine and vinorelbine all had significant genotoxicity, as assayed by the wing Somatic Mutation and Recombination Test (SMART) of Drosophila. All the three drugs caused increments in the incidence of mutational events and somatic recombination. An effect of Vinca alkaloids that may also be important in their anti tumour activity is their antivascular action. It has been shown that vincristine and vindesine are able to reduce the capillary network formation by HUVEC cells cultured on Matrigel at non-cytotoxic concentrations, while vinblastine and vinorelbine produce anti-angiogenic effects by direct cytotoxicity [185]. Vinflunine seems to have an antivascular activity consistently superior to that of vinorelbine [25]. UPTAKE AND EXTRUSION ANIMAL CELLS

OF

VINCA

ALKALOIDS

IN

Vinca alkaloids are lipophilic molecules that can readily cross membranes by simple diffusion [186]. Experiments performed with several human cancer and tissue cell lines have shown in all cases rapid uptake of every one of Vinca alkaloids [23, 187-189]. Uptake is thought to occur by diffusion although energy dependence or independence of the uptake is seldom mentioned in reports. However, for instance in cultured human promyelocytic leukemia HL-60/C1 cells it has been shown that rates of uptake of vinblastine were unaffected by depletion of cellular adenosine triphosphate, reinforcing that uptake is not mediated by an energydependent system [189].

842

When incubated with human hepatocytes, vinorelbine was the most rapidly and intensely accumulated Vinca alkaloid followed by vinblastine, vindesine and vincristine, as would be suggested by the lipophilicities of the molecules [187]. Vinflunine is even more ipophUic than vinorelbine and accumulates fester inside cells [23]. Although uptake is considered to occur by diflusion, the Vinca alkaloids accumulate inside animal cells to concentrations many times higher than extracellular concentrations. Vinblastine and vincristine have been shown to accumulate more than 100 fold in cultured human promyelocytic leukemia HL-60/C1 cells [189]. Addition of 10 nM vinblastine to the culture medium of HeLa cells resulted in a 40 fold accumulation, while addition of 100 nM vinblastine resulted in a 31 fold accumulation [182]. In BS-C-1 cells, 32 nM vinblastine accumulated 284 fold [183]. This intracellular accumulation is thought to result, at least in part, from the binding of the drugs to tubulin and microtubules [172], For example, the maximum vinblastine intracellular levels observed in HeLa cells are similar to the intracellular levels of tubulin [182]. However, the existence of other intracellular reservoirs for drug accumulation is not discarded and, recently, some evidence has been obtained that suggest that vinorelbine and vinflunine may be sequestered inside the ceE by other mechanism than binding to tubulin [23]. The authors characterized uptake of 1 nM vinblastine, 3 nM vinorelbine and 30 nM vinflunine by HeLa cells. The concentrations used for each drug were the ones inducing the same effect in cells, i.e., a 50% inhibition of mitosis. As predicted from their lipophilicity, uptake rate was much higher for vinflunine, followed by vinorelbine, and the peak concentration for the three drugs was 4.2 pM for vinflunine (140 fold), 1.3 uM for vinorelbine (430 fold) and 130 nM for vmblastine (130 fold). In these conditions, mitosis was blocked but microtubules were not disassembled. Since micromolar concentrations of vinorelbine and vinflunine significantly reduce microtubute polymerization in vitro [181], the authors suggest that not all intracellular vinflunine and vinorelbine is available to bind to tubulin and must be sequestered in other cellular reservoirs such as membrane compartments [23]. In animal models, concentrations in tissues can also exceed those in plasma, and it has been observed that, in certain tissues, the drugs are retained for prolonged periods, sometimes related to the specific therapeutic indications of each drug [190]. In mice, vinblastine is

843 843

selectively retained in genetic tract and lymphatic tissues, a fact that may be the basis of the activity of this alkaloid against malignant transformations with an origin in these tissues [191]. Likewise, in rats, the lungs were among the organs with higher accumulation of vinorelbine, which is used in the treatment of small cell lung cancer [190]. This differential retention may result in some cases from different relaxation times of the binding of the drug to different tissue isotypes of tubulin, but, in other cases, it seems that the tissues not retaining the alkaloids possess effective means of extruding the drug when plasma levels decrease [191]. Meaning that extrusion of the Vinca alkaloids from animal cells may occur not only by diffusion but also as a result of a more efficient transport mechanism. In many cases, the Vinca alkaloids are just released by diffusion after exposure to the drug ends [189], with a rate dependent on the strength of their binding to tubulin and/or on the release rate from other intracellular sequestration mechanisms. However, as stated above, some tissues possess a more effective way of extruding the drugs, namely cancer cells that have become resistant to chemotherapy. In fact, the best characterized mechanism of resistance of cancer cells to chemotherapy drugs, like the Vinca alkaloids, is the phenomenom known as multidrug resistance (MDR), which is due to the overexpression of the mdrl gene coding for the membrane localized P-glycoprotein, that actively pumps the drugs out of the cell [15, 192]. P-glycoprotein is constitutively overexpressed in various normal tissues including the renal tubular epithelium, the adrenal medulla, the liver, and the blood brain barrier, where it is thought to protect cells from toxic agents/xenobitotics [193, 194]. P-glycoprotein belongs to the family of ABC transporters (see section "Accumulation ..." above) and it is localized in the plasma membrane, being able to extrude from the cell a variety of structurally diverse drugs, drug conjugates and metabolites. Extrusion of these compounds by P-glycoprotein is ATP-dependent and can take place against considerable concentration gradients [192]. P-glycoprotein expression may occur in tumour types derived from tissues that normally express the protein, like renal cell cancer, but its overexpression may also be induced by the treatment with anticancer drugs. All Vinca alkaloids used in cancer therapy can induce the expression of P-glycoprotein and the associated multidrug resistance phenotype, due to the capacity of P-glycoprotein to pump out of the cell a

844

number of unrelated anticancer drugs, preventing therapeutic intracellular concentrations to be achieved [15]. METABOLISM OF VINCA ALKALOIDS IN ANIMAL CELLS Vinca alkaloids are metabolized primarily by the liver, and metabolites are eliminated by biliary excretion [15]. When incubated with human hepatocytes in suspension, vinblastine, vincristine, vindesine and vinorelbine are rapidly taken up and intensely metabolized by the cells in a number of unidentified products [187]. On the other hand, the capacity of cancer cells to metabolize these drugs is usually limited. For example, HPLC analysis of extracts of human promyelocytic leukaemia HL-60/CI cells incubated with growth-inhibitory concentrations of labelled vinblastine and vincristine indicated little or no metabolism of either drug by cells or culture fluids [189]. In the liver, the only and possibly main enzymatic system that has been shown to be involved in metabolism of Vinca alkaloids is the cytochrome P450 monooxygenase CYP3A4 [195-197]. The large interpatient variability in the sensitivity to the Vinca alkaloids has been frequently associated to individual disparities in the levels of CYP3A4. Recently, it has been shown that tumour CYP3A4 may also contribute to the development of drug resistance during chemotherapy [196]. Another enzyme that has been implicated in the metabolism of vincristine in acute myeloblastic leukaemia (AML) cells is myeloperoxidase. The fact that AML is resistant to vincristine has been related to the presence of mieloperoxidase, which is able to catalize the vincristine's oxidative breakdown [198, 199]. The compounds resulting from metabolism of the Vinca alkaloids are little characterized. The main hepatic metabolite of vinblastine, vincristine, vinorelbine and vinflunine seems to be, for each of the compounds, the respective 4-O-deacetyl alkaloid. Other metabolites have also been detected but only very seldom were structurally characterized [15, 191, 200-203].

845 845

THE VINCA ALKALOIDS FROM PLANTS TO ANIMALS - THE EVOLUTIONARY LINK The Vinca alkaloids metabolism and transport in the producing plant cells and in the treated animal cells illustrate some interesting aspects of how evolution can be winding and parsimonious in the solutions it creates. Plants possess an incredibly diverse biosynthetic capacity leading to the production of a myriad of compounds that, although not having an apparent function for fundamental life processes (growth, development and reproduction), seem to have vital roles as mediators of ecological interactions, being very important for the survival of plants. This chemical wealth is the basis of the use of plants in medicine, and is still largely unexplored. One example of application of the so called plant secondary metabolites are the terpenoid indole alkaloids of Catharanthm roseus, used in cancer therapy, and known as the Vinca alkaloids. In the plant, the biosynthesis of the Vinca alkaloids involves more than 20 enzymatic steps including several cytochrome P450 monooxygenases and one class III plant peroxidase. Removal of the toxic alkaloids from the cytoplasm to the vacuole of plant cells is made by an uncharacterised transport mechanism that we suggest may be an ABC transporter, as already shown for several other plant secondary metabolites. In human and model animal cells, metabolism of the exogenously applied Vinca alkaloids is carried out by a cytochrome P450 monooxygenase and in some cases by myeloperoxidase, and removal of the toxic alkaloids from the cytoplasm to the extracellular compartment is made by an ABC transporter. The mechanisms involved in metabolism and transport of the Vinca alkaloids in animals are thought to have developed, at least in part, as a defence against the toxic compounds present in the plants that animals eat [204]. The similarities between mechanisms involved in production and accumulation of toxic defence compounds in plants, and metabolism and transport of the same compounds as xenobiotics in animals, mean that essential building blocks of such complex and divergent organisms as plants and animals have a very ancient common origin, and that, through evolution, they were sometHnes recruited to functions that oppose to each other in nature. The P450 superfemiry, for example, is found in all groups of organisms, including Archae, and is believed to have originated in an

846

ancestral gene that existed over 3 billion years ago [205]. The ABC transporter superfamily is also referred as one of the biggest multigene families and it has been shown to exist in bacteria, fungi, plants and animals [161, 194, 206]. Single ancestor genes have thus suffered major duplication events and evolved to result in a panoply of functions. To construct the amazing diversity of life, evolution has sometimes played with a small number of pieces to construct the biochemical survival strategies inherent to that diversity. ABBREVIATIONS D4H DAT PRX G10H NMT OMT ORCA SGD SLS STR T16H TDC TIA

desacetoxy vindoline 4-hydroxylase acetylcoenzyme A : 4-O-deacetylvindoline 4-0acetyltransferase peroxidase geraniol 16-hydroxylase S-adenosyl - I-methionine : 16-methoxy - 2,3-dihydro-3hydroxytabersonine - iV-methyltransferase S-adenosyl - Z-methionine : 16-hydroxytabersonine - 16O-methyltransferase octadecanoid responsive Catharanthus AP2-domain protein strictosidine p-D-glucosidade secologanin synthase strictosidine synthase tabersonine 16-hydroxylase tryptophan decarboxylase terpenoid indoe alkaloid

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©

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THE CHEMISTRY OF OLEA EUROPAEA Armandodoriano Bianco, Alessia Ramunno Scuola di Specializzazione in Chimica e Tecnologia delle Sostanze Organiche Natvrali - Universita di Roma "La Sapienza ". Piazzale Aldo Moro 5, Roma, Italy. ABSTRACT: This review summarises the chemistry of Olea europaea, in particular of hydrophilic components, which are seen as the key compounds responsible for several properties, identified as the organoleptic characteristics of foods derived from O. europaea and as the biological properties attributed to this plant, like ipotensive, anti-oxidising, etc. In fact, two groups of polar compounds can be identified in O. europaea: terpenenoids, as oleuropein; phenols, as hydroxy-phenyl ethanol derivatives; and compounds related to these two categories. Oleuropein and its metabolites are terpenoids present in all parts of plant and represent the chemotaxonomic markers of O. europaea. Tyrosol, hydroxytyrosol and related compounds represent the main characteristic phenolic fraction of O. europaea and are recognised as very powerful anti-oxidising agents. The chemistry of oleuropein and of other phenolic components of O. europaea is described, in relation also with the biological activity of these compounds. O. europaea represents, in fact, a key plant in the economy of Mediterranean region, and olives are the key component of Mediterranean diet. Olives, olive oil in particular, represent a unique food in the scenario of Mediterranean region and they certainly have contributed to the evolution of civilisation of this part of the world. 1. Introduction 2. Chemistry of Olea europaea 2.1. Molecular composition 2.2. Molecular modifications 3. Biological activity and Pharmacology of Olea europaea 3.1. Antioxidative and radical scavenging effects of olive phenols 3.2. Effects of oleuropein on gastric mucosa 3.3. Effects of olive components on glycaemic and blood pressure controls. 3.4. Antimicrobial activity 4. Bibliography

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1. INTRODUCTION Olea europaea is a typical plant of Mediterranean region created, according to the classical tradition, by Athena, the Greek goddess of Knowledge. The history of olive-tree is a fascinating story. We could think that an olive branch announced to Noe that the Deluge was finished (Genesis 8, 10-12). An olive-wreath had been reward for the winner in Olympia's games in Greece and was present in lustral ceremonies in Rome. Olive-tree was not only the symbol of Justice (Psalms 54,10) and Knowledge (Siracide 24, 14) for Jews, but was also the image of prosperity and beauty (Geremia 11,16). Honour is given to men and gods by the olive oil (Giudici 9, 9). We can find olive-tree and olive oil in every important moment of Jesus Christ's history. Jesus prayed in the Garden of Olives, Maddalena (Luca 7, 36-56) as Maria of Betania (Giovanni 12,1-3) covered Jesus's feet with olive oil. Islam also considers olive-tree a sacred plant (Holy Koran, sura XCV, 1; sura LXXX, 29; sura XVI, 11). The olive has followed the evolution of the civilisation, starting from the Middle East to the Mediterranean region until the New World. Olive still symbolises the Peace. Literature too considers it. Dante in the Divina Commedia talks about tiie olive for three times: twice in the Purgatory and once in the Paradise but never in the Hell. So this plant appears to be at the borderline between research and history. Why? In our opinion, it is because olive tree is a living organism, having a rather deep significance. Following these considerations, it is easy to understand that studying O. europaea is not a simple molecular recognition but rather it needs a widespread research. 2. CHEMISTRY OF OLEA EUROPAEA In the O. europaea, we may contemporaneously observe two aspects of natural products chemistry: the study of molecular structure (the compounds present in the olive), and the study of the chemical reactivity (as we will describe later, these same compounds are more or less modified during the technological process of olive oil production).

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In the olive, two main groups of compounds are present: fats, as glycerides and lipids, and phenols and related substances. This article discusses about phenols and their related polar compounds that are typical of this plant and that have been until now slighted respect to those constituting the main part of the olive, such as glycerides and fatty acids. Before 1960, one of main components of olive, the oleuropein, was studied in Roma. Panizzi's research group established the structure of this monoterpene glucoside, the leader of the group, that only several years later was identified as secoiridoids. Panizzi's group has isolated two other compounds: the oleuropeic acid that is a monocyclic monoterpene, and its saccharose derivative. That was the beginning of the scientific discovery of the olive tree. In fact, in the pioneering researches of Panizzi, the polar and phenolic fraction of olive was forgotten. In 1993, we began to face up the isolation of olive glycosidic fraction from a systematic point of view, using a specific protocol for polar compounds, and succeeded in the isolation of many glycosidic components present in olive. In the olive, there are two main compounds, besides that reported in the literature. The first one is oleuropein, the first secoiridoid isolated in 1960 by Panizzi in Roma, as mentioned above. The second one is the cornoside that is a hemiquinone glucoside, structurally and biogenetically related to hydroxy-tyrosol that is the principal free phenol in the olive. It should be remembered that, depending on the olive cultivar, the oleuropein can be prevalent against the cornoside, or it could be the contrary, until reaching the equality. With the same protocol, many minor components were also isolated. Some of them are new compounds, while other known. Without dealing here with the problems of isolation and purification, we will list their molecular structures that generally were identified using all possibilities offered by NMR, coupled with chemical manipulation. Firstly, we report on the three hydroxy-tyrosol glucosides that are present in the plant. Other minor glycosidic components are glucosides of tyrosol. In addition, we isolated the verbascoside, a phenyl-propanoid glycoside that is characterised by the presence of hydroxy-tyrosol moiety. Different analogues of oleuropein, as the isomer of oleuropein at the double bond in 8-9 position, were isolated. We have found also polar, but non-glycosidic compounds, i.e. free tyrosol and hydroxy-tyrosol and the aglycone of cornoside, halleridone. Finally, one more thing to mention is that phenols, in the olive, are not only linked to a polar substrate, but to non-polar ones

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as well. Recently, we have isolated an oleil derivative of tyrosol that is present in significant amounts in the olive. 2.1. Molecular Composition The molecular composition of O. europaea, with respect to the phenolic and polar fractions, is quite characteristic, being based on the presence of a phenolic moiety that can be inserted in a terpenoid skeleton, as in oleuropein and related secoiridoids, or may be alone with the presence or absence of a sugar unit. 2.1.1. Oleuropein. Oleuropein 1 is the first secoiridoid whose structure was recognised in 1958-65[l-3], but only several years later it was classified as secoiridoid, when this class of monoterpenoids was constituted.

Figure 1. Structure of oleuropein 1

Oleuropein structure was determined by Panizzi et al.[2] on the basis of the results obtained from an accurate degradation of this compound. Only the absolute configurations of C-l and C-5 remained undetermined, as the cis/trans configuration of C-8/C-9 double bond. Hinouye determined some years later [4], the absolute configuration of chiral centres of the secoiridoid oleuropein 1, relating 1 to the iridoid asperuloside 2. Bisdeoxy-acetyl asperuloside 3, prepared by hydrogenolysis from asperuloside 2, was opened by a series of oxidative steps to the two

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secoiridoid dimethyl esters 4a and 4b» having racemie C-8 centre with hydroxyl in both R (4a) and S (4b) configurations. Dehydration of these two secondary alcohols afforded two possible olefins: 5-ateohol 4b afforded the lf-olefin 5b, while Jl alcohol 4a gave the Z-olefin 5a.

Figure 2. Absolute configuration of Oleuropein 1

A simple work up on oleuropein 1, transesterification with methanol of ester function at C-7 afforded the J£-olefin 5b, so demonstrating the absolute configuration of the C-8/C-9 double bond, as well as all chiral centres of oleuropein 1. A similar approach was used for the partial synthesis of oleuropein 1 [5] that was depicted in Figure 3, The chiral starting product was a

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glucosidic iridoid, the bisdeoxy-asperuloside 6. The partial synthesis starts with the protection of compound 6 with benzyl groups that can easily be eliminated at the end of the synthesis in mild hydrogenolytic conditions that do not interfere with ester functions of oleuropein. After protection, compound 6 is osmilated to diol 7. COOCHj

COOCH, 3 !

COOCH,

RO

HO OH

Figure 3. Partial synthesis of oleuropein 1.

This last compound was selectively oxidised at the vicinal diol function with sodium periodate and successively with Jones's reagent, affording the desired acid 8. Direct esterification of compound 8 with dioxy-phenyl-ethanol 14, having phenolic functions protected with benzyl moieties, gave the key intermediate secoiridoid 9. The last step of the synthesis is the stereoselective reduction of 9 that afforded the alcohol 10

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as a single product, with the desired (S) absolute configuration at C-8 centre. The compound 10 was then dehydrated, giving the C-8/C-9 double bond with the correct configuration. The final removing of the protecting benzyl groups by hydrogenolysis afforded the oleuropein 1. Oleuropein 1 has been a recognised chemo-taxonomic marker of O. europaea; however its presence in olives is attributed to the ripening stage of fruits. Our recent experimental data were achieved for some Spanish, Portuguese and Italian cultivars, which were examined in different stages of ripening (green, cherry and black) [6,7]. These data confirmed the previously obtained results [8,9], with a decrease of oleuropein 1 during olive maturation and a contemporary increase of oleuropein derivatives. 2.1.2. Cornoside. Cornoside 11 is a glucoside that could be biogenetically related to hydroxy-tyrosol, the main phenolic component of O. europaea.

Figure 4. Cornoside 6

Its presence in olive was reported for the first time in 1993 by Scarpati et al. [10], who isolated 11 from leccino cultivar. Successively Bianco et al. demonstrated that cornoside 11 (Fig.4) constitutes, together with oleuropein 1, one of the main glucosidic phenolic components present in O. europaea [6,7]. Depending on the olive cultivar, the oleuropein 1 can be prevalent against the cornoside 11, or it could be the contrary, until reaching the equality [6,7]. Oleuropein 1 appears to be present in larger quantities in olives original of Italy and Greek (27-28%) compared to the quantities (18.521%) detected in olives of Spain and Portugal [7]. In addition, 1 appears to be the main glucosidic component of olives original of Italy ("Taggiasca" "Carolea" and "Cassanese" cultivars) and Greece ("Thasos" and "Conservolia" cultivars). The main glucosidic component of olives, originated from Spain and Portugal, appears to be, on the

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contrary, cornoside 6 (24.5% in "Hojiblanca" and 23.5% in "Douro" cultivars) [7]. The determination of the profile of the phenolic fraction was performed for the first time by Bianco et al. [7] by nuclear magnetic resonance of proton, on different olive cultivar samples, appropriately selected. Obviously, the proposed NMR determination of the phenolic profile is not an alternative to HPLC procedures, but constitutes a rapid, alternative methodology to examine the phenolic contents in relation to the main components. In fact, the sensibility of NMR technique does not evidence components that are present in quantities less than 5% of the total. Comoside 11 was first isolated from Forsythia genus of Oleaceae by Jensen et al. [11]. Its presence in O. europaea and its structure were demonstrated by spectroscopical methods, examining, in addition to NMR date of 11, date, of the compounds mixture also obtained by enzymatic hydrolysis of 11 (Fig. 5) [10].

Figure 5 Enzymatic hydrolysis of cornoside 11

The aglycon 12 of cornoside 11 is in fact in equilibrium with an addition product 13 that is known as rengyolone [12] or halleridone [13], and it seems to be a natural compound and not an extraction artefact. We consider comoside 11 to represent, together with oleuropein 1, the chemotaxonomic marker of oleaceae, being present in several species and always constituting the main glucosidic component [7], 2.1.3. Tyrosol and Hydroxy-tyrosol Glucosides. Tyrosol and hydroxy-tyrosol glucosides (Fig.6) constitute the principal part of minor phenolic components of olive. In fact, hydroxy-tyrosol 14 and tyrosol 18 are present in all parts of O. europaea. It seems that glucose links ail hydroxyls of phenol moiety without an evident

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preference. This can be related to the necessity of allowing a high hydro solubility to this phenolic fraction of olive, as in the general concept of glycosylation in natural products. Hydroxy-tyrosol glucosides were isolated from different olive cultivars, and their relative quantities seem to be in relation with the olive organ considered [14]. 1 OH

14 OH HO

OH

OH

Figure 6. Hydroxy-tyrosol glucosides

Compound 15 was first identified in Osmanthus asiaticus, a plant of Oleaceae family [15]. Compound 16 was isolated in Primus grayana, a plant of rosaceae family [16] and in Ricciocarpus natans, Ricciaceae [17]. Compound 17 was assumed to be present in O. europaea [18,19], on the basis of chromatographic considerations and was identified [14], together with 15 and 16[14], in carolea cultivar. Structures of compounds 15-17 were determined by spectroscopical method, essentially 'H- and 13C-NMR spectroscopy. Comparison between 13 C-NMR data of glucosides 15-17 and hydroxy-tyrosol allowed to determine the site of glucosylation and therefore the structure of these compounds. Tirosol glucosides (Fig. 7) are also present as minor components in olive, but their relative quantities do not seem related to the olive organ considered [20].

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Compound 19 is also known as salidroside. The isolation of salidroside from O. europaea had been already described [21-22] but needed an unambiguous identification.

Figure!. Tyrosolglucosides

The structure of 19 was determined by proton and carbon magnetic resonance experiments. 'H-NMR and I3C-NMR spectra unambiguously determined the nature of the aglyconic and the glucosidic parts of the molecule and the relative positions of glucose moiety. In particular, the 'H-NMR spectrum revealed, besides the aromatic protons resonances in the range 6.69-7.15 ppm, the -CH2-CH2- methylene part as triplets centred at 2.80 and 3.75 ppm. Moreover, the spectrum showed a doublet at 4.38 ppm due to the anomeric proton of glucose moiety that produces resonance signal in the range 3.10-3.70 ppm. The site of glycosylation was determined, comparing the 13C-NMR spectrum of compound 19 to the tyrosol one. In the 13C-NMR spectrum of 19, the deshielding of the primary alcoholic function (about 10 ppm) and the shielding of the C-lp (about 1 ppm) typical of the glycosidation effect were noticed. Compound 20 [23] was also detected in comparable quantities with respect to 19, and its structure was determined, as did for 19, by spectroscopical methods. 2.1.4. Oleuropein Related Compounds. Several minor oleuropein related compounds were isolated in olive, reported in Figure 8. Compound 21, demethyl-oleuropein, is a hydrolysis product of conjugated methyl ester present in oleuropein. The presence of this compound increases during the olive ripening [6-9]. Compound 22, ligstroside, is very similar to oleuropein from which it differs for the presence of a tyrosol unit instead of hydroxy-tyrosol [24].

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Compound 23, oleoside (see Figure 9), is the dimethyl ester, corresponding to oleuropein, where methyl alcohol esterifies the carboxyl group at C-7 also [25]. COOMe HO

XJ

COOH

l

4

21

OH

Figure 8. Oleuropein related compounds

In compound 24, oleuroside, a modification at the level of monoterpenoid unit is present. In fact, the C-8/C-9 double bond of oleuropein is shifted to C-8/C-10 position, as in the secologanin. The occurrence of 24 in O. europaea seems to be in contrast with the biogenetic trend of this plant that appears to be devoted to the a molecular structure that was classified as oleuropein-type [26,27]. MeO

COOMe

HO

0

Y ° COOMe

HO

23

24

Figure 9. Oleuropein related compounds (continue)

There are also some non-glucosidic iridoids related to oleuropein and have been isolated from O. europaea. Two of them are aldehydes 25 and 26 (see Figure 10) that probably arise from the hydrolysis of the

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glucosidic moiety of oleuropein with a subsequent rearrangement of the obtained aglycone [28]. OOCH3 25 R=H R'=CH3 26 R=CH3 R'=H

Figure 10. Oleuropein related compounds (continue)

Two other non-glycosidic secoiridoids (see Figure 11) have been isolated from O. europaea. Compound 27 is the elenolic acid, described by Panizzi et al. [2], whereas compound 28 is its methyl ester whose total synthesis was accomplished by MacKellar et al [29], so demonstrating the absolute configuration of these two compounds. OOCH3 27 R=OH

OHC'X 6

28R=OCH3

H3O 'H Figure 11. Oleuropein related compounds (continue)

The o-diphenolic compound 29 (Figure 12) was obtained from ripe black olives by Scarpati et al. [30] and successively reisolated by PaivaMartinsetal. [31].

29 Figure 11 Oleuropein related compounds (continue)

These authors demonstrated that in unripe green olives, oleuropein 1 is present as the major o-diphenolic compound, while in ripe olives, demethyloleuropein 21 predominates. Both these glucosides disappear from olive juice, as they are hydrolysed by native P-glucosidases.

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Demethyloleuropein aglycon rapidly eliminated the carboxyl group, giving 29. 2.1.5. Tyrosol Related Compounds and Other Phenolics. Recently, the major C6-C2 phenolic compounds found in the vegetation water of fruits of O. europaea, Dritta and Cipressino cultivars, were 4-hydroxyphenylethanol 18 and 3,4-dihydroxy-phenylethanol 14, A further hydroxylated compound, the 3,4-dihydroxyphenylglyeol 30 appears to be the 2-hydroxy-derivative of 14. This latter compound (see Figure 13), the formal metabolite of norepinephrine, is first reported as a major component of the olive phenolic fraction by Bianchi et al [32].

30

Figure 13. 2-hydroxy-tyrosol

Phenolic fraction of O. europaea is also constituted by lignans and related compounds. Recently, two new lignan glucosides, (+)-l-acetoxypinoresinol 4"-Me ether 4'-(3-D-glucoside and (+)-l-hydro3y-pinoresinol 4'-P-D-glucoside, together with 2 known glucosides, (+)-acetoxypinoresinol 4-P-D-glucoside and esculin, have been isolated from the bark of O. europaea africana [33]. Flavonoids are also present. De Laurentis et al. [34] isolated and identified a series of flavonoid compounds from the dried leaves of blooming cultivars of O. europaea: hesperidin, rutin, luteolin-7-Oglucoside, apigenin, apigenin-7-O-glucoside, quercetin, kaempferol 2.1.6. Oleuropeic Acid and Oleuropeil Saccharose. In the early researches on O .europaea, [35], Panizzi demonstrated that oleuropeic acid 31, the 4-(l.hydroxyisopropyl)-l-cycloexene-l-carboxylic acid, occurs in the root bark of 0. europaea, mainly as a sucrose ester, i.e. the 6-O-oleuropeil saccharose 32 (see Figure 14). The structure of 31 was determined by comparison with an authentic sample [36-38]. The demonstration of the position of ester linkage in oleuropeil saccharose 32 was achieved with a combination of enzymatic and chemical reactions [39].

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K>i°

HO

32

31

HO

OH

Figure 14 Oleuropeic acid and oleuropeil saccharose

Alkaline hydrolysis of 32 afforded saccharose and oteuropeic acid, indicating the nature of the glycosidic moiety of 32. Selective hydrolysis of interglycosidic linkage of saccharose with invertase allowed to isolate free fructose together with glucose esterified with oleuropeic acid The esterification site was revealed through periodate oxidation and confirmed by NMR data that showed the classical esterification downfield shin for the protons geminal to the primary alcoholic function of glucose. 2,1.7. Verhascoside. Verbascoside 33, whose structure is reported in Figure 15, is present in O. europaea, as in other oleaceae, demonstrating the ubiquous occurrence of this compound in nature [40]. J3H

33 HO Figure 15. Verbascoside

Verbascoside revealed also a significant antioxidant activity in vitro, indicating that this compound boosts the antioxidant activity of O. ewopaea derivatives.

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2.1.8. Phenolic Esters, The less polar fraction of O .europaea appears to be constituted by phenols esterified by a fat acid moiety, Esterified phenols are not completely soluble in water medium where they give rise to a micellar phase. The main and first component isolated from this fraction resulted to be the oleil ester of tyrosol 34, shown in Figure 16.

Figure 16.1-oleoyl-tyrosol

The structure of 1-oleyltyrosol [41] was determined by simple chemical and spectroscopic methods. The nature of the fatty acid moiety was deduced, besides spectroscopic considerations, by alkaline hydrolysis that allowed to isolate oleic acid. Alkaline hydrolysis allowed to isolate also tyrosol thereby determining the phenolic component The site of esterification was determined by analysing of the 'H-NMR spectrum of this phenolic ester, in which the usual deshielding of about 1 ppm of primary alcoholic protons appeared to give a triplet at 6 4.60; the 13 C-NMR spectrum also shows a deshielding of a carbon of the primary alcoholic function of tyrosol of about 2 ppm typical of esterification

effect 2.2. Molecular Modification Give the molecular composition of the olive's polar fraction, there began an investigation of the molecular modifications of these compounds. Also because this chemical problem is strictly linked to that of the production of the oil and of other foods derived from olive. We in fact know mat the olive is the key-plant in the agrifood system of Mediterranean region countries. Now-a-days the olive oil is a functional food, because it contains not only the fat material, but also high quality compounds, such as phenols. And these compounds come out

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from a complex sequence of chemical reactions that we began to analyse step by step. The particular aspect of the olive oil is the extraction procedure from fruits of the olive. Even if this procedure has undergone several technological innovations, its ground scheme is always the same. We can distinguish three different phases of this extraction: the first one consists in the pounding of the olive; the scutching follows, it consists in making a mix of the olive's paste with water for a variable time (generally for half an hour) at 30-40 degrees. The last step is the separation, with different methods, generally by pressure, of liquid phase from solid residue, and successively of the oil phase from the water phase. This allows to realise a series of several enzymatic reactions. In the first fragmentation's phase of the olives, all the substances present in the fruit are together with the available enzymes. Efficient hydrolases exist in the olive that cleave both glycosidic and ester bonds, letting reactive structures to be free, as the 1,5-dialdehydic functions, inside the oleuropein structure. We observed quickly the hydrolysis of oleuropein, making an NMR study, consisted in putting some drops of the liquid obtained by cutting a ripe olive. On the contrary, in the second phase, the scutching, non-enzymatically catalysed reactions are prevalent, essentially acid catalyses, because in the pounding step, hydrolysis of ester functions produced free carboxylic groups. Last step of olive oil preparation is similar to the process of phase separation from the solid and liquid parts in the first section, and successively from the fat and aqueous phases. These separations are not very accurate and we obtain a product, the olive oil, saving little quantities of water that transfer the phenolic fraction from the olive to the oil. The study of these reactions allowed to acquire a series of data on the chemical modification of oleuropein that is particularly rich in functional groups, and of phenol present in O. europaea. 2.2.1. Chemical Modification of Oleuropein. Enzymatic hydrolysis of glucosidic function allows to free aglycon that exists in different forms, which we revealed by following the hydrolysis of oleuropein 1 in NMR tube [42-43]. The hemiacetalic 35a structure (see Figure 17) is formed as a consequence of the hydrolysis of glucose that appeared to be in equilibrium with the dihaldeidic structure 35b. In the

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same hydrolytic medium, the hydration of the C-3 formyl occurred giving rise to a compound 35c.

~V^

CHO 35b

OH

CHO 35c

OH

25/26

Figure 17. Oleuropein aglycon rearrangements

Aglycon 35 gives rise also to rearrangement products; the more important ones are 25-26 that are derived by the addition of the enolic form of aglycon to the exocyclic double bond. Gariboldi et al. [28], however, seem to demonstrate in our paper that these compounds are natural ones, deriving from a biogenetic pathway as that followed by Bianco et al [42-43] by enzymatic hydrolysis in NMR tube. After the hydrolysis of esters functions, the hydrolysed aglycons 36a and 37a are, as above, in equilibrium with the dihaldeidic forms, 36b and 37b. A series of non-enzymatic reactions may be observed, as the decarboxylation of the beta-carbonyl acid, with the formation of the decarboxylated aglycon 38a in equilibrium with 38b, (see Figure 18).

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COOH

COOH ^"CHO

^_

CHO

CHO 38a

OH

38b

^CHO

37b

Figure 18. Oleuropein aglycon rearrangements (continue)

Figure 18 shows compounds that are transferred in the oil. Some people insists on searching oleuropein in the oil, cutting of compounds that are more abundant, thinking that oil preparation should be a simple transfer of compounds from olive to oil, instead of a complex series of enzymatic and non-enzymatic reactions. The presence of the various forms of oleuropein aglycon was demonstrated by their isolation from olive oil [42- 43,23]. 2 2 . 2 Chemical Modification ofCornoside. A simple transformation undergoes the cornoside 11 (see Fig 19) that consists in the formation of aglycone 12, followed by two different rearrangements [10,23]. HO /

\ HO

Figure 19. Cornoside rearrangements

This usually happens in mild acid conditions. These two rearrangements are: the first one is hydroxy tyrosol 14, that arises from a

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transposition of tertiary hydroxyl and the second one is halleridone 13, that derives from the addition of a primary alcoholic function to the unsaturated carbonyl function. These reactions were observed in NMR tube. 3. BIOLOGICAL ACTIVITY AND PHARMACOLOGY OF OLEA

EUROPAEA Epidemiological studies in the Mediterranean region evidenced that a diet rich in grain, legumes, fruits and vegetables, wine and olive oil has beneficial effects on human health. In fact, these foods are rich in antioxidant vitamins, flavonoids and polyphenols that play an important role in prevention against cancer and coronary heart disease. It has been now recognised that the phenolic profile of the foods, along with high intakes of the monounsaturated fatty acids, as oleic acid mainly, confers its health-promoting properties to the Mediterranean diet. In fact, olive oil is the main source of unsaturated acids and polyphenols that constitute a complex mixture in olive fruits and in its derived products. In addition, in O. europaea fruits, phenolic profile and content are important factors to consider in order to evaluate virgin olive oil quality. To remain that they are also partly responsible for autoxidation stability and organoleptic characteristics of olive oil. Elevated use of extra-virgin olive oils, which are particularly rich in these phenolic antioxidants, as well as squalene, oleic and other unsaturated acids, should afford significant protection against cancer (colon, skin, breast), coronary heart disease and ageing by inhibiting oxidative stress [44]. In Europe, epidemiological data demonstrated that mortality from breast and colorectal cancer is considerable by lower in countries where olive oil consumption is high (such as Greece, Italy and Spain) than in those where the consumption is low (such as Scotland, England and Denmark) [45]. Pharmacological studies were mainly focussed on oleuropein that represents the key phenolic compound in O. europaea, and on related phenolic compounds such as tyrosol and hydroxy-tyrosol. Therefore, the following discussion will be focussed on reported biological activity of these compounds.

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Oleuropein and hydroxy-tyrosol, derived from oleuropein hydrolysis [46], possess several biological properties certainly mediated by their antioxidant and tree radical-scavenging ability (antimicrobial, hypotensive, vasodilatator and hypoglycaemic activities) [47]. 3.1 Antioxidative and Radical Scavenging Effects of Olive Biphenols In the last years, natural products have been used as antioxidative, melanogenesis inhibitors and sunscreen [48]. Lipid peroxidation is related to aging, membrane damage, heart disease, stroke and cancer in living organism. This oxidative mechanism could be stopped by the addition of synthetic anti-oxidants, but now it has been recognised that natural antioxidants are safe compared to the synthetic compounds [49]. Well-known natural anti-oxidants are represented by ubiquinones, tocopherols and related compounds, flavonoids, cinnamic acid derivatives, Hcopene and related tetraterpenoids, and also by phenolic compounds [50]. The antioxidative effect of oleuropein and hydroxy-tyrosol (see Fig 20) was investigated by Saija et al. [51], in a model system consisting of dipalmitoyl-phosphatidyl-choline/linoleic acid unilamellar vesicles and a water-soluble azo-compound as a free radical generator.

OLEUROPEIN

^ , ^7\L0H

HYDROXYTYROSOL

Figure 20. Oleuropein and hydroxy-tyrosol

This model system studies the antioxidant potential against the attack of oxygen radicals on biomembranes from aqueous phase [52]. Antioxidant effects of olive phenols depend on their interaction with model membranes [53], e.g., oleuropein interacts with DMPC (dimyristoyl-phosphatidyl-choline) membranes. Oleuropein contains a sugar moiety needed to prevent drug access to lipid membranes [54]. In

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fact, lipophilicity of drugs is evidently related to their incorporation with lipids in the model membranes. The interaction observed between oleuropein and DMPC liposomes may be due to the introduction of lipophilic molecules into the ordered structure of the lipid bilayer [55].

Figure 21. lipid bilayer

Drug molecules act as a spacer in such a structure causing a destabilisation of the lipid mosaic. The modification of the fluidity of the model membrane is an important factor for cell membranes functions (Figure 21). Oleuropein appears to interfere with some biological processes such as lipoprotein oxidation, platelet aggregation, platelet and leukocyte eicosanoid production and cardiovascular control too. As previously described, oleuropein and hydroxy-tyrosol are characterised by a catechol moiety that appears to be needed for their scavenger and antioxidant activities. In fact, it was demonstrated that these compounds prevent thermally initiated autoxidation of methyl linoleate in homogenous solutions [56], protect LDL from oxidation [57] and inhibit production of

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isoprostanes and other markers of lipid peroxidation, occurring during LDL oxidation [58]. The free radical-scavenging capacity of oleuropein and hydroxy-tyrosol was tested, using some radical generators. As a radical initiator, AAPH (2,2'-azobis (2-amidinopropane)-hydrochloride) was used for peroxyl radicals generation, because it is a common free radical found in the body [59] and has often been used in several antioxidant activity assays [60]. It appears to be slightly less reactive than OH-radical [61]. R-+O 2

•

ROO • +LH

• ROOH + L •

L +O2

-LOO-

LOO-+LH

• LOOH + L •

2 LOOLOO-+InH LOO- +In R-N=N -R

—

•

ROO •

N o radical products

• LOOH + In _^ No radical products •

? p -

Table 1. R-N=N-R radical initiator, LH linoleic acid, L linoleic radical, LOO linoleic peroxy radical, InH inhibitor.

Other experimental studies carried out with a different radical, DPPH {l,l-diphenyl-2-picrylhydrazyl radical), revealed that oleuropein and hydroxy-tyrosol elicit a good, concentration-dependent, scavenging effect. Incubation of DPPC/LA {dipalmitoylphosphatidylcholine/linoleic acid) LUV with AAPH increased the accumulation of LOOH (linoleic peroxy acid) formed from LA peroxidation. When the tested biphenols were added, a reduction in the amount of LOOH formed was observed and thus oleuropein proved more effective than hydroxy-tyrosol [62]. The cited works demonstrated that oleuropein and hydroxy-tyrosol are potent antioxidants against lipid peroxidation in phospholipid bilayers, induced by aqueous oxygen radicals. These results may be very interesting, because biphenols could have important applications in human diseases caused by free radical damage. Polyphenols are clearly present in the technological products of olive, such as olive oil. The phenolic contents are related to the peculiar procedure of oil production. In fact, this manufacturing process consists in

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traditional steps that began with washing the olives to remove dirt and other rubbish adhered to the fruit. Then, olives are crushed and the juice is homogenised before pressing. Olive oil is produced under pressures and successive filtration to separate oil from water. After centrifugation, extravirgin olive oil (VOQ) is obtained. High quality oils are bottled directly, but low quality oils (high acidity) are processed once more to obtain refined virgin oil (RVO). Finally, the oil extracted from the residual juice or husk with organic solvents, such as hexane, yields a low-quality refined husk oil (RHO). Several experimental data supported that VOQ contains a higher concentration of three phenolic antioxidants classes, simple phenols, secoiridoids and lignans, and squalene than RVO and seed oils. For these reasons, in the Mediterranean region, where olive oil is an essential constituent of the diet, there is a lower incidence of cancer and heart disease [63]. Probably olive oil components may reduce oxidative stress via inhibition of lipid peroxidation, an interesting mechanism responsible for diseases such as cancer, heart ailment and ageing. The soluble fat should have chemopreventive effects against breast cancer and other diseases. Recent epidemiological studies demonstrated that the components of dietary olive oil might have an important role in the disease prevention, over all against breast and colorectal cancer development [64]. Therefore, it is necessary to establish the olive oil components that are responsible for their protective effects. Once again, there is a significant difference in the concentration of phenolic compounds between VOQ and RVO. Secoiridoids in olive oils is higher in VOQ than in RVO and the concentration of lignans is higher in VOQ than in RVO. The antioxidant effects of natural phenols in olive oils was studied by Papadopoulos [46], adding extracts of a VOQ, having polyphenols, to a sophisticated bleaching oil, washed-out of phenolic antioxidants and evidenced a significant inhibition of auto-oxidation over time in comparison with samples without such an addition. On the other hand, it has revealed that this effect is more pronounced in hydroxy-tyrosol than caffeic acid and protocatechuic acid Reactive oxygen species are associated to xanthme/ipoxanthine system. In fact, the oxidative stress caused by hydrogen peroxide and xanthine oxidase is repressed in the presence of hydroxy-tyrosol, though, unexpectedly, it was inactive in concentrations under 500 umol/L. The

882

antioxidant effect of hydroxy-tyrosol is probably due to a dihydroxy substituted phenol ring [65]. Xanthine is one of our organism amino acids and may be catabolised by the xanthine oxidase. This enzyme can attack the amino acid in artery walls so that cholesterol could deposit to heal the lesions. Using the hypoxanthine/xanthine oxidase model to generate reactive oxygen species and comparing VOQ and RVO oils with seed oils, it has been evidenced that scavenging of the hydroxyl radical was higher in olive oil extracts than those of seed oils. In addition, the samples of studied olive oils are also found to be potent inhibitors of xanthine oxidase activity. It is important to underline that phenolic compounds purified from olive oil have a chief antioxidant power than vitamin E and dimethylsulphoxide, standard free radical scavengers both in vitro and in vivo. CO OH .OH

m' 2,5-DHBA

URIC ACID Figure 22. Salicylic acid and its hydroxylated xanthine/hypoxanthine oxidase system.

metabolites produced

by

The studies carried out by Owen [65] revealed not only the difference between the oils in the phenolic fraction composition, but also the degree of diphenol (2,5-dihydroxybenzoic acid and 2,3-dihydroxybenzoic acid)

883

produced by hydroxyl radical attack on salicylic acid. This experiment was executed monitoring the concentration of each phenolic component, the hydroxylation of hypoxanthine and at last the hydroxylation of salicylic acid. Thus, the olive oil samples analysed were dissolved in a phosphate buffer and the end products of the enzyme or free radical reactions were quantitated (see Figure 22). In the olive oil extract, especially in VOQ, lignans were also identified. Experiments carried out on animals have been shown to inhibit cell growth in cancer of skin, colon, and breast. Probably, the lignans anticancer activity is due to the structural similarities with oestradiol and the synthetic antioestrogen tamoxifen; as a result, the lignans have been evidenced to inhibit MCF-7 human breast carcinoma cells increase, induced by oestradiol. Other studies have revealed the squalene existence in the olive oils. The activity of this compound is resulted one of the principal protective agents against skin cancer, probably due to the scavenging singlet oxygen generated by ultraviolet light [66]. These results are confirmed by studies that show a lower incidence of this neoplasm in the Mediterranean population. Furthermore, olive cake, the material left after compression of the fruits, contains triglycerides, oleanane derivatives and some phenolic compounds. Using BHT (butylated hydroxytoluene), ascorbic acid and a-tocopherol as standards for comparison, Amro et al [67] carried out some experimental tests to identify the olive compounds responsible for antioxidant and radical scavenging activities. After a testing period, fraction containing oleuropein showed a significant antioxidant activity, even if the auto-oxidation of the representative fraction of material cake of tested olive was less than those treated with BHT [67]. Moreover, reducing power of olive compounds contained in the examined fractions was greater compared to ascorbic acid, which is known as a good reducing agent. In this case, only fractions containing ferulic acid, cinnamic acid and caffeic acid showed a reducing power higher than the ascorbic acid, because these compounds may act as electron donors and react with free radicals to convert them into more stable products. Table 2 contains the IC5o values of the compounds isolated from olive cake butanol extract and their scavenging effects. The IC» values of BHT, ascorbic acid and a-tocopherol are 37,0 ug/ml, 28,4 ug/ml and 34,2 ug/ml, respectively.

884

Protocatechuic acid

ICso 27ug/ml

Coumaric acid Ferulic acid Cinnamic acid Caffeic acid

IC50 135ug/ml IC50 4,5ug/ml IC50 7 ug/ml ICs, 4,5 ug/ml

Oleuropein

IC50 25 ug/ml

Table 2 ICm of phenolic component ofO. Europaea

These results showed that oleuropein has a good antioxidative activity. The beneficial health effects of Mediterranean diet are probably related to non-nutrient components present in foods, such as olive oil, as well as wine and others. In fact, oleuropein and hydroxy-tyrosol, both green and black tea components and gallic acid inhibit androstenedione 6-phydroxylase activity. This enzyme is a CYP3A marker of human liver microsomes and has an important role in the metabolism of xenobiotic substrates [68]. Metabolic reactions occur in the hepatic microsomial cytochromes P450 (CYP) that represent an important source of oxygen radicals, such as superoxide anions, O2", and hydrogen peroxide, H2Q2. In feet, more enzymes needed for chemical compounds metabolism are in the lipophilic membrane of the hepatic reticuloendoplasmatical system. Microsomial oxidation reactions of chemical and natural products occur in an interesting enzymatic system that includes cytochrome P450, cytochrome P450 reductase, NADPH and O2. Cytochrome P450 oxidation cycle has been described in the following Figure 23. Enzymes of cytochrome P450 are haem-proteins related to NADPHcytochrome P450 reductase. All these biological factors are needed for the oxidative reactions. CYP1, CYP2, and CYP3 constitute the P450 enzyme family. Assays carried out in human and in rat liver microsomes by some olive oil phenols demonstrated inhibition of cytochrome P450 activity, specifically of CYP3A and CYP2C11 markers and of reactive species generation.

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Figure 23. Oxidative reactions of cytochrome P4S0

In human liver microsomes, it was evidenced that inhibitors of reactive oxygen species generation are, sequentially, gallic acid, hydroxy-tyrosol and 3,4-dihydroxyphenylacetic acid; while, in rat liver microsomes, the most potent inhibitors were, in the growing order, gallic acid, caffeic acid, pyrogallol, oleuropein, 3,4-dihydroxyphenylacetic acid and hydroxytyrosol. The presence of phenolic hydroxyl groups, for the scavenging radical activity, seems very important, which 3,4-dimethoxyphenethyl alcohol does not for DPPH scavenging activity. For these reasons, a classification of antioxidants has been done: those that inhibit the generation of reactive oxygen species, and those that scavenge the reactive oxygen species generated. Some results evidenced that human CYP3A have a very good capacity to generate reactive oxygen species; therefore, there were interesting results in examining the effects of dietary phenols, such as oleuropein and hydroxy-tyrosol, known CYP3A inhibitors [69].

886

However, it is chiefly underlined that olive oil phenols, such as hydroxy-tyrosol, and others food-derived compounds, such as gallic acid, work as free radical scavengers. Recently, free radicals have been correlated to several diseases for human health. Hydroxy-tyrosol (DPE: 3,4-dihydroxyphenylethanol) is a liposoluble and hydrosoluble compound present in high concentration in extra virgin olive oil, in free or esterified form, such as oleuropein aglycone [42-43]. Several chemical and epidemiological studies confirmed that olive oil's beneficial effects are related to high concentration of oleic acid and the presence of vitamin and non-vitamin antioxidants, such as DPE. This compound shows different biological actions; one of them is the inhibitory effect on peroxynitrite dependent DNA base modification and tyrosine nitration. Furthermore, DPE counteracts cytotoxicity, caused by reactive oxygen species, ROS, in Caco-2 cells and in erythrocytes [70]. In particular, the Caco-2 cells imitate, in vitro, the food-intestinal tract interaction. In fact, these cells have often been used in laboratories to study the molecular mechanism of DPE intestinal transport. To evaluate DPE transepithelial transport cultured Caco-2 cells were used. In this system, cells grow and differentiate on a membrane of polycarbonate, in which the luminal part of the polarised epithelium is oriented. During the experiment, adding [14C] DPE to the compartment, a transepithelial flux of DPE was observed. Moreover, other data suggest that DPE is quantitatively absorbed following its oral administration. The only characterised DPE metabolite was the HMPE (3-hydroxy-4-methoxyphenylethanol), a methylated product of intestinal COMT (catechol-O-methyltransferase) activity. In vitro, DPE, in the presence of a methyl donor S-adenosylmethionine and purified COMT from porcine liver, has a Km lower than that of endogenous substrate, such as dopamine. For these reasons, DPE may be a favoured substrate for COMT in vivo. The obtained data revealed, first of all, the high ease of use of DPE and the relationship between nutritional positive effects of olive oil and the high content of DPE and its precursor, oleuropein aglycone [71].

887

3.2 Effects of Oleuropein on Gastric Mucosae Oleuropein, the major active substance contained in O. europaea, was shown to inhibit prostaglandins biosynthesis and release (see Figure 24). On the other hand, the drug possesses anti-inflammatory action and very interesting effects on gastric mucosae [72]. Prostaglandins (PG) belong to eicosanoid family of poly-unsaturated fatty acids oxygenated products. These compounds show great biological activity; in fact, prostaglandins are chemical mediators that are released during allergic and inflammatory processes. PG, in particular PGE, inhibits the gastric acid secretion. In fact, PGE and its analogues reflect a pronounced protection against steroids and NSAIDs-induced (non-steroidal anti-inflammatory drugs) gastric ulcers. Acetylsalicylic acid (ASA), the pioneer of NSAIDs group, is distinguished from others, as it blocks COX (cyclooxygenase) with irreversible acethylation reaction (Figure 24). Acetate

ASA

^JX (attive)

Acethytated COX (inattive)

Figure 24 Cox's acetylation by ASA

PGE2 and PGF2a stimulate synthesis of protective mucus in stomach and in small intestine. When ASA is administrated, prostaglandins and prostacyclins synthesis is blocked and acid secretion is increased, reducing mucosae protection. COX has two different isoforms: COX-1, expressed in most tissues and implicated in the regulation of normal homeostatic functions such as gastric acid secretion; and COX-2, induced often by inflammatory processes. As a result, COX-2 is implicated in the production of pro-inflammatory eicosanoids (see Figure 25). In fact, one of the therapeutic actions of ASA et similia NSAIDs has been the inhibition of COX activity to reduce pro-inflammatory eicosanoids [73].

888

Arachidonic acid

OOH PGG,

* PGEsmthase

PGE, , PGE S-dietoredutase

COOH

Figure 25. Prostaglandins and tromboxans biosynthesis

In one of the test animals groups, ASA, in 1% carboxymethylcellulose (CMC) suspension, was administrated orally at 150 mg/Kg/24h. In three other test groups, O. europaea extract was given respectively at 25, 50 and 100 mg/Kg/24h one hour before ASA ingestion. The treatment lasted for three days (see Table 3). During this period, they were only allowed to take water. Twenty four hours after the last treatment, all animals were sacrificed and their stomachs removed to evaluate the ulcers developed. Histologic examination has shown that most of the recorded lesions were antral gastritis, hypertrophic gastritis and fundus cystic dilatation. In all these cases, inflammatory signs have been noted, but pre-treatment with O. europaea extract resulted in preventing the lesions induced by ASA.

889

Treatment

Number rats

of Mean scores

% of rats

Ulcer index

presenting ulcers

ASA 150 mg/Kg/24h. for 3

10

1,3

70

91

10

0,8

60

48

10

0,6

60

36

10

0,6

50

30

days ASA 150 mg/Kg/24h. for 3 days + Olea eur. 25 mg/Kg/24h 50 mg/Kg/24h 100 mg/Kg/24h

Table 3. Results of digestive tolerance study

This effect doesn't seem to be dose dependent, especially in animal groups treated by 25 and 50 mg/Kg/24h of the extract, where histologic examination respectively showed 5 and 6 regeneration signs out of 10 surface of epithelium. Probably, this fact corresponds to the activation of epithelium cells multiplication. In group treated with higher doses (100mg/Kg/24h), histologic study indicated a normal mucosae. Since oleuropein was demonstrated to antagonise acetylcholine and E2 prostaglandin's actions on smooth muscle, the anti-inflammatory activity of O. europaea extract was investigated. Moreover, as compounds with oleanic structure, contained in O. europaea leaf, exert anti-ulcer activities, so they were studied for their effect on the rat gastric mucosae. 3.3 Effects of Olive Components on Glycaemic and Blood Pressure Controls The effects of oleuropein on NO release in cell culture and its activity on nitric oxide synthase (iNOS) were also studied. NO (nitric oxide) is a reactive free radical, characterised by numerous important biological functions. Nitric oxide is also an important chemical mediator for vertebrates [74].

890 NH,

NHOH NADPH *O,

NH3 ARGINYL

,

NADP+

XJ.—„ NOS A

L-NMMA

NH 3 N-0-HYDRO XYARGINYL

NOS

NO

CITRULLINE

NITRIC OXIDE

Vasodllatation Immunity regolation Inhibition of platelet aggregation

Figure 26. Nitric oxide activity

NO is both cytostatic and cytotoxic for some pathogens, such as Plasmodium falciparum, Schistosoma mansoni, Leishmania major and Toxoplasma gondii [75]. It is generated by nitric oxide synthase that, in murine macrophages, is induced by LPS (lipopolysaccharide). During incubation of murine macrophagic cells line with LPS and oleuropein, there was a marked production of nitrites. In fact, some reported data show that oleuropein increases the response of macrophages to bacterial lipopolysaccharides, probably due to an increase in iNOS activity. Oleuropein activity on NO production is very interesting and may resolve some pathological problems such as platelet aggregation, thrombosis, vasorelaxation, etc [76]. In fact, NO exerts several effects on cardiovascular system, exhibits vasorelaxant activities and reacts with superoxide to form peroxynitrite (see Figure 26). Flavonoids, phenolic compounds occurring ubiquitously in vascular plants, show a broad spectrum of pharmacological effects. There are a

891

number of medicinal plants used for their anti-inflammatory, spasmolytic, anti-carcinogenic, vessel stabilising or diuretic actions. It is well known that flavonoids interfere with several enzymes, such as adenosine deaminase, cAMP phosphodiesterase, trypsin or cytosolic amino peptidase. There was carried out a screening of flavonoids, phenylacrylic acids and various hydroxylated phenyl acetic acids, urinary metabolites of flavonoids, against three metallopeptidases, containing Zn as cofactor, to study their mechanism of activity in vitro [77]. These ectoenzymes, neutral endopeptidase (NEP), angiotensinconverting enzyme (ACE) and amino peptidase N (APN) are located at the outer membrane of different cells, especially in mammal atrium, are endowed with natriuretic and diuretic properties, and are capable to release the vassal musculature. APN is constituted by 151 amino acids and is synthesized in the cardiac atrial cells; the atrial straining seems to be the most important factor entangled in ANP liberation. This peptide increases in some pathological conditions, such as congested heart failure, renal failure and insufficient ADH secretion. GIU386

Glu-352

Hta-3«5

J

^

Tyr-471

Figure 27. ANP (amino peptidase N).

In fact, ANP increases elimination of Na and urinary flux, glomerular filtering, thus inhibits the secretion of renin, vasopressin and aldosteron. These actions cause an arterial pressure reduction (see Figure 27).

892

NEP inactivates a series of renal and CNS-active peptides, such as substance P, bradykinin, enkephalins and atrial natriuretic factor. It has been well underlined that inhibitors of NEP should be useful in the treatment of pain because of having a large spectrum of activities similar to that of opioid analgesics. In fact, inhibitors of NEP protect the endogenous atrial natriuretic factor degradation and enhance the typical renal effects of ANF on diuresis and natriuretic response (see Figure 28). Converting enzyme is a dipeptidyl-carboxypeptidase that catalyses dipeptide separation from carboxylic terminal of a series of peptides. Its important substrates are angiotensin I that is transformed in angiotensin II, and bradykinin that is inactive (see Figure 28). Angiotensinogen

Kininogen jRcnin

t-PA

/

Kallikrcin/

Angiotensin I *

\ CAGE

* Bradykinin

I

~

Angiotensin II

AT,

receptor

•Vamconttrictioa •EmtoUwlin production

—

[ Degradation products

A T 2 receptor

Bj receptor

B 2 receptor

•V»««

LIVER

Figure 29. Triglyceride (TG) absorption transport and metabolism.

As reported in the Figure 29, fat is absorbed and packaged as chylomicrons (CM), and lipoprotein lipase (LPL) releases TG to yield the CM remnants. Very low density lipoproteins (VLDL) synthesised by the liver are broken down by LPL to yield intermediate density lipoprotein (DDL) and ultimately low-density lipoprotein (LDL). High-density lipoprotein clears cholesterol from the cell through lecithin-cholesterol acyltransferase (LCAT). 3.4 Antimicrobial Activity Oleuropein, the secoiridoid responsible for the bitter taste of olives, was studied in vitro for its antimicrobial activity. Recently, it was investigated against Mycoplasma hominis, Mycoplasma fermentas, Mycoplasma pneumoniae, Mycoplasma pirum [87]. O. europaea is a plant resistant to microbe and insectan attack. In particular, oleuropein has been shown to inhibit or stop the growth time of a serial of bacteria and microfungi [88]. In general, this compound is used as a food additive.

896

Oleuropein and its aglycone inhibit Lactobacillus plantarum, Pseudomonas fragi, Staphylococcus carnosus, Enterococcus faecalis, Bacillus cereus, Salmonella enteritidis and, moreover, a serial of fungi [89]. The phenolic compounds act on the exoprotein secretion of the Staphylococcus aureus too [90]. In vitro, there has been observed the possible antimicrobial activity of oleuropein against human pathogenic bacteria. Moreover, the phenolic compound also confirmed its action on gram-positive and gram-negative bacterial strains such as Salmonella spp., Vibrio spp. and Staphylococcus aureus [91]. Experimental data indicated that M. pneumoniae, M hominis, Mfermentans and M. pirum are vulnerable to oleuropein [92]. Phenolic compounds, indicated as surface-active agents, are able to denature microbial proteins and are generally harmful [93]. These moieties damage the cell membranes or the cell peptidoglycan, causing escape of cytoplasmic constituents such as proteins, glutamate or potassium and phosphate ions [94]. In fact, bacterial cell is characterised by three important structures: cellular wall, cytoplasmic membrane and cytoplasm (Figure 30).

Figure 30. Bacterial membrane.

The phenolic compounds actions are concentration dependent and at sublethal concentrations enhance the leakage of cell constituents [95]. In particular, oleuropein causes a significant outflow of glutamate, potassium and inorganic phosphate ion form, e.g., E. coli; the same

897

compound has no influence on the velocity of glyeolysis but decreases the ATP contents of the cells. Gleuropein is more toxic for gram-positive than for gram-negative, because the glycosidic group impedes the drug penetration on the outer membrane or the contact on the target site [96]. The o-diphenol system is certainly responsible for antimycoplasmal activity of oleuropein. It is difficult to control such mycoplasma! infections with suitable broadspectrum antibiotics. Moreover, tetracycline and erythromycin, two important chemotherapeutic agents, as well as quinolones, are needed for the efficacy of an integral host immune system to eliminate the mycoplasmas. Therefore, the recent studies on activity carried on oleuropein indicate that this phenolic compound might be considered as an interesting and potential new natural drug for the treatment of mycoplasmal infections. In fact, oleuropein showed also activity against M. fermentans and M hominis strains, naturally resistant to erythromycin and tetracyclines [97]. Moreover, several studies carried out on olive fermentation showed that naturally occurring antimicrobial substances are needed to be removed in order to initiate fermentation [98]. In particular, at effective concentrations of oleuropein, the supplements affected both spore germination and subsequent increase. The inhibition appears during the transformation of the phase bright spore to a phase dark form. It was demonstrated that the addiction of oleuropein at various stages during the germination caused inhibition of the outgrowth of the germinated spores. During the development, important changes occur in the initiation of RNA, protein and membrane syntheses [99]. In fact, adding phenols to bacteria, sporulated or not, also affected membrane synthesis or inactivated cellular enzymes [100]. As reported before, oleuropein attacks the cytoplasmic membrane, destroying its permeability and causing outflow of intracelMar constituents such as glutamate, potassium and phosphorus. This action is crucial for me spores while the effectiveness of germination inhibitors may depend upon their capability to permeate the spore coat and may obstruct germination-promoting sites [101]. Furthermore, an interesting bactericidal activity of oleuropein against nine strains of Lactobacillus plantarum isolated from green olive fermentation brines was observed [102]. Lactobacillus plantarum [103] is a facultative heterofermentative, asporogenous, gram-positive rod and is responsible for Spanish-style

898

green olive fermentation. During ibis process, by opportune acidity, Lactobacittus plantarum metabolises sugars eluted from olives in brines. Moreover, phenolic compounds are responsible for the stoppage or the variability in lactic acid fermentation, and some phenolics related to oleuropein cause delay or inhibition in the growth of various bacteria, including Lactohacillus plantarum. Several experimental studies have demonstrated mat the morphological characteristics of these bacteria change after heat-treated or untreated oleuropein incubation, such as cells become longer in size and wider. These changes are observed after 30-60 minutes of incubation in both oleuropein solutions. Probably, oleuropein promotes disruption of the lactobacillus peptidoglycan. Acknowledgements We thank to all members of my team: researchers, PhDs, technicians and students. Financial support of MIUR. and CNR is acknowledged. 4. BIBLIOGRAPHY [1] LPanizzi, MX.Scarpati, G.Oriente, Ricerca Sci, 28,994 (1958). [2] LPanizzi, M.L.Scarpati, G.Oriente, Gazz.Chim.Ital, 90,1449 (1960). [3] LPanizzi, M.L.Searpatis C.Trogolo, Gazz.Chim.ltal, 95,1279 (1965). [4] Rlnjouye, T.Yoshida,S.Tobita, K.Tanaka, T.Nishioka, Tetrahedron Letters, 2459 (1970). [5] A.Bianco, G.Naccarato, P.Passacantilli, G.Righi, M.L.Scarpati, J.NatProd, 55,760 (1992). [6] A.Bianco, N.Uccella, Food Research International, 33,475 (2000). [7] LBastoni, A.Bianco, F.Piccioni, N.Uccella, Food Chemistry 73,145151 (2001). [8] J.P Donaire., A.J Sanchez., J.Lopez-Gorge, L.Recalde, Phytochemistry, 14,1167 (1975). [9] MJ.Amiot, A,Fleuriet, J.J.Macheix. Phytochemistry, 28,67 (1989). [10]A.Bianco, R. Lo Scalzo, MLScarpati, Phytochemistry, 32, 455 (1993).

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[11] S.R. Jensen, A. Kjaer, B.J. Nielsen, Acta Chem. Scand, 27, 367 (1973). [12] H. Endo, H. Hikino, Can. J. Chem., 62,2011 (1984). [13] I. Messana, M. Sperandei, G. Multari, C. Galeffi, G.B. MariniBettolo, Phytochemistry, 23,2617 (1984). [14] A.Bianco, R.A. Mazzei, C. Melchioni, G. Romeo, M.L. Scarpati, A.Soriero, N. Uccella, Food Chem., 63,461 (1998). [15] MSugiyama and M.Kikuchi, Chem.Pharm.Bull, 40,325 (1992). [16] H.Simomura, Y.Sashida, T.Adachi, Phytochemistry, 26,249 (1987). [17] S.Kunz, HBecker,Phytochemistry, 31,3981 (1992). [18] A.Vazquez Roncero, E.Graziani Costante, RMaestro Duran, Grasas yAceites, 25,269 (1974). [19] A.Vazquez Roncero, RMaestro Duran, E.Graziani Costante, Grasas

yAceites,25,341 (1974). [20] A. Bianco, C. Melchioni, A. Ramunno, G.Romeo, N.Uccella, Natural Products Research, 18,29-32. (2004). [21] M.L. Scarpati; A. Soriero; F. Veri. La Chimicae I'lndustria (Milano) 1997, 79(8), 1049 [22] A. Troshchenko; G.A. Kutikova. Khim. Prir. Soedin 1967, 3, 244; CAN 67:114346. [23] Data to be published. [24] Y.Asaka, T.Kamikawa, T.Kubota, H. Sakamoto, Chemistry Letters, 2, 141 (1972). [25] RT.LaLonde, C.Wong, A.I.M.Tsai, J.Am.Chem.Soc, 98(10), 3007 (1976). [26] ELKuwajima, T.Uemura, K.Takaishi, K.Inoue, HJnouye, Phytochemistry, 27(6), 1757.(1988). [27] A.Bianco, The Chemistry of Iridoids, in, Atta-ur-Rahman Ed., Studies in Natural Products Chemistry, Elsevier, Vol 7 p. 439 (1990). [28] P.Gariboldi, G.Jommi, L.Verotta, Phytochemistry, 25(4), 865 (1986). [29] F.A.MacKellar, RC.Kelly, E.E.van Tamelen, C.Dorschel, JAm.Chem.Soc, 95,7155 (1973). [30] R. Lo Scalzo,M.L. Scarpati, /. Nat. Prod., 56(4), 621 (1993).

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©

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THE CHEMISTRY OF THE GENUS CICER L. PHILIP C. STEVENSON1*2 & SHAZIA N. ASLAM1 x

Natural Resources Institute, University of Greenwich, Chatham, Kent ME4 4TB, UK. 2 Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey, TW9 SAB, UK. ABSTRACT: The genus Cicer L. consists of 43 species of annual and perennial herbs in the monogeneric tribe Cicereae (Leguminosae). The genus affords considerable interest because C. ariettnum, the most well studied and most vulnerable species to disease and pests, is the chickpea, an important crop and food for resource-poor farmers, especially in Asia. More than 200 natural products have been identified from the genus. This chapter describes the chemistry of Cicer and the function of those compounds with known biological activities especially in the context of micro-organisms and insects that are natural pests and diseases of the cultivated crop. Some compounds occur constitutively in healthy chickpea plants that present inbuilt defence to infection by a range of pathogens and insects. For example, although the pterocarpans, maackiain and medicarprn occur constitutively they also increase in concentration when the plant is attacked by a fungus, nematode or bacterium. These induced compounds (phytoalexins) are either biosynthesised de novo or as recent work suggests are accessible from glycosides stored in vacuoles. While these phytoalexins occur in all species of Cicer a few produce a wide array of isoflavonoids including recently discovered compounds from the rare isoflav-3-ene and arylbenzofuran classes all of which show varying degrees of biological activity against pathogens of chickpea and all are inducible. The chemical ecology of the plant especially with relevance to agricultural applications is discussed. We also describe their distinct occurrence among Cicer species and indicate how knowledge about their biosynthesis can inform the taxonomy of the genus. The recent synthesis of the arylbenzofuran cicerfuran from novel stilbenes precursors has been achieved along with several analogues. Their importance beyond the defensive value to the plant has become apparent through biological activity testing against different micro-organisms and shows that these compounds may provide valuable novel activity against bacteria, fungi and protozoans. The chapter will also discuss some aspects of chemical synthesis of flavonoids of structural relevance to Cicer L.

1-INTRODUCTION The genus Cicer L. occurs in the monogeneric tribe Cicereae in the sub-family Papilionoideae of the Leguminosae. It contains 43 species of annual and perennial herbs [1] with a geographic distribution from the Himalayas to the Ethiopian Highlands, and has centres of diversity across central Asia, An isolated species, C. canariense occurs on the Canary Islands in the Atlantic Ocean [2]. The genus affords considerable research interest since one species, C. arietinum L., the cultivated species

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known variously as chickpea, garbanzo and channa, is an important food crop and so has been well studied, although the chemistry of 22 other species in the genus has also been investigated [ 3 - 6 ] . A summary of the plant chemistry of Cicer was published in 2001 [7]. Interestingly, C. arietinum is not known in the wild [8] but is a major source of human and domestic animal food, particularly in the semi-arid tropics where its production is concentrated [9] and it provides a considerable portion of the daily diet of some of the world's poorest people. It is especially important in South Asia, where it comprises a large part of a primarily vegetarian diet. It is also a highly versatile crop providing gram flour, the principle ingredient for many biscuits, breads, sauces and sweets as well as its more familiar role in vegetable dishes. Because it is used in such a wide variety of geographical locations it is considered the world's third most important pulse crop after dry beans (Phaseolus vulgaris L.) and dry peas (Pisum sativum L.) [10]. Its value as a crop has focused interest on the genus and much of the work on related species has been conducted in the hope that phenotypic characters can be found that, if transferable to the cultivated species, might enhance productivity. Perhaps the most important of these characters is natural resistance to agricultural pests, especially fungal diseases. More than 50 species of fungus have been reported to occur on chickpea [11], although only a few of these have the potential to cause serious crop damage and thus pose a threat to production, notably Aschochyta blight (Aschochyta rabiei), Botrytis Grey Mould (Botrytis cinerea) and Fusarium wilt (Fusarium oxyspomm f. sp. ciceri). Chickpea is also attacked by various insect pests most notably the pod borer Helicoverpa armigera (Lepidoptera : Noctuidae) and some work has focused on identifying chemical components in Cicer that influence food choice of this insect as well as the determination of components that affect the behaviour of ovipositing adults [12,13]. Some of these studies have also led to the identification of chemotaxonomic markers that support recently proposed sub-generic systematic restructuring and groupings within the genus [6]. Over 200 compounds have been identified so far in the genus although approximately 120 of these are simple hydrocarbons and relatively ubiquitous in the plant kingdom. Nonetheless they may be important as possible recognition chemicals and mediators for oviposition by insect pests [14]. This chapter lists all the known compounds that have been isolated from the genus Cicer under 5 sections in the text. It also ascribes a biological activity to the compounds where applicable to the ecology of Cicer. Where possible, semi-systematic names have been

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used along with the trivial names since IUPAC approved names for many compounds are too cumbersome and are rarely used in the literature. Section 6 describes the biosynthesis of the flavonoids, the main group of natural products in Cicer, and section 7 highlights some areas of synthesis relevant to structures in Cicer, notably those describing the synthesis of the aryl benzofurans. 2- ALIPHATIC ACIDS. One of the most characteristic physical attributes of Cicer is the profuse exudation from leaf hairs of some species especially C. arietinum. The composition of this exudate is highly acidic (pH 6)-O- a-galactopyransoyl-(l-* 2)-1-D4-0-methyl-c/jzVo-inositol (228) scopoletin (7-hydroxy-6-methoxycoumarin) (229) umbelliferone (7-hydroxycoumarin) (230) 2-methyl-2,3,4-trihydroxybutanoic acid-l,4-lactone ACKNOWLEDGEMENTS. The authors acknowledge grants from the UK Higher Education Funding Council for England. Also, we are indebted to Dr. Nigel Veitch and Professor David Hall for advice in the preparation of this manuscript.

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[101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119]

Rembold, H.; Wallner, P.; Nitz, S.; Kollmannsberger, H.; Drawert, F.; Journal of Agriculture and Food Chemistry, 1989, 37, 659-662. Rembold, H.; Kohne, A.C.; Schroth, A.; Journal of Applied Entomology, 1991, 112, 254-262. Semwal, A.D.; Sharma, G.K.; Arya, S.S.; Food Chemistry, 1996, 57, 233-239. Gallardo, M.; Bueno, M ; Angosto, T.; Gallardo, E.; Matilla, A.J.; Phytochemistry, 1992,31, 2283-2287. Modi, A.T.; McDonald, M.B.; Streeter, J.G.; Seed Science And Technology, 2000, 5,115-127. Quemner, B.; Brillouet, J-M.; Phytochemistry, 1983, 8, 1745-1751. Luk'yanchikov, M.S.; Khimiya Prirodnykh Soedinenii, 1992, 282-283. Mandavia, M,K,; Khanm N,A.; Gajera, H.P.; Allelopathy Journal, 2000, 7, 8592. Peterson, J.K.; Harrison, H.F.; Jackson, D.M.; Snook, M.E.; Hortscience, 2003, 38, 1129-1133. Sharan, M.; Taguchi, G.; Gonda, K.; Jouke, T.; Shimosaka, M.; Hayashida, N.; Okazaki, M.; Plant Science, 1998,132, 13-19. Ford, C.W.; Phytochemistry, 1981, 20, 2019-2020. Veitch, N.C.; Grayer, R.J.; In Flavonoids: Chemistry, Biochemistry and Applications, Andersen, O.M. and Markham, K.R., Eds.; CRC Press, Boca Raton, 2005, {In press) Yamamoto, S.; Aizu, E.; Jiang, H.; Nakadate, T.; Kiyoto, I.; Wang, J.C.; Kato, R.; Carcinogenesis, 1991,12 , 317-323. Li, T.; Satomi, Y.; Katoh, D.; Shimada, J.; Baba, M.; Okuyama, T.; Nishino, H.I; Kitamura, N.; Cancer Letters, 2004, 207, 27-35. Barz, W.; Welle, R. In Phenolic Metabolism in Plants, H.A. Stafford & R.K. Ibrahim, Eds. Plenum Press, New York, 1992, pp 139-164. Ladizinsky, G.; Adler, A.; Theoretical and Applied Genetics, 1976, 48, 197-201. Ahmad, F.; Gaur, PM.; Slinkard, AE, Theoretical and Applied Genetics 1992, 83 620-627. Ahmad, F.; Slinkard, A.E.; Theoretical and Applied Genetics, 1992, 84, 688-692. Smirnoff, P.; Rotman, M.; Ladizinsky, G; Biochemical Sytematics and Ecology, 1981,9, 23. Tayyar, R.I.; Waines, G.; Theoretical and Applied Genetics, 1996, 92, 245-254. Kazan, K.; Muehlbauer, F.J.; Plant Systematics and Evolution, 1991,175, 11-21. Tayyar, R.I.; Lukaszewski, A.J.; Waines, G.; Genome, 1994, 37, 656-663. Seferoba, I.V.; ZhurnakBotanische, 1996, 8, 96. Dewick, P.M., In The Flavonoids: Advances in Research since 1986, ed. Harborne, J.B.; Chapman and Hall, 1993, p. 117. Dewick, P.M., In The Flavonoids: Advances in Research, Harborne, J.B. and Mabry, T.J. Eds.; Chapman and Hall, 1982, p. 535. Kinoshita, T.; Tetrahedron Letters, 1997, 38, 259-262. Clemens, S.; Barz, W.; Phytochemistry, 1996, 41, 457-460. Banerji, A.; Goomer, N. C ; Synthesis, 1980 , 874-875 Bois, F. Beney, C. Mariotte, A-M.; Boumendjel, A.; Synlett, 1999, 1480-1482. Deshpande, S. R.; Mathur, H. H.; Trivedi, G. K.; Synthesis, 1983, 835. Tanaka, H.; Stohlmeyer, M; Wandless, T. J; Taylor, L. P; Tetrahedron Lett. 2000, 41, 9735-9739.

956 [120] [121] [122] [123]

Laschober, R.; Kappe, T.; Synthesis, 1990, 387-388. Kamara, B.I.; Brandt, E. V.; Ferreira, D.; Tetrahedron, 1999, 55, 861-868. Al-Maharik, N'; Botting, N. P.; Tetrahedron, 2004, 60, 1637-1642 Farkas, L.; Gottsegen, A.; Nogradi, M.; J. Chem. Soc. Perkin Trans. 1,1974, 305312. [124] Pelter, A.; Foot, S. Synthesis, 1975, 326. [125] Gandhidasan, R.; Neelakantan, S.; Raman, P. V.; Synthesis, 1982, 1110-1111. [126] van Aardt, T. G.; van Rensburg, EL; Ferreira, D.; Tetrahedron, 2001, 57, 71137126.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. Vol. 33 © 2006 Elsevier B.V. B.V. All rights rights reserved. ©

957

NEW RESEARCH AND DEVELOPMENT ON THE FORMOSAN ANNONACEOUS PLANTS YANG-CHANG WU Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan To honor one of the pioneers of Taiwanese natural product chemistry-late Professor Sheng Teh Lu

INTRODUCTION The Annonaceae, a pantropic family, is well developed in the tropics and the subtropics of both new and old world. Only a few species are distributed in warm temperature (Asiminid) and the islands of the Pacifica. There are ca 130 genera and over 2200 species in the world, and the greatest concentration of genera and species is in Indo-Malaysian area of Asia [1]. Economically this family is an important source of edible fruits, edible oils, raw material for perfumery, and folk medicine for various purposes [2]. In Taiwan, there are 21 species (8 genera) of Annonaceous plants, where three of them are native: Fissistigama glaucescens (Hance) Merr.; F. oldhami (Hemsl.) Merr.; and Goniothalamus amuyon (Blanco) Merr. [3]. Phytochemistry research on Annonaceae in Taiwan began in 1934. An alkaloid, annomontine, was isolated from Annona montana by Nozoe [4]. There were some alkaloids reports in 1970s by T. S. Yang et al. [5] which then continued by our group in 1980s. Most of the early studies focused on alkaloids. Afterwards, many non-alkaloids, Annonaceous acetogenins, diterpenoids, styrylpyrones and so on also were isolated from various species. Some of them showed interesting biological activities. This review discusses recent investigations on the chemistry and pharmacological activities of four major groups: alkaloids, annonaceous acetogenin, diterpenoids, and styrypyrones.

958 Table. 1. The Investigated Condition of Formosan Annonaceous Plants

unripe fruit

fru ts

'

'eaves stem

bark

root

Annona O

A. cherimola Mill. 0

A. glabra L.

O O

A. artemoya (A. cherimola X A. squamosa)

0

A. montana Macf.

O

O

A. muricata L.

0

0

A. reticulata L.

O

0

A. squamosa L.

O

O

O

0

o

0

o

A. purpurea L.

0

0

Artabotrys O

A. uncinatus (Lam.) Merr.

o

Cananga Cananga odorata (Lam.) Hook. F. & Thomas

O

0

Fissistigma F. glaucescens (Hance) Merr. F. oldhami (Hemsl.) Merr

O

0

0

0

0

0

0

O

o

O

0

Goniothalamus G. amuyon (Blanco) Merr. Polyalthia P. longifolia Benth. et Hook. F. P. longifolia Benth. et Hook. F. 'pendula' P. suberosa Hook. f. P. liukiuensis Hatusima Rollinia R. mucosa Bail

0

Uvaria U. rufa Bl

O means the phytochemical research was done or ongoing by our lab.

0

0

959

NITROGEN-CONTAINING CONSTITUENTS OF FORMOSAN ANNONACEOUS PLANTS Annonaceous plants in Taiwan contained a large amount of alkaloids. Among the alkaloids, mostly are benzylisoquinoline-type ones. General benzylisoquinoline alkaloids are a large group of natural products consisting of more than thousands defined structures and also showed diverse biological functions including the analgesics [6], muscle relaxant [7], anti-tumor [8], and antibiotic activity [9]. The biosynthetic of general benzylisoquinolines begins with decarboxylation, or^o-hydroxylation, and deamination of L-tyrosine into both dopamine and 4-hydroxyphenylacetaldehyde (4-HPPA). Norcoclaurine synthase catalyzes the condensation of dopamine and 4-HPAA to form (5)-norcoelaurine, which represents the first committed pathway intermediate with a benzylisoquinoline nucleus [10,11].

deamination |

COOH

Tyrosine

Dopamine

t hydroxylation|

decarboxylation | HO

H2N COOH

-*.

Hi

1 DOPA

* Norcoclaurine synthase

Benzylisoquinoline plays an important role as a biosynthesis centre for Annonaceous alkaloids such as isoquinolones, protoberberines,

960

proaporphines, aporphines, phenanthrene, aristolactam and litebamine alkaloids.

isoquinolone

morphinandienone

tetrahydroprotoberbenne

tetrahydrobenzylisoquinolme

dioxoxaporphine

litebamine

aristolactam

Six benzylisoquinolines were isolated from Formosan Annonaceous plants. Reticuline (A-l), the most frequently occurring

961

benzyltetrahydroisoquinoline, along with coclaurine (A-2) and iV-methylcoclaurine (A-3) were isolated from A. squamosa [12]. (+)-Orientaline (A-4), together with two new compounds, annocherine A (A-5) and annocherine B (A-6), were isolated from the stems of A. cherimola [13,14],

H A-l, Rj= CH 3 , R2=OH, R3=OCH3

A-S, R=CH3 A-6, R=H

A-2, R t = H, R2= H, R3= OH A-3, R,= CH3, R2= H, R3= OH A-4, R,= CH3, R2=OCH3, R3=OH

Simple Isoquinolones No biogenetic details of simple isoquinolone were elucidated, but it is likely to yield the isoquinolones from the oxidation of benzylisoquinolmes. There are a few isoquinolones in the Annonaceae. Only three simple isoquinolones, doryphornine (A-7), cherianoine (A-8) and thalifoline (A-9), were isolated from the stems of A. cherimola [14,15]. CH 3

H R A-7

A-8, R=H A-9, R=OCH 3

Protoberberines and Tetrahydroprotoberberine Protoberherines: The protoberberine alkaloids are the most widely distributed benzylisoquinoline alkaloids. Hundreds of alkaloids of this series are discovered that they were yielded from the tetrahydroisoquinoline catalyzed by BBE [16], the berberine bridge

962

enzyme. Berberine has general antimicrobial properties [16]. It inhibits reverse transeriptase [17], intercalates with DNA [18], and inhibits aldose reductase [19,20]. Five protoberberines were presented in Formosan Annonaceous plants. Among them, palmatine (A-10) was isolated from G. amuyon [21]. Fissisaine (A-11), eolumbamine (A-12), and dehydrodescretamme (A-13) were isolated from F. balansae (materials collected in Mainland China) [22]. (-)-8-Oxopolyalthiaine (A-14) was derived from P. longifolia [23].

A-10 A-11 A-12 A-13

Ri OCH3 OCH 3 OH OCH 3

R2 OCH3 OCH3 OCH 3 OH

Rj H OH H H

R4 OCH3 OH OCH3 OH

A-14

Tetrahydroprotoberberines (4H-protoberberines): There are four 4H-protoberberines described among Annonaceous plants in Taiwan: Discretamine (A-15) was obtained from F. glauscense [21]; Tetrahydropahnatine (A-16), from G. amuyon [A16], thaipetaline (A-17), from F. balance [22]; and (-)-Kikenamine (A-18), from the stems of A. cherimola and F. balance [13,22]. Most of these 4H-protoberberines are 2,3,9,10-oxygenated derivatives and only A-17 was 2,3,4,9,10oxygenated derivative.

A-15 A-16 A-17 A-18

Ri R2 OH H OCHj H OH OH OCHj H

R3 OH OCH, OH OH

963 963

Proaporphines Proaporphines is a small group of compounds among the Formosan Annonaceous plants; it was regarded as the direct precursor of aporphines [16]. In previous studies, proaporphines demonstrated antivirus [24], anesthetic and weak anticancer activities [16]. Three proaporphines, including stepharine (A-19), glaziovine (A-20) and promucosine (A-21), were extracted from the leaves of A. purpurea [25,26]. Among them, A-21 was the first naturally occurring TV-esterficated proaporphine.

R,

R2

A-19 OCH3

CH3

A-20 OH

CH3

A-21 OCH3 COOCH3

Aporphinoids Aporphines: Aporphines, the simplest derivatives of the tetrahydrohydrobenzylisoquinoline, are the most abundant alkaloids in Formosan Annonaceous plants. There are several different biogenetic pathways of aporphines [16]. In view of the related derivatives, the biogenetic origin of aporphine alkaloids in Annonaceae could be regarded as benzylisoquinolines or proaporphines, which result in the different substitution of aporphines. The 1,2,9,10- and 1,2,10,11- substituted aporphines were synthesized via the ortho-para or ortho-ortho phenolic coupling, respectively. The 1,2- or 1,2,10- substituted ones were formed directly from proaporphines [16]. Most aporphine alkaloids are toxic. They exhibit antagonistic effects to dopamine. Many of them have anticonvulsant activity or induced convulsions in animals [27,28] and cytotoxic activity [29]. They are distributed in all the known Formosan Annonaceous species. Thirty-three aporphines (A-22-A-54) were isolated and eleven of them were identified as new compounds. Among these aporphines, iV-methoxycarbonyl aporphines (A46-A-54), isolated as novel //-substituted aporphines, were firstly described by our lab. Their structures and the occurrences are listed in the following table.

964

Table, 2. The Structures of Aporphines, (* New compound)

Compounds A-22 annonaine

A-23 anolobinc

A-24 artabonatine* A-25 asimilobine

-OCH2O-

-OCH2O-

-OCH2OOH

OCH3

R,

R,

R,

R»

H

H

H

H

H

H

H

OCHj

H

H

R>

Occurrence

Ref.

H

A. cherimala

13

A, glabm

30

H

OCH3

OH

H

H

H

H

H

H

H

H

H

H

A-26 calycine

-OCH2O-

H

H

H

OCHj

H

OH

A-27 crebanine

-OCHjO-

H

H

OC

OGH3

H

H

G. amuyon

2i

P. longifolia

23

A. cherimala

13

F. glauscetice

32

F. oldhamii

21

A. uncinatus

33

F. oldhamii

21

A, uncinatus

33

F. oldhamii

34

F. glaucescens 21

H, A-28 glaucine

OCH3

OCHj

H

H

H

OCH3

OCH3

H

CH3

A. purpurea

A-29 isocorydine

OCH3

OCH3

H

H

H

H

OCH,

OH

CH3

A. eherimofa

13

A. purpurea

26

A-30

laurotetanine

A-31 Hrinidine A-32

michelahine

A-33 iV-ibnnylanonaine* A-34 iV-fonnylpurpureine*

25

OCHj

OCH3

H

H

H

OH

OCH3

H

H

A, cherimala

13

OCH3

OH

H

H

H

H

H

H

CHj

A. purpurea

26

H

OH

H

H

H

H

H

A. cherimola

13

C. adorata

35

-OCHjO-

-OCH2OOCH3

OCHj

H

H

H

H

H

H

CHO

A. gtabra

30

OCH3

H

H

OCH3

OCHS

H

CHO

A. purpurea

25

965 A-35 A'-mediyllaurotetanine

H

H

H

OH

OCHj

H

CH,

A. cherimafa

13

H

H

H

H

H

H

CHj

A. cherimah

13

H

H

H

H

H

F. otdhamii

21

OCH3

H

H

H

-OCHjO-

H

H

A, gtabra

30

A-39 norisocorydine

OCH, OCH,

H

H

H

H

OCH3

OH

H

A. cherimala

15

A-40

nornuciferine

OCH3

OCH3

H

H

H

H

H

H

H

A. glabra

31

A-41 norpurpureine

OCH3

OCH3

OCH, H

H

OCHj OCH,

H

H

A. purpurea

26

OH

OCH3

H

H

H

OCHj OCH,

H

CH,

P. longifatia

23

A-43 thabaicalidine

OCH3

OCH3

OH

H

H

OCH3

OCH,

H

CH,

A. purpurea

25

A-44 thalicimidine

OCH3

OCH,

OCHj

H

H

OCHj

OCH3

H

CH3

A, purpurea

26,36

H

OH

H

H

H

H

CH,

A, cherimola

13

C, odorata

35

A-36 /V-methylasimilobine A-37

norannuradhapurine

A-38 nordometicme

A-42

predecentrinc

A-45 ushisunine

A-46 romucosine*

OCH, OCH3 OH

OCHj

-OCH2OOH

-OCH2O-

-OCH2O-

OH OCHj

F. giuucescvm 21

H

H

H

H

H

H

COOCH, R. mucma

37

A-47 romucasine A*

OH

OCH3

H

H

H

H

H

H

COOCH3 R. mucma

38

romucosine B*

OH

OCH3

Cl

H

H

H

H

H

COOCH3 & mucasa

38

A-49 romucosine C*

OCH3

OCH3

H

H

H

H

OH

H

COOCH3 /?, mucma

38

A-50 romucosine D*

OCH3

OCHj

H

H

H

H

OCH3

H

COOCH, R. mucosa

38

romucosine E*

OCH3

OCH3

H

H

H

H

H

H

COOCH, R. mucosa

38

A-52 romucosine F*

OCH,

OCH3

Cl

H

H

OCHj

OCH3

H

COOCH, R, mucosa

26

A-53 romucosine G*

OCHj

OCH,

OCH3

H

H

OCH3

OCH,

H

COOCH3 A. cherimala

14

romucosine H*

OCHj

OCH,

H

H

H

H

OCH3

OH

COOCH, R. mucasa

14

A-48

A-51

A-S4

Aporphine-N-oxides: Aporphine-iV-oxides, the iV-oxidation products of aporphines, were also isolated. Four Aporphine-iV-oxides were obtained from P. longifolia, including (+)-O-methylbulbocapnine-a -iV-oxide (A-S5), (+)-O-methylbulbocapnine- (3-iV-oxide (A-56), (+)-JV-methyl nandigerine-p-iV-oxide (A-57), (+)-oliveroline-P-iV-oxide (A-58). A-55-A-57 were isolated as new compounds [39].

966

A-55 A-56 A-57 A-58

R, R2 H OCH3 H OCH3 H OCR, OH H

R.i

OCH3 OCH3 OH H

orientation ofiV-oxide a

P P P

orientation of 6a-H a a a

P

Oxoaporphines: Oxoaporphines, the other oxidation products of aporphines, were also widely distributed among Annonaceous plants. Fourteen oxoaporphines were isolated and three of them were new compounds. Among them, liriodenine (A-59) is the most ubiquitous and isolated from several species of Formosan Annonaceous plants: A. cherimola [13], A. montana [40], A. reticulate [41], A. squamosa [12], R. mucosa [37], A. uncinatus [42], F. glauscense and G. amuyon [21]. Annolatine (A-60) was isolated from A. montana [40]. Artherospermidine (A-61) was isolated from A. cherimola [13] and A. uncinatus [42]. Atherospermidine A (A-62) was isolated from A. uncinatus [42]. Fissiceine (A-63) was yielded from F. glaucesens [32], kuafumine (A-64) whereas oxocrebanine (A-65) was isolated from F. glauscense [21,32,43]. Oxoanolobine (A-66) and oxoasimilobine (A-67) were isolated from A. cherimola [13,15]. Oxonantenine (A-68) was derived from A. reticulate [12]. Oxoglaucine (A-69) was isolated from A. cherimola [13] and A. purpurea [25,26]. Oxonuciferine (lysicamine, A-70) was isolated from A. cherimola [13], A. glabra [30], A. purpurea [25,26], and C. odorata [35,44]. Oxopurpureine (A-71) yielded from in A. purpurea [25,26], and oxoxylopine (A-72) from A cherimola [13] and F. glaucescens [32].

967

Table. 3. The Structures of Oxoaporphincs. (* New compound)

Ri A-59 A-60

R2

-OCH 2 OOCH 3

OH

R3

R4

R5

R*

H

H

H

H

H

OCH,

OCH3

H

A-61

-OCH 2 O-

OCH3

H

H

H

A-62

-OCH2O-

CH3

H

H

H

A-63

-OCH 2 O-

H

OH

OCH3

H

A-64

-OCH2O-

OCH,

OCH,

OCH,

H

A-65

-OCH2O-

H

OCH3

OCH3

H

A-66

-OCH 2 O-

H

H

OH

H

A-67

OH

OCH3

H

H

H

H

A-68

OCH3

OCH3

H

H

OCH,

OCH3

H

H

A-69

-OCHjO-

-OCH2O-

A-70

OCH3

OCH,

H

H

H

H

A-71

OCH,

OCH3

OCH3

H

OCH,

OCH3

H

H

OCH3

H

A-72

-OCH2O-

6a,7-Dehydroaporphines: 6a,7-Dehydroaporphines is a small group of aporhinoids with only four compounds were isolated from A. purpurea, including 7-formyl-dehydrothalicsmidine (A-73), 7-hydroxydehydrothalicsmidine (A-74), 7-hydroxy-dehydroglaucine (A-75) and dehydrolirinidine (A-76) [25,36].

968

A-73 A-74 A-75 A-76

R, OCH3 OCH3 OCH3 OCH3

R2 OCH3 OCH3 OCH3 OH

R3 OCH3 OCH3 H H

R4 OH CHO OH H

R5 R6 O C H , OCH3 OCH3 OCH3 OCH3 OCH3 H H OCH3

CH3O.

CH,

CH 3

HO'' A-79

A-80, R=OCH 3 A-81,R=OH

OCH,

A-83

A-82

CH3O

A-85, R O H A-86, R O C H 3

A-84

969

Besides the typical aporphinoids, several aporphinoids with interesting structures were also isolated. Two /?-quinone-aporphines, (-)-fissilandione (A-77) and (-)-norfissilandione (A-78), were only isolated from F. balansae [22]. Artacinatine (A-79) and artabonatine C-F (A-80-A-83), were isolated from A. uncinatus [42,45]. Annobraine (A-84) was isolated from A. glabra [30]. Noraristolodione (A-85) and norcepharadione B (A-86) were isolated from F. balansae [22]. Phenanthrenes Most phenantherenes here are aminoethylphenantherene derivatives (open aporphines). There are eight phenantherenes distributed in two genera of Formosan Annonaceous, Annona and Fissistigma. Fissicesine (A-87), fissicesine-A^-oxide (A-88), atherosperminine (A-89), iV-noratherosperminine (A-90), and 7V-methylatherosperminium (A-91) were isolated from F. glauscense [21,46]. Argentinine (A-92) and annoretine (A-93) were isolated from A. montana [40]. Thalicpureine (A-94) was yielded from the leaves of A. purpurea [25]. Among these isolates, A-87~A-88 and A-91~A-93, were isolated as new compounds and A-93 as a novel litebamine-type skeleton which was firstly described by our lab [47,48].

A-87 , R=N(CH3) A-88 , R=N+(CH3)2O"

A-91

A-89, R=CH3 A-90, R=H

OCH

CH3I

A-92

A-93

A-94

970 970

Aristolactams Aristolactams were regarded as intermediates in the biosynthetic pathway of aristolochic acid, which was produced by aporphines via oxidation process. Eleven aristolactams were isolated from Fissistigma. Piperolactam A (A-95), piperolactam C (A-96), aristolactam Allla (A-97), aristolactam BII (A-98), and agoniothalactam (A-99) were isolated from F. balansae [49]. Aristolactam FII (A-100), stigmalactam (A-101), aristolactam All (A-102), enterocarpam I (A-103), and velutinam (A-104) were isolated from F. oldhamii [49]. Aristolactam Bill (A-105) was isolated from both the species [49].

Table. 4. Structures of Aristolactams in Annonaceae

R.

R:

R3

R4

R5

R«

A-95

H

OCH3

OH

H

H

H

A-96

OCH3

OCH3

OCH3

H

H

H

A-97

H

OH

OCH,

OH

H

H

A-98

H

OCH3

OCH3

H

H

H

A-99

H

OCH3

OCH3

OH

H

H

A-100

OCH,

OH

OCH3

H

H

H

A-T01

OCH3

OCH3

OCH3

OH

H

H

A-102

H

OH

OCH,

H

H

H

A-103

H

OH

OCH3

H

OCH3

OCH,

A-104

OCH,

OCH3

H

H

OH

H

A-105

H

OCH,

OCH3

OCH3

H

H

971

Amides Eight acyl-amides, cherinonaine (A-106), a novel dimeric acyl-amide, along with dihydro-feruloytyramine (A-107), Af-m-caffeoyltyramine (A-108), A^nms-feruloymethoxytyramine (A-109), N-cisferuloymethoxytyramine (A-110), jV-fraHs-feruloytyramine (A-lll), iV-frvms-caffeoyltyramine (A-112), and iV-p-coumaroyltyramine (A-113) were isolated from ,4. cherimola [50,51]. A - l l l was also isolated from C. odorata.

CH 3 O.

CH 3 O'

A-107

H

,-OMe "OH

HO

HO

OMe

A-108

A-109, *= trans A-110, *~cis

OH

A-lll, R=OCH3 A-112, R=OH

A-113

Azafluorenes As an observation of our previous studies, azafluorenes were only distriuted in Polyalthia. The biogenetic origin of azafluorenes was proposed: it arises from the degradation of oxoaporphines into 1-azaanthraquinolone and then the loss of CO group converts the 1-azaanthraquinolone into azafluorenes [52]. There are four azafluorenes-

972

polylongine (A-114), darienine (A-115), polyfothine (A-116), and isooncine (A-117)- isolated from P. longifolia [39,53].

Ref. 47

R, A-114

R2

R3

A-115 OCH3 OCH3

OH

A-116

H

OCH3

OCH3

A-117

H

OCH3

OH

Other Alkaloids An indole alkaloid, cheritamine (A-118), and perlolidine (A-119), which possessed a novel skeleton, were isolated from A. cherimola [50] and A. squamosa [54], respectively. Rollipyrrole (A-120), a propentdyopent derivative, was isolated from R. mucosa [55]. Glaucenamide (A-121), a terpene alkaloid, was isolated from F. glaucescens [32]. Cananodine (A-122), a guaipyridine sesquiterpene alkaloid, was isolated from C. odorata [44]. Uncinine (A-123), a (3-utenolide alkaloid, was isolated from A. uncinatus [45]. A-118-A123 were isolated as new compounds. One morphinandienone, O-methylflavinantine (A-124), was isolated from F. oldhamii [21]. Adenosine (A-125) and uridine (A-126) were isolated from A. cherimola [13,15]. Cleistopholine (A-127) was isolated from C. odorata [35]. Squamolone (A-128) was isolated from A cherimola [13] and A purpurea [26].

973 H (CH 2 ) 2 2 -

2

O

A-118

O.

NH2 A-121

A-119

O

O

CH, H3O

A-122

NH2

A-123

OH

O

o

Ho'

A-125

A-126

A-128

Bioactivities of Annonaceous Alkaloids Cytotoxicity: In our published studies, several alkaloids were screened including benzyl isoquinolines, protoberberines, aporphines, and amides. Palmatine [56], norannuradhapurine (A-37) [56], kuafumine (A-64) [43], liriodenine (A-59) [56], atherosperminine (A-89) [56], argentinine (A-92) [40], annoretine (A-93) [40], A^rans-femloytyramine (A-111) [44,57], A^-^ran^-caffeoyltyramine (A-112) [57], A^-p-coumaroyltyramine (A-113)

974

[57], cananodine (A-122) [44] and cleistopholine (A-127) [44] were cytotoxic. Among them, liriodenine was the most potent alkaloid against P-388 (murine lymphocytie leukaemia), KB (human nasopharyngeal carcinoma), and HT-29 (human colon carcinoma) cells [56]. Antiplatelet Aggregation Activity: Alkaloids from Annonaceous plants were reported to inhibit platelet aggregation. According to our previous studies, more than twenty compounds were examined and most of the alkaloids were found inhibiting arachidonic acid (A.A) induced platelet aggregation [41]. Phenanthrene-alkaloids inhibited platelet aggregation initiated by four inducers (arachidonic acid, PAF, collagen, and thrombine) [58] whereas iV-oxide derivatives of aporphines showed slightly reduced efficacy as compared to the parent aporphines [59]. 6a,7-Dehydroaporphines showed better performance than the aporphines and three of them specifically inhibited platelet aggregation induced by PAF [25,36]. Recent studies of iV-methoxycarbonyl aporphines also revealed significant activities [38]. Cardiovascular Activity: Vasorelaxation can be induced by liriodenine (A-59) [60,61], isocorydine (A-29) [41], coclaurin (A-2) [58], iV-methyleoclaurine (A-3) [58], atherosperminine (A-89) [58] and iV-methylatherosperminium (A-91) [58]. Liriodenine was also found to have many bioactivities including antiarrythmic efficacy [62] and antimuscamic activity [60,61]. Antimicrobial activity was displayed by phenanthrenes, benzylisoquinolines and aporphines [63]. (-)-Discretamine (A-15) was examined as an antagonist of a-adrenoceptor, (XiD-adrenoceptor and 5-HT receptors [64,65]. Trachealis relaxation was induced by atherospermidine (A-61) [65].

ANNONACEOUS ACETOGENINS Annonaceous acetogenins are a bio-potent class of natural compounds isolated from Annonaceae plants. Since the first bioactive Annonaceous acetogenin, uvaricin, was found from the roots of Uvaria accuminata (Annonaceae) by Mad et al. in 1982 (Fig. 1) [66], more than 350 Annonaceous acetogenins have been isolated in the last two decades. Generally, it has been reported that Annonaceous acetogenins possess a broad spectrum of bioactivities, such as anticancer, antiparasitic, insecticide, and immunosuppressive effects.

975 OAc

erythro

trans | trans threo

O

Fig. (1). Uvaricin

The common structural features of Annonaceous acetogenins are a terminal y-lactone ring (Fig. 2) and a terminal aliphatic side chain connected with some oxygen-bearing moieties, such as one to three tetrahydrofuran rings, several hydroxyls, acetoxyls and/or ketones (Fig. 3). Depending on the substitutes in the long aliphatic chain, Annonaceous acetogenins were classified into several subtypes, including 1) acetogenins with mono-tetrahydrofuran (THF) ring, 2) ones with adjacent bis-THF rings, 3) ones with non-adjacent bis-THF rings, 4) ones with non-adjacent THF rings or tetrahydropyran (THP) rings, 5) ones with adjacent tris-THF rings, and 6) miscellaneous, which includes those substituting for epoxide or/and double-bond [67].

Fig. (2). Types of the terminal y-lactone rings systems in Annonaceous acetogenins.

976 976 OH

Mono-THF

OH

OH

O

ring Bl

B2

B3

B4

Bis-adjacent THF ring

OH

Non-adjacent

,

THF ring B5 Tri-adjacent

OH

O.

.0.

THF ring

OH

HO.

OH

THP rings

B7

B8 O

Epoxide B9

Bll

BIO

977 Linear (double

OH bond

or diol)

Fig. (3). Types of the oxygen-bearing moiety systems in the aliphatic side chain of Annonaceous acetogenins

All Annonaceous acetogenins contain multiple stereocenters, the elucidation of which often presents stereochemical problems. Because of their waxy nature, Annonaceous acetogenins do not form crystals suitable for X-ray crystallographic analysis. Relative stereochemistries of ring junctions have typically been determined by comparison of natural compounds with synthetic model compounds, and such methods are proven invaluable to the study of acetogenins. Recently, the absolute stereochemistries of the carbinol centers of acetogenins were determined with the help of synthetic model compounds and high field nuclear magnetic resonance (NMR) analysis of their methoxyfluoromethylphenylacetic acid (MPTA) esters (Mosher esters) [68]. In the past two decades, our laboratory has focused on the investigation of the bioactive constituents in the family plants in Taiwan. A series of acetogenins (AA01-AA75) were isolated from two genera of Formosan Annonaceous plants, Annona and Rollinia. Among them, twenty-seven acetogenins were published as new compounds [69]. Most of them include one or two tetrahydrofuran ring(s), an a»(3-unsaturated-y-lactone as the main structure with functional groups such as -OH, =0, C=C, and adjacent diols on the long chain. According to the structural features, Formosan Annonaceous acetogenins are classified as 1) acetogenins with mono-tetrahydrofuran (THF) ring, including six subtypes (Al-Bl, A1-B2, A2-B1, A2-B2, A3-B1, and A7-B1), 2) ones with adjacent bis-THF rings, including four subtypes (A1-B3, A2-B3, A2-B4, and A3-B1), 3) ones with non-adjacent bis-THF rings, including three subtypes (A1-B5, A2-B5, and A3-B5), and 4) miscellaneous, including those substituted with epoxide and/or double-bond (A1-B9/B11, A2-B9, and Al-Bl 2). The bioactivities of these compounds have been studied and will be discussed later. The following sections will introduce the Annonaceous acetogenins from Formosan Annonaceous plants according to their structural classification.

978

Annonaceous Acetogenins with Mono-Tetrahydrofuran (THF) Ring Mono-THF compounds are the largest group of Annonaceous acetogenins. This class of acetogenins possesses a THF ring with one or two flanking hydroxyls and various terminal lactone rings. To date, thirty-eight mono-THF acetogenins were isolated from Formosan Annonaceous plants. They were classified into six subtypes according to the terminal lactone rings and the number of the flanking hydroxyls, including 1) Al-Bl, six acetogenins with a terminal oc,p-unsaturated y-lactone ring and two hydroxyls flanking with the THF ring (AAOl-07), 2) A1-B2, two acetogenins with a terminal a,|3-unsaturated y-lactone ring and one hydroxyl flanking with the THF ring (AA08, 09), 3) A2-B1, fifteen acetogenins with a terminal a,P-unsaturated y-lactone ring with a hydroxyl at C-4 and two hydroxyls flanking with the THF ring (AA10-24), 4) A2-B2, nine acetogenins with a terminal a,P-unsaturated y-lactone ring with a hydroxyl at C-4 and one hydroxyl flanking with the THF ring (AA25-33), 5) A3-B1, five acetogenins with a ketolactone terminal and two hydroxyls flanking with the THF ring (AA34-39), and 6) A7-B1, one acetogenin with a terminal oc,P-unsaturated y-lactone ring flanking with a THF ring and two hydroxyls flanking with the THF ring (AA40). These compounds are listed as follows (see Table 1). Among these compounds, muricins A and B were isolated and elucidated as stereoisomers. Their absolute configurations were determined through analyses of their Mosher ester derivatives. Muricin B (AA30) is the first example of Annonaceous acetogenin that possesses a hydroxyl group of the S-configuration substituted at C-4. Muricin C is the first example of an Annonaceous acetogenin in which the THF ring begins with another odd position at C-l 7. Muricins D and E are the first Annonaceous acetogenins that possess a C33 skeleton. Table S. Mono-THF Acetogenins Isolated From Formosan Annonaceous Plants

No

Compound Name

THF and Hydroxyl Positions

THF Relat. Config.

Molecular Formula

Molecular Weight

thltlth

CSSHMO,

564

thltlth

C37HftsOs

592

thltlth

CJJHMO,

580

la Al-Bl without free OH group AA01

Solamin

AA02

Reticulatacin

lb Al-Bl with free OH group AA03 Longifolicin

mono-THF 15-20 mono-THF 17-22

mono-THF

979

AA04

Corossolin

AA05

ei'.v-Corossolin

AA06

Corossolone

AA07

c/'.s-Corossolone

10, 13-18 mono-THF 10,15-20 mono-THF 10, 15-20 mono-THF 10=O, 15-20 mono-THF 10=O, 15-20

thltlth

CJSHMOS

580

th/c/th

C35H64O6

580

thltlth

C3.sH62Ofi

578

th/c/th

C35H62O6

578

t/th-th

CJSHMCX

580

t/th-th

C35H64O6

580

thltlth

C35H64O8

612

thltlth

C35HMO7

596

thltlth

C35HMO7

596

thltlth

C 35 H 62 O 7

594

thltlth

C.WHMO7

596

thltlth

C35HMO7

596

thlclth

C35H64O7

596

thltlth

C 3 7Hfis07

624

thltlth

CH^O,

624

thlclth

C57H68O7

624

thltlth

C35H62O7

594

thltlth

C.,5HMO7

596

thltlth

C35H64O6

580

thltlth

CJSHMO?

594

thltlth

C35HHO8

612

t/er-th

1 CJSHMO?

596

2 A1-B2 with free OH group AA08

Vluricin H

AA09

Muricin I

mono-THF -19,24,25 mono-THF -19,24,25,28=29

3 A2-B1 with free OH groups AA10

Annomonicin

AA11

Xylopianin

AA12

Annoreticuin*

AA13

Annoreticuin-9-one*

AA14

Gonithalamicin

AA15

Annonacin

AA16

Cis-annonacin

AA17

Xylomaticin

AA18

Annomontacin

AA19 AA20

Cis-annomontacin* Annonacinone =Annonacin-10-one

AA21

Rolliacocin *

AA22

Murisolin

AA23

Muricin G*

AA24

Annomurilin*

mono-THF 4,8, 13, 15-20 mono-THF 4,8,15-20 mono-THF 4,9, 15-20 mono-THF 4,9=0,15-20 mono-THF 4, 10, 13-18 mono-THF 4, 10, 15-20 mono-THF 4, 10, 15-20 mono-THF 4, 10, 15-20 mono-THF 4, 10, 17-22 mono-THF 4, 10, 17-22 mono-THF 4, 10=O, 15-20 mono-THF 4, 11, 15-20 mono-THF 4, 15-20 mono-THF 4, 10, 15-20,23=24 mono-THF 4, 16-21,28,29

4 A2-B2 with free OH groups AA25 iMuricatetrocin A

mono-THF

I

980

AA26

Muricatetrocin B

AA27

Muricin E*

AA28

Muricin D*

AA29

Muricin A*

AA30

Muricin B*

AA31

Annocatalin*

AA32

Muricin C*

A A3 3

Muricin F*

4,-16, 19,20 mono-THF 4,-16,19,20 mono-THF 4,-16,22,23 mono-THF 4,-19,22,23 mono-THF 4,-19,26,27 mono-THF 4,-19,26,27 mono-THF 4,-19,28,29 mono-THF 4,-21,24,25 mono-THF 4,-21,24,25,28=29

tler-th

C35H64O7

596

tlth-th

C35HMO7

596

tlth-th

C35H64O7

596

tlth-th

CJSHMO,

596

tlth-th

C3 5 H M O 7

596

tlth-th

C35H&1O7

596

tlth-th

C35H64O7

596

tlth-th

C35H62O7

594

thltlth

C35H64O6

580

thltlth

C35H64O7

596

thltlth

C35HMO7

596

thltlth

C35H62O7

594

thltlth

C3 5 H 62 O 7

594

thltlth

C35HMOoside2j_19=20 epoxy 17-18 {eposide), 21=22

-

C35H62O4

546

c

CMH^OJ

558

c

C37HMO3

558

984

A1-B12 AA70

Cohibin A

AA71

Artemoin-D*

AA72

Artemoin-C*

AA73

Artemoin-B*

AA74

Artemoin-A*

linear 15, 16, 19=20 linear 15,16 linear 17, 18 linear 19,20 linear 21,22

th-c

C«H64O 4

648

C35HG6O4

550

C 3 5 H«O 4

550

C35H66O4

550

C 3 5 H«O 4

550

C22H36O6

396

—

-

Miscellaneous AA75

Rollicosin*

terminal lactone 15,16-19(19=0)

th

Chemotaxonomy In our investigation of the cytotoxic constituents of Formosan Annonaceous plants, the Annonaceous acetogenins were isolated from five species of this family, Annona muricata, A. reticulata, A. atemoya, A. montana, and Rollinia mucosa. Most of the mono-THF acetogenins were isolated from A. montana and A. muricata, while most of bis-THF acetogenins were isolated from A. reticulata, A. atemoya and R. mucosa (see Table 5). A. atemoya is a hybrid species of A. squamosa and A. cherimola. Compared with the reviews by McLaughlin et al. and Cave et al. [67], in which there was no report on the isolation of bis-THF acetogenins from A. muricata or A. montana, it clues that the genus Annona could be separated into two subgenus by the evidence of the chemotaxonomical researches; one of which takes a biogenetic pathway to generate mono-THF acetogenins and the other takes a resembling but different pathway to generate bis-THF acetogenins. Table 9. Annonaceous Acetogenins Isolated from Formosan Annonaceous Plants

No. AA53 AA54 AA31

Compound Annona muricata A reticulata name Annocatacin A* leaf Annocatacin B* seed Annocatalin* leaf

AA49

Annoglaucin

AA18 AA10

Annomontacin Annomonicin r/.?-Annomontac in* Annomurilin*

AA19 AA24

A. atemoya

Rollinia mucosa

A. montana

unripe fruit seed

seed leaf seed leaf

seed

A. cherimola

985 AA15

Annonacin cir-Annonacin Annonacinone AA20 =Annonacin-10one AA12~~~1 Annoreticuin* Annoreticuin-9AA13 one* AA40 Aromin AA71 Artemoin-D* AA72 Artemoin-C* AA73 __] Artemoin-B* AA74 Artemoin-A* Asinricin AA50 AA51

Bullatacin

AA61

Bullatalicin