Studies in Natural Products Chemistry: Bioactive Natural Products Part L

Studies in Natural Products Chemistry Volume 32 Bioactive Natural Products (Part L)

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

Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol.

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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) Studies in Natural Products Chemistry: Cumulative Indices Vol. 1-30 Bioactive Natural Products (Part L)

Studies in

Natural Products Chemistry Volume 32 Bioactive Natural Products (Part L)

Edited by

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

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FOREWORD Natural product chemistry continues to expand to exciting new frontiers of great importance in medicine. Advances in spectroscopic techniques such as NMR and mass spectroscopy coupled with new developments in high throughput screening techniques have opened up new horizons for discovery of bioactive substances. The pharmaceutical industry has been using these developments to a great extent which is reflected from the growing number of patents based on pharmacophores derived from natural sources. Natural product chemistry offers some significant advantages in comparison to combinatorial synthetic methods now being increasingly used for preparing bioactive compounds of different structural types. The wide range of structures found in terrestrial and marine organisms offer exciting opportunities for the discovery of new pharmacophores which can reveal novel mechanisms to tackle diseases. Volume 32 of "Studies in Natural Products Chemistry" contains 23 comprehensive articles written by international authorities in various fields of natural product chemistry ranging from immunosuppressant and antimalarial compounds to bioactive substances useful in cancer and neural diseases. It is hoped that the present volume, which is the 32nd of the Series, which I initiated in 1988 will again be of great interest to research scientists and scholars working in the exciting field of new drug discovery.

I would like to express my thanks to Mr. Liaquat Raza and Ms. Qurat-ul-Ain Fatima for their assistance in the preparation of the index. I am also grateful to Mr. Wasim Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.

Atta-ur-Rahman Ph.D. (Cantab.), Sc.D. (Cantab.) Federal Minister/ Chairman Higher Education Commission Government of Pakistan

July 2005

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Vll

PREFACE The study of natural products, or "Nature's Combinatorial Library" has had a long history as a source of drugs. In the anticancer area, for example, vinblastine and vincristine, etoposide, paclitaxel (Taxol™), docetaxel, topotecan, irinotecan, the anthracyclines, the bleomycins and the mitomycins are all clinically used natural products or natural product derivatives. In addition to these compounds other natural products or natural product analogs are in advanced clinical trials as anticancer agents, including several epothilones and the halichondrin analog E7389. A review of all new small chemical entities introduced as drugs between 1981 and 2002 showed that 33% of these were natural products or derivatives of natural products, and the figure rises to 49% if synthetic compounds based on natural product models are included (Newman et al., J. Nat. Prod. 2003, 66, 1022). In spite of this impressive record of success, the study of natural products as potential Pharmaceuticals or agrochemicals has lost some favor, particularly within the pharmaceutical industry, as resources were diverted to the newer technique of combinatorial chemistry and other new areas of research. Fortunately, the study of bioactive natural products continues to flourish in universities, research institutes, and selected pharmaceutical companies around the world, and this latest volume in the wellestablished series "Studies in Natural Products Chemistry" bears eloquent testimony to the continued vitality of natural products research. The opening chapter sets the stage with a review of the synthesis of the immunosuppressant FR901483. One of the potential problems with natural products as Pharmaceuticals is that of drug supply, but this chapter demonstrates that moderately complex compounds can be synthesized efficiently. An even more dramatic example of the use of synthesis is that of the halichondrin analog E7389 referred to above, and drug supply is thus less of an issue than it was in the past. The marine environment has become an important source of new structures and new activities, and this is reflected in the next three chapters, which review bioactive natural products from South African marine invertebrates, bioactive marine sesterterpenoids, and antimalarial leads from marine organisms. The ready accessibility of plants ensures that these sources of bioactivity will continue to be thoroughly investigated, and there are chapters covering saponins, iridoids, sesquiterpenoids, cucurbitacins, phthalides, polyisoprenylated benzophenones, and simple benzophenones. Several chapters review the constituents of specific plant genera or families; these include Trypterygium wilfordii, Erythrina, Aristolochia, and the Solanaceae family. It is interesting to note that even well-known compounds and compound classes can provide novel bioactivities. Thus the iridoid geniposide can inhibit angiogenesis, certain cucurbitacins have anti-inflammatory properties, and both garcinol and some withanolides have cancer chemopreventive activity. It is also encouraging to note that a derivative of the well-known compound triptolide is in Phase I clinical trials as an anticancer agent. Of course, not all bioactivities are beneficial; many Illicium sesquiterpenoids are neurotoxic, and aristolochic acid from Aristolochia sp. is responsible for the symptoms of Chinese herb neuropathy recognized in 1992. Microbial sources are making an increasingly important contribution to bioactive natural products, and these sources are represented by chapters on griseofulvin and other

vi halogenated compounds and on bioactive alkaloids from fungi, and by a chapter on metabolites from extremophiles collected in the Berkeley acid mine waste pit. The use of extremophile organisms opens up exciting possibilities for new structures and new activities; in this case novel polyketide-terpenoid metabolites were isolated from a Penicillium sp. with selective cytotoxicity in the National Cancer Institute's 60-cell line screen. The remaining three chapters cover an assortment of topics, from studies of plants used in Bantu medical and magic practices to metabolites from oomycete phytopathogens to isoflavones as functional food components. The variety of the compounds and activities covered in this volume is renewed evidence of the structural and pharmacological diversity of natural products and of the strength of natural products research. This work thus celebrates the truth, to paraphrase Mark Twain, that "reports of the death of natural products research have been greatly exaggerated". The reader is invited to join in the celebration.

David G. I. Kingston Department of Chemistry Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061, USA

IX

CONTENTS Foreword

v

Preface

vii

Contributors

xi

Synthesis of immunosuppressant FR901483 and biogenetically related TAN1251 alkaloids JOSEP BONJOCH AND FAIZA DIABA

3

Bioactive natural products from southern african marine invertebrates MICHAEL T. DAVIES-COLEMAN

61

Bioactive marine sesterterpenoids SALVATORE DE ROSA AND MAYA MITOVA

109

Antimalarial lead compounds from marine organisms ERNESTO FATTORUSSO AND ORAZIO TAGLIALATELA-SCAFATI

169

Bioactive saponins with cancer related and immunomodulatory activity: Recent developments MARIE-ALETH LACAILLE-DUBOIS

209

Chemical and biological aspects of iridoid bearing plants of temperate region NEERAJ KUMAR, BIKRAM SINGH, V.K. KAUL AND P.S. AHUJA

247

Iridoids and secoiridoids from Oleaceae JOSE A. PEREZ, JOSE M. HERNANDEZ, JUAN M. TRUJILLO AND HERMELO L6PEZ

303

Pharmacological activities of iridoids biosynthesized by route II MARINA GALVEZ, CARMEN MARTIN-CORDERO AND MARIA JESUS AYUSO

365

Chemistry and neurotrophic activity of .seco-prezizaane- and anislactone-type sesquiterpenes from Illicium species YOSHIYASU FUKUYAMA AND JIAN-MEI HUANG

395

New insights into the bioactivity of cucurbitacins JOSE LUIS RIOS, JOSE M. ESCANDELL AND M. CARMEN RECIO

429

Griseofulvin and other biologically active halogen containing compounds from fungi T. REZANKA AND J. SPIZEK

471

Bioactive alkaloids of fungal origin HIDEO HAYASHI

549

Chemistry and biological activities of naturally occurring phthalides GE LIN, SUNNY SUN-KIN CHAN, HOI-SING CHUNG AND SONG-LIN LI

611

Chemistry and biological activity of polyisoprenylated benzophenone derivatives OSMANY CUESTA-RUBIO, ANNA LISA PICCINELLI AND LUCA RASTRELLI

671

The benzophenones: Isolation, structural elucidation and biological activities SCOTT BAGGETT, EUGENE P. MAZZOLA AND EDWARD J. KENNELLY

721

Bioactive compounds from Tripterygium wilfordii RENSHENG XU, JOHN M. FIDLER AND JOHN M. MUSSER

773

Bioactive natural compounds from medico-magic plants of bantu area BLANDINE AKENDENGUE, GUY JOSEPH LEMAMY, HENRI BOUROBOU BOUROBOU AND ALAIN LAURENS

803

Bioactive non-alkaloidal constituents from the genus Erythrina RUNNER R.T. MAJINDA, CORNELIUS C.W. WANJALA AND BENARDF. JUMA

821

Chemical constituents and pharmacology of Aristolochia species TIAN-SHUNG WU, AMOORU G. DAMU, CHUNG-REN SU AND PING-CHUNG KUO

855

Chemistry and bioactivity of withanolides from South American Solanaceae ADRIANA S. VELEIRO, JUAN C. OBERTI AND GERARDO BURTON

1019

Bioactive secondary metabolites related to life-cycle development of oomycete phytopathogens MD. TOFAZZAL ISLAM AND SATOSHI TAHARA

1053

Bioprospecting in the Berkeley PIT: Bioactive metabolites from acid mine waste extremophiles ANDREA A. STIERLE AND DONALD B. STIERLE

1123

Isoflavones as functional food components F.R. MARIN, J.A. PEREZ-ALVAREZ AND C. SOLER-RIVAS

1177

Subject Index

1209

XI

CONTRIBUTORS P.S. Ahuja

Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India

Blandine Akendengue

Departement de Pharmacologie, Faculte de Medecine, Universite des Sciences de la Sante, B.P. 7464 Libreville, Gabon

Maria Jesus Ayuso

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

Scott Baggett

Department of Biological Sciences, Lehman College and The Graduate Center, The City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, USA

Josep Bonjoch

Laboratori de Quimica Organica, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028Barcelona, Spain

Henri Bourobou Bourobou

Centre National de Recherche Scientifique et Technologique Herbier National du Gabon, B.P. 13354, Libreville, Gabon, France

Gerardo Burton

Departamento de Quimica Organica and UMYMFOR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2 Ciudad, Universitaria C1428EGA Buenos Aires, Argentina

Sunny Sun-Kin Chan

Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, P.R. China

Hoi-Sing Chung

Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, P.R. China

Osmany Cuesta-Rubio

Instituto de Farmacia y Alimentos (IFAL), Universidad de La Habana, Ave. 23, No. 21425, CP 13600 La Lisa, Ciudad de La Habana, Cuba

Amooru G. Damu

Department of Chemistry, University, Tainan, Taiwan

Michael T. DaviesColeman

Department of Chemistry, Rhodes University, Grahamstown 6140, South Africa

National

Cheng

Kung

Xll

Salvatore De Rosa

Istituto di Chimica Biomolecolare del C.N.R. Via Campi Flegrei, 34, 80078 Pozzuoli (NA), Italy

FaTza Diaba

Laboratori de Quimica Organica, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028Barcelona, Spain

Jose E. Escandell

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

Ernesto Fattorusso

Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli "Federico II", Via D. Montesano, 49, 1-80131, Naples, Italy

John M. Fidler

Pharmagenesis Inc., Palo Alto, CA 94304, USA

Yoshiyasu Fukuyama

Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 7708514, Japan

Marina Galvez

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

Hideo Hayashi

Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan

Jose M. Hernandez

Instituto de Bio-Organica "Antonio Gonzalez", Avda. Astrofisico Francisco Sanchez, 2, 38205, Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez 3, 38205, La Laguna, Tenerife, Spain

Jian-Mei Huang

Beijing University of Chinese Medicine, Beijing 100029, China

MD. Tofazzal Islam

Laboratory of Ecological Chemistry, Graduate School of Agriculture, Hokkaido University, Kita-Ku, Sapporo 0608589, Japan

Benard F. Juma

Department of Chemistry, University of Botswana, P/Nbag UB 00704, Gaborone, Botswana

V.K. Kaul

Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India

xi

Edward J. Kennelly

Department of Biological Sciences, Lehman College and The Graduate Center, The City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, USA

Neeraj Kumar

Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India

Ping-Chung Kuo

Department of Chemistry, University, Tainan, Taiwan

Marie-Aleth LacailleDubois

Laboratoire de Pharmacognosie, Unite de Molecules d'Interet Biologique, UMIB EA 3660, Faculte de Pharmacie, Universite de Bourgogne, BP 87900, 21079 Dijon Cedex, France

Alain Laurens

Laboratoire de Pharmacognosie, UMR 8076 CNRS, Faculte de Pharmacie, Universite Paris XI, rue JeanBaptiste Clement, 92296 Chatenay-Malary, France

Guy Joseph Lemamy

Departement de Chimie-Biochimie, Faculte de Medecine, Universite des Sciences de la Sante, B.P. 7464 Libreville, Gabon, France

Song-Lin Li

Institute of Nanjing Military Command for Drug Control, No. 293, Zhongshan Eastern Road, Nanjing 210002, P.R. China

GeLin

Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, P.R. China

Hermelo Lopez

Instituto de Bio-Organica "Antonio Gonzalez", Avda. Astrofisico Francisco Sanchez, 2, 38205, Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez 3, 38205, La Laguna, Tenerife, Spain

Runner R.T. Majinda

Department of Chemistry, University of Botswana, P/Nbag UB 00704, Gaborone, Botswana

F.R. Marin

Departamento de Quimica-Fisica Aplicada (Area de Tecnologia de Alimentos), Facultad de Ciencias, Universidad Autonoma de Madrid, 2804, Madrid, Spain

Carmen Martin-Cordero

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

National

Cheng

Kung

XIV

Eugene P. Mazzola

University of Maryland-FDA Joint Institute for Food Safety & Applied Nutrition, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA

Maya Mitova

Institute of Organic Chemistry with Centre Phytochemistry, B.A.N., 1113 Sofia, Bulgaria

John H. Musser

Pharmagenesis Inc., Palo Alto, CA 94304, USA

Juan C. Oberti

Departamento de Quimica Organica and IMBIV, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, 5000 Cordoba, Argentina

Jose A. Perez

Instituto de Bio-Organica "Antonio Gonzalez", Avda. Astrofisico Francisco Sanchez, 2, 38205, Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez 3, 38205, La Laguna, Tenerife, Spain

J.A. Perez-Alvarez

Departamento de Technologia Agroalimentaria (Division de Tecnologia de Alimentos), Escuela Politecnica Superior de Orihuela, Universidad Miguel Hernandez de Elche, 03312, Orihuela (Alicante), Spain

Anna Lisa Piccinelli

Dipartimento di Scienze Farmaceutiche, Universita di Salerno, Via Ponte Don Melillo, 84084, Fisciano, Salerno, Italy

Luca Rastrelli

Dipartimento di Scienze Farmaceutiche, Universita di Salerno, Via Ponte Don Melillo, 84084, Fisciano, Salerno, Italy

M. Carmen Recio

Departament de Farmacologie, Facultat de Farmacia, Universitat de Valencia, Vicent Andres Estelles s/n. 46100 Burjassot, Valencia, Spain

T. Rezanka

Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20, Prague 4, Czech Republic

Jose Luis Rios

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

Bikram Singh

Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India

of

XV

C. Soler-Rivas

Departamento de Quimica-Fisica Aplicada (Area de Tecnologia de Alimentos), Facultad de Ciencias, Universidad Autonoma de Madrid, 2804, Madrid, Spain

J. Spizek

Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20, Prague 4, Czech Republic

Andrea A. Stierle

Department of Chemistry and Geochemistry, Montana Tech of the University of Montana, Butte, Montana

Donald B.Stierle

Department of Chemistry and Geochemistry, Montana Tech of the University of Montana, Butte, Montana

Chung-Ren Su

Department of Chemistry, University, Tainan, Taiwan

Orazio TaglialatelaScafati

Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli "Federico II", Via D. Montesano, 49, 1-80131, Naples, Italy

Satoshi Tahara

Laboratory of Ecological Chemistry, Graduate School of Agriculture, Hokkaido University, Kita-Ku, Sapporo 0608589, Japan

Juan M. Trujillo

Instituto de Bio-Organica "Antonio Gonzalez", Avda. Astrofisico Francisco Sanchez, 2, 38205, Universidad de La Laguna, La Laguna Tenerife, Spain: and Instituto de Productos Naturales y Agrobiologia, CSIC, Avda. Astrofisico Francisco Sanchez 3, 38205, La Laguna, Tenerife, Spain

Adriana S. Veleiro

Departamento de Quimica Organica and UMYMFOR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2 Ciudad, Universitaria C1428EGA Buenos Aires, Argentina

Cornelius C.W. Wanjala

Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya

Tian-Shune Wu

Department of Chemistry, University, Tainan, Taiwan

Rensheng Xu

Pharmagenesis Inc., Palo Alto, CA 94304, USA

National

National

Cheng

Cheng

Kung

Kung

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Bioactive Natural Products

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

SYNTHESIS OF IMMUNOSUPPRESSANT FR901483 AND BIOGENETICALLY RELATED TAN1251 ALKALOIDS JOSEP BONJOCH and FAIZA DIABA Laboratori de Quimica Organica, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028-Barcelona ABSTRACT: The review covers the synthetic studies of FR901483 and the biogenetically related TAN 1251 alkaloids.

1. INTRODUCTION The immunosuppressant FR901483 (1, Figure 1) was isolated from the fermentation broth of Cladobotrym sp. No. 11231 by a Fujisawa group in 1996 [1]. The structure was determined X-ray crystallographically, and the absolute configuration was not assigned until Snider achieved the enantiocontrolled total synthesis in 1999 [2]. From a structural point of view, the most conspicuous feature of 1 is an azatricyclic ring system consisting of the combination of the morphan and indolizine nuclei sharing the piperidine ring, namely, 5-azatricyclo[6.3.1.01>5]dodecane. Furthermore, there is a phosphate ester residue, which is essential for the activity of FR901483. OMe

HO. 8

i-J

2J^/3 '1

..

'NHMe

OMe

2

. 10 1

MeHN

(HO) 2 P-O

OMe

OPO(OH)2

FR901483

Figure 1. On the left, FR901483 structure with the replacement nomenclature numbering used in this review. On the right, FR901483 structure showing the heterocyclic system numbering (octahydro-l//-7,10a-methanopyrrolo[l,2-a]azocine) used by several authors.

FR901483 exerts potent immunosuppressive activity in vitro and significantly prolongs graft survival time in the rat skin allograft model, apparently by inhibition of purine nucleotide biosynthesis. This compound is likely to function by a different mechanism from that of cyclosporin A or tacrolimus (FK506), an important feature given the drug-associated side effects of both drugs. It is thought that the role of

CO2H

OH OH

Adenylsuccinate synthetase

inosine monophosphate

H

0H

OH Adenylsuccinate lyase

adenylsuccinate

OH OH adenosine monophosphate

Scheme 1. Adenosine biosynthesis

FR901483 in suppressing the immune system results from an antimetabolite activity whereby adenylosuccinate synthetase and/or adenylosuccinate lyase are inhibited. These enzymes function as key catalysts in the de novo purine nucleotide biosynthetic pathway. Addition of adenosine or deoxyadenosine (but not deoxyguanosine, deoxycytidine, uridine or thymidine) results in elimination of the immunosuppressive activity of FR901483. Thus, FR901483 may inhibit one of the key steps for adenosine biosynthesis (Scheme 1). The TAN1251 series of compounds have a novel tricyclic skeleton containing a l,4-diazabicyclo[3.2.1]octane and a spiro-fused cyclohexanone. It can be believed that they are biosynthetically related to the FR901483, which is probably biosynthesized from modified tyrosine dimer I by oxidative coupling to close the pyrrolidine ring and further elaboration to provide keto aldehyde II (Scheme 2). An intramolecular aldol reaction of the keto aldehyde will lead to the tricyclic skeleton of FR901483, while dienamine formation from the secondary amine and aldehyde will provide TAN1251C (4), which can be isomerized to TAN1251A (2) or reduced to TAN1251D (5).

RO

OMe

'NHMe

P.

-nitrobenzenesulfonylchloride, followed by treatment of the resulting nosylate with CsOAc [16] to yield an acetate, which after reduction with LiAlH4 gave the same alcohol 15 (Scheme 9). Inversion of the configuration at C(9) was accomplished by an initial reaction of alcohol 15 with/?-nitrobenzenesulfonylchloride to selectively form the nosylate of the less hindered equatorial alcohol. The axial alcohol at C(7) was protected as the TBDMS ether and the resulting compound was treated with CsOAc to afford the desired acetate 16 (69%) together with the readily separable elimination product 17. Elaboration of 16 to desmethylamino FR901483 was accomplished by hydrolysis of the acetate to give the alcohol, which was converted to the corresponding dibenzyl phosphite ester [17], which in turn, was oxidized to the phosphate ester 18. Hydrolysis of the TBDMS group and debenzylation of the diphosphate ester afforded the FR901483 analog 19, which was converted to the dipotassium salt to avoid confusion in NMR analysis due to totally or partially protonated samples in the tertiary amine.

12

1./>NO2PhSO2CI 2. CsOAc, 18-crown-6

LiAIH 4 ,THF -78 °C to 65 °C

ep/-13

13

3. LiAIH4

15 ,Ar 1./>NO2PhSO2CI 2. TBDMSOTf 15

TBDMSO,.

3. CsOAc, 18-crown-6 OAc 17

16 1. K2CO3, MeOH 2. Tetrazole, (BnO)2PN(/-Pr)2

,Ar

™

TBDMSQ,

3. m-CPBA OPO(OBn)2

18 Scheme 9. Completion of the synthesis of 19

3.1.2 Synthesis of(-)-FR901483 When Snider started the synthesis of enantiopure FR901483, its absolute configuration was unknown, but he estimated that it might originate from L- rather than D-tyrosine and hence started the enantiocontrolled synthesis of FR901483 using (S)-hydroxylamine 10. The latter was prepared from iV-Boc-L-tyrosine through methylation, cleavage of the Boc group, and conversion of the resulting methyl (5)-O-methyltyrosine to hydroxylamine 10 by Grundke's procedure [18] in 73% yield (Scheme 10). The synthesis was pursued using the methodology developed in the desmethyamino series. The stereoselectivity (6:1) in the formation of isoxazolidine 8 was slightly lower than in the racemic series, probably due to the change of the ethyl ester of rac-10 to the less demanding methyl ester in 10. Hydrogenolysis of the 6:1 mixture of isoxazolidine 8 and the diastereomer gave the lactam 7 and its epimer. The two diastereoisomers were separated after conversion of the corresponding tosylates to the azide 23 and its epimer. Next, 23 was converted to iV-methylcarbamate 24 by hydrogenolysis in the presence of Boc2O [19] and then methylation [20]. Reduction of the ester in 24 with

13 O2Me

EtO2C,,

T9

O,Me

NH 2

NHOH

Ss/Nk^--Ar / \ T | ] CO2Me

1. anisaldehyde

OMe

2. m-CPBA 3.NH2OH.HCI

, (86%)

2. (77%)

(73%)

Ar

45psi H2, Pd/C, AcOH

1-TsCI ^

CO2Me

. NaN3 (78%)

\ < j S " Af f ]I I CO2Me O

X

n 23

Ar = p-OMeC6H4

1. KOf-Bu,f-BuOH 25 °C, 30 min

1.LiBH 4 , THF 2. HCI, AcOH, H2O (97%) N(Boc)Me

3. Boc2O, Et3N (95%) 4. Dess Martin

2. TFA, CH 2 CI 2

6 (PG = Boc) OMe

OMe

NHMe

25 (36%)

OMe

NHMe

26 (16%)

27 (5%)

Scheme 10. Preparation of ketoaldehyde 6 and its aldol reaction

UBH4 followed by an acid hydrolysis gave a deprotected keto alcohol, in which the secondary amine was again protected with BOC2O. Finally, the aldehyde intermediate 6 was obtained using the Dess Martin reagent. The aldol reaction of 6 proceeded analogously to that of the model ketoaldehyde 12, although in lower yield. Treatment of crude 6 with KtBuO in ?-BuOH followed by a cleavage-step of the Boc group provided 41% of a unseparated mixture of the desired aldol adduct 25 and the bad regioisomer 27, compound 26 being isolated in 16% yield. The use of NaOMe in MeOH in this process did not improve the yield of the desired compound 25.

14

Reduction of the 7:1 mixture of 25 and 27 with LiAlH4 afforded the corresponding diol mixture, which after benzyloxycarbonylation and purification give carbamate 28 (52% from 25). For the inversion of the equatorial alcohol at C(9), 28 was chemoselectively converted to its nosylate and the axial alcohol at C(7) protected as the triethylsilyl ether to give 29. The use of TBDMS protecting group, used in the synthesis of 19, was discarded due to its reluctance to undergo cleavage for steric reasons. Compound 29 was treated with CsOAc and 18-crown-6 to displace the nosylate to give acetate 30, obtained together with the undesired elimination compound 31. The remaining steps to reach FR901483 were carried out in virtually the same conditions that were developed in the synthesis of 19. Nevertheless, some processes had to be modified to deal with the problems caused by the presence of the methylamino group at C(3). The acetate 29 was hydrolyzed with K2CO3 in MeOH and then treated with OMe

OMe 1.LiAIH4,THF -78°C->65°C

HQ,

1.p-NO2PhSO2CI

•NHMe 2. Et3N, CbzCI

2. TESOTf

(52%) 28

25

.OMe

,Cbz

CsOAc, 18-crown-6

OMe

TESQ,

OAc

29

30 (70%)

31 (20%)

R = SO 2 PhNO 2 OMe 1. K2CO3, MeOH 1.TBAF 2. HCI

2. (BnO)2PN(/Pr)2, tetrazole; then f-BuOOH

Cbz OPO(OBn)2

32

Scheme 11. Snider's synthesis of (-)-FR901483

3. H2, Pd

FR901483.HCI

15

(BnO)2PN(/-Pr)2 and tetrazole to yield the corresponding phosphite ester. This was converted to the phosphate 32 with MBuOOH [21] instead of mCPB A since the latter reagent easily causes the oxidation of the tertiary amine to the corresponding amine oxide. Finally, hydrolysis of the TES group with TBAF, protonation of the tertiary amine with excess of HC1 and a hydrogenolysis process that allows debenzylation of the phosphate ester as well as deprotection of the secondary amine provided the monohydrochloride salt of 1. In summary, Snider developed the first synthesis of (-)-FR901483 (1) in 2% overall yield from O-methyltyrosine methyl ester in 22 steps establishing the absolute configuration of the natural product. The strategy relies on a 1,3-dipolar cycloaddition from a nitrone, followed by transformation of the resulting bicyclic isoxazolidine into the azaspiranic ring and then by an intramolecular aldol reaction to give the tricyclic ring of the target. The synthesis is completed with adjustments of the oxidation level and configuration as well as the formation of the phosphate unit. 3.2 The approaches of Sorensen and Ciufolini. Synthesis of l-azaspiro[4.5]decan-8-ones from two tyrosine units The other two enantioselective syntheses of FR901483 reported by Sorensen and Ciufolini (sections 3.2.2 and 3.2.3) as well as an approach to the demethylamino derivative by Wardrop (section 4.1) have in common the elaboration of the l-azaspiro[4.5]decan-8-one intermediates by means of an oxidative azacyclization from a nitrogen-tethered phenol derivatives. In the following section an overview of this process is given. 3.2.1 The hypervalent iodine oxidation of nitrogen-tethered phenol derivatives, a straightforward entry to l-azaspiro[4.5]decanediones Phenolic oxidation with organohypervalent iodine reagents [22] constitutes a powerful tool for the synthesis of functionalized 1azaspirodecanes, a process which formally involves generation of an aryloxenium ion and intramolecular capture of this intermediate by Nnucleophiles. An alternative way to achieve the same type of azabicyclic compounds is a dearomatizing spirocyclization triggered by an electrophilic center pendant to the aromatic ring, such as an Nacylnitrenium ion. A particularly direct synthetic entry to subtarget IV would materialize if a phenolic amide or amine such as Ilia or Hlb could be induced to undergo oxidative cyclization to spirodienone IV, which could then be reduced to valuable synthetic intermediates. (Scheme 12).

16

Hr

III

%

Phl(OAc)2 [DIB]

path a; X = O path b; X = H, H

IV

Scheme 12. The spirocyclization of nitrogen-tethered phenols

The desirability of the transformation of Scheme 12 was recognized as early as 1987, when Kita published a pioneering study of the oxidation of phenolic amides with iodobenzene bis(trifluoroacetate) (PIFA) [23]. However, this intramolecular spirocyclization fails due to the propensity of the nucleophilic oxygen atom of the amide to intercept the electrophilic intermediate arising through activation of the phenol (Scheme 13). The preferential formation of spirolactones is probably due to an electronic effect. In most cases non-nucleophilic solvents are required to prevent solvent participations [24]. Bn

Phl(OCOCF3)2 [PIFA]

-i

H,0

Scheme 13. Oxidative Ospirocyclization of phenolic amides

The transformation depicted in Scheme 12 using synthetic equivalents of phenol-tethered amides (path "a") was first carried out successfully by Ciufolini [25,26], who later applied this methodology to his synthesis of FR901483 (3.2.3). Variations of this process starting from JVmethoxyamides were reported by Kikugawa [27-30] and Wardrop (4.1 and 5.3). These transformations involve formation of jV-acylnitrenium ions rather than aryl oxidation and subsequent trapping. On the other hand, a more straightforward route (path "b") using amines was reported for the first time by Sorensen [5] in his FR901483 synthesis (3.2.2). Ciufolini went on to establish an efficient oxidative spirocyclization of phenolic sulfonamides [31]. Ciufolini was able to harness the effects responsible for the reactivity of the oxygen atom of an amide in the oxidative cyclizations by engaging an oxazoline, in which the nitrogen atom acts as the nucleophile [25].

17

Thus, oxidation of oxazoline derivatives of phenolic compounds 33 with IBD in trifluoroethanol leads to spirocyclic amides 34 [26]. The low yield of compound 34b is attributable to the carbamate carbonyl of 33b competing effectively with the oxazoline nitrogen to capture the electrophilic intermediate obtained by activation of the phenol (Scheme 14).

Phl(OAc)2 [DIB] OAc TFE

a b c

H NHBoc NHTs

R, H Bn Bn

Yield 42% 22% 41%

Scheme 14. Oxidative spirocyclization of phenolic oxazolines

On the other hand, the azaspirocyclization of N-acyl-Nalkoxynitrenium ions, which can be generated under mild conditions by the treatment of iV-methoxyamides with iodine (III) reagents, constitutes a very efficient synthetic entry to l-azaspiro[4.5]decanediones.

OMe Phl(CF3CO2)2 [PIFA]

1. f-BuOCI 2. Ag2CO3/TFA

a. inCH 2 CI 2 (72%) ref 10 b. in TFEA (80%) ref 28

(83%) ref27

OMe

Phl(TsO)(OH) [HTIB] (82%) ref 29

OMe

PIFA (77%) ref 30, isolated the OMe A/-Phth derivative

NPhth

38

Scheme 15. Synthesis of azaspirodecanediones from ,/V-methoxyphenylamides

In 1989 Kikugawa reported the intramolecular ipso attack of a nitrenium ion generated from the JV-chloro-./V-methoxyamide of anisole 35, to give the l-methoxy-l-azaspiro[4.5]decadienone 36 in 83% yield [27]. The same author went on to improve this process, which had already been done by Wardrop [10]. Both described the cyclization of the JV-methoxyamide 35 using PIFA in trifluoroethanol (TFEA) [28] and

18

CH2CI2 [10] in good yields (Scheme 15). Kikugawa has also reported the cyclization of the 7V-methoxy-(4-halophenyl)amides 3 7 with [hydroxy(tosyloxy)iodo]benzene (HTIB), the fluoro derivative resulting in the best yield of azaspirodecadienone 36 (82%) [29]. Moreover, Kikugawa has described the cyclization of both 4-methoxy and 4-fluoro derivatives of the corresponding jV-phtalimide analogs (i.e. 38) using the hypervalent iodine reagents PIFA and HTIB, respectively, the azaspirodienone being isolated in 77% and 79% yield [30]. It is worth noting that Wardrop has also successfully explored the Nacylnitrenium route applied to a- and ^-substituted 3-(methoxyphenyl)N-methoxypropionamides (e.g.39) [32]. Using PIFA as the oxidant agent, a 71-face selective azaspirocyclization was found, compound 40 being isolated with an excellent diastereoselectivity (Scheme 16).

PIFA

OMe

MO

^

0 M e

». -78 °C (75%, dr >96%)

Scheme 16. yV-acylnitrenium ion-promoted diastereoselective spirocyclization

Last but not least, the oxidative cyclization of phenolic secondary amines (cf. Illb -> IVb, X = iV-alkyl) is possible, but it remains problematic. This challenging transformation may rightfully be termed the "Sorensen reaction" as it was introduced in the synthesis of FR901483 by this author (see 3.2.2). Yields are often unsatisfactory, and the spirocyclic targets IVb are accompanied by a host of byproducts, although Honda [33] has successfully applied this transformation in his synthesis of TAN1251A (5.5). Such shortcomings are magnified when the amino group in the substrate is primary (Illb, X = NH), rendering the transformation entirely unsuitable for multistep synthetic operations. Ciufolini [31] reported that sulfonamide substrates (Illb, X = NSO2R) can be used to efficiently carry out these hitherto problematic oxidative transformations. Treatment of homotyramine sulfonamides with iodobenzene diacetate (DIB) in hexafluoroisopropanol induces oxidative spirocyclization in high yield, although if a protected amino substituent is included, the process proceeds less efficiently (Scheme 17).

19

Oxidative azaspiroannulation IVb

TS

>r H Phl(OAc)2 (CF3)2CHOH (60%)

Scheme 17. Oxidative spirocyclization of phenolic sulfonamides

3.2.2 Sorensen's synthesis via oxidative cyclization of an amino-tethered phenol Sorensen's synthetic analysis of FR901483 is shown in Scheme 18. The cornerstone of his strategy is the idea that exposure of aminophenol 41 to an appropriate oxidant, such as iodobenzene diacetate, could give rise to 42. This appealing ring-closure would create the azaspiro[4.5]decane substructure of target 1 and would not be complicated by the formation of diastereoisomers. His goal was to effect intramolecular C-N bond N-, 3

per1 Oxidative azaspiroannulation

HQ,,

Aldol to

cyclization

Scheme 18. Sorensen's retrosynthetic analysis of FR901483

FR901483(1)

20

formations with substrates possessing the natural oxidation state at C(3). To construct the remaining ring and C(7) stereocenter of 1, Sorensen used an intramolecular aldol reaction that had been precedented in the syntheses of 2-azabicyclo[3.3.1]nonanes from amide derivatives [2,34], but not from (3-aminoaldehydes. Finally, from 44 Sorensen explored an uncommon strategy for introducing the conspicuous phosphate ester of 1. The starting materials for the synthesis were the known methyl (S)-Omethyltyrosine and the protected aminoaldehyde 45, which was prepared following the multi-step sequence depicted in Scheme 19 (69% yield from the known amino ester intermediate). The protecting group of choice was the Fukuyama-type nitrosulfonamide [35], readily cleavable but stable in the oxidative-cyclization reaction.

1.NO 2 C 4 H 4 SO 2 CI(88%) 2. Mel,K 2 CO 3 (91%) 3. AICI 3 , EtSH (92%) OMe

Scheme 19. Synthesis of phenol 45

The reductive amination between aldehyde 45 and methyl 0-methylL-tyrosine ester gave the amino phenol 41, which incorporates all carbon atoms of the synthetic target FR901483. The crucial transformation in the synthetic pathway was the following oxidative cyclization of aminophenol 41. After much experimentation, it was found that exposure of a solution of 41 in hexafluoro-2-propanol to iodobenzene diacetate at room temperature resulted in the formation of azaspiro[4.5]decadienone 42 in 51% yield (based on 70% consumed 41). The outcome of this ring closure is noteworthy because it constitutes the first example of this type of oxidative cyclization starting from an amino compound (Scheme 20). After changing the protecting group of the exocyclic nitrogen atom, the dienone moiety of 42 was reduced to an epimeric mixture of alcohols 46 using Raney nickel under a hydrogen atmosphere. A reduction of the methyl ester with LiAlH4 in 46 produced a mixture of diols that could subsequently be converted by Swern oxidation into a single ketoaldehyde 43 in which the a-amino aldehyde unit is stereochemically stable [36]. For the closure of tricyclic skeleton of 1 by an aldol cyclization, Sorensen was mindful of the work of Snider and was able to convert 43 into the desired compound 44 in 34% yield using NaOMe in MeOH. When 43 in THF was treated with pyrrolidine and AcOH, the equatorial C(7) epimer of 44 was formed in 51% as the main product.

21

Owing to its rigid and concave architecture, hydroxyketone 44 was converted into diol 47 in a completely diastereoselective fashion by hydrogenation. To achieve a synthesis of 1 from 47, inversion of stereochemistry at C(9) and production of a C(9) monophosphate ester were required. It was possible to accomplish both objectives in a single step by a Mitsunobu reaction between 47 and dibenzyl hydrogen phosphate [37]. Despite its modest yield, this reaction was achieved in the presence of a free C(7) hydroxyl group, and directly produces the natural stereochemistry and the phosphate ester moiety at C(9). Hydrogenolysis of the dibenzylphosphate 48 followed by cleavage of the Boc group afforded FR901483 (1) as its hydrochloride salt.

CO2Me

(51 %)

LUAIH4

Ar

CO2Me

2. Swern (90 %)

H2, Raney Ni (92 %)

(BnO) 2 PO 2 H, (C6H4CI)3P, HO,,,

HO,,, HO.

DIAD, Et3N (37%)

K JVIe Bo c

OP(O)(OBn)2 47

Scheme 20. Sorensen's synthesis of (-)-FR901483

48

1.H 2 , Pd/C 2. HCI (81

FR901483.HCI

(D

22

In summary, Sorensen completed a concise synthesis of FR901483 (16 steps from methyl ester of O-methyltyrosine, 1.5% overall yield) by a strategy that for the first time featured an oxidative cyclization of a phenolic secondary amine (obtained by a reductive coupling of two tyrosine units). The other notable aspect of his synthesis is the one-step conversion of a hydroxyl group to a phosphate unit with an inversion of configuration that shortens the global sequence in comparison with other routes. 3.2.3 Ciufolini synthesis via oxidative cyclization of an oxazolinetethered phenol The synthesis of Ciufolini, published in 2001 [6], is based on the strategy he had previously developed to assemble the spirobicyclic skeleton through an oxidative cyclization of phenolic oxazolines (see 3.2.1). The strategic plan for the synthesis of FR901483 (1) is outlined in Scheme 21 and starts with the same disconnection C(7)-C(8) as used by Snider and Sorensen, involving a retroaldol process which leads to aldehyde 6. This advanced intermediate could be prepared from the spirodienone 49 available from an adequate oxazoline such as 50, which comes from two tyrosine units. IVIH(PG)

50

Scheme 21. Ciufolini's retrosynthetic analysis of FR901483

The construction of the suitable oxazoline 50 requires compounds 51 and 52, both of which may be made from L-tyrosine. For the coupling of amino alcohol 52 and carboxylic acid 51, the Vorbriiggen protocol [38] was the method of choice, since it leads to the desired heterocycle in one step and tolerates an unprotected phenolic function in 51 (Scheme 22). In the key step of this synthesis, oxazoline 50 underwent DIB oxidation/acetylation to 49 through a novel process, which has been mentioned in section 3.2.1. The nature of the protecting group applied to the lateral group in 50 is crucial for the success of the cyclization step. In particular, the Boc blocking group is unsatisfactory because it competes effectively with the oxazoline for the electrophilic intermediate produced

23

through DIB activation of the phenol. The choice of a tosylamide form instead of the nitrosulfonamide for the protection of the pendant amino group allows the iV-protecting group to be retained until the spirolactam reduction step. Although acetylation of the primary product of oxidative cyclization also results in 7V-acylation of the tosylamide, it is unimportant, because both acetyl groups are removed simultaneously at a later stage of the synthesis. Conversion of dienone 49 to the corresponding cyclohexanone was carried out by hydrogenation in the presence of PtC>2, while other hydrogenation catalysts (Pd or Rh) also provided variable quantities of a rearomatized product. A straightforward series of reactions advanced intermediate 53 to keto aldehyde 6, which constitutes the substrate for the crucial aldol cyclization leading to the intermediate 55. Ciufolini optimized the regio- and diastereoselectivity for this stage using sodium methoxide in 90% aqueous methanol, bridged compound 55 being obtained in 44%. It should be noted that aldehyde 6 is fairly resistant to epimerization at C(6), minimizing the probability of formation of aldol isomers possessing the undesired C(6) (.^-configuration. This stereochemical stability, first recorded by Snider, is consistent with observations by Myers and Garner concerning the configurational stability of amino acid-derived aldehydes [36]. The final sequence that produced the first Ciufolini approach to FR901483 commenced with reduction of the ketone in the acetyl derivative of 55 to the corresponding diol 56. The shape of the molecule disfavors the approach of reducing agents from the top face of the ketone, so that the desired C(9) axial carbinol is not directly available. The reduction was achieved by the use of L-selectride, in the hope of obtaining at least some of the correct carbinol diastereomer, but the reaction occurred with complete diastereocontrol in favor of the equatorial alcohol 56. Inversion of the C(9) configuration was achieved by Snider's method, via />-nitrobenzenesulfonate ester, and the resulting acetate 57 was obtained in a satisfactory 73% yield, the elimination product also being isolated (12%). Global deprotection/reduction of 57 was achieved in high yield by vigorous LiAlH4 treatment, and the emerging secondary amino group was protected as a benzyl carbamate prior to selective phosphorylation of the C(9) carbinol, without protection of the rather hindered C(7) alcohol. Final hydrogenolysis of all benzyl groups in 58 in the presence of aqueous HC1 provided the bis-hydrochloride of 1. Sorensen's synthesis of 1 demonstrated the feasibility of a Mitsunobutype inversion of the configuration of the C(9) carbinol in substrates similar to 59. This allowed Ciufolini to simplify his own synthesis as shown in Scheme 22 (on the right). Thus, LiAlH4 reduction of compound 55 produced a 6:1 mixture of equatorial, 59, and axial amino alcohols.

24

1.Phl(0Ac)2 CF3CH2OH 2.Ac2O, Py (41 %)

lyie

TPAP, NMO

first generation

-prenyloxybenzaldehyde [70] gave a mixture of aldol products, which were treated with MsCl. The resulting unseparated mesylates were submitted to an elimination process to steroselectively give the Z isomer. Reduction of the lactam with A1H3 gave an improved yield of the corresponding enamine, which finally was deprotected to provide (-)-TAN1251A. In summary, the enantioselective total synthesis of (-)-TAN1251A was accomplished by Wardrop in 17 steps and with 3% overall yield, the key step being the N - m e t h o x y -iV-acylnitrenium ion-induced spirocyclization. H

MeO,c \ HN-OMe

1.CIC0 2 Me 2. NaOH

LNaOH

3. Me2SO4

2. MeONH2.HCI, DCC HOBT, NMM

(76%) O MeOPhl(OCOCF3)2, CH2CI2, MeOH;

"

then H2O

2. (CH2OH)2

(69%)

0 M e

1.LiAIH4 2. CICO2Bn 3. Zn, AcOH 4. BrCH2CO2Bn

(86%) (43%)

lyie /H Cbz

BnQ >=O

1. LDA, ArCHO (80%) 2. MsCl; then Kf-BuO (75%)

1. H2, Pd/C 2. DPPA

3. AIH3 4. HCI

(57%)

148

119

Scheme 45. Wardrop's synthesis of (-)-TAN1251A

(61%) (-J-TAN1251A (2)

51

5.4 Ciufolini's synthesis of (+)-TAN1251C Ciufolini published in 2001 [6b] the synthesis of enantiopure TAN1251C (4) using an advanced intermediate of his FR901483 synthesis (3.2.3). The point of divergence of both synthesis is the keto lactam 54 (Scheme 22). The synthesis of 4 involves ten initial steps to achieve alcohol 54 from tyrosine (16% overall yield) and pursues with the change of the alkyl side chain on the phenolic hydroxyl group (Scheme 46). Thus, Odemethylation of 54 followed by prenylation and treatment of the resulting lactam with LiAlH4 gaves azaspiranic compound 149. Protection of the secondary amine and oxidation of both alcohols using TPAP/NMO lead to the keto aldehyde 150. Treatment of the latter with Cd/Pb couple [78] allows the deprotection of the secondary amine as well as the cyclization process to provide (+)-TAN1251C (4).

(10 steps, 16% overall yield)

see Scheme 22

Me-"1

OMe

1. BBr3 2. Prenyl bromide 3. LiAIH4 OH 149

CHO 1. TrocCI (51%, from 54)

Troc ,NI\/|e

RQ-

2. TPAP, NMO (63%)

Cd/Pb couple NH 4 OAc (79%)

150

(+)-TAN1251C (4)

Scheme 46. Ciufolini's synthesis of (+)-TAN1251C

In summary, the second enantiocontrolled synthesis of (+)-TAN1251C requires a maximum of 16 linear steps from L-tyrosine (4% overall

52

yield). The process not only uses the same methodology developed in the Ciufolini's FR901483 synthesis but even utilizes a common synthetic intermediate, which is a polyfunctionalized azaspirodecane (i.e. 54) that is obtained by aromatic oxidation of an oxazoline phenolic derivative to achieve the spirolactam unit. 5.5 Honda's Approach to (-)-TAN1251A In 2002, Honda developed a fruitful new synthetic entry to the known azatricyclic compound 119, which has led to a synthesis of (-)TAN1251A [33]. The retrosynthetic pathway is shown in Scheme 47, the synthetic target being the intermediate 119, which had previously been converted into TAN1251A by Kawahara and Nagumo [63] and Wardrop [64]. The strategy employed by Honda to form the spirocenter involved a hypervalent iodide-promoted phenol oxidation reaction, as in the Wardrop and Ciufolini syntheses, but on the more advanced intermediate 151, with the piperazine ring already incorporated. Compound 151 could be prepared by a coupling of L-tyrosine and glycine derivatives, the former acting as the chiral source to construct the TAN 1251A platform, followed by a lactamization process. Q

JVIe

ref63, 64

L-Tyrosine

TAN1251A

Glycine

119

152

151

Scheme 47. Honda's approach to the synthesis of (-)-TAN1251A

The starting material for the synthesis (Scheme 48) was the tyrosine aldehyde 154, which was readily prepared from the methyl ester of Ltyrosine. Reaction of the latter with ethyl chloroformate followed by Obenzylation of the phenol gave ester 153, which was reduced, reprotected at the nitrogen atom, and treated with the Dess-Martin reagent to give aldehyde 154. The construction of the piperazine ring was carried out through a reductive amination of 154 with glycine methyl ester, followed by deprotection of the nitrogen and lactamization of diamino ester 155. Hydrogenolysis of the benzyl ether group afforded the amino phenol 151 required for the next crucial transformation.

53

At this point, Honda focused on the critical step of the synthesis, the oxidative transannular cyclization to close the pyrrolidine ring, which involves the simultaneous construction of the quaternary center of the target. The oxidative cyclization of 151 in 2,2,2-trifluoroethanol with bis(acetoxy)iodobenzene (DIB) gave the desired spirocompound 152 in 43% yield, which was increased up to 69% by using hexafluoroisopropanol as the solvent. The successful cyclization leading to 152, featuring an unprotected aminophenol derivative, is the second time Sorensen's reaction (2.2) has been used. To achieve the Wardrop intermediate 119 and hence the formal synthesis of (-)-TAN1251A, the reduction of dienone 152 and an acetalization process were required. Although difficulties were initially encountered in obtaining the reduced compound by catalytic hydrogenation, Honda found that the dienone could be converted to the ketone using triethylsilane in the presence of CuCl and DPPF (20% mol of each) [79]. Finally, acetalization afforded the known intermediate 119. In summary, Honda reported a formal synthesis of (-)-TAN1251A in which a hypervalent iodine-promoted cyclization of a tethered secondary amine and phenol was used in the key step for the elaboration of the tricyclic skeleton.

NH2CH2CO2Me, NaCNBH3 2. BnBr (77%)

OBn

2. Boc2O 3. Dess Martin

OBn

2. TFA

153

(73%)

154

(83%)

H

Phl(OAc)2, (CF3)2CHOH (69%)

r^HVIe

1.Et3SiH, CuCI(60%)

TAN 1251A (2)

2. (CH2OH)2 (66%)

119

Scheme 48. Honda's formal synthesis of (-)-TAN125lA

54

5.6 The Kawahara-Nagumo approach to (-)-TAN1251A from a proline derivative In 2002, four years after publishing the first paper describing the synthesis of racemic TAN 1251 A, Kawahara and Nagumo reported a formal synthesis of the same antimuscarinic compound but in its enantiopure form [62]. Unlike the previous enantioselective syntheses where tyrosine was the chiral source, Kawahara and Nagumo chose a proline derivative as the starting material. The retrosynthetic pathway is outlined in Scheme 49 and starts with the simplification of the TAN 1251A again to the known intermediate 119, whose first conversion into the alkaloid had been reported by the same authors [63]. The target compound 119, in turn, can be prepared from 126 also following their known protocol. Compound 126 should be obtained from 156 via installation of an azide group and N-alkylation. Disassembly of the cyclohexanone ring from 156 in a retrosynthetic sense leads to keto aldehyde 155, which may be prepared by alkylation of proline 154 followed by some functional group interconversion steps.

Boo-

119

126

156

155

154

Scheme 49. Proline-based approach to (-)-TAN1251A by Kawahara and Nagumo

The synthesis starts with the alkylation of the trans-4-hydroxy-Lproline derivative 154 with 4-iodo-l-butene using 2.5 eq of LDA to afford 157 (67%) and its epimer (15%). Each one is useful for the synthesis of 156 since after hydrogenation of 159 the stereogenicity at spirocenter is lost. The sequence 157 -> 156 was carried out separately in both epimeric mixtures obtaining similar yields. The synthetic sequence, which is depicted in Scheme 50 for the major epimer 157, involves conversion of the latter to aldehyde 158 through a reduction-oxidation process, followed by Wacker oxidation of the terminal alkene to give the keto aldehyde 155. Aldol condensation and succesive elimination process in the ketol intermediate gave cyclohexenone 159, whose hydrogenolysis followed by reaction with ethylene glycol and a desilylation step afforded hydroxy acetal 156. Alcohol 156 was converted to the corresponding azide through a Mitsunobu-type process, and after

55

removal of the Boc group and alkylation of the secondary amine, polyfunctionalized azaspiranic compound (+)-126 was achieved. The synthesis was then pursued using the same methodology already described by these authors in the racemic series. Compound 126 was converted into tricyclic amide (+)-119 by the sequence of catalytic hydrogenation, saponification of the ester group, lactam ring formation and methylation. Since Wardrop had reported the conversion of (+)-119 into (-)TAN 1251A using the procedure already described by Kawahara and Nagumo in the racemic series, the enantiocontrolled synthesis of (+)-119 achieved by these authors constitutes a new formal synthesis of 2. This approach is noteworthy for its use of a proline derivative as the starting material and the elaboration of the carbocyclic ring by an aldol reaction. TBDPSQ

LDA, HMPA, 4-iodo-1-butene

^ N

TBDPSQ 1.DIBAH 2. TPAP, NMO

(67%)

(80%)

Boc 154

.OTBDPS

.OTBDPS TBDPSQ C H 0

Boc

Wacker oxidation

Boc—I 0H,

(92%)

158

1.H 2 , Pd(OH)2

1. DPPA, DEAD 2. CF3CO2H

Bo


ATLANTIC OCEAN 0 21) "E

30'F;

500 KILOMETRES

Fig. (1) Map of southern Africa indicating the three bio-geographical zones, ocean currents and sites of major marine invertebrate collections (A = Cape Peninsular, B = Tsitsikamma Marine Reserve, C = Algoa Bay, D = Coffee Bay, E = Aliwal Shoal, F = Sodwana Bay, G = Ponto do Ouro)

Marine Natural Products Research in Southern Africa - A Brief Historical Perspective Regions of high species diversity and endemism can offer rich rewards for marine natural products chemists in search of bioactive compounds with possible medicinal properties. The potential for South Africa's marine resources to provide a source of new pharmaceuticals was first recognized over thirty years ago in a South African government report entitled "Drugs from the Sea" that concluded with the following statement, "Very little attention has yet been paid in South Africa to the recovery of drugs from the sea. This field offers exciting and rewarding challenges to South African scientists. Once research brings down the unit cost, the sea may offer a vast potential for the production of drugs for South Africa [6]." Prior to this report, South African contributions to the

64

chemistry of marine organisms were modest and comprised a series of research publications emanating from the National Chemistry Research Laboratory in Pretoria describing the characterization of highly unsaturated fatty acids and alcohols from fish oils [7-9] and the pioneering marine algal polysaccharide structural research of Nunn and co-workers at Rhodes University [10-12]. The first studies in South Africa of the chemistry of southern African marine invertebrates began at the University of Cape Town in 1977 under the direction of Elsworth and Cragg who focussed on the natural products chemistry of selected marine organisms collected around the Cape peninsular, the acknowledged dividing line between the cold temperate west coast and the warm temperate south east coast marine fauna and flora [5]. Their initial research involved a GC-MS study of the complex sterol profiles of two oceanic and three intertidal species of annelid worm [13] the ribbed mussel Aulacomya ater, sea stars Marthasterias glacialis and Henrica ornate, and the sea cucumber Cucumeria frauenfeldi [14, 15]. Interestingly, South African marine worms and coelenterates also attracted the attention of international researchers at this time and Mazzanti and Piccinelli compared the occurrence of indole and imidazole compounds in six species of southern African annelid worms and six species of coelenterates [16]. In contrast to the increased global interest in marine natural products as a source of potential pharmaceuticals in the 1980s, the natural products chemistry of southern Africa's marine resources was largely ignored for most of this decade. However, in the last fifteen years, this situation has been reversed. Four research groups have provided a significant insight into the chemistry of biologically active natural products from southern Africa's marine fauna and flora and the contributions of the research groups led by George Pettit, Yoel Kashman, the late John Faulkner and myself, predominate in the following review. BIOACTIVE METABOLITES FROM SOUTHERN AFRICAN MARINE WORMS (PHYLUM HEMICHORDATA) Cephalostatins The most significant group of bioactive metabolites to be isolated from southern African marine worms have undoubtedly been the powerful cell growth inhibitors, cephalostatins 1-17 (1-17), isolated by Pettit et al. from

65

the marine tube worm Cephalodiscus gilchristi (Class Pterobranchia) initially collected off the south east of southern Africa in 1972 and again in 1981 [17-24]. The cephalostatms are closely related to the ntterazines isolated by Fusetani et al. from the Japanese ascidian Ritterela tokioka [25]. The trisdecacyclic pyrazine structure of cephalostatin 1 (1) was eventually solved by X-ray analysis many years after the initial collections of C. gilchristi [17]. The structures of the related cephalostatins 2-4 (2-4) essentially followed from comparison of the spectroscopic data with those of 1 [18]. All four compounds exhibited "7

Q

1

similar exceptional ED50 values in the range 10" -10" |J.gmL~ in the US National Cancer Institute's (NCI's) P-388 lymphocytic leukaemia cell line screen.

1 2 3

R1 = R = H R1 = OH, R2 = H R1 = OH, R2 = Me H

OH

66

Conversely, cephalostatins 5 and 6 (5, 6), in which ring C of the left hand steroid unit is aromatized, exhibited reduced activity in the PS system ( 1 0 - 1 0 ugmL") suggesting the importance of C - D' ring junction and possibly C-22' spiroketal structural integrity to the cytostatic properties of the cephalostatins [19]. OH

5 6

R = Me R =H

Further clues pertaining to the structure activity relationships within this intriguing group of metabolites were provided by the isolation of further compounds in this series from C. gilchristi. Cephalostatins 7-9 (79) and 1-4 displayed almost identical activity and selectivity (TI50 values of 0.1 -1 nM) against a series of cancer cell lines (including non-small cell lung HOP 62, small cell lung DMS-273, renal RXF-393, brain U-251 and SF-295, leukaemia CCRF-CEM, HL-60 and RPM1-8226) in the NCI's in vitro 60 cell line primary screen [20]. Not unexpectedly, concurrent screening of cephalostatins 5 and 6 revealed that these two compounds only exhibited modest activity against two cell lines, CNS U251 and renal SN12K1 [20]. The conclusion drawn from this profile of activity was the apparent importance of the pyridizine right hand side unit to the bioactivity of the cephalostatins, and that minor changes in the left hand side E' and F' rings does not significantly reduce this activity [20].

67

HO

A re-collection (450 kgs wet weight) of C. gilchristi in 1990, for possible pre-clinical development of 1, led to the isolation and identification of seven more cephalostatin analogues. The differential cyctotoxicity of cephalostatins 10 and 11 (10-11), in the NCI's anticancer screens mirrored that of 1 [21]. Further compounds isolated from this re-collection included the first symmetrical cephalostatin, cephalostatin 12 (12), and the C-l' hydroxylated derivative of 12, cephalostatin 13 (13) [22]. Interestingly, while 12 and 13 showed substantial growth inhibitory activity against many of the NCI's cancer cell lines, the GI50 values obtained from these assays were on average

68

orders of magnitude (400 and 1000 nM respectively) higher than those of 1 (1 nM). OH

R ! = H , R2 = OMe

OH

13 R = OH

Comparative evaluation of 1 and cephalostatins 14 and 15 (14, 15) in the NCI in vitro primary screen again revealed a reduced average GI50 cytotoxicity (100 nM and 68 nM respectively) for 14 and 15 compared to 1 [23]. The final pair of cephalostatins (16 and 17) isolated from C. gilchristi exhibited the same cyto toxic profile as the majority of the other cephalostatins with the panel averaged GI50 values of 16 (lnM) and 17 (4nM) comparable to that of 1 (lnM) [24].

69

14 R = H 15 R = Me

17

Cephalostatin 1 (repeatedly the most active in the series) has recently been shown to induce a novel pathway of receptor-independent apoptosis of leukaemia cells at nanomolar concentrations [26]. Given the unique profile of activity of 1 in the NCI 60 cell line screen, this compound was identified in 2000 as a Rapid Access to Intervention Development (RAID) project by the NCI [27]. Unfortunately, preclinical development

70

of 1 was hampered by the poor availability of this compound from natural sources (ca. lOOmg of 1 isolated from one ton of C. gilchristii) and although numerous cephalostatin analogues have been synthesized [28], there are relatively few syntheses reported for the naturally occurring cephalostatins [29]. Fuchs et a/.'s synthesis of 1 (from the abundant plant derived steroid, hecogenin acetate) provides possibly the best route for the production of multi-gram amounts of the cephalostatins to alleviate the supply problem [30]. BIOACTIVE METABOLITES FROM SOUTHERN AFRICAN MARINE SPONGES (PHYLUM PORIFERA) Although, marine sponges are a prolific component of both inter-tidal and sub-tidal southern African marine ecosystems they are poorly described and the number of species occurring along the southern African coast is unknown [31]. It is noteworthy that our recent investigations of the natural products chemistry of southern African members of the sponge family Latrunculiidae have contributed to the discovery of five new species and the raising of two new genera [32,33]. Spongiostatins Pettit et al. initiated the discovery of potential anti-cancer compounds from southern African marine sponges with the isolation of the potent spongiostatins 4 - 9 (18 - 23)[34-36] from the bright orange "wallsponge" Spirastrella spinispirulifer (Family Spirastrellidae), a large, conspicuous sponge common on sub-tidal reefs along both the cool and warm temperate south west and south east coasts of southern Africa [31]. Spongiostatins 4-9 are related to the first three compounds isolated in this series, spongiostatin 1-3 (24-26)[37,38] isolated from a different species of sponge, Spongia sp. (Family Spongiidae) collected off the Republic of the Maldives, a group of islands in the northern Indian Ocean. The family of spongiostatin compounds can be broadly divided into two structural groups with the presence or absence of a tetrahydrofuran ring in the macrocycle defining the difference between these two groups. The members of the first group (18, 20, 24-26) without a tetrahydrofuran ring differ from each other in the substitution pattern at C-5, C-15 and C-50

71

while those in the latter group (19, 21-23) only differ in substitution at C5 and C-50.

OMe

OH

H I OH OH

^ C l , R2 = Ac, R 3 = H 18 20 R1 = R3 = H, R2 = Ac 24 R1 = Cl, R2 = R3 = Ac 25 R1 = H, R2 = R3 = Ac 26 R1 = Cl, R2 = H, R3 = Ac

The structures of 24 and 25 were found to be consistent with the structures of altohyrtin A and C respectively, isolated from an unrelated Hyrtios sponge, and reported by Kobayashi and Kitagawa [39, 40]. Initial discrepancies in the assignment of the absolute stereochemistry of the spongiostatins and altohyrtins were eventually resolved in 1997 by the first total synthesis of altohyrtin C/spongiostatin 2 by Evans et al. [41], followed by the total synthesis of altohyrtin A/spongiostatin 1 by the Kishi group [42]. The structures of compounds 18, 20, 24 - 26 presented here are drawn with the revised stereochemical assignments [43]. Although these revised assignments could probably be extrapolated to compounds 19, 21 - 23, the original stereochemistry proposed by Pettit et al. is retained here.

72

.0 OMe

H 1 OH OH

OH

19 R = C 1 , R = H 21 R1 = H, R2 = H 22 R1 = H, R2 = Ac 23 R1 = Cl, R2 = Ac The trace amounts of the spongiostatins (ca. 10"7% yield) present in S. spinispirulifer required increasingly excessive and environmentally controversial recollections of this sponge (e.g. 2409 kgs) during the 1980s for the investigation of their potent in vitro activity (Table 1) [34]. The antineoplastic mechanism of action of the spongiostatins is well established. Spongiostatin 1 blocks microtubule assembly through inhibition of the glutamate-induced polymerization of tubulin [44]. Microtubules maintain cell shape and are an integral part of the mitotic spindle necessary for cell division. The collapse of microtubule assembly therefore halts cell division and leads ultimately to cell death [45]. The competition provided by the plethora of tubulin inhibitors presently in clinical development [45] and a paucity of spongiostatin 1 has hampered the preclinical development of this compound [46]. Paterson et a/.'s synthesis of 24 (1% yield from 33 steps) presently provides the most economic solution to the supply problem [47].

73 Table 1 T h e isolated yields of spongiostatins 1 - 9 and their cytoxicity in the NCI's 60 cell line screen and as an inhibitor of tubulin polymerization [43].

Yield

Compound

Spongiostatin 1

3.5xlO' 6 % (13.8mg)

GIso NCI 60 cell line screen

ICso inhibition of tubulin polymerization

1.3xl0"10M

5.3xlO" 6 M

Spongiostatin 2

1 xlO" % (4.3mg)

8.5xl0-'°M

4.6xlO" 90% inhibition at a concentration of 50 JJM) were comparable to those observed, vide supra, for the structurally related tsitsixenicins.

AcO

114 Tetraprenyltoluquinols Of the soft corals present on the coral reefs of Kwazulu-Natal and southern Mozambique, three genera, Sinularia, Sarcophyton and Lobophytum predominate [90]. Two soft corals, Sinularia dura (Family Alcyoniidae) and a Nephthea sp. (Family Nephtheidae) collected in Sodwana Bay yielded four tetraprenyl-toluquinols (116 - 119) [104]. The

95

structures of these compounds were established from spectroscopic data with nOe correlations providing the relative stereochemistry of the dioxabicyclooctane moiety in the side chain of sindurol (116). Acid hydrolysis of nephthoside (118) gave the aglycone, 117, and D-arabinose. The stereochemistry of the pentose was established from its optical rotation. Sindurol was twice as toxic to P-388 mouse leukaemia cells (IC50 - 2.1p,M) when compared with compounds 117-119 (IC50 = 5 |oM) [104]. AcO

OH

118 R = H 119 R = Ac

Sarcoglane and Flaccidoxides Two common in-house bioassays to screen for metabolites exhibiting general cytotoxicity, the fertilized sea urchin egg cytotoxicity assay and the brine shrimp lethality assay, were used to identify bioactive diterpene metabolites in two soft coral species collected from Sodwana Bay and Ponto do Ouro respectively. Sodwana Bay specimens of Sarcophyton

96

glaucum (Family Alcyoniidae) yielded the tricyclo-[7,5,010'14]-tetradecane diterpene sarcoglane, (120) [105] while Ponto do Ouro specimens of Cladiella kashmani (Family Alcyoniidae) gave the known flaccidoxide (121) [106], and the related cembrane diterpene (122) [107], in addition to flaccidoxide-13-acetate (123) [108]. The structures of all these diterpenes were solved by analysis of spectroscopic data. The relative stereochemistry of 120 followed from coupling constant analysis and nOe data while a combination of Mosher's method and molecular modelling studies established the absolute stereochemistry of 121.

OH

120

121

R1 = OH, R2 = OAc

122 R1 = H, R2 - OH 123 R ! =R 2 BIOACTIVE METABOLITES FROM SOUTHERN AFRICAN MARINE MOLLUSCS (PHYLUM MOLLUSCA) In southern Africa, nearly three-quarters of the 381 known species of marine opisthobranch molluscs belong to the order Nudibranchia [5]. In contrast to shelled marine molluscs (prosobranchs), nudibranchs reflect an evolutionary trend towards the complete loss of the shell [109]. Forgoing the obvious protection against predation provided by a shell, nudibranchs are generally brightly coloured as a warning to potential predators that they are non-palatable. The non-palatability of nudibranchs is attributed to bioactive natural products, which they selectively sequester from their diet and store in glands lining their mantle tissue [109]. Accordingly, nudibranchs prey on a variety of organisms rarely fed upon by other animals (e.g. sponges and octocorals) because of the toxic metabolites produced by these organisms. Therefore natural products derived from nudibranchs generally have an implied bioactivity and the structures of 27

97

metabolites isolated from three nudibranch species collected off the coast of southern Africa are presented here. Variabilins and Nakafurans There are no reports of natural product investigations of nudibranchs collected along the west coast of southern Africa. The endemic nudibranch Hypselodoris capensis is a colourful member of the family Chromodorididae and the linear p-substituted sesterterpenes (18R)variabilin (124) [110], 22-deoxyvariabilin (125) [111] and 22-deoxy-23hydroxymethylvariabilin (126) in addition to the sesquiterpenes nakafurans 8 and 9 (127, 128) [112], were isolated from specimens of H. capensis collected in the Tsitsikamma Marine Reserve [113]. Compounds 124 -126 were also isolated from one of//, capensis' dietary sponges (Fasciospongia sp.) . A further investigation of the sponges preyed upon by H. capensis established a Dysidea sponge as the source of the nakafurans sequestered by the nudibranch [114]. Standard spectroscopic techniques established the structures of the compounds isolated from H. capensis. Variabilin is a common metabolite of Dictyoceratid sponges and possesses anti-microbial [115], anti-tumor [116], anti-inflammatory [117] and icthyotoxic [118] properties. Nakafurans 8 and 9 have also been shown to be icthyotoxic [112].

o

124

R1 = OH, R2 = Me, R3 = a-Me

125

R 1 = H , R 2 = R3 = Me

126

R1 = H, R2 = CH2OH, R3 = Me

127

128

98

Millecrones and Millecrols The endemic nudibranch Leminda millecra is the single representative of the family Lemindidae occurring off the southern African coast. There have been two investigations of the natural products sequestered by this species. The first investigation, by Pika and Faulkner, of L. millecra specimens collected from Coffee Bay yielded the sesquiterpenes millecrone A (129), millecrone B (130), millecrol A (131) and millecrol B (132) [119]. The structures of compounds 129 - 132 were delineated from NMR and mass data while their relative stereochemistry was delineated from nOe data. Of the four metabolites only millecrone A inhibited the growth of Candida albicans (50|ag/disk). Millecrone B was active against Staphylococcus aureus and Bacillus subtilus (50|j.g/disk) and millecrol B was only active against the latter bacterium at similar concentrations. A diversity of soft coral spicules (from Alcyonium foliatum, A. valdivae and Capnella ihyrsoidea) found in the digestive glands of the Coffee Bay specimens of L. millecra suggested a possible soft coral source for the sequestered metabolites 129-132 [119].

130

131

132

Sesquiterpenes, Triprenylquinones and Triprenylhydroquinones L. Melinda is particularly abundant in Algoa Bay and specimens collected from this region of the southern African coast yielded a plethora of diverse metabolites including 129, 130, isofuranodione (133)[120], (+)-8hydroxycalamenene (134)[121], algoafuran (135), cubebenone (136) and seven closely related triprenylquinones and hydroquinones (137 - 143) [122]. The structural elucidation of these compounds was accomplished from spectroscopic data. Gas chromatographic analysis of several octocorals preyed upon by L. millecra in Algoa Bay confirmed that the

99

sea fan Leptogorgia palma was the source of 130 and 136 and an unidentified Alcyonium soft coral was the origin of 129 [122]. OH

OAc

o 133

135

134

o

136

OH

138 139 R = OH

o 140

141

100

Hamiltonins, Lati unculins and Spongiane Diterpenes Four unusual chlorinated homo diterpenes, hamiltonins A-D (143 - 146), the sesterterpene hamiltonin E (147) and the toxins latrunculin A and B (148, 149) [123], were isolated from the brightly coloured dorid nudibranch Chromodoris hamiltoni (Family Chromodorididae) collected from the Aliwal shoal [124]. Since latrunculin A and B were initially shown to cause concentration dependent changes in cell shape and actin organization [125], they have now become the most widely accepted tools for exploring the inhibition of actin polymerization in molecular biology [126].

o o

147

101

149

148

Latrunculin B was also present in extracts of C. hamiltoni collected from the reefs off southern Mozambique. The latter specimens of C. hamiltoni also yielded two spongiane diterpene lactones (150, 151) [127]. No evidence of the hamiltonins was found in extracts of the Mozambique specimens of C. hamiltoni and possibly reflects geographical variation in the organisms that make up C. hamiltonVs diet in this region of the southern African coast. The structures of both spongiane diterpenes and the hamiltonins A -E were determined from spectroscopic data while the absolute stereochemistry at C-16 in 147 was established by comparison of its CD data with those of related compounds [127].

AcO,

12 H

OAc

OAc OAc

150 151 A 12

152 R = H 153 R = OAc

102

Diterpenes Finally, only one member of the marine pulmonate gastropod family Timusculidae, Trimusculus costatus, occurs along the intertidal zone in the warm temperate region of southern Africa. Two bioactive diterpene acetates 152 and 153 were isolated from this species and their structures and relative stereochemistry established from recourse to nOe and other spectroscopic data [128]. Both compounds were found to deter feeding of Pomadasys commersonni, a common, omnivorous intertidal and subtidal southern African fish at natural concentrations (ca. 2.5 mg/pellet) [128]. ABBREVIATIONS CD ECE HMBC NCI nOe SKB SIO RAID RU

= = = = = = = = =

circular dichroism endothelin converting enzyme heteronuclear multibond coherence National Cancer Institute nuclear Overhauser effect SmithKline Beecham Scripps Institution of Oceanography Rapid Access to Intervention Development Rhodes University

ACKNOWLEDGEMENTS I thank Dr Gordon Cragg of the Natural Products Branch at the NCI for helpful discussions and Professor Douglas Rivett and Dr Rob Keyzers for helpful editorial comments. Financial support from the South African National Research Foundation and the Department of Environmental Affairs and Tourism is gratefully acknowledged. REFERENCES [1] [2] [3] [4]

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

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BIOACTIVE MARINE SESTERTERPENOIDS SALVATORE DE ROSA1 AND MAYA MITOVA2 Istituto di Chimica Biomolecolare del C.N.R. Via Campi Flegrei, 34; 80078 Pozzuoli (NA), Italy. Institute of Organic Chemistry with Centre of Phytochemistry, B.A.N., 1113 Sofia, Bulgaria. ABSTRACT: Terpenoids are b y far t he 1 argest c lass of natural products. Within this class of compounds, the sesterterpenes form a rare group of isoprenoids, which occur in widely differing source. Marine organisms have provided a large number of sesterterpenoids, possessing novel carbon skeleton and a wide variety of biological activities. The more significant sesterterpenoids from marine organisms, which show biological activities, are reported and they are grouped in a biogenetic sequence. Natural products that do not contain 25 carbon atoms but are obviously sesterterpene derivatives are also included. The anti-inflammatory activity is the most relevant among the biological activities observed for marine sesterterpenoids. The high potential for some of these products suggested that they could be developed as drugs for the treatment of inflammation. The different directions that can be taken to obtain quantities of secondary metabolites are reported.

INTRODUCTION Natural products play a dominant role in the discovery of leads for the development of drugs for the treatment of human diseases. It should be realised that the bioactive compounds, which are synthesised in nature to protect a given organism, had been selected from a wide variety of possibilities and were under the pressure of evolution for several hundreds of million years to reach an optimal activity. The terpenoids (isoprenoids) are a class of secondary metabolites that may be formally considered to be constructed from the five-carbon isoprene unit [1]. The terpenes have been classified primarily on the basis of their number of isoprene units (monoterpenes Cio, sesquiterpenes C15, diterpenes C2o, sesterterpenes C25, triterpenes C30 and carotenoids C40) and then on their carbon skeleton. The monoterpenes, sesquiterpenes, diterpenes and sesterterpenes contain the isoprene units linked head to tail, while the triterpenes and carotenoids contain two C15 and C20 units, respectively 1 inked in the middle tail to tail. Several thousand terpenes

110 have been isolated and they are by far the largest class of natural products. Within this class of compounds, the sesterterpenes form a rare group of isoprenoids, which occur in widely differing source and have been isolated from terrestrial fungi [2], plants [3] and insects [4] as well as from marine organisms [5,6], mainly from sponges and nudibranches. Marine organisms have provided a large number of sesterterpenoids, possessing novel carbon skeletons different from those present in terrestrial species. Several sesterterpenoids isolated from marine organisms have shown a wide variety of biological activities. The aim of this contribution is to review the more significant sesterterpenoids from marine organisms, which show biological activities, emphasising those compounds with a potential industrial application. In this review the structures will be covered in a biogenetic sequence and also include natural products that do not contain 25 carbon atoms but are obviously sesterterpene derivatives, such as degraded sesterterpenes with 21-24 carbon atoms, and alkylated sesterterpenes with 26-27 carbon atoms. In addition, some of our own results on anti-inflammatory activity of marine metabolites have been reported. Likewise, some data on the different directions that can be taken to obtain secondary metabolites have been included in the final section to suggest alternative production of marine metabolites and to highlight the possible future importance of marine biotechnology in the production of large quantities of marine secondary metabolites. Previous specific reviews on sesterterpenoids have beenpublished [7-10]. Furthermore, several reviews on terpenoids [ 1113] and on marine organisms [5-6,14-16] all contain material on sesterterpenoids. LINEAR SESTERTERPENOIDS After the isolation of all-^ra«5-geranylnerolidol (1) and geranylfarnesol (2) from the phytopathogenic fungus Cochliobolus [17] and from the wax of the insect Ceroplastse albolineatus [18], a large number of acyclic (absence of formation of any additional carbon-carbon bonds compared with a linear combination of isoprene units) sesterterpenoids were isolated from marine organisms. Furanosesterterpenes are a prominent class of metabolites mainly isolated from marine sponges of the family Thorectidae. The less elaborate component of this interesting group is furospinosulin-1 (3), first isolated from Ircinia spinosula [ 19] and later

Ill

from several Dictyoceratida species, including the South African Fasciospongia sp. [20], the Australian Thorecta sp. [21] and Californian Spongia idia [22] that contains also the oxidized derivative, idiadione (4). OH

,CH 2 OH

O

5a A 12 ' 13 R = 5b A 12 ' 13 R = SO3H; 6b A 1 3 1 5 R = SO3H Both compounds 3 and 4 showed activity atlO |a.g/ml [22] in the Anemia salina bioassay, which gives results that correlate well with cytotoxicity in cancer cell lines such as human epidermoid carcinoma KB, murine lymphoma P388 [23], mouse lymphoma L5178y and murine lymphoma L1210 [24]. Minale and co-workers [25] reported in 1972 the isolation from the sponge /. oros of ircinin-1 (5a) and ircinin-2 (6a), both containing two furan rings and a conjugated tetronic acid moiety. Before the isolation of ircinins, only four others sesterterpenoids were known. Subsequently, it was reported that the mixture of both ircinins inhibited potently mouse ear oedema after topical application, with an ID50 of 51

112 |j.g/ear. Ircinin was found to be a good inhibitor of human recombinant synovial phospholipase A2 (PLA2) (IC50 3.1 \iM) and 5-lipoxygenase (IC50 1.3 \xM), and it was not cytotoxic on human neutrophils at all concentrations tested (0.1-100 (J.M) [26]. These results demonstrate that ircinin is a novel marine inhibitor of PLA2 with a potent topical antiinflammatory profile and they suggest a potential role of ircinin as an inhibitor of inflammatory processes. Recently, the anti-inflammatory activity has been recorded in many sesterterpenes, with a mechanism of action different from those of nonsteroidal, anti-inflammatory drugs (NSAID). The inflammatory response has been shown to be involved in a diverse array of pathological conditions such as arthritis, gout, psoriasis, bee stings and many chemically induced oedemas. The inflammatory response is mediated by the b iosynthesis o f eicosanoids from arachidonic acid (arachidonic a cid cascade), as well as other autacoids released locally in response to an irritant. Arachidonic acid is primarily stored in the sn-2 position of membrane phospholipids. The hydrolysis of the ester at this position is specifically catalysed by PLA2. Lipoxygenase, cycloxygenase and cytochrome P-450 are enzymes from arachidonic acid cascade [27]. The most commonly used non-steroidal, anti-inflammatory agents, indomethacine and the salicylate, inhibit the cycloxygenase pathway but the use of these inhibitors is associated with an increased risk of gastrointestinal bleeding and renal complications. Thus, the inhibition of release of arachidonic acid by PLA2 has become an attractive target for investigation. The development of marine specific inhibitors of PLA2 offers the prospect for a new generation of anti-inflammatory drugs without side effects, derived from non-selective inhibition of constitutive enzymes. From the sponge /. variabilis was isolated variabilin (7a), a furanosesterterpene containing a tetronic acid moiety, which showed antimicrobial activity against Gram-positive bacteria Staphylococcus aureus [28] and Sarcina lutea [29]. After the preliminary observation of Tiberio in 1895 [30] and Fleming in 1929 [31] that a metabolic product of the mould Penicillium notatum inhibited the growth of a staphylococcal culture, and the introduction of penicillin in the treatment of bacterial infections several antimicrobial drugs were produced. The introduction of antimicrobial drugs for the control of infection is the biggest achievement in the history of medicine.

113

Unfortunately, many bacteria acquire resistance to one or more of the antibiotics to which they were formerly susceptible. Since most antibacterial agents interact with a specific protein or cellular component, modification of the target is a common means by which resistance can be conferred. Pharmaceutical companies are actually developing new antimicrobial agents against resistant bacteria.

7 a R = OH; 7 b R = OSO3H; 7cR = H; Later was reported that variabilin (7a) is a good inhibitor of human secretory and cytosolic PLA2 with anti-inflammatory activity [32] and shows in vitro antiviral activity against Herpes simplex (HSV) and Polio vaccine (PV1) viruses [33]. The high cytotoxicity against the BSC cell line exhibited by variabilin severely limits its potential usefulness as antiviral agent [33].

Thereafter, several compounds closely related to ircinins and variabilin have been isolated [5,6]. Fusetani and co-workers [34] reported the isolation of two compounds, dehydroderivative of ircinin (8) and an isomer of variabilin (9) from the Japanese sponge Cacospongia scalaris. Both compounds inhibited the cell division of fertilised starfish eggs at a concentration of 1.0 |j.g/ml. This assay is a variation on the test with sea urchin embryos, which can detect DNA and RNA synthesis inhibitors, microtubule assembly and protein synthesis inhibitors, the common leads

114

for the development of anticancer drugs [35]. From the Australian sponge, Thorecta sp. was isolated the rare 22-deoxy-variabilin (7c) that inhibited the growth of S. aureus at 100 |j.g/disk, Bacillus subtilis at 50 u.g/disk and Candida albicans at 100 )ug/disk in a standard agar plate assay [21]. From sponges of the genus Ircinia, collected in the Northern Adriatic Sea, together with the previously described ircinin-1 (5a), ircinin-2 (6a) and variabilin (7a), the corresponding sulphates 5b-7b were isolated. The sulphated derivatives 5b-7b showed greater activity in the A. salina bioassay (LC50: 1.72 and 1.22 |^g/ml, ircinins and variabilin sulphated, respectively), than the corresponding non-sulphated compounds 5a-7a (LC50: 2.38, 2.73 and 2.10 \iglm\), being less toxic in the fish {Gambusia affinis) lethality test (LC50: 5b-6b 5.09, 7b 9.50, 5a 3.35, 6a 3.03 and 7a 3.15 fxg/ml) [36]. Less common are those examples of this class of compound in which the tetronic acid moiety is nonconjugated. Palinurin (10a) was the first example of this class of compound, isolated from /. variabilis [37]. Subsequently, from an Australian Ircinia sp. was isolated a hydro derivative (11) of palinurin, which inhibits the growth of B. subtilis [38]. OR

10a R = H; 10bR=SO 3 H;

O

Additionally, three metabolites of this class of compounds, spongionellin (12), dehydrospongionellin (13) [39] and okinonellin B (14) [40], were isolated from a Japanese sponge of genus Spongionella and were shown to inhibit the cell division of fertilised starfish eggs at 2.0-5.0 u.g/ml. From an Australian Dysidea sp. was isolated isopalinurin (15) that possessed inhibitory properties against the protein phosphatase enzyme in chicken forebrain [41].

115

OH

Palinurin (10b) and fasciculatin (16b) sulphates, together with the previously described palinurin (10a) and fasciculatin (16a), were isolated from the Tyrrhenian sponges /. variabilis and /. fasciculata, respectively. Yet again the sulphated derivatives were more active in A. salina bioassay (LC50: 10b 3.04, 16b 1.44,10a 7.56,16a 2.03 fig/ml) and less toxic in the fish lethality test (LC50: 10b 2.30, 16b 2.20, 10a 1.67, 16a 1.04 ng/ml) [42].

o 16a R = H; 16bR = SO3H Several compounds closely related to palinurin and spongionellin have been isolated [5,6] that showed moderate antimicrobial and cytotoxic activities.

116

Sponges of the genus Sarcotragus are a rich source of sesterterpenes with both conjugated and nonconjugated tetronic acid moieties [43,44]. Sarcotins G (17) and H (18) are closely related to ircinin-1 (5a) and - 2 (6a), except that a furan ring is replaced by a 5-methoxy-2(5H)-furanone moiety. These compounds showed cytotoxic activity with IC50 values 5.010.0 |a.g/ml against five human tumour cell lines (lung carcinoma A549, ovarian carcinoma SK-OV-3, skin carcinoma SK-MEL-2, central nervous system carcinoma XF498 and colon carcinoma HCT15) [44], while sarcotin F (19), an oxidised derivative of palinurin (10a) showed less cytotoxic activity (IC50 7.6-24.1 fig/ml) in the same panel of cell lines [44].

OCH,

12

17 A 1 2 ' 1 3 ; 18 A 1 3 ' 1 5 H3CO

OH

Unusual trifuranosesterterpene acids, hippospongins A-C, were isolated from the Australian Hippospongia sp., which are speculated to be biosynthetically related to the C25 tetronic acids and the C21 furanoterpenes. Only hippospongin A (20) showed a mild antimicrobial activity, inhibiting the growth of S. aureus at a concentration of 200 ug/disk [45]. An a,(3-unsaturated. y-lactone linear sesterterpene (21) was isolated from the Caribbean sponge Thorecta horridus that exhibited inflammatory activity both in vivo inducing paw oedema and in vitro inducing release of histamine [46].

117

From a sample of the sponge Fasciospongia cavernosa, collected in the bay of Naples (Italy), were isolated two linear sesterterpenes, cacospongionolide D (22) with a p-hydroxybutenolide moiety, and luffarin-V (23) with two y-butenolide functionalities in the molecule. Cacospongionolide D showed a potent activity (LC50 0.1 (J.g/ml) [47] in the A. salina bioassay, and a moderate ichthyotoxicity to G. affinis (LC50 2.54 ng/ml) in the fish lethality assay. Luffarin-V was less active (LC50 1.72 |ag/ml) in the A. salina bioassay.

O

24 An unusual sesterterpenoid acid (24) with a tetrahydropyran ring was isolated from the Indonesian sponge Hippospongia sp. that inhibited the human Ras-converting enzyme (hRCE), with an IC50 value of 10 |J.g/ml [48]. The Ras signalling pathway has emerged as an important target for the development of anticancer drugs. Ras is a membrane bound G protein that functions as a molecular switch in a network of signalling pathways, controlling cell differentiation and proliferation. Mutated Ras genes, encoding activated Ras proteins, have been identified in approximately 30% of all human cancers. The approaches to therapeutic intervention in

118

the Ras signalling have focussed on the development of inhibitors that block the lipid modification needed for proper Ras membrane localisation (farnesyl transferase inhibitors) or to finding inhibitors of proteolytic processing of Ras (RCE protease inhibitors) [49]. Linear, closely related difuranoterpenes containing 21 carbon atoms have been found to occur in large amounts in the sponge of the genus Spongia. All of them possess the same carbon skeleton, and oxidation in the central chain accounts for all their differences. The first two C21 compounds, ninetin (25) and dihydroninetin (26) isolated from S. nitens [50] possess a y-lactone ring in the central part of the chain. S. officinalis and Hippospongia communis contain several C21 fiiranoterpenes in large amounts [51,52]. From a specimen of S. officinalis, collected in the Northern Adriatic Sea, together with furospongin-2 (27), previously reported from the same sponge collected in the Tyrrhenian Sea [51], its three isomers (28-30) were isolated. These C21 fiiranoterpenes (27-30) showed high activity (LC50 0.09-1.60 |u.g/ml) in the A. salina bioassay [53].

The most widely distributed component of this group, furospongin-1 (31), which possesses interesting biological activities, was first isolated from the Mediterranean S. officinalis, and the closely related H. communis [51], few years later, was isolated from two Australian sponges, Phyllospongia radiata and P. foliescens [54], Furospongin-1 showed analgesic [55] and antispasmodic activities [56]. Furthermore, furospongin-1 reduced, at yM concentration, the tissue levels of ATP and had no significant effect on ATP breakdown in bovine mitochondria, showing that its antispasmodic action has a mechanism different from that of the mitochondrial ATP synthase inhibitor, oligomycin [56].

119

O

Linear C21 furanoterpenes commonly occur in Spongiidae or Thorectidae sponges with structurally-related sesterterpenic tetronic acids from which they are biosvnthesised by the loss of a four-carbon fragment [14]. This hypothesis has raised considerable interest [15] and has received some experimental support from the degradation of linear conjugated furanosesterterpenic tetronic acids to C21 furanoterpenic carboxylic acids by oxidation in basic aqueous solution [57]; and from the isolation of C21 furanoterpenic carboxylic acids, ircinin-3 (32) and ircinin4 (33), related to ircinin-1 and ircinin-2, from the sponge /. Oros [58]; the acid 34 related to variabilin (7a) isolated from sponge of genus Sarcotragus [59]; and the acid 35 together with the C20 aldehyde 36 both related to fasciculatin (16a) [57]. The C21 furanoterpene acid 34 showed antiviral (HSV and PVl) and cytotoxic activities comparable with that of variabilin [33]. Further evidences of the hypothesis of Minale were obtained by the isolation of three chlorinated C24 norsesterterpenes (3739), closely related to ircinin-1 (5a) and ircinin-2 (6a), from the sponge /. oros, collected in the Northern Adriatic Sea [60]. In fact, we may suppose

120 that 37 and 39 are the first stage of degradation of the tetronic acid, through introduction of a chlorine atom by action of a chloroperoxidase, then hydrolysis of the lactone ring and subsequent decarboxylation to produce keto-chlorohydrins, which can easily be degraded giving the C21 terpenes. The norsesterterpenes, 37-39, showed less cytotoxic activity (LC50 8.2-8.6 |J.g/ml) than ircinin-1 (5a) and ircinin-2 (6a) (LC50 2.4 and 2.7 ng/ml), in the A. salina bioassay [60].

COOH 32 A 12 ' 13 ; 33 A 13 ' 15 COOH

COOH

O -,-

» 10,11 r,

37 A

TT

->o A 10,11

n

Cl

A

R = H;11 12 38 A R = Ac; 39 A ' R = H

Untenospongins A (40) and B (41), isolated from an Okinawan species of Hippospongia exhibited potent coronary vasodilating activity, markedly inhibiting KCl induced contraction of rabbit coronary artery with IC50 values of 10"6 and 2 x 10~6 M, respectively [61]. The norsesterterpenoids, sarcotins N (42), O (43) and e«?-kurospongin (44), isolated from Sarcotragus sp., showed moderate cytotoxicity, with

121 IC50 values 3.0-30.0 |a.g/ml, against a panel of five human tumour cell lines (A549, SK-OV-3, SK-MEL-2, XF498 and HCT15) [62]. OH OH

40

•o

OCH3

OH

O

O

Rhopaloic acids A-C (45-47), three unusual norsesterterpenes isolated from a Japanese species of Rhopaloeides, selectively inhibited the gastrulation of fertilised eggs of the Starfish Asterina pectinifera at jaM level. The minimum inhibitory concentrations of 45-47 were 0.5, 0.4 and 0.2 fjJVI, respectively [63]. Rhopaloic acid A, which was synthesised [64], exhibited also potent cytotoxicity against human myeloid K562 cells (IC50 0.04 fimol/1), human leukaemia MOLT4 cells (IC50 0.05 (imol/1), and L1210 cells (IC50 0.10 jxmol/1) [65]. Furthermore, rhopaloic acids A-C together with rhopaloic acids D-G (48-51) were isolated from an Indonesian Hippospongia sp.. Rhopaloic acids A-E showed a RCE protease inhibitory activity with IC50 values of ~ 10 |J.g/ml [48]. Compounds 45-49 were more active in the cell-based assay against colon tumour L0V0 cells (IC50 ~ 1 Mg/ml) than in the enzyme assays, suggesting

122

that the cytotoxic effect of the compounds might result from hitting more than one molecular target [48].

OH

51

123

Muqubilone (or aikupikoxide A) (52), a norsesterterpene peroxide acid, isolated from the Red Sea sponge Diacarnus erythraeanus showed in vitro antiviral activity against herpes simplex virus type 1 (HSV-1) with IC50 of 30.0 |J.g/ml [66], and cytotoxic activity with an IC50 > 1 Mg/ml, against three type of cancer cells, including P388, A549 and human colon carcinoma HT29 [67].

MONOCARBOCYCLIC SESTERTERPENOIDS Marine sponges of genus Luffariella (Thorectidae; Dictyoceratida) are a rich source of monocarbocyclic sesterterpenoids and most of them possess interesting bioactivities. In 1980 and 1981, Scheuer and coworkers [68,69] reported the isolation of manoalide (53), seco-manoalide (54), (6E)- (55) and (6Z)-neomanoalide (56a) from the Palauan sponge L. variabilis, which showed interesting antimicrobial activity against Gram positive bacteria Streptomyces pyogenes, S. aureus and B. subtilis [68,69]. Later, Kobayashi and co-workers [70] reported, from the Okinawan sponge Luffariella sp., the isolation of manoalide (53), (6E)- (55) and (6Z)-neomanoalide (56a) that showed cytotoxic activity against L1210 cells (IC50 0.032, 9.8 and 5.6 |ig/ml for 53, 55 and 56a, respectively), and only manoalide was active against KB cells with an IC50 value of 0.3 |ag/ml [70,71]. Manoalide is the first compound of this group to be reported, characterised by cyclisation that is reminiscent to those of the carotenoids and one or two potentially reactive rings, yhydroxybutenolide ring and a 8-lactol ring (a-hydroxy-dihydropyran ring) or its derivative. Subsequently, it was found that manoalide showed molluscicidal activity towards Biomphalaria glabrata at 1.5 ppm [72], analgesic activity at 50 mg/kg in the phenylquinone test, and antiinflammatory activity in the induced inflammation of the mouse ear, with a potency greater than that of indomethacine and less than that of hydrocortisone [73]. The most important finding has been that manoalide is the inhibitor of various secreted forms of PLA2 a t n M concentration

124 [74-77]. It was suggested that the binding of manoalide to PLA2 is irreversible and involves initial formation of a Schiff base (imine) between a lysine residue on PLA2 and the aldehyde group of y-hydroxybutenolide, than a second lysine reacts with the aldehyde group of cthydroxy-dihydropyran ring to produce an adduct in which the manoalide is irreversibly bound to PLA2 [75,78]. Over 140 citations concerning manoalide recorded in MEDLINE show the high interest pointed to this compound. Eight total syntheses have been reported [79-86]. Secomanoalide (54), which is the geometrical isomer of manoalide, has similar potency and efficacy in the inhibition of bee venom PLA2, suggesting that the inhibition reaction is not dependent on a rigid geometrical relationship between the aldehyde group and the second lysine residue [75].

56a A

6>7

55 A 6J £, R = OH Z,R = OH;56b A 6 ' 7 Z,R; OAc

From the Western Pacific sponge L. variabilis was isolated dehydromanoalide (57) that showed a marked decrease in inhibition of bee venom PLA2 (IC50 0.28 \xM) [76, 87].

125 In 1992, Konig and c o-workers [72] reported the isolation of Z-2,3 dihydro-neomanoalide (or luffariolide C) [88] (61a), its 24-acetyl derivative (61b), 6Z-24-acetoxy-neomanoalide (56b) and Eneomanoalide-24-al (58), from an Australian sponge of genus Luffariella. All these compounds showed antibacterial activity against Escherichia coli, B. subtilis and Micrococcus luteus, in a TLC bioautographic test [72].

.0

59 HO

61a R = OH; 61b R = OAc Kobayashi and co-workers [70] reported, from the Okinawan sponge Luffariella sp., the isolation of several sesterterpenoids related to manoalide, named luffariolides A-J (59-67). All luffariolides showed cytotoxic activity against L1210 cells (IC50 1.1-4.5 ng/ml) and only luffariolides F (64) and G (65) exhibited weak activity also against KB

126

cells [70,71,88]. Luffariolides H (66) and J (67) showed antimicrobial activity against S. aureus, with minimum inhibitory concentrations (MIC) of 16.7 and 33.3 ng/ml, respectively, B. subtilis (MIC, both 8.4 |ig/ml) andM luteus (MIC, both 8.4 ng/ml) [88].

62 R = H, OH; 63 R = O

HO CHO

67

127

Faulkner and co-workers reported the isolation of luffariellolide (68) from a Palauan sponge Luffariella sp., which was a potent antagonist of topical induced inflammation in the mouse ear, but it was less potent than manoalide (53) inhibitor of bee venom PLA2 with an IC50 value of 1.6 x 10~7 M. Luffariellolide is a partially reversible inhibitor of bee venom PLA2, because it lacks one of the two masked aldehyde groups that appears to be responsible for the irreversible reaction of manoalide with lysine residue of PLA2 [89].

From the Fijian sponge Fascaplysinopis reticulata were isolated two sesterterpenoids related to luffariellolide, zso-dehydro-luffariellolide (69) and dehydro-luffariellolide diacid (70). Zso-dehydro-luffariellolide inhibited at 1 mg/ml 81% of the HIV-1 reverse transcriptase activity [90] and reduced the activity of p56lck tyrosine kinase at 0.5 mM to 45% in ELISA based assays [91]. Hyrtiolide (71) was isolated from the Fijian sponge Hyrtios erecta together with its correlated /so-dehydro-

128

luffariellolide. Hyrtiolide showed weak antifungal activity towards Ustilago violaceae [91]. Muqubilm [92] (or prianicin A) [93] (72), a norsesterterpene peroxide acid, isolated from the Red Sea sponges, Prianos sp. [92-94] and Diacarnus erythraeanus [66] showed antimicrobial activity against Streptococcus beta haemolytic (MIC 2.5 |j.g/ml), S. aureus (MIC 12.0 |u.g/ml) and Corynebacterium diphteriae (MIC 3.0 |ug/ml) [93], and it displayed potent in vitro activity against Toxoplasma gondii at a concentration of 0.1 foM without significant toxicity [66]. Furthermore, muqubilin totally inhibited the cell division of fertilised sea urchin eggs at 16 )J.g/ml [94]. .EnZ-muqubilin (72), 2-e/n-muqubilin (73) and deoxydiacarnoate B (121) (see bicyclic section) were isolated from the New Caledonian sponge Diacarnus levii [95]. The mixture of all three compounds showed cytotoxicity against both chloroquine sensitive and resistant strains of Plasmodium falciparum, the human parasite responsible for the most severe cases of malaria [95].

-O 2 XOOH

72;

73 2-epi

COOH

COOH

COOH

The finding of new antimalarial drugs, particularly those against multiresistant P. falciparum, is extremely important, because in the last

129

years the malaria has regained its status as an extremely important threat to the human health. It is estimated that, in regions where malaria is endemic, each year about 1.5 million of people die from this disease. Tasnemoxides A-C (74-76), closely related to muqubilin, were isolated from the Red Sea D. erythraeanus, and showed moderate cytotoxicity (IC50 > 1 ng/ml) against three cancer cell lines including P388, A549 and HT29 [96]. In order to provide sufficient manoalide for continued pharmacological evaluation, F aulkner and coworkers m ade an e xtensive c ollection of L. variabilis, from different locations in Palau. From a small number of specimens of L. variabilis were isolated two new metabolites, luffariellin A (77) and Luffariella B (78) in place of manoalide and seco-manoalide [97]. Despite the different carbon skeleton, the functional groups in luffariellins A and B are identical with those in manoalide and secomanoalide, respectively, and they showed almost identical antiinflammatory properties. Both luffariellins were potent antagonists of topical induced inflammation in the mouse ear, and inhibitors of bee venom PLA2. with an IC50 value of 5.6 x 10'8 M and 6.2 x 10'8 M, for luffariellins A and B, respectively [97].

HO" CHO

78 Hippospongin (79), isolated from the Okinawan sponge Hippospongia sp., is an unusual sesterterpene containing an isolated cyclohexenofuran ring and a tetronic acid moiety, which showed antispasmodic activity (5 x 10" M), abolishing the contractile responses to carbachol and histamine on the guinea-pig ileum [98]. Further sesterterpenes (80 and 81) and two

130

norsesterterpene (82 and 83), related to hippospongin, were isolated from the Okinawan sponge Ircinia sp.. The norsesterterpenes 82 and 83 were more cytotoxic (IC50 < 1 fig/ml) than the sesterterpenes 80 and 81 (IC50 > 1 fig/ml) against KB cells [99]. An additional norsesterterpene, untenic acid (84) was isolated from an Okinawan sponge Spongia sp., which activates sarcoplasmic reticulum Ca2+-ATPase [100].

HO.

79 HO. 12 -

O"

80 A 1 2 ' 1 3 £; 81 A 12 ' 13 Z O

R

OH

82 R = H; 83 R = C1

O

COOH

84 From the Caribbean sponge Cacospongia linteiformis were isolated cyclolintemone (85) [ 101] and its 3-deoxy derivative (86) [ 102] with a novel rearranged monocarbocyclic skeleton, l-alkyl-l,2,6-trimethyl-2cyclohexene ring system. Both compounds were ichthyotoxic at 10 ppm to G. affinis, and showed antifeedant activity at a concentration of 30 |a.g per cm of food pellets against the fish Carassius aurantus [101,102]. Furthermore, cyclolinteinone showed anti-inflammatory activity, inhibiting the nuclear transcription factor-KB binding activity, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) enzymes,

131

and it was capable of controlling the excessive production of both prostaglandin (PGE2) and nitric oxide (NO) [103].

85 R = O; 86 R = H2

OSO3Na

Halisulphate 2 (87), a sulphated sesterterpene with a monocarbocyclic skeleton related to cyclolinteinone, was isolated from the Californian sponge Halichondria sp. [104]. Halisulphate 2 showed anti-microbial activity against S. aureus, C. albicans and B. subtilis at 20 jag/disk, it inhibited mouse ear oedema after topical application and was an inhibitor of PLA2 [104]. BICARBOCYCLIC SESTERTERPENOIDS Sesterterpenoids with a bicarbocyclic skeleton in many instances show structures reminiscent of the clerodane and labdane diterpenoids. Palauolide (88), isolated from an unidentified Palauan sponge, is structurally a classical example of clerodane type [105]. From the Palauan sponge Fascaplysinopsis sp. was isolated palauolol (89) that maybe a biosynthetic precursor of palauolide [106]. Palauolide (88) and palauolol (89), both containing a functional group y-hydroxybutenolide ring, inactivate bee venom PLA2 with 85% and 82% inhibition for 88 and 89, respectively, at 0.8 |u.g/ml [27,106], and showed anti-microbial activity against B. subtilis and S. aureus at 10 u.g/disc [105,106]. From the Palauan sponge Thorectandra sp. were isolated palauolide (88), palauolol (89) together with their derivatives, named thorectandrols A-E (90-94). Compounds 88-94 were tested for antiproliferative and cytotoxic activities against 12 human tumour cell lines originated from breast, CNS,

132

colon, lung, ovarian and renal carcinoma, leukaemia and melanoma. Palauolol (89) was active in all the cell lines with IC50 in the range 0.5-7.0 (J.g/ml, while palauolide (88) showed a decrease in activity in all the cell lines with IC50 7.7-53 jxg/ml. Thorectandrols A-D were weakly active with IC50 over 30 fj.g/ml, whereas thorectandrol E was not cytotoxic to any of the cell lines at the maximum dose tested [107,108].

O

92 R = OAc; 93 R = H 94 R = OH

90R = H;91 R = OAc AcO

95 Luffalactone (95) from the Pacific Luffariella variabilis is a sesterterpene with a labdane type skeleton, related to manoalide (53). Luffalactone showed 52% inhibition of oedema in the mouse ear assay at 50 |ng/ear [87], In order to find compounds related to cacospongionolide (155) (see tricyclic section) [109], we have investigated other Mediterranean horny sponges belonging to the family Thorectidae. From a specimen of Fasciospongia cavernosa, we isolated, in good yields, an isomer of cacospongionolide, named after for uniformity cacospongionolide B (96)

133

[110]. Structural differences between the two compounds are due to the absence of the cyclopropane ring and the presence of an exomethylene group. There are two varieties of F. cavernosa, one is massive, and the second is encrusted. The massive form is very common along the Adriatic coasts and Aegean Sea, while the encrusted form is distributed in all the Mediterranean Sea. Normally, from specimen of the massive form were isolated only one or two correlated metabolites, while from specimen of the encrusted form were isolated a complex mixture of cacospongionolides: cacospongionolides D (22) [47], E (97) [111] and F (98) [112] that was recently synthesised [113], and related metabolites, such as 25-deoxycacospongionolide B (99) [114] and cavernosolide (151) [115].

The isolation of several related constituents from individual specimens of F. cavernosa confirms the peculiarity of the sponges belonging to the family Thorectidae. In fact, similar variation of related metabolites was observed for the sponges Luffariella variabilis, L. geometric and Thorectandra excavatus [5]. The structures of cacospongionolides are similar to that of manoalide (53) (see monocyclic section). The differences between the two compounds, apart the non-polar region, are due to the lack of the hydroxyl group at C-24 in cacospongionolide. This lack renders the cacospongionolides more stable than manoalide. Despite the absence of the C-24 hemiacetal function, cacospongionolides showed potent inhibitory activity on recombinant human synovial PLA2 similar to that of manoalide, while a lower inhibitory activity was shown on other secretory PLA2S [116].

134

As cacospongionolide (155), cacospongionolide B (96) showed a high cytotoxicity (LC50 0.25 M-g/ml), in the A. salina bioassay. It was moderately ichthyotoxic to G. affinis (LC50 1.05 (J.g/ml) and showed a high antibacterial activity against the Gram-positive bacteria B. subtilis and M. luteus, with an MIC value of 0.78 fig/ml, comparable with that of gentamycin [110]. Further pharmacological screening revealed that cacospongionolide B is a new inhibitor of PLA2; preferentially inhibiting the human synovial PLA2 (IC50 4.3 |aM), and pancreatic PLA2 (IC50 4.0 }iM), and its potency on the human synovial enzyme was comparable to that of the reference inhibitor, manoalide (IC50 3.9 |oM). This activity was confirmed in vivo on a model of chronic inflammation, the established adjuvant-induced arthritis. Cacospongionolide B was less active than indomethacine, an NSAID. Nevertheless, the stomachs of the animals treated with this NSAID showed redness and perforations, while these toxic effects were absent in the rats treated with cacospongionolide B [116]. Furthermore, it has been shown that cacospongionolide B inhibited nuclear factor-^B (NF-A:B)-DNA binding activity and nuclear translocation of this transcription factor. The NF-&B pathway has emerged as an important target for the development of drugs against chronic inflammatory disorders and cancer. Moreover, cacospongionolide B is able to downregulate the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), resulting in decreased production of the two important mediators of inflammation process detected in high levels in rheumatoid synovial tissues, nitric oxide (NO) and prostaglandin E2 (PGE2). Cacospongionolide B also reduced the mRNA expression of the major factor in the development of chronic inflammatory conditions, tumour necrosis factor- a (TNF-a) [117]. The use of cacospongionolide B as inhibitor of the PLA2 is covered by patents [118]. Recently, Snapper and co-workers reported the total synthesis of cacospongionolide B and its enantiomer [119]. The examination of SPLA2 inhibition with synthetic variants of cacospongionolide B revealed that the inhibition is enantioselective, i.e. the natural product is a more potent inhibitor of bee venom SPLA2 (IC50 49 \iM) than the unnatural enantiomer (IC50 106 |i.M). Moreover, the inhibition is notable for synthetic precursor possessing the furan group (IC50 76 |uM) in place of y-hydroxybutenolide moiety. These results suggest that the y-hydroxybutenolide moiety is not the sole structural feature of the natural product involved in SPLA2 inhibition [119].

135

All cacospongionolides isolated showed more or less similar biological activities. In particular, as anti-inflammatory agents, they preferentially inhibited bee venom and human synovial PLA2 in the |uM range (Table 1). Cacospongionolide E (97), however, was the most potent inhibitor towards human synovial PLA2, showing higher potency than the referenced compound monoalide [111]. Our results confirmed the suggestion [76] that the pyranofuranone part interacts with PLA2 enzymes, but that the hydrophobic region of the molecule, which can be partially linear (manoalide) or cyclic (cacospongionolides), may facilitate this interaction. These results demonstrate that cacospongionolides are a novel class of marine metabolite inhibitors of PLA2 with a potent topical anti-inflammatory profile and a high antimicrobial activity and this suggests a potential role of cacospongionolides as drugs. Table 1.

Effect of Different Cacospongionolides on a Panel of Secretory PLA 2 a [lll]

PLA2 Enzymes

Cacospongionolide (155)

N. naja venom %I(10nM)

3.5

Pancreas %I (10 uM) IC50 (uM)

Human synovial %I(10uM) IC50 (uM)

RAP+zymosan %I(10nM) IC50 (uM)

Bee venom %I (10 uM) IC50 (uM)

14.1 N.D.

90.7 3.0

21.8 N.D.

96.3 2.3

Cacospongionoiide B (96)

0.0

64.2 4.0

86.7 4.3

36.9 7.8

35.4 N.D.

Cacospongionolide E (97)

0.0

5.3 N.D.

96.7 1.4

65.1 N.D.

94.8 2.8

Manoalide (53)

17.0

32.3 N.D.

93.2 3.9

38.4 N.D.

62.5 7.5

a IC» values were determined for those compounds that reach 50% inhibition at 10 uM; N.D. = not determined.

A number of carbobicyclic sesterterpenoid sulphates were found, including halisulfate 1 (100), isolated from Halichondria sp. [104]; halisulfates 8-10 (101-103), isolated from the Australian sponge Darwinella australensis [120]; hipposulfates A (104) and B (105), isolated from the Okinawan Hippospongia metachromia [121] and sulfircin (106) that was isolated as its N,N-dimethylguanidinium salt, from a deep-sea member of the genus Ircinia [122]. Halisulfate 1 (100) is an mhibitor of human 12-lipoxygenase (12-HLO) (IC50 1.0 \xM) and 15HLO (IC50 0.9 uM) [123]. 12-HLO is involved in the development of

136

psoriasis and controlling cancer cell proliferation, while 15-HLO in the development of atherosclerosis and tumourigenesis. Halisulfates 9 (102) and 10 (103) inhibited cell division of the fertilised eggs of the sea urchin Strongylocentrotus intermedius (IC50 50 and 35 |J.g/ml for 102 and 103, respectively) [120]. Hipposulfates B (105) showed cytotoxic activity with an IC50 of 2.0 (ig/ml against four human tumour cell lines, A549, P388, melanoma MEL28 and HT29 [121]. ,OSO3Na

OSO 3 Na

106

105 Sulfircin (106) showed activity against the fungal pathogen C. albicans with a MIC of 25 ng/ml [122].

137

A new class of sesterterpenes in which the middle three units of a penta-isoprenoid chain cyclised into a bicyclic system, leaving the first and the last isoprenoids to substitute the decaline moiety, was isolated from sponges of genus Dysidea and Irdnia. From the Palauan Dysidea sp. was isolated dysideapalaunic acid (107) that inhibited the aldose reductase [124]. An inhibitor of aldose reductase is expected to prevent neuropathy or cataract as a complication of diabetes. These diseases are caused by the accumulation of sorbitol in the peripheral nerve or the crystalline lens, as a result of enzymatic reduction of glucose by the aldose reductase in the sorbitol cycle [125,126]. The absolute stereochemistry of dysideapalaunic acid was established by its total synthesis [127,128]. COOH

COOH

OH COOH

Kohamaic acids A (108) and B (109) were isolated from the Okinawan Ircinia sp.. They exhibited cytotoxicity against P388 cells, with IC50 values of >10 (32%) and 2.8 |a.g/ml, respectively. Kohamaic acids are closely related to dysideapalaunic acid (107), but they have different stereochemistry at C-15 [129]. Dysidiolide (110), isolated from the Caribbean sponge D. etheria, is a potent inhibitor of the human cdc25A protein phosphatase (IC50 9.4 \xM), a potential target for anticancer therapy. Moreover, dysidiolide inhibited growth of the A549 (IC50 4.7 \xM) and P388 (IC50 1.5 pM) cells [130]. The interesting biological activities and the rare structural features of dysidiolide prompted several

138

researchers to undertake its total synthesis [131-139]. From D. cinerea were isolated two new inseparable metabolites, bilosespens A (111) and B (112). The mixture of both bilosespens showed cytotoxic activity with an IC50 of 2.5 (J.g/ml against four human tumour cell lines (A549, P388, MEL28andHT29)[140]. Carbobicyclic norsesterterpenoids, containing cyclic peroxides were isolated from four sponge genera, Mycale, Latrunculia, Sigmosceptrella and Diacornis. From a Thai Mycale sp. were isolated two related norsesterterpenoids 1,2-dioxanes, mycaperoxides A (113) and B (114), which showed significant cytotoxicity (IC50 0.5-1.0 |J.g/ml) against the cell lines P388, A549 and HT29 and displayed antiviral activity (IC50 0.25-1.0 |u.g/mi) against vesicular stomatitis virus and herpes simplex virus type-1 [141]. XOOH

COOH

HOOC

117 Trunculins A-E are norsesterterpene peroxides isolated from Latrunculia brevis [142,143]. Only trunculins A (115), B (116) [142] and E (117) [143] inhibited the growth of S. aureus, B. subtilis and C. albicans when tested at 100 mg/disk in the standard disk assay. From Sigmosceptrella laevis were isolated sigmosceptrellins A-C (118-120) that w ere i chthyotoxic (LD 5 ng/ml) a gainst L ebistes reticulatus [144]. Together with eM/-muqubilin (72) and 2-e/?/-muqubilin (73) (see

139 monocyclic section), from the New Caledonian sponge Diacornus levii was isolated the antimalarial agent deoxy-diacarnoate B (121) [95]. From a specimen of/7, cavernosa collected in the Aegean Sea, together with cacospongionolides B (96) and F (98), was isolated a new C21 terpene £-lactone (122), closely related to the cacospongionolide B, by the loss of four C atoms, through an oxidative rupture of the y-hydroxybutenolide ring [145]. XOOH -^\x

w

v^-n^/

—*s^

COOH

11816S,17S 11916R, 17S 120 16S, 17R

/ ''*"

121

N

'

122

This new compound, named cavernolide (122), showed antiinflammatory activity and exhibited specific inhibition of human synovial PLA2 in a concentration-dependent manner with an IC50 value of 8.8 |oM. Cavernolide was less potent in this assay than the referenced inhibitor manoalide (IC50 3.9 |j.M). In addition, this compound reduces TNF-a production, iNOS and COX-2 expressions [146]. TRICARBOCYCLIC SESTERTERPENOIDS Marine sponges are a rich source of tricarbocyclic sesterterpenoids with a cheilanthane skeleton, which seems to be derived from geranylfarnesol by a cyclisation initiated at the isopropylidene group that is typical of triterpenes. Luffolide (123), an anti-inflammatory compound, is a classic example of this class of compounds. The hydrolysis of phosphatidyl choline by bee venom PLA2 is completely inhibited by luffolide at a concentration of 3.5 |iM [147]. Further bioactive metabolites with cheilanthane skeleton were isolated from sponges of genus Spongia, Cacospongia, Petrosaspongia, Fasciospongia, and Ircinia and from the nudibranch Chromodoris. Spongianolides A-F (124-129) possessing a y-hydroxybutenolide moiety, were isolated from a Spongia sp. [148]. The absolute

140 stereochemistry of spongionohde A was established by its total synthesis [149]. CHO OAc

OAc

HO

123 O

HO

OH

126 16R,R = OCOCH3 127 16S,R = OCOCH3 128 16R, R = OCOCH(OH)CH3 129 16S, R = OCOCH(OH)CH3 130 16R, R = H 133 16R,R = OH 134 16S, R = OH

131 16R 132 16S Spongianolides A-E inhibited protein kinase C (PKC) at IC50 20-30 \\M, moreover, compounds 124-127 potently inhibited (IC50 0.5-1.4 JJM) the proliferation of the mammary tumour cell line MCF7 [148]. Simultaneously, from the Caribbean sponge Cacospongia linteiformis were isolated the spongianolides C a n d D (126 and 127) designated as lintenolides A and B, which showed high antifeedant activity against the fish C. aurantus (30 \xg per cm2 of food pellets) and ichthyotoxicity to G. affinis (10 ppm) [150]. Further, lintenolides C-G (130-134) were isolated from the Caribbean sponge Cacospongia cf. linteiformis [ 151,152]. All lintenolides A-G inhibited the growth of murine fibrosarcoma WEHI 164, murine monocyte/macrophage J774, bovine endothelial GM7373 and P388 cell lines (Table 2) [152].

141 Table 2.

Cytotoxicity of Different Lintenolides Against a Panel of Tumour Cells [152)

Mean IC» (ng/ml) Cell line:

WEHI164

J774

P388

GM7373

Lintenolide A (126)

0.92

0.36

0.098

0.085

Lintenolide B (127)

3.1

0.71

0.30

0.34

Lintenolide C (130)

50.0

23.4

2.7

0.22

Lintenolide D (131)

46.5

10.9

19.0

25.0

Lintenolide E (132)

53.3

30.7

125.0

0.021

Lintenolide F (133)

8.8

0.94

0.90

1.6

Lintenolide G (134)

3.2

1.70

0.037

0.30

From the New Caledonian sponge Petrosaspongia nigra were isolated several tricarbocyclic sesterterpenoids petrosaspongiolides A-J (135-144) [153,154] and M-R (145-149) [155]. From a Vanuatu Sponge sp., a 21hydroxy derivative of petrosaspongiolide P (150) was isolated [156]. All these compounds are biogenetically derived from luffolide (123). Petrosaspongiolides A-J exhibited cytotoxicity (IC50 0.5-14.8 iag/ml) against human bronchopulmonary non-small-cell-lung carcinoma cell line (NSCLC-N6) [154]. Petrosaspongiolides M-R (145-149) inhibited different preparations of PLA2 by irreversibly blocking these enzymes, particularly human synovial and bee venom, with IC50 values in the micromolar range. These compound displayed a much lower activity (or no activity at all) towards porcine and Naja naja PLA2 enzymes. The most potent compound, petrosaspongiolide M (145) (IC50 1.6 and 0.6 LIM for human synovial and bee venom PLA2 enzymes), was slightly more active than manoalide (53) (IC50 3.9 and 7.5 |aM) under the same experimental conditions. Petrosaspongiolide P (147) was more selective, inhibiting human synovial PLA2 (IC50 3.8 \xM) to a greater extent that bee venom PLA2 (37.9% inhibition at 10 \M) [155]. Furthermore, petrosaspongiolide M was able to reduce in a dose-dependent fashion.

142 PGE2, TNFa, LTB4 levels [157], and it has shown to modulate the expressions of COX-2 and iNOS by interfering with NF-kB [158].

OAc

136 R = CH 3 143 R = CH2OAc

135 R] = CH3, R2 = OAc 137 R, = CH2OAc, R2 = OAc 138 R, = CH2OH, R2 = OAc 139 R, = CHO, R2 = OAc 140 R, = COOH, R2 = OAc 141 R, = CH2OH, R2 = OH 142 R, = COOH, R2 = OH

OAc CH 2 COOH

OAc

HO

O

145 24S,R = H 146 24S,R = OAc 151 24R,R = H

HO

147 R = H 148 R=OAc 150 R = OH

HO

O

149

Besides, petrosaspongiolide M was capable of reducing the morphine withdrawal at 10" M [159]. The 21-hydroxy derivative of petrosaspongiolide P (150) inhibited human synovial PLA2 at 10 |jM with a value of IC50 5.8 (JM, showing a slightly lower potency but higher selectivity towards this enzyme than the referenced inhibitor m anoalide [156]. Cavernosolide (151), isolated from the Tyrrhenian sponge Fasciospongia cavernosa, is the 24 epimer of petrosaspongiolide M (145)

143

and showed high cytotoxicity (LC50 0.37 |ag/ml) in the A. salina bioassay and a moderate ichthyotoxicity (LC50 0.75 (a.g/ml) to G. affinis [115].

o 152

154

Suvanine (152), isolated from the sponge Coscinoderma mathewsi, has a cheilanthane skeleton with different stereochemical features and contains both sulphate and furan rings [160-162]. Suvanine was found to facilitate neuromuscular transmission in the indirectly stimulated rat hemidiaphragm preparations. Suvanine was also an acetyl cholinesterase inhibitor, and similar properties were exhibited by the suvanine sodium salt [161]. Besides, the suvanine sodium salt showed antithrombin and antitrypsin activity with IC50 of 9 and 27 |ug/ml, respectively [162]. Furthermore, suvanine was ichthyotoxic towards goldfish at 10 (J-g/ml, and exhibited 90% inhibition of sea urchin egg cell division at 16 ng/ml [160]. Inorolide C (153) was isolated from the nudibranch Chromodoris inornata. It was shown to inhibit the proliferation of KB (IC50 6.4 (ag/ml) and L1210 (IC50 1.9 ng/ml) cells [163]. From the Okinawan sponge Hyrtios erectus was isolated hyrtiosal (154), possessing a novel rearranged tricarbocyclic skeleton (hyrtiosane) [164]. Its structure was confirmed by total synthesis [165]. This compound exhibited in vitro antiproliferative activity against KB cells with an IC50 of 3.0 ug/ml [164]. In 1988, we reported the isolation and structural elucidation of a new tricarbocyclic sesterterpene [109], bearing a y-hydroxybutenolide moiety, from the Dictyoceratide sponge, Fasciospongia cavernosa, erroneously classified a s Cacospongia mollior, collected in the North Adriatic Sea. We named this compound after cacospongionolide (155), on the basis of the erroneous classification of the sponge [110]. Cacospongionolide was reported as a potent inhibitor of human synovial and bee venom PLA2 (Table 1) [111]. Besides, cacospongionolide showed high cytotoxic

144 activity (LC50 0.1 fig/ml), in the A. salina bioassay, very high inhibition (75%) in the crown-gall potato disc assay, an antitumoural like test [109].

155 From the Caribbean sponge Cacospongia linteiformis was isolated lintenone (156) with a new tricarbocyclic skeleton, which contains fused cyclohexane, cyclopentane and cyclobutane rings. Lintenone exhibited high antifeedant activity against the fish C. aurantus (30 p,g per cm2 of food pellets), ichthyotoxicity to G. affinis (10 ppm) and moderate toxicity in A salina assay (LC50 109 ppm) [166]. CHO CHO

157 = CH2OH, A13.= 13 158 = CH 2 OH,A'° = 13 161 R = COOH, A = Z 162 R = C O O H , A13I J = £

R 159 160 163 164

R = CH 2 OH ,A 1 3 = Z R = CH 2 OH , A 13 = £ R = COOH, A 13 = Z IJ R = COOH, A13 =E

From the New Caledonian sponge Rhabdastrella globostellata were isolated two isomalabaricane sesterterpenes, aurorals 1 and 2 (157 and 158) and the corresponding trinor-sesterterpenes aurorals 3 and 4 (159 and 160) [167]. From the Okinawan sponges Rhabdastrella (Jaspis) stellifera were isolated the corresponding oxidised compounds jaspiferals C-F (161-164) [168]. Since jaspiferals C-F were isolated together with the related triterpenes stelliferins A-F [169] and nortriterpenes jaspiferals AB [ 168], we can suppose that also aurorals 1-4 and jaspiferals C-F are degraded triterpenoids. Aurorals, which differ from jaspiferals by the presence of a primary alcohol group at C-4 position, exhibited higher

145 cytotoxic activity on the KB cells. The mixtures of aurorals 1-2 (157 and 158) and jaspiferals C-D (161 and 162) showed ID50 values of 0.2 and 5.5 jj-g/ml, respectively. The mixtures of aurorals 3-4 (159 and 160) showed moderate activity on KB cells with an IC50 of 8.0 M-g/ml, while jaspiferals E-F (163 and 164) were inactive until 10 |o.g/ml [167]. Furthermore, the mixtures of jaspiferals C-D, and jaspiferals E-F exhibited cytotoxicity against L1210 cells with IC50 values of 4.3 and 3.1 fj.g/ml, respectively [168]. Besides, jaspiferals E-F showed antifungal activity against Trichophyton memtagrophytes (MIC 50 (ig/ml) [168]. Halorosellinic acid (165) possessing an ophiobolane skeleton was isolated from the cultural broth of the marine fungus Halorosellinia oceanica. Compound 165 showed moderate antimalarial activity with IC50 value 13 |u.g/ml and weak antimycobacterial activity with MIC 200 Hg/ml [170].

HOOC

COOH

CH2CH2COOH

NCH2COOH ~CH2CH2COOH

167 Rt = CH3, R2 = CH2COOH 168 R! = CH2OAc, R2 = CH2COOH 170 R[ = CH3, R2 = CH2CH2SO3H From the New Caledonian Petrospongia nigra, together with the previously reported petrosaspongiolides A-J (135-144) was isolated a pyridium alkaloid 23-norsesterterpene named petrosaspongiolide L (166) that showed cytotoxic activity against NSLC-N6 cells with IC50 value of 5.7 (j.g/ml. Petrosaspongiolide L could be considered a condensation product with ammonia of a 16-keto, 18-al precursor, derived from petrosaspongiolide K (209) (see tetracyclic section) [154]. Four

146 pyridinium alkaloids, spongidines A-D (167-170), related to petrosaspongiolide L, were isolated from the Vanuatu Spongia sp.. These compounds inhibited mainly the human synovial PLA2 at 10 |oM and they were devoid of significant cytotoxic effect on human neutrophils at concentration up to 10 pM [156]. TETRACARBOCYCLIC SESTERTERPENOIDS The main group of marine tetracarbocyclic sesterterpenoids is of those with a scalarane skeleton, which appears to be of the same origin as cheilanthane and is formed by closely biosynthetic process involving additional cyclisation. Metabolites of this class have been reported from marine sponges of the order Dictyoceratida and their predator nudibranches [5, 6]. The first example of this group was scalarin (171), isolated from the sponge Cacospongia scalaris bearing a yhydroxybutenolide moiety. [171].

171 R = a-OAc 172R=p-OAc 173 R = p-OH

174

175

A number of 19-deoxy, 20-deoxo, 12-O-deacetyl and 12-epimers were isolated [5,6]. From the Japanese Spongia sp. were isolated \2-episcalarin (172), 12-0-deacetyl-12-epi-scalarin (173), 12-e/?z-deoxoscalarin (174) and 12-0-deacetyl-19-deoxyscalarin (175) [172]. These compounds exhibited selective cytotoxicity against four tumour cell lines, being more active on L1210 cell line (IC50 13.2, 2.3, 2.1 and 1.6 jig/ml for 172-175, respectively) and less active on A549, KB and HeLa cell lines with an IC50 of the range 14.3-29.4 |ag/ml [172]. 12-0-deacetyl-19-deoxyscalarin (175), first isolated from the sponge Hyrtios erecta, showed also

147

cytotoxicity against P388 cells with IC50 of 2.9 |ig/ml [173]. Moreover, compound 175 showed antitumour activity in vivo on sarcoma-180implanted mice with an increase of lifespan (ISL) of 50.3% at 5 mg/kg intraperitoneal administrations. This activity is more potent than of a positive control, 5-fluorouracil (ISL: 32.9%) at the same dose [172]. 12£/>/-acetylscalarolide (176), isolated from the Spanish C. scalaris, showed significant cytotoxic activity towards a panel of four tumour cell lines (Table 3) [174]. 12-O-acetyl-16-O-methylhyrtiolide (177), with an additional methoxy group at C-16 exhibited cytotoxicity against L1210, A549, KB and HeLa cell lines with IC50 values of 2.2, 5.3, 15.6 and 5.3 fag/ml, respectively [172]. AcO

r

OAC

Heteronemin (178), first isolated from the sponge Heteronema erecta [175], was toxic to A. salina and gametes of the giant kelp Macrocystis pyrifera at 10 |J.g/ml and also immobilised the larvae of the red abalone Haliotis rufescens at 1 fig/ml [22]. Furthermore, heteronemin showed antituberculosis activity, inhibiting the growth of Mycobacterium tuberculosis with an MIC of 6.25 |ag/ml [176]. Salmahyrtisol B (179), isolated from the Red Sea Hyrtios erecta [177], is related to scalarafuran (180), isolated from Spongia idia, a compound toxic to A. salina at 10 Hg/ml, [22]. Salmahyrtisol B showed cytotoxic activity with an IC50 > 1 Hg/ml against P388, A549 and HT29 cells [177]. Generally, scalarane sesterterpenoids are not functionalised on A- and B-rings. A structure-activity study showed that an oxygen-bearing substituent at C-3 of scalaranes, together with the presence of hydroxyl groups at C-12 and C-19, leads to increase of antitumour activity [178]. Accordingly, salmahyrtiol C (3-oxo-12-O-deacetyl-12-epi-deoxyscalarin) (181), first isolated from the Japanese H. erecta [178] and subsequently from the Red Sea H. erecta [177], exhibited potent cytotoxicity against P388 (IC50 of 14.5 ng/ml) and human gastric carcinoma MNK-1 (IC50 of

148

57.7 ng/ml), MNK-7 (IC50 of 56.0 ng/ml) and MNK-74 (IC50 of 36.8 ng/ml) cells. Intraperitoneal administration of 181 (0.5-8.0 mg/kg) on mice with P388 leukaemia increased the mean survival time (10.7-15 days) and ISL (24.4-74.4%) dose-dependently [178]. 12-Deacetoxy-21acetoxyscalarin (182), isolated from the Japanese H. erecta, showed cytotoxic activity against P388 cells with IC50 value of 0.9 )J.g/ml [179]. OH

HO

HQ, '-r-0

= CH 3 , R2 = CH2OH = CH2OH, R2 = CH3 From the Maldivian H. erecta were isolated sesterstatins 1-3 (183-185) that showed cytotoxic activity against P388 cells with IC50 value of 0.46, 4.2 and 4.3 ng/ml, respectively [180]. Additional 3- (186 and 187) and 19-oxygenated scalaranes (188 and 189) were isolated from the nudibranch Chromodoris inornata that showed cytotoxic activities against L1210 (IC50 6.6, 0.95, 4.1 and 0.35 ng/ml for 186-189, respectively) and KB (IC50 22.8, 5.2, 21.0 and 3.1 ng/ml for 186-189, respectively) cell lines [163]. Scalaradial (190) and its 12-deacetoxy derivative (191) are two classical examples of compounds with a 1,4-dialdehyde moiety. Scalaradial (190) was isolated from two species of Cacospongia, C. mollior[ 181] and C. scalaris [174]; 1 2-deacetoxyscalaradial (191) was isolated from C. mollior [182]. The majority of terpenoids, containing an unsaturated 1,4-dialdehyde functionality, are intensely pungent [183] and

149 generally are very versatile repellents [184]. This activity was explained by their interaction with vanilloid receptors [185]. However, scalaradial (190) was tasteless and showed antifeedant activity at a concentration twice the sesquiterpene polygodial (192) [186]. The antifeedant activity of 12-deacetoxyscalaradial (191) was similar to that reported for 192, and moreover 12-deacetoxyscalaradial was hot to the taste. These results showed that the molecular size was not a restrictive factor in these activities and pointed out the specific importance of the substituent at C12 in 190 and 191, or in the equivalent C-l position of a supposed polygodial derivative [182]. CHO CHO

186 R = p-OAc 187 R = a-OAc

188R = CH2OH 189R = CH2OAc

AcO CHO

190 R = OAc 191 R = H

193R = OH 194 R = OAc, \8-epi

CH2OH CHO

CHO

192

195

196 Rx = OH, R2 = (3OH 197 R, = OAc, R2 = pOH 198 Ri = OH, R2 = aOAc

In 1991, de Carvalho & Jacobs [187] reported the potent activity of scalaradial (190) against bee venom PLA2 (IC50 0.07 |u.M). They observed that scalaradial completely inactivated the enzyme by a two-step mechanism, involving apparent non-covalent binding followed by covalent modification. Subsequently, we observed that scalaradial showed

150

a topical anti-inflammatory activity on ear oedema in mice, with an ID50 of 172 p,g/ear comparable with that of indomethacine. It is a potent inhibitor of several PLA2, with a high selectivity for human recombinant synovial PLA2 (IC50 0.5 |oM). Moreover, scalaradial showed cytotoxic effects on human neutrophils at concentrations of 5 ^M [26]. Many other scalaranes were screened in the bee venom PLA2 a ssay but a 11 showed less activity than scalaradial. From the Japanese C. scalaris was isolated deacetylscalaradial (193) that showed interesting cytotoxic activity against L1210 cells with an IC50 value of 0.58 |ug/ml [188]. Scalaradial (190) and deacetylscalaradial (193) were shown to act on both R- and Ctype vanilloid receptors [185]. From the C. scalaris, collected in the Southern Coast of Spain, were isolated 18-epz-scalaradial (194) and 19dihydroscalaradial (195). Both compounds showed significant cytotoxicity towards four tumour cell lines (Table 3) [174]. Table 3. Cytotoxicity of Compounds 176,194,195,199,206-208 Against a Panel of Tumour Cells [174]

Mean IC5o (ng/ml) Cell line:

P388

A549

HT29

MEL28

12-ep/-acetylscalarolide (176)

1.0

2.0

2.0

2.0

18-epi-scalaradial (194)

0.2

0.2

0.2

0.5

19-dihydroscalaradial (195)

2.0

2.0

2.0

2.5

16-acetylfuroscalarol (199)

2.5

5.0

2.5

10.0

norscalaral A (206)

1.0

1.0

1.0

2.0

norscalaral B (207)

2.0

2.0

2.0

2.0

norscalaral C (208)

1.2

2.5

5.0

2.5

From the Japanese H. erecta were isolated two sesterterpenoids (196 and 197) [179] related to scalarolbutenolide (198), isolated from the Mediterranean Spongia nitens [189]. Compounds 196 and 197 were cytotoxic against P388 cells with IC50 values of 0.4 and 2.1 p.g/ml, respectively [179]. These compounds cannot strictly be considered as

151

scalarane, because they show different arrangements of the carbons C-24 and C-25. 16-Acetylfuroscalarol (199), with moderate cytotoxicity (Table 3), isolated from the Spanish C. scalaris [174] and 12-0-acetyl-16-0deacetyl-12,16-episcalarolbutenolide (200), cytotoxic against L1210 (IC50 2.4 |J.g/ml) and KB (IC50 7.6 |ag/ml) cell lines, isolated from the nudibranch C. inornata [163], showed the same carbon skeleton of scalarolbutenolide. From the Indonesian Phyllospongia sp. were isolated two sesterterpenes (201 and 202), which exhibited cytotoxicity against KBcellsatl0ng/ml[190]. AcO

O

203 R = OAc 204 R = OH

205

206 R = 16(3-OH 207R=16a-OH

Tetracarbocyclic norsesterterpenoids a re extremely rare and are only isolated from sponge of subclass D ictyoceratida. Hyrtial (203), isolated from H. erecta, was the f irst 2 5-norscalarane to be reported. It showed anti-inflammatory activity at 50 |ig/ml close to the activity of indomethacine [191]. From the Okinawan sponge H. erecta were isolated 12-deacetylhyrtial (204) and its A17 isomer (205) that showed cytotoxic activity against KB cells with IC50 values of 10.0 and 2.82 fag/ml, respectively [192]. Norscalarals A-C (206-208) isolated from the Spanish C. scalaris showed cytotoxicity against four tumour cell lines (Table 3) [174]. Petrosaspongiolide K (209), isolated from the New Caledonian

152

Petrosaspongia nigra, was the first reported 23-norscalarane. Petrosaspongiolide K showed cytotoxic activity (IC50 1.3 p.g/ml) against NSCLC-N6 cells [154]. Scalarane sesterterpenoids also include alkylated derivatives, called homoscalaranes with methylations at C-20 or C-24 and bishomoscalaranes with methylations at C-20 and C-24 and rarely at C-23 and C-24 [193]. HOOC

AcO

209

208

AcO

CHO O

210

A series of 24-methylscalaranes were isolated from the Palauan sponges Dictyoceratida sp. and Halichondria sp. [194]. Only compound 210 was shown to have significant inhibitory activities (IC50 0.5 |i.g/ml) on the platelet aggregations caused by adenosine 5'-diphosphate, collagen, or arachidonic acid [194]. Another group of related compounds were isolated from the Australian sponge Lendenfeldia sp., as only the compound 211 was the inhibitor of platelet aggregation [195]. Further 24homoscalaranes were isolated from L. frondosa, and only the compound 212 exhibited moderate anti-inflammatory activity, inhibiting 35% of bee venom PLA2 at 8 JJM [196]. AcO AcO.

CHO O

AcO. 'OH

213 R = 214R = Ac

Four 24-homoscalaranes (213-216) that exhibited 30-95% inhibition of the growth of KB cells at 10 fj.g/ml were isolated from the Indonesian Phyllospongia sp. [190]. From the Pacific nudibranch Glossodoris sedna were isolated several scalarane and homoscalarane compounds, but only

153

compound 217 was ichthyotoxic at 0.1 ppm against G. affinis and inhibited mammalian cytosolic PLA2 (IC50 18.0 |u.M) [197]. Foliaspongin (218), a 20,24-dimethylscalarane derivative, isolated from the sponge Phyllospongia (Carteriospongia) foliascens, showed anti inflammatory activity [198,199]. HO

AcO AcO,

CHO O

HO

CHO O

A

215 Rj = OH, R2 = H 216 R, = OMe, R2 = OMe AcO

R

219 R = CHO 220 R = H AcO

222 R = 24a-Me 223 R = 240-Me

CHO O

OR

224 R = H 226 R = CH3CHOHCH2CO227 R = CH3CH2CHOHCH2CO228 R = CH3CH2CO229 R = CH3CO-

230 R = CH3CH2CH(OCOCH3)CH2CO231 R= CH3CH2CH(OCOCH2CH3)CH2CO232 R = CH3CH(OCOCH3)CH2CO-

Subsequently, several bishomosesterterpenoids were isolated from P. foliascens, collected in different seas. From the Neo Guinean sponge C. foliascens were isolated several bishomosesterterpenoids, but only

154

compounds 219-221 showed ichthyotoxic effects towards L. reticulatus at LD50 of 5, 20 and 40 mg/1, respectively [200]. Phyllactones A (222) and B (223), with moderate cytotoxicity against KB cells (IC50 20.0 fig/ml), were isolated from the Chinese P. foliascens [201]. From the Indonesian Phyllospongia sp. were isolated two 20,24-dihomoscalaranes (224 and 225) that showed cytotoxicity against KB cells at 10 |u.g/ml [190]. From the Australian Strepsichordaia lendenfeldi, together with the alcohol 224, were isolated four different acyl derivatives (226-229) and three esters with the same skeleton and different acyl groups (230-232). All these compounds exhibited potent cytotoxicity against both P388 and A549 cell lines (Table 4) [202]. Table 4.

Cytotoxicity of Compounds 224,226-232 Against a Panel of Tumour Cells [2021 Mean IC5o (ng/ml)

Cell line:

224

226

227

228

229

230

231

232

P338

0.1

0.23

0.5

0.67

0.91

0.12

0.12

0.2

A549

0.1

0.66

0.5

0.67

0.88

0.25

0.21

0.2

From the Red Sea Hyrtios erecta, together with hyrtiosal (154), previously reported [164], was isolated salmahyrtisol A (233), a furan sesterterpene with a new tetracarbocyclic skeleton. The coexistence of the unusual sesterterpenes 233 and 154 is noteworthy from the biosynthetic viewpoint and maybe hyrtiosal is the logical biosynthetic intermediate for salmahyrtisol A. Salmahyrtisol A showed cytotoxic activity with an with IC50 > 1 M-g/ml against three type of cancer cells including P388, A549 andHT29[177]. Suberitenones A (234) and B (235), isolated from the Antarctic sponge Suberites sp., are two sesterterpenoids with an unprecedented carbon skeleton. Suberitenone B inhibited (IC50 10 |umol/ml) the cholesteryl ester transfer protein (CETP), which mediates the transfer of cholesteryl ester and triglyceride between high-density lipoproteins and low-density lipoproteins. Many studies have found an inverse correlation between levels of high-density lipoproteins and incidence of atherosclerotic cardiovascular diseases. Therefore, CETP inhibition is considered to be a good target for the development of an effective agent against atherosclerotic diseases [203].

155

From the Japanese nudibranch Chromodoris inornata were isolated two sesterterpenes, inorolides A (236) and B (237) with a new carbon skeleton. Both compounds exhibited cytotoxic activity against L1210 (IC50 1.9 and 0.72 ^g/ml for 236 and 237, respectively) and KB (IC50 3.4 and 2.2 |J.g/ml for 236 and 237, respectively) cell lines [163].

AcQ

P

238R = C1 239 R = Br

OH

OH

241 R, = OH, R2 = H 242 R, = H, R2 = OH 244 R, = R2 = H

From the marine fungus Fusarium helerosporum were isolated two groups of sesterterpenes, neomangicols A-C (238-240) [204] and mangicols A-G (241-247) [205], both with unusual carbon skeleton that constitutes two new classes of rearranged sesterterpenes. Neomangicols A (238) and B (239) were found to be active against a variety of cancer cell lines. Neomangicol A was most active against MCF7 and human colon carcinoma CACO2 cell lines, displaying IC50 values of 4.9 and 5.7 |aM, respectively. Neomangicol B was less active having a mean IC50 value of

156

27 )j.M across the entire panel (versus 10 \xM for neomangicol A). Neomangicol B displayed antibacterial activity similar to that of gentamycin, against the Gram-positive bacterium B. subtilis [204]. Mangicols A-G (241-247) showed weak cytotoxicity with IC50 values ranging from 18 to 36 |a.M in the 60 cell lines panel. Mangicols A and C inhibited mouse ear oedema (81 and 57% reduction in oedema, respectively) at 50 (j.g per ear. These values are consistent with the potencies of the anti-inflammatory agent, indomethacine [205].

RO

243 R, = OH, R2 = H 245 R, = H, R2 = OH 246 R, = R2 = H

247

OR

248a R = H 248b R = Ac

Aspergilloxide (248a), a sesterterpene epoxide diol with a new carbon skeleton was isolated from the marine fungus of the genus Aspergillus. It showed little cytotoxicity towards HCT116, but its acetate derivative (248b) inhibited HCT116 cell line at 61 \xM [206]. PENTACARBOCYCLIC SESTERTERPENOIDS Although numerous marine sesterterpenoids have been found, only a few sesterterpenoids possessing a pentacarbocyclic skeleton have been isolated. Disidein (249a) and two halogenated related derivatives (250a, 251) were isolated from the Mediterranean sponge Dysidea pallescens [207-208]. The stereochemistry of disidein was determined by X-ray analysis of the acetyl derivatives of bromo-disidein (250b), which shows the same carbon skeleton of scalarane. The triacetyl disidein (249b) showed moderate analgesic activity [55]. From the Neo Guinean sponge Phyllospongia foliascens, together with bishomoscalarane derivatives (see tetracyclic section), was isolated a related compound (252) with an additional cyclobutane ring. This

157

compound showed ichthyotoxic effects towards L. reticulatus at the LD50 of5mg/l[200]. R1Q /^OFL

249a Rj = R 2 = H; 249b R{ = Ac, R 2 = H 250a Rj = H, R 2 = Br; 250b R, = Ac, R 2 = Br 251 Rj = H, R 2 = Cl AcO

252

253

Phyllofenone B (253), an additional bishomoscalarane derivative with a pentacarbocyclic skeleton was isolated from P. foliascens. It showed cytotoxicity against P388 cells with IC50 value of 5.0 |ig/ml [209]. PRODUCTION OF MARINE COMPOUNDS Although the marine environment is a plentiful source of interesting new products with pharmaceutical potential, only a few of these marine natural products have reached the stage of commercial production. Arabinofuranosyladenine (ara-A, isolated from the Gorgonian Eunicella cavolini) [210] is the unique marine secondary metabolite currently in clinical use and is one of most potent antiviral drugs [211]. The second one is avarol, a sesquiterpene hydroquinone isolated from the sponge

158

Dysidea avara [212,213] currently commercialised as a cream against skin disorder. Words such as "promising" and "potential" dominate the literature on marine natural products, while papers describing successful application of these products remain scarce. In fact, patent applications are less than 10% of the total number of papers published on marine natural products. The number of patent applications on marine natural products is very little when compared with those of terrestrial origin. The limited availability of larger quantities of a particular organism as starting material for extraction of the compounds is one of the major causes for the low attractiveness of such secondary metabolites for commercial utilisation. Furthermore, the isolation of large quantities of these compounds from animal tissues is unacceptable because of its devastating impact on the natural environment. Four different approaches can be undertaken to obtain bioactive marine secondary metabolites in bulk amounts: 1- Chemical synthesis 2- Aquaculture 3- Cultivation of marine organisms in bioreactor 4- Cell culture. Chemical Synthesis Generally, pharmaceutical companies need a strong patent position before starting the long and expensive path, of a drug development, and they prefer compounds that can be synthesised. This approach has successfully been undertaken specially for those compounds with a potential industrial application, but very often, for the high structural and stereochemical complexity of the metabolite, the synthesis includes many steps with low yield and it is not commercially realistic. Aquaculture The first attempt for i n situ a quaculture of c ommercial marine sponges (bath sponges) was made in Adriatic Sea in 1870, but no detailed statement of the methods employed was reported. Smith [214] first reported the description of cultivation of sponges in the late 19th century. Subsequently, Moore [215] described the procedures for the cultivation of sponges. The technique exploits the capacity of sponges to regenerate them and to form new colonies even only by small fragments. Then, the large-scale commercial sponge aquaculture was developed in several

159

countries [216-218]. Farming of sponges in a sustainable manner for the production of bioactive compounds has recently been started both in New Zealand [219] and Mediterranean Sea [220]. Cultivation of Marine Organisms in Bioreactor Aquaculture has the disadvantage that the growth rate of sponges is dependent on in situ conditions, which cannot be controlled. Therefore, some researchers have considered the possibility of producing sponge biomass under controlled condition. The main difficulties are the supply of an adequate food source and the accumulation of waste products. Recently, Osinga and co-workers [221] reported growth of the sponge Pseudosuberites andrewsi in a closed system, using the microalgae Chlorella sorokiniana and Rhodomonas sp. as food source. These two microalgae were selected, because it was microscopically observed, on fresh material, that these algae were ingested and digested by the sponge cells. The high growth rates observed for this sponge suggest a promising future for cultivation of sponges in closed systems. Cell Culture The high proliferation capacity of sponge cells suggests that it should be easily feasible to establish their cell cultures in vitro. Then, in analogy to the production of bioactive metabolites from fungi and bacteria, the production of secondary metabolites will be accomplished in a bioreactor using sponge cells in culture. In the last few years, there has been developed the production of axenic sponges cell culture, but until now, only the maintenance of sponge cells in vitro has been achieved [222224]. Primary cell cultures have been obtained from several sponges, with a low cell density in the cultures. This low proliferation can be explained in the culture condition utilised and/or in the experimental approach to establish the culture condition. The lack of in-depth knowledge of the nutritional requirements of marine sponges maybe one question to settle. Recently, we have reported that by optimising some physical parameters (pH, temperature, light) and supplementing the commercial medium with different compounds, such as cholesterol, fatty acids, glucose, it was possible to promote the sponge cell proliferation [225,226]. It has been observed that the single cells in suspension did not proliferate readily [223], because they loose telomerase activity and hence

160 their potency for cell division [227]. The formation of multicellular aggregates from dissociated single sponge cells regain telomerase activity, and with this their growth potential. These aggregates were termed primmorphs [228,229]. Another promising method is the fragmentation of intact sponges. Brummer and co-workers [230] reported the in vitro cultivation of sponge fragments without further dissociation and reaggregation. There are same limitations in the cultivation of sponge fragments. In fact, only species with high capability of wound healing can be used for fragmentation [230]. In all methods, cell culture, primmorphs and fragmentation, morphological changes indicate that the culture conditions may not be optimal. Further ecological parameters have to be involved in the optimisation of culture conditions and sponge bioreactor design. Recent studies have demonstrated the ability of sponge cell cultures to produce secondary metabolites [231,232]. If an appropriate growth medium and bioreactor system for primmorphs can be developed, this system may have promising biotechnological potential. ACKNOWLEDGMENTS One of the authors (M. Mitova) gratefully acknowledges a Marie Curie Research Training Grant of the European Community programme, "Quality of Life and Management of Living Resources" contract QLK5CT-2001-50974.

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Ohta, S.; U no, M.; Y oshimura, M.; H iraga, Y.; I kegami, S.; Tetrahedron Lett., 1996, 37, 2265-2266. El Sayed, K.A.; Hamann, M.T.; Hashish, N.E.; Shier, W.T.; Kelly, M.; Khan, A.A.; J. Nat. Prod., 2001, 64, 522-524. Youssef, D.T.A.; Yoshida, W.Y.; Kelly, M.; Scheuer, P.J.; J. Nat. Prod., 2001, 64, 1332-1335. de Silva, E.D.; Scheuer, P.J.; Tetrahedron Lett, 1980, 21, 1611-1614. de Silva, E.D.; Scheuer, P.J.; Tetrahedron Lett, 1981, 22, 3147-3150. Kobayashi, J.; Zeng, C.-M.; Ishibashi, M.; Sasaki, T.; J. Nat. Prod, 1993, 56, 436-439. Tsuda, M.; Shigemori, H.; Ishibashi, M ; Sasaki, T.; Kobayashi, J.; J. Org. Chem., 1992, 57, 3503-3507. Konig, G.M.; Wright, A.D.; Sticher, O.; J. Nat. Prod, 1992, 55, 174-178. Jacobs, R.S.; Culver, P.; Langdon, R.; O'Brien, T.; White, S.; Tetrahedron, 1985, 47,981-984. Bennet, C.F.; Mong, S.; Clark, M.A.; Kruse, L.J.; Crooke, S.T.; Biochem. Pharmacol., 1987, 36, 2079-2086. Glaser, K.B.; Jacobs, R.S.; Biochem. Pharmacol., 1986, 55, 449-453. Glaser, K:B.; de Carvalho, M.S.; Jacobs, R.S.; Kernan, M.R.; Faulkner, D.J; Mol. Pharmacol., 1989, 36, 782-788. Lombardo, D.; Dennis, E.A.; J. Biol. Chem., 1985, 260, 7234-7240. Potts, B.C.M.; Faulkner, D.J.; de Carvalho, M.S.; Jacobs, R.S.; J. Am. Chem. Soc, 1992,774,5093-5100. Katsumura, S.; Fujiwara, S.; Isoe, S.; Tetrahedron Lett, 1985, 26, 5827-5830. Katsumura, S.; Fujiwara, S.; Isoe, S.; Tetrahedron Lett, 1988, 29, 1173-1176. Garts, M.E.; Tallman, E.A.; Bonfiglio, J.N.; Harcourt, D.; Ljungwe, E.B.; Tran, A.; Tetrahedron Lett, 1986, 27, 4533-4536. Bury, P.; Hareau-Vittini, G.; Kocienski, P.; Dhanak, D.; Tetrahedron, 1994, 50, 8793-8808. Pommier, A.; Kocienski, P.J.; J. Chem. Soc. Chem. Commun., 1997, 1139-1140. Pommier, A.; Stepanenko, V.; Jarowicki, K.; Kocienski, P.J.; J. Org. Chem., 2003, 1000 ng/mL). The lack of activity of manzamine F provided the first information on structure-activity relationships within this class of compounds, highlighting the key role of the eight membered ring, where the differences between the inactive manzamine F and the active manzamine A are confined. The reduction of the double bond and/or the insertion of a ketone group on the adjacent carbon is evidently deleterious for the antimalarial activity.

6

32

Fig. (7) Chemical structure of the inactive manzamine F (32).

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Additional information on structure-activity relationships came with the isolation of «eo-kauluamine [33, Fig. (8)] a very complex molecule constituted by two manzamine units dimerized through ether linkages between the eight-membered rings [45]. Although also this molecule, like manzamine F (32), lacks the double bond in the eight-membered ring, it demonstrated to possess the same antimalarial activity of manzamine A.

OH

34

Fig. (8) Chemical structure of the active weo-kauluamine (33) and the inactive 12,34oxamanzamine A (34).

The lack of antimalarial activity for 12,34-oxamanzamine A [34, Fig. (8)] (IC50 = 5000 ng/mL) [42] indicates that the C-12 hydroxy, the C-34 methine or the conformation of the eight-membered ring are of key importance for the antimalarial activity. Authors proposed that these data, combined to the lack of activity of manzamine F, suggest that the ability of the C-34 allylic carbon to form a stabilized carbocation may play a

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critical role in the biological activity of the manzamine alkaloids against the malaria parasite. Manzamines have also been reported to be antiinflammatory, antifungal, antibacterial and antitubercolosis agents and to exhibit activity against AIDS opportunistic pathogens (e.g. Cryptosporidium parvum and Toxoplasma gondii) [42, 45-47]. In order to correctly evaluate their antimalarial potential, it should be noted that, apart from the relatively narrow therapeutic index, a major weak points of these compounds is constituted by their extremely complex structures. Although the complete synthesis of manzamines has been recently described [48], obviously, it will not be able to provide the adequate amounts of compounds for complete clinical studies and, hopefully, for introduction in therapy. Thus, the recently initiated efforts aimed at the microbial production of manzamines could represent the better chance for the development of these unique molecules as antimalarial drugs.

,-OH

Fig. (9) Chemical structures of the active lepadin E (35) and the inactive lepadin D (36)

Lepadins constitute a class of recently discovered antimalarial marine alkaloids. These molecules are decahydroquinoline derivatives bearing a linear eight-carbon chain isolated from two marine invertebrates of Australian origin, Clavelina lepadiformis [49] and Didemnum sp. [50]. Lepadin E (35) [Fig. (9)] exhibited antimalarial activity in the high nanogram range (IC50 = 400 ng/mL) while its close analogue lepadin D (36) [Fig. (9)] is almost completely inactive (IC50 = 6100 ng/mL). This

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marked difference of activity highlights the importance of the 2£-octenoic acid ester functionality in place of the secondary alcohol. The mechanism of action of these molecules has not been investigated; however, it could be, in some extent, related to that of the structurally similar aromatic quinoline compounds, as chloroquine. Authors have proposed that the conformationally mobile side-chain could serve to stabilize non-bonding interactions with heme, or with any other "receptor" molecule [50]. However, an exclusive pharmacokinetic effect of the alkyl side chain cannot be excluded. Complete synthesis of lepadins has been accomplished [51, 52]. A class of alkaloids whose chemical structure appears to be related to that of lepadins have been isolated from sponges belonging to the genus Oceanapia [53, 54]. These molecules, called phloedictynes, are 1,2,3,4tetrahydropyrrolo-[l,2-a]-pyrimidinium derivatives bearing at C-6, in addition to an OH group, a variable-length alkyl chain and at N-l a four/five methylene chain ending in a guanidine group, while at C-7 a thioethylguanidine chain may be present or not (37) [Fig. (10)].

Fig. (10) Structural variety of phloedictynes (37) and of the active compound phloedictyn 5,7i (38)

Recently, phloedictynes have been tested against the chloroquineresistant FGB 1 strain of the malaria parasite Plasmodium flaciparum and

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some of them, particularly phloedictyn 5,7i (38) [Fig. (10)], exhibited a good activity (IC50 = 300 ng/mL) with cytotoxicity at concentrations 50fold higher [54]. Although this activity is not exceptional, the simple structure of phloedictyns, that can be obtained through complete synthesis with relative ease [55], encourages further studies. Hopefully, the importance of the different functional groups and the optimal length of the alkyl chains will be thus estimated. For the above classes of marine alkaloids the antimalarial activity is several orders of magnitude higher than the cytotoxic activity and thus, although their mechanism of action has not been determined, it should be likely ascribed to a specific action on Plasmodium. This could not be the case of homofascaplysin A (39) [Fig. (11)], a/?carboline-indole alkaloid isolated from the sponge Hyrtios erecta [56]. Indeed, homofascaplysin A (39) showed activity against chloroquineresistant P. falciparum strains with an IC50 of about 20 ng/mL, but its toxicity toward rat skeletal muscle myoblast cells was estimated to be less than 1 |a.g/mL, and thus the selectivity index of this molecule is very narrow.

39 Fig. (11) Chemical structures of homofascaplysin A (39) and of ascididemnin (40)

A similar reasoning applies to ascididemnin (40) [Fig. (11)] and to 6bromoaplysinopsin (41) [Fig. (12)]. The first is a pyridoacridone alkaloid isolated from several marine sponges, whose antiparasitic activity against P. falciparum falls in the same concentration range required for the cytotoxic activity [57]. The second is a simple indole derivative, first isolated in 1985 [58], recently re-obtained from the sponge Smenospongia aurea [59], whose activity against the D6 clone of P'. falciparum has IC50 = 340 ng/mL with a selectivity index of only 14. Interestingly, compound

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41 showed also a high affinity for human serotonin 5-HT2C and 5-HT2a receptors. s-s

H3ca 42 Fig. (12) Chemical structures of 6-bromoaplysisnopsin (41) and of lissoclinotoxin A (42)

Analogously, lissoclinotoxin A (42) [Fig. (12)], a sulfur-containing alkaloid isolated from the tunicate Lissoclinum perforatum [60], showed high activity against P. falciparum but was later found to be a DNAdamaging agent [61] at very low concentrations and, thus, its use as an antimalarial agent cannot be proposed. Heptyl prodigiosin (43) [Fig. (13)] is another antimalarial alkaloid isolated from a tunicate. Precisely, this pigment was purified from a culture of a-proteobacteria isolated from a marine tunicate and showed an antimalarial activity similar to that of quinine against the chloroquine sensitive strain P. falciparum 3D7 with an in vitro activity that was about 20 times the in vitro cytotoxic activity against mouse lymphocytes. When this molecule was tested in vivo, a single administration of 5 mg/Kg significantly extended the survival of P. berghei ANKA strain-infected mice but, unfortunately, the same dose caused sclerotic lesions at the site of injection [62].

OCH-,

43

Fig. (13) Chemical structures of the antimalarial pigment heptylprodigiosin (43)

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CYCLOPEROXIDES The artemisinin inspiration The sweet wormwood Artemisia annua (Compositae), also named qinghao, has been used in Chinese folk medicine for 2000 years, originally as a treatment for haemorrhoids, but starting from the III century also to treat fevers. In 1972, after activity-guided fractionation, the sesquiterpene derivative artemisinin (in China named qinghaosu: "the active principle of qinghao") (44) [Fig. (14)] was isolated; later its structure was elucidated and it was shown to possess a potent antimalarial activity [5, 63]. This molecule soon appeared to constitute a major breakthrough in the antimalarial therapy because of: i) its nanomolar activity on chloroquine-resistant P. falciparum strains (higher than the activity on chloroquine-sensitive ones) even on cerebral malaria; ii) its fast action; iii) the absence of detectable toxicity at therapeutic doses.

45 Fig. (14) Chemical structures of artemisinin (44) and of its semisynthetic derivatives artemether (45) and artesunate (46)

Artemisinin (44) is a structurally complex cadinane sesquiterpene lactone bearing an endoperoxide group embedded in a 1,2,4-trioxane ring. With its unique juxtaposition of peracetal, acetal and lactone functionalities, it has very much to interest organic chemists. Totally synthetic routes to artemisinin have been developed [64], but their complexity suggests that they will very unlikely supplant the natural extract as drug source.

190

An intense scientific activity has been carried out entailed to the chemical derivatization of artemisinin. The aim was to obtain compounds with better solubility, higher stability, and thus with increased formulation characteristics, and, possibly devoid of the neurotoxic side effects detected for the natural molecule [65]. These efforts soon resulted in the recognition that the endoperoxide linkage is an essential feature for antimalarial activity, given that the acyclic diol and the ether (1,3-dioxolane) analogues of artemisinin were completely devoid of activity [66]. Consequently, the lactone group became the main site for chemical variations that bore the preparation of the oil-soluble artemether (45) [Fig. (14)] and the water-soluble artesunate (46) [Fig. (14)]. Although these molecules are now used for treatment of severe malaria with the support of the World Health Organization, unfortunately, they still possess neurotoxic activity. As a result of the continuing synthetic studies, several artemisinin derivatives, some of which surpass the parent compound in antimalarial potency, have been prepared [67] but many of them show toxicity or have unfavourable pharmacokinetic features. An essential requirement to design optimized artemisinin derivatives would be a perfect knowledge of the mechanism of its antimalarial activity. Unfortunately, still today our knowledge appears incomplete. While the crucial importance of the peroxide pharmacophore is no longer questioned, basically two different mechanisms of action, not completely antithetic, are now on the ground.

H ?

Fe"PPIX

alkylation of protozoan biomolecules

Fig. (15) A schematic view of the postulated mechanism of action of artemisinin (44)

191

According to the first hypothesis, artemisinin (or its analogues) would interact, within the parasite food vacuole, with the iron center of the heme unit released during the digestion of hemoglobin. The interaction of artemisinin with the heme ferrous iron would cause the cleavage of the peroxide bridge and the formation of alkoxy radicals that, after several rearrangements, would result in the formation of free C-centered radicals. These should be toxic to the parasite because they should alkylate not better defined "sensitive" macromolecular targets [Fig. (15)]. This hypothesis was based on the evidence that, in several experimental models, artemisinin reacts with iron ions and in particular it interacts strongly with hemin (ferriprotoporphyrin IX) and its ferrous form (ferroprotoporphyrin IX) to give covalent adducts [68]. However, two different recent evidences have weakened this postulated mode of action: i) it has been recently demonstrated that, once in the parasite cell, artemisinin only scarcely accumulates within the food vacuole and, thus, a key role of its interaction with heme is unlikely [69]; ii) some artemisinin derivatives that are extremely active as antimalarials show very low tendency in vitro to form carbon radicals [70]. The second mechanism hypothesized for artemisinin is based on the interaction with a specific target. This has been identified as a Ca2+dependent ATPase specific of P'. falciparum (PfATP6), a trans-membrane protein associated with the parasite endoplasmic reticulum [69]. However, it is still not clear whether artemisinin reacts with this target as it is (and, therefore, the peroxide bridge exerts its key role concomitantly with the binding), or it needs a foregoing reaction with an iron-containing molecule that, however, should not be heme [70]. Further experiments would be required to gain more insights into the mechanism of action of the cycloperoxide-containing antimalarial agents. The isolation of different antimalarial cycloperoxides from natural sources can evidently help in this task. Indeed, it could provide additional information about the structural features required to the carbon backbone of a cycloperoxide-containing antimalarial agent. With luck, this research could afford new natural compounds whose antimalarial activity is higher than that of artemisinin. In this context, with the inspiration of artemisinin, several research groups are currently engaged in the isolation

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of cycloperoxide-containing compounds from terrestrial plants and active compounds as yingzhaosu A (47) [Fig. (16)] have been obtained and some semisynthetic derivatives, as arteflene (48) [Fig. (16)], have also been prepared.

Fig. (16) Chemical structures of yingzhaosu A (47) and of the semisynthetic derivative arteflene (48)

In the next two sections we will give a survey of the contribution in this field coming from marine sources. Indeed, a number of cyclic peroxides have been isolated from marine organisms and some of them have been tested for antimalarial activity. For clarity, we decided to divide these molecules in two categories according to their postulated (and only in few cases unambiguously demonstrated) biogenetic origin: polyketide derivatives and terpene derivatives. Polyketide derivatives Marine sponges belonging to the family Plakinidae contain a series of simple cycloperoxide derivatives that have been identified as polyketide metabolites possessing six- or five-membered 1,2-dioxygenated rings (1,2-dioxane or 1,2-dioxolane, respectively). A further variation is represented, in some cases, by the presence of a 3-methoxy substitution, building a peroxyketal group. The parent compound of this group of secondary metabolites is plakortin (49) [Fig. (17)] that was isolated more than 25 years ago from Plakortis halichondroides [71]. This interesting secondary metabolite, whose polyketide skeleton suggests the involvement of butyrate units in the biogenesis, has been recently re-isolated in remarkable amounts from

193

the Caribbean sponge Plakortis simplex [72]. In the same study the absolute configuration of the four stereogenic carbons of plakortin has been determined by means of chemical derivatization and reaction with chiral auxiliaries; in addition, a closely related analogue, named dihydroplakortin (50) [Fig. (17)] has been obtained [72].

o

-v-

y

49

O^O/^COOCH3

50

Fig. (17) Chemical structures of plakortin (49) and dihydroplakortin (50) At the time of its first isolation, plakortin was found to be a weak antibacterial agent, while a recent study has finally disclosed the antimalarial potential of this molecule [73]. Using the pLDH assay, plakortin (49) and dihydroplakortin, (50) were assayed against D10, chloroquine-sensitive strain, and W2, chloroquine-resistant strain of P. falciparum. The two compounds showed a good activity, that was more potent on the W2 strain (IC50 = ab. 250 ng/mL on D10; ab. 180 ng/mL on W2). In addition, the two compounds proved to be not cytotoxic in vitro [72]. Interestingly, in the same investigation [73] the structurally related, even more sterically hindered, five-membered cycloperoxide plakortide E (51) [Fig. (18)] was found to be inactive.

OOCH 3

51 Fig. (18) Chemical structure of the inactive plakortide E (51)

The chemical structure of these two antimalarial leads is remarkably simple and thus they could constitute a good probe to establish structureactivity relationships, to check the currently postulated mechanisms of

194

action for antimalarial peroxides and to prepare semisynthetic or totally synthetic derivatives. In this regard, a synthetic study toward this class of cyclic peroxides has recently appeared [74]. Some 1,2-dioxane derivatives structurally related to plakortin have been isolated from Plakinidae sponges and tested for their antimalarial activity. Plakortide F (52) [Fig. (19)] has been isolated from a Plakortis sp. [75] and it has been shown to possess an antimalarial activity that is slightly lower (about one half) compared to that of plakortin: IC50 = 480 ng/mL on D10; ab. 390 ng/mL on W2; however, unless plakortin, this molecule was found to be consistently cytotoxic since the IC50 of toxicity against human colon carcinoma and mouse lymphoma cells is only about double (IC50 = 1.25 \iglmL) than the concentration of the antimalarial activity.

Fig. (19) Chemical structures of plakortides F (52), K (53), and L (54).

A moderate antimalarial activity was also recently reported for plakortide K (53) [Fig. (19)], an 1,2-dioxane derivative substituted at position 3 with an a,(3 unsaturated ketone, isolated from a Jamaican sponge Plakortis sp. [76]. This molecule showed activity against W2 P. falciparum strain with IC50 = 570 ng/mL and a selectivity index > 8.4. Interestingly, plakortide L (54) [Fig. (19)], a closely related analogue lacking the carbonyl function, was completely inactive. Two additional plakortides, named plakortide O (55) [Fig. (20)] and plakortide P (56) [Fig. (20)], have been isolated from Plakortis halichondrioides and tested for antimalarial activity against P. falciparum [77]. These compounds showed a very low activity with an IC50 = 8 (j,g/mL for plakortide O and

195

an IC50 > 50 ug/mL for plakortide P. In addition, these molecules showed toxicity in vitro toward several cell lines at lower concentrations.

55

56

Fig. (20) Chemical structures of plakortides O (55), and P (56).

It should be noted that all these plakortides have a close structural similarity with plakortin and, therefore, their lower level of antimalarial activity can be utilized to gain useful information about the structureactivity relationships within this class of simple cycloperoxide derivatives. The main differences among these compounds are ascribable to the stereochemistry. Indeed, while in the structure of plakortin the most hindered chains attached to the 1,2-dioxane ring are in cis orientation, in the other analogues a trans orientation is present. Most likely, these latter molecules experience a more problematic approach of the cycloperoxide group to its target. However, the chemical structure of the side chains must be also important, as indicated by the marked difference of activity between plakortides K (53) and L (54) and between plakortides O (55) and P (56).

H H3C00C

v°"V 0 C H 3

CH 3

57

58

Fig. (21) Chemical structures of peroxyplakoric acids A3 (57) and B3 (58) methyl esters

Further information on the structure-activity relationships come from data on synthetic and natural 3-alkoxy-l,2-dioxene and 3-alkoxy- 1,2dioxane (both peroxyketals) derivatives that were shown to possess a very good antimalarial activity. In this class of molecules, the alkoxy

196

substituent at position 3 could partly mime the non-peroxidic oxygen atom of the 1,2,4-trioxane ring of artemisinin. The methyl esters of peroxyplakoric acids A3 (57) [Fig. (21)] and B3 (58) [Fig. (21)], isolated from Plakortis sp., showed a very good antimalarial activity against P. falciparum with IC50 = 50 ng/mL and a good selective toxicity index (about 200) [78]. Through the syntheses of some analogues of these active compounds, some conclusions about the structural requirements within these classes of antimalarials were drawn. For example, compound 59 [Fig. (22)] proved to be almost completely inactive, whereas compound 60 [Fig. (22)] retained the in vitro activity of peroxyplakoric acid B3 methyl ester, indicating the importance of the side chain for the antimalarial activity [79]. PCH3 H3COOC

59

60

Fig. (22) Two synthetic analogues of peroxyplakoric acids methyl esters.

When compound 60 was examined through an in vivo system against P. berghei infection, it showed little antimalarial potency because of lability in mouse serum. This undesired finding was demonstrated to be due to the hydrolysis of the ester function to the inactive carboxylic acid. Indeed, the monoethyl amide analogue of 60, that is stable to hydrolysis in the serum, showed a good in vivo activity [80]. Finally, the low antimalarial activity observed for two additional marine cycloperoxides strictly related to peroxyplakoric acid B3 methyl ester, namely chondrillin, (61) [Fig. (23)] [81], and muqubilone, (62) [Fig. (23)] [82], provides other interesting suggestions. The insertion of a double bond within the 1,2-dioxane ring is evidently detrimental for the activity, while the presence of the methoxy group at C-3 exerts a pivotal role in the determination of the antimalarial activity for this group of molecules. Most likely, simple 1,2-dioxane molecules, that, like plakortin, are consistently active, possess other features that are able to compensate the lack of the methoxy group.

197

H3COOC

61 0-0

O

CH-,

62 Fig. (23) Chemical structures of chondrillin (61) and muqubilone (62).

Terpene derivatives Terpene derivatives containing a peroxide group are frequently isolated from natural organisms and marine sources make no exception. Unfortunately, only very few of these molecules have been tested for their antimalarial activity. Sigmosceptrellin A, (63) [Fig. (24)], is a norsesterterpene derivative that showed activity against P. falciparum with IC50 = 470 ng/mL on D6 clone and 420 ng/mL on W2 clone [18] and low toxicity. Interestingly, the C-3 epimer of 63, named sigmosceptrellin B, (64) [Fig. (24)], proved to possess an activity four times lower in the same test with an IC50 = ab. 2000 ng/mL [82]. This is a good demonstration of the importance of relative stereochemistry to determine the antimalarial activity in the series of 1,2-dioxane derivatives.

o

63 64 Fig. (24) Chemical structures of sigmosceptrellin A (63) and and of its C-3 epimer sigmosceptrellin B (64).

198

Methyl-3-epinuapapuanoate, (65) [Fig. (25)], a norditerpene derivative isolated from the New Caledonian sponge Diacarnus levii [83], showed in vitro activity against chloroquine-resistant strains of P. falciparum with IC50 = 1.2 ug/mL [84]. When the molecule was tested against P. berghei in vivo, at the concentration of 25 mg/Kg, a 56% growth inhibition was observed.

65 Fig. (25) Chemical structure of methyl-3-epinuapapuanoate (65)

MISCELLANEOUS COMPOUNDS In this section we have grouped all the marine secondary metabolites that possess a certain antimalarial activity and do not fall in one of the preceding groups, namely they do not contain an isonitrile or a cycloperoxide group and they are not alkaloids. The antimalarial activity of these molecules is generally very low, falling in the jag/mL range; however, since almost all are not cytotoxic, their activity against Plasmodium should be intended as specific and could be used, at least in principle, to elaborate optimized derivatives. Halorsellinic acid (66) [Fig. (26)] is an ophiobolane sesterterpene isolated from the marine fungus Halorsellinia oceanica that showed in vitro antimalarial activity with IC50 =13 ug/mL [85]. (5)-Cucurphenol (67) [Fig. (27)] is a sesquiterpene phenol isolated from different marine sponges belonging to the genus Didiscus [86]. This molecule exhibited a series of biological activities including potent antifungal activity against Candida albicans, inhibition of the protonpotassium ATPase with a possible application to treat peptic ulcers, and in vitro antimalarial activity with MIC of 3.6 ug/mL against the D6 clone of P. falciparum and of 1.8 ug/mL against the W2 clone.

199 HOOC

66

Fig. (26) Chemical structure of halorsellinic acid (66)

Another phenol-containing antimalarial marine metabolite is 15oxopuupehenol (68) [Fig. (27)]. This molecule, isolated from sponges of the genus Hyrtios, is a representative of a distinctive family of sponge metabolites comprising also the quinol-quinone pair of avarol and avarone and biogenetically originating from the junction of a sesquiterpene with a C6-shikimate moiety. Compound 68 exhibited in vitro activity against P. falciparum with MIC of 2.0 ug/mL against the D6 clone of P'. falciparum and of 1.3 |xg/mL against the W2 clone [87].

o.

67

x

x

68

Fig. (27) Chemical structures of cucurphenol (67) and 15-oxopuupehenol (68)

Gorgonians are well known as the sources of unique diterpenes, some of them belonging to unprecedented chemical classes. Some of these molecules have shown a moderate antimalarial activity. Briarellins are a class of eunicellin diterpenes isolated from the gorgonians Pachyclavularia violacea and Briareum polyanthes [88].

200

Among them, briarellin L (69) [Fig. (28)] exhibited activity against P. falciparum with IC50 = 8.0 ug/mL, while, interestingly, the closely related analogue briarellin J, (70) [Fig. (28)], differing only for the lack of an acetoxy group, is practically inactive.

Fig. (28) Chemical structures of the moderately active briarellin L (69) and of the inactive briarellin J (70)

Bielschowskysin, (71) [Fig. (29)], is a diterpene very recently isolated from the Caribbean gorgonian Pseudopterogorgia hallos possessing a highly oxygenated tetracyclic structure based on a previously undescribed ring system [89]. This molecule was shown to exhibit antimalarial activity against P. falciparum with IC50 = 10.0 ug/mL, however, compound 71 exhibited toxicity toward human cancer cell lines at lower concentrations. PHH s-Q

Fig. (29) Chemical structure of bielschowskysin (71)

CONCLUSIONS The marine environment contains compounds that could serve as useful lead structures for the development of new classes of antimalarial drugs.

201

As seen from the preceding discussion, the number of marine lead compounds is lower than that of leads coming from terrestrial plants. This is not the result of an intrinsic "poverty" of the marine sources, while, more likely, it reflects the relatively small number of research group working on marine chemistry. Nevertheless, the antimalarial marine molecules can efficiently integrate the panel of lead compounds isolated from terrestrial sources with new chemical backbones and, sometimes, with "typically marine" functional groups (as isonitriles). In Table 1 we have summarized the antiplasmodial and the cytotoxic activities possessed by the most important antimalarial marine leads encountered in this review. Among them, some isonitrile containing diterpenes, as well as some manzamines and polyketide cycloperoxides emerge as the most promising candidates to future developments. In this regard, the possibility of producing some of these molecules through bacterial cultivation combined to genetic engineering could increase the possibilities of their full pharmacological evaluation and their possible introduction in therapy. As far as the marine antimalarial cycloperoxides, it should be outlined that, in addition to the few molecules tested, many other cycloperoxide-containing molecules have been described from marine sources but they have never been tested for their antimalarial activity. At least nine sponge genera have been recognized to be cycloperoxide producer (or, alternatively, to host cycloperoxideproducing symbiont microorganisms). Terpene cycloperoxides have been described from Prianos, Sigmosceptrella, Latrunculia, Mycale, and Diacarnus sponges; polyketide cycloperoxides have been described from Chondrilla, Xestospongia, Plakinastrella, and Plakortis sponges. These organisms constitute a casket that could keep a treasure: a cycloperoxide compound with antimalarial activity comparable to that of artemisinin and, possibly with simpler structure and better solubility. In the last two or three years we have assisted to a flowering of researches in the field of malaria, and in particular in the field of marine antimalarials. For example, the proposal of a new mechanism of action for artemisinin dates back only to 2003 and the discovery of the antimalarial potential of marine molecules as manzamines and plakortin is a result of the last two or three years. Thus, in conclusion, we are

202

confident that in the near future more marine antimalarials will be disclosed and, hopefully, some of them could also start the long way to become a drug. Table 1. Summary of the antiplasmodial (against D6 and W2 clones) and cytotoxic activities of the main antimalarial marine leads. W2 D6 Cytoa Compound Source IC50(in IC50(in toxicity ng/mL) ng/mL) IC50 (in ng/mL) 142 Axisonitrile-3 Axinella 16.7 >20,000 cannabina (11) 3.2 Isocycloamphilectane Cymbastela 2.5 4300 hooperi (17) Acanthella sp. 0.4 700 Kalihinol A (25) 8.0 Manzamine A Haliclona sp. 4.5 1200 (33) Didemnum sp. 400 Lepadin E > 20,000 (35) Oceanapia 300 Phloedictyn 5,7i 15,000 fistulosa (38) Dihydroplakortin Plakortis 250 180 >20,000 simplex (50) Peroxyplakoric Acid Plakortis sp. 50 10,000 B3 methyl ester (58) Sigmosceptrellin A Sigmosceptrella 470 420 >20,000 sp. (63) Use as reference: 50.5 3.8 17,400 Chloroquine (ref. [331) 4.1 Artemisinin 0.71 >20,000 (ref. [32]) 1 All sponges except Didemnum sp. (a tunicate).

203

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

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BIOACTIVE SAPONINS WITH CANCER RELATED AND IMMUNOMODULATORY ACTIVITY: RECENT DEVELOPMENTS MARIE-ALETH LACAILLE-DUBOIS Laboratoire de Pharmacognosie, Unite de Molecules d'lnteret Biologique, UMIB EA 3660, Faculte de Pharmacie, Universite de Bourgogne, BP 87900, 21079 Dijon Cedex, France ABSTRACT: Saponins are natural glycosides of steroid or triterpene which exhibited many different biological and pharmacological actions: e.g. immunomodulatory, antitumor, antiinflammatory, molluscicidal, antiviral, antifungal, hypoglycemic, hypocholesterolemic, to mention just a few. The aim of this review is to summarize recent advances on the bioactivity of saponins related to cancer and immune system, which has attracted a great attention during the last five years. INTRODUCTION Saponins are an heterogenous group of natural products both with respect to structure and properties offering a great molecular and biological diversity. They are structurally composed of a triterpenoid or steroid aglycone moiety and quite complex oligosaccharidic substituents. There has been an increase in the interest of biological effects of saponins which were evaluated by many in vitro and in vivo test systems [1-3]. They are often related to their membrane interacting properties, resulting in potential toxic or specific biological effects which have been reviewed (antiviral, analgesic, antifungal, antibacterial, hypocholesterolemic, hypoglycemic, antitumor, immunoadjuvant etc [1-3]. Hovewer the application of these secondary metabolites as successful therapeutic agents is still very much limited. They are used as wound healing (asiaticoside), veinotonic (aescin, ruscogenin glycosides), antiinflammatory (glycyrrhizin, aescin), expectorant (senegosides). Since some compounds display antitumor activities in association with modification of the immune system, there is no clear distinction between these activities. Hovewer we will report the last research developments on saponins having cancer related and immunomodulatory activity. Some of these compounds have interesting structural features, that may be used as lead structures for the development of further semi synthetic derivatives. The discussion will also focus on the significant achievements

210

in the understanding of their mechanism of action and structure-activity relationships. I. CANCER RELATED ACTIVITY Advances in the treatment and prevention of cancer will require the continued development of novel cancer preventive and therapeutic agents. Cancer chemoprevention is related to the administration of agents to prevent the initiational (mutational) or promotional events that occurs during the processus of neoplastic development (carcinogenesis). The initiation involves the direct action of the carcinogen on target cells (or after metabolic activation) whereas promotion and / or progression means that the initiated cells are stimulated to proliferate. Inhibition of mutagenesis and inhibition of the tumor promotion/ progression have been used as screening methods for the discovery of potent chemopreventive agents. Chemotherapeutic agents in the contrary are administered in order to kill the formed tumor. In vitro cytotoxic or growth inhibitory activity on tumor cells as well as in vivo antitumor activity on transplanted tumors in animals are useful test systems for the discovery of potential antitumor agents. We will report here recent advances in the discovery of saponins as potent chemopreventive, cytotoxic, and antitumor agents. I.I. Chemopreventive activity Antimutagenic activity The bioassays are based on in vitro application of saponins to bacterial or more recently to mammalian cells treated with known mutagens. A fraction, PCC100 (50-250 p-g/ml) consisting of a mixture of group B soyasaponins (Fig. l)and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). These results showed for the first time the antimutagenic activity of soyabean saponins in mammalian cells [4]. Another study on soyasaponin I (1) could bring a potential explanation of the mechanism of action. It was found to be a potent and specific sialyltransferase inhibitor in the concentration range 5-100 |iM. Many studies demonstrated that hyperialylation, which is observed in certain pathological processes, such as oncogenic transformation is associated with enhanced sialyltransferase (ST) activity [5]. Kaikosaponin III (2) from Pueraria thunbergiana (Leguminoseae) showed a potent

211

R1O

2

2

R2

R3 H H

(1)

Soyasaponinl

rha - gal - glcA-

OH

(1Z7) (77)

Soyasaponin II

rha - 2 ara- 2 glcA-

OH

(128) (129)

Soyasaponin IV

2

gal- glcAara- 2glcA2 glc - gal- 2glcA-

Soyasaponinl II Soyasaponin V

rha - 2gal - 2glcA2 2 gal -*glcArha-*gal

(75) (76) Soyasapogenol A

(130)

Soyasaponin A1

(132)

H 2

dehydrosoyasaponi n I

(134)

2

gal - glcA-

OH

H H H

OH

OH

OH

H

gb - ara0-

OH

. 3araO-

OH

2

2

H

3

gb - gal - glcA-

Soyasaponin A2

(133)

H

OH

rha - 2 ara0g rha - 2glcO-

H

Soyasapogenol B

(131)

OH

gto

2

tha - gal - glcA-

=0

Fig. 1 Soyasaponins

COOR 2

CH 2 OH

CH 3 2

2

R =rha- gal- glcA(2) kaikasaponin III

R1

4 (5) (6)

(73) (74)

kalopanaxsaponin kalopanaxsaponin kalopanaxsaponin kalopanaxsaponin saphdoside C hederagenin

A I B H

R2

H rha - 2 araH xyl - 3 rha- 2 ararha- 2 ararha- 2 gb-' xyl - rha- ararha - 2 gb-' aleglc - 4 xyl- 3 rha- 2 araH H

H

antimutagenicity by using the Ames test. At 1 mg/plate, it decreased the number of revertants of Salmonella typhymurium TA100 by 99% against Aflatoxin Bl (AFB1), but by 75% against N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) [6]. In this assay, hederagenin (74) and its glycosides (3-6) exhibited potent antimutagenic activities against aflatoxin Bl (1.5 |a,g/plate), kalopanaxsaponin A (= a-hederin) (3) being the most

212

active (59% inhibition at 0.5 fig/ml, p10"5M) also induced necrosis as demonstrated by annexin V staining [55],

CH 2 OH

R1

OH

(82) saikosaponin d

(102) songarosaporin c

R2

glc— fucglc

Gypenosides, triterpenoid saponins from Gymnostemma pentaphyllum were reported to induce apoptosis in human hepatoma cells [56] through the up-regulation of the apoptotic - inducing proteins (Bax and Bak) and down regulation of Bcl-2 leading to release of mitochondrial cytochrome c and activation of caspase cascade [56]. Avicins D (11) and G (12), acacic acid glycosides having a monoterpenic acid substituent at C-21 were shown to inhibit the growth of several cancer cells, avicin G being more active (IC50 0.12-1.49 (ig/ml, p

(92) trigonoside II

H

ara-xyl- xyl3

(93) trigonoside III (94) astraversiarine XV

0 K>gB— o

R
20 ng/mL):

Catalpol Verbenaceae Bouchea fluminensis

Stimulating. Regulator of digestive functions. Antiinflammatory

Lamiide

Stachytarpheta cayennensis

Ipolamiide

Verbena officinalis

Hastatoside Verbalin

Anti-inflammatory: • Active (p.o.) in carrageenin-induced paw edema test. Cancer chemopreventive: • Strong inhibitor of rat-brain phospholipid peroxidation. Anti-inflammatory: • Active (p.o.) against carrageenan-induced mouse paw edema. • Active as inhibitor on histamine and bradykinin induced contractions of guinea-pig ileum. Anti-inflammatory: • Active (v.o.) against carrageenan-induced mouse paw edema. Anti-inflammatory: • Active (v.o.) against carrageenan-induced mouse paw edema. • Active (v.t.) against TPA-induced mouse ear edema

[128] [128] [129]

[130] [130] [30]

OS

382

DISCUSSION Many reviews have dealt with the distribution, structure, properties and biosynthesis of iridoids and secoiridoids [131-137] without distinguish between the biosynthetic pathway route. In our case, some of the most studied activities, as anti-inflammatory, antitumoral-chemopreventive, and protective by iridoids biosynthesized by route lla, have been discussed. Anti-inflammatory and analgesic activities: Different assays related with inflammation have been developed on different iridoids. Some iridoids appear to be efficient on subacute processes but not or only slightly active in acute processes. On paw and ear edema, has been established different relationships: Recio and cols.[30] have published the modest relevance of iridoids as systemic antiinflammatory agents. However, some of them, are very active when were locally administrated. This could be due to the weak acidic nature of the iridoids, that makes them more lipophilic in acidic environments such as inflamed tissues. About the structure-activity relationship, they conclude that, the double bond between C-7 and C-8 is responsible for the activity and an OH group at C-5 may increase the activity. The absence or presence of an extraannular carboxyl or carboxymethyl group at C-4 is not relevant, but the conversion of COOH to COOMe increased the topical activity. On the other hand, the facts that can reduce the anti-inflammatory activity could be the oxidation of the double bond to an epoxy group, or an OH-group at C-8. We have realized that other iridoids biosynthesized by route II have not been studied on inflammation tests, and it should be interesting in order to confirm this structure activity relationship. These iridoids might be geniposide, which is similar to bartsioside but with a COOMe group on C4. This assay could find out if COOMe really increase the topical antiinflammatory activity. Testing the activity of ipolamiide, which have an OH-group on 5 and 8, and comparing with lamiide which have OH on 5, 7 and 8 could help to know the real influence of OH radicals on the activity. Besides, only few of these iridoids have been studied further to find their mechanism of action. For example, Bermejo and cols.[125] have

383

studied the effect of some of these iridoids on the arachidonic acid metabolism. Aucubin showed a significant effect as LTC-4 inhibitor, higher than harpagoside and harpagide. Harpagoside and 8-acetylharpagide inhibited PGE-2-release on macrophages. Aucubin, bartsioside, 8-acetylharpagide and harpagoside have shown also TXB-2 inhibition. These results confirm the hypothesis that the anti-inflammatory activity is due to the double bond between C-7 and C-8 with additional OH-radicals in the body of the molecule. Moreover, the substitution with an additional moiety at C-8 is a positive chemical feature for inhibition of TX-synthase activity, conclusion opposite to Recio and cols. [30] where the OH on C-8 reduced the topical activity. Despite of what Bermejo and cols. [125] have published in relation to the unfavorable effect that the presence of a foreign moiety at C-6 have on the anti-inflammatory effect, we think that this conclusion is not clear, since the compounds that they compare do not only have that modification, but also they have oxidized the double bond between C-7 and C-8 as an epoxy group, fact that makes it to loose the activity, as have been described previously [30]. For all these reasons, we also suggest to investigate the inhibitory activity of TX-synthase of lamiide, ipolamiide, loganin and loganic acid, as well as geniposide and derivatives to confirm the structure-activity relationship. In addition, this test could justify the antithrombotic activity showed by geniposide and derivatives [64]. Anti-tumoral and chemopreventive activities: Only few of the iridoids biosynthesized by route II have been assayed for antitumoral or chemopreventive activities. These compounds showed higher cancer chemopreventive than antitumoral activity. The iridoids that stand out against the rest are aucubin, geniposide and derivatives, and picroliv. Aucubin is chemopreventive through different mechanisms: first, it inhibited phase I enzymes, as P-450 [33], avoiding the activation and transformation of procarcinogens to carcinogens agents. It has been proved that the responsible of this activity is its genine, aucubigenin. Secondly, it activated apoptosis, increasing IkB levels, through IkB degradation inhibition and therefore non active NF-kB cannot translocate to the nucleus [29]. It also inhibited antigens-induced TNF-a and IL-6

384

production [29]. This compound has not shown a cytotoxic activity on different tumor cell lines as A-431, BC-1, HT, Lu-1, Mel-2, U-373, Raji human Burkitt lymphoma, Hep-3B, E-367, RN-6, S-180, B-16, L-929 and 3T3 [127], except for Hep G2 cells [28] and a weak activity against leukemic cells [32]. Aucubigenin has shown DNA- and RNA-polimerase inhibition activity [28]. The low cytotoxicity of aucubin is interesting for the chemoprevention point of view. Geniposide and derivatives have also shown very interesting antitumoral and chemopreventive properties: In vivo studies have shown that geniposide with its derivatives, geniposidic acid and genipine decrease the growth of the implanted tumor by ascitic cells, and have a synergic effect with X-irradiation. Besides, geniposide and geniposidic acid are able to decrease undesirable radiation damage to the hematological tissue after radiotherapy [73]. Geniposide inhibits the tumorigenesis induced by different carcinogens, as TPA, benzo-a-pyrene, or Epstein-Barr virus [65, 138]. It can induce phase II enzymes like glutathion-S-transferase (GST) or glutamil-cistein synthetase, helping to the carcinogens detoxification [139]. At the same time, they inhibit phase I enzymes (e.g. cytochrome P-450 and others microsomal monooxygenases), that can activate the transformation of procarcinogens [140]. Besides, it showed an antioxidant activity able to inhibit the lipidic peroxidation, and antimutagenic activity [51], due to the capacity to protect the DNA from the carcinogens-adduct formation, as aflatoxin Bi [139, 141]. Geniposide inhibits enzymes like mieloperoxidase (MPO) or ornithine-decarboxylase (ODC) that are involved on oxidative and tumoral processes [138]. Recently, it has been published the capacity of geniposide to inhibit the angiogenesis, another step of the tumor formation process [142]. Genipin showed a higher chemopreventive activity as inhibitor of induced-formation of carcinogens than geniposide [65]. However,genipin also elicit negative effects like anti-apoptotic [143], mutagenic and genotoxic activities that induce cellular death [144]. A synthetic derivative of geniposide obtained by acetilation, pentaacetyl-geniposide, has also shown cancer chemopreventive properties against some carcinogens [65, 145, 146] and antitumoral activity due to its ability to inhibit DNA and RNA synthesis decreasing therefore the C6 glioma cell line growth rate in vitro and in vivo [147, 148]. This compound also induces apoptosis by different pathways, activating pro-apoptotic

385

signals as p-53 and c-myc, inhibiting Bcl-2 [149] or through the activation of protein kinase C-delta (PKC-6) [150]. Since there are not references about apoptosis and geniposide, we could think that the acetylation of geniposide exert apoptosis activation. We suggest future studies of apoptosis with geniposide. Picroliv, a mixture of the iridoids picroside I and kutloside, has been extensively studied as a chemopreventive agent. It showed antioxidant activity [104, 105], inhibition of lipid peroxidation [87, 108-113], inhibition of VEGF and HIF-1 expression in glioma cells [102], (proteins related to the cell cycle that are over-expressed on tumors), and anti-tumor promoting against different carcinogenesis models [101, 111]. It also down-regulated AP-1 and oncogenes [112]. Picroliv was also able to inhibit sarcoma development, and to reduce transplanted solid tumors, inhibited topoisomerases I and II and different enzymes implicated on the regulation of the cell cycle as, PKC or TK [101, 102]. Picroside and kutloside structures are very similar to catalpol, except for a cinammoyl or vanilloyl radical. Catalpol has not been deeply studied from the antitumoral point of view, and we think that it could be convenient to do it. Catalpol is very close to aucubin with the only difference of the oxidized double bond to an epoxy group. Comparing both of them, we could know the influence of this group on the antitumoral activity. There are others iridoids, as 8-acetylharpagoside that only showed a weak cytotoxicity on A431 [45]. The anti-inflammatory activity also is positive for the chemoprevention, because of the capacity of inhibiting different interleukines release contributes to the chemopreventive activity. Despite most of the compounds did not shown antioxidant activity against free radicals, they were able to inhibit the lipid peroxidation induced by different carcinogens and toxics, also related to thier, also hepatoprotective effect [87, 108-113, 128]. Hepatoprotective and injury protective activities: Some of the iridoids have been tested in vitro and in vivo against different hepatic toxics as a-amanitin or carbon tetrachloride. Aucubin and picroliv are the only iridoids assayed against a-amanitin [24-28], the toxic

386

compound of Amanita sp being both of them active. As seeing on the formula, one of the differences between them is that the C-7-C-8 double bond of aucubin, is oxidized as an epoxy group in picroliv iridoids. Therefore, the activity may be due to the radical OH in C-6 and CH2-OH on C-8. Related to carbon tetrachloride hepatotoxicity, aucubin is active only on in vivo assays [27], but not in vitro [49]. This fact also is observed in other iridoids as loganin or catalposide [49], suggesting that the active agent could be a metabolite. The iridoid that has shown most important hepatoprotective activity is picroliv [88-98], and we emphasize that it could be interesting to assay catalpol in the showed assays for picroliv, and study the influence of these radicals on the hepatoprotective activity. Picroliv has been also assayed against aflatoxin Bi [81-87]. There is some controversy about the effect that geniposide has at the hepatic level. Some authors showed that its hepatotoxicity is due to the increase of transaminase levels [67, 151], whereas recent references defend the protective effect on the liver of this compound [139-141, 152, 153]. Despite genipin is hepatotoxic [67, 151], it is able to increase the glutathion-transferase (GST) levels [139]. This data is surprising, since some of the plants, rich on geniposide are used in popular medicine for treating hepatic diseases. Recently, it has been published that genipin may enhance the bile acid-independent secretory capacity of hepatocytes, effect that make consider genipin as a potent therapeutic agent for a number of cholestatic liver diseases [154]. All these results suggest to work deeply on the hepatic effect of geniposide and derivatives. The protective effects of iridoids are not only at the hepatic or antitumoral levels, but also, may act on neurons or on other tissues against hypoxia. Aucubigenin, catalpol, gardenoside, geniposide and genipin have shown neuritogenic properties, since they are able to induce the neuronal differentiation on PC12h cells through activation of cellular cycle pathways activation as protein-kinase A or cAMP [36, 37]. In addition, genipine is active against Alzheimer's disease, because it can protects the hippocampal neurons from amieloid beta-protein toxicity. [70]. Other iridoids as harpagoside and harpagide and derivatives have attenuated the glutamate-induced neurotoxicity on rat cortical neurons [121]. Catalposide also has been tested as a protector against oxidative injury of Neuro A2 cells, being active by protection of hydrogen peroxide-induced cell death by zinc protophrphyrin IX [41].

387

Picroliv has also shown also a protective effect on different cells and tissues damaged by ischemic injury [102, 110, 112, 117-119], although this compound has not been studied as a neuritogenic agent. Other activities: It is also interesting to comment other activities as the immunoenhancing, anti-allergic, antiasthmatic or healing properties of some of the iridoids biosynthesized by route II. Aucubin, at concentrations lower than 5 \ig/mL significantly stimulated the proliferation of human peripheral blood mononuclear cells and enhanced the secretion of IFN-gamma [63], able to activate non-specific cytocidal activity in macrophages towards a variety of intracellular parasites and neoplastic cells, and also has a direct antiviral action. Geniposide has anti-asthmatic properties, because its inhalation is able to decrease the tight junction permeability of trachea and to produce the reestablishment of the integrity of the tissue affected by the antigens exposure [155]. Picroliv has shown anti-allergic properties, through the inhibition of passive cutaneous anaphylasis in mice and protection of mast cells from degranulation, although, this effect is not due to a direct blocking of the histamine receptor [115]. Picroliv is also able to increase T cell response against infections by Mycobacteria sp. [114] Aucubin, geniposide and geniposidic acid have promoting effects on collagen synthesis and turnover rate in the stratum corneum [52-54]. This activity has made geniposide subject of clinical investigation as a crosslinking agent [66]. In conclusion, we could say that on a wide sense, the dialdehydes generated by the metabolism and hydrolysis of the aglycones of iridoids, could be in part responsible for the described activities. The non-saturated carbonyl group can react with the SH groups, from amino acids and nucleic acids, causing different effects as the cellular growth inhibition [38, 77]. It seems that the pharmacological activities showed by different iridoids are due to their genine, like aucubin. The structural similarity between the aglycone and glutaraldehyde suggests a similar mechanism of enzyme inhibition through protein cross-linking by Schiff reactions.

388

ABBREVIATIONS p.o. p.t. TNF-a IL-6 NF-kB LPS iNOS NO EBV-EA TPA A431 DPPH PLA(2) PKA EGF 20-MC INF DMBA NDEA IGF-I IGF-IR PGE2 LTC4 TXB2 HSV-1 VSV BC-1 HT Lu-1 Mel-2 U-373 Hep-3B E-367 RN-6 S-180 B-16 L-929

Administrated orally. Administrated topically. Tumoral necrosis factor Interleukine Nuclear factor k-B Lipopolysaccharide inducible Nitric oxide synthase. Nitric oxide. Epstein-Barr virus early antigen 12-0-tetradecanoylphorbol-13 -acetate Human epidermoid carcinoma 1,1 ,-diphenyl-2-picrylhydrazyl radical Phospholipase A-2 Protein-kinase A Epithelial growth factor. 20-methylcholanthrene Interferon 7,12-dimethylbenz(a)anthracene N-Nitrosodiethylamine Insuline growth factor Insuline growth factor receptor Prostaglandin E-2 Leukotriene C-4 Thromboxane B-2 Herpes simplex type 1 Vesicular stomatitis virus Human breast cancer Human fibrosarcoma Human lung cancer Human melanoma Human glioma Human hepatocarcinoma Rat neuroblastoma Rat neurinoma Mouse sarcoma Mouse melanoma Mouse conective tissue

389

3T3 HeLa

= =

Mouse fibroblast Human epitheloid cervical carcinoma

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

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CHEMISTRY AND NEUROTROPHIC ACTIVITY OF S£C0-PREZIZAANE- AND ANISL AC TONE-TYPE SESQUITERPENES FROM ILLICIUM SPECIES YOSHIYASU FUKUYAMA3 and JIAN-MEI HUANGb "Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan and bBeijing University of Chinese Medicine, Beijing 100029, China ABSTRACT: This review focuses mainly on our findings of chemistry and neurotrophic activity of the seco-prezizaane- and anislactone-type sesquiterpenes. The genus lllicium is the only member of the family Illiciaceae. From a chemical and biological point of view, the lllicium species are known to be rich in biosynthetically unique seco-prezizaane-type sesquiterpenes, some of which have been known to exhibit neurotoxic activity. Our continuing chemical studies of the lllicium species such as lllicium anisatum, I. tashiroi, I. merrillianum and /. jiadifengpi have resulted in the isolation of a number of sesquiterpenes, most of which belong to both of secoprezizaane- and anislactone-type sesquiterpenes. Chemical studies on unique sesquiterpenes, such as illicinolide, tashironin, merrilactone A, and jiadifenin isolated from the above plants, allow us to further categorize .seco-prezizaane-type sesquiterpenes into six sub-groups according to their carbon skeletons: anisatin-subtype, pseudoanisatin-subtype, majucin-subtype, minwanensin-subtype, cycloparvifloralonesubtype, and a//o-cedrane-type. On the basis of chemical correlation of these subtypes, plausible biosynthetic pathway for all the subtypes of seco-prezizaane-type and anislactone-type sesquiterpenes is discussed. In addition, some sesquiterpenes such as merrilactone A, 11-debenzoyltashironin and jiadifenin are shown to have potent neurite outgrowth promoting activity in primary cultured rat cortical neurons.

INTRODUCTION The genus lllicium belongs to the family Illiciaceae and is an evergreen shrub or tree. About 40 species have been disjunctively distributed in the eastern North America, Mexico, the West Indies and the eastern Asia. The highest concentration of species is in the northern Myanmar and the southern China where nearly 35 species have been described [1]. The genus is comparatively primitive and has close affinities with the Magnoliaceae. Therefore, lllicium was classified into the family Magnoliaceae in much of the early taxonomic literature [2] but was subsequently excluded from the Magnoliaceae by Smith on the basis of floral morphology and vegetative anatomy [3] and assigned familial rank as the Illiciaceae.

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The fruits of the Illicium species are distinctive star-shaped follicles which emit a characteristic refreshing flavor. The fruits of /. vernum Hook, in particular, are the source of the only economically important product derived from the genus Chinese star anise, which is widely used as a spice for flavoring food and beverages. Hence, essential oils have been the primary subject of chemical research on Illicium species and the presence of volatile phenols have been reported as constituents of various parts of all Illicium studied so far. On the other hand, the fruits of /. anisatum, Japanese star anise, have been known to be very toxic for several centuries. Many researchers had been involved in the attempt to isolate the toxic substance since the middle of the 19th century. In 1952, Lane et al. succeeded in the isolation of the pure toxic principle named anisatin (1) [4] for the first time. Its complete structure was later established by Yamada and Hirata [5]. Since anistatin (1) and neoanisatin (la) were recognized as the unprecedented sesquiterpene dilactone containing an unusual P-lactone, several other members of the toxic Illicium species have also been the subject of chemical investigation mainly for Fig. (1). Structure of anisatm(l) ^ P Ur P 0Se ° f clarifying relationship and neoanisatin (la) between structure and toxicity, thus resulting in the isolation of a number of unusual sesquiterpenes and neolignans. Kawano et al., who had continued to investigate the toxic substance in /. anisatum [6], succeeded in systematic studies on chemical components in Illicium plants. Later on, Schmidt and our group have joined in chemical and biological studies on Illicium plants. A number of unique seco-prezizaane-type sesquiterpenes or so called Illicium sesquiterpenes have been reported exclusively by the above three groups, and the occurrence of Illicium sesquiterpenes has been found limited to the genus Illicium. The results of previous research have culminated in classifying them into further subgroups on the basis of a lactone-type as follows: anisatin-subtype, pseudoanisatin-subtype, minwanensin-subtype, majucin-subtype, pseudomajucin-subtype, and cycloparvifloralonesubtype. Also classification of two main types, anislactone-type and a//o-cedrane-type, which consist of rare carbon skeletons, differs from seco-prezizaane-type. This review surveys first the reported secoprezizaane-type sesquiterpenes in terms of the formation of a lactone, and secondly focuses particularly on our chemical studies and lastly on the neurotrophic activities of the sesquiterpenes isolated from /. tashiroi, I. minwanense, I. merrillianum, and /. jiadifengpi.

397

Seco-Prezizaane-Type Sesquiterpenes Anisatin-Subtype

The presence of a very toxic anisatin is noticed in many Illicium plants, such as /. majus [7], /. floridanum [8], /. minwanense [9] and /. merrillianum [10]. Anisatin-subtype sesquiterpenes are characterized by having two types of a 13,14-|3-lactone and a 11,7-6-lactone. The previously reported anisatin-type sesquiterpenes are shown in Fig. (2). All of them except for 5 and 8 belong to neoanisatin derivatives lacking the hydroxyl group at the C-3 position. 1-Hydroxyneoanisatin (2), 6deoxy-1-hydroxyneoanisatin (3), 2-oxo-6-deoxyneoanisatin (9), and an epimeric mixture of 10 and 11 were isolated from /. majus [11, 12], whereas 2a-hydroxyneoanisatin (4) and 2a-hydroxyanisatin (5) were isolated from the pericarps of/, anisatum [13] and /. merrillianum [14], respectively. It should be noted that veranisatins A (6), B (7) and C (8) were isolated as new convulsants in a small amount from the non-toxic Chinese star anise (/. verum Hook, f.) which has been used as a basic spice and also in traditional Chinese and Japanese medicines. They have caused severe convulsions and death at 3 mg/kg (p. o.) in mice [15, 16]. Compound 9 also exhibited picrotoxin-like convulsion and its LD50 was 1.46 mg/kg in mice (i. p.) [11]. HO

HO

RN, H O O 2 R, = R4= OH, R2 = R3 = H 3 R 1 = O H > R 2 = R3 = R4 = H 4 R! = R3 = H, R2 = R4 = OH 5 Rt = H, R2 = R3 = R4 = OH

6 R, = H , R2 = CH2OCH3 7 R, = H, R2 = CO2CH3 8 R = OH R = l > 2 CO2CH3

10 R, = H, R2 = CH3 11 R! = CH3, R2 = H

Fig. (2). Anisatin-type sesquiterpenes 2 - 1 1

Pseudoanisatin-Subtype

Pseudoanisatin was first isolated as a nontoxic compound from /. anisatum by Lane et al [4]. The wrong structure of pseudoanisatin was first proposed on the basis of the spectral data [17], but later it was revised as 12 with a 7-membered 11,14-lactone by an X-ray crystal

398

HO. HO

R, HO 12 R, 13 R, 14 R, 1SR, 16 R, 17 R,

= H, R2 = R3 =OH = R2 = H, R3 = OH = OH, R2 = R3 = H = R2 = OH, R3 = H = R3 = H, R2 = OH =H, R2 = =O, R3 = OH

18 R, = R2 = OH 19 R, = H, R2 = OH 20 R, = OH, R2 = H

23

Fig. (3). Pseudoanisatin-type sesquiterpenes 12-24

26a

27 R, = R3 = OH, R2 = R4 = H

29

Fig. (4). Ketone and acetal equilibrium of pseudoanisatin-type sesquiterpenes 12 and 25 - 26, and acetal sesquiterpenes 27 - 29

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structure determination [18]. Another pseudoanisatin-type sesquiterpenes, 6-deoxypseudoanisatin (16) [19, 22] and l a hydroxypseudoanisatin (18) [20] occurred in the seeds, fruits and leaves of /. anisatum. Recently, another deoxypseudoanisatins, 3dexoxypseudoanisatin (13), 2|3-hydroxy-3,6-dideoxypseudoanisatin (14) and la-hydroxy-3-deoxypseudoanisatin (19) were isolated from /. merrillianum [21], and (2iS)-hydroxy-6-deoxypseudoanisatin (15), 3oxopseudoanisatin (17) and la-hydroxy-6-deoxypseudoanisatin (20) were isolated from /. miwanense [22]. 3,6-Dideoxy-10hydroxypseudoanisatin (21) bearing a hydroxy group at the C-10 position was first found in the fruits of /. merrillianum [24] along with 2,10-epxoy-3-dehydroxypseudoanisatin (22) having an ether linkage between C-2 and C-10. The unusual structure of 22 was established by X-ray crystallographic analysis [25]. Another group of unusual sesquiterpenes having a 1,4 ether linkage and a 14,15-6-lactone named 1,4-epoxy-6-deoxypseudoanisatin (23) and neodunnianin (24) were isolated from /. dunnianum [30]. Later, the structure of 23 was revised to be the same as that of 20 by comparing the spectral data of both. Some pseudoanisatin-type sesquiterpenes were lately found to coexist as ketone and acetal equilibrium such as pseudoanisatin (12) and cyclopseudoanisatin (12a), parviflorolide (25) and cycloparviflorolide (25a) [26], merrillianolide (26) and cyclomerrillianolide (26a) [10] as shown in Fig. (4). It is interesting that 8a-hydroxy-10deoxycyclomerrillianolide (27) [21], 2a-hydroxycycloparviflorolide (28) [14] and an acetal form of 6-deoxypsudoanisatin (29) [23] exist exclusively even in methanol-G?4. However, we have no evidence to rationalize what kind of factors play key role in a favorite equilibrium between a keto and an acetal form in pseudoanisatin-subtype on the basis of the global minimum energy calculated by MM2. The structures of the sesquiterpenes, which belong to pseudoanisatin-subtype having a hydroxyl group at the C-7 position, seem to be confused. The structure of 7-deoxy-7(3-

HO

R,Cf

HO

OR, 30 R, = R 3 == H, 31 R, = Bz, R2 = 32 R, = Ac, R2 = 33 R, = Bz, R2 -

R2 = O H OH, R3 = H OH, R3 = Bz K3 - M

30a R, = R3 = H,R 2 = OH 31a Rj = Bz, R 2 == OH.R3 = H 32a R, = Ac, R 2 == OH =Bz 33a Ri = DZ, K.7= = R 3: = H

Fig. (5). Revised structures of pseudoanisatin-type sesquiterpenes 30-33

OH 30b

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hydroxypseudoanisatin [13] from the seeds of /. anisatum was first elucidated as pseudoanisatin-type sesquiterpene 30, then revised as an 11,3-6-lactone 30a and finally corrected as an 11,7-5-lactone 30b [Fig. (5)] [8], which belongs to the minwanensin-type. Other Illicium plants, /. dunnianum, I. tashiroi and /. merrillianum elaborated dunnianin (31), 6deoxydunnianin (33) [27], and isodunnianin (32) [28], respectively. The structures of these compounds were assigned on the basis of NMR spectral data and by comparison with spectral data of pseudoanisatin (12) and dunnianin (31). Schmidt et al. [29] reported that dunnianin (31) and 7-deoxy-7|3-hydroxypseudoanisatin (30) were isolated from the American star anise, /. floridanum, and their structures were reinvestigated by NMR spectroscopic analyses as well as X-ray crystallographic analysis of dunnianin. As a result, the structures of 31 and 30 were revised as 31a and 30a, which consist of an 11, 3-6-lactone instead of an 11, 14-e-lactone. In comparison with their spectral data, the occurrence of the very large geminal coupling constant (near 20 Hz), accounting for the presence of a 6-lactone ring, and the appearance of H3 as a doublet (about 5Hz) with essentially no coupling with H-2|3 were found to be characteristics of sesquiterpenes having an 11, 3-6-lactone. In light of the spectral data of the other pseudoanisatin-type sesquiterpenes 32 and 33, the structure of 32 and 33 may be revised as 32a and 33a. However, the structure of 30a was revised one more time as 30b in 1999. The 11, 3-6-lactone ring was revised to a 5-lactone closed between C-ll and C-7. However, the vicinal coupling constant of about 0 Hz between H-2|3 and H-3 was not consistent with such a structure. The X-ray crystallographic analysis proved that the structure of 30b was correct and the aforementioned spectral discrepancy could be attributed to a strong intramolecular hydrogen bond between the OH group attached to C-14 and C-3 [8]. Pseudoanisatins derivatives, which have been checked to this point, are non-toxic substances unlike anisatin, but it is worthy to note that isodunnianin (32) not only promote neurite outgrowth in primary cultured fetal rat cortical neurons at 10 uM but also increase the choline acetyltransferase activity [28]. Miwanensin-Subtype

The opening of the spiro (3-lactone of the anisatin-type sesquiterpene leads to the minwanensin-type. The structure of minwanensin (34) from the pericarps of/, minwanense was elucidated [9] and later revised by X-ray crystallographic analysis of its pbromobenzoyl derivative [23], as shown in Fig. (6). This type of compounds, such as 3-acetoxy-14-«-butyryloxy-10-deoxyfloridanolide (35), 14-acetoxy-3-oxofloridanolide (36), 13-acetoxy-14-(«-

401

butyryloxy)floridanolide (37), debenzoyldunnianin (30b) and \A-O-nbutyrylfloridanolide (38), were also found in the fruits of /. floridanum [8] and /. merrillianum [24], respectively. Recently, two C-l epimeric keto forms, (15) and (li?)-miwanenones (39, 40) were isolated from /. miwanense [21]. Additionally 3,4-dehydro-compounds having a hydroxyl group at C-10, 3,4-dehydro-13,14-dihydroxyfloridanolide (41) [31] and 3,4-dehydrofloridanolide (42) [24] also occurred in the above two plants. The structure of minwanensin is considered to be similar to that of anisatin, but it does not show toxicity to mouse at a dose of 50 mg/kg (p. o.). It has no neurotrophic effect either. This suggests that the presence of a (3-lactone in a molecule may be responsible for neurotoxicity.

HO

RiO

34 R, =H, R2 = OH, R3=H 35 R, =Ac, R2 = OCO/iPr, R3= OH

36 R, = = O, R2 = OAc, R3=H 37 R, = H, R2 = OCOnPr, R3= OAc 38 R, = H, R2 = OCOnPr, R3= H

HO Ho.

39 R, = CH3, R2 = H 40 R, = H, R2 = CH3

41 R = OH 42R = H

Fig. (6). Minwanensin-type sesquiterpenes 34-42

Majucin-Subtype

A number of new majucin-type sesquiterpenes having a y-lactone ring as shown in Fig. (7) were found in the pericarps of /. majus, belonging to one of the Chinese Illicium plants. Majucin (43) was the first to assign structure by extensive spectroscopic analysis and

402

comparing its NMR data with tnose of anisatins as well as the data of neomajucin (44) established by an X-ray diffraction method [32, 7]. More majucin-type sesquiterpene lactones, such as (2S*)hydroxyneomajucin (45), 2-oxoneomajucin (48), 2,3-dehydromajucin (49), (2i?*)-hydroxy-3,4-dehydroneomajucin (51), (15*)-2-oxo-3,4dehydroneomajucin (52), (li?*)-2-oxo-3,4-dehydroxyneomajucin (53) and (IR*, 101S'*)-2-oxo-3,4-dehydroneomajucin (54), were reported [7, 34]. Particularly, it should be noted that compound bearing the (10S1*)hydroxyl group is only 54 among the anisatin-like sesquiterpenes. 6Deoxy-neomajucin (46), isolated from the seeds of/, anisatum, is the

R2

43 44 45 46 47

R, R, R, R, R,

= = = = =

H, R2 R3 R3 R2

R 2 = R 3 = OH, R, = H = H, R 3 = OH, R4 = H =OH, R 2 = H, R4 = H = H, R 2 = OH, R4 = H = H, R 3 = H, R 4 = Bz

HO

HO

'.

51

52 R, = CH3, R2 = H 53 R, = H, R2 = CH3

54

CO 2 CH 3

HZ

55

Fig. (7). Majucin-type sesquiterpenes 43-55

first example of the majucin type found in the Japanese star anise [19]. /. angustisepalum also contains the majucin-type sesquiterpene like 10benzoyl ester of neomajucin (47) [33]. In the course of searching for neurotrophic compounds in /. jiadifengpi, we could isolate jiadifenin (55) and 51 as active substances together with a new sesquiterpene, 1,2dehydroneomajucin (50) [35]. In particular, 55 is the first example of a majucin-type sesquiterpene with an oxo-function at the C-10 position. However, since jiadifenin consists of an inseparable equilibrated mixture

403

with regard to the C-10 acetal carbon, its structure elucidation remained ambiguous on the basis of the spectral data. In order to confirm and establish the absolute structure of 55, a synthesis of 55 was attempted starting from 2-hydroxy-3,4-dehydroneomajucin (51), which was the main sesquiterpene isolated from /. jiadifengpi. HO. -0.14 - 0 . 0 ^ MTPAO

( + 0.09

+ 0.13 Fig. (8). A6 (S-R) values (ppm) for MTPA ester derivatives of 51

First, the modified Mosher's method [36] was applied to clarify the absolute configuration of 51 which had never been determined. The A 6 (S-R) value as shown in Fig. (8) enabled us to assign the configuration for C-2 as S. Next, the C-2 hydroxyl group of compound 51 was oxidized with Dess-Martin reagent to give ketone 52 in 81% yield, which previously was obtained as a natural product from Illicium majus [7]. Treatment of 52 with DBU in benzene caused an epimerization on the C-l carbon, resulting in a thermodynamically more stable product 53 [34]. It is noted herein that the chemical conversion of 51 to 52 and 53 can assign the absolute configurations of 52 and 53 as (lS)-2-oxo-3,4-dehydroneomajucin and (li?)-2-oxo-3,4dehydroneomajucin, respectively. It is anticipated to spontaneously produce compound 55a if the C-10 hydroxyl group is oxidized to ketone. Thus, 53 was subjected to Swern oxidation, giving rise to unexpected oxetane 56. This unusual reaction was reasonably rationalized based on the generally accepted mechanism of Swern oxidation as indicated by the following. A sulfoxonium intermediate A abstracts more acidic H-l from H-10, and then the formed carbanion attacks on the oxygen of the sulfoxonium species in B, resulting in the formation of 56. However, Dess-Martin reagent could oxidize the C-10 hydroxyl group to ketone without problem and a usual work-up provided the expected acetal 55a in good yield. Adding methanol to the reaction mixture led this oxidation to the direct formation of jiadifenin (55) as an equilibrated mixture, which was identical in all respects with the natural mixture [35]. It should be noted that compound 56 exhibited neurotrophic activity at 1 \iM, compared with that of 51.

404

HO,

O 52

51

53

56 HO.

CO2H

52

55a

55

a

Scheme l. Synthesis of jiadefenin (55) from 51 "Reagents and conditions: (a) Dess-Martin reagent, CH2C12, r. t., 81%; (b) DBU, benzene, 80°C, 74% (c) (COC1)2, DMSO, CH2C12, -78°C; (d) Et3N, -78°C - 0°C, 34%; (e) Dess-Martin reagent, dioxane, r. t , 70%; (f) MeOH, r. t., 90%.

Pseudomajucin-Subtype

Sesquiterpenes belonging to pseudomajucin-subtype as shown in Fig. (9) feature a y-lactone ring closed in a 11,4-manner. Typical ones are pseudomajucin (57) and its 7-O-(3-D-glucoside (58), which were isolated from the pericarps of /. majus [37]. The structure of 57 was established using X-ray crystallographic analysis. The glucoside linkage position in 58 was determined as C-7 by glycosylation shift (2.6 ppm) at C-7 compared with that of 57. Another group of novel

405

sesquiterpene lactones, (6i?)-pseudomajucin (60), (6i?)pseudomajucinone (61) and merrillianin (62), were isolated from the pericarps of/, merrillianum, and their structures were elucidated on the basis of the spectral data [38]. It should be noted that a methanol solution of 60 coexists with (6i?)-pseudomajucinone (61) in keto/acetal equilibrium, whereas the crystals solely consist of keto-type 61. In fact, treatment of 60 with trimethylorthoformate and methanol in the presence of Amberlyst R 15 afforded a sole 7-O-methylated product 60a, which was then reacted with 4-bromophenyl isocyanate and 1,8diazabicyclo[5.4.0]undec-7-ene as a base in toluene to give rise to the/?bromophenylcarbamate 60b as a single crystal, thus suitable for X-ray analysis. These results allowed us to assign the absolute configurations of the chiral centers of 60b as IS, 2R, 4S, 5S, 6R and 9R, and thereby 60 could be determined as (67?)-pseudomajucin. Accordingly, 61 turns out to be (6i?)-pseudomajucinone having the same absolute configuration as that of 60. Moreover, 61 was crystallized from an ethyl acetate solution although 60 and 61 coexisted as an equilibrated mixture in solvent. Thus, the stereochemistry of 61 was unambiguously established by Xray crystallographic analysis. The structure of 61 was obviously identical with the keto-form of 60 as shown in Fig. (9). Although some pseudoanisatin-type sesquiterpenes have been reported to occur as an

10

R,O 12

12 57 R, = R2 = H 58 R, = H, R2 = Glucose 59 R, = COnPr, R2 = H

60 R, = R2 = H 60a R, = H, R2 = Me 60b R, = CONHp-BrPh, R2 = Me

OH

63 Fig. (9). Pseudomajucin-type sesquiterpenes 57 -62

406

60b

acetal/keto equilibrium [10, 26], it is the first example that each absolute structure of pseudomajucin-type sesquiterpenes (60 and 61) coexisting in an acetal/keto equilibrium has been established independently by Xray crystallographic analysis as shown in Fig. (10). In contrast with (6£)-pseudomajucin (57) existing as a sole acetal-form, its 61 6i?-form 60 readily reaches to keto-form 61 in equilibration. Fig. (10). The ORTEP drawings of 60b and 61 Although we attempted MM2 calculations to compare the global minimum energy between 60 and 61, no reasonable explanation could be provided for 60 and 61 coexisting as an acetal/ketal equilibrated mixture, as well as for (6S)pseudomajucin (57) not being so. Merrillianin (62) is a unique seco-prezizaane-type sesquiterpene with an unprecedented dilactone having a seven-membered 14,7-lactone, which may be biosynthesized from pseudomajucin (57) or an unknown pseudomajucin-type sesquiterpene 63 by oxidative cleavage of the C6/C-7 bond. The oxidative cleavage of the C-6/C-7 bond in 63 or 57 should lead to 62. However, the oxidative cleavage of the C-6/C-7 bond of pseudoanisatin-type sesquiterpenes also may afford a similar skeleton, which takes a spatially revised relationship of two lactone rings in comparison with 62. Thus we attempted an oxidative cleavage of a pseudoanisatin-type sesquiterpene, 1 a-hydroxy-3-deoxypseudoanisatin (19), which we previously isolated from /. merrillianum [10]. Oxidative cleavage of 19 with sodium periodate in ether and water

407

,OH

(O)

57

NaIO4

HO \5

OH

ether/H2O r.t. 88%

19 Scheme 2. Oxidative cleavage of the C-6/C-7 bond of 19

nicely provided the product 64 in good yield (Scheme 2). The structure of 64 was elucidated by extensive analysis of spectroscopic data. Especially, the NOESY spectrum of 64 was carefully compared with that of 62. The NOESY spectrum of 64 revealed the presence of an additional correlation between H-8a and H-12, and no correlation between H-8p and H-15. Hence, the y-lactone ring in 64 is fused down onto C-4 and C-9 opposite to that of 62. This chemical result not only confirms the configuration of the lactone rings in 62, but also supports that a plausible biosynthetic precursor of 62 may be either the unknown pseudomajucin 63 or pseudomajucin (57). Merrillianin (62) is the first example of the C-6/C-7 seco-pseudomajucin-type sesquiterpene, which can be regarded as biogenetically significant for a variety oilllicium sesquiterpenes [38]. Cycloparvifloralone-Subtype

In 1999, new cycloparvifloralone-type sesquiterpenes were reported to consist of a unique acetal-hemiacetal and/or ortholactone structure as shown in Fig. (11). Cycloparvifloralone (65) occurred in the leaves of/, parviflorum, and (11)7,14-ortholactone-14-hydroxy-3-

408

65 R, = R2 = H 66 R, = OH, R2 = H 67 R, = H, R2 = OH HO,

68

HO

f HO HO

'-

OH

71 Fig. (11). Cycloparvifloralone-type sesquiterpenes 65-72

oxofloridanolide (70) was isolated from the fruits of /. floridanum [26], whereas /. merrillianum yielded a number of this type of sesquiterpenes, such as 2a-hydroxycycloparvifloralone (66) [14], 3ahydroxycycloparivifloralone (67) [39], merrillianone (68) [10], 1,2dehydrocycloparvifloralone (69) [39], (ll)7,14-ortholctone-3ahydroxyfloridanolide (71) [39] and merrilliortholactone (72) [14]. All of these possess a hitherto rare ring system with a cage-like acetalhemiacetal and/or an orthoester structure. As cycloparvifloralone-type sesquiterpenes contain an acetal-hemiacetal or an orthoester group in a molecule, it can be anticipated that they are probably equilibrated between an acetal-hemiacetal and an aldehyde-ketone or between an orthoester and a lactone. However, neither of the aldehyde-ketone form or the lactone for 65-72 has been detected by the NMR spectra even in a protic solvent such as methanol-6?4. This means that the presence of compounds 65-72 in solution is considerably favored over that of their 11,7- or 11,14-lactone forms in keto/acetal and/or lactone/orthoester equilibrium. It is generally accepted that they should coexist with each corresponding aldehyde-ketone or lactone form. In fact, acetylation of 68 under normal conditions afforded diacetate 68b as a single product. The absolute structure of 68b was unambiguously established by X-ray crystallographic analysis [14]. This result indicates that compound 68 is equilibrated with aldehyde-ketone 68c through hemiacetal-ketone intermediate 68a. It should be noted that recently 8-

409

deoxymerrilliortholactone (73) and its lactone form 74 were found as an inseparable equilibrated mixture in/, merrillianum [21].

CHO

68

HO-.J

HO

'-

OH

73 Fig. (12). Acetal and aldehyde, and ortholactone and lactone equilibrium of cycloparvifloralone-type sesquiterpenes 68, 73 and 74

/4//0-Cedrane-Type Sesquiterpenes New carbon skeletal sesquiterpenes, which are not able to take their place with the known subclasses of seco-prezizaane-type sesquiterpenes typical of Illicium plants, have been found as natural products, as shown in Fig. (13). A new skeletal sesquiterpene named tashironin (75) was isolated first from /. tashiroi [40] and later found with 11-O-debenzoyltashironin (76) in /. merrillianum [39]. The structure of tashironin (75) was elucidated by extensive analysis of spectroscopic data. Tashironin consists of a 2oxatricyclo[4.3.1.04'9]heptane skeleton, which belongs to a unique tetracyclic sesquiterpene possessing the very rare a//o-cedrane skeleton. Following our report on the isolation of tashironin, three tashironin congeners, debenzoyl-7-deoxo-la,7a-dihydroxytashironin (77), debenzoyl-7-deoxo-7a-hydroxytashironin (78) and debenzoyl-7-deoxo7a-hydroxy-3-oxotashironin (79), were isolated from I.floridanum [31]. Although anisatin (1) and its related seco-prezizaane-subtypes such as pseudoanisatin (12) have been postulated to be biosynthesized

410

from a tricarbocyclic precursor, a//o-cedrane [41], there is no evidence to support this plausible biosynthetic route leading to seco-prezizaane skeleton. It has been still obscure because no tricarbocyclic sesquiterpene made up by the same carbon skeleton as a//o-cedrane has been found in the Illicium species. Thus it should be emphasized that tashironin (75) possesses a precise carbon skeleton corresponding to the a//o-cedrane skeleton, which can be regarded as a biogenetic key for an intermediate leading to all the Illicium sesquiterpenes, i.e. anisatin-, pseudoanisatin-, majucin-, miwanesnin-, pseudomajucin- and cycloparvifloralone-subtypes. Other new skeletal sesquiterpenes named illicinolide A (80) and B (81) were isolated from /. tashiroi [44, 45]. The structure of illicinolide A was elucidated on the basis of the spectral data and then its absolute configuration was established by X-ray crystallographic analysis of the />-bromobenzoyl derivative 80a [44]. The structure of Illicinolide B was assigned as 6o>hydroxyillicinolide A by spectral data compared with those of illicinolide A and its absolute structure was determined by applying the CD dibenzoate rule to the /?-bromobenzoyl derivative 81a [45]. While illicinolides A and B are likely to be closely related to the previously reported anisatin (1) and majucin (43), the structural feature containing y-lactone ring closed between C-7 and C-9 is rather similar to noranisatin [46], an oxidatively degraded product of anisatin.

O VOH

75 R = Bz 76R = H

77 R, = OH, R2 = H 78 R, = R2 = H 79 Rx = H, R2 = =O

Fig. (13). /4//o-cedrane-type sesquiterpenes 75-81

80 80a 81 81a

R, R, R, R,

OCH3 = R2 = R3 =H = R2 = /7-BrBz, R3 = H = R2 = H, R3 = OH = R2 =/?-BrBz, R3 = OH

411

It is generally accepted that anisatin could be biosynthesized from an acorane through a tricarbocyclic precursor, a//o-cedrane, after breaking the bond between C-6 and C-ll as shown in Scheme 3 [47]. Tashironin should be derived directly from a//o-cedrane through oxidation of the C-ll in a cationic intermediate A. On the other hand, the highly oxygenated abnormal structure of illicinolides A (80) and B (81) may be rationalized biogenetically by assuming that the C-ll HO

Seco-prezizaane

HO' HO

j

OH

Pseudoanisatin (12)

Acorane

HO Tashironin (75)

HO HO' OCH3 Illicinolide A (80) Scheme 3. Plausible biosynthesis of seco-prezizaanes, tashironins and illicinolides through a common key biosynthetic intermediate, a//o-cedrane

412

carbon would originate from the C-11 in the normal anisatin skeleton by breaking both the C-ll/C-7 and C-ll/C-10 on a tetracyclic intermediate B which could be made up through a bond formation between C-ll and C-14 from a//o-cedrane as outlined in Scheme 3. Thus, the isolation of tashironin, illicinolide, anisatin, and their related sesquiterpenes from the same source is of considerable significance and throws light on the biogenesis oi Illicium sesquiterpenes. Anislactone-Type Sesquiterpenes In 1989, anislactones A (82) was isolated first as a minor component from the fruits of /. anisatum [48]. The structure of anislactone A was established by X-ray crystallographic analysis, whereas anislactone B (83) was determined to be an epimer with regard to the hydroxyl group attached at the C-7 position [49]. Both compounds have a unique carbon-skeleton which has never been recorded as of natural compounds. Anislactones are most likely to be from the majucin-subtype sesquiterpene because they bear a y-lactone ring typical of majucin. According to structural similarity, the biosynthesis of anislactones was proposed to be presumably derived from the majucin-type sesquiterpene as follows: the ring construction occurs between C-7 and C-8 in the majucin-type compound, followed by the bond formation of C-6 and C-8, and then hydroxylation at C-8. In this case, C-7 should become a C-8 methyl group. Although this seems to be a better explanation for converting from the majucin-type compound to anislactones, the inversion of the C-9 configuration as well as the origin of the C-8 methyl group still have remained ambiguous. Herein, we propose a new term "anislactone" for this type of sesquiterpenes since anislactones should take a position independent of the previously known Illicium sesquiterpenes. We found that anislactone B (83) was the main component in Illicium merrillianum. Following the isolation of 83 and 82, other new anislactone-type sesquiterpenes named 7-0- actetylanislactone B (84) [10], merrilactones A (85) [51], B (88) [50] and C (86 and 87) [50] were obtained from the pericarps of /. merrillianum. In particular, merrilactone A (85) possesses an oxetane ring in feature with two kinds of y-lactones and has an interesting neurotrophic property such as promoting neurite outgrowth in primary cultured rat cortical neurons. Its structure has been elucidated first to be a unique sesquiterpene bearing two y-lactones and an oxetane ring by extensive analyses of spectral data and then substantiated by X-ray crystallographic analysis. Further, its absolute configuration has been established by applying the modified Mosher's method.

413

0 82 R, = H, R2 = OH 83 R, = OH, R2 = H 84 R, = OAc, R2 = H

85

OAc

0

HO

86 R, = OH, R2 = H 87 R, = H, R2 = OH 86a R, = OCH3, R2 = H

88

Fig. (14). Anisalactone-type sesquiterpenes 82-88

On the other hand, merrilactone C (86 and 87) was obtained as an inseparable mixture with a ratio of 5 : 1 due to a lactol ring. In fact, this mixture was treated with trimethylsilyl diazomethane in methanol to give a sole methylated product 86a. Fortunately, 86a gave single crystals suitable for X-ray analysis. The ORTEP drawing of 86a as shown in Fig. (15) reflects that the methoxyl group attaches Fig. (15). The ORTP drawing of 86a downward at the C-14 position, making up the same convex-shaped structure as 84.

414

Merrilactones A (85) and B (88) are presumably derived from anislactone B (83) by cross-esterification between the C-l and C-4 hydroxyl groups. Thus intramolecular transesterification was attempted to preliminarily obtain 88 from 83. At first, 83 was subjected to acidic conditions at room temperature, but no reaction occurred. Next, heating a solution of 83 in methanol-water (1:1) in the presence of sodium hydroxide, followed by acidification, afforded a mixture of anislactone A (82) and two diastereomers (89 and 90) of 88 in 26%, 12%, and 14%, respectively (Scheme 4). Contrary to our expectations, none of 85 and 88 was found in the products. The structural assignments for 89 and 90 were unambiguously done by extensive analyses of their 2D NMR data, indicating the plane structures of 89 and 90 being the same as 88.

.OH

l)NaOH MeOH/H 2 O(l:l) reflux, 16h

HO

O OH

HO 90 (14%) yK

83

,o

O

89(12%)

Scheme 4. Chemical conversion of anislactone B (83) into other anislactones

415

88

89

90

Rg. (16). Selected NOES Y of 88, 89 and 90

However, their NMR data were not identical with one another. The 2D NOESY of 89 and 90 as shown in Fig. (16) clarified them to be epimers of 88 with respect to C-l and C-7. It turns out that two hydroxyl groups at the C-l and C-7 positions take ^-configurations in 89, whereas they take a (3- and an a-configuration in 90, respectively. This reaction probably involves a series of retro-aldol reactions and subsequent aldoltype ring construction as shown in Scheme 5. When 83 was treated with base, the (3-hydroxyl ester moiety initiated a retro-aldol mediated C6-C7 bond cleavage to give A, which underwent an intramolecular aldol condensation to yield anislactone A (82). Additional formation of p-hydroxyaldehyde B under basic conditions caused another retro-aldol mediated C1-C9 bond cleavage to give C, which in turn brought about consecutive ring closures (D, E) by an aldol condensation, thereby giving rise to 89 and 90 after acid work-up. Taking this mechanism into consideration, the conversion of 83 to 89 and/or 90 is favorable when the C-l hydroxyl group takes a ^-configuration, whereas the a hydroxyl group at the C-1 position rather leads to anislactones 82 and/or 83 than 88 due to the ring strain of y-lactone [51]. Thus it is concluded that merrilactone B (88) is not an artifact, but a natural product. Next, our attention focused on the preparation of merrilactone A (85), because an available amount of 85 was very limited to further biological studies. We envisioned a way to utilize anislactone B (83), a large amount of which could be easily obtained from lllicium merrillianum. Our synthetic plan for 85 starting from 83 involved three-step procedure i.e. dehydration, epoxidation and ring expansion (Scheme 6) [51]. At first, a solution of 83 in neat trifluoroacetic acid was refluxed to bring about the lactone transformation to the C-4

416

hydroxyl group and the dehydration of the C-l hydroxyl group, giving rise to 91 in 90% yield. Then, epoxidation of 91 with mchloroperoxybenzoic acid afforded a separable mixture of the desirable

A

90 Scheme 5. Possible mechanism for transformation of anislactone B (83) to 82, 89, and 90 upon treatment of base via sequential retro-aldol and aldol reactions

417

• < % < \o

OH 'v% n

mCPBA CH 2 C1 2

92 (64%)

rt, 36h

91

83

93 ( 4 % )

p-TsOH, dry CH 2 C1 2

92

»rt, 24h, 7 8 %

85

Scheme 6. The synthesis of merrilactone A (85) from anisalctone B (83)

a-epoxide 92 and the unnecessary (3-epoxide 93 in 64% and 4% yield, respectively. High stereoselectivity of epoxidation could be rationalized due to a favorable attack of the peroxyacid from less hindered convex face of 91. Finally, 92 was treated with ptoluenesulfonic acid to give 85 in 78% yield, which was identical in all respects with natural merrilactone A. Thus, we have established a practical preparation of merrilactone A from anislactone B, and thereby have been able to prove the absolute stereochemistry of anislactones A (82) and B (83) to be the same as that of merrilactone A (85) [51]. After our paper was published, Danishefsky et al. reported the total synthesis of merrilactone A [52]. The last two steps of their synthesis of 85 essentially utilized our procedures. Anislactone-type sesquiterpenes are composed of a new type of carbon skeleton and their occurrence is limited only to /. anisatum and /. merrillianum. As these rare natural products feature the presence of a y-lactone ring closed between C-5 and C-6, they are most likely to be biogenetically derived from the majucin-type sesquiterpenes having a ylactone at the same positions. Kouno proposed that anislactones came from the majucin-type compound by the ring contraction between C-7

418

and C-8, followed by the bond formation of C-6 and C-8 [49]. However, this biogenetic hypothesis is not able to reasonably explain the inversion of the C-9 configuration and the origin of the C-8 methyl group in the anislactones. As shown in Scheme 7, it is generally accepted that a tricyclic carbon skeleton, a//o-cedrane A, turns into secoprezizaanes such as anisatin, pseudoanisatin, miwanensin, majucin, pseudomajucin and cycloparvifloralone after breaking the C6-C11 bond of A or the C7-C11 bond of the prezizaane B, which is also derived from A. Co-occurrence of tashironin (75) and its congeners suggests that an intermediate A plays an important role in the biosynthesis of secoprezizaane-type sesquiterpenes. Herein, we propose an alternative biosynthetic pathway leading to anislactones from A as shown in Anisatin Pseudoanisatin miwanensin Majucin Pseudomajucin Cycloparvifloralone

jeco-Prezizaane C

t Tashironin Illicinolide

Prezizaane B ^//o-cedrane A

Anislactone Merrilactone

Scheme 7. Plausible biosynthetic route of anislactone-type sesquiterpene via a commnon intermediate, a//o-cedrane

419

Scheme 7. The bond cleavage between C-10 and C-ll in A gives rise to a bicyclical carbon skeleton D, which repeats the breaking of the C6C7 bond and then the five-membered ring construction between C-6 and C-10, resulting in the formation of anislactone-type carbon skeleton E. This biogenetic hypothesis seems to have no contradiction in explaining the inversion of the C-9 configuration and the origin of the C-8 methyl group. Thus, a//o-cedrane A can be regarded as a significant intermediate for the biosynthesis of all of the Illicium sesquiterpenes. Biological Activity Neurotoxic Activity

Anisatin (1) and neoanisatin (la) are convulsive toxic principles in /. anisatum and regarded as picrotoxin-like potent phytotoxins. The neuropharmacological study of anisatin demonstrates that its convulsive toxicity is probably due to a potent non-competitive GABA antagonist [53]: but, at the present time, which structural part of anisatin is of significance to cause convulsive activity has remained equivocal. A systematic study of structure and toxicity-relationship has not been carried out because of the limited available quantity of compounds, although a number of various anisatin-related compounds have been known as natural products. The toxicity of representative compounds to mice (i. p.) was examined, and compared with that of anisatin (1) and neoanisatin (la) [7, 54]. Among anisatin related compounds, veranisatins A (6), B (7) and C (8), isolated from non-toxic Chinese star anise (/. vernum), caused convulsions and death at 3 mg/kg (p. o.) in mice [15], whereas 2a-hydroxyneoanisatin (4), a positional isomer of the potent neurotoxic anisatin, induced no anisatin/picrotoxin-like convulsions and dramatically decreased the neurotoxicity in mice in

Table 1.

Lethality induced by Illicium sesquiterpenes

Sesquiterpenes

LD50 (mg/kg)

Sesquiterpene

LD50 (mg/kg)

anisatin (1)

1.03*

2-oxo-6-dehydroxyneoanisatin (9)

1.46

neoanisatin (la)

1.62*

(2S*)-hydroxyneomajucin (45)

>40

veranisatin A (6)

40

majucin (43)

>40*

(2/?)-hydroxy-3,4-dehydroxyneomajucin(51)

>40

neomajucin (44)

12.2*

(15}-2-oxo-3,4-dehydroxyneomajucin (52)

>40

pseudoanisatin (12)

>100

(l.ft)-2-oxo-3,4-dehydroxyneomajucin (53)

>40

minwanensin (34)

>50

*Litchfield-wilcoxon method

420

comparison with 1 and la. This is due to an unfavorable interaction of the 2-OH group with the receptor or merely by its high polarity which impairs transport to the target on the basis of comparison of the three dimensional molecular shape and electrostatic properties of active and inactive seco-prezizaane type sesquiterpenes [55]. Other anisatin-type 2-oxo-6-dehydroxyneoanisatin (9) and majucintype neomajucin (44) are also very toxic [7, 11], whereas majucin (43) and its analogues 45, 48, 51, 52 and 53 could not produce any appreciable behavioral changes at dose up to 40 mg/kg [34] as summarized in Table 1. As neomajucin (44) was recognized as a toxic compound, the presence of a spiro (3-lactone moiety in anisatin is not likely to be absolutely responsible for the convulsive toxicity. We have to wait for further investigation in order to prove this kind of problem associated with relationship between structure and toxicity in the Illicium sesquiterpenes. Recently, thirteen seco-prezizaane-type sesquiterpenes were investigated for their structure-activity relationships in GABA receptors to housefly-head and rat-brain membranes. Veranisatin A (6) was found to be the most potent inhibitor in both membranes, followed by anisatin. It is interesting that pseudoanisatin (12) which is not neurotoxic for rats and mice shows a high selectivity for binding to the GABA receptor in housefly membranes. In fact, both anisatin (1) and pseudoanisatin (12) exhibited moderate insecticidal activity against German cockroaches [56]. Neurotrophic Activity

Neurotrophic factors are a subset of biologically active proteins, which are involved in the survival of developing neurons and in the maintenance of mature neurons throughout life [57, 58]. A role of neurotrophic factors in the course of neuronal development is well understood by the example of nerve growth factor (NGF), which enhances neurite outgrowth and maintains cell viability. Such discoveries have raised the hope that NGF may be possible in medicinal treatment of neurodegenerative diseases such as Alzheimer's disease [59]. However, these trophic proteins cannot penetrate into the target brain through the blood-brain barrier due to their high molecular weight, and also bioavailability and stability are problems to be overcome Thus, some endogenous small molecular compounds that are able to mimic the biological effect of the natural neurotrophic factors, or to stimulate their synthesis and secretion, might be promising candidates for pharmaceutical agents of various neurodegenerative diseases. Sex hormones, thyroid hormones, vitamin D and their derivatives are already known to affect survival and differentiation of dissociated mouse embryo brain in cultures or in cultured rat septal neurons. However, application of these hormones to patients with normal hormone function

421

would result in a multitude of undesired effects since they also affect the function of most organs. However, few searches for exogenous NGFlike compounds have been carried out [60, 61]. Thus we have started exploring small molecular compounds from plants-derived natural products having a typical neurotrophic property, which can enhance neurite outgrowth and increase choline acetyltransferase (ChAT) activity in primary cultured fetal rat cortical neurons [62]. Along this line our efforts have resulted in the discovery of novel neurotrophic natural products [63]. HO.

CO,R O

HO 75 R = Bz 76R = H

85

Fig. (17). Illicium sesquiterpenes having neurotrophic activity

As illustrated in the previous chapters, the Illicium plants are shown to be rich in biosynthetically unique seco-prezizaane-type sesquiterpenes and prenylated C6-C3 compounds, some of which were found to exhibit neurotrophic activity [28, 64, 65]. Thus some of seco-prezizaane-type sesquiterpenes which have no neurotoxic action may exhibit an intriguing neurotrophic property. By using the cultures of fetal rat cortical neurons we have evaluated whether Illicium sesquiterpenes isolated by us have neurotrophic properties or not. The neurotrophic sesquiterpenes are shown in Fig. (17). Isodunnianin (32) was found not to be neurotoxic but to have a neurotrophic property such as enhancing neurite outgrowth in primary cultured neurons in the serumcontaining medium at 10 ^M as well as increasing choline acetyltransferase activity at 10 days after seeding [28]. However, under

422 (b)

(a)

M

450 400

313.9

321 1

3» 300 250 200 I.^ll

too 50

Irfl

a 2B2.5

ill conrol hFCF

IIKJ

331.3

330

5

282.5 M

•

j

300 ISO

z a

200 ISO 100 50 D

III oofirol

0.0)

0.1

1.0

10

Fig. (18). Enhancement of neurite outgrowth of rat cortical neurons by compounds 51 and 55 in primary cultured rat cortical neurons; (a) (2S)-hydroxy-3,4-dehydroneomajucin (51); (b) jiadifenin (55); Data are represented as mean + SE (n = 80). Student's test; -kirP 50 uM), Fig. (12); and cucurbitacin D (IC50 = 1.36 (J.M) vs its dihydro-derivative, cucurbitacin R (IC50 > 50 uM). Moreover, the presence of an acetoxyl group at C-25 only modified the potency of the compounds, e.g. cucurbitacin E (IC50 0.18 uM) vs its dihydroderivative, cucurbitacin I (IC50 — 0.95 uM); and cucurbitacin B (IC50 = 0.30 uM) vs its dihydro-derivative, cucurbitacin D (IC50 1.36 uM). Finally, all the compounds tested inhibited actin polymerization in formyl-Met-Leu-Phe (fMLP)-stimulated neutrophils, but did not modify Ca2+ flux or inhibit protein kinase C (PKC) in TPA-activated JY cells. In conclusion, the authors hypothesized that minor changes in the sidechain, but not in the A-ring, markedly affect the potency level for cell adhesion inhibition. This is caused by the disruption of the cytoskeleton, which itself is a consequence of the inhibition of actin polymerization [51].

449

Ito et at. [65], isolated cucurbitacins D and F from Elaeocarpus mastersii and studied their cytotoxicity against a series of human cancer cell lines. Of the two compounds, cucurbitacin D showed higher activity against human lung cancer (Lul), human colon cancer (Col2), human oral epidermoid carcinoma (KB), hormone-dependent human prostate cancer (LNCaP), human telomerase reverse transcriptase-retinal pigment epithelial cells (hTERT-RPEl), and human umbilical vein endothelial cells (HUVEC), with a range of ED50 values from 0.01 to 0.06 u.g/ml. In contrast, the range of cucurbitacin F was from 0.1 to 1.9 ug/ml.

Fig. 12. Chemical structure of cucurbitacin L

Cucurbitacin E inhibited the cell growth of different tumor cell lines, showing remarkable activity in both primary prostate carcinoma explants and immortalized prostate carcinoma cells. It was also found to be a potent inductor of disruption of the actin cytoskeleton; indeed, in a comparative study with similar analogues, the anti-proliferative activity was shown to be directly correlated to the disruption of the F-actin cytoskeleton. Still, the appearance of microtubules remained unaffected [66]. Cucurbitacin E also inhibited a wide range of cancer cell lines with an IC50 range from 13 nM (LOX IMVI melanoma cells) to 295 nM (OVCAR-5 ovarian carcinoma cells). When its anti-proliferative effects were compared with those of a series of cucurbitacin congeners on PC-3 cells (prostate carcinoma), a high correlation between inhibition of cell growth and F-actin disruption was demonstrated. Again, the influence of the side-chain in the chemical structure was clearly supported [66]. Moreover, the same authors [67] demonstrated that cucurbitacin E preferentially inhibits proliferating vs quiescent endothelia. In fact, this compound inhibited the log-phase of ECV and HUVEC endothelial cells at 12 nM and 13 nM, respectively, whereas confluent cells were inhibited at 170 nM and 76 nM, respectively. Cucurbitacin E thus has potential as

450

an anti-angiogenic agent in the treatment of tumor vasculature, especially since proliferating endothelial cells are more sensitive to it than the confluent, low-turnover endothelial cells; however, an in vivo study to evaluate the therapeutic index has yet to be undertaken. Cucurbitacin I suppressed the levels of phosphotyrosine signal transducer and activator of transcription 3 (STAT3) in v-Src-transformed NIH 3T3 cells and human adenocarcinoma A549 cells (IC50 0.5 pM). Moreover, it not only increased mouse survival, but also inhibited growth of human and murine tumors in mice, affecting tumors with high levels of constitutively activated STAT3 while not inhibiting tumors with low levels of activated STAT3 [68]. STAT3 is a key signal transduction protein that, after phosphorylation, plays a dual role of transducing biological information from cell surface receptors to the cytoplasm and translocating to the nucleus where gene expression is regulated. As this protein plays a pivotal role in human tumor malignancy, compounds which suppress its activity may have great potential as anticancer agents [68,69]. Picracin (cucurbitacin Q) and deacetylpicracin (cucurbitacin O) from Picrorhiza scrophulariaeflora, Fig. (13), inhibited phytohemagglutinininduced T-lymphocyte proliferation in a dose-dependent manner with an IC50 of 1 uM. This effect cannot be due to the cytotoxicity of the compounds since in specific experiments the authors demonstrated that neither cucurbitacin exhibits toxic effects for IC50 values up to 50 uM. The mechanism of action seems to be an interference with the cytoskeleton and subsequent abrogation of proliferative signal transduction, which in turn inhibits T-lymphocyte proliferation [70].

0C0CH3

Fig. 13. Chemical structures of picracin (cucurbitacin Q) and deacetylpicracin (cucurbitacin 0)

The methanol extract of Kageneckia oblonga showed a high cytotoxicity against P-388 murine leukemia, A-549 human lung

451

carcinoma, and HT-29 colon carcinoma (IC50 = 2.5 (j.g/ml), but the isolated cucurbitacins showed either weak cytotoxicity (23,24dihydrocucurbitacin F) or none at all (3p-(p-D-glucosyloxy)-16a,23otepoxy-cucurbitan-5,24-diene-ll-one) [13]. The authors compared the cytotoxic effect of 23,24-dihydrocucurbitacin F with that previously reported for cucurbitacin F [71]. Whereas the former had only a weak effect (IC50 = 5 jag/ml), the second exhibited strong cytotoxicity against KB (IC50 = 0.074 ng/ml) and P-388 cell lines (IC50 = 0.04 ng/ml). The authors hypothesized that toxicity increases when a double bond at C23—C-24 is present [13]. However, since the presence of an acetoxyl group at C-25 increases toxicity against non-cancerous cells [43], there is probably more than one structural requirement for increased cytotoxicity. The inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA were examined as a preliminary evaluation of the potential antitumor-promoting activities for eleven cucurbitacins isolated from Bryonia dioica (bryonioside A-G, cabenoside D, bryoamaride, bryodulcosigenin, and bryosigenin). All of these cucurbitacins showed potent inhibitory effects in this test, with the inhibition of induction ranging from 88 to 100% at 1 x 103 mol ratio/TPA, while also preserving the high viability (60-70%) of the Raji cells used in the experiment [9].

OH HO HO

Fig. (14). Chemical structures of cayaponosides B and C2

452

In a similar screening, Konoshima et al. [72] studied the inhibitory effects of twenty-four 29-nor-cucurbitacin glucosides isolated from the roots of Cayaponia tayuya and found that five of them, cayaponosides B, B3, D, D3b, and C2, exhibited significant inhibitory effects on EBV activation induced by the tumor promoter TPA. Moreover, two of the cucurbitacins shown to be active in vitro, cayaponosides B and C2, Fig. (14), inhibited mouse skin tumor promotion in a two-stage in vivo carcinogenesis test.

= R HO-\_^---V--V OH CH 2 OR

CH 2 OR

HO

Fig. (15). Chemical active cucurbitacin-glycosides

Using assays involving EBV-EA activation and two-stage carcinogenesis of skin tumors, the same authors [73] had previously studied the inhibitory effects of nine cucurbitacins isolated from

453

Hemsleya panacis-scandens, eight from Hemsleya carnosiflora, and four from Cowania mexicana. Of the tested cucurbitacins, scandenoside R6, scandenoside R7, carnosifloside III, Fig. (15), 23,24-dihydrocucurbitacin F, 25-acetyl-23,24-dihydrocucurbitacin F, 2-0-P-D-glucopyranosyl23,24-dihydrocucurbitacin F, and cucurbitacin F showed significant activity, inhibiting EBV-EA activation by 85% at 10"3 mol ratio/TPA. Cucurbitacin F and its glucoside both exhibited remarkable anti-tumor promotion effects in a two stage in vivo carcinogenesis test on mouse skin papillomas. Cucurbitacins I, D, and B, along with tetrahydrocucurbitacin I (cucurbitacin R) were found to inhibit the incorporation of radioactive precursors into DNA, RNA, and protein in HeLa S3 cells. The ID50 values of the cucurbitacins, which indicate inhibition of macromolecule biosynthesis, were close to their respective ED50 values, which indicate inhibition of cell proliferation. The authors [58] established a relationship between the capacity of cucurbitacins to inhibit the biosynthesis of DNA, RNA, and protein in HeLa S3 cells and the inhibitory effect on the proliferation of these cells. The inhibitory effects of cucurbitacins on the biosynthesis of cellular macromolecules, as well as the inhibition of cellular growth, originate from a common, as yet unknown target of cucurbitacin activity. However, a correlation has been established between the growth-inhibitory activity of cucurbitacins and dexamethasone, a fact which implies a glucocorticoid mechanism for the former. Notwithstanding, Witkowski et al. [58] assert that the inhibitory effects of cucurbitacins on biosynthesis involve a mechanism resembling the immediate extragenomic effects of glucocorticoids, and are thus not receptor-mediated. Using previously reported data on twenty-four cucurbitacins studied by the National Cancer Institute (NCI), Van Dang et al. [74] established a relationship between chemical structure and cytotoxicity by comparing the data concerning the toxicity against KB cells (nasopharynx human carcinoma) with that concerning toxicity against animals. The most relevant structural features for cytotoxicity are: the presence of an a,(3unsaturated ketone in the side chain (Table 3), a free 16a-0H group in the cucurbitane skeleton, and the presence of an acetoxyl group at C-25 (Table 4). Moreover, the presence of a keto or hydroxyl group at C-2 / C3 and the stereoisomery of the OH group at C-3 were found to modify dramatically the cytotoxic potency (Table 5).

454 Table 3. Influence of C-23 — C-24 substitutions in the cytotoxicity [74] Cucurbitacins

Substitutions

ED5o ng/ml (M)

Cucurbitacin B 23,24-Dihydrocucurbitacin B

A23 C-23,24-dihydro

0.002 (9 xltr 12 ) 2(3.5x10-')

Cucurbitacin Q 23,24-Dihydrocucurbitacin Q

A23 C-23,24-dihydro

30(53x10'') 2900 (5 x 10"6)

Table 4. Influence of C-25 substitutions in cytotoxicity [74] Cucurbitacins

Substitutions

ED.,,, ng/ml (M)

Cucurbitacin B Cucurbitacin D

C-25 (acetoxyl) C-25 (hydroxyl)

0.002 (9 x lO"12) 2 (4 x 10"9)

Cucurbitacin E Cucurbitacin I

C-25 (acetoxyl) C-25 (hydroxyl)

0.00005 (9 x 10"14) 6(11 x 10"')

Table 5. Influence of C-2 and C-3 substitutions in cytotoxicity [74] Cucurbitacins

Substitutions

ED50 ng/ml (M)

Cucurbitacin B Isocucurbitacin B

C-2 (OH) C-3 (O) C-2 (O) C-3 (OHcc)

0.002 (9 x 10"12) 400 (7 x 10'7)

Cucurbitacin D Isocucurbitacin D 3-e/)/-isocucurbitacin D

C-2 (OH) C-3 (O) C-2 (O) C-3 (OHcc) C-2 (O) C-3 (OHP)

2 ( 4 x 10-') 30(58x 10"') 200 (4 x 10'7)

As can be seen above, minor structural modifications not only change the cell cytotoxicity, but also affect the toxicity in animals (Table 6). Thus, while unsaturated C-l cucurbitacins clearly increased both the cytotoxic potency as well as the toxicity in animals (cucurbitacin E vs cucurbitacin B), the saturation of C-23 decreases the toxicity in both cells and animals (cucurbitacin I vs cucurbitacin L).

455 Table 6. Influence of C-l, C-23, and C-25 substitutions on cell and animal toxicities [74] Cucurbitacins Cucurbitacin Cucurbitacin Cucurbitacin Cucurbitacin

E B I L

Substitutions

ED50 (ng/ml)

Toxicity (mg/kg)

A1 A23 C-25 (acetoxyl) C-l,2dihydro A23 C-25 (acetoxyl) A1 A23 C-25 (hydroxyl) A1 C-23,24 dihydro C-25 (hydroxyl)

0.00005 (9 x 10"'4) 0.002 (9 xlO'' 2 ) 6(11 x 10"') 300 (6 x 10"7)

10 2 2 12.5

Finally, blocking C-2, C-3, and C-l6 hydroxyls has been found to reduce the toxicity in all the known cases (Table 7). Comparative data of the cytotoxicity of cucurbitacin Q vs that of its triacetyl-derivative showed a spectacular difference in cytotoxicity, with the former being much more potent than the latter.

Table 7. Influence of C-l, C-2, and C-16 acetylation on cell toxicity [74] Cucurbitacins Cucurbitacin P Cucurbitacin P 1,2,16-triacetate Cucurbitacin O 1,2,16-triacetate

Substitutions

ED 5 0 ng/ml

C-23,24 dihydro C-23,24 dihydro A23

500 45000 20000

On the basis of the data described above, Van Dang et al. [74] used a computer-aided drug design (CADD) to establish a quantitative electronic structure-activity relationship (QESAR) between cytotoxic cucurbitacins and other cytotoxic natural products, including maytansinoids and quassinoids, with the aim of designing new cucurbitacins as future therapeutic agents against cancer. The authors evaluated the pharmacophore of these groups and designed some theoretically active compounds. Of these, the C-25 tygloyloxy derivative seems to be the most effective, Fig. (16), with a theoretical therapeutic index 1727 times higher than that of cucurbitacin E, the most active of the 25-acetyl-derivatives studied.

456

Fig. (16). 25-Tygloyloxy cucurbitacin I (25-tygloyloxy,25-deacetyl cucurbitacin E)

Effects of cucurbitacins as adaptogens and on the immune system As was described above, Panossian et al. [57] demonstrated the adaptogenic activity of Bryonia alba roots in preclinical and clinical trials. The same authors [75] studied the potential mechanism responsible for these adaptogenic effects, focusing on the potential activity of cucurbitacin R-diglucoside, one of the constituents of the active extract. This compound had previously been found to increase the working capacity of mice, and also to increase the survival of mice infected with Staphylococcus aureus as well as that of X-ray irradiated rats. It also reduced stomach ulcers in immobilized rats [75]. In fact, cucurbitacin R-diglucoside protects against stress-induced alterations of eicosanoids in blood plasma and stimulates the adrenal cortex to adapt the organism to stress. Panossian et al. [75] demonstrated that cucurbitacin R-diglucoside increases corticosteroid secretion by stimulating the adrenal cortex, modulating corticosteroid release until optimal levels are obtained, thereby protecting the adrenal cortex from hypotrophy. Moreover, cucurbitacin R-diglucoside modifies the metabolism of eicosanoids, increasing the production of PGE2, which is sub-produced in times of stress. PGE2 has a cytoprotective influence on the gastrointestinal epithelium, which is clearly damaged by stress. On the other hand, cucurbitacin R-diglucoside inhibited the biosynthesis of the pro-inflammatory mediators from LOX such as LTB4 and 5-hydroxy6£,8Z,llZ,14Z-eicosatetraenoic acid (5-HETE), which activate chemotaxis of neutrophils, lysosomal enzyme release, vascular permeability, and superoxide anion generation. Moreover, it inhibited the NADPH-dependent enzymatic and ascorbate-induced non-enzymatic lipid peroxidation. However, cucurbitacin R-diglucoside had no effect in

457

the in vitro assays when these were carried out on isolated leukocytes of immobilized rats, probably because stress significantly suppresses the 5LOX and 12-LOX activity of leukocytes by a mechanism mediated by the increase of corticosterone formation. A previous pretreatment with cucurbitacin R-diglucoside decreased 12-LOX activity, but increased that of 5-LOX. This finding indicates that the systemic effects of cucurbitacin R-diglucoside as an adaptogen occur at a central rather than at a peripheral level [75]. Cucurbitacins B, D, and R were assayed as immunomodulators on mitogen concanavalin A-stimulated IL-2 dependent murine lymphoblasts (IL-2 BL) and mitogen concanavalin A-stimulated murine spleen cells (ConA SC), but the activity was of little interest due to the pattern of activity and its combination with the results of the compounds' influence on growth of permanent cell lines, described above. One interesting finding, however, concerned the activity of cucurbitacin R, the free form of the aforementioned compound, which gave IC50 values of 1.0 and 0.46 (ig/ml against IL-2 BC and ConA SC, respectively [64]. Effects on insects and plant parasites Many secondary metabolites found in plants deter phytophagous invertebrates, sometimes even modifying insect growth and development if included in the diet [8]. Natural products can often act as insecticides via different pathways, as is the case with the analogues of insect juvenile hormones produced by plants. Thus, some derivatives of these analogues are used as commercial insecticides while others act as ecdysteroid antagonists [8]. Ecdysteroids are steroidal hormones responsible for controlling molting and metamorphosis in insects, thereby contributing to their normal development and probably that of other invertebrates as well. Analogues of ecdysteroids, the so-called phytoecdysteroids, occur in some plants, but there is another parallel group of compounds made up of known antagonists of the ecdysteroid receptor [24]. Of this latter group, the cucurbitacins form a widely-cited subset [23]. Cucurbitacin B, for example, is a known antagonist of 20-hydroxyecdysone [25,26], and is responsible for the antagonistic activity of a methanolic extract of Iberis umbellata (Cruciferae) [26] and Physocarpus opulifolius (Rosaceae) [4],

458

which prevents the 20-hydroxyecdysone-induced morphological changes in the Drosophila melanogaster Bn permanent cell line. CH2OH

Carnosoflogenin A

CH2OH

Camosoflogenin C CH 2 OR

Carnosifloside II CH 2 OR

Carnosifloside VI Fig. (17). Chemical structure of cucurbitacins from Hemsleya camosiflora

Four of the seven cucurbitacins assayed in the Drosophila melanogaster Bn bioassay exhibited antagonistic activity [8]. Carnosoflogenin A and C, as well as carnosifloside II and VI, Fig. (17), all isolated from Hemsleya camosiflora, showed weak antagonistic activity at 0.1 mM. The ED50 values obtained to produce a 50% reversal of the reduction in A405 brought about by 5 x 10~8 M 20hydroxyecdysone were 3.4 x 10"4 M and 1.2 x 10"4 M, respectively. These effects were lower than that reported for cucurbitacins B and D,

459

which show activity in the 0.1 uM range. In the last case, the activity was associated with the presence of an a,P-unsaturated C-22 ketone. In the cucurbitacins isolated from Hemsleya carnosiflora, however, the activity was associated with the presence of a trans-A24 double bond. Moreover, dihydrocucurbitacin F and 25-acetoxy-dihydrocucurbitacin F, both isolated from the same source and possessing an oxo-function but lacking a double bond, showed only weak antagonistic activity, with ED50 values in the range of 3 x 10"5 M.

Hexanorcucurbitacin D

OCOCH3

HO1

Cucurbitacin F

Cr'

>A3'-4' 15 (Z>A 3 ' 4 '

New azaphilones named isochromophilones III-VI (18-21) were isolated from the culture broth of Penicillium multicolor FO-3216 as inhibitors of ACAT. Their structures were elucidated by NMR and other spectroscopic analyses. The IC50 values of isochromophilones (17-20) (ACAT) activity in an enzyme assay using rat liver microsomes were calculated to be 110, 50, 50 and 120 uM, respectively [79]. The compound 19 also inhibited the activity of cholesteryl ester transfer protein (CETP) with an IC50 value of 98 uM. The compounds 18, 20 and 21 weakly inhibited the activity of CETP in 300 uM. Antimicrobial and cytotoxic activities of 18, 20 and 21 were tested. They inhibited the growth of Staphylococcus aureus, Bacteroides fragillis, and Pyricularia oryzae at 50 ng/disk. However, they did not

484

inhibit the growth of Bacillus subtilis, Micrococcus luteus, Mycobacterium smegmatis, Escherichia coli, Pseudomonas aeruginosa, Xanthomonas oryzae, Acholeplasma laidlawii, Candida albicans, Saccharomyces sake, Aspergillus niger and Mucor racemosus in the same concentration. The IC50 values of 18, 20 and 21 against the growth of B16 melanoma cells in vitro were 33, 36 and 30 uM, respectively.

R H Ac H

18 19 20

A No No Yes

AcO

21

New isochromophilones VII-VIII (22) and (23) were isolated from the culture broth of Penicillium sp. FO-4164. Both isochromophilones inhibited DGAT activity (assayed in vitro using rat liver microsomes) with IC50 values of 20.0 and 127 uM and ACAT activity with IC 50 values of 24.5 and 47.0 uM, respectively [80]. Antimicrobial activity was tested at a concentration of 10 u.g/paper disk. Both isochromophilones showed antimicrobial activity against Bacillus subtilis, Mycobacterium smegmatis, Micrococcus luteus and Pyricularia oryzae. But no antimicrobial activity was observed against the following microorganisms: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Saccharomyces sake, Mucor racemosus and Aspergillus niger.

485

Isochromophilone IX (24), a novel GABA-containing metabolite, was isolated from a cultured fungus, Penicillium sp. [81]. Cl

AcO'

The azaphilone (25) produced by Penicillium sclerotiorum active in assays for the detection of antagonists of the endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors has been identified. Data for the inhibition of endothelin-1 (ET-1) and endothelin-3 (ET-3) binding in the ET(A) and ET(B) receptor assays, respectively, have been reported for this series. Compound 25 was more selective for the rabbit ET(A) receptor than for the rat ET(B) receptor. The IC50 value for (25) was 9 uM in an assay based on binding of ET-1 to rabbit ET(A) receptors. In an assay based on the binding of ET-3 to the rat ET(B) receptor compound 25 exhibited IC50 of 77 uM. This compound demonstrated an antagonistic behavior in a secondary assay based on blockade of ET-1 stimulated arachidonic acid release from rabbit renal artery smooth muscle cells, when present at concentrations greater than or equal to 30 uM [82].

A novel brominated azaphilone derivative, 5-bromoochrephilone (26) and known derivatives, were isolated from the culture broth of a producing organism Penicillium multicolor, fermented in a medium

486

containing potassium bromide. Nineteen azaphilone-related compounds isolated from the above strain and from other fungi were tested for the inhibition of gpl20-CD4 binding, and the structure-activity relationship was discussed. 5-Bromoochrephilone was found to be the strongest inhibitor (IC50, 2.5 uM). A halogen atom at C-5, a proton at C-8 and a diene structure in C-2 side chain of 6-oxoisochromane ring are necessary for gpl20-CD4 binding [83]. The results in Table 3 indicate that the halogen atom at C-5 and the orientation from C-8 to C-10 in the isochromane ring of azaphilones, in addition to the diene structure in C-2 side chain, are very important for the inhibition of gpl20-CD4 binding. Table 3. Inhibitory activities of azaphilones on gpl20-CD4 binding. Inhibitor isochromophilone I isochromophilone II isochromophilone III isochromophilone IV isochromophilone V tetrahydroisochromophilone I sclerotiorin rubrorotiorin 5-bromoochrephilone rotiorin luteusin A chaetoviridin A chaetoviridin B

IC50 (uM) 6.6 3.9 48 96 14.6 >260 >250 >240 2.5 >240 9.4 >230 140

The effect of thirteen different fungal azaphilones on cholesteryl ester transfer protein activity was tested [84].

487

A new secondary metabolite, 8-O-methylsclerotiorinamine (27), was isolated from a strain of Penicillium multicolor, and its structure was established using NMR spectroscopy and chemical evidence. The metabolite significantly inhibited the binding between the Grb2-SH2 domain and the phosphopeptide derived from the She protein and also blocked the protein-protein interactions of Grb2-Shc in cell-based experiments, with IC50 values of 5.3 and 50 uM, respectively [85].

27 OMe

Four new azaphilones, named helicusins A, B, C and D (28-31), were isolated from Talaromyces helicus. These four new azaphilones showed weak monoamine oxidase-inhibitory effects [86].

28 R

29 R -

Three new azaphilones (32-34), named luteusins C, D and E, were isolated after a 21-h surface cultivation of the ascomycete Talaromyces luteus (anamorph of Penicillium vonarxii) on rice, together with luteusins A and B, previously known as inhibitors of monoaminooxidase [87]. The new compounds had no MAO-inhibitory activity [88]. The potential to inhibit MAO is lost after conversion of hydroxyl group in position C-8 to oxo-group and hydrogenation of the side chain double bonds.

488

Falconensins, i.e. azaphilones esterified by chloroorselinic acid, A (35), B (36) and C (37), were isolated from mycelial extracts of the Venezuelan soil fungus ascomycete Emericellafalconensis [89]. Itabashi et al. [90] later isolated falconensone H (43) together with falconensins A, B, C and D (38) from the same culture. Their chemical structures are shown below.

35 R, - H, R2 = R3 = Cl, R4 = Me 37 R, = Ac, R2 = R3 = Cl, R4 = Me 39 R, = R3 = H, R2 = Cl, R4 = Me 4OR,=R3 = R4 = H , R 2 - C I 42 R, = R 4 - H , R 2 - R 3 = C 1 F>3

Further falconensins, i.e. falconensins E (39), K (40), L (41), M (42) and N (44), were isolated also from Emericella falconensis. Three new azaphilone derivatives designated falconensins E, F and G were isolated from the mycelium of Emericella falconensis, along with falconensins AD and H. The structures of new falconensins were established by spectroscopic investigation and chemical correlations. The absolute stereochemistry of falconensins A-G was also established [91].

489

38 R, = Ac, R2 - Rj = Cl, R, = Me 41R|=R 3 =R4=H,R 2 -C1 44R 1 -R 4 -H,R 2 =R 4 -C1

Six new hydrogenated azaphilones designated falconensins I-N were isolated as minor components from mycelia of Emericella falconensis and/or E. fruticulosa along with nine azaphilone derivatives, falconensins A-H. The structures of falconensins I-N were determined by spectroscopic investigation and chemical correlation [92]. Chaetoviridines A (45), B (46), C (47) and D (48), also belonging to the azaphilone group, can be isolated from the fungus Chaetomium globosum var. flavoviride grown on wheat. Spectral data show that chlorine-containing azaphilones are involved with a conjugated y-lactone connected angularly with the azaphilone unit. Chemical structure of chaetoviridin A was determined by NMR, and so the absolute configuration has been established [93]. The red pigment chaetoviridin A contains an unsaturated y-lactone ring, whereas the yellow chaetoviridins B, C and D contain a saturated y-lactone ring as shown. Chaetoviridin A was shown to inhibit monoaminooxidase and growth of P. oryzae at 2.5 ug/ml.

490

O

46R=H 47 R=OH

OH

48

OH

The fungal metabolite, sclerotiorin (49) was first isolated independently by two groups from Penicillium sclerotiorum [94,95] and later from P. multicolor [95]. The 7-epimer (50) is also a known metabolite of P. hirayamae [96]. Sclerotiorin was isolated for the first time from lichen mycobiont Pyrenula japonica [97]. The related rubrorotiorin (51) was also found in P. hirayamae [98].

49 50 7-epimcr

Chlorofusin (52) was one of the major components produced by fermentation of Fusarium sp. 22026. The DELFIA-modified ELISA was used to guide to the purification of inhibitors of the p53/MDM2 interaction from these fermentation extracts. Chlorofusin was the most abundant inhibitory compound. Titration of purified chlorofusin in the

491 DELFIA-modified ELISA gave an IC50 of 4.6 uM. In simultaneous crossscreen testing, chlorofusin was inactive at a concentration of 4 uM in the TNFa:TNFa receptor protein-protein interaction, which was configured in the same format as the primary assay. The compound showed no cytotoxic effects against Hep G2 cells at a concentration of 4 uM. At a test concentration of 7.3 uM, chlorofusin did not exhibit any antimicrobial activity against the following test strains: Escherichia coli, Staphylococcus aureus, Serratia marcescens, Bacillus subtilis, Klebsiella pneumoniae, Proteus vulgaris, Candida albicans, Cryptococcus neoformans and Aspergillus niger [99].

Cl

OH

J ^

CONH 2

52 H 2 NOC

Indoles Indole-alkaloid isoprenoid was isolated from extracts of Penicillium crustosum grown on rice. This compound, designated thomitrem (53), contains a 18(19)-double bond and lacks the characteristic penitrem 17(18)-ether linkage [100]. Penitrems are a group of tremorgenic mycotoxins produced by a variety of Penicillium and Aspergillus species, amongst which Penicillium crustosum is generally regarded as the most important producer of this group of mycotoxins. They have been reported to intoxicate animals. Generally, penitrem A is considered to be the most significant of the series of P. crustosum mycotoxins, which includes

492

penitrems B, C, D, E and F, and other related metabolites such as PC-M4, PC-M5, PC-M51 and PC-M6.

,OH

Cl 53

A fungus, Penicillium crustosum, is known to produce the tremorgenic mycotoxins penitrems A, C, F (54-56), when grown in surface culture [101-103]. All of these tremorgens have a common core structure composed of an indole moiety, biosynthetically derived from tryptophan, and a diterpenoid unit from four mevalonate-derived isoprenes. Penitrems are also produced by other Penicillium species and by Aspergillus sulphureus. This group of metabolites is capable of eliciting tremors in vertebrates, and some specific members have also shown insecticidal activity. For example, compound 54 shows convulsive and insecticidal activities against Bombyx mori, Spodoptera frugiperda, and Heliothis zea, and its use as an insecticide was patented in 1990. Natural penitrem analog 6-bromopenitrem E [104] also exhibits insecticidal activity.

,OH

54 R, = OH, 23a,24a-epoxide 55 R, - H 56 R, = H, 23a,24a-epoxide

Twenty-five Penicillium species were isolated and mycotoxins produced by several of these species, including penitrems A-F, were detected. The levels of penitrem A in these samples were in the range 357500 ug/kg [105]. HPLC and diode array detection were used to confirm

493

the chemical structure of the mycotoxins, e.g. penitrem A and ochratoxin A in extracts from three mycotoxigenic fungi (Penicillium crustosum, Penicillium glabrumlspinulosum, and Penicillium discolor) that dominated on Castanea sativa nuts sold in Canadian grocery stores [106]. Another paper includes data for detection and dereplication of >400 fungal metabolites using MS/ESI+ methods [107]. Sporidesmins, the series of sulfur-containing physostigmine-like metabolites, were isolated from Sporidesmium bakeri A (57), B (58) and C (59), a fungus that causes facial eczema and liver damage in farm animals, e.g. New Zealand sheeps [108-110]. Later studies identified sporidesmins D (60) [111], E (61) [112], F (62) [111], G (63) [113,114], H (64) [115]) and J 65) [115] from P. chartarum. Protonated [M+H]+ and deprotonated [M-H]" ions were observed in positive and negative ion ESI modes, respectively [116]. In a further paper, complexation of sporidesmin A, with metals, was used for its analysis [117].

MeO

O

OMe 57 R = OH, n= 2 58 R = H, n= 2 61 R = OH, n= 3 63R = OH, n=4

MeO OMe

59

494

,,,iv\\SMe

MeO OMe 60

MeO

MeO OMe

OMe

Me

64

62

MeO 65

Saccharothrix aerocolonigenes produces an indolocarbazole antitumor agent rebeccamycm (66) under submerged fermentation conditions. Adding D,L-6-fluorotryptophan to culture of S. aerocolonigenes induces the formation of two novel indolocarbazoles, fluoromdolocarbazoles A (67) and B (68). Feeding A^-5-fluorotryptophan to culture of S. aerocolonigenes induces the production of a novel indolocarbazole, fluoroindolocarbazole C (69). These fluoroindolocarbazoles have been isolated from culture broth and purified to homogeneity by vacuum liquid chromatography and column chromatography. All three fluoroindolocarbazoles are more potent than rebeccamycin against P388 leukemia via ip route in murine model [118].

495

66 67 68 69

Ri

R2

R3 R4

Cl H H H

H F F H

H

H H F

Me Me

H H

Antraquinones Like the fungal and lichen xanthones, anthraquinones, which are also produced both by lichens and fungi, are derived from extended polyketides by cyclization. Several chlorinated compounds were described. The structures of novel topoisomerase I inhibitors, topopyrones A and B (70 and 71), were elucidated by spectral analysis of the chemical derivatives. It was suggested that topopyrone B is converted from topopyrone A [119]. Topopyrones A and B selectively inhibited recombinant yeast growth dependent on the expression of human topoisomerase I with IC50 values of 1.22 and 0.15 ng/ml, respectively. The activity and selectivity of 71 were comparable to those of camptothecin. The relaxation of supercoiled pBR322 DNA by human DNA topoisomerase I was inhibited by these compounds, however, they did not inhibit human DNA topoisomerase II. Both topopyrones were cytotoxic to all tumor cell lines when tested in vitro. Topopyrone B has potent inhibitory activity against herpesvirus, especially varicella tester virus (VZV). It inhibited VZV growth with EC50 value of 0.038 ug/ml, which is 24 fold stronger than that of acyclovir (0.9 ug/ml). Both topopyrones were inhibitory against Gram-positive bacteria.

496

OH

OH

O

O

"OH

Aspergillus ustus produces several novel pentacyclic metabolites, the austocystins, two of which, i.e. A (72) and C (73), contain chlorine [120]. OMe

O

72 Rj = Me, R 2 = H

The fungus Cercospora beticola, which is a highly destructive disease of sugar beets worldwide, has been shown by several groups to produce a series of highly intricate metabolites, beticolins 1 (74) (= cebetin A), 2 (75), 3 (76), 4 (77), 6 (78), 8 (79) and cebetin B (not shown), the latter of which is bis-M% complex of cebetin A [121-126]. Following some initial confusion regarding the complexity of structures and the fact, that beticolin 2 and cebetin A are in equilibrium, the situation now appears to be in order [125,126].

497

ci HO,

HO,

OH

O

OH 76R = Me;a-COOMe 77 R - CH2OH; a-COOMe 78 R = Me; P-COOMe 79 R - CH2OH; 0-COOMe

The cheese mold Penicillium nalgiovensis produces nalgiolaxin (80), which appears to be the first natural chlorinated anthraquinone isolated [127,128], although fragilin (81) was the first fully characterized [129]. A summary of the known chlorinated anthraquinones (80-88) is given below.

R,0

498 No. Ri

R2

80 81 82 83 84 85 86 87 88

Me Me H H Me Me

Me

OH OH OH OH OH OH OH OH

R3

Rt

R5

MeCHOH H H H H H

H H H

Cl - H OH - H OH H

OH OH OH OH

OMe OMe OMe OH OH

Several chlorinated metabolites closely related to anthraquinones are also known. For example, the aspen tree fungus Phialophora alba which protects the tree against attack by the decaycausing fungus Phellinus tremulae produces anthrone, in addition to other derivatives [130]. Anthrone was found in cultures of Aspergillus fumigatus [131], and the corresponding bromo compound was formed in the presence of bromide [131]. The novel bis-anthrones, flavoobscurin A (89), B (90) and B2 (the latter two are rotational isomers) were produced by Anaptychia obscurata [132]. OH Cl

Cl

Cl

Cl

OH

Macrocycles Several relatively large cyclic peptides have been found to contain halogen. Islanditoxin (91) which was isolated in 1955 from a culture of

499

Penicillium islandicum is a chlorine-containing peptide whose structure was determined later. This organism also produced cyclochlorotine (92), which is an infectant of yellowed rice [133]. The fungus Metarhizium anisopliae produces the chlorohydrin cyclic peptide (93) [134]. Cyclochlorotine, a hepatotoxic mycotoxin, was also isolated from Penicillium islandicum.

Monorden (94) and the novel resorcylic acid lactones pochonins A (95), B (96), C (97), D (98) and E (99) were isolated from cultures of the clavicipitaceous hyphomycete Pochonia chlamydosporia var. catenulata strain P 0297. Fermentation of P 0297 in bromide-containing culture media led to a shift in secondary metabolite production and yielded monocillins (compounds without bromine in the molecule) as major metabolites besides monorden (94) as well as the novel compounds pochonin F and a monocillin II glycoside as minor metabolites, Fig. (1). Most of these compounds showed moderate activities in a cellular replication assay against herpes simplex virus and against the parasitic protozoan Eimeria tenella. In contrast to the structurally related zearalenone derivatives, none of the metabolites of strain P 0297 was

500

found to be active in a fluorescence polarization assay for the determination of modulatory activities on the human estrogenic receptor ERbeta[135].

95R = H 96R = OH

OH

98R-H 99R-OH

—B— pHvittK —*- MonDtden (mgfll

1.0-

-O-

Mycdialdiywd(h
50 >50 >50 >50 25 6.2 6.2 6.2

A new chlorinated depsidone (maldoxone 120) and a new spirocyclohexadienone (maldoxin 121) have been isolated from the culture medium of an as yet unidentified Xylaria species. Their role in the grisan-depsidone biosynthetic pathway was discussed [147]. OH COOMe

Cl

OMe OMe 120

MeOOC

Terpenes Over 30 sesquiterpene aryl esters have been isolated from Armillaria spp.; of these, eight are esterified with chlorinated orsellinate. The honey mushroom, A. mellea, produces 70 mg of the chlorinated compound armillaridin (122) per kg dry mycelium [148], while liquid cultures of A. ostoyae contain the chlorinated compounds, melledonal C (123) (50 mg/1) and melleolide D (35 mg/1) (124) [149]. Clitocybe elegans is the only other genus for which chlorinated sesquiterpene aryl esters have been reported [150], including melledonal D (125), which has not yet been detected in Armillaria spp. The pathogenic basidiomycete, Armillaria causes root disease in both coniferous and deciduous trees. Armillaria

506

spp. were the most frequently isolated fungi associated with root in living spruce and balsam fir trees in Ontario, Canada [151]. It is not known yet what makes some of the Armillaria spp. such virulent parasites, but secondary metabolites are thought to be the major cause. In a test for fungi or phytotoxicity of fourteen Armillaria metabolites, four of them chlorinated, the toxicity decreased with increasing hydrophobicity, e.g. methylating the hydroxy group of the orsellinate and/or adding a chlorine atom [152]. Sonnenbichler et al. [149] found that increased amounts of melledonal C (123) and other nonchlorinated sesquiterpene aryl esters were produced in cultures of A. ostoye growing in the presence of an antagonistic fungus or host plant cells [149,152]. The biosynthesis of the chlorinated metabolite was enhanced up to 5-fold upon antagonization [149], indicating that the physiological purpose of the sesquiterpene aryl esters is their antibiotic activity.

R4

122 123 124 125

Ri

R2

CHO CHO CHO CH2OH

H OH OH OH

R3 H H OH H

R4

R5

H OH OH OH

Me Me Me Me

Two novel sesterterpenes, neomangicols A (126) and B (127), were isolated from the mycelial extract of a marine fungus belonging to the genus Fusarium. The carbon skeleton of the neomangicols is undescribed and constitutes a new class of C25 rearranged sesterterpenes. Neomangicol A was most active against MCF-7 (human breast carcinoma) and CACO-2 (human colon carcinoma) cell lines, displaying IC50 values of 4.9 and 5.7 uM, respectively. Neomangicol B was less active, having a mean IC50 value of 27 uM across the entire cell panel (versus 10 uM for neomangicol A), while neomangicol B displayed antibacterial activity similar to that of the known antibiotic, gentamycin, against the Gram-positive bacterium Bacillus subtilis [153].

507

HO

HO,

126 R = Cl 127R = Br

The fungal metabolite ascochlorin (128), which is produced by Ascochyta viciae [154-156], was simultaneously isolated as "LL-Z1272y from Fusarium sp. [157] and as "ilicicolin D" from Cylindrocladium ilicicola, a fungus of dead beech leaves [158,159]. Other metabolites from these fungi include: LL-Z1272a (129), LL-Z1272X, (130) [157,158], and cylindrochlorin (131) [160]. The fungus Nectria coccinea also produces several of these metabolites, including the new chloronectrin (132) [161]. The hydroxy analog (133) was produced by Ascochyta viciae [162]. The tobacco pathogen Colletotrichum nicotianae produces colletochlorin B (134), C (135), and D (136) [163-165]. The hypolipidemic active metabolites, ascofuranone (137) and ascofuranol (138), have been isolated from Ascochyta viciae [166]. The fungus Acremonium luzulae also produces ascochlorin (138) [167], while Strobilurus tenacellus and Mycena spp. contain the new strobilurin B (139) [168].

508

CHO

128R = H 130 R = OAc OH

129 CHO

CHO

CHO 132 R = Ac 133 R = H

509

OH

136 CHO OH

CHO

138 X = H, OH COOMe

MeO OMe

510 The strobilurins were first isolated from Strobilurus tenacellus [169]. The chlorinated strobilurin B (140) has been isolated from three genera and seven species, e.g. Mycena alkalina, M. avenacea, M. crocata, M. vitilis, Xerula longipes, X. melanotricha, and S. tenacellus. Chlorinated oudemansin B (141) is produced by X. melanotricha [170]. The chlorinated and nonchlorinated strobilurins and the closely related oudemansins are new respiration inhibitors, binding to cytochrome b. Thirteen strobilurins and three oudemansins have been isolated so far. Their high antifungal activity against phytopathogenic fungi and insects and their low toxicity toward mammals and bacteria make them attractive lead compounds for the synthesis of agricultural fungicides [171]. Most fungi that produce strobilurins and oudemansins grow on wood. Oudenmansiella mucida also produces a nonchlorinated strobilurin on sterilized beech wood. Therefore, it appears that the strobilurins play a role in securing nutrient resources for the producers from competing fungi [171]. The aromatic ring and the benzylic carbon atom in the strobilurins and oudemansins are derived from the shikimate pathway, whereas the side chain is built up from acetate units [171]. MeCX

V

Y "V^ ^^ "V' JDMe

MeC

141

Aromatic Compounds Two strains of the basidiomycete, Bjerkandera adusta produces in static liquid culture, phenyl, veratryl, anisyl, and chloroanisyl metabolites as well as a series of compounds not previously known to be produced by Bjerkandera species. A new metabolite, for which the name bjerkanderol B (142) was given, has been proposed. Experiments with static liquid cultures supplied with 13C6- and 13C9-Z-phenylalanine showed that all

511

identified aromatic compounds (with the exception of phenol) could be derived from L-phenylalanine. For the aryl propane diols, the 13C label appeared only in the phenyl ring and the benzylic carbon, suggesting a stereoselective resynthesis from a C7 and a C2-unit, likely aromatic aldehyde and decarboxylated pyruvate, respectively. For both strains, cultures supplied with Na37Cl showed incorporation of 37C1 in all identified chlorometabolites. The compounds have been reported to exhibit important physiological functions in this white rot fungus. Possible mechanisms for their formation through the newly discovered compounds have been discussed [172]. OH

OH

MeO'' 142

A new D-glucose-6-phosphate phosphohydrolase (G6Pase) inhibitor (143), CJ-21,164 was isolated from the fermentation broth of the fungus Chloridium sp. The structure was elucidated to be a novel tetramer of the salicylic acid derivatives by spectroscopic analyses. The compound inhibited G6Pase in rat liver microsomes with an IC50 of 1.6 uM. Glucose output from hepatocytes isolated from rat liver was inhibited when it was present in the incubation medium, consistent with the role of this compound as a G6Pase inhibitor. It dose-dependently inhibited G6Pase, with an IC50 value of 1.6 uM. At a concentration of 133 uM, this compound inhibited the rate of glucose output stimulated with 25 nM glucagon by 81 %. Hepatocytes incubated with this compound did not show significant cytotoxicity at these concentrations, suggesting that the reduction in glucose output might not be a consequence of cytotoxicity [173].

512

!OOH

OH

OH

OMe

143

In the search for new, naturally occurring, anti-angiogenic compounds, it was found that a culture broth of an unidentified fungal strain B90911 exerted inhibitory activity on capillary-like tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Active compounds were isolated by bioassay-guided separation and their structures were identified to be two new asterric acid derivatives, i.e. 3-chloroasterric acid (144) and 3,5-dichloroasterric acid (145), by spectroscopic analyses. These compounds significantly inhibited (10 ug/ml) the VEGF-induced tube formation of HUVEC, suggesting that asterric derivatives could be useful for further study as anti-angiogenic agents [174]. COOMe

OMeMeOOC 144 R, = H, R2 = Cl 145 R! = R2 = Cl

Simple naphthalene metabolites (146 and 147) were produced by Verticillium lamellicola [175] and (148) found in the fungus Scolecobasidiella avellanea.

513 COOR,

O

COOR,

Cl

146 R! = H, R2 = Me 147 R, = Me, R2 = H

148

The structures of two novel fungal antibiotics, isolated from a Pterula species that interfere with the NADH: ubiquinone oxidoreductase and inhibit the respiration of eucaryotes, were determined by spectroscopic techniques. The compounds, pterulinic acid (149) and pterulone (150), contain a 1-benzoxepin ring system and are chlorinated [176]. In the serial dilution assay, both compounds showed antibacterial activities at concentrations up to 100 ug/ml {Acinetobacter calcoaceticus, Escherichia coli, Salmonella typhimurium, Bacillus brevis, Bacillus subtilis, Micrococcus luteus, etc.). They exhibited weak cytotoxicity activities toward L1210 and BHK cells but showed moderate activity toward HL60 and HeLa cells. In contrast, 150 was not cytotoxic against L1210, HL60, BHK, and HeLa cells at concentrations up to 100 jig/ml and has phytotoxic activities. The germination of Lepidium sativum and Setaria italica was inhibited at concentrations 10-50 ug/ml. Both compounds neither showed nematocidal activity against Meloidogyne incognita and Caenorhabditis elegans, nor hemolytical effects at concentration up to 100 ug/ml. HOOC

149 150

Antibiotic aspirochlorine was originally isolated from Aspergillus tamarii [177], later from A. flavus [178] and A. oryzae [179], and shown

514

to have the novel structure (151) by X-ray crystallography and total synthesis [180]. Armillaridin (152) is a novel phenolic sesquiterpene containing a cyclobutane ring that is produced by Armillaria mellea [181]. Later work with this organism revealed the related metabolites: melleolide D (153) [182], melledonals B (154), C (155) [183] and armillaricin(156)[184].

151

OMe

OMe

In one of the few studies of marine fungi, the novel chloriolins A-C (e.g. the structure of chloriolin B, 157) have been reported from a cultured, unidentified fungus associated with the sponge Jaspis johnstoni [185].

515

1 157

Coumarins and Isocoumarins One of the most ubiquitous of all fungal metabolites is the isocoumarin derivative ochratoxin A (158), which was first isolated from Aspergillus ochraceus [186,187] and later from Penicillium viridicatum [188], P. cyclopium, P. commune, P. variabile, P. purpurescens [189], Aspergillus melleus and A. sulphureus [190]. The related ochratoxin C (159) is produced by Aspergillus ochraceus [191], and 4-hydroxyochratoxin A (160) was isolated from cultures of Penicillium viridicatum [192]. When ochratoxin A was administered in the diet, hepatocellular tumors, renalcell tumors, hepatomas and hyperplastic hepatic nodules were observed in male mice. Incidence of and mortality from urothelial urinary tract tumors have been correlated with the geographical distribution of Balkan endemic nephropathyin. Ochratoxin A has been detected in moldy cereals including wheat, maize, rye, barley and oats, peanuts, coffee beans, bread, flour, rice, peas and beans from 0.03 to 27.5 ppm. Residues of ochratoxin A have been detected in samples of meat from animals slaughtered immediately after consuming contaminated feed and were detected at levels of 10 to 920 ug/kg in sausage, ham and bacon samples. The quantification of ochratoxin A, at levels within the range 0.25-10 ng/ml from wine by HPLC-fluorescence detection, was described [193]. RP-HPLC - fluorescence method for the detection of ochratoxin A in wine with a detection limit of 0.05 ng/ml was also published [194]. A stable isotope dilution assay by LC-MS/MS was developed for quantification of the ochratoxin A by using [Ds]-ochratoxin A as internal standard with a low detection and quantification limits of 0.5 and 1.4 ug/kg, respectively [195]. The LC-MS/MS method (ESI and APCI) was also applied to the analysis of contaminated coffee samples by ochratoxins A and B with absolute minimum detection limit around 10-20 pg per injection. Fragment ions from the [M+H]+ and [M+Na]+ ions of

516

both ochratoxins were monitored in the multiple reaction monitoring mode [196].

159 R, -Et, R 2 - H 160 R, - H, R2 - OH

The metabolite profiles of three different isolates of Penicillium nalgiovense from cheese were analyzed. The novel isocoumarin metabolite, dichlorodiaportin (161) was isolated from the cultures of P. nalgiovense along with further metabolites. The reason isocoumarins occur naturally and the possible toxicity or health benefits of these compounds in cheese and other relevant food products should be investigated [197]. MeO OH

The structures of four new biologically active nematocidal halogenated dihydroisocoumarins (162-165) isolated from submerged cultures of the wood-inhabiting ascomycete Lachnum papyraceum have been elucidated by spectroscopic methods. The compounds are structurally related to lachnumon and mycorrhizin A, which are also produced by the fungus [198]. The brominated metabolites isolated from L. papyraceum in this investigation are further examples of how bromine can be introduced into normally chlorinated fungal metabolites when bromide salts are added to the culture medium. Besides obtaining derivatives that are useful for QSAR studies and for assessing the importance of the halogen atom for the biological activity of the metabolites, the shifts in the secondary metabolism of the fungus induced by the addition of bromide to the

517

culture medium may also be helpful during studies of the biosynthesis of mycorrhizins. R,O

O

Ri

162 163 164 165

R2 H OH OH Me H

H H H

R3

Br Cl Cl Cl

R4

(R)-Me (S)-Me (R)-Me (R)-Me

Chlorflavonin (166) was discovered in the culture broth of Aspergillus candidus [199-201]. A biosynthetic study of this fungal metabolite indicates that it is a true metabolite and is synthesized de novo by this microbe [202]. It was also detected in Aspergillus candidus and A. campestris [203]. The fungus Monilinia fructicola produces chloromonilinic acids A (167) and B (168) derived from chloromonilicin [204], to be discussed later. OH

166

The strain of Actinamadura spiralis isolated from a soil sample produced new antibiotics, pyralomicins (169-175). Pyralomicins inhibited the growth of Micrococcus luteus and Escherichia coli at the concentration of 0.2-25 ug/ml by agar dilution method. Pyralomicin la (169) and pyralomicin 2a (173) did not show any acute toxicity in mice at 100mg/kgip[205].

518 OH

OR,

169 170 171 172

Ri

R2

R3

R4

H H H Cl

Cl Me Cl Cl

Me Cl Me Me

Me Me H H

OH

OR4 OH

173 174 175

Ri

R2

R3

R4

H H H

Cl Me Cl

Me Cl Me

Me Me H

A new antifungal agent, CJ-19,784 (176) was isolated from the fermentation broth of a fungus, Acanthostigmella sp. This compound inhibits the growth of pathogenic fungi, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus with IC50 values of 0.11, 20 and 0.54 ug/ml, respectively. Compound 176 and 3'-Cl analog did not exhibit significant activity against HeLa cells. It is well known that the production of halogenated microbial metabolites depends on the presence of halogen atoms in the fermentation medium. For example, chlortetracyclin and chloromonilicin are produced well under the fermentation in chlorine-containing media, and the chlorine atom is easily exchanged for a bromine atom by replacing the media with bromine-containing media. It is very interesting that

519

compound 176 but not 3'-Cl analog was produced as a major product under the fermentation in chlorine-containing medium (0.2 % sodium chloride). This suggests that the fungus Acanthostigmella sp. would have unknown biosynthetic mechanisms enabling prior use of the bromine atom [206]. OH

.OMe

OMe

A novel inhibitor of STAT6 activation, named as TMC-264 (177), was discovered from the fermentation broth of Phoma sp. TC 1674. TMC-264 suppressed expression of IL-4 driven luciferase and germline C epsilon mRNA with IC50 values of 0.3 uM and 0.4 uM, respectively. It exhibited a potent inhibitory activity against tyrosine phosphorylation of STAT6 with an IC50 value of 1.6 uM, whereas weakly inhibited tyrosine phosphorylation of STAT5 with an IC50 value of 16 uM, but did not inhibit the phosphorylation of STAT1 up to 40 uM. TMC-264 blocked formation of the complexes between phosphorylated STAT6 and STAT6 oligonucleotides in a dose-dependent manner, while it did not affect the formation of phosphorylated STAT1/STAT1 oligonucleotides complexes. These results suggested that TMC-264 selectively inhibited IL-4 signaling by interfering both with phosphorylation of STAT6 and binding of the phosphorylated STAT6 to the recognition sequence [207]. The structure was elucidated on the basis of NMR analyses of normal abundance and biosynthetically 13C enriched TMC-264 (177) [208].

520

OMe

MeO

177 O

In the process of screening microbial extracts from endophytic Cytospora sp., three novel compounds cytosporins A, B and C (178) were found as specific inhibitors of angiotensin II binding to receptors of AT2. The IC50 of component A in the biochemical assay was found to be approximately 25-30 uM in ATi and 1.5-3 uM in AT2. These results were obtained by blocking one of the two angiotensin II binding sites in rat adrenal glands. Further analysis suggested reversible inhibition by this compound, and thus it appears to be a competitive inhibitor [209].

178

Miscellaneous Compounds In addition to the numerous fungal and lichen metabolites discussed in the preceding sections, there are several others that do not easily fit into the well-defined structural categories. Several other chlorinated aliphatic metabolites are produced; a volatile organohalogen, l-chloro-5-heptadecyne (179) was detected in an edible wild milk cap Lactarius spp. [210]. Lepiochlorin (180), an antibacterial lactol, was isolated from liquid cultures of a Lepiota sp., a fungus cultivated by gardening ants [211]. Another aliphatic halogenated compound with an interesting tnchloromethyl group was isolated from the mycelium of the fungus Resinicium pinicola in 120 mg/kg yield [212].

521

The compound, pinicoloform (181), showed antibiotic and cytotoxic activities. -(CH 3 ) 10 — 179 OH

181

Kaitocephalin (182) was isolated from Eupenicillium shearii and protected chick primary telencephalic and rat hippocampal neurons from kainate toxicity at 500 uM with EC50 values 0.68 uM and 2.4 uM, respectively, without showing any cytotoxic effect. Although a wellknown AMPA/KA antagonist CNQX with a quinoxalinedione skeleton effectively protected chick primary telencephalic neurons from kainate toxicity with EC50 value 0.53 uM, it exhibited cytotoxicity against chick primary telencephallic and rat hippocampal neurons at the concentrations of 20 uM and 2 uM, respectively. Kaitocephalin also protected chick primary telencephalic and rat hippocampal neurons from AMPA/cyclothiazide (500 uM/50 uM) toxicity with EC50 values 0.6 and 0.4 uM, respectively. Kaitocephalin is the first AMPA/KA antagonist from nature, consisting of a quite different skeleton from other known AMPA/KA antagonists [213]. H

COOH

COOH

NH,

The salt-water culture of Aspergillus ochraceus separated from the Indo-Pacific sponge Jaspis coriacea has yielded two new chlorine containing polyketides, chlorocarolide A (183) and B (184). These compounds have an overall structural analogy to penicilic acid whose biosynthesis has been intensely studied. The structures and stereochemical features of the chlorocarolides were reported [214].

522

O'

183 7R* 8R* 184 7S* 8S*

Three new chlorine-containing compounds (185-187) together with penicillic acid were obtained from a marine-derived fungus Aspergillus ostianus isolated from a marine sponge at Pohnpei. Compound 185 inhibited the growth of R. atlantica at 5 ug/disc (inhibition zone 12.7 mm), while 186 and 187 were active at 25 ug/disc (10.1 and 10.5 mm, respectively). The growth of E. coli and S. aureus was also inhibited by these compounds. Compounds (185-187) did not inhibit the growth of S. cerevisiae and M. hiemalis even at 100 ug/disc. Compound 185 was the most potent among the three new components. Thus, the position of Cl affects the activity of these compounds [215].

R,

185R, = C1,R2 = OH 186 Kx = OH, R2 = Cl

187

Two new compounds named aranochlors A (188) and B (189) were detected as minor components from the fermented broth of the fungal strain Pseudoarachniotus roseus [216]. Fermentations were carried out in shake flasks as well as in laboratory fermenters. For the isolation of 188 and 189 six batches of each 100 liters were processed. Both compounds were present both in the culture filtrate and the mycelium. Both aranochlors exhibited antibacterial and antifungal activities. The

523 minimum inhibitory concentrations (MIC) of 188 and 189 required to inhibit a variety of bacterial and fungal strains are listed in Table 5. Table 5. Inhibitory activities of aranochlors against microorganisms. Test Organism Staphylococcus aureus Bacillus subtilis Micrococcus luteus Escherichia coli Pseudomonas aeruginosa Candida albicans Saccharomyces cerevisiae Aspergillus niger

different

MIC (ug/ml) Aranochlor A Aranochlor B 3.12 1.56 3.12 3.12 0.39 0.39 25 50 >200 >200 >200 >200 1.56 6.25 >200 >200

189

The red pigments, auxarconjugatins A and B (190,191) were isolated from mycelia of Auxarthron conjugatum, an ascomycetous fungus belonging to the Onygenaceae, in which the causative fungi of severe mycoses are also found [217].

524

OMe 190 R=H 191R = Me

Selective growth inhibition of IL-6 dependent cells was detected in fermentation extracts of a fungal strain, which was characterized as Sporothrix species. An active metabolite, 192 termed chlovalicin was isolated. Chlovalicin dose-dependently inhibited the growth of IL-6 dependent MH60 cells (IC50, 7.5 uM) in the presence of 0.2 U/ml IL-6 and, to a lesser extent, the growth of B16 melanoma cells (IC50, 38 uM). This compound did not show any antimicrobial activity at a concentration of 1 mg/ml [218].

192

The structure determination of lachnumon (193), brominated derivatives of lachnumon (194) and mycorrhizin (195) and brominated derivatives of mycorrhizin A (196) was described. The compounds, which exhibit similar antimicrobial and nematocidal activities as their chlorinated analogues, were isolated from extracts of cultures of the ascomycete Lachnum papyraceum to which CaBr2 had been added [219].

525

MeO' O

Br

193 R = H

195 R = H

194R = C1

196R = C1

A novel fungal metabolite, Sch 202596 was discovered from the fermentation of a fungal culture Aspergillus sp. [220]. The fungus, Aspergillus sp. was isolated from the tailing piles of an abandoned uranium mine in Tuolemene County, California. Compound 197 was revealed to be a new spirocoumaranone by spectroscopy, related to the griseofulvin family of compounds. In the in vitro galanin receptor GALRl assay compound (197) exhibited inhibitory activity with an IC50 value at 1.7 uM. Cl

MeOOC

MeOOC

197

HIOH

"OH

The structure and absolute configuration of microline, a new metabolite isolated from culture filtrates of Gilmaniella humicola, has been shown to be (198) by spectral data, chemical transformations and Xray analysis [221].

526

H

The structures of mikrolin and mycorrhizinol, two new metabolites isolated from culture filtrates of Gilmaniella humicola, have been shown to be (199) and (200), respectively, by spectral and chemical studies [222].

OH

OH

O 199

200

The structure and configuration of gilmaniellin, a metabolite of Gilmaniella humicola, has been shown to be (201) by X-ray analysis [223].

527

OH

O

201

The novel substances designated as ICM0301 A-H were isolated from the culture broth of Aspergillus sp. F1491 [224, 225]. ICM0301s inhibited the growth of human umbilical vein endothelial cells induced by basic fibroblast growth factor with IC50 values of 2.2-9.3 ug/ml. ICM0301C (202) and ICM0301D (203) showed significant anti-angiogenic activity at a concentration lower than 101 ug/ml in the angiogenesis model using rat aorta cultured in fibrin gel. ICM0301s showed very low cytotoxicity against various tumor cells. The taxonomy of the producing organism, and the fermentation, isolation and biological activities of ICM0301s were described. The structures were elucidated by spectroscopic analyses. ICM0301C and D have a substituted decalin skeleton containing one chlorine atom.

528

OH

H 202 R = Me 203 R = H

Vertihemipterin A, the ascochlorin glycoside, and its aglycone, 4',5'dihydro-4'-hydroxyascochlorin, and a new analog, 8'-hydroxyascochlorin, were isolated from the fermentation broth of the pathogenic fungus Verticillium hemipterigenum [226]. Structures of these compounds were elucidated by spectroscopic methods. Cytotoxic activities of these ascochlorin analogs were evaluated. All compounds were tested for their cytotoxic activities against three cancer cell-lines, KB, BC-1 and NCIHI 87, as well as Vero cells. The compounds exhibited significant cytotoxicities against all cell lines, e.g. for compounds 204-206, see Table 6.

529

Table 6. Cytotoxic activities of compounds 204-206. Cytotoxicity (IC50, ug/ml) Compound KB BC-1 1 NCI-H178C Verod 19 8.4 7.9 6.9 204 >20 38 >20 >20 205 2.2 3.4 206 2.7 1.4 1 oral human epidermoid carcinoma; human breast cancer; a

;

human small cell lung cancer; d African green monkey kidney fibroblast OH

OHC

204 R = - O 205 R = OH

OHC

OH

206

Trichodermamides A and B (207), two modified dipeptides, have been isolated from cultures of the marine-derived fungus Trichoderma virens [226]. Trichodermamide B displayed significant in vitro cytotoxicity against HCT-116 human colon carcinoma with an IC50 of 0.32 ug/ml. This metabolite also exhibited moderate antimicrobial activities against amphoterocin-resistant C. albicans, methacillin-resistant S. aureus, and vancomycin-resistant E. faecium with MIC values of ca. 15 ug/ml against

530

all three strains. Trichodermamide A was completely inactive in all of these bioassays, suggesting that the chlorine atom is an essential part of the pharmacophore. Chlorination is known to play an essential role in the activity of numerous, structurally diverse natural products including the antibiotics vancomycin and chloramphenicol and the antitumor compounds cryptophycin and rebeccamycin [227]. In the case of trichodermamide B (207), the chlorohydrin moiety at C4 and C5 might be a precursor of an epoxide, which could be the biologically active molecular form of this molecule. .OMe

Cl

Or

T^

OMe

207

The spiroxins 208-210 were purified from the culture extract of a marine-derived fungus, isolated from a soft orange coral collected from the waters near Vancouver Island, Canada [228]. Their unique bisnaphthospiroketal structures were established by spectroscopic methods. In addition to cytotoxicity, these compounds showed antibiotic activity and were active in a mouse xenograft model against human ovarian carcinoma. The mechanism of action of these compounds was shown to be due, in part, to their effect on DNA. Spiroxin A (208) showed some activity against Gram-positive bacteria but only marginal activity against Gram-negative bacteria. Compound 208 showed antitumor activity in nude mice against ovarian carcinoma (59 % inhibition after 21 days) at 1 mg/kg/dose given IP on day 1, 5 and 9 post staging. In a cytotoxicity assay, 208 exhibited a mean LC50 value of 0.09 ug/ml against a panel of 25 diverse cell lines. In evaluating its probable mechanism of action, it was observed that in the presence of e.g. 2-mercaptoethanol, 208 caused a concentration-dependent nicking of pBR322 DNA, suggesting that the compound partly exerts its cytotoxicity effect through a single-stranded DNA cleavage. Cytotoxicity of quinones has been attributed to DNA

531

modification, alkylation of essential protein thiol groups, oxidation of essential protein thiol groups by superoxide radicals or a combination of these mechanisms. The oxidation state of the spiroketal carbon, a masked ketone, could allow the spiroxins to behave chemically as quinone epoxides, possibly causing DNA cleavage under reducing conditions via an oxidative stress mechanism involving the formation of thiol conjugates. Thus, a variety of mechanisms may play a role in spiroxinmediated cytotoxicity.

o

OH

208 R, = H, R 2 + R3 = O 209 R, = Cl, R 2 + R 3 = O 210 R, = Cl, R 2 = OH, R3 = H

Gymnastatins A-E have been isolated from a strain of Gymnascella dankaliensis originally separated from the sponge Halichondria japonica [229]. Cytotoxic activities of compounds 211-215 were examined in the P388 lymphocytic leukemia test system in a cell culture [230]. The results showed that three of the compounds (211-213) exhibited potent cytotoxic activity and two (214 and 215) exhibited weak cytotoxic activity (ED50 0.018, 0.108, 0.106, 10.5 and 10.8 ug/ml, respectively). Gymnastatin A of these compounds showed strongest cytotoxicity. This evidence suggested that conjugated ketones were important for the enhancement of cytotoxicity in gymnastatin analogs, and hence the cytotoxic activity of compound 213 resulted from a conjugated ketone, which might be derived from compound 213 in the test system. Pericosines A (216) and B have been isolated from a strain of Periconia byssoides, originally separated from the sea hare Aplysia

532

kurodai, and their structures have been established based on spectral analyses. Pericosine A exhibited significant cytotoxicity (ED50 0.12 ug/ml) in the P388 lymphocytic leukemia test system in cell culture.

Cl

Cl

RHN 211aR 1 =OH,R 2 = 211bR!=H,R 2 = O

R=

OMe

i

\

RHN

OMe

212

213

214b R, = H, R 2 = O H

215a R, = OH, R 2 = H 215b R, = H, R 2 = O H

533

The isolation and structure determination of a new chlorinated benzophenone antibiotic, pestalone (217), is described [232]. The new compound was produced by a cultured marine fungus only when a unicellular marine bacterium strain, CNJ-328, was cocultured in the fungal fermentation. The fungus, isolated from the surface of the brown alga, Rosenvingea sp., collected in the Bahamas Islands, was identified as an undescribed member of the genus Pestalotia. Pestalone (217) exhibits moderate in vitro cytotoxicity in the National Cancer Institute's 60 human tumor cell line screen (mean GI50 = 6.0 uM). More importantly, pestalone showed potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MIC 37 ng/ml) and vancomycin-resistant Enterococcus faecium (MIC 78 ng/ml). The potency of this agent toward drug-resistant pathogens suggests that pestalone should be evaluated in more advanced, whole animal models of infectious disease.

OH HO,

OMe

COOMe 216 217

Structures of three novel compounds designated monordens C to E (218-220), isolated from the fermentation broth of amidepsine-producing fungus Humicola sp. FO-2942, were elucidated by spectroscopic evidence [233].

534

o.

220

FR225659 and four related (221-225) compounds are novel gluconeogenesis inhibitors that consist of a novel acyl-group and three abnormal amino acids [234]. Spectroscopic analysis concluded that FR225659 is an N-acyl tripeptide consisting of a novel acyl, a 3-chloro-4hydroxyarginine, a 3-hydroxy-3-methylproline, and a dehydrovaline. They were isolated from the culture broth of Helicomyces sp. No. 19353 and were purified by chromatography. These compounds inhibited the glucagon-stimulated gluconeogenesis of rat primary hepatocytes and had hypoglycemic effects in two different in vivo models [235].

535

N

Ri R2 R3 OH Me 221 OH OH Et 222 OH OH H Me 223 224 OMe OH Et 225 OMe OH Me Two new biologically active cyclopentenones, VM 4798-la (226) and VM 4798-lb (227) were obtained [236] as a 3:1 inseparable mixture from fermentations of Dasyscyphus sp. A47-98. The mixture of the two isomers showed cytotoxic and weak antibacterial and antifungal properties (e.g. Micrococcus luteus, Mycobacterium phlei, Candida parapsilosis, Rhodotorula glutinis, Aspergillus ochraceus and Zygorhynchus moelleri). In the serial dilution assay, 226 and 227 inhibited the growth of fungi and bacteria at 10-100 ug/disk. 226 and 227 caused a 50 % lysis of HeLa S3-, HL 60- and L1210-cells at a concentration of 10 ug/ml. The cytotoxic activity on Jurkat cells is quite significant with a 50 % lysis at 1.4 uM. The 3:1 mixture of 226 and 227 completely inhibited the incorporation of the appropriate radioactive precursors into DNA, RNA and proteins in Jurkat cells at a concentration of 1.9 uM. This is suggested to be a consequence of the breakdown of the mitochondrias membrane potential.

536

COOMe HO 227

CONCLUSION Fermentation of produced strains, purification, isolation and biosynthesis of griseofulvin were discussed in this review. In addition, compounds similar to griseofulvin were described. Griseofulvin is among the oldest antibiotics and a few that have been successfully used in the treatment of fungal diseases of the skin, nails, and hair. It inhibits e.g. Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Microsporum audouini, M. canis, M. gypseum, and Epidemophyton floccosum and decreases growth of Aspergillus spp. and Phialophora spp. Usually, topical application is not sufficient and has to be accompanied by peroral application. In addition to its therapeutic uses, griseofulvin and its derivatives are interesting with respect to their biosynthesis, which has some specific features including introduction of the halogen atom in a reaction catalyzed probably by a halogen peroxidase, similarly to other chlorine containing antibiotics such as chlorotetracycline and chloramphenicol. It would be worth investigating, whether targeted genetic modification of production strains would lead to new derivatives of griseofulvin and related compounds and, possibly also to hybrid antibiotics with better biological activity or physico-chemical properties. In addition to antimicrobial activity, many compounds referred here exhibit other interesting biological activities, such as nematocidal, cytotoxic, antitumor and antiangiotensic etc. that might be used in the therapeutic practice. ACKNOWLEDGEMENTS This work was supported by the Grant Agency of the Czech Republic (grant no. 204/01/1004) and by the Institutional Research Concept no. AV 0Z 5020 903. The authors wish to express their thanks to Mrs G.

537

Brou5kova for administrative help. Excellent technical assistance of Mr. M. Rezanka (student of Faculty of Science of the Charles University, Prague) and Mr. P. Rezanka (student of Institute of Chemical Technology and Faculty of Science of the Charles University, Prague) is gratefully acknowledged. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18]

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

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Bioactive Alkaloids of Fungal Origin Hideo Hayashi Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan ABSTRACT: In order to obtain fungal isolates, which produce bioactive compounds, random screening was carried out using okara (an insoluble residue of the whole soybean homogenate) as a cultural medium. We observed three kinds of activities against silkworms: insecticidal activity, convulsive activity and paralytic activity. A soil isolate, Penicillium simplicissimum ATCC 90288, produced novel insecticidal indole alkaloids that we designated as okaramines. Okaramines were found to be produced by not only this strain but also other strains belonging to P. simplicissimum. The isolate, Aspergillus aculeatus KF-428 also produced two okaramine congeners: okaramines H and I. These data strongly supported the fact that okaramines are widely produced by fungi. Till now, eighteen okaramine congeners have been isolated; their biogenesis and structure-activity relationships are described in this review. We also describe the results of synthetic studies for the okaramines J and N. The isolate, Penicillium expansum MY-57 produced five insecticidal compounds: the new communesin congeners, communesins D, E, and F, and the known communesins A and B. Convulsive compounds, verruculogen and penitrems, were produced by the isolates P. simplicissimum MF-24 and P. simplicissimum ATCC 90288, respectively, indicating that our convenient bioassay system with silkworms could be used to search for convulsive compounds. Novel convulsive compounds, brasiliamides A, B, C, D, and E, were found in the cultural media of Penicillium brasilianum JV-379. Finally, chance observation led to the isolation of new paralytic compounds, asperparalines A, B, and C, from Aspergillus japonicus ATCC 204480. Asperparalines have unique structures consisting of a bicyclo[2,2,2]diazaoctane core and a spirosuccinimide moiety.

INTRODUCTION Combinatorial chemistry has become a powerful methodology for the construction of new compounds. Natural products, however, also have enormous potential as a source of new compounds. In particular, numerous useful microbial products have been isolated as antibiotics, herbicides, fungicides and enzyme-inhibitors. Moreover, microorganisms have provided various compounds with diverse bioactivities, such as immunomodulatory, antitumor and antihelmintical activities [1].

550

Many efforts have been made to identify strains producing insecticidal components. In the 1960s, piericidins [2,3] and aspochracin [4,5] were isolated from Streptomyces sp. 16-22 and from Aspergillus ochraceus, respectively. In the 1970s, milbemycins were isolated as insecticides and acaricides from Streptomyces hygroscopicus subsp. aureolacrimosus [6-8]. Avermectins were isolated from Streptomyces avermitilis and developed as antiparasitic agents [9]. In the 1980s, our group also isolated a new insecticidal compound, A^-norphysostigmine, from Streptomyces sp. [10]. Spinosyns A, B, C and D (formerly known as A83543A-D), which were isolated from Sacchropolyspora spinosa in 1991, were found to possess potent mosquito larvicidal activity [11]. Spinosyns have also been used for crop defense [12]. However, we should emphasize that, among the various microbial products, only a few, such as the above-described avermectins and spinosyns, have practical applications. In the 1980s, our group began to screen microbes for insecticidal compounds that could be used in practice, or become lead compounds for the generation of new carbon skeletons. As a result, various strains were found to exhibit insecticidal, convulsive and paralytic activities against silkworms. This chapter deals with the procedures used for isolation of bioactive strains and their active principles. An overview of their chemical structures, activities, synthesis, and structurally related compounds is also given. INSECTICIDAL COMPOUNDS Various media were used for culturing fungi, and the fungal secondary metabolites are known to depend on the cultural conditions, such as the medium constituents and temperature. In our study, okara, which is an insoluble residue of whole soybean homogenate and a waste material in tofu (soybean curd) production, was used as a culture medium. This is the first time that this material has been used for culturing fungi. Fungal strains isolated from soil samples in the usual manner were cultured with okara media for about 10 days. The okara and mycelia were soaked in acetone, and aliquots of both acetone extracts were added to an artificial silkworm diet. Third instar silkworm larvae were introduced into a Petri dish containing the artificial diet, and the mortality rate was determined 24 h after initiating the administration. Using this screening method several hundred isolates were checked for their activity, and two strains, Penicillium simplicissimum Thom ATCC 90288 (originally AK-40) and Penicillium expansum Link MY-57, exhibited the insecticidal activity.

551

Okaramines Discovery of Okaramines A (1) andB (2)

The material from an acetone extract of okara fermented with P. simplicissimum ATCC 90288 was partitioned between ethyl acetate and water. The ethyl acetate layer thus obtained exhibited insecticidal activity. The ethyl acetate fraction was repeatedly chromatographed on silica gel with a hexane-acetone mixture, and then on alumina with a hexane-ethyl acetate mixture. Crystallization of the 50% ethyl acetate eluate and of 60-80% ethyl acetate eluates gave two active compounds, which were named okaramines A (1) and B (2), respectively [13-16]. The molecular formula of okaramine A (1) was determined to be C32H32N4O3 by HR-EIMS together with 13C- and !H-NMR spectra, implying nineteen degrees of unsaturation. The 'H-NMR spectrum is shown in Fig. (1). The 13C-NMR spectrum indicated that 1 had two amido carbonyl carbons and twenty olefmic carbons, suggesting that 1 was a heptacyclic compound. The UV absorption maximum at 374 nm indicated the presence of an indole chromophore with an expanded conjugation. Precise analysis of ' H - ' H COSY and HMBC spectra led to the planar structure of 1. Acetylokaramine A (3), giving a good crystalline structure for X-ray analysis, and the structure thus established is shown in Fig. (2). Okaramine A (1) was shown to be composed of two moieties, i.e. 3a-hydroxy-A^-(reverse-prenyl)-l,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid and 6,6-dimethyl-7/7-3,6dihydroazocino[5,4-6]indole-2-carboxylic acid. These two moieties formed a diketopiperazine ring, resulting in the formation of 1.

10.0

UllKlJJL 8.0

7.0

6.0

JL 5.0

4.0

Fig. (1). 300 MHz 'H-NMR spectrum of okaramine A (1) in acetone-rf6

3.0

2.0

1.0

552

1, okaramine A R = H 3, acetylokaramine A R = Ac

Fig. (2). ORTEP drawing of acetylokaramine A (3)

Okaramine B (2), C33H34N4O5, seemed to be an analog of 1. The H-NMR spectrum shown in Fig. (3) and 13C-NMR data strongly suggested that the azocinoindole moiety in 1 existed unchanged in 2. The essential difference between the l H-NMR spectra of 1 and 2 consisted of the absence of a vinyl group and a methine proton, and the appearance of an ethylidene group. Okaramine B (2) also contained an additional !

_JU ,

8.0

,

1

.

1

1

7.0

1

.

.

1

1

6.0

.

1

1

.

1

,

5.0

r—1

1

1

1

1

1

4.0

Fig. (3). 270 MHz 'H-NMR spectrum of okaramine B (2) in DMSO-rf6

1

1

3.0

_prxn

r-

2.0

1.0

553

hydroxyl group and a methoxyl group. To determine the connectivity of each functional group, a long-range 2D !H-13C COSY experiment was carried out for 2, and the results indicated that C-ll was bound to C-8a, and that an azetidine ring was newly formed. In addition, it was shown that two hydroxyl groups were located at C-2 and C-3a, and a methoxyl group at C-3. The relative stereochemistry was confirmed by the nuclear Overhauser effect difference spectra of 2, indicating the a-orientation for 2-OH, 3a-0H and H-ll and the p-orientation for 3-OCH3. The absolute configurations at C-2, C-3a and C-8a of 2 were deduced to be the same as those in 1 by comparing the CD spectrum of 2 with that of 1. Okaramine B (2) has two moieties, a hexahydropyrroloindole and a dihydroazocinoindole, and it is of particular interest that the pyrroloindole part is condensed with a newly formed azetidine ring.

"OH

2, okaramine B

4, okaramine C

Discovery of Okaramine C (4)

The unique structures of okaramines A (1) and B (2) led us to investigate whether okaramines or similar compounds were produced by other strains. First, we examined four strains belonging to P. simplicissimum, i.e. IFO 5762, AHU 8065, AHU 8402 and MF-24, and found that strain AHU 8402 showed the highest activity [17]. From an okara medium fermented with P. simplicissimum AHU 8402, three insecticidal compounds were isolated. Two of them were identified as 1 and 2, and the third one seemed to be a new, related compound, which was thus named okaramine C (4). Okaramine C (4), C32H36N4O3, proved to be a tetrahydro-derivative of 1. The ]H-NMR and 1H- H COSY spectra indicated that 4 had an additional reverse-prenyl group, an exchangeable proton and a -CH2-CH< group instead of -CH=CH- and -CH=CCH-CH2OH, strongly suggesting that a secondary methyl group at C-12 in 2 had been replaced in 5 by a hydroxymethyl group. All spectral data and acetylation of 5 led to the conclusion that 5 was 12-hydroxyokaramine B [18]. Okaramine E (6), C32H32N4O4, showed IR and UV spectra quite similar to those of 5, indicating, that 6 had the same functionalities and conjugated systems as 5. In the H-NMR spectrum of 6, signals assigned to a hydroxymethyl group at C-12 were observed, suggesting that the substitution on the azetidine ring in 6 was the same as that in 5. The NMR data of 6 also showed the existence of a dihydroazocinoindole moiety and a 1,2-disubstituted benzene ring. Compared with 5, the signals of two hydroxyl groups at C-2 and C-3a, a methine group at C-3a, and a methoxyl group at C-3 had disappeared, and signals attributable to a hydroxyl group and a -CH2-CFK linkage were newly observed, suggesting that 6 had a hydroxyl group at C-2 or C-3 a. This assumption helped complete the molecular formula for 6. The location of the hydroxyl group was concluded to be at C-3a from a comparison of the chemical shifts of C-3b and C-8a between 5 and 6. This conclusion and the configuration of the hydroxyl group were supported by the similarity in the chemical shifts of C-2, C-3 and C-3a between 6 and 1 [18]. The molecular formula of okaramine F (7), C32H30N4O4, was determined by HR-EIMS, and was identical to the molecular formula of okaramine E (6), but with two fewer hydrogen atoms. In the 'H-NMR spectrum of 7, almost all the signals observed in 6 were found, with the exception that the signals assigned to H-2 and H2-3 in 6 were replaced by one singlet signal, indicating that the bond between C-2 and C-3 had become unsaturated. Another possibility that a newly introduced double

555

bond was located between C-3 and C-3a was ruled out by a comparison between the UV spectra of 6 and 7. Okaramines generally show UV absorption at around 380 nm, which is characteristic of the azocinoindole ring, but 7 showed an absorption maximum at 402 nm, strongly suggesting that the double bond was between C-2 and C-3 [18]. The molecular formula of okaramine G (8) was C32H34N4O3, indicating that 8 has two more hydrogen atoms than okaramine A (1) and two less than okaramine C (4). From an inspection of the 'H- and 13 C-NMR spectra of 8, 8 was found to possess a 3a-hydroxy-A^-(reverseprenyl)-l,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid moiety, a 2,3-disubstituted indole and a diketopiperazine ring. The spectra indicated an additional reverse-prenyl and another exchangeable proton in 8, both of them resulting from the reductive cleavage between N-3' and C-4'. This structure was confirmed by HMBC experiments. From the NOESY experiments, the conformation of 8 was deduced to be quite different from that of 1—namely, 2-(reverse-prenyl)indole moiety in 8 turned around the C-l'-C-llb' axis and a reverse-prenyl group at C-6a' was closer to a carbonyl at C-12' [19].

«OH

OCH-,

5, okaramine D

6, okaramine E

"'OH

7, okaramine F

"OH

8, okaramine G

556 Discovery of Okaramines H (9) and I (10)

In further screening microbes for insecticides, an isolate Aspergillus aculeatus Iizuka KF-428 was obtained from a soil sample. This strain also exhibited the insecticidal activity when grown on okara, and three active principles were isolated. Two of them were identified as okaramines A (1) and B (2). The third one also seemed to be an okaramine-related compound and was named okaramine H (9) [20]. Okaramine H (9) had a molecular formula of C32H32N4O3, which was identical to that of okaramine A (1). The 1H- and C-NMR spectra of 9 were very similar to those of 1, suggesting the existence of azocinoindole and pyrroloindole moieties in 9. Signals assignable to a prenyl group in 9 were observed instead of the signals assigned to a reverse-prenyl group in 1. In long-range *H and 13C shift-correlated 2D-NMR experiments, the signal of C-7a was correlated with H2-IO, H-6, and H-4, revealing that the prenyl group was located at C-7. The orientations of hydrogen atoms at C-2 and C-8a and of a hydroxyl group at C-3a in 9 were considered to be the same as those of 1, because of the similarity between chemical shifts and coupling constants of H-2 and H-3 with those of 1. Okaramine H (9) might be formed through an aza-Claizen type rearrangement in which a reverse-prenyl group at N-8 of 1 is transferred to C-7 via the formation of a six-membered ring [20]. An inactive okaramine-related compound was isolated from the okara culture of A. aculeatus KF-428. This compound, okaramine I (10), had a molecular formula of C27H24N4O3. The ! H- and 13C-NMR spectra of 10 were the same as those of depentenylokaramine A, which was formed by hydrogenolysis of 1 with Pd/C [15, 20].

"OH

"OH

9, okaramine H

10, okaramine I

557 Discovery of Okaramines J (11), K (12), L (13), M (14), N (15), O (16), P (17), Q (18), andR (19)

Okaramines have attracted considerable attention due to their molecular complexity and intriguing biogenesis. We thoroughly searched the fermented material of P. simplicissimum ATCC 90288 for new okaramine congeners, with the result that nine members of the okaramine family were isolated. Okaramine J (11) had a molecular formula of C32H36N4O3, which is identical to the molecular formula of okaramine C (4). The ' H-NMR spectrum of 11 is shown in Fig. (4). The critical differences were the absence of one of two reverse-prenyl groups that were observed in 4 and the appearance of a new prenyl group and one exchangeable proton coupled to the methine proton at C-8a. The HMBC spectrum of 11 indicated that the prenyl group was bound to C-7 in the pyrroloindole ring [21]. Okaramine K (12), C32H34N4O3, had a molecular formula identical to that of okaramine G (8) [21]. The essential difference between the 'H-NMR spectra of 8 and 12 was the absence of a reverse-prenyl group

JL 10.0

pplll -

9.0

8.0

7.0

6.0

5.0

4.0

Fig. (4). 270 MHz 'H-NMR spectrum of okaramine J (11) in acetone-rf6

3.0

2.0

1.0

558

and the appearance of a prenyl group and one exchangeable proton. The HMBC spectrum of 12 indicated that the prenyl group was bound to C-7 in the pyrroloindole ring. Okaramine K (12) had 'H- and 13C-NMR spectra each consisting of signal accompanied by a 1/9-fold weaker signal of the same multiplicity. This suggested that 12 occurred as a 9:1 mixture of isomers. Furthermore, two isomers were obtained in pure form by HPLC, although each pure isomer was found to rapidly convert into a mixture of the original composition. To assign the stereoisomer, we carried out NOESY experiments on 12. The correlation observed between H-N3' and H-ll' from the major isomer supports the idea that the major isomer has the (Z) configuration at the C-l'=C-2' bond. On the other hand, the minor isomer of 12 was shown to have the {E) configuration at the same bond. It has been reported that aplysinopsin-type indole alkaloids, which are structurally similar to echinulin, underwent photoisomerization in a solution under either UV irradiation or ordinary daylight [22]. Therefore, 12 underwent facile photoisomerization in a solution under UV irradiation to become appreciably enriched by the (E) configurational isomer {ZIE = 6/4). Interestingly, the {ZIE) ratio of the stereoisomers of 12 reverted to a mixture of the original composition in one or two days at room temperature under condition of laboratory daylight. These facts can be interpreted as indicating that (Z)-12 is more thermodynamically stable. However, 8 did not exist as a mixture of {ZIE) configuration because of the steric repulsion between H-ll' of the indole nucleus and I3-CH3, I4-CH3.

iOH

•"'OH

O

11, okaramine J

12, okaramine K

Okaramine L (13) had a molecular formula of C32H36N4O3, which is identical to the formulae of okaramines C (4) and J (11). In the 'H-NMR spectrum of 13, the signal at H-N8 that was observed in 4 had disappeared, and a new signal assigned to the benzene ring of the

559

pyrroloindole moiety was observed. Careful comparison of the 'H-NMR spectrum of 13 with that of 11 revealed that methylene protons at C-10 of the prenyl moiety were shifted upfield, suggesting that this moiety was located at N-8. This consideration was confirmed by HMBC experiments, in which correlation was observed between H2-IO and both C-7a and C-8a, and between H-8a and C-10 [21]. Okaramine M (14) had a molecular formula of C29H30N4O3. The presence of a reverse-prenyl group was established by the NMR spectra. The presence of an acetyl group was also indicated in the NMR spectra. The placement of the reverse-prenyl group at C-3a in the indoline moiety was confirmed by HMBC correlations between C-3a and each of I3-CH3, I4-CH3, and H-ll. The placement of the acetyl group at N-8 was indicated by the fact that signals of H-8a and H-7 were recognized at a lower-field position than those of the corresponding protons of the other okaramines. Furthermore, ! H and 13C long-range correlation between H-8a and an acetyl carbon was observed [21]. Okaramine N (15) had a molecular formula of C32H34N4O3, indicating that 15 had two more hydrogen atoms than okaramine A (1). The precise analysis of NMR experiments suggested that the C-l'=C-2' double bond o -"OH

13, okaramine L

14, okaramine M

MOH

••'lOH

15, okaramine N

16, okaramine O

560

was saturated, and this assumption was supported by the fact that 15 lacked the UV absorption at 374 run present in 1. The results of the NOESY experiment indicated that the relative configurations at C-2, C-2', C-3a, and C-8a of 15 were all of c/s-type [23]. Okaramine O (16) had a molecular formula of C32H34N4O4, which was identical to the formula of 15, but with one more oxygen atom. The NMR spectra of 16 indicated the presence of an oxymethine group and a hydroxyl group, and the location of the hydroxyl group was also determined to be at C-l'. The relative configurations at C-2, C-2', C-3a, and C-8a of 16 were determined to be the same as those of 15 on the basis of the NOESY experiment. The hydroxyl group at C-l' was determined to have an a-orientation on the basis of the NOESY experiment and the lR-lH coupling constants [23]. Okaramine P (17), C32H34N4O4, had the same molecular formula as 16. The ^ - N M R spectrum of 17 is characterized by the disappearance of the reverse-prenyl signals in 16 and the appearance of a prenyl group. The HMBC spectrum indicated that the prenyl group was connected to C-7. The relative stereochemistry of 17 was the same as that of 16 at all chiral

"OH

"OH

18, okaramine Q

17, okaramine P

19, okaramine R

561

centers, as judged by the NOESY and the 13C-NMR chemical shifts [23]. Okaramine Q (18) had a molecular formula of C32H32N4O4. The UV spectrum (A,max 234, 288, 376 nm) indicated the presence of an indole chromophore with an expanded conjugation. The ^ - N M R spectrum of 18 resembled that of okaramine B (2), except for the absence of the methoxyl proton signal in 2 and the presence of signals due to isolated methylene protons. A precise comparison between the NMR spectra of 18 and 2 led to the conclusion that 18 is a demethoxyl derivative of 2 [23]. Okaramine R (19) appeared to possess the molecular formula of C32H32N4O4 by HREIMS, suggesting the presence of an additional oxygen atom as compared with okaramine A (1). The ^-NMR spectrum of 19 was identical to that of 1, with the exception that 19 lacked a methine signal at C-8a, and showed a new amide proton signal. In the 13 C-NMR spectrum of 19, the signal at C-8a that was obviously observed in 1 also disappeared, and a new signal assigned to an amide carbonyl

24

562

carbon was observed. The 'H-NMR signals for 3a-0H and H2-3 were correlated with this carbonyl carbon signal in the HMBC experiments. These data indicated that the carbonyl must be at C-8a, forming an oxyindole moiety [23]. Possible okaramine precursors, i.e. cyclo (Trp-Trp) (20), cyclo (2-(reverse-prenyl)-Trp-Trp) (21), cyclo (A^reverse-prenyl)-frp-Trp) (22), cyclo (2-(reverse-prenyl)-Trp-2'-(reverse-prenyl)-Trp) (23), and cyclo (Arl-(reverse-prenyl)-Trp-2'-(reverse-prenyl)-Trp) (24), were isolated during the course of the investigation of okaramine congeners [21]. Three of these compounds, 21, 22, and 24, are new compounds. cyclo(2-(Reverse-prenyl)-Trp-2'-(reverse-prenyl)-Trp) (23) was synthesized by Schkeryantz and coworkers as a precursor of gypsetin [24], but it was isolated for the first time from natural sources in our study. One year after our findings, Kozlovsky and coworkers reported the isolation of fellutanines A, B, C, and D from Penicilliumfellutanum [25], which were found identical to the compounds 20, 22, and 23, respectively. Absolute Configuration of Okaramines

In order to clarify the absolute configuration of okaramines, we determined the absolute stereochemistry of the above-mentioned derivatives of cyclo (Trp-Trp) (20). Acid hydrolysis of 20 gave L-tryptophan, which was identified by comparison with standard D,L-tryptophan samples by chiral HPLC analysis. Thus, the absolute configuration at C-2 was proved to be S [21]. The absolute stereochemistries of 21, 22, 23, and 24 were defined by a CD comparison with cyclo (L-Trp-L-Trp) (20). Furthermore, acid hydrolysis of 23 afforded L-tryptophan through the loss of a reverse-prenyl side chain [26, 27]. Hydrolysis of 21, 22, and 24 also gave L-tryptophan. Accordingly, these results elucidated the absolute stereochemistries of 21, 22, 23, and 24 as those depicted [21]. The stereochemistries of okaramine C (4), okaramine J (11), okaramine K (12), okaramine L (13), and okaramine M (14), including the absolute configurations, were then established [21]. The absolute configuration at C-2' of 4, 11, 12, and 13 was determined to be S by chiral HPLC analysis of the acid hydrolysate of each of these compounds. NOESY and NOE difference experiments were carried out to define the stereochemistry of 11. Because NOEs were observed between H-2 and H-2', between H-2 and H-8a, and between H-8a and 3a-OH, the absolute stereochemistry of 11 was determined. Based on NOESY and NOE difference data, the absolute stereochemistry of 11 was found to be identical to those of 4 and 13. The relative configurations at C-2, C-8a,

563

and C-3a in 12 were also determined based on the NOESY and NOE difference experiments. It was assumed that the absolute stereochemistry of 12 was the same as those of 4, 11, and 13, because all these compounds are produced by the same strain. The chiral HPLC analysis of the acid hydrolysate of okaramine M (14) revealed the presence of L-tryptophan. The absolute configuration at C-2' was determined to be S. The V( H ,H) coupling observed between H-2 and H-2' of 14 is consistent with the cis relationship between these protons. Thus, C-2 of 14 had the S configuration. In the NOE difference spectra of 14, significant NOEs were observed between H-2 and H-2', and between H-2 and H-8a. In addition, NOE enhancement was observed for 13-CH3, 14-CH3, and H-ll upon irradiation of H-8a. Therefore, the absolute stereochemistry of 14 was determined. The absolute stereochemistries of okaramines N (15), O (16), P (17), Q (18), and R (19) were considered to be the same as those of other okaramines because of biogenetic consideration. Insecticidal Activity of Okaramines

Insecticidal activities of okaramines and their derivatives are summarized in Table 1 using the LD50 values. Acetylokaramine A (3) showed the same activity as okaramine A (1). Okaramine C (4), whose azocine ring is cleaved, was also as active as 1, suggesting that the azocine ring moiety does not play an essential role in exhibiting the activity. On the other hand, okaramine G (8), whose azocine ring is also cleaved, showed much less activity than 1. This reduction in activity seems to have been caused by the conformational change in 8. Okaramines H (9) and I (10) exhibited no activity, indicating the importance of a reverse-prenyl group at N-8. Okaramines J (11), K (12), and L (13) exhibited no activity. This Table 1. Insecticidal Activity of Okaramines against Silkworms. Compound

1LD50 (ng/g diet)

okaramine A (1) acetylokaramine A (3) okaramine B (2) okaramine C (4) okaramine D (5) okaramine E (6) okaramine F (7) okaramine G (8) okaramine H (9) okaramine I (10) okaramine J (11)

8 8 0.2 8 20 >100 >100 40 >100 >100 >100

okaramine K. (12)

>100

Compound

LD50 (Hg/g diet)

okaramine L (13) okaramine M (14) okaramine N (15) okaramine O (16) okaramine P (17) okaramine Q (18) okaramine R (19) 4',5'-dihydroxyokaramine B (25) r,2',4',5'-tetrahydroxyokarmaine B (26) derivative of okaramine B (27) derivative of okaramine B (28)

>100 >100 >100 >100 >100 8 >100 6 80 >100 >100

564

fact also strongly suggested that the reverse-prenyl group at N-8 was very important and could not be substituted by a prenyl group. Okaramines N (15) and O (16) showed no activity, indicating that the resulting conformational change of the azocine ring moiety must be one reason for the reduction in activity. The LD50 values of okaramines B (2) and D (5) were 0.2 and 20 fxg/g diet, respectively, indicating that the hydroxylation at C-12 had drastically reduced their activity. Okaramines E (6) and F (7) exhibited no activity at a dose of 100 ug/g diet, suggesting that the functional groups in the pyrroloindole moiety play an important role in the insecticidal activity. To determine the effects of the azetidine and azocine ring moieties on the activity, chemical modification of 2 was carried out. Hydrogenation of 2 over 10% Pd/C in acetic acid provided 4',5'-dihydrookaramine B (25), l',2',4',5'-tetrahydrookaramine B (26), and two azetidine opened-ring compounds (27 and 28) [28]. Dihydrookaramine B (25) and tetrahydrookaramine B (26) had LD50 values of 6 (ag/g diet and 80 |ag/g diet, respectively, indicating that the reduction of activity was due to conformational change of the azocine ring moiety. Because two azetidine opened-ring derivatives (27 and 28) showed no activity, the azetidine ring was suggested to be the essential component responsible for the activity. The silkworm is a useful insect rather than a pest, and thus it was necessary to determine whether okaramines would also exhibit activity against harmful insects. Okaramines A (1) and B (2) were tested against various harmful insects. As a result, the most active of the 18 okaramines, compound 2 exhibited the same activity against the second instar larvae of the beet armyworm (Spodoptera exigua) as against silkworms, and thus this compound was considered to have potential use in practical applications. Biosynthetic Pathway

Biogenetic consideration of the structures of okaramines and related compounds hitherto isolated leads to the plausible biosynthetic scheme for okaramines outlined in Fig. (5). The basic framework of okaramines is derived from two molecules of L-tryptophan and two isoprene units. The sequence of events in the biosynthesis of okaramines is of crucial importance to the following discussion. According to our proposal, cyclo (L-Trp-L-Trp) (20) derived from L-tryptophan is biosynthetically considered to be an efficient precursor of okaramines. The formation of cyclo (2-(reverse-prenyl)- L-Trp-L-Trp) (21), cyclop1-(reverse-prenyl)L-Trp-L-Trp) (22), and cyclo (A^1-(reverse-prenyl)-L-Trp-2'(reverse-prenyl)-L-Trp) (24) is thought to arise via prenylation of 20.

565

Okaramine C (4) is derived from 24 by intramolecular cyclization and further oxidation at C-3a (Fig. (5) part 1). Intramolecular cyclization of 4 forms a tetrahydroazocine ring, leading to okaramine N (15). Oxidation at C-l' of 15 gives okaramine O (16), which yields okaramine A (1) through dehydration between C-l' and C-2'. On the other hand, aza-Claisen rearrangement of a reverse-prenyl group in 4, 16, and 1 leads to okaramines J (11), P (17), and H (9), respectively (Fig. (5) part 2). Intramolecular cyclization of 1 forms an azetidine ring, resulting in the formation of a biosynthetically significant postulated intermediate (29). Oxidation of this intermediate leads to okaramines Q (18) and E (6). Subsequent modification of 18 leads to okaramine B (2) and okaramine

=x, 0=

I cyclization

OCS.N^

19

H

A^.

4

Fig. (5). Proposed biosynthetic pathway for okaramines (part 1)

566

D (5) successively. Okaramine D (5) could be formed from the intermediate via 6 (Fig. 5 part 3). Removal of a reverse-prenyl group from 1 leads to okaramine I (10).

rearrangement

HN'

Hi'hrWoH CU.N;

jhydroxylation

I desaturation

HN

Hl'hpH'IOH

VN

H

"

rearrange -ment 17

16

I dehydration [d

Idehydration

HN

Hl-rrrl'lOH ^ J rearrange -ment

HI'hrH'IOH O^N^J , cleavage N"-"O

Fig. (5). Proposed biosynthetic pathway to okaramines (part 2)

I

rearrangement l]

567

HI'hpj-WOH

T^

elimination

1

Postulated intermediate i 29

v hydroxylation

Fig. (5). Proposed biosynthetic pathway to okaramines (part 3)

Synthetic Study of Okaramines N (15) andJ (11)

In 2003, Corey and coworkers described a remarkably simple synthesis of okaramine N (15) that took advantage of the new and

568

powerful Pd-promoted construction of the tetrahydroazocinoindole subsystem (Fig. (6)) [29]. (5)-A^-Boc-tryptophan methyl ester (30) was converted to the known indoline (31). Introduction of a reverse-prenyl group and oxidation furnished 32. The removal of the Boc-protecting group from 32 and saponification gave an amino acid. Schotten-Baumann acylation of the amino acid with FmocCl afforded

a) NaBH4CN, AcOH b) i) CuCl, ;-Pr2NEt, 2-acetoxy-2-methyl-3-butyne ii) DDQ iii) H2, Pd/C, quinoline c) i) SOC12 ii) LiOH iii) FmocCl d) 3-methyl-2-butenal,NaBH4 e) i-Pr2NEt, bis(2-oxo 3-oxazolidyl)phosphinic chloride f) Pd(OAc)2 g) Et2NH h) A'-methyltriazolidinedione Fig. (6). Synthesis of okaramine N (15) by Corey [29]

569

reverse-prenylated indole (33). 7V-Alkyl tryptophan methyl ester (34) was acylated with 33 to afford a tetracyclic intermediate (35). Treatment of 35 with Pd(OAc)2 provided tetrahydroazocinoindole (36). Exposure of 36 to excess diethylamine in THF resulted in Fmoc cleavage and cyclization to furnish diketopiperazine (37). The bisindole (37) underwent highly

11 Anth = 9-anthracenyl a) i) AnthSO2Cl, Et3N ii) tert-butyl isourea b) i) NBS, Et3N ii) 3,3-dimethyldioxirane iii) NaBH c) 1,1 -dimethylpropargyl bromide, CuCl, ;-Pr2NEt d) H2, Pd/Al2O3 e) TFA f) TMSOTf, 2,6-lutidine g) PyBop, Et3N h) Al/Hg i) i) KOH/MeOH ii) HBTU, ]pyrroloindole to a C-prenylated derivative (Fig. (7)). Hexahydro[2,3-£]pyrroloindole (40) was obtained by oxidative Witkop cyclization of L-tryptophan tert-buty\ ester. The alkylation of 40 afforded alkyne (41). The resulting alkyne (41) was hydrogenated to afford alkene (42). Treatment of 42 with TFA produced rearranged 43, indicating that this transformation was a charge-accelerated, aza-Claisen rearrangement. Removal of the tert-butyl ester provided acid (44). The indole C-2 reverse-prenylated derivative (45) was made in four steps from L-tryptophan according to the procedure described for the total synthesis of gypsetin [24]. Coupling 44 and 45 afforded pentacycle (46). Reductive removal of the anthracenylsulfonamide protecting group afforded 47. The methyl ester was hydrolyzed to the free amino acid, which underwent cyclization under peptide-coupling conditions to give okaramine J (11). Okaramine-Related Compounds

Okaramine A (1) is a novel heptacyclic compound containing a hexahydropyrroloindole moiety and a dihydroazocinoindole moiety. The azocinoindole moiety has been reported to constitute only two compounds: a metabolite (48) of Aspergillus ustus [31] and cycloechinulin (49) produced by A. ochraceus [32] (Fig. (8)). One of the structural characteristics of the okaramine family is the presence of a reverse-prenylated hexahydro[2,3-&]pyrroloindole moiety. Some related

H 3 CO H

48 Fig. (8). Compounds with azocinoindole moiety

49, cycloechinulin

571

compounds are shown in Fig. (9). Brevianamide E (50) was isolated from the culture medium of Penicillium brevicompactum by Birch and Wright in 1970 [33]. Amauromine (51) was isolated as a vasodilator from the culture broth of Amauroascus sp. No. 6237 by Takase and coworkers in 1984 [34, 35]. A family of new compounds, including ardeemin (52), iV5-acetylardeemin (53), and 15b-hydroxy-iV5-acetylardeemin (54), were isolated from the fermentation broth and mycelia of a strain of Aspergillus fischeri var. brasiliensis by Karwowski and coworkers in 1993 [36. 37]. A^5-Acetylardeemin (53) potentiates the cytotoxicity of the anticancer agent vinblastine in multi-drug resistant human tumor cells [36]. In 1994, Shinohara and coworkers isolated gypsetin (55) as an inhibitor of acyl-CoA:cholesterol acyltransferase from the cultured broth of Nannizzia gypsea var. incurvata IFO 9229 [38, 39]. In 2000, Kozlovsky and coworkers reported the isolation of fellutanine D (56) from the cultures of Penicillium fellutanum; and since then it has been reported cytotoxic [25].

50, brevianamide E

51, amauromine

o

o

52, ardeemin R = H 53, A^-acetylardeemin R = Ac

H

54, 15b-hydroxy-7V5acetylardeemin

OH

55, gypsetin

56, fellutanine D

Fig. (9). Compounds with hexahydro[2,3-6]pyrroloindole moiety

572

Each of the tryptophan metabolites shown in Fig. (10) has prenyl groups, reverse-prenyl groups, and a diketopiperazine ring. Echinulin (57), which contains a tryptophan moiety, was isolated by Birch and Farrar in 1963 [40]. Neoechinulin (58), which contains a dehydrotryptophan moiety, was also isolated as a pigment from the same molds that produced 57 by Barbeta and coworkers in 1969 [41]. Neoechinulins A (59), B (60), and C (61) were isolated as ivory crystals, yellow crystals, and yellow crystals, respectively, from sugar beet molasses cultures of Aspergillus amstelodami by Dossena and coworkers in 1974 [42]. Neoechinulins D (62) and E (63) were also isolated from the neoechinulins A-, B-, and C-producing strains by Marchelli and coworkers in 1977 [43]. Cryptoechinulin A, which is identical to compound 61, was isolated in small amounts from cultures of A. amstelodami along with a large quantity of 57 by Cardillo and coworkers in 1974 [44], and cryptoechinulin G (64) was isolated from the same strain by Gatti in 1978 [45]. In 1976, Nagase and coworkers isolated isoechinulins A (65), B (66), and C (67) from the course of their search

59, neoechinulin A

60, neoechinulin B

61, cryptoechinulin A neoechinulin C

62, neoechinulin D

Fig. (10). Structures of echinulin family (part 1)

573

for indole metabolites in the mycelia ofAspergillus rubber [46]. In 1999, Fujimoto and coworkers reported the isolation of tardioxopiperazines A (68) and B (69) as immunomodulatory constituents from an Ascomycete Microascus tardifaciens [47]. It is noteworthy that all compounds shown in Fig. 10 have a reverse-prenyl group at C-2 in the indole ring.

63, neoechinulin E

65, isoechinulin A

67, isoechinulin C

64, cryptoechinuline G

66, isoechinulin B

68, tardioxopiperazine A

69, tardioxopiperazine B Fig. (10). Structures of echinulin family (part 2)

574

Communesins Identification of Communesins A (70) and B (71) and Discovery of Communesins D (72), E(73), and F (74)

The acetone extract of okara fermented with Penicillium expansum Link MK-57 was found to exhibit the insecticidal activity against silkworms. The acetone extract of okara fermented with this strain was purified by solvent extractions, column chromatography, HPLC, and crystallization to yield five active compounds—i.e., two known compounds, communesins A (70) and B (71), and three new ones, communesins D (72), E (73), and F (74) [48]. The structures of the known compounds, 70 and 71, were assigned by comparing their physicochemical properties and spectral data with those reported in the literature [49]. o. ,o

70, communesin A

71, communesin B

Communesin D (72) was obtained as colorless needles and gave a protonated molecular ion [M+H]+ at m/z 523.2687 by HRFABMS, consistent with the molecular formula of C32H34N4O3. The UV spectrum showed an absorption maximum at 266 nm, suggesting that 72 had the same chromophore as 71. The 1H- and 13C-NMR data are similar to those for 71, indicative of the presence of a 1,2-disubstituted benzene and a 1,2,3-trisubstituted benzene ring moieties. The NMR data also strongly suggested that 72 had the same carbon skeleton—including the seven-ring system—as 71. Communesin D (72) was also found to have a (2£,4£)-2,4-hexadienoyl moiety by the ^ - N M R signals. The methyl signal at N-15 in 71 was not observed in 72, and a new signal assignable to an aldehyde proton was observed at 5H 8.91 (1H, d, J= 0.5). This fact, together with the difference in molecular formula between 71 and 72, suggested that the methyl group in 71 was substituted by a formyl group in 72. Key HMBC correlations between H-l' and C-6, and between H-5 and each of C-6, C-4, and C-8a, clearly established the location of this formyl group as N-15 and allowed the planar structure of 72 to be fully assigned [48]. Communesin E (73) had a molecular formula of C27H30N4O2, as

575

determined by HRFABMS and NMR data, suggesting that 73 was a demethyl compound of 70. The 'H-NMR spectra of 73 and 70 were nearly superimposable, but 73 lacked the signal for an iV-methyl observed in 70, indicating that 73 was an N-\5 demethyl derivative of 70. In addition, the presence of an acetyl group at N-16 and a 2-methyl-l,2-epoxypropyl moiety at C-ll was also suggested by the 'H-NMR data. Consequently, 73 was elucidated to be the A^5-demethylcomrnunesin A [48]. Communesin F (74) was found to have the molecular formula of C28H31N4O from the HRFABMS and NMR data. The ^ - N M R (Fig. (11)) and 13C-NMR spectra of 74 differed from those of 70 only by the absence of the epoxyl group and the appearance of a double bond, consistent with the difference in molecular formula between 74 and 70. The whole structure of 74, including the heptacyclic skeleton, an acetyl group at N-16 and a methyl group at N-15, was determined by a precise analysis of the ^ H COSY, HSQC, HMBC, and NOESY spectra of 74 [48]. The relative stereochemistry of 72, 73, and 74 was deduced to be the same as that of 70 and 71 at all chiral centers on the basis of the close similarity of the spectral parameters, especially the 13C-NMR chemical shifts, with the corresponding values for 70 and 71.

H

H

H r I

CHO

72, communesin D

73, communesin E

74, communesin F

^O

576

.ill 7.0

6.0

I i JIAL 5.0

4.0

3.0

PpTTT

2.0

Fig. (11). 400 MHz 'H-NMR spectrum of communesin F (74) in CDC13

The insecticidal activity of communesins A (70), B (71), D (72), E (73), and F (74) against third instar larvae of silkworms was examined by an oral administration. The LD50 values for 71 and 74 were 5 and 80 [ig/g of diet, respectively. Communesins A (70), D (72), and E (73) exhibited lower insecticidal activity than did 71 and 74, with the LD50 values for 70, 72, and 73 being 130, 130, and 200 ug/g of diet, respectively.

Biosynthetic Pathway of Communesins

May and coworkers proposed the plausible biosynthetic root to communesin A (70) shown in Fig. (12) (part 1) [50]. A quinone methide (75) derived from tryptamine and the related natural product, aurantioclavine (76), undergo a Diels-Alder reaction to form a polycyclic intermediate (77). This highly twisted lactam (77) should be easily cleaved by the residual primary amine to produce spiro lactam (78). Reduction of 78 and aminal closure afford a common intermediate (79) of communesins. Epoxidation and acylation of 79 afford 70. Expansion

577 H,N HOOC

Tryptamine

•N

R 76 R = H (aurantioclavine) Quinone methide R = Me imine

NH epoxidation al d N H ] . I acylation

79 Fig. (12). Plausible biosynthetic pathway to communesins (part 1) [50]

of this pathway suggests the pathway to communesins B (71), D (72), E (73), and F (74) (Fig. (12) part 2.). Acylation of 79 affords 74. On the other hand, epoxidation and acylation afford 71. Elimination of a methyl group at N-15 of 70 generates 73, while oxidation of a methyl group at N-15 in 71 generates 72. Communesin-Related Compounds

Communesins A (70) and B (71) were originally isolated from the mycelia of a strain of Penicillium sp. adhering to the marine alga, Enteromorpha intestinalis, and reported to exhibit cytotoxic activity in the P-388 lymphocytic leukemia test system in cell cultures [49]. The ED50 values for 70 and 71 are reported to be 3.5 and 0.45 u-g/ml, respectively, in the test system.

578

9t,. b NH

^

I

H

NH

^

epoxidation

H

^

acylation

79 acylation

Jdemethylation

74

Fig. (12). Plausible biosynthetic pathway to communesins (part 2)

Communesin-related compounds are shown in Fig. (13). Jadulco and coworkers quite recently isolated communesins B (71), C (80), and D (72) from the fungus Penicillium sp. derived from the Mediterranean sponge Axinella verrucosa [51]. These three communesins have been shown to exhibit moderate antiproliferative activity in several bioassays performed on different leukemia cell lines. Nomofungin (81), which had a pyran oxygen instead of an NH in 71, was isolated from the fermentation broth of an unidentified endophytic fungus obtained from the bark of Ficus microcarpa [52]. Later synthetic studies of nomofungin revealed that this compound was identical to 71 [53, 54]. Perophoramidine (82) was isolated from the Philippine ascidian Perophora namei [55]. Perophoramidine is a hexacyclic substructure of

579

80, communesin C

81, nomofungin

82, perophoramidine Fig. (13). Communesin-related compounds

the core heptacyclic ring system of communesins; it exhibits cytotoxicity toward the HCT116 colon carcinoma cell line and induces apoptosis via PARP cleavage. CONVULSIVE COMPOUNDS Verruculogen Identification of Verruculogen (83)

During the course of searching for okaramine-related compounds OH :

o OHM

H3CO

83, verruculogen

580

produced by strains belonging to P. simplicissimum, we observed interesting convulsive activity against silkworms in a strain of P. simplicissimum MF-24. The purification guided by the convulsive effect on silkworms led to the isolation of an active principle. The active principle, C27H33N3O7, was identified as verruculogen (83) [56]. Verruculogen (83) was originally isolated from the culture of P. verruculosum as an agent responsible for the tremor producer activity in mice or 1-day old cockerels [57]. Verruculogen (83) caused convulsive activity in the silkworms at a dose of 0.1 ug/g diet. Verruculogen-Related Compounds

H3CO

84, acetoxyverruculogen

85, fumitremorgin B o H3C0

H3CO

86, fumitremorgin A OH

=

o

OH 1

87, fumitremorgin C o

H-,CO

88, 12,13-dihydroxyfumitremorgin C Fig. (14). Verruculogen-related compounds

91, demethoxyfumitremorgin C

581

Verruculogen-related compounds are summarized in Fig. (14). In 1982, Uramoto and coworkers reported the isolation and structural elucidation of acetoxyverruculogen (84) from P. verruculosum as a tremorgenic metabolite [58]. In 1974, Yamazaki and coworkers reported the planar structure of fumitremorgin B (85) [59], which had been isolated as one of two toxins (fumitremorgins A and B) from Aspergillus fumigatus, growing on rice and miso (soybean paste) [60]. The structures of fuitremorgins A (86) and B (85) were determined in 1980 [61, 62]. These two compounds cause severe tremors and convulsion in experimental animals. Fumitremorgin C (87), the simplest member of the fumitremorgin family, was isolated from A. fumigatus by Cole and coworkers in 1977 [63]. Hermkens' group reported the total synthesis of 87 in 1988 [64], and Hino's group also reported the synthesis of 87 in 1989 [65]. Abraham and Argmann described the isolation of 12,13-dihydroxyfumitremorgin C (88) from A. fumigatus DSM 790 [66]. In 1995, Cui and coworkers reported the isolation of trypanostatins A (89) and B (90) from a marine fungal strain of A. fumigatus BM939 [67]. Trypano statins completely inhibit the cell-cycle progression of tsFT210 cells in the G2/M phase [68]. Cui and coworkers also isolated demethoxyfumitremorgin C (91), 83, 85, 87, and 88, showing the co-occurrence of these compounds, and 89 and 90 in the secondary metabolite of the strain BM939 [69, 70]. These findings suggested the OR 7

O

H 3 CO

89, tryprostatin A R = OCH3 90, tryprostatin B R = H

94, cyclotryprostatin C Fig. (15). Tryprostatin-related compounds

92, cyclotryprostatin A R = H 93, cyclotryprostatin B R = CH3 o o

95, cyclotryprostatin D

582

possible intermediacy of 91 in the biogenesis of the verruculogen and fumitremorgins. In 1997, Cui and coworkers also isolated cyclotryprostatins A (92), B (93), C (94), and D (95) as new inhibitors of the mammalian cell cycle from the same strain, A. fumigatus BM939 [71]. The structures of the tryprostatin family are shown in Fig. (15). Penitrem A and 6-Bromopenitrem E Identification of Penitrem A (96) and Discovery of 6 Bromopenitrem E (98)

As mentioned earlier, P. simplicissimum ATCC 90288 produced insecticidal okaramines. Moreover, this strain induced the same effect on the silkworms as verruculogen (83). The acetone extract obtained from the mycelia and media of this strain was concentrated and the aqueous residue was extracted with dichloromethane. The dichloromethane extract was partitioned between hexane and methanol, containing 10% water. The activity was found only in the lower layer. The active ethyl acetate extract obtained from the lower layer was chromatographed on silica gel with a hexane-ethyl acetate mixture. The 40-60% ethyl acetate eluates were rechromatographed on silica gel with a hexane-chloroform mixture. HPLC of the active 90-100% chloroform eluates on a Capcell pack Cig column, using 65.7% aqueous methanol with a flow rate of 1.0 ml/min, yielded two active compounds, AC 1 and AC 2. The convulsive principle AC 1, C37H44CINO6, was determined to be penitrem A (96) by means of the spectral data (MS, UV, IR, ! H-, and C-NMR) [72], which were indistinguishable from those reported previously for 96 [73]. The convulsive principle AC 2, C37H44BrNO6, showed spectroscopic characteristics quite similar to those of 96. The only difference between the two principles was that AC 2 had a bromine atom in the place of the chlorine atom of 96. The !H-NMR spectrum of AC 2 is shown in Fig. (16). In the 13C-NMR spectrum of 96, signals assignable to C-6 and C-7

OH Cl

H

96, penitrem A

H

98, 6-bromopenitrem E

583

UlUUu ppm •*—r- levistolide A (111) > senkyunolide P (115) > riligustilide (113) [301]. Subsequently these four phthalide dimers were claimed in a Japanese patent to produce vasodilatory effects on KC1 and noradrenaline contracted rat mesenteric arteries [146]. Nevertheless, in general, the anti-spasmodic potencies of several phthalides in different isolated tissue preparations are relatively weak and their vasorelaxing properties in vitro often fail to translate to hypotensive effects in vivo. The potential benefits of phthalides as anti-hypertensive agents remain to be verified.

652 Table 5. Vasorelaxing Effects of Phthalides in Various Isolated Vascular Preparations

Phthalide

Butylidenephthalide (20) Butylidenephthalide (20) Butylidenephthalide (20) Butylidenephthalide (20) Z-Butylidenephthalide (22)

Butylphthalide (30)

Animal

Dog

Dog

Dog

Dog

Rat

SHRsp1

Tissue

Contractile Agent

EC 5 0

References

(jiM)

Coronary artery

PGF2(
7>8>10b/?-hexahydro-lH-5,10c5ethanonaphtho[l,2-c:7,8-c']difuran-3,10-dione (106), Z,Z'3.3\8.8'-Diligustilide (109), Z,Z'- 6.8',7.3'-Diligustilide (110), Levistolide A (111), Riligustilide (113)

"Please refer to Table 1 for references

Clinical Indications As documented in the Chinese Pharmacopoeia, Danggui and Chuanxiong are mainly used to relieve pain induced by the so-called "blood stagnation syndrome" [359]. The manifestation of their analgesic actions, according to the TCM theory, is related to the facilitation of blood circulation and removal of blood stasis [359]. In TCM practice, Danggui is primarily prescribed for the treatment of gynaecological disorders such as irregular menstruation, amenorrhea, and dysmenorrhea, while Chuanxiong is commonly used for the treatment of migraine and headache [360, 361]. These two herbs are also used as remedies for asthma, stroke and angina pectoris [360, 361]. Gaoben is usually used as a pain-killer for headache-like symptoms [362]. Clinical Evaluations Despite the growing interest in the pharmacology of naturally occurring phthalides, to date butylphthalide (30) is the only single phthalide rather than in a mixture being studied in clinical trials. In late 2002, a phase III clinical trial was conducted in China to investigate the therapeutic potential of butylphthalide (30) in treating cerebral ischemia [321, 363]. The details of the clinical trial have not been published yet, but the investigators claimed that phthalide 30 elicited "obvious therapeutic effects with minimal adverse reaction in 590 patients with acute cerebral ischemia" [363].

655 Other clinical studies, which include only a few case reports, were all carried out using the extracts of phthalide-containing TCM herbs [364-368]. However, in most of these studies, the general and fundamental clinical trial designs employing features such as randomization, double blinding, placebo control, and proper statistical analysis were rarely employed. Rhizoma Chuanxiong (3 g/day for 14 days) was claimed to ameliorate symptoms of acute cerebral infarction [364]. In a separate study by the same group of investigators in China, Rhizoma Chuanxiong (1 g/day for 1 -2 years) was reported to improve symptoms of transient cerebral ischemic attack [365]. Nevertheless, which symptoms were examined and the details of the scoring system adopted were not documented in either study. Crude oil extract from Radix Angelicae Sinensis (10-30 mg) was reported to alleviate spasm-mediated abdominal pain under various clinical conditions in 151 out of 162 patients (93%) in China, and this effect was comparable to that of 0.3 mg atropine (34 out of 35 patients, 97%) [366]. An in-patient study was conducted in China to study the effect of Radix Angelicae Sinensis (62.5 g, i.v./day for 10 days) to treat chronic obstructive pulmonary diseases [367]. Despite the decrease in the plasma levels of several detrimental factors (such as angiotensin-II and digitalis-like factor), pulmonary function as determined by PaO2 (arterial oxygen tension), PaCO2 (arterial carbon dioxide tension) and SaO2 (arterial oxygen saturation) did not improve. The potential estrogenic effects of Radix Angelicae Sinensis (4.5 g/day for 24 weeks) were investigated in 71 postmenopausal women in a randomized, double-blinded, placebo-controlled clinical trial conducted in the United States of America [368]. Endometrial thickness and menopausal symptoms as measured by the Kupperman index were improved equally in the herb-treated and placebo groups. Maturation value and number of superficial cells in vaginal smears were not affected in either group. The authors concluded that Radix Angelicae Sinensis, when used alone, was not more effective than placebo in treating menopausal symptoms. Concluding Remarks Although from the modern medical science perspective, phthalide-containing herbs remain to be established as clinically effective, the parallels between the pharmacological actions of different phthalides and the traditional clinical indications of these herbs underscore their potential therapeutic benefits. Structurally simple and biologically active with diverse beneficial properties, the phthalides constitute an ideal lead class of compounds for chemical modification to enhance their promising pharmacological activities. On the other hand, we enthusiastically anticipate that with the recent growing interest in natural products and herbal medicines, the therapeutic benefits of phthalides and phthalide-containing herbs will soon be established scientifically, and these ancient traditional medicines will make important contributions to modern medicinal science.

656 ACKNOWLEDGEMENTS The authors greatly acknowledge Dr. Hugh A. Semple (Director, Scientific and Regulatory Affairs, Kinetana Group Inc., Edmonton, Alberta, Canada, and Adjunct Professor in the College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada) for his critical comments and improvements to the language proficiency in the preparation of this article. Abbreviations 5-HT

Serotonin (5-Hydroxytryptamine)

6-keto-PGF 2 a

6-Keto-prostaglandin F 2 a

AA

Arachidonic acid

BBB

Blood-brain barrier

BK C a

Large-conductance calcium-activated potassium channel

EC 5 0

Effective concentration to achieve 50% of maximal response

ED50

Effective dose to achieve a response in 50% of the population

GABA

y-Aminobutyric acid

IB M X

3-Isobutyl-l-methylxantine

IC50

Effective concentration to achieve 5 0 % inhibition of a response

i.c.v.

Intracerebroventricular

i.g.

Intragastrical

i.p.

Intraperitoneal

i.v.

Intravenous

K ATP

ATP-activated potassium channel

L-NAME

A^-nitro-L-arginine methyl ester

MCAO

Middle cerebral artery occlusion

NMDA

jV-Methyl-£>-aspartate

NOs

N O synthase

NSCC

Non-selective cation channel

ODQ

l-H[l,2,4]Oxadiazolo[4,3-a]quinoxalin-l-one

PAF

Platelet-activating factor

PaCO 2

Arterial carbon dioxide tension

PaO 2

Arterial oxygen tension

657 PDE

Phosphodiesterase

PGI2

Prostacyclin

p.o.

Per os (oral)

PRP

Platelet-rich plasma

rCBP

Regional cerebral blood flow

SaO2

Arterial oxygen saturation

sGC

Soluble guanylate cyclase

SHRsp

Spontaneous hypertensive stroke-prone rat

SOCC

Store-operated calcium channel

TCM

Traditional Chinese medicine

TXA2

Thromboxane A2

TXB2

Thromboxane B2

VOCC

Voltage-operated calcium channel

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

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CHEMISTRY AND BIOLOGICAL ACTIVITY OF POLYISOPRENYLATED BENZOPHENONE DERIVATIVES OSMANY CUESTA-RUBIot, ANNA LISA PICCINELLI§, LUCA RASTRELLI § ' Instituto de Farmacia y Alimentos (IFAL), Universidad de La Habana, Ave. 23, No. 21425, CP 13600 La Lisa, Ciudad de La Habana, Cuba. *Dipartimento di Scienze Farmaceutiche, Universita di Salerno, Via Ponte Don Melillo, 84084, Fisciano, Salerno, Italy. ABSTRACT: Plants of the family Clusiaceae or Guttiferae, in particular those belonging to the genera Clusia, and Garcinia, produce a series of oxidized and polyisoprenylated benzophenone derivatives, some of which are structurally complex and biologically active. From a biogenetic point of view, these compounds may be considered as benzophenones in which the acetate derived benzene ring is modified by intervention of isoprenyl groups. Several compounds belonging to this class have shown a wide range of biological activity such as antimicrobial, antifungal, anticarcinogenic and anti-HIV inhibitory activities. In this chapter we shall review the chemistry and biological activity of the polyisoprenylated benzophenone derivatives isolated from the genera Clusia, Garcinia, Vismia, Allanblackia, Moronobea, Symphonia, Hypericum, Tovomita, Tovomiptosis and Ochrocarpus.

INTRODUCTION Clusiaceae (Guttiferae) is a family almost exclusively tropical in distribution and comprises about 40 genera and 1200 species most of which are woody [1]. Extensive phytochemical studies have shown Clusiaceae to be a rich source of secondary metabolites including xanthones, triterpenoids, flavonoids, lactones and organic acids. In addition plants of this family produce a series of oxidized and polyisoprenylated benzophenones (PBDs), some of which are structurally complex and biologically active. From a biogenetic point of view, these compounds are thought to be of mixed shikimate and acetate biosynthetic origin in which the acetate

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derived benzene ring is modified by intervention of prenyl groups. Structural modifications and alkylation of this ring results in the formation of a complex bridged bicyclic or tricyclic system. This situation has permitted to obtain both simple (SBDs) and complex molecules as bicyclo[3.3.1]-nonane and adamantyl derivatives. Genera Garcinia and Clusia have been reported as the main sources of PBDs but these compounds have been also isolated from other genera including Vismia, Tovomita, Allanblackia, Symphonia, Hypericum, Moronobea , Tovomiptosis and Ochrocarpus recently. Bronianone, a yellow pigment present in the stem wood of G. hombroniana was the first member of polyisoprenylated benzophenone derivatives isolated from Clusiaceae [2]. This compound contains maclurin moiety, the 2,4,6,3',4'-pentahydroxy benzophenone (2). The structure proposed initially was revised and suggested as (1) finally. OH HO.

OH

(2) Maclurin

(1) Bronianone MeO

O

OH

(3) Marupone Fig.(l). Simple benzophenone derivatives and maclurin.

PBDs seem to be derivatives of maclurin or another benzophenone derivative modified in the A ring by intervention of prenyl groups. B ring can be unsubstituted or contain up to 2 phenolic groups. PBDs have shown different biological properties but, probably the three most important are the cytoprotection against HIV-1 in vitro of guttiferones [3,4], antimicrobial properties [5-11] and cytotoxic activity found in diverse nucleus [12-16]. The presence of keto-enolic equilibrium in most of them seems to play an important role on account of when this possibility disappears, a lower potential is often observed.

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Occurrence About 100 PBDs have been isolated from different genera of the family Clusiaceae. Initially, these products were associated with genera Clusia and Garcinia, nowadays their distribution is associated with other 8 genera. Simple and complex structures have been isolated from genera Vismia, Symphonia, Hypericum, Tovomita, Allanblackia, Moronobea, Tovomiptosis and Ochrocarpus too. Resin is a rare reward limited to a few tropical genera like Clusia whose flowers produce floral resins in many species. The viscous liquid is collected by bees and used as a nest construction material. In this context some PBDs have been reported in tropical propolis [11,17]. Investigation of the chemistry of the floral resins revealed that they are composed of almost pure polyisoprenylated benzophenones [18,19]. The analysis quantitative by RP-HPLC of 11 floral resins of Clusia species belonging to the sections Chlamydoclusia, Cordylandra, Phloianthera and Polythecandra, by Porto et al. [20] described the correlation between the chemical composition and the taxonomic sections. In the sections Chlamydoclusia and Polythecandra, the floral resins have bicyclo-[3.3.1]nonane benzophenone derivatives as major constituents and simple benzophenone derivatives as minor components, while section Phloianthera floral resins have these two compound types in almost equal amounts. In section Cordylandra were isolated only compounds possessing the bicyclo-[3.3.1]-nonane benzophenone structure. It is very interesting to note the high percent of prenylated benzophenone derivatives in some natural sources. Floral resins, latex, leaves, and fruits can contain up to 70 % of these compounds [19,20]. Simple benzophenone derivatives (SBDs) Some PBDs show up to 6 isopentenyl groups although no complicated arrangements are observed in these structures. Many of them are true examples of polyisoprenylated benzophenones because the acetate derived ring shows aromatic properties. We shall consider this group as simple benzophenone derivatives and most of them are included in Table 1. Marupone (3), isolated from trunk wood of Moronobea pulchra Ducke, seems to be the first example of SBDs in genus Moronobea [21]. Its structure was deduced on spectral and chemical evidences. The allocation of the geranyl group to C-3 was based on UV spectroscopy data

674 Table 1. Simple polyisoprenylated benzophenones isolated from Compounds Bronianone (1) Marupone (3) Vismiaphenone A (4) Vismiaphenone B (5) Vismiaphenone C (6) Vismiaphenones D-G (7-10) Iso- vismiaphenone B (11) Myrtiaphenones A-B (12-13) Clusiaphenone A (14) Clusiaphenone B (IS) Clusiaphenone C-D (16-17) Kolanone(18) Tovophenones A-B (19-20) Tovophenone C(21) Grandone (22) Machuone (23) Weddellianones A (24)

Weddellianones B (25) Lanceolatone (26)

Hilarianone (27) Vismiaguianones A-E (28-32) Pseudoguttiaphenone A (33) Nemorosinic acid A (34) 3-geranyl-2,4,6-trihydroxybenzophenone (36) 4,6,4'-trihydroxy-2,3'-dimethoxy-3-prenylbenzophenone (37) Garciosaphenone (38) Cudraphenones A-D (39-42)

plants of Clusiaceae Sources G. hombroniana M. pulchra V. decipiens V. guaraminangae V. decipiens C.ellipticifolia G. myrtifolia V. guaraminangae G. pseudoguttifera V. cayennensis V. decipiens C.ellipticifolia G. myrtifolia G. pseudoguttifera C.ellipticifolia Csandiensis C.sandiensis C.ellipticifolia G .kola Tovomita mangle Tovomita brevistaminea Tovomita brevistaminea C. grandiflora C.sandiensis C. weddelliana C. lanceolata C. pana-panari C. burchellii C. fluminensis C. hilariana C. paralicola C. pernambucensis C. weddelliana C. pana-panari C. lanceolata C. burchellii C. fluminensis C. hilariana C. pana-panari C. paralicola C. pernambucensis C. hilariana V. guianensis G. pseudoguttifera C nemorosa Tovomita krukovii G. multiflora G. speciosa Cudrania chinchinensis

References 2, 21 22 23 22

26,28 24 23 31 25 22

26,28 24 31

26,28 27 27 26 5 29 30 30 19 27 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 12 31 32 6 33 34 13

considering the absence of UV AICI3 shift and the formation of two cyclization products upon acid treatment, which requires the presence of the geranyl group ortho to both hydroxyls.

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14 SBDs closely related have been reported from the genera Vismia, Garcinia, and Clusia: vismiaphenones A-G (4-6), isovismiaphenone B (11), myrtiaphenones A-B (12,13) and clusiaphenones A-D (14-17). All of them can be considered as 2,4,6-trihydroxy-3,5-diisopentenyl benzophenone or 2,4,6-trihydroxy-3,5-diisopentenyl-4-hydroxy benzophenone derivatives. Three compounds named vismiaphenone A (4) and B (5) and isovismiaphenone B (11) were isolated from the berries of V. decipiens Schlecht-Clam [22]. Their structures are very closely related and all of them showed one unsubstituted aromatic ring. Structures were deduced from their spectral and chemical data. The proposed structure for vismiaphenone A was confirmed by acid-catalysed cyclization and cyclodehydrogenation with DDQ (2,6-dichloro-3,5-dicyanbenzoquinone). The presence of a 2,2-dimethylchromene ring and the loss of one hydroxyl group in the NMR spectra of vismiaphenone B and iso-vismiaphenone B indicated cyclization to a pyran ring of the prenyl group. Vismiaphenone C (6) was isolated from the root bark of V. guaraminangae together with the known vismiaphenone A (4). The UV, MS (m/z 105) and NMR data indicated one unsubstituted ring, two equivalent chelated hydroxyl groups, and a methoxy group. Consequently vismiaphenone C was considered an isomer of vismiaphenone A. The structure was confirmed by acid-catalysed cyclization of the side chains [23]. Myrtiaphenones A (12) and B (13) were isolated from hexane extract of G myrtifolia [24]. NMR data of myrtiaphenone A showed the presence of two OMe groups, a hydrogen-bonded phenolic OH group (8 10,92), two isoprenyl groups and a benzoyl group also identified from the intense peaks at m/z 105 and 77. The structure of 6-hydroxy-2,4-dimethoxy-3,5diprenyl benzophenone was assigned to myrtiaphenone A. This compound was synthesized previously but, it was not identified as a natural product. Myrtiaphenone A can be considered as a methyl derivative of the known compound vismiaphenone C. All NMR data of myrtiaphenone B (13) were in agreement with the structural characteristics of prenylated benzophenones. The resonances at 8 6.40 and 5.32 shared a J constant of 10 Hz, typical of a cis double bond, and a gem-dimethyl group indicated the presence of a chromene ring. Myrtiaphenones are very closely related from a structural point of view. Myrtiaphenone A can be easily converted in myrtiaphenone B by cyclo dehydrogenation with DDQ.

676 OR, (4) Vismiaphenone A, R1=R2=H, R3=CH3, R4=H (6) Vismiaphenone C, R,=R3=H, R2=CH3, R4=H (7) Vismiaphenone D, R,=R3= H, R2=CH3, R4= OH (12) Myrtiaphenone A R,=R2=CH3, R3=H, R4=H (15) Clusiaphenone B, R,=R2= R3=R4=H

OR,

(5) Vismiaphenone B, R,=R2=R3=H (8) Vismiaphenone E, R,= R2=H, R3=OH

(11) Isovismiaphenone B, Ri=R2=H

(9) Vismiaphenone F, Ri=CH3, R2=H, R3=OH

(13) Myrtiaphenone B, Ri=H, R2=CH3

O

O

(10) Vismiaphenone G

OH

(14) Clusiaphenone A OH

OH

OH (16) Clusiaphenone C Fig. (2). Simple benzophenone derivatives closely related

OH

O

(17) Clusiaphenone D

Fuller et ah, following anti-HIV bioassay-guided fractionation isolated four new prenylated benzophenones, vismiaphenones D-G (7-10), from extracts of leaves of V. cayennensis [25]. All these compounds showed to be very similar to vismiaphenones A-B (4, 5) and myrtiaphenones A-B

677

(12, 13). The main difference was associated with the benzoyl group. It was identified as a p-disubstituted aromatic ring on the basis of evidences observed in the NMR spectra and mass spectral fragmentation (m/z 93, 121). Vismiaphenone F (9) and myrtiaphenone A (12) showed a common characteristic in their 'H NMR spectra: the anomalous chemical shifts of the aryl methoxyl groups (8 3.32 and 3.19 respectively) vicinal to the benzoyl group. Authors have suggested that this feature is likely due to anisotropic shielding produced by benzoyl group. A similar effect was detected in myrtiaphenone B where the gem-dimethyl group falls in the shielding zone of the benzoyl group. The proton resonance spectrum of vismiaphenone G (10) revealed that one prenyl group had undergone epoxidation of the olefinic bond (ABX spin system). This uncommon structural characteristic among natural prenylated benzophenone derivatives is the sole difference between vismiaphenone G and vismiaphenone D (7). Clusiaphenone A-D (14-17), isolated from the genera Clusia, are SBDs closely related [26-28]. All these compounds presented one unsubstituted aromatic ring. Clusiaphenone A, isolated from the fruits of C. ellipticifolia and C. sandiensis, and clusiaphenones C-D, isolated from the fruits of C. ellipticifolia, have the two isoprenyl group ciclizated. The marked diamagnetic shift (A5= - 0.4 ppm) displayed by the a-chromene proton in the *H NMR spectra of clusiaphenones C-D agrees with a peri relationship between the phenolic hydroxyl and the chromene ring. From the biogenetic point of view, clusiaphenones A-D may derive from the same precursor (2,4,6-trihydroxybenzophenone) by attachment of two isopentenyl chains and successive cyclodehydrogenation and/or cyclization. The dried and powered fruit pulp of G. kola was extracted with light petroleum (bp 40-60 °C) [5]. A yellow-brown precipitate, kolanone (18), was obtained after standing. The base peak at m/z 105 (C7H5O) observed in the mass spectrum suggested that one of the benzophenone rings was unsubstituted. NMR data evidenced the presence of two 3-methyl-2butenyl groups and a geranyl unit, confirmed by mass spectrum which showed ions for the loss of C9H17 and C10H17 as well as for the loss of C 5 H 8 and C5H9. Three polyisoprenylated benzophenones closely related and named tovophenones A-C have been reported in Tovomita species. Spectral data of these compounds were consistent with a polyisoprenylated benzophenone with one w-hydroxyphenyl group and a lavandulyl chain (C10H17). Tovophenones A (19) and B (20) were isolated from the roots of

678

T. mangle [29]. A lavandulyl chain (CioHn) was identified by the presence of two terminal methylene groups and verified by one-step cleavage of the double bonds with OSO4-HIO4. Tovophenone B showed to be a derivate of tovophenone A where a pyran moiety is generated by arrangement of a prenyl rest. Tovophenone B was not considered an artefact formed during the process of purification carried out because it was observed in the crude extract. However, many PBDs have showed to be unstable in solutions and the process of structural transformation can occur rapidly. A new study permitted to obtain the known tovophenones A and B and the new compound tovophenone C (21) [30] from T. brevistaminia. This compound showed a dihydrofuran ring with a hydroxyisopropyl group substituent and, like tovophenone B, can be also considered as a tovophenone A derivative. Grandone (22) was obtained from the floral resins of C. grandiflora as its dimethyl derivative. It contains three 3-methyl-2-butenyl groups and constitutes the first SBD isolated from floral resins of Clusia spp [19]. Machuone (23) was found in the fruits of C. sandiensis together with clusiaphenones A-B, and clusianone [27]. Structure was established on spectral basis and two tautomers were present in a c.a 4:1 ratio. Machuone is closely related to grandone. Initially the main PBDs isolated from floral resins of Clusia spp. showed to be bicyclo-[3.3.1]-nonane derivatives but a new study demonstrated the presence of several SBDs in these sources. Structures of weddellianone A (24) and B (25), lanceolatone (26) and hilarianone (27) were established on spectroscopic evidences of their methyl derivatives obtained after treatment of fresh floral resins with diazomethane [20]. All the structures were represented with a 1,3,5-triketone system because of the treatment before mentioned don't facilitate the determination of the exact structures. On the other hand, many natural SBDs have shown to have methoxyl groups in their structures and then in the methyl derivatives above mentioned is not possible to establish the origin of the methoxyl groups certainly. A study of V. guianensis led to isolation of new SBDs [12]. Vismiaguianones A-E (28-32) were identified as benzophenone derivatives characterized by the presence of an unsubstituted benzoyl group. Vismiaguianones A-C showed an additional ring suitable to cyclization between a hydroxyl and isoprenyl group at C-5. Vismiaguianone A has a hydroxy-dimethyl-dihydropyran ring, while vismiaguianones B and C have a dihydrofuran ring with a hydroxyisopropyl group substituent.

679

OH

OH OH (20) Tovophenone B

O

(21)Tovophenone C

O

OH

(23)Machuone

(24) Weddellianone A

(26) Lanceolatone Fig. (3). Simple benzophenone derivatives from (20) to (27).

(25) Weddellianone B

(27)Hilarianone

680

The position of ring C on the acetate derived ring was based on the presence of only one hydrogen-bonded proton in the H NMR spectrum of vismiaguianones A and C (§H 12.60 and 12.71, respectively) in respect to two weakly hydrogen-bonded hydroxyl protons in the H NMR spectrum of vismiaguianones B (5H 9.82 and 8.14). The hydroxyl groups were hydrogen-bonded to the carbonyl group of the benzophenone moiety Vismiaguianones D and E exhibited an additional aromatic ring which is associated with a phenylpropanoid rest. IR bands (1773 and 1774 cm"1) and 13 C NMR chemical shifts of the signals ascribed to esther groups (5 166.9 and 165.0), corresponding to each compound, were in agreement with the 5-lactone function observed for the first time in benzophenone derivatives. Pseudoguttiaphenone A (33), isolated from heartwood of G. pseudoguttifera together myrtiaphenone A-B and vismiaphenone C, is a 4methyl- vismiaguianone A derivative [31]. Pseudoguttiaphenone A could be biogenetically derived from vismiaphenone C via a cyclization between a hydroxyl and isoprenyl group and epoxidation in the 2,2dimethylchroman ring. A phytochemical study of fruits of C. nemorosa led to isolation of nemorosinic acid A (34) and B (35) which contained an oxidized lavandulyl chain characterized by the presence of a carboxyl group [32]. Both sets of NMR spectra indicated the presence of two components in a ca 3:2 and 1:1 ratio respectively which demonstrated the existence of a keto-enolic equilibrium in these compounds. However, nemorosinic acid A was identified as a PBD and nemorosinic acid B as an alkylarylketone. The structural relationship observed between these two compounds was firmly established on spectral evidences. Other SBDs found in family Clusiaceae were also included in Table 1. i.e. 3-geranyl-2,4,6-trihydroxybenzophenone (36) [6], 4,6,4'-trihydroxy2,3'-dimethoxy-3-prenylbenzophenone (37) [33], garciosaphenone (38), a digeranylbenzophenone isolated from trunk bark and stems of G. speciosa [34]. Four new SBDs, cudraphenones A-D (39-42), were isolated from the roots of Cudrania cochinchinensis (Moraceae) [13]. The ring B of these compounds showed a different substitution pattern with respect to PBDs isolated from Clusiaceae. Cudraphenones A-D presented a 3-hydroxy-2prenyl substitued ring B and only one isoprenyl chain on the ring A. The methyl protons of the 2,2-dimethylpyran ring of cudraphenone C appeared at a higher field position than those of cudraphenone A. The upfield shifts were caused by the anisotropic effect of the ring B.

681 OH

OH

HO

OH

0

(30)Vismiaguianone C

(29) Vismiaguianone B

(28)R=H Vismiaguianone A (33)R=CH3 Pseudoguttiaphenone A

OH

(32)Vismiaguianone E

(31) Vismiaguianone D

R:

(34) Nemorosinic acid A

CO,H (35) Nemorosinic acid B

HO

xo O

°" OCH3

(37) 4,6,4-trihydioxy-2,3-dimethoxy-3-prenyl benzophenone OH

O

0

R

R=Geranyl (38) Garciosaphenone

R—Geranyl (36)3-geranyl-2,4,6-trihydroxybenzophenone

(40) Cudraphenone B R,=R3=H, R2=C5H, (39) Cudraphenone A

(42) C

"d

Fig. (4). Simple benzophenone derivatives from (28) to (42)

(41) Cudraphenone C

682

Bicyclo-[3.3.1]-nonane derivatives Elucidation of the structures of xanthochymol (43) and isoxanthochymol (44), the first members of the bicyclo-[3.3.1]-nonane benzophenone derivatives was very important in the chemistry of these secondary metabolites. Their structures were established by chemical transformations and spectroscopic means which included X-ray analysis [35,36]. Initially, xanthochymol and isoxanthochymol were isolated from G. xanthochymus fruits by Karanjgoakar et al. [37]. Both structures were deduced on the basis of spectral evidences. The structure of isoxanthochymol was firmly established by an X-ray crystallographic analysis but, the structure of xanthochymol was suggested by comparison with the first one. Dreyer also reported the presence of xanthochymol in the mature fruit of C. rosea [38], the structural analysis was developed considering the structure mistakenly identified as xanthochymol in the previous report [37]. Blount and Williams revised the structure of xanthochymol employing spectroscopic methods that included an X-ray crystallographic method [35]. Finally, the structure of xanthochymol was deduced as (43). Significant differences in the 'H NMR spectra of xanthochymol and isoxanthochymol included the presence of signals due to two terminal methylene groups in xanthochymol and two methyl groups on a satured carbon (§ 0.93 and 1.24) in the isoxanthochymol spectrum. These findings are confirmed by 13C NMR spectra which showed two signals at S 109.8 and 113.5 for two terminal methylene carbons (-C(Me)=CH2) and two signals at 5 123.9 and 124.2 for methine carbons of two trisubstituted olefin groups (-CH=CMe2) [35]. Xanthochymol is converted in isoxanthochymol by acid-catalysed reactions and this method has been frequently used in order to define or compare these structures. These structures presented the same relative stereochemistry associated with the bicyclo moiety. New exhaustive chemical studies confirmed the above structures mentioned [7,36]. As xanthochymol, many PBDs isolated from Guttiferae show a structure based on the bicyclo-[3.3.1]-nonane-2,4,9-trione system (Table 2).

683 Table 2. Bicyclo-[3.3.1]-nonane derivatives. Compounds Xanthochymol (43)

Isoxanthochymol (44)

Cambogin=isogarcinol (45)

Camboginol=garcinol (46)

18-O-methyl isogarcinol * (47) 18-O-methyl garcinol * (48) Guttiferones A-D (49,53,50,51) Guttiferone A Guttiferone G (52) Guttiferone E (54)

Guttiferone F (55) Clusianone (56)

7-epiclusianone (57) Nemorosone (58)

Hydroxy nemorosone (59) 7-epinemorosone (60) Spiritone (61)

Insignone (62)

Sources C. rosea G. subelliptica G. xanthochymus G. pyrifera G. staudtii G. mannii G. ovalifolia G. purpurea G.ovalifolia G. subelliptica G. xanthochymus G. purpurea G.cambogia G. indica G. assigu G. purpurea G.cambogia G. indica G. assigu G. assigu G. assigu S. globulifera G. livingstonei G. macrophyl/a G. macrophylla G. ovalifolia C. rosea G. pyrifera A. stuhlmannii C. spiritu-santensis C. lanceolata C. pana-panri C. weddelliana C. fluminensis C. burchellii C. paralicola C. pernambucensis C.congestiflora C.sandinensis G. assigu H. sampsonii Rheedia gardneriana C. rosea C. grandiflora C. insignis C. nemorosa C. nemorosa C. nemorosa C. insignis C. renggerioides C. spiritu-sanctensis C. fluminensis C, burchellii C. pernambucensis C. weddelliana C. insignis

References 3 7 37 16 54 57 58 62 3,58 7 37 7,86 43 41,42 44 7,62 43 41,42 44 44 44 3 3 15 15 3 3 16 4 19,20 20 20 20 20 20 20 20 45 27 44 54 10 19 19 19 19 19 18 18 18 20 20 20 20 20 20

684 Table 2. Bicyclo-|3.3.1]-nonane derivatives. Compounds Scrobiculatones A and B (63,64) Plukenetiones D-E (65-66) Plukenetiones F-G (67-68) Chamone I and II (69,70) Aristophenones A-B (71) Propolone A (72) Sampsoniones K-M (73-75) Hyperibones A-I (76-84) Ochrocarpinones A-C (85-87) 15,16-dihydro-16-hydroperoxyplukenetione F (88) Garcinielliptone I (89) * Possible structural mistake

Sources C. scrobiculata C. plukenetii C. plukenetii C. havetiodes var. stenocarpa C.grandiflora

G.aristata propolis H. sampsonii H. scabrum O. punctatus C. havetiodes var. stenocarpa 0. punctatus G. subelliptica

References 20 47 47 52 9 49 11 51 8 14 52 14 53

We proposed to group PBDs that present a bicyclo-[3.3.1]-nonane2,4,9-trione system in accord with benzoyl moiety position: type A if it is on C-l, type B if it is on C-3 and finally type C if it is on C-5, Fig.(5) [40]. The structures previously reported as type C, nemorosone and 7epinemorosone, have been corrected and all polyisoprenylated benzophenones derivatives with a bicyclo-[3.3.1]-nonane-2,4,9-trione system isolated, therefore, are derivatives of type A or B [18,19]. In this work, we use this classification and a unique numbering system in order to facilitate the structural comparisons. Type A: R]= benzoyl group or derivative. R2,R3,R4: prenyl groups Type B: R2= benzoyl group or derivative. Ri,R3, R4:prenyl groups Type C :R3= benzoyl group or derivative. R], R2, R-t prenyl groups R5 = CH 3 or prenyl groups

*This type has not been observed

OH Fig. (5). Bicyclo-[3.3.1]-nonane derivatives. Type A-C

These PBDs isolated so far from 7 genera, presented several common NMR spectroscopic features which have been firmly established. 1. All benzophenone derivatives show an unconjugated carbonyl at C-9 (5 207-210) belonging to bicyclo moiety. 2. All compounds possess one or two aliphatic methyl singlets (5 1.0-1.6) that correlate in HMBC with an aliphatic quaternary carbon at C-8.(5 47-51)

685

3. All derivatives present a sole aliphatic methylene group (CH26) in the bicyclo and one of their protons is usually observed as double doublet (8 1.9-2.2) in *H NMR spectrum. 4. Most of them show an aromatic AMX or AA BBX system in the B ring. AA BB system has been also observed. 5. Gem-methyl group at C-8 shows two ranges of 13C chemical shifts and they seem to be associated with the configuration at C-7. If 3 JH6ax-H7=10-13 Hz, ranges will be 8 15-17 and 5 22-24. If 3JH6ax-H7= 78 Hz, ranges will be 8 22-25 and 8 26-28 (Table 3). The bicyclic ring system requires the group at C-l (benzoyl or 3methyl-2-butenyl), and the prenylated chain at C-5 to be in an equatorial orientation and then there are only two stereochemical possibilities associated with the last one. Prenyl rest at C-7 has been observed both axial and equatorial, thus 4 absolute configurations are the maxima number of possibilities for this bicyclo. Structures of xanthochymol (43), cambogin (45) and clusianone (56) have been firmly defined by X-ray analyses [35,41,42]. When the molecular models of isogarcinol and clusianone are compared they showed to have the same relative configuration in the bicyclic rest (considering only the bridgehead asymmetrical carbons). Xantochymol contains another stereochemical possibility that originates the fusion of rings. These results suggest that the arrangements are not stereospecifics and any absolute stereochemistry can be observed. From de latex of G. cambogia, cambogin and camboginol were isolated in large quantities (5.5 and 37% respectively) [43]. Their structures (45) and (46) were elucidated respectively by chemical and spectral means which included NMR spectra (no bidimentional techniques). These compounds are very closely related to xanthochymol (43) and isoxanthochymol (44) respectively. All NMR, physical and chemical data suggested they are optical antipodes. Camboginol (46) was converted to cambogin by refluxing with benzene solution containing traces of HC1 or CF3COOH. Thus the absolute configuration of cambogin was easily deduced by comparison with camboginol.

686

(+) form

(44) Isoxanthochymol (45) Cambogin=Isogarcinol (optical antipode)

(43) Xanthochymol

(47) 18-0 methyl isogarcinol

(46) Camboginol-Garcinol (48) 18-O-methyl garcinol (54)Guttiferone E (optical antipode) (55)Guttiferone F( C-23 epimer)

(52) Guttiferone G. Ri=R2= 3-methyl-2-butenyl (53) Guttiferone B. R,=geranyl R2=CH3

(49) Guttiferone A

\ ^ Y ^ ~ ^ ^

(50) Guttiferone C

^

(51) Guttiferone D

Fig.(6). Bicyclo-[3.3.1]-nonano derivatives from (43) to (55).

A study of the hexane extract of the fruit of G. indica indicated the presence of garcinol and isogarcinol and suggested a modification for the structure of camboginol on UV spectral evidences [41]. Initially both products were suggested as type A PBDs but, a new study about the structure of isogarcinol based on X-ray crystallographic analysis permitted to corrected them and garcinol and isogarcinol were included as type B PBDs. They represent the same structures of camboginol and cambogin respectively [42]. Fuller et al. have suggested precedence for the names

687

camboginol and cambogin based on chronological and structural accuracy standpoints. Besides, the name garcinol has been also attributed to an aryl benzofuran isolated from Garcinia spp. From G. assigu collected in Papua, New Guinea, the 18-O-methyl ethers of isogarcinol (47) and garcinol (48) were isolated. Compounds were identified on the basis of NMR data and by comparison with the known garcinol and isogarcinol and the authors suggested the same relative stereochemistry in all these compounds [44]. The same type of Cotton effect by CD analysis was observed both in isogarcinol and garcinol with respect to the above mentioned methylated derivatives. However, both 3 JH6ax-H7= 13.9 Hz (prenyl group at C-7 in eq. position) and the 13 C chemical shifts assigned to gem-methyls at C-8 in the O-methyl derivatives are not in agreement with the stereochemistry of isogarcinol determined by X-ray analysis. Considering the X-ray study, isogarcinol exhibits a prenyl group at C-7 in axial position (chair conformation) and the NMR data of 18-O-methyl ether of isogarcinol suggest a prenyl group at C-7 in eq. position (chair conformation). Probably, the PBDs founded in G. assigu are O-methyl derivatives of the C-7 epimers of isogarcinol and garcinol respectively. Guttiferones A, C and D (49-51) isolated, from the extract of ground S. globulifera roots, [3] and guttiferone G (52), isolated from G. macrophylla [15], are the unique bicyclo-[3.3.1]-nonane derivatives that exhibit only one aliphatic methyl singlet belonging to bicyclo moiety. In these cases one isopentenyl group was attached on one of the alyphatic methyl carbons. The same relative configuration was suggested for guttiferones A, C and D. Guttiferone B (53), obtained from the same source [3] showed an oppositive configuration at C-7. A 10.4 Hz coupling constant between H-6 and H-7 determined a diaxial orientation between them and thus the 2,2dimethyl allyl group at C-7 was equatorial. Guttiferone E (54), isolated from G. ovalifolia and C. rosea [3], showed 'H and 13C NMR spectra identical to those of camboginol. However, its optical rotation [a]o = + 101°, was opposite in sign to that reported of camboginol [OC]D = - 125°. It was converted to isoxanthochymol by acid-catalyzed cyclization. Guttiferone E is thus the optical antipode of camboginol and a double bond isomer of xanthochymol. Guttiferone F (55) constitutes the unique PBD isolated from genus Allanblackia so far [4]. Its structure is very closely related to camboginol (46) and guttiferone E (54 (+) camboginol), two PBDs isolated from genus Garcinia and Clusia. Physical and spectroscopic data suggested that guttiferone F is the C-23 epimer of camboginol or

688

guttiferone E. Chemical evidences obtained by acid-catalyzed conversion of guttiferone F to 23-epi-cambogin permitted to the authors the verification of the epimeric configuration at C-23. De Oliveira et al. have published three main studies about chemical composition of Clusia's floral resins [18-20]. They have treated the floral resins with diazomethane in order to facilitate the separation of the major components. The first two works led to postulate the existence of the following PBDs in Clusia spp: grandone (22), clusianone, nemorosone, hydroxynemorosone, 7-epinemorosone and nemorosone II. Except grandone all of them showed to be bicyclo - [3.3.1]-nonane derivatives. Clusianone had been isolated both from the roots of C. congestiflora [44] and from fruits of C. sandinensis [27], later McCandlish et al. reported an X-ray diffraction analysis of clusianone [45] that defined the structure as (56), in which a type B nucleus and the equatorial 3-methyl-2butenyl group at C-7 were firmly established. The C-7 epimer (57) of clusianone (7-epiclusianone) has been successively isolated from Rheedia gardneriana [10]. Clusianone reported in two different works, [27 and 19] showed differences of C chemical shifts that suggest an epimer relationship between these compounds i.e. gew-methyl group at C-8 (Table 3). NMR data of structures proposed for nemorosone [19] and nemorosone II [18] isolated by De Oliveira et al. were identical with those of O-methyl derivatives obtained employing nemorosone isolated from Clusia rosea in a new study [40]. Nemorosone as it is in the nature was fully characterized by NMR spectroscopy techniques that included nOe difference spectroscopy experiments on the natural product. After comparison of the H and C NMR chemical shifts of the natural product nemorosone and its methyl derivatives with O-methyl nemosorone and O-methyl nemorosone II previously described, we proposed that only there exist one nemorosone. The O-methyl nemorosone (type C) and O-methyl nemorosone II (type A) previously isolated from Clusia spp. are the O-methyl tautomers of the same natural product that is named nemorosone (58). A structure type A for nemorosone was confirmed on the basis of nOe difference spectroscopy experiments. Saturation at the frequency of the Me-33 axial gave positive increments at the aromatic protons signals (H-12 and H-16) of the benzoyl group. In the same way, irradiation of the H-6 eq. showed an interaction with methylene protons at C-22 and when the latter protons were irradiated, interactions with methylene protons at C-6 and Me-25 were observed. These results prompted us to define that

689

nemorosone is a type A PBD. An analogous situation was proposed for hydroxy nemorosone (59) but it has been isolated only by De Oliveira et al. up to date. Nemorosone was identified as the major component from the floral resin of C. rosea (48%), C. grandiflora (69 %), C.insignis (43 %) and C. nemorosa (38 %) [19]. Table 3. "C chemical shifts of gem-methyls at C-8 in bicyclo-[3.3.1|-nonane derivatives. Compounds h 13C (gem-methyls at C-8) Me ax.: Me eq. Methyl Clusianone 15.9 :22.4 Hidroxynemorosone 16.1 :24.4 Clusianone* 22.5:27.0 Methyl-O-7-epinemorosone 23.9:27.2 Methyl-O-nemorosone 15.7:23.4 Guttiferone F 23.2 :27.3 Guttiferone B 16.5:23.8 Guttiferone E 23.2 : 27.3 Isoxanthochymol 22.7:27.1 O-methyl Chamone I 16.4 :24.7 Chamone II 16.6:24.7 Propolone A 15.9:23.7 Aristophenone * 15.8:23.7 Nemorosone* 15.6:23.2 Plukenetione D (acetate) 23.3 :27.1 Plukenetione E (acetate) 22.0 :26.0 Plukenetione F 23.5 :27.3 Plukenetione G 22.5 :26.8 18-O-methyl isogarcinol 16.3 :23.9 18-O-methyl garcinol 16.3 :22.6 15,16-dihydro-16-hydroperoxyplukenetione F 23.6:26.8 Sampsonione K 22.2 :26.8 Sampsonione L 22.2:26.8 Sampsonione M 24.0 :26.5 Xanthochymol 23.2:27.5 Insignone 23.9:27.2 Spiritone 15.8:24.1 Hyperibone A 16.1.: 23.9 Hyperibone B 16.0:23.9 Hyperibone C 16.5 :24.7 Hyperibone D 16.5 : 23.3 Hyperibone E 16.9:23.9 Hyperibone F 17.0:23.8 Hyperibone G 16.5 : 23.3 Hyperibone H 25.1:26.8 Hyperibone I 23.9:27.3 Ochrocarpinone A 15.9:23.8 Ochrocarpinone B 16.5:23.2 * Only one tautomer has been considered

References 19 19 27 18 18 4 3 3 35 9 9 11 49 40 47 47 47 47 44 44 52 51 51 51 35 20 20 8 8 8 8 8 8 8 8 8 14 14

A similar misassignment was suggested for 7-epinemorosone (60) but due to the lack of authentic sample the observation was only a hypothesis [40]. Bittrich et al. reinvestigated the structure of 7-epinemorosone

690

employing the O-methyl derivative isolated from Tovomitopsis saldanhae. Finally the structure was corrected and considered as type A BPD [46]. From Clusia spp. spiritone, insignone and scrobiculatones A and B were isolated [20]. Their structures (61-64) were established on spectral evidences which included 2D NMR techniques. Comparison of 13C chemical shifts assigned to gem-methyl group at C-8 of spiritone and 7epiclusianone [19] suggested that the stereochemistry between them is very closely related. When the same comparison was done between insignone and 7-epinemorosone, similar 13C chemical shifts were observed between above mentioned groups and then insignone and 7-epinemorosone show a similar stereochemistry respect to bicyclo moiety. On the other hand, scrobiculatones A and B seem to be very closely related to nemorosone (58) on the basis of the same analysis. Henry et al. as part of their phytochemical studies of Caribbean Guttiferae examined the extract of C. plukenetii [47]. The investigation permitted to isolate plukenetiones D and E as acetyl derivatives and plukenetiones F and G. Plukenetiones D and E (65,66) are a tautomeric pair of PBDs. The NMR data of regioisomeric pair plukenetiones F and G (67,68) provided evidence for a 2,2-dimethyl-2H-pyran moiety. The unusually high field signal observed for one methyl of pyran ring of plukenetione F may be due to shielding effects from the phenyl group. This effect is absent in plukenetione G. Although, the absolute configurations of plukenetiones D and E have not been determined Henry et al. suggested that plukenetiones D/E and 7epinemorosone are the same product. This consideration is logic but, these products could be enatiomers in like manner guttiferone E and camboginol. Grossman and Jacobs [48] developed a comparative study among PBDs isolated from Clusiaceae in order to clarify some structural aspects but, structures mistankely identified until that moment were included, i.e: Omethyl nemorosone [19] and O-methyl-7-epinemorosone [18]. This situation conditioned comparisons between compounds that nowadays are considered to have the same structure (i.e. O-methyl nemorosone [19] and O-methyl nemorosone II [18] are the methylated tautomers of the same compound named nemorosone (58). Therefore, the conclusions obtained should be considered carefully. Particularly, the conformations associated (chair or twist-boat) with the more saturated ring in bicyclo-[3.3.1]-nonane derivatives. Some authors have suggested that the conformation of the mentioned ring depends of the configuration at C-7 [18,40,47,48]. When prenyl group at C-7 is equatorial predominates the chair conformation and

691

when the isopentenyl group is in axial position (chair conformation) the twist-boat conformation has been suggested as predominat on the basis of the existence of two 1,3-diaxial interactions between the isopentenyl group at C-7 and C-2 and C-4 if the chair conformation is conserved. These observations are logics but, it is also possible to consider that both conformations are represented at the equilibrium in similar quantities. We developed a semiempirical computational procedure for conformational search and energy minimization (AMI, MOPAC v6) in order to clarify this point [49]. The results assessed the chair as the predominant conformation for stereoisomer R (equatorial isoprenyl group at C-7). However, the twist boat and the chair conformers of S stereoisomer (axial isopentenyl group at C-7 in chair conformation) showed to have an energy difference < 0.7 Kcal/mol, suggesting that both conformations are represented at the equilibrium. Keto-enolic equilibrium observed in PBDs is associated to the process of conversion between tautomers which has been evidenced by two sets of NMR signals in compounds as nemorosone (58), clusianone (56), aristophenones (71) and xerophenones A and B (95,96). Equilibriums showed in Fig. (8) are suggested on the basis of that the velocity of conversion between conformers is faster than that between tautomers. On the other hand, all PBDs analysed by X-ray diffraction methods present a chair conformation both when isopentenyl groups occupies an axial or equatorial position [27,40,49,50]. Examination of the reported values of vicinal coupling constant for a series of PBDs gave two ranges. In the case of nemorosone (58) (as it is in nature or in O-methyl derivatives) and guttiferone B (53), the coupling constant between H-6 axial and H-7 has a value of 10-13 Hz, suggesting that the 3-methyl-2-butenyl substituent occupies an equatorial position in the predominant chair conformation [3,18,40]. On the other hand, there are other examples where the coupling constant between H-6 axial and H-7 reaches a value of 7-7.5 Hz. That is the case for the compounds guttiferone A and F (49,55), plukenetione E acetate (66) and plukenetione G (68) [3,47]. All of them possess one isopentenyl group in axial position (if the chair conformation is considered) but their 3J values are not characteristic of the chair conformation of cyclohexane derivatives (Jaa=10-12 Hz, Jae=Jee=2-5 Hz). This result can be justified if both chair and twist-boat conformations contribute to the vicinal coupling constant observed.

692

(64) Scrobiculatone B. R=C 5 H 9 eq*.

(56)Clusianone R=C 5 H 9 eq*

(67) Plukenetione F. R= C 5 H 9 ax •

(57) 7-epiclusianone R=CsH 9 ax*

(58) Nemorosone R(= R 3 = H, R 2 =eq*. (59) Hydroxy nemorosone R,= H., R 2 = eq*., R 3 = OH (60) 7-epinemorosone R ( - R 3 - H, R 2 - ax*. (65) a Plukenetione D R,= CH 3 CO, R 2 =ax*., R 3 =H (66) b PUkenetione E R,= CH 3 CO, R 2 =ax*., R 3 =H R 2 - C5H9

(63) Scrobiculatone A. R=C 5 H 9 eq*. (68) Plukenetione G. R=C 5 H, ax*.

(70) Chamone II R=C 5 H 9 eq*

(69) Chamone I R=C 5 H 9 eq* *axial or equatorial in chair conformation

(62) Insignone

Fig. (7). Bicyclo-[3.3.1]-nonane derivatives from (56) to (70).

(61)Spiritone

693

Fig. (8). Tautomers and conformers in bicyclo-(3.3.1)-nonane drivatives

Lokvan et al. studied the chemical composition of the trunk latex of C. grandiflora [9]. After treatment with diazomethane three PBDs were obtained: the known nemorosone and two new compounds closely related named chamones I and II (69,70). Comparison with the spectral data of nemorosone established that chamone I contained an additional prenyl group and a terminal methylene carbon. The relative configuration of chamone I and nemorosone was determined using ID NOESY pulse sequences. Selective irradiation of the methoxyl group at C-2 permitted to suggest the chair conformation in the B ring and the same relative configuration for the mentioned compounds. The NMR data and the lack of reactivity with diazomethane suggest that the C-3 isopentenyl group has ring closure at the C-2 enolic hydroxyl in chamone II. A study of the fresh fruits of G. aristata led to isolation of tautomeric pair of PBDs, aristophenones A and B [49]. Aristophenone (71) was presented in CDCI3 as a tautomeric pair in a ratio 1:1, as evidenced by two sets of NMR signals, the consequence of the presence of the enolizable 1,3-diketone system. *H and 13C chemical shifts of the 12 olefinic methyl groups were assigned. Ideally, the protons of each methyl group could be irradiated and the nOes recorded of those are cis to the corresponding methylene groups (13, 25 and 30). However, selective saturation and NOE detection is difficult when both target and enhanced signals are situated as close together as they are in a tautomeric pair in a ratio 1:1. Another approach to this problem involves C chemical shifts. The shifts of methylene carbons in conjugated unsaturated fatty acids are sensitive to whether they are situated in CM (Z) or trans {E) configurations. The former

694

are shielded (ca. 5 27.5) relative to the latter (ca. 8 32.5) due to stericcompression effects. Methyl carbons exhibit the same type of geometrical characteristics in corresponding systems. If we examine the three pairs of olefinic methyl groups (13, 14; 25, 26; and 30, 31), we see that methyls 13, 25, and 30 are cis to methylene 10, 22, and 27, respectively, while methyls 14, 26, and 31 are trans to these groups. From these discussions the chemical shifts of the cis methyl carbons were shielded relative to those of trans methyls. The chemical shifts of the directly attached methyl protons could then be determined by means of the HSQC experiments. The *H and 13C chemical shifts of the 12 olefinic methyl groups [49] were reported in Table 4. Table 4 . ' H a n d Position 13 14 25 26 30 31

1

C chemical shifts of olefinic methyl groups la 8 13 C 5'H 17.7 1.58 25.3 1.73 18.0 1.75 26.1 1.59 17.7 1.53 26.0 1.81

lb 8 13 C 17.5 25.6 17.9 25.7 17.1 26.2

I 'H .62 .71 .68 .65 .46 .82

The tautomeric mixture was acetylated employing Ac2O/pyridine in order to facilitate the structural analyses and two tri-acetylated compounds were obtained. Aristophenone is similar to clusianone the main difference was associated with the different substitution of phenolic ring. Propolone A (72) was isolated in large quantities from an ethanol extract of Cuban propolis [11]. This finding and the isolation of nemorosone from C. rosea has permitted to suggest the role of this plant in the chemical composition of Cuban propolis. However, propolone A has not been reported as a component of Clusia rosea's floral resin, the main Clusia sp. distributed in Cuba. Thus, propolone A can be a derivative of nemorosone and the conversion would occur whereas the process of conservation and treatment of propolis samples and extracts are carried out. Some authors have reported that natural PBDs are unstable mainly in solutions and our experiences are in agreement with those observations. On the other hand, Porto et al. noted the structural similarities between the PBD isolated from floral resins of C. scrobiculata scrobiculatone A (63) [20] and propolone A. However, the first one has not been isolated from floral resins of C. rosea so far.

695

30

31

O

OH \>

°\

1T 0

Prenyl 502 Rheedia gardneriana

^Y\ T OH

Prenyl

[6971]

736 Compound

Structure Prenyl HO

MW|

Species

Ref

502

Clusia insignis, Clusia nemorosa, Clusia renggerioides

[59]

502

Clusia spiritusanctensis, Clusia sandiensis, Garcinia assugu

[37, 45, 58]

502

Carcinia dulcis

[27, 64]

502

Clusia hilariana

[68]

502

Garcinia kola

[72]

502

Clusia lanceolata

[68]

1

82

—Prenyl

7-Epi-nemorsone

Prenyl Prenyl 83

Prenyl

V

Clusianone

Prenyl OH

84

Garciduol B

Prenyl

85

Hilarianone

fl fl 0

0 Prenyl

86

Kolanone

0

87

Lanceolatone

OH Prenyl

737

u

Compound

Structure Pren yi

MW

Species

Ref

Y

502

Clusia grandiflora, Clusia insignis, Clusia nemorosa, Clusia rosea

[58, 73]

//.o

88

Nemorosone

[ 0

-^—Prenyl

\

T

Prenyl Prenyl

89

Nemorosone II

I

,1

S 0

^—Prenyl

Y

Clusia grandiflora, 502 Clusia renggerioides, Clusia rosea

[59, 74]

Prenyl Prenyl Preny 90

Nemorosonol

{

Plukenetione D

Clusia mulliflora, Clusia nemorosa

[46. 75 76]

502

Clusia plukenelii

[67]

502

Clusia plukenetii

[67]

502

Propolis from Cuba

[77]

II

OH

91

502

\J>—Prenyl ff 0 Prenyl

I

-^—Prenyl

V \ o[5 Pren yi

rV 0H Prenyl

92

Plukenetione E

l

Y

j—Prenyl

II

0

0 yi

/} '\Jj 93

Propolone A

Pren

">C/Prenyl

— o=r^ |

0

738 Compound

Structure Prenyl

MW

Species

Ref

94

Weddellianone A

502

Clusia weddelliana

[68]

95

28,29-Epoxyplukenetione A

516

Clusia havetioides var. stenocarpa

[66]

518

Clusia nemorosa

[58, 59]

518

Ochrocarpos punctatus

[78]

518

Ochrocarpos punctatus

[78]

518

Clusia plukenetii

[67]

Prenyl Prenyl

96

Prenyl

Hydroxynemorosone

Prenyl

97

Ochrocarpinone B

Prenyl

98

Ochrocarpinone C Prenyl

Prenyl

99

Plukenetione B / 0

Prenyl

739 Compound

100

MW

Structure

- -Prenyl

Sampsonione B

Species

518 Hypericum sampsonii

Ref

[12]

OH Prenyl

101

Sampsonione G

518

Clusia havelioides var sienocarpa, Hypericum sampsonii

[II. 66]

Prenyl

0

Prenyl Prenyl 102

Sampsonione L

518 Hypericum sampsonii

[13]

Pren1

103

Xerophenone A

Clusia plukenetii, Clusia portlandiana

[67, 79]

518 Clusia portlandiana

[79]

528 Hypericum sampsonii

[11]

518 Prenyl OH

104

O

Xerophenone B

O

105

OH

Sampsonione H Geranyl

106 Nemorosinic acid A

530 O

OH

Clusia nemorosa

[80]

740 Compound

Structure

107 Nemorosinic acid B 0

MW

Species

Ref

530

Clusia nemorosa

[80]

534

Clusia havetioides var. stenocarpa, Ochrocarpos punctatus

[66, 78]

534

Garcinia aristata

[81]

534

Garcinia aristata

[81]

534

Ochrocarpos punctatus

[78]

O

OOH

108

15,16-Dihydro 16hydroperoxyplukenetione F

OH

109

Prenyl

Aristophenone A Prenyl O

OH Prenyl

110

Aristophenone B Prenyl O

111

O

Ochrocarpinone A

Prenyl

Prenyl

112

542 Hypericum sampsonii

Sampsonione E

O (/

Geranyl

[II]

741 Compound

113

Plukenetione C

114

Spiritone

Structure

MW

Species

Ref

550

Clusia havetioides var. stenocarpa, Clusia plukenetii

[66, 67]

556

Clusia spiritusanctensis

[68]

iPrenyl

Prenyl Prenyl

Prenyl

115

Insignone

560

Clusia insignis

[68]

116

Chamone II

568

Clusia grandiflora

[74]

Prenyl

742

Compound

117

Structure

MW

Hypersampsone F

Species

Ref

568 Hypericum sampsonii

[IV]

568 Hypericum sampsonii

[11. 17]

570

[74]

Prenyl Prenyl

118

Sampsonione D

Geranyl Prenyl Prenyl 119

Chamone I

Clusia grandiflora

Prenyl

120

Hypersampsone D

570 Hypericum sampsonii

[17]

570 Hypericum sampsonii

[IV]

570

[68]

0

0 0 Geranyl

121

Hypersampsone E O O

Geranyl

Prenyl

122

Weddellianone B

Prenyl Prenyl

Clusia weddelliana

743

Compound

123

Structure

33-Hydroperoxyisoplukenetione C

MW

Species

Ref

582

Clusia havetioides var. stenocarpa

[66]

OOH

124

Sampsonione I

125

Sampsonione J 0

o

584 Hypericum sampsonii

[14]

584 Hypericum sampsonii

[14]

586 Hypericum sampsonii

[12]

586 Hypericum sampsonii

[II]

586 Hypericum sampsonii

[11]

586 Hypericum sampsonii

[13]

Geranyi Prenyl

126

Sampsonione A 0

127

Sampsonione C

°

128

OH Geranyi

0

Geranyi

Sampsonione F Geranyi Prenyl Prenyl

129

Sampsonione K

Prenyl

744 Compound

Structure Prenyl

MW

Species

Ref

Geranyl 130

Sampsonione M

OH

131

Prenyl

N < '

OH

OH

[8284]

602

Garcinia indica, Garcinia ovalifolia, Garcinia xishuanbannanansis

602

Garcinia pyrifera, Garcinia subelliptica

90]

602

Garcinia cambogia

[91]

[85-

Prenyl

Garcinol (camboginol) 11

135

Garcinia assugu, larcinia pedunculala

Prenyl

133 Cycloxanthochymol

134

602

Prenyl

132 (+)-Isoxanthochymol

OH

[13]

.Prenyl

(-)-Isoxanthochymol (Isogarcinol, cambogin)

1

586 Hypericum sampsonii

iPrenyl

Guttiferone A Prenyl O

OH

Calophyllum brasiliense, Garcinia intermedia, Garcinia livingstonei, 92602 Garcinia 94] macrophyUa, Symphonia globulifera

745

Compound

Structure OH

136

Preny[

MW

Guttiferone E Prenyl

OH

137

Prenyl

Species

Ref

Prenyl Clusict rosea, Garcinia assugu, 602 Garcinia huillensis, Garcinia ovalifolia, jarcinia pedunculata

[37, 83, 88, 95]

,\ Prenyl

Guttiferone F

602

Allanblackia sluhlmannii

[96]

Prenyl

OH

138

Prenyl

602

Xanthochymol

OH

139

Prenyl.

Prenyl

Clusia rosea, Garcinia indica, Garcinia mannii, Garcinia ovalifolia, Garcinia staudtii, Garcinia xanthochymus, Garcinia xishuanbannanansis, Rheedia madrunno

97100]

%v \Prenyl

Pedunculol

604 Garcinia pedunculata

[83]

616

Garcinia assugu

[37]

616

Garcinia assugu

[37]

Prenyl

OMe

140

Prenyl

Garcinol, 13-Omethyl ether Prenyl

OMe

141

Prenyl_ O,

^Prenyl

Isogarcinol, 13-Omethyl ether I Prenyl

746 Compound

142

Bronianone

Structure Geranyl

Geranyl .0

HO

MW

Species

670

Garcinia hombronlana

670

Symphonia globulifera

[88]

670

Symphonia globulifera

[88]

670

Symphonia globulifera

670

Garcinia macrophylla

Ref

101]

Preny!

0 OH 143

0

Preny[

.Geranyl

Guttiferone B Geranyl O

OH Prenyl

144

Guttiferone C

Prenyl OH

145

Prenyl.

Guttiferone D

Prenyl

OH 146

Prenyl,

''^tf

^Geranyl

Guttiferone G

[93]

''Prenyl O

OH

Benzophenones and xanthones are known to co-occur in the Clusiaceae [1], and the garciduols (74, 75, 84) are three benzophenonexanthone dimers [27,64]. The cudraphenones (33, 37, 42, 37), a group of benzophenones isolated from the Moraceae family, are prenylated on the A- and B-rings [47]. These are the only four compounds that are prenylated on both the A- and B-rings. Ten benzophenone glycosides, nine O-glycosides (56-58, 60, 62, 6768, 71-72) and one C-glycoside (61) have been isolated from five different plant species. None of these are prenylated, and the glycoside substituent is attached to either the A- or B-ring [39,54-57,60,63].

747

The most common polyprenylated benzophenones have a bicyclo[3.3.1]nonane B/C-ring system. Approximately 38 bicyclo[3.3.1]nonane benzophenones have been isolated. A typical bicyclo[3.3.1]nonane benzophenone is xanthochymol (138), which is discussed in detail below. The floral resin of twelve Clusia species has yielded at least fourteen (66, 79-80, 82, 85, 87-89, 93-94, 96, 114-115, 122) bioactive bicyclo[3.3.1]nonane polyprenylated benzophenones. These compounds have the isoprenyl and benzoyl residues attached at different positions on the bicyclo[3.3.1]nonane ring [58,59,68,73,102]. In addition, the floral resin is used by pollinating bees for nest construction [77]. Four isolated PPBs, with the bicyclo[3.3.1]nonane system, contain peroxide bonds: 15,16-dihydro-16-hydroperoxyisoplukenetione F (108), ochrocarpinone A (111), plukenetione C (113), and 33hydroperoxyisoplukenetione C (123) [66,78]. The tautomeric benzophenone pair xerophenone A (103) and B (104) present an interesting variation in benzophenone chemistry by having a 7-membered C-ring and an oxo bridge between carbons 4 (C-ring) and 10 (B-ring). These compounds feature an oxatricyclo[4.3.1.1]undecane-7,9dione system [79]. A number of PPBs occur as tautomeric pairs; examples include aristophenone A and B (109, 110) [81] and plukenetione D (91) and E (92), which were isolated after acetylation [67]. Plukenetione A (76) was the first PPB isolated with a adamantyl skeleton and an methylpropenyl group [65]. Sampsonione D (118) and I (124) each have one isopentenyl side chain replaced by an isopropenyl moiety [11,13,14]. Grossman et al. details the relationships of the plukenetiones, nemorosone II, and sampsoniones [103]. They showed that plukenetiones B, D, and E are diastereomeric to nemorosone II and sampsonione G. Nemorosonol (90), isolated from Clusia nemorosa fruits, has a novel tricyclo[4.3.3.0]decane acetate-derived B-ring [75]. This compound's structure was determined by X-ray crystallography [76], and is the only isolated benzophenone with a tricyclo[4.3.3.0]decane system. There has been considerable confusion in the nomenclature and structural elucidation of garcinol/caboginol (134) and isoxanthochymol/isogarcinol (131). Their naming history is discussed by Bennett et al. [1] and by Fuller et al. [96]. The synonyms for these compounds are listed in their respective entries in Table 2. As noted by Fuller et al., the common name garcinol refers to more then one

748

compound. In addition, from a structural and chronological standpoint the common name caboginol has precedence for 134. Details on the structural elucidation of these compounds has been described [104-110]. ISOLATION OF BENZOPHENONES Multiple chromatographies are needed to purify benzophenones, and a variety of normal and reversed-phase solvent systems and solid phases including column chromatography over silica gel, reversed-phase, and Sephadex LH-20, as well as preparative TLC and HPLC have been employed. Isolation methods for selected benzophenones are given, and our experiences with the activity-guided isolation of xanthochymol (138) from G. xanthochymus fruits are discussed. Typically, the roots, leaves, fruits, wood, or floral resin are extracted with a single solvent, or solvents, of increasing polarity including CeH6 [75], hexane [67,81], CH2C12 [96], acetone [45], petroleum ether [111], MeOH [96], and/or EtOH [13,47]. After in vacuo concentration, the residue is resuspended in water and sequentially partitioned with solvents of increasing polarity, including w-hexane, CeH6, CH2CI2, EtOAc, and BuOH. After partitioning, extracts are passed over silica gel, either open column [67,83] or vacuum-liquid chromatography (VLC) [27], usually with mixtures of hexane-EtOAc [49,50] or CHCh-MeOH. Separation via Sephadex LH-20 using isocratic systems of MeOH [27], CH 2 Cl 2 -Me0H [88], CHCh-MeOH [29,37], or a gradient solvent system of CH2C12 -> CH2Cl2-MeOH, [93] has also been employed. After initial separation benzophenone-enriched fractions are rechromatographed (using preparative TLC, column chromatography, or HPLC) over a variety of stationary phases (in order of decreasing times employed) including silica gel, C,8, Sephadex LH-20, C8 [88], diol [88], MCI gel CHP-20P [55], and Toyopearl HW-40 [55]. Usually, multiple chromatographies over the same stationary phase or a combination of stationary phases are used to purify benzophenones. Recrystallization has been described in a few publications [54,81,83]. Isolation methods for selected benzophenones are given below. The anti-HIV guttiferones A-D (135, 143-145) were isolated from Symphonia globulifera by extracting with CH 2 Cl 2 -Me0H and then MeOH. The combined organic extracts were partitioned with EtOAc and, after in vacuo concentration, were passed over a diol column, eluted with CH 2 Cl 2 -Et0Ac-Me0H. The HIV-active fractions were combined and rechromatographed over a diol column, eluted with CH2C12. Next, the

749

HIV-active fractions were combined and rechromatographed over a column, eluted with 9:1 MeOH-H2O and 100% MeOH. Final purification was achieved by C8 HPLC using M e O H ^ % H 2 O-0.01% TFA [88]. Sampsoniones A-M (100-102, 105, 112, 118, 124-130) were isolated from whole air-dried Hypericum sampsonii which was extracted with 95% EtOH. The EtOH extract was concentrated under reduced pressure and partitioned between CH2CI2 and H2O. The CH2CI2 phase was chromatographed over silica gel, eluted with hexane-EtOAc mixtures. The sampsonione enriched fraction(s) were rechromatographed over silica gel, eluted with hexane-CHCb-acetone mixtures. Individual sampsoniones were isolated by preparative TLC or VLC over Cis [1114]. The vismiaphenones (35, 44, 45, 53) were isolated using Sephadex LH-20 with 1:1 CH 2 Cl 2 -Me0H, followed by normal-phase HPLC (17:3 hexane-'PrOH) with a cyano column [15]. Percolation with hexane [67] or extraction with CI-bCh-MeOH, then MeOH [67] were used in isolating plukenetiones B-G (77, 78, 91, 92, 99, 113). After extraction, Henry et al. subjected the hexane extract to repeated chromatography over silica gel using Me2CO-hexanes mixtures to yield plukenetiones B-G [67]. Chaturvedula et al. isolated the related benzophenone 15,16-dihydro-16-hydroperoxyisoplukenetione F (108) from Ochrocarpos punctatus by fractionation over Sephadex LH-20 with n-hexane-EtOAc, followed by preparative reversed-phase TLC and reversed-phase HPLC Chaturvedula et al. used a similar procedure to isolate the ochrocarpinones A-C (97, 98, 111) from O. punctatus [78]. The benzophenone glycoside iriflophenone-4-OP-D-glucopyranoside (58) was isolated from Davallia solida by chromatography of the nBuOH layer over Sephadex LH-20, eluted with MeOH [56]. The first fraction was further purified by preparative cellulose TLC and then reversed-phase HPLC with MeOH-H2O to yield 58. Porto et al. methylated a Clusia floral resin extract, chromatographed the derived compounds over silica gel, rechromatographed using preparative argentation TLC (5% silver nitrate), and thereby isolated seven polyisoprenylated benzophenones [68]. Lokvam et al. [74] and de Oliveira et al. [59] also methylated a crude extract before isolating chamones I, II (119, 116), and nemorosone II (89) from Clusia species. The isolation of xanthochymol (138), shown in Fig. (3), illustrates typical methods used to purify benzophenones. Two partitioning methods were developed in the course of our laboratory work with Garcinia

750

xanthochymus. The first method dissolved the MeOH extract in 9:1 H2OMeOH and partitioned sequentially with hexane and EtOAc. This was a less-than-optimum system because the benzophenones, biflavonoids, and xanthones were found in both organic phases. An optimized method resuspended the dried MeOH extract in 100% water and partitioned sequentially with CHCI3 and EtOAc concentrating the benzophenone and xanthones in the CHCI3 partition and the biflavonoids into the EtOAc layer, Fig (3). After partitioning, the CHCI3 layer was separated over Sephadex LH20 and eluted with MeOH. The benzophenone-enriched fraction was chromatographed repeatedly over re versed-phase (2:8-0:1 H2O-MeCN, 5% steps) to yield two novel benzophenones, the known benzophenone aristophenone A (109) and fractions A and B, each a mixture of benzophenone double-bond isomers [112]. Fraction A, was a mixture of 136 and 138, Fig. (3C), and fraction B was a mixture of 131 and 133. Repeated attempts to separate these fractions using normal-phase and reversed-phase preparative TLC; column chromatography over Sephadex LH-20, silica gel, C\%, polyamide, and cyano columns; and HPLC over Ci8, C8, cyano, phenyl, and silica columns were unsuccessful. Other researchers have encountered difficulties in separating benzophenone double-bond mixtures consisting of 136 and 138 and related compounds [88,90]. After a protracted method development using various types of argentation (silver) chromatography systems, compounds 136 and 138 were isolated with a quaternary solvent system (40:10:1.25:0.2 hexaneEtOAc-95% EtOH-TFA) over normal-phase TLC impregnated with a 10% solution of AgNO3, Fig. (3D and 3E). This procedure was also used to separate 131 from 133 [112]. The separation of G. xanthochymus was monitored by HPLC (described below) and TLC. Two d 8 TLC systems, 1:1 and 15:85 10 mM ammonium acetate-MeCN, were used to combine collected fractions. After development, compounds were visualized with 1% vanillin in acidified EtOH. After heating, benzophenones turned greenyellow. These two TLC systems proved very useful in monitoring the separation of benzophenones in our studies on G. xanthochymus and for the dereplication of a number benzophenones in other Clusiaceae species. Table 2 lists analytical and preparative HPLC methods developed for the isolation and quantification of benzophenones. Most methods have utilized reversed-phase Cig columns with mixtures of MeCN or MeOH and H2O, with or without an acid modifier. Exceptions include

OUh

C

138 and 136

U.H" Ei.tr

^

D

O 10-

PP5"

138

u.w

*

mmJ

QCrJft-

|

I

E

frfljfr

136

• 1

OI>HV

4t.U(V

Fig. (3). Isolation of xanthochymol (138). A: EtOAc partition, B: CHCI3 partition; HPLC system, gradient: 9:1 10 mM ammonium acetate—MeCN to 100% MeCN (see text for details), PDA extracted at 254 nm C Fraction A, before Ag-TLC D. Isolated xanthochymol (138) E. Isolated guttiterone E (136); HPLC system, gradient 1 1 1 0 mM ammonium acetate—MeCN to 100% MeCN in 26 mm at 1 mL/min

752

Nucleodex p-PM [90], C8 [88], and cyano [15] columns used in three different methods. During our isolation work with G. xanthochymus two HPLC methods were developed. The HPLC methods used a Phenomenex Luna Cig (5 jum, 250 x 4.6 mm) column and a solvent system of A = 10 mM ammonium acetate and B = MeCN. In the first method the initial conditions were 9:1 A-B, and a linear gradient was initiated until minute 45. The final solvent mixture was 0:1 A-B. The column was held at 100% B until minute 55, and then the initial conditions (9:1 A-B) were reinitiated at minute 56. The column was equilibrated for 10 minutes before the next injection. In the second system the initial conditions were 1:1 A-B, and a linear gradient was initiated at minute 4 until minute 26. The final solvent mixture was 0:1 A-B. Sample chromatograms are shown in Fig. (3). Both HPLC systems were used to track compounds isolated from G. xanthochymus fruits and to dereplicate benzophenones in a number of other Clusiaceae species. Table 2: Published HPLC Methods Used for the Isolation and Quantification of Benzophenones Compound(s) Analyzed 48-50 69 93 70,71 97,98 6, 53, 55, 59 109,110 108, 111 136,138 136', 138" 117,120,121 131,135,136 135,144,145 132,133, 136,138 Compound(s) Analyzed 66, 88, 96 88 79,80,85,87,94,112, 114,115 "isolated as a mixture

Isolation Methods Column; Solvent System MetaChem, Intersil ODS-3 (8 urn, Cm, 250 x 20 mm) 10 mL/min; isocratic 4:1 MeOH-H 2 O; gradient 70:30 to 90:10 MeOH-H 2 O in 30 minutes Column not reported; 9:1 MeOH-H 2 O Waters ^Bondapack C, 8 2 mL/min; 9:1 MeOH-H 2 O Column not reported; 88:12 MeOH-H 2 O Shimadzu ODS C, 8 (250 x 10 mm); 70:30 MeCN-H 2 O Dynamax-cyano (4.1 x 30 cm), 80 mL/min; 17:3 hexane-'PrOH Waters .uBondapack C l g 2 mL/min; 9:1 MeOH-H 2 O Shimadzu ODS C 18 (250 x 10 mm); 75:25 MeCN-H 2 O Nucleodex P-PM (5 fim, 250 x 10 mm) at 0 °C; 52.5:47.5:0.1% MeCN-H 2 O-TFA Rainin Dynamax (1.0 x 25 cm); 97:3 MeCN-H 2 O Cosmosil 75 C, 8 Prep; 9:1 MeOH-H 2 O and 1:0 MeOH-H 2 O Rainin Dynamax (1.0 x 25 cm); 24:1 MeCN-H 2 O Rainin Dynamax (1.0 x 25 cm); MeOH^t% H 2 O-0.01% TFA Phenomenex Luna C, 8 (5 fim, 250 x 4.6 mm) 1 mL/min; gradient 9:1 10 mM ammonium acetate-MeCN to 100% MeCN in 45 minutes Quantification Methods (column; Solvent System) Waters Novapak C18 (4 ^m, 3.9 x 150 mm) 1 mL/min; gradient 60:40 to 100:0 MeCN-H 2 O in 60 minutes (quantified as Me esters) Column not reported; gradient 50:50 to 100:0 MeOH-AcOH 2% in 15 min Waters Novapak Cu (4 fim, 3.9 x 150 mm) 1 mL/min; gradient 60:40 to 100:0 MeCN-II 2 O in 60 minutes (quantified as Me esters)

Ref.

[52] [621 [77] [62] [78] [15] [811

US] [90] [88] [17] [881 [88] [112]

[59] [102] [68]

753

STRUCTURAL ELUCIDATION OF BENZOPHENONES The structures of benzophenones have been established by UV, IR, MS, and most extensively, by ID and 2D NMR. The structures of a few benzophenones have been determined by X-ray crystallography including: nemorosonol (88) [76], 7-epi-clusianone (81) [71], xanthochymol (138) [113], and (-)-isoxanthochymol (131) [104,114]. Chemical tests with FeCU [47] or Gibbs reagent [27] and acetylation [27] or methylation [42] are used to show the phenolic nature of benzophenones. IR has been useful in showing that benzophenones contain hydroxyl groups, both conjugated and nonconjugated ketone groups, and aromatic C=C bonds. Xanthochymol (138), to our knowledge, is the only benzophenone analyzed, in detail, for its electron impact MS fragmentation behavior [110]. Reported MS losses for benzophenones include an mlz 105 (C6H5CO+) for a unsubstituted phenyl ketone A-ring [44,65], mlz 137 (C6H5 O2-CO"1") for a 3,4-dihydroxybenzophenone moiety, and mlz 68 (CsHg) for a prenyl-type group. The positive electrospray ionization (ESI) mass spectrum, Fig. (4), of xanthochymol (138) showed a base peak at [M + H] + = mlz 603 and ,M ao 75: JO

s.l

ut.11 3>lm

2»£5_"3°z a , * , 300

150

UO

ISO

»"• «0

+i»«r| J t f »

i i

<SO

150

SOJ

Fig. (4). Positive ESI mass spectrum of xanthochymol (138)

754

losses at mlz 467, loss of the 10 carbon side chain attached at position 8 or the loss of the 3,4-dihydroxybenzophenone moiety. The mlz 411 ion likely represents an additional prenyl group loss. Additional fragments were observed at mlz 343, 286, and 233 likely corresponding to additional losses of 1, 2, or 3 prenyl, or parts of prenyl, groups. The negative ESI mass spectrum (not shown) only displayed a base peak at [ M - H T = ;»/z601. The structures of most benzophenones have been determined by 1D ('H, 13C, and DEPT) and 2D (COSY, HSQC, HMBC, and NOESY) NMR experiments. The majority of benzophenone NMR spectra have been recorded in CDCI3 and CD3OD. Benzene-c/6, mixtures of benzened(, and CDCI3 [75], or pyridine-ds have also been used [88,93]. The aforementioned solvents were used to resolve overlapping signals of studied compounds. Deuterated TFA (0.1%) has also been used to increase the rate of keto-enol interconversion in benzophenones. We now turn our attention to the structural elucidation of xanthochymol (138). The 500-MHz *H spectrum of xanthochymol (138) is displayed in Fig. (5). The chemical shifts, coupling data, multiplicity, COSY, and HMBC correlations are shown in Table 4. Because of the tautomeric nature of benzophenones, NMR spectra are sometimes acquired after the addition of deuterated TFA (0.1%). We found this to be essential when using CDCI3, but not with CD3OD. However, carbon spectra recorded with CD3OD-TFA displayed sharper signals for C-l, 3, 4, 8, and 10. To aid in the structural determination of xanthochymol (138), we divided the proton spectrum into three regions. Region one, from 5 0-3.0, contains numerous overlapping signals, but yields some valuable information, specifically the number of methyl groups. Due to overlapping signals, the exact number of methyl groups is not conclusive, but 7 or 8 is a good approximation. Furthermore, the methyl signals appear as two groupings, two upfield signals at 8 1.01 and 1.17 and 5-6 signals between 5 1.5-1.8. The two upfield signals are characteristic of geminal-dimethyl protons on a sp3 carbon. The number of methyl groups from 5 1.5-1.8, along with data from the next region (5 4.0-5.2) indicates the number and type (isopent-2-enyl versus isopent-3-enyl) of prenyl groups in xanthochymol (138). The 'H spectrum of xanthochymol (138) displays signals for a least one olefinic proton at ca. 5 5.00 (indicating two isopent-3-enyl groups) and two signals for terminal methylene protons, indicating the number of isopent3-enyl groups. Due to overlapping 'H signals, DEPT-135 and HSQC experiments are needed to confirm the number and type of prenyl groups.

Methyl groups

Methine and methylene groups

AMX spin system

Terminal methylenes

Olefinic protons

uw I

• • • • I • • • • I ' ' ' ' I • • • • I • ' • . I • ' ' • I ' • • • I • • ' ' I ' • ' ' I ' • ' • I ' • • ' I • • • • I ' ' ' ' I ' ' ' ' I

7 . 5

7 . 0

6 . 5

6 . 0

5 . 5

5 . 0

4 . 5

4 . 0

3 . 5

3 . 0

Fig. (5). 'H spectrum of xanthochymol (138) recorded at 500 MHz in CD,OD

2 . 5

2 . 0

1 . 5

756

The last region (8 6.0-8.0) reveals one structural fragment. In polyprenylated benzophenones, the aromatic A-ring typically has either a 3- or 5-spin pattern. Xanthochymol (138) clearly displays an aromatic 3spin system consisting of protons at 5 7.22 d (J = 2.1 Hz), 7.00 dd (2.1, 8.1 Hz), and 6.72 d (8.1 Hz). Even though the ] H spectrum is crowded, three important structure features are obtained: i) the type of aromatic Aring; ii) the approximate number and types of prenyl groups; and iii) the presence of an aliphatic geminal-dimethyl group. The 13C, DEPT-135, and DEPT-90 experiments showed a nonconjugated ketone at 5 209.8, flanked by two quaternary bridgehead carbons (8 59.5 and 68.9), an enolized 1,3-diketone [8 117.9 and 194.5 (2x)], a methylene carbon at 8 42.7, and a methine carbon at 8 47.0 which, together with a quaternary carbon at 8 50.4, established the bicyclo[3.3.1]nonane system in xanthochymol (138). The DEPT-135 showed five sp2 methines and six aliphatic methylenes. The number of methine carbons, five sp2 and two aliphatic, was confirmed by a DEPT90 experiment. The carbon and DEPT experiments further indirectly confirmed the AMX 3-spin system with 13C quaternary aromatic resonances at 8 128.7, 145.3, and 151.4, and the number of prenyl groups as four (two isopent-2-enyl and two isopent-3-enyl groups). The HSQC spectrum, displayed in Fig. (6), unequivocally establishes the number and type of protonated carbons in xanthochymol (138). Even though the DEPT-135 experiment displays the number of CH3, CH2, and CH carbons, the HSQC spectrum resolves the following: i) an olefinic CH proton is under the H2O signal, thus 138 contains two isopent-2-enyl groups; ii) xanthochymol (138) has two terminal methylenes, and, therefore, two isopent-3-enyl groups; and iii) the exact number of CH3, CH2, and CH carbons. Therefore, the HSQC spectrum establishes that 138 contains eight methyl, eight methylene, and seven methine carbons. Resonances for C-H pairs are listed in Table 3. Table 3. Protonated Carbons of Xanthochymol Based on the HSQC Spectrum CH

CH,

CH3

"H .01 .17 .52 .63 .67 .71 .71 .75

5 C 26.4 22.2 17.2 16.8 25.0 17.3 21.8 25.4

S

H 1.9 2.0

1.3,1.5 2.05, 2.25 2.08,2.28 4.52 4.65 2.55,2.73

S

C

35.8 36.7 32.1 42.7 29.8 112.5 109.4 26.0

S

H

1.52 2.6

4.90 5.05 6.72 7.00 7.22

"C 47.0 43.7 124.1 120.4 114.0 124.2 116.2

-J

Cn

-

u> -

•I

O

I

o o

I

I • " 1X1

CO

o

o

o

I "

o

o

Fig. (6). HSQC spectrum of xanthochymol (138) recorded at 500-MHz in CD,OD

l in

758

The COSY spectrum connects 3 multi-spin networks in xanthochymol (138). Analysis of the COSY spectrum shows one olefinic proton coupled to a methylene group. The second olefinic proton is also coupled with a second methylene group, and an additional methylene and methine group. Also, a CH2-CH-CH2-CH2 coupled spin system, possibly connecting two prenyl groups, is observed. The three observed proton spin systems are: i) 5 5.05 (18), 2.73 (17A), and 2.55 (17B); ii) 8 4.92 (25), 2.28 (24A), 2.08 (24B), 1.52 (6), 2.25 (7A), and 2.05 (7B); and iii) 5 2.0 (29AB), 2.6 (30), 1.5 (34A), 1.3 (34B), and 1.9 (35 AB). At this point, using the 'H, 13C, DEPT, COSY, and HSQC spectra, we have tentatively identified five structural fragments shown in Fig. (7). We now use the HMBC spectrum, Fig. (8), to confirm these structural fragments and to assemble the complete structure of xanthochymol, Fig. (9). The HMBC spectrum, Fig. (8), confirms the following substructures: i) HMBC correlations from H-12 (5 7.22) to C-13 (5 145.3) and C-14 (5

X

Fig. (7). Structural fragments of 138 deduced by ] H,

I3

\

C , DEPT, COSY, and HSQC spectra

151.4) and from H-15 (5 6.72) to C-13 (5 145.3) and C-14 (5 151.4) confirms the 3,4-dihydroxybenzophenone moiety, and a HMBC cross peak from H-12 (5 7.22) to C-10 (5 194.8) connects the AMX system to the carbonyl carbon at C-10 (5 194.8), Fig. (7A); ii) HMBC cross peaks from H-7B (5 2.05) to C-l (5 194.5) and from H-6 (5 1.52) to C-7 (5 42.7), C-5 (5 50.4) confirmed the bicyclo[3.3.1]nonane system, Fig. (7B); and iii) the geminal-dimethyl protons which displayed HMBC cross peaks to each other, Fig. (7C); iv) HMBC cross peaks from the methyl groups to the olefinic carbons and correlations from the CH2 groups to their respective olefinic carbons confirmed the two isopent-2-enyl groups, Fig. (7D); and v) HMBC cross peaks from H-29 (5 2.0) to C-30 (5 43.7) and C-31 (5 147.9), from H-35 (5 1.9) to C-34 (5 32.1), C-30 (5 43.7), and C-37 (5 109.4), and from H-33 (5 1.63) to C-30 (5 43.7) confirmed the two isopent-3-enyl groups, Fig. (7E), are connected at C30.

Fig. (8). HMBC spectrum of xanthochymol (138) recorded at 500 MHz in CD,OD

760

Finally, the HMBC spectrum is used to connect the structural fragments to the bicyclo[3.3.1]nonane' system and complete the structure of xanthochymol, Fig. (9). Observed HMBC cross peaks from H-17B (5 2.55) to C-3 (5 194.5) and from H-7B (5 2.05) to C-24 (5 29.8) placed the isopent-2-enyl groups at C-4 (5 68.9) and C-6 (5 47.0). HMBC cross peaks from Me-22 and Me-23 to C-4, 5, and 6 (5 68.9, 50.4, 47.0, respectively) placed the geminal-dimethyl group at C-5. An HMBC correlation was observed from H-29 to C-l, thus the two isopent-3-enyl groups, Fig. (7E) were placed at C-8. Therefore, using ID and 2D NMR experiments we assigned the structure in Fig. (9) to xanthochymol. 27

Fig. (9). Arrows denote key HMBC cross peaks and bold lines indicate key COSY correlations for xanthochymol (138)

1 For clarity the stereochemistry of the bicyclo[3.3.1]nonane is not shown, as not to confuse it with the bolded COSY cross peaks.

761 Table 4. ] H, 13C, DEPT, COSY, and HMBC NMR Data for Xanthochymol (138) in CD3OD H" DEPT" C COSY" HMBC"

(5, mult. J= Hz) (8) 1 194.5 2 117.9 3 194.5 68.9 4 S 50.4 1.52, m 47.0 6 7 2.05,2.25, m (12.8) 42.7 8 59.5 9 209.8 10 194.8 11 128.7 12 7.22, d, (2.1) 116.2 13 145.3 14 151.4 15 6.72, d, (8.1) 114.0 16 7.00, dd, 2.1,(8.1) 124.2 17 2.55, 2.73, m 26.0 18 5.05, t, (5.5) 120.4 19 134.7 20 1.75 25.4 21 1.71 17.3 22 1.17 22.2 23 1.01 26.4 24 2.08,2.28 29.8 4.92, 124.7 25 26 132.6 1.67 25.0 27 1.52 17.2 28 2.0, m 36.7 29 2.6, m 43.7 30 147.9 31 4.52, brs 32 112.5 1.63 16.8 33 34 1.3, 1.5, m 32.1 1.9, m 35.8 35 145.9 36 4.65, brs 109.4 37 1.71 21.8 38 •Recorded at 500 MHz; "Recorded at 300 MHz

C C

c c c

CH CH2 C C C C CH C C CH CH CH2 CH C CH, CH, CH, CH, CH2 CH C CH, CH, CH2 CH C CH2 CH, CH2 CH2 C CH2 CH,

7,24 6

5, 7, 23, 24 1,24

16

10, 13, 14, 16

16 12, 15 18 17

11, 13, 14 12, 14 3,18,19

18,19,21 18, 19,20 5, 6, 23 4, 5, 6, 22 6,25 24

30 29,34

30,35 34

25, 26, 28 25, 26, 27 1,30,31

32 30,31,32 30 30, 34, 36, 37 35,38 35,36,37

BIOLOGICAL ACTIVITIES Isolated benzophenones display a number of biological activities, Table 5. Reported biological activities are grouped into eight categories: antioxidant, antibacterial, antifungal, antiviral, cytotoxic, molluscicide, trypanocidal, and additional. Of the 146 isolated benzophenones only 49, (33%) have been evaluated for their biological activity. This section discusses the various biological activities of isolated benzophenones.

762 Table 5. Reported Biological Activities for Benzophenones Antioxidant Compound 7 13 17 83 88 131

Assay

DPPH (1,1 -diphenyl-2-picrylhydrazyl)

Result ICso = 66.3/iM IC5O = 5.3^M IC 50 = 7.8/jM IC 5 0 >100//M IC50 = 44.1/iM ICso=13.3//M

134

ICsu = 10.2/iM

139 140 131 134

IC a , ICso Weak Weak

134

Compound

86

89 119 93

Peroxide determination Hypoxanthine/xanthine oxidase system Fenton reaction system H 2 O 2 /NaOH/DMSO system Antibacterial Assay/organism Bacillus subtilis Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pneumoniae Candida albicans Paenibacillus larvae Paenibacillus alvei Paenibacillus larvae Paenibacillus alvei Streptomyces chartrensis, Streptomyces violochromogenes

131 132 133 134 138

Methicillin-resistant Staphylococcus aureus (MRSA)

134

Helicobacter pylori

138

Compound 81 86 93

Compound 13 83 131 134 140

Staphylococcus aureus Escherichia coli Streptococcus faecalis Klebsiella pneumonia Antifungal Assay/organism Cladosporium sphaerospermum A range of organisms Candida albicans, Candida tropicalis, Pseudomonas aeuruginosa Antiviral Assay

Epstein-Barr virus early antigen

> 100 > 100 activity activity

Ref. [29] [371 [1021 [371 [37. 115,116] [37] [117]

Suppressed the superoxide, hydroxyl, and methyl radicals

[115, 118]

Result 14.2 ± 0.2 mmh 15.0±0.2mm b 14.4 ±0.1 mmb 14.0 ± 0.2 mmb 14.1 ±0.1 mm' 19.5 mm b 2.3 mm' 19.7 mm" 10.0 mm"

Ref.

50"

[77]

12-25/jg/mL 25/ig/mL* 25/jg/mL" 6.25 - 25/jg/mL 3.1 -12.5/ig/mL

[89]

Complete inhibition at 31.5 /jg/mL at 6h and 3.9 fig/mL at 12h Active Inactive 0.78 fig/mL* 1.56 ^g/mL11

[72]

[74] [74]

[37,119] [87] [120]

Result Inactive Activite

Ref. [1211 [72]

16.2"

[77]

Result

52.7±2.3® 52.7±2.3® 56.0*1.9® 47.0 ±2.5® 54.9 ±2.6®

16nmol 16nmol 16 nmol 16nmol 16nmol

Ref.

[37]

763 Antiviral (cont.) Compound 141 46 53 55 59 132 135 136 137 141 142 143 117 120 121

Compound 33 37 42 47 48 49 50 63 64 69 70 73

Assay

Anti-HIV

Anti-HBeAg secretion on MS-G2 hepatoma cell line Cytotoxic Assay HSC-2 human oral squamous cell HGF normal human gingival fibroblasts HSC-2 human oral squamous cell HGF normal human gingival fibroblasts HSC-2 human oral squamous cell HGF normal human gingival fibroblasts HSC-2 human oral squamous cell HGF normal human gingival fibroblasts

KB cell line

131/133" 136/138" 97 98 108 111 135 144 124 125 88

A2780 human ovarian cells

P388 cell line HeLa human cervix carcinoma Hep-2 human larynx carcinoma PC-3 prostate cancer U251- central nervous system HL-60 leukemia cells

134

Compound 81

K562 leukemia cells NB4 leukemia cells U937 leukemia cells Molluscicide Assay/organism Biomphalaria glabrata

Result 60.1 ±2.1 @16nmol Inactive Inactive EC51, ~ 11 /jg/mL Inactive Inactive ECso = 1 -10/jg/mL EC 50 = 1 -10,ug/mL ECso = 23 ^g/mL EC 5 o=l-lO/ig/mL EC5() = 1 -10^g/mL EC SO =1 -10^g/mL 10,ug/mL 10/ig/mL 10/ig/mL

Ref.

Result CC50 = 0.17mM CCSI, = 0.43 mM CC50 = 0.036 mM CCa, = 0.090 mM CC50 = 0.092 mM CC50 = 0.19mM CC50 = 0.052 mM CC5l, = 0.19mM ECso > 20 ^g/mL EC5IJ > 20 /jg/mL ECso > 20 //g/mL ECso = 2.4 ± 0.9/ig/mL EC51> = 3.3 ± 1.5/ig/mL ECso = 10.0 /ig/mL (inactive) ECso = 9.0 ^g/mL (inactive) EC50 = 8.2 fig/mL (inactive) IC50 = 5.8 fiM IC50 = 10/iM ICso = 7.4±O.2/jg/mL ICso = 8.2 ± 0.2 /ig/mL ICs,, = 8.4±0.6/(g/mL lCso = 6.9±0.3^g/mL ICso = 6.8 ,ug/mL ICso = 8.0 ^g/mL EDso = 6.9/(g/mL ED50 > 30 /jg/mL (inactive) ICso = 3.3 MM \C5a = 3A/iM

Ref.

ICso = 7.2 /iM ICso = 3.9 juM ICso = 9.42 ftM ICso = 5-20 MM ICso = 5-20/;M ICso = 5-20 MM ICso = 5-20 / J M

Result Inactive

[15]

[88]

[17]

[47]

[52]

[62] [90] [78]

[93] [14]

[102] [1221 [82]

Ref. [121]

764 Trypanocidal Compound

Assay/organism

81

Trypanosoma cruzi

135

Epimastigotes Trypomastigotes

Result LC5o = 518 fiM active in vitro, (not in vivo) in mice 100/iM 83 fiM

Ref. [121] [92]

Additional Compound 7 13 17 81 10 49 131/133' 136 138 131 134 138 138

Assay Brine shrimp lethality

Testosterone 5a-reductase DNA strand-scission activity Microtubule disassembly inhibition Apoptosis detected by Western blot analysis

Result LD 5U > 100/JM LD5,, = 43.1 fiM LD 5U > 100,uM LC 50 = 49.7 MM IC 50 > 1.00 mM 43 ± 12% nicked at 2.5 /ig/mL Inactive IC S 0 =1.5//M IC50 = 2 ^ M Apoptosis activation of caspase-3

Male F344 rats using a aberrant crypt foci (ACF) bioassay w/ azoxymethane (AOM)

No CNS effect at 1/5 LD 50 (LD 5 ,,= lOOOmg/kgi.p.) Significantly inhibited AOM induced ACF formation

Apoptosis induction in HL-60 cells

EC5,, = 8.4^M

Cardiovascular effects in cats

Ref. [29] [1211 [35] [52] [90]

[82] [120] [123]

[116] Reduced NO production 49, 87, and 92% at 2.5, 5, and 1 0 ^ M , respectively Ulceration induction in rats by Significantly prevented adverse [118] affects indomethacin "tested as a mixture; bzone of inhibition; ctotal inhibition, dminimum inhibitory concentration, "concentration required to eliminate 99% of organism 134

Griess reaction

A number of benzophenones have been assayed for their cytotoxicity towards ovarian [78,93], leukemia [14,82], and CNS [102] cancer cell lines. In addition, the guttiferones (132 and 135-142) have displayed potent cytotoxicity toward leukemia [82] and ovarian cancer cell lines [37,93]. Garcinol (134), also a guttiferone, has been evaluated for a number of biological activities. Garcinol was found to neutralize the superoxide anion, methyl, and hydroxyl radicals in a variety of antioxidant assays [118] and displayed anti-glycation activity in a fructose-BSA assay [115]. Sang et al. [116] studied the reaction mechanism between the stable free radical l,l-diphenyl-2-picrylhydrazyl (DPPH) and 134. They also isolated the DPPH/garcinol oxidation products [116] and tested 134 in a NO generation, apoptosis, and H2O2 antioxidant assays. Two animal studies have been conducted on garcinol (134). The first study used F344 male rats and administered 134 at 0.01% or 0.05%. Garcinol provided significant in vivo protection against the development

765

of aberrant crypt foci (ACF) [123]. Another study, using male Wistar rats, evaluated a G. cambogia extract against indomethacin induced gastric ulcers [124]. They concluded that G. cambogia extract prevented gastric ulcer formation and maintained the rats at a near normal state, but the bioactive constituents were not identified. However, benzophenones 131 and 134 have been isolated from G. cambogia fruits [91]. An extract of G. cambogia, sold in the US as a dietary supplement, has also been evaluated as a weight loss aide [125]. Hydroxycitric acid, rather then a benzophenone, is believed to be the compound responsible for the weight loss properties of G. cambogia. Other reported activities for 134 include a bactericidal effect on Helicobacter pylori [119], strong cytotoxicity [82,122], and additional antibacterial activities [89]. The guttiferones (132 and 135-142) displayed partial cytoprotection toward HIV-1 infection in human lymphoblastoid CEM-SS cells; however, no decrease in viral replication was observed [88,96]. The vismiaphenones were also assayed for their anti-HIV activity in the NCI primary HIV screen; only 55 was active [15]. Guttiferone A (135) and 7-epi-clusianone (81) were assayed against Trypanosoma cruzi, the etiologic agent of Chagas' disease, and both were found to be active [92,121]. Xanthochymol (138) and guttiferone E (136) displayed outstanding activity in a in vitro microtubule disassembly assay [90]. Kolanone (86), nemorosone II (89), propolone A (93), chamone I (119), and xanthochymol (138) displayed significant antimicrobial and antifungal activity against a variety of pathogenic yeasts and bacteria [72,74,77,120]. Compounds 131-134 and 138 displayed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) at 3.1 - 12.5 ,ug/mL, nearly equal to the antibiotic vancomycin [89]. CONCLUSION Benzophenones are an interesting class of compounds displaying much structural diversity and numerous bioactivities. By far, the richest sources of benzophenones have been Clusiaceae species. However, of the approximately 37 genera and 1610 Clusiaceae species [126], only a few genera/species have been exhaustively analyzed. It is likely that a number of novel bioactive benzophenones are still undiscovered.

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ABBREVIATIONS ACF CH2C12 CHCb COSY DEPT-135 DPPH ESI EtOH geranyl group HMBC HPLC HSQC IR Me2CO MeCN MeOH MS NMR PPB prenyl group TLC VLC

Aberrant crypt foci Methylene chloride Chloroform Correlation spectroscopy Distortionless enhancement by polarization transfer 1,1 -diphenyl-2-picrylhydrazyl Electrospray ionization Ethanol -CH2-CH=C(Me)-CH2-CH2-CH=C(Me)2 Heteronuclear multiple-quantum correlation High-performance liquid chromatography Heteronuclear single-quantum correlation Infrared spectroscopy Acetone Acetonitrile Methanol Mass spectroscopy Nuclear magnetic resonance Polyprenylated benzophenone -CH2-CH=C(Me)2 Thin-layer chromatography Vacuum-liquid chromatography

ACKNOWLEDGEMENTS Scott Baggett was supported by NIH-NCCAM National Research Service Award #F31-AT00062. This research was supported by funds from the NIH-National Institute of General Medical Sciences SCORE award S06GM08225 and the Professional Staff Congress of The City University of New York (PSC-CUNY) award 669662. Kurt Reynertson is thanked for his careful review of this manuscript. REFERENCES

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

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BIOACTIVE COMPOUNDS FROM TRIPTERYGWM WILFORDII RENSHENG XU, JOHN M. FIDLER AND JOHN H. MUSSER Pharmagenesis Inc., Palo Alto, CA 94304, USA ABSTRACT: Tripterygium wilfordii (Celastraceae) is a wild shrub distributed in southern China. It was used in Traditional Chinese Medicine as an insecticide for killing fly larvae 'maggots and Oncomelania snails, a vector of Schistosoma japonicum. In 1969, the decoction of the radix of T. wilfordii was first used in China to treat patients with rheumatoid arthritis (RA); although efficacious, side effects were observed. Subsequently, a partially purified extract called "multiglycoside of the radix" (TWG) was used to treat autoimmune diseases including RA, lupus erythematosus, chronic nephritis and hepatitis. TWG appeared to have fewer side effects compared to the T. wilfordii decoction. In China, TWG is also used to treat various skin disorders, such as psoriasis and leprosy. In an unrelated area, TWG was found to have male spermicidal antifertility activity. Thus far, more than 46 diterpenoids, 20 new triterpenoids, 26 alkaloids and other small molecules have been isolated and identified from the plant. Among them the most potent are triptolide type compounds, which show immunosuppressive, anticancer and antifertility activities. The derivative of triptolide, PG490-88, is being evaluated in phase I clinical trials as an anticancer agent. The natural products structural chemistry of T. wilfordii has been well defined with several total syntheses of triptolide reported.

INTRODUCTION Traditional Chinese Medicine (TCM) is one of the most developed time-honored medicines in the world. It has a long history of use in practice and has been carefully recorded in ancient books. Shennong-Bencao (Shennong herbs) is a compendium book published back to A.D.200 [1]. The characteristics and medical applications of 356 medicines have been described in the book. Now TCMs have been developed to include 10,000 herbs and are popular in China for treating different kinds of diseases. The development of TCM with modern science and technology has allowed the exploration of many new drugs; some of them have

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been acknowledged worldwide. The famous antimalarial artemisinin (qinghausu)[2] and anti-dementia huperzine A [3] are examples. Recently the high efficiency of immunosuppressive activities of Tripterigyum wilfordii (Celastraceae) has attracted the attention of scientists. The herb was recorded early in "ShengnonBencao" as a toxic agent and used as an insecticide for killing fly larvae 'maggots' and oncomelania snails, a vector of Schistosoma japonicum [1,4]. According to TCM, TW radix activates blood circulation and relieves blood stasis, counters inflammation and relieves edema, purges internal heat and eliminates toxins. In 1969, the decoction of the radix was first used in a hospital in Fujian to treat rheumatoid arthritis (RA) patients and showed a high level of efficacy [16], but side effects were also observed. Later, the purified extract of xylem from its roots, the so-called "Reigongten-doudai" (multiglycoside of the herb, TWG), was developed as an over-the-counter drug popularly used in China to treat various immunosuppressive diseases (e.g., RA, lupus erythematosus, chronic nephritis and hepatitis) [5]. Studies on the plant further characterized its biological activities and chemistry. The following is a summary of these studies, with a focus on the chemistry of compounds extracted from Tripterygium wilfordii.

EXTRACTS OF THE TW PLANT AND THEIR BIOACTIVITIES There are several forms of purified extract reported in the literature: TWG/T2, EA and PG27. For TWG/T2, the xylem of the roots was extracted by water, then the chloroform extraction of the water extract was passed through silica gel columns, eluted with chloroform and subsequently with chloroform with 10% alcohol, and the later elution was concentrated, dried and used as TWG [6]. EA was the ethyl acetate extract of the xylem[7]. For PG27, the roots were extracted with alcohol, and the concentrated dichloromethane extract of the evaporated alcohol extract was passed through the silica gel column and eluted with CH2CI2 and CH2CI2: MeOH 95:5; the concentrated and dried late elution

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fractions were used as PG27 for bioassays [8]. All of these extracts show immunosuppressive activity in vitro and in vivo. TWG inhibited Concanavalin A-induced proliferation of mouse spleen cells and thymocytes in a dose-related manner. The expression of interleukin-2 (IL-2) receptors by Concanavalin Aactivated spleen cells was completely inhibited by adding TWG, but IL-2 production was not completely suppressed [9]. T2 at 0.1-1 |ig/ml inhibited antigen- and mitogen-stimulated proliferation of T cells and B cells, IL-2 production by T cells, and immunoglobulin production by B cells. T2 did not affect IL-2 receptor expression by T cells, IL-1 production by monocytes, or the capacity of monocytes to present antigen. Inhibition could not be accounted for by nonspecific toxicity. These results supported the conclusion that T2 exerts a powerful suppressive effect on human immune responses, and this action might account for its therapeutic effectiveness in RA [10]. The immunosuppressive activity of PG27 was demonstrated by prolonging rat heart and kidney allograft survival. PG27 administered intraperitonially to Lewis recipients for 16 days at 1030 mg/kg/day significantly increased the median survival time of Brown Norway heart allografts from 7 to 22 days. Oral administration was also effective, in prolonging the cardiac allograft survival time to > 100 days when PG27 treatment was given for 52 days. At a dosage of 20-30 mg/kg/day, PG27 significantly extended the median survival time of Brown Norway kidney allograft recipients from 9 up to 77 days, and 30 mg/kg/day of PG27 for 52 days extended survival beyond 200 days. PG27 combined with cyclosporin (CsA) significantly enhanced heart and kidney allograft survival, even at doses of CsA that were ineffective when administered alone, providing evidence of synergy between PG27 and CsA. PG27 combined with CsA substantially prolonged hamster-to-rat cardiac xenograft survival, again showing synergy, as well as completely inhibiting xenoantibody production. PG27 also suppressed graft-versus-host disease in murine allogeneic bone marrow transplantation [6,11,12]. TWG exhibited a significant anti-inflammatory effect on acute agar-induced edema of the rat paw and suppressed carrageenaninduced inflammation in vivo. The number of exudate cells and the concentration of Prostaglandin E2 (PGE2), nitrite and TNF-oc in the

776

exudates obtained from TWG treated animals were significantly reduced (by 69%, 78%, 57% and 77%, respectively), compared to that from vehicle-treated animals. Cyclooxygenase-2 mRNA was markedly suppressed in the air pouch lining tissue of TWG treated rats (p > 0.001). In contrast, PGE2 content of the kidney and stomach, and the production of PGE2, nitrite and TNF-a by spleen cells, were not affected by treatment with TWG [5, 13]. TWG has an anti-spermatogenesis action similar to that of gossypol. The fertility of Wistar rats given 10 mg/kg of TWG daily was reduced after four weeks of treatment and completely lost after treatment for eight weeks. The spermatocyte density and activity was remarkably reduced (P < 0.01), but neither the organs nor sexual activity changed during treatment. There were degenerative changes in the seminiferous tubules and sperm of male rats when they were treated with TWG at 30mg/kg for 35 and 80 days. The number of spermatocytes was also subsequently reduced as well as its susceptibility [14,15]. In 1983, Yu reviewed 144 cases of clinical use of TWG to treat rheumatoid arthritis. The total effective rate was 93.3% (17.6% remission, 37.5% effective and 38.2% improvement). The course of treatment was three months with an oral dose of 1-1.5 mg/kg, and the maximum daily dose was 90 mg given in three doses. Side effects included gastrointestinal disturbances, irregular menstruation and amenorrhea in female patients and gynecomastia in males. Side effects subsided after withdrawal of the drugs [5]. Another report showed various degrees of improvement by TWG treatment in 24 of 26 cases of lupus erythematosus (92.3%)[16]. TWG 1 mg/kg/day orally was used to replace azathioprine in tripledrug therapy (CsA, prednisone and azathioprine) for 10 renal transplant recipients; all of the recipients have been examined up to 3-10 months with good renal function. Side effects including mild gastrointestinal reaction were acceptable. It was concluded that TWG was more effective than azathioprine [17]. Recently similar clinical reports appeared in the literature [18 -20]. CHEMICAL COMPONENTS OF THE TW PLANT Kupchan et. al., were first to study the chemical components of the plant. Three new diterpenoids, triptolide 1, tripdiolide 2 and

777

triptonide 4 were separated from the extract of the roots, collected from Taiwan. All of them have three specific epoxides and an a,punsaturated y-lactone ring in their structures. The 14-P-hydroxyl group in triptolide and tripdiolide is hydrogen bonded to the C-9,11 epoxide oxygen atom. The authors cited this relationship to explain the antileukaemic biological activity of these compounds[21 - 23]. Since then 46 diterpenoids have reportedly been separated from the plant or its variety, growing in Japan, Tripterigium wilfordii Hook fil. var. regelii Makino. Among them 12 (compounds 1-12) belong to the triptolide type, 21 (16-36) are of the triptophenolide type and 10 (37-46) belong to the triptoquinone type (cf. table 1 & fig.l)[2144]. Table 1.

New Diterpenoids from TW plant. Name

Formulae

M.P. ("C)

(<X]D (solvent)

References

1

Triptolide

C 2 0 H24O 6

226-227

-154°(CH 2 C1 2 )

21

2

Tripdiolide

C20H24O7

210-211

-138°(CH 2 CI 2 )

21

3

Tripterolide

C 2 oH 24 0 7

225-228

4

Triptonide

C 20 H 22 O 6

251-252

5

Triptolidenol

C20H24O7

193-194

25

6

16-OH-triptolide

C20H24O7

230-232

27

7

Tripchloride

C20H25O6CI

230-231

26

24 -175°(CH 2 C1 2 )

21

8

Triptotetraolide

C2oH26Og

258-260

28

9

Isotriptetraolide

C 2 oH 2 fi08

250-252

29

10

Triptriolide

CMH26O7

260-262

30

11

Tripdioltonide

C 2 oH 2 40(i

222-224

26

12

13,14-epoxide-9, 11,12-trihydroxytriptolide

C 20 H 26 O 7

268-270

26

13

Tripterifordin

C20H30O3

255-256

-46.6°(CHC13)

31

14

Tripterinin

CMH30O4

244-245

1.86°(CHCI 3 )

32

15

13-t77/'-manoyl oxide18-oic acid

C20H32O3

33

16

Triptonolide

C 20 H 22 O 4

34

778 able 11. Contd....)

Name

Formulae

M.P. (°C)

17

Triptophenolide (Hypolide)

C 2 oH 24 03

232-234

35

18

Triptophenolide methyl ether

C 2 ,H 2 6 O 3

152-154

35

[<X1D (solvent)

References

19

Neotriptophenolide

C2,H26O4

189-191

35

20

Isoneotriptophenolide

C2IH26O4

185-187

36

21

Triptonoterpene

C20H28O2

153-155

22

Triptonoterpene methyl ether

C21H30O3

209-211

189.5 (MeOH)

25

23

Neotriptonoterpene

C 2 ,H 30 O 3

205-207

95.1° (MeOH)

38

24

Triptobenzene A*

C 2 oH 2 80 3

amorphous

89.3° (CHCI3)

39

25

Triptobenzene B*

C 2 oH 3 o0 2

amorphous

18.3° (MeOH)

39

26

Triptobenzene C*

C20H26O4

amorphous

-7.4° (CHCI3)

39

27

Triptobenzene D*

C20H26O2

174-177

48.1°(CHC13)

39

28

Triptobenzene E*

C 2 oH 24 04

amorphous

29

Triptobenzene F*

C20H24O4

amorphous

39.0°(CHCl3)

39

30

Triptobenzene G*

C20H24O4

amorphous

31.7°(CHC13)

39

31

Triptobenzene H

C 2 iH 2 8 O 4

amorphous

171°(CHCI3)

40

32

Triptobenzene 1

C20H24O4

amorphous

33

Triptobenzene L

C20H30O2

amorphous

34

Triptobenzene M

C20H28O3

35

Triptobenzene N

C 2 oH 2 60 3

36

Triptonoterpenol (Triptonodiol)

C 2 iH 3 o0 4

210-212

37

Triptoquinone A*

C20H24O4

179-182

128°(CHC13)

42

38

Triptoquinone B*

C20H26O4

amorphous

167"(CHO13)

42

39

Triptoquinone C*

C20H28O4

amorphous

-63" (CHC13)

42

40

Triptoquinone D*

C 2 oH 2 g0 3

amorphous

26.1°(MeOH)

42

41

Triptoquinone E*

C 2 oH 2 60 3

121-123

30.3° (MeOH)

42

42

Triptoquinone F*

C 2 oH 2 60 4

207-209

80.5° (CHCh)

42

43

Triptoquinone G*

C 2 oH 2 60 5

amorphous

35.0° (CHC13)

42

25

39

40 30.4° (MeOH)

41

amorphous

42.4° (MeOH)

41

amorphous

-42.3° (MeOH)

41 38,41

779 (Table 1. Contd....) Name

Formulae

M.P. (°C)

[<X|D (solvent)

References

44

Triptoquinone

C 20 H 22 O 4

55-58

121.6°(CHCI3)

43

45

Triptotin A

C 20 H 22 O 6

187-188

68.7°(CHC13)

44

46

Triptotin B

C20H26O6

amorphous

91.8°(CHC13)

44

* Isolated from Tripterygium wilfordii var. regelii Mankino R2

14

1 2 3 4 5 6

H H H H OH H

CH3 CH3 CH3 CH3 CH3 OH

P-OH H P-OH p-OH P-OH a-OH =0 H p-OH H p_OH H

Ri

R2

21

H

OH

22

H

OCH3

23 OH

0CH3

OH

24 Ri=O; R2 = CH2OH; R3=2H OH

28 R, = OH; R2 = CH3

25 R i = < n . R2 = CH3; R 3 = 0

29 R, = H; R2-CH2OH

26 R t - 0 ; R2-CH2OH;

30 epimerof29

R3-0

780 (Fig. l.Contd....) HO,

OH

HOOC

H OH

\

1

1=*6

R, R; R3 R4 R5 R

32

33 P-OH H H 34 0 H OH 35 0 H H 36 0 OH H

H H2 OH H H2 OH H 0 OH OMcH, OH

HOOC

Fig. 1. Structures of new diterpenoids from TW plant (1).

There are about 20 alkaloids separated from the herb. Most of them are three-ring sesqueterpenes linked with a pyridine through two ester groups. Many of these alkaloids have immunosuppressive activities {c.f. table 2 & fig. 2) [45-55].

781 Table 2.

Alkaloids from TW plant Name

Formulae

M.P. (°C)

[<x]o (Solvent)

References

47

Wilfordine

C43H4,O,8N

175-176

5° (CHC13)

45

48

Deacetylwilfordine

C 4 ,H4,Oi 8 N

182-184

49

Wilforine

C 43 H 4 ,O, 8 N

170-171

30°(Me 2 CO)

45

50

Deacetylwilforin e(Wilforzine)

C 4 ,H 47 O 17 N

177-178

6° (Me 2 CO)

47

51

Wilfornine (2Nicotinylwilforine)

C 42 H 4g Oi 8 N 2

187-189

52

Wilforgine

C 4 iH, 7 Oi,N

211-212

53

Wilformine (Euonine)

C 38 H 47 O 18 N

155-157

45

54

Wilforjine (Deacetyl wi 1 formine)

C36H45O17N

156-158

22

55

Wilfortrine

C 4 ,H 47 O 20 N

235-237

56

Deacetylwilfortrine

C 39 H 45 O,,N

179-182

48

57

Wilforidine

CWR.SO.KN

176-178

49

58

Wilfordlongine

Cj(,H45Ol8N

179-181

50

59

Isowilfortrine

C 4l H4 7 O 20 N

329-331

51

60

Wilfordconine

C 4 ,H 47 0 2 oN

192-193

52

61

Neowilforine

C 43 H,9O| 7 N

156-157

62

Celafiirine

C 2 ,H 27 O 3 N 3

154-155

-1P(CHC1 3 )

54

63

Celacinine

C 25 H,,O 2 N 3

203-204

-19°(CHC1 3 )

54

64

Celabenzene

C 23 H 2 ,O 3 N 3

156-158

0° (CHC13)

54

65

Wilforcidine

C 36 H 38 O 8 N 2

188-189

55

66

Triptofordinine A-1 *

C41H43O12N

193-194

56

67

Triptofordinine A-2*

C 4 ,H 43 O, 2 N

94-95

56

48

45,48

25° (Me 2 CO)

10°(Me 2 CO)

45,48

46,47

53

* isolated from Tripterygium wilfordii var. regclii Mankino

Approximately 20 new triterpenoids {cf Fig. 3) have been separated from the TW plant or its variety {cf 5-7) [56-65]. Among them, tripterine or celastrol type compounds can be separated from other celastrus species of the

782

R4

AcO Benzoyl Benzoyl Benzoyl Benzoyl Nicotinyl Furanoyl Ac H Furanoyl Furanoyl H

R,0

Ac Ac Ac H Ac Ac Ac Ac Ac Ac Ac

OH OH H H H H H H OH

OH OH

.OCOQH;

58 R, = Ac; R2 = H 59 R| = Ac R2 = Furanoyl 60 R, = Fuanoyl R 2 =H

64 R =

67

R=-

Fig. 2. Structures of alkaloids from TW plant.

same family of plants. Celastrol type compounds show immunosuppressive and anti-inflammatory activity. In addition, sesquiterpenoids (cf. Fig. 4) are reported to be separated from the TW plant and its variety [66-68] (c.f. fig. 8). Among all of the components isolated from the TW plant, the most potent compounds, with immunosuppressive, anti-

783

HO :::|-|3 in the presence of CF3CH2OH at -5°C afforded 118 and its diastereomer in a significantly higher ratio 38:1 as well as higher yield (ll%){cf. fig. 8). BIOACTIVITY OF CHEMICAL COMPONENTS FROM TW PLANT Tao et al. [80] have described the immunosuppressive components in TWG or T2 and EA as triptolide 1 and tripdiolide 2. The concentration of each compound in the extract was 0.36 (ig of 1 and 0.68 |lg of 2 in 1 mg of T2 and 1.08 ng of 1 and 0.31 |ig of 2 in 1 mg of EA. Fidler et al. [11,12] indicated that 1 (PG490 and its semisynthetic succinate ester prodrug of 1, PG490-88), as well as PG27, was efficacious as an immunosuppressive agent and prevented allograft rejection after cardiac transplantation. PG27 contained 0.36 % of 1. PG490-88 at 0.335 mg/kg/d significantly increased cardiac allograft mean survival time from 7 to 47 days, and significantly increased cardiac allograft survival when used in combination with CsA. Yang et al. [81] demonstrated 1 is the major active component in the plant extract. It induced caspasemediated apoptotic death of T cell hybridomas and peripheral T cells, but not thymocytes. In a comparison of several epoxide

792

Fig. 8. Schema of enantioselective total synthesis of triptolide.

diterpenoids extracted from the herb, the immunosuppressive activity order according to the therapeutic index (TI) was: triptolidenol 5 (30.7)> tripchlorolide 7 (16.7) > 16-hydroxytriptolide 6 (15.8)> triptolide 1 (13.7) > tripdiolide 2 (8.8)> triptonide 4 (7.5) [82]. In the same study, the anti-inflammatory activity order was: triptriolide 10 (>19) > 1(17) > 5(9.6) > 7 (9.0) > 2 (7.3) > 6 (6.6) > 4 (5.9).

793

Triptolide 1 (PG490) inhibits early cytokine gene expression in Jurkat T cells, effectively suppresses T lymphocyte activation, and is a potent inhibitor of interleukin (IL-2) transcriptional activation [83,84]. Compound 1 blocks IL-2 transcriptional activators Nuclear Factor of Activated T-cells (NF-AT) and Nuclear Factor-KB (NF-xB) in Jurkat cells. However, 1 inhibits the stimulation-dependent enhancement of DNA-binding activities at the NF-AT, but not at the NF-/tB, target DNA sequence and thus inhibits transactivation but not DNA binding of NF-*B. 1 inhibits NF-xB transcriptional activation without inhibiting nuclear NF-*B DNA-binding activity in a variety of cell types [82-86]. Inhibition of Activator Protein 1 (AP-1) transcriptional activation by 1 [85] involves inhibition of AP-1-specific DNA binding activity, similar to that observed for binding to the NF-AT target DNA sequences [82]. T-cell activation involving CD28 costimulation is sensitive to inhibition by 1, but resistant to CsA and FK506. The capacity of 1 to suppress costimulation-triggered T cell activation is consistent with the use of triptolide-based treatments in ailments that are resistant to CsA treatment and are known to involve T lymphocyte costimulation. Among these states are GVHD, systemic rheumatic and other autoimmune diseases, and allotransplant rejection [8792]. In 1981, Zhang et al. [93] first examined the antitumor activity of 1. Compound 1 injected i.p. at 0.25 mg/kg markedly prolonged the survival time of L6i5-bearing mice (by more than 140%) and inhibited the growth of S37, HCS and W256 by 38%, 47% and 51%, respectively, at 2 mg/kg. Recently it was reported that 1 inhibited the proliferation and colony formation of tumor cells at extremely low concentrations (2-10 ng/ml) and was more potent than taxol [94]. In vivo, treatment of mice with 1 for 2-3 weeks inhibited the growth of xenografts formed by four different tumor cell lines (B16 melanoma, MDA-435 breast cancer, TSU bladder cancer and MGC80-3 gastric carcinoma), indicating 1 has a broad spectrum of activity against tumors that contain both wild-type and mutant forms of p53. In addition, 1 inhibited experimental metastasis of B16F10 cells to the lung and spleen of mice. Importantly, tumor cells that were resistant to taxol attributable to the over expression of multidrug resistant gene 1 were still sensitive to the effects of triptolide [94]. PG490-88, the ester prodrug of 1,

794

retarded growth and caused regression of nude mouse xenograft tumors from a range of human tumor cell lines (H23 non-small cell lung cancer, COLO205 and HT-29 colon carcinoma, and PC-3 and DU145 prostate cancer) with both wild-type and mutant forms of p53 [95 and unpublished observations, Fidler J.M.; Li K.]. Compound 1 blocked NF-KB activation and sensitized tumor necrosis factor (TNF-oc)-resistant tumor cell lines to TNF-ccinduced apoptosis [85]. Compound 1 acted in synergy with CPT-11 (irinotecan), a topoisomerase I inhibitor, by blocking the S/G2/M arrest induced by CPT-11. As with doxorubicin, inhibition of p21 induction by CPT-11 drove cells into apoptosis rather than growth arrest [94]. PG490-88 enhanced the efficacy of the chemotherapeutic agents taxol, vinorelbine, 5-fluorouracil and CPT-11 and demonstrated enhanced anti-tumor activity with nonsmall cell lung cancer, colon carcinoma, prostate cancer and fibrosarcoma in nude mouse tumor xenografts [94 and unpublished observations, Fidler J.M.; Li K.]. Moreover, PG490-88 acted in synergy with CPT-11 to cause tumor regression [95]. PG490-88 is being evaluated in phase I clinical trials [96]. In cells treated with 1, the expression of p53 increased but the transcriptional function of p53 was inhibited. Additionally, 1 induced accumulation of cells in S phase, blocked accumulation of cells in G2/M induced by the topoisomerase II inhibitor doxorubicin, and prevented doxorubicinmediated induction of p2i wafl/cipl ; a p53-responsive gene. These data suggested that 1 enhanced apoptosis in tumor cells by blocking p21-mediated growth arrest [97]. 1 had dose-dependent effects on both normal and cancer-derived primary cultures of human prostatic epithelial cells. Whereas low concentrations of 1 inhibited cell proliferation and induced a senescence-like phenotype, higher concentrations induced apoptosis that was unexpectedly associated with nuclear accumulation of p53. Paradoxically, levels of the p53 target genes, p 21 wafl/cipl and hdm-2, were reduced, as was bcl-2. It was suggested that 1 might be an effective preventive as well as therapeutic agent against prostate cancer and might activate a functional p53 pathway in prostate cells [98]. In a different therapeutic area, compound 13 showed anti-HIV replication activity in H9 lymphocyte cells with an EC50 of 1 (ig/ml [31]. Many of the diterpene quinoid compounds showed significant inhibitory activities for IL-loc and IL-1(3 release from

795

LPS-stimulated peripheral blood mononuclear cells compared with the reference compound prednisolone. The inhibition by compounds 37-43 was 30-70% at a concentration of 1 x 10"5 g/ml, and for prednisolone at 3 x 10"7 g/ml was 87% for IL-la and 76% for IL-lp. At the same concentration triptophenolide compounds did not show any inhibition [42]. When several diterpenoids and triterpenoids, isolated from the herb, were examined for antifertility activity, 1 showed the most potent in vitro spermicidal activity [99]. In contrast, 4 had the most potent male in vivo antifertility effect in the modified MB-50 assay, and it was therefore suggested that 4 might be a good candidate to be developed into a male antifertility agent. The triterpenoid tripterine (celastrol) is reported to have anti-inflammatory and immunosuppressive activities. Celastrol inhibited the response of mouse splenocytes to SRBC both in vitro (at 0.1 - 1.0 |ig/ml there was a 27.1-75.3% inhibition) and in vivo (at 1-2 mg/kg qd x 6, there was a 30.9-44.3% inhibition) with either primary or secondary stimulation. This compound was also found to significantly inhibit cotton pellet-induced granuloma growth in rats and to depress the delayed-type hypersensitivity (DTH) reaction of mouse skin to dinitrochlorobenzene [100]. Celastrol showed potent inhibition of LPS-induced NF-KB activation in murine macrophage RAW264.7 cells transfected with an NF-KB reporter gene construct and on nitric oxide (NO) production in LPSstimulated RAW 264.7 cells. The IC50 value for NF-KB activation was 0.27 uM and for NO production was 0.23 uM [101]. In low nanomolar concentrations, celastrol was found to suppress the production by human monocytes and macrophages of the proinflammatory cytokines TNF-a and IL-p\ Celastrol also decreased the induced expression of class II MHC molecules by microglia. In macrophage lineage cells and endothelial cells, celastrol decreased induction but not constitutive NO production. Low doses of celastrol administered to rats significantly improved their performance in memory, learning and psychomotor tests. The potent antioxidant and anti-inflammatory activities of celastrol and its effects on cognitive functions suggest that the drug may be useful to treat neurodegenerative diseases accompanied by inflammation, such as Alzheimer's disease [102].

796

Most of the sesqueterpene alkaloids showed depressant effects on humoral immunity using the hemolysin reaction as an index. Wilfortrine 55 (160 mg/kg mg/kg/d x 9d, i.p.) suppressed the graft-vs.-host reaction (GVHR). 53 (80 mg/kg/d x lOd, i.p.) showed marked inhibitory effects on the DNCB-induced DTH reaction on mouse skin. Both alkaloids (80 mg/kg/d x 4d, i.p.) significantly decreased the clearance rate of charcoal particles and the weights of spleen and thymus [103]. However, the potency of the suppressive activities was not comparable with that of triptolide diterpenoids TISSUE CULTURE AS A SOURCE OF TRIPTOLIDE To avoid the limitation of plant sources, several groups initiated studies of tissue culture from the TW plant as a source of triptolide 1. Kutney's group first studied TW tissue culture. The shake-flask and stirred fermenter batch cultures of TW cell tissue line TRP 4a grown on modified PRL-4 medium were harvested after 6 weeks. Cell cultures were extracted and separated by column chromatography. Compounds 1 and 2 were separated in yields that were 3 and 16 times greater, respectively than those observed in the plant itself. Additionally, dehydroabietic acid, celastrol, oleanoic acid, polpunonic acid, p-sitosterol and other compounds were separated from the culture [104]. Zhu and his group cultivated tissue induced from TW leaves and stems, on the 67-v medium containing 0.1 mg/L, 2,4-D and 0.1 mg/L KT for 7 weeks. After separation of the cultivated material, the yield of 1, 2 and 4 was 12 times more than that in the plant (9.184 mg/L) [105]. CONCLUSION The chemistry of components from the TW plant has been well characterized. The biological activity of these components is under study for the development of clinically useful therapeutic agents. Among the components, triptolide type compounds are being developed for treatment of autoimmune diseases as well as cancer, and other components such as celastrol-type compounds and alkaloids also have prospects for further development. All of these are examples derived from the investigation of Traditional Chinese

797

Medicines, where there is a wealth of promising compounds. Further exploration with modern scientific technology will lead to the discovery and development of even more useful medicines to treat cancer and other diseases in the future.

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Sheng-nong Bencai-Jing (Compendium of Chinese Herbs), Publisher of Ancient Books of Traditional Chinese Medicine, Beijing, 1982; 309310. Balint G.A., Pharmacology & Therapeutics, 2001, 90, 261-265. Zhang S.L., New Drugs & Clinical Remedies, 1986, 5, 260-266. Nanjing College of Pharmacy; Zhongcaoyao Xue (Chinese medicines), Jiangsu People Publisher, Nanjing, 1976, 624-626. Yu D.Y.; J, Trad. Chin. Med. 1983, 3, 125-129. Xia Z.L.; Deng F.X.; Chin. J. Pharm., 1994, 25, 136-137. Tao X.; Ma Y.M.; Lipsky P.E.; Inflamm. Res., 1999, 48, 139-148. Wang J.; Xu R.S.; Jin R.L.; Chen Z.Q.; Fidler J.M.; Transplantation, 2000, 70, 447-455. Kuang Y.D.; Zhang H.; Qing W.Z.; Shanghai J. Immunology, 1988, 8, 250-253. Tao X.; Laurie S.D.; Lipsky P.E.; Arthritis & Rhematism, 1991, 34, 1274-1281. Wang J.; Xu R.S.; Jin R.L.; Chen Z.Q.; Fidler J.M.; Transplantation, 2000, 70, 456-464. Fidler J.M.; Ku G.Y.; Piazza D.; Xu R.S.; Jin R.L.; Chen Z.Q.; Transplantion, 2002, 74, 445-457. Zheng J.R.; Xu L.F.; Wang D.H.; Gao J.W.; Ada Academiae Medicinae

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

803

BIO ACTIVE NATURAL COMPOUNDS FROM MEDICO-MAGIC PLANTS OF BANTU AREA BLANDINE AKENDENGUE1*, GUY JOSEPH LEMAMY1, HENRI BOUROBOU BOUROBOU2, ALAIN LAURENS3 1

Departement de Pharmacologie, Faculte de Medecine, Universite des Sciences de la Sante, B.P. 7464 Libreville, Gabon. 2 Centre National de Recherche Scientifique et Technologique, Herbier National du Gabon, B.P. 13354, Libreville, Gabon. Laboratoire de Pharmacognosie, UMR 8076 CNRS, Faculte de Pharmacie, Universite Paris XI, rue Jean-Baptiste Clement, 92296 Chatenay-Malabry, France. ABSTRACT: Ethnobotanical studies conducted in three Bantu countries, namely Central Africa, Gabon and Equatorial Guinea, led us to collect, among other medicinal plants, eighteen medico-magic plants belonging to thirteen families. Seven are trees (Distemonanlhus benthamianus, Erythrophloeum guineense, Guibourtia tessmanii, Monodora myristica, Musanga cecropioides, Polyalthia suaveolens, and Tetrapleura tetraptera), four are shrubs (Lippia multiflora, Microdesmis zenkeri, Tabernanthe iboga, and Securinega microcarpd), six are herbs {Aframomum stipulation, Costus afer, Costus lucanusianus, Pennisetum purpureum, Piper umbellatum, and Scoparia d ulcls) and two are liana {Adenia gracilis, and Adenia lobata). These plants are used for various activities such as antalgic, febrifuge, aphrodisiac, antiparasitic, neurotonic, emetic or oxytocic. Active compounds obtained from some of these medico-magic plants are reviewed. Among these active molecules are indole alkaloids such as ibogai'ne, tabernanthine and voacangine from Tabernanthe iboga; isoquinoline alkaloids such as oliverine from Polyalthia suaveolens; diterpenoids such as scopadulcic acid B, scoparic acid A and scopadulin from Scoparia dulcis; triterpenoids such as aridanin from T. tetraptera. Other natural active compounds isolated are coumarins, flavones, saponins and benzoxazolinones. These products showed various biological activities such as antiviral, antimicrobial, antiparasitic, cytotoxic, stimulating of central nervous system, analgesic, anti-inflammatory, H+, K+-ATP-ase inhibitor, (3-glucuronidase inhibitor, hypotensive and bradycardisant. Keywords: bioactive, medico-magic, Bantu

INTRODUCTION The Bantu are the people in Africa who call a human being a Muntu (singular; plural: Bantu) in their language. They live in an area that • : correspondence to Blandine Akendengue ([email protected])

804 extends from the Equator all the way to the Cape of Good Hope in the south. The common root of Bantu languages has been demonstrated [1,2]. Medicinal plants used by Bantu people have been reported herein [3,4]. The Bantu regard bad luck in life, marriage problems, childlessness, bad crops, d eterioration o f 1 iving c onditions, p rofessional p roblems, s uch a s illness, just as well as diseases which require treatment, like fever or tuberculosis. Plants are used in all these contexts to resolve problems and to cure. The sign of God is looked for in the plant, whether it is called upon for help or is kept for strengthening by the spirits; it is personalised and regarded as a treasure. Man lives in harmony with his remedies. In accordance with his cultural conception, he creates reciprocal relationships between him and the plant. These relationships become stronger during illness and weaker in times of health but never disappear. Medico-magic plants include also the esoteric part. They are not just consumed and thrown away when they seem useless. Rather, their use is subject to rules and regulations marked by the typical characteristics of magic: manipulation of certain materials, which can be learned and is backed by the firm conviction to be able to achieve or change something for the benefit of the user. There are procedures in all Bantu societies where the same plant can be used both as remedy and magic. Bantu medicine displays some magical character. The administration of medicomagic plants follows a defined ritual and above all, for the healer and the patient, it is clear that the efficiency of these magic plants is conditioned by the respect of rituals. We registered various ethnic groups' concepts of the effects and uses of medicinal plants in a cultural and social context. These species, which have therapeutic properties, can be used in specific rituals (Aframomum stipulatum, Tabernanthe iboga), or to free someone from a magic spell (Guibourtia tessmanii, Polyalthia suaveolens) or bring luck (Scoparia dulcis). G. tessmanii, especially, is an endemic big tree of primary big forests of Cameroon and Gabon [5]. T. iboga, the most famous in Gabon, is prized by the natives for its hallucinogenic and aphrodisiac properties. Traditional healers use iboga for its stimulating properties to enhance their psychic powers, increase inspiration and stave off fatigue. The strength of this study is that it combines both fieldworks and results from scientific investigations of listed plants which include biological activities of extracts and active compounds. Traditional uses of plants and extracts activities are presented, and active products obtained from some of these plants are reviewed. Fieldworks have been conducted among traditional healers in Central Africa (Bambari and Bangui areas) and in Bata and Malabo areas of Equatorial Guinea [3] by one of us (B.A.). Identification of plants was made via vernacular names and with the help of botanists. In Gabon, ethnobotanical studies have been led in the Estuaire and Ngounie provinces by the same author and by H.B.B. who also identified the plants. Eighteen medico-magic plants have been collected. Nevertheless,

805

all Bantu medico-magic species are not presented in this paper. Traditional uses are listed in Table 1. Some plants have been quoted for treating different diseases. Five species have been recognised to possess a cicatrizant activity (Costus afer, C. lucanusianus, Erythrophloeum guineense, Musanga cecropioides, and Piper umbellatum). Costus species seem to be known also for their pectoral properties. Four plants are known for their antalgic activity (A. stipulatum, Monodora myristica, Musanga cecropioides, P. umbellatum), whereas three are febrifuge (Lippia multiflora, P. umbellatum, Tetrapleura tetraptera). Two plants are aphrodisiac; among them T. iboga is widely used as hallucinogen during rites and as a tool by hunters to increase awareness and allow them to remain very still for prolonged periods. The traditional healers also showed antihelmintic, antimalarial, antirheumatic, antifungal, emetic and oxytocic plants. The other cited diseases are cold, dermatosis, otitis, epilepsy, and gonorrhoea. The majority of listed plants showed a biological activity; some activities being associated with the empirical use. Lyophilizated preparations of C. lucanusianus showed a uterine relaxant activity producing a complete inhibition of oxytocin-induced contractions. This uterine relaxant activity was reversible. Barium chloride-induced contractions were not modified [6,7]. Chewing sticks of Distemonanthus benthamianus induced improvement of children's gingival health and oral hygiene during a clinical study [8]. The extracts and essential oil of L. multiflora showed in vitro antimalarial activities against FcBl chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum. The essential oil inhibited the growth of parasite by 50%, 24 and 72 hours after administration at dilutions of 1/12,000 and 1/21,000, respectively. Tested on a highly synchronised culture, the essential oil inhibited growth mostly at the trophozoite-schizont step, indicating a potential effect on the first nuclear division of the parasite [9,10]. The essential oil of L. multiflora also showed pediculocidal and scabicidal activities against body lice, head lice and scabies' mites [11] as well as antibacterial activity. The Gram-negative bacteria were the most sensitive [12].

806 Table 1. Bantu medico-magic plants use Family and specie

Use (route)

Annonaceae Monodora myristica Dun.

Constipation, headaches (Ea)

Polyalthia suaveolens Engl. et Diels

Antiparasitic, anti-inflammatory (E)

Caesalpiniaceae Distemonanthus benthamianus H. Baill.

Dermatosis (E)

Erythrophloeum guineense Don.

Cicatrizant (E)

Guibourtia tessmanii (Harms) J. Leonard

Antiseptic (E)

Apocynaceae Tabernanthe iboga Baill.

Neurotonic, hallucinogen, aphrodisiac (Ib)

Crassulaceae Kalanchoe crenala Haww.

Otitis, cold, antifungal (E), epilepsy (1)

Euphorbiaceae Microdesmis zenkeri Pax.

Oxytocic (E)

Securinega microcarpa (Blume) Pax et H.

Aphrodisiac (1)

Graminae Pennisetum purpureum Schum.

Dermatosis (E)

Mimosaceae Tetrapleura tetraptera Taub.

Febrifuge, antirheumatic (E), emetic (I)

Moraceae Musanga cecropioides R. Br. Tedlie

Antalgic, oxytocic, cicatrizant (E)

Passifloraceae Adenia gracilis Harms

Medico-magic (E)

807

Adenia lobata Engler

Cold (E)

Piperaceae Piper umbellatum L.

Antalgic, febrifuge, cicatrizant (E)

Scrophulariaceae Scoparia dulcis L.

Emetic, antihelmintic, oxytocic, gonorrhoea (I)

stomach

disorders,

Verbenaceae Lippia multiflora Mold.

Febrifuge, antimalarial (E)

Zingiberaceae Aframomum stipulatum K . Schum

Antalgic , tonic (E)

Costus afer Ker-Gawl.

Antirheumatic, cicatrizant (E), pectoral (1)

Costus lucanusianus J. Braun et K. Schum

Antihelmintic, pectoral (I), cicatrizant (E)

" E: external I : internal b

The essential oils from the leaves of A. stipulatum and that of the seeds of M. myristica also exhibited in vitro antibacterial activities. Tested by the diffusion method, the both essential oils (5 ul per disc) inhibited the growth of bacteria with a 10-18 mm zone diameter of inhibition area [13]. Alcoholic extract of T. tetraptera also showed antibacterial activities with an MIC of 4mg/mL against Pseudomonas aeruginosa and Staphylococcus aureus, whereas aqueous extract only inhibited the growth of S. aureus [14]. T. tetraptera extract also exhibited molluscicidal activities against Bulinus globosus, Schistosoma mansoni, S. bovis, and Lymnaea natalensis [15-18]. An anticonvulsant activity was observed with the fruit volatile oil of T. tetraptera. Intraperitoneally (i.p.) administered in mice, T. tetraptera oil (0.4 mL per mouse) protected 78 % of them against leptazol-induced convulsions [19]. Analgesic effects were observed with aqueous extracts of P. umbellatum and M. cecropiodes. Administered in rats (i.p.), P. umbellatum aqueous extract brought about an ataxia with a decrease in the animal's watchfulness, which lasts about 48 hours. A fall of rectal temperature and a decrease of spontaneous motor activity as well as an increase of analgesic activity were also observed [20]. The aqueous extract of the leaves of M, cecropiodes did not affect ache-induced

808

contractile response, but produced significant inhibition of the twitch and pendular movement of the rat and rabbit smooth muscles and reduced writhing induced by acetic acid in mice [21]. Aqueous and ethanolic extracts of S. dulcis and essential oil of L. multiflora also showed analgesic activities as well as anti-inflammatory effects. Aqueous and ethanolic extracts of S. dulcis prolonged the sleeping time induced by pentobarbital in mice; ethanolic extract but not water extract reduced writhings induced by acetic acid in mice and paw oedema induced by carrageenin in rats [22]. The essential oil of L. multiflora (2, 4 and 8 mL/kg) showed a dose-dependent analgesic effect on acetic acid-induced writhing in mice. At 8mL/kg, the essential oil also antagonised hyperexia induced by Brewer's yeast [23, 24]. Crude alkaloid extracts of M. myristica and Polyalthia suaveolens trunk barks collected in Gabon exhibited in vitro antiplasmodial activities against Kl chloroquineresistant strains of P. falciparum with IC50 values of 7.65 and 4.08 ug/mL respectively [25]. The active compounds listed in this paper have been isolated from the following species: L. multiflora, M. myristica, P. suaveolens, S. dulcis, T. iboga, and T. tetraptera. These products showed various biological activities such as antiviral, antimicrobial, antiparasitic, cytotoxic, stimulating of central nervous system (CNS), analgesic, antiinflammatory, H+, K+-ATP-ase inhibitors, P-glucuronidase inhibitors, hypotensive and bradycardisant. These compounds belong to the following groups: alkaloids, terpenes, coumarins, flavones, saponins and benzoxazolinones. ALKALOIDS Ibogai'ne (1), ibogaline (2), iboxygaine (3), noribogaine (4), and tabernanthine (5), five T. iboga indole alkaloids, showed a tremorproducing potency to mice when given subcutaneously (s.c.) and intravenously (i.v.). Pharmacokinetics parameters obtained in mouse brain after i.v. injections showed that the tremor-producing activity depends more on chemical structure than on lipid solubility of tested alkaloids. A methoxy group in the position of R2 (Figure 1) enhances tremorigenic potency, tabernanthine and ibogaline (s.c.) being the most active with ED50 values of 5 umol/kg and 8 umol/kg respectively. ED50 values of 35 umol/kg and 80 umol/kg were observed with ibogaine and iboxygaine respectively, noribogaine being less active [26]. Tabernanthine antagonised the impairment of brain catecholamines turnover observed at simulated high altitude but did not change the endogenous levels of brain dopamine and noradrenaline in rats at normal atmospheric pressure or in hypoxia [27]. Ibogaine (1), tabernanthine (5), voacangine (6) and conopharyngine (7) (two others iboga alkaloids), when given i.p. to mice, showed a CNS stimulating activity in the course of a habituation test using psychogalvanic reaction (PGR). Structural parameters were also

809 Alkaloids Indole alkaloids:

IBOGAINE(l) IBOGALINE (2) 1BOXYGAINE (3) NORIBOGAINE (4) TABERNANTHINE (5)

Rl

R2

R3

CH3O CH3O CHjO OH

H CH3O H H CH3O

H H OH H

Zetler, Singbartl, and Schlosser (1972)

OMe

COOCH,

VOACANGINE (6) CONOPHARYNGINE (7)

R= H R = CH3O

Bert, Marcy, Quermonne, Cotelle, and Koch (1988)

810 Aporphine

OMe

OLIVERINE (8)

Cave, Guinaudeau, Leboeuf, Ramahatra, Razafindrazaka (1978) [56]

Terpenoids

Monoterpenoid

CARVACROL (9) Bruneton(1993)

811 Diterpenoids

SCOPADULCIC ACID B (13): R = COOH SCOPADULCIOL(16) : R = CH2OH Hayashi, Asano, Mizutani, Takeguchi, Kojima, Okamura, and Morita (1991)

HOOC

SCOPARICACIDA(17)

Hayashi, Kawasaki, Okamura, Tamada, and Morita (1992)

812

HOOC

SCOPADULIN (18)

Hayashi, Kawasaki, Miwa, Taga, and Morita (1990)

Triterpenoid

COOH

NHCOCH

ARIDANIN (20) Adesina, and Reisch (1985) [57]

813 Flavones

GIUM

OH

8-HYDROXYTRICETIN 7-GLUCURON1DE (21) Kawasaki, Hayashi, Arisawa, Morita, and Berganza (1988)

OMe OMe

OMe

HYMENOXIN (23)

Hayashi, Uchida, Hayashi, Niwayama, and Morita (1988)

Coumarin

SCOPOLETIN (24) Ojewole, and Adesina (1983)

814 Miscellaneous

MeO. -N — H

6-METHOXYBENZOXAZOLINONE (25)

Chen, and Chen (1976) Fig. (1). Chemical structures of bioactive compounds.

observed in this test. IbogaTne was more active than tabemanthine, suggesting that a methoxy group in the Rl position seems very favourable for a CNS stimulating activity. Voacangine (6) and conopharyngine (7) were less active, indicating that the presence of a methoxycarbonyl group (Figure 1) is unfavourable [28]. Neither a locomotor hyperactivity nor stereotypy was observed, suggesting a non-amphetaminic-like stimulating activity and a direct cortical effect. These results are in accordance with the traditional use of plants as neurotonic. On isolated rat duodenum, ibogame and tabemanthine increased the hypertonic effect following the addition of calcium ions to the organ previously decalcified [29,30]. Tabemanthine showed a bradycardisant activity [31]. The anti-addictive properties of ibogaine have been described. Animal studies and noncontrolled observations in humans indicated that ibogaine significantly affects drug dependence phenomena such as drug withdrawal and intake of addictive drugs, ibogame attenuated many symptoms of naloxoneprecipitated withdrawal in morphine-dependent rats [32]. An intraperitoneal injection of ibogaine (40 mg/kg, single dose) in rats that previously r eceived a c ocaine s elf-administration p roduced a s ignificant decrease o f cocaine i ntake, w hich remained unaltered for m ore t han 4 8 hours. However, a more prominent inhibitory effect was observed in animals treated repeatedly with ibogaine (40 mg/kg i.p.), once a week for 3 consecutive weeks. Since the half-life time of ibogaine is short, these results indicated that the compound or its metabolite is a long-lasting interrupter of cocaine dependence [33]. IbogaTne is not itself addictive but it is claimed to cure the addiction of several hard drugs such as cocaine and heroine but even alcohol and nicotine. Oliverine (8), an isoquinoline alkaloid isolated from P. suaveolens collected in Cameroon, exhibited after 24 hours of incubation a microfilaricidal activity at 10-100 (xg/mL, but the compound showed a minimal lethal dose of 8 mg/kg of body weight in mice [34].

815

TERPENOIDS Monoterpenoids Carvacrol (9), isolated from a hexane extract of L. multiflora leaf collected in Nigeria, exhibited tremendous antimicrobial activity [35]. The essential oil of L. multiflora showed pediculocidal and scabicidal activities when tested comparatively with benzyl benzoate. A 20% v/v preparation of Lippia oil applied to scabietic subjects for 5 consecutive days gave 100% cure compared with 87.5% cure obtained for benzyl benzoate at the same concentration. The scabicidal activity of L. multiflora essential oil seems probably due to the presence of terpineol (10), a- and P-pinene (11; 12) which are known to be lethal to body and head lice [11]. Terpineol and a-pinene were also isolated from the oil of M. myristica fruit collected in Cameroon, as well as carvacrol isolated from the oil of M. myristica seeds collected in Nigeria [36]. Carvacrol has been suggested to be responsible for the antibacterial activity of the oil of M. myristica seeds collected in Democratic Republic of Congo [13]. The antibacterial activity of phenols, especially carvacrol, has been reported for others medicinal plants too [37]. Diterpenoids Various active diterpenoids have been isolated from S. dulcis, a herb growing in tropical and subtropical areas, used to treat stomach disorders, hypertension and blennorhagia. Scopadulcic acid B (13), a tetracyclic terpenoid isolated from 70 % ethanolic extract of a Paraguayan specie of S. dulcis, and its derivates diacetyl scopadol (14) and scopadulcic B methyl ester (15), as well as scopadulciol (16) isolated from a Taiwanese specie, prevented gastric secretion by inhibiting the H+, K+-adenosine triphosphatase (ATP-ase), which is responsible for the H+ ions secretion into the stomach [38-40]. Scopadulcic acid B and its debenzoyl derivate, diacetyl scopadol, had no effect on the related enzyme Na+, K+-ATP-ase. The inhibition mechanism of scopadulcic acid B involves the failure of both H+, K+-ATP-ase and the K+-dependent dephosphorylation step of the enzyme without any effect on the phosphorylation step [39]. A labdanetype diterpenoid, scoparic acid A (17), isolated from an ethanolic extract of S. dulcis collected in Paraguay, inhibited p-glucuronidase by binding specifically with the enzyme [41]. Scopadulcic acid B and scopadulin (18), another tetracyclic diterpene also isolated from a chloroform-soluble part of the 70 % ethanolic extract of Paraguayan specie, exhibited in vitro and in vivo antiviral activities against Herpes Simplex Virus Type 1 [42, 43]. Scopadulcic acid B inhibited the viral replication with an in vitro therapeutic index of 16.7. Single-cycle replication experiments showed that the compound interfered with considerably early events of virus growth. Orally (p.o.) or i.p. administered immediately after virus inoculation, scopadulcic acid B effectively prolonged both the appearance

816 of herpetic lesions and the survival time at a dose of 100 and 200 mg/kg per day [42]. In vitro and in vivo antitumour and cytotoxic activities were also observed with scopadulcic acid B. The compound showed an in vitro cytotoxic activity against human tumour tissues with IC50 values of 0.068-0.076 ug/mL. The normal human tissues were relatively more resistant, an IC50 of 0.245 ug/mL being observed against Chang Liver cells. The 5-fluorouracil (5-Fu), used as reference, exhibited in the same conditions against tumour tissues and Chang Liver cells IC50 values of 6.0-6.8 ug/mL and 11.1 (xg/mL respectively. In mice, inoculated with Ehrlich cell suspension, at 25 mg/kg (i.p.) the cytotoxic activity of scopadulcic acid B was lower than that of 5-Fu with respectively, 12.5% and 50% survival attained at 45 days after tumour implantation without detectable tumour ascites [44]. Scopadulcic acid B was proved to be a potent antitumour promoter as well as various natural terpenoids, inhibiting the effects of tumour promoter 12-O-tetradecanoylphorbol-13acetate (TPA) in vitro and in vivo. The compound inhibited TPAenhanced phospholipid synthesis in cultured cells, and suppressed in mice the promoting effect of TPA on skin tumour formation initiated with 7,12dimethylbenz[a]anthracene [45]. Triterpenoids An ethanolic extract of S. dulcis collected in Brazil as well as glutinol (19), a triterpenoid from which it has been isolated, showed analgesic activity p.o in mice and rats, reducing writhings induced by acetic acid. They exert analgesic effects through a peripheral mechanism. The ethanolic extract and glutinol also showed anti-inflammatory effects, inhibiting the carrageenin-induced paw oedema and pleurisy. Glutinol mainly exerts his action during the early phase of acute inflammatory process [22]. Aridanin (20), a triterpenoid glycoside isolated from T. tetraptera fruits collected in Nigeria showed a molluscicidal activity against Biomphalaria glabrata with a LQo of 0.88 ug/mL, Bayluscide® (70 % niclosamide), the reference, exhibiting in the same conditions a LC50 of 0.35 ug/mL [46]. At low concentrations of 0.125 to 1.0 ppm, aridanin caused a significant reduction in the egg production and growth of B. glabrata and Lymnaea columella, indicating that the compound could control schistosomiasis if used in slow-release formulations [47]. Aridanin was also active against Schistosoma mansoni and S. bovis miracidia, 100% mortality or immobility being observed after 30 min (50 ug/mL) or after 5 min exposure time at a dose of 100 ug/mL. At a low concentration of 0.25 ug/mL, aridanin reduced the production of cercariae in snails, revealing that the compound acts against the transmission of schistosomiasis at different stages of the schistosome development [16].

817

FLAVONES Two flavonoids, 8-hydroxytricetin 7-glucuronide (21) and isovitexin (22), also isolated from ethanolic extract of S. dulcis collected in Paraguay, were found to be P-glucuronidase inhibitors. The two compounds showed mild inhibitory activity against P-glucuronidase from bovine liver. The inhibitory activity of 8-hydroxytricetin 7-glucuronide was one-tenth of that of the well-known P-glucuronidase inhibitor, glucosaccharo-l:4-lactone. Tested in the presence of a large amount of bovine serum albumin (BSA), 8-hydroxytricetin 7-glucuronide showed almost the same degree of inhibition in the presence of 400 times of BSA, suggesting that it inhibits the activity of P-glucuronidase even in the presence of other proteins [48]. Hymenoxin (23), another flavone isolated from a chloroformic soluble fraction of ethanolic extract of S. dulcis, showed an in vitro cytotoxic effects against human tumour tissues such as HeLa 229, HeLa S3 and against normal tissues such as Chang liver and intestine cells. Like with scopadulcic acid B, a difference of susceptibility between the human cancer tissues and those from normal tissues was observed with hymenoxin, the rD50 against HeLa 229 cell growth and Chang liver cell growth being 0.097 ug/mL and 0.510 ug/mL respectively [49]. COUMARIN Hypotensive and neuromuscular actions were observed with scopoletin (24), a coumarin isolated from the fruit of T. tetraptera collected in Nigeria. Administered at 10-100 mg/kg i.v, the compound reduced the arterial blood pressure of anaesthetised rats. At a dose of 10"6-10"3 M, scopoletin produced negative chronotropic and inotropic responses in guinea-pig isolated atria, inhibited acetylcholine-induced contractures of the toad rectus abdominis muscle, and depressed electrically-evoked twitches of the chick isolated biventer-cervicis muscle and rat isolated phrenic-nerve hemidiaphragm muscle preparations. The pharmacological effects o f s copoletin w ere not a ltered b y a tropinisation [51]. S copoletin relaxes the smooth muscles and reduces electrical stimulation-evoked and exogenous, noradrenaline-evoked contractions of muscle preparations. Ojewole suggested that scopoletin probably produces hypotensive action in laboratory animals through its smooth muscle relaxant activity, dilating blood vessels, and by acting as a non-specific spasmolytic agent [50]. SAPONINS Saponins and ethanolic extracts of T. tetraptera stem bark, collected in Ivory Coast, inhibited in vitro the luteinising hormone-releasing, hormone (LHRH)-induced LH release. The decrease in the amount of immunoassayable hormone was both time and dose dependent [52,53].

818

MISCELLANEOUS The 6-methoxybenzoxazolinone (25) isolated from S. dulcis showed a hypotensive activity [54]. Hypotensive effects of methanolic extract of L. multiflora leaves collected in Ivory Coast seem to be due to a caffeic ester isolated from a phenolic fraction. The compound induced a marked and long-lasting tensional fall: at 2mg/kg i.v, the decrease in systolic arterial blood pressure was high as- 40% and lasted for more than 15 minutes [55]. ABREVIATIONS i.p. i.v. p.o s.c. E I ATP-ase BSA CNS 5-Fu IC ID LC TPA

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

intraperitoneal intravenous per os subcutaneous external internal adenosine triphosphatase bovine serum albumin central nervous system 5-fluorouracil inhibitory concentration inhibitory dose lethal concentration 12-O-tetradecanoylphorbol-13 -acetate

ACKNOWLEGMENTS The authors gratefully acknowledge the International Centre for Bantu Civilizations (CICIBA) and European Union Bantu Cultural Program for financial support of fieldwork. They also thank the Ministry of Culture of Central Africa and the Ministry of Information, Tourism, Culture and Art of Equatorial Guinea for arranging meetings with the traditional healers. They are grateful to Dr. Kosh Komba, Mr. Boris Belle, Mr. Etienne Mokili and Mr. Bernard Ndonazi for having provided information in Central Africa. They also owe thanks to Mrs. Anasthasie Adomba and Mr. Odembet of Gabon for having shared their knowledge with them. Finally they would like to thank Mr. Cornelio Essono and all the traditional healers of Equatorial Guinea for information. REFERENCES [1] [2]

Clist, B.; Lanfranchi, R. In Ratines Bantu/Bantu roots. Obenga, Th.; Souindoula S., Eds; CICIBA: Libreville, 1991, pp. 29-45. Obenga, Th. In Ratines Bantu/Bantu roots. Obenga, Th.; Souindoula S., Eds; CICIBA: Libreville, 1991, pp. 121-150.

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Hayashi, T.; Okamura, K.; Kakemi, M.; Asano, S.; Mizutani, M.; Takeguchi, N.; Kawasaki, M.; Tezuka, Y.; Kikuchi, T.; Morita, N.; Chem Pharm Bull, 1990, 35(10), 2740-2745. Asano, S.; Mizutani, M.; Hayashi, T.; Morita, N.; Takeguchi, N.; The Journal of Biological Chemistry, 1990, 265(36), 22167-22173. Hayashi, T.; Asano, S.; Mizutani, M.; Takeguchi, N.; Kojima, T.; Okamura, K..; Morita, N.; JNat Prod, 1991, 54(3), 802-809. Hayashi, T.; Kawasaki, M.; Okamura, K.; Tamada, Y.; Morita, N.; Tezuka, Y.; Kikuchi, T.; Miwa, Y.; Taga, l.;JNat Prod, 1992, 55(12), 1748-1755. Hayashi, K.; Niwayama, S.; Hayashi, T.; Nago, R.; Ochiai, H.; Morita, N.; Antiviral Research, 1988, 9, 345-354. Hayashi, T.; Kawasaki, M.; Miwa, Y.; Taga, T.; Morita, N.; Chem Pharm Bull, 1990, 35(4), 945-947. Hayashi, K.; Hayashi, T.; Morita, N.; Phytotherapy Research, 1992, 6, 6-9. Nishino, H.; Hayashi, T.; Arisawa, M.; Satomi, Y.; Iwasima, A. Oncology, 1993, 50, 100103. Adewunmi, C O ; Awe, S.O.; Adesina, S.K.; Planta Med, 1988, 54(6), 550-551. Adewunmi, C O ; Furu, P.; Madsen, H.; Phytotherapy Research, 1989, 3 (3), 81 -84. Kawasaki, M.; Hayashi, T.; Arisawa M.; Morita, N.; Berganza L.H.; Phytochemistry, 1988, 27(11), 3709-3711. Hayashi, T.; Uchida, K.; Hayashi, K.; Niwayama, S.; Morita, N.; Chem Pharm Bull, 1988, 36(12), 4849-4851. Ojewole, J.A.O.; Adesina, S.K.; Planta Med, 1983, 49,46-50. Ojewole, J.A.O.; Adesina, S.K.; Planta Med, 1983, 49, 99-102. Benie, T.; El Izzi, A.; Tahiri, C ; Duval, J.; Thieulant, M.L.; C R Soc Biol, 1987, 181, 163167. El Izzi, A.; Benie, T.; Thieulant, M.L.; Duval, J.; Planta Med., 1990, 56, 357-359. Chen, CM.; Chen M.T.; Phytochemistry, 1976, 15, 1997-1999. Pam Huu Chanh; Koffi, Y.; Pam Huu Chanh, A.; Planta Med, 1988, 54(4), 294-296. Cave, A.; Guinaudeau, H.; Leboeuf, M.; Ramahatra, A.; Razafindrazaka, J.; Planta Med, 1978, 33, 243-250. Adesina, S.K.; Reisch, J.; Phytochemistry, 1985, 24(12), 3003-3006.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

821

BIO ACTIVE NON-ALKALOID AL CONSTITUENTS FROM THE GENUS ERYTHRINA RUNNER R.T. MAJINDA1*, CORNELIUS C. W. WANJALA2, BENARD F. JUMA1 Department of Chemistry, University of Botswana, P/Bag UB 00704, Gaborone, Botswana. 2Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya ABSTRACT: The genus Erythrina is very rich in secondary metabolites particularly of the flavonoids class. A literature survey of non-alkaloidal secondary metabolites from Erythrina showed the presence of flavanones, flavonols, chalcones, cinnamoylphenols, stilbenoids, isoflavones, isoflavans, isoflavanones, pterocarpans, isoflav-3-enes, 3-phenoxychromones, coumastans, 3-phenyl-coumarins, lignans, cinnamate esters, simple phenolics, triterpenes, sesquiterpenes, long-chain carboxylic acids, and long-chain alcohols. The documented bioactivities of some of the isolated metabolites range from antimicrobial, anti-inflammatory, inhibition of platelet aggregation, tyrosinase inhibition, phospholipase A2 (PLA2) inhibitors, cyclooxygenase inhibitors, antioxidant, inhibitors of NA/H + exchange system, phospholipase C inhibitors, behavioural depression, muscle relaxation, (3-adrenergic inhibition, diuretic, anticancer, cytotoxic, DNA-repair properties, oestrogenic or proestrogenic activities, antitrypanosomal, antiplasmodial, and anti-HIV activities. The bioactivity profile represented the various classes fairly reasonably but it became apparent that a number of these isolates have not yet been tested for biological activity. A very close agreement between ethnomedical use of the various Erythrina extract preparations (Table 1) and the results of biological activity (Table 2) was found, with the reported activities of pure isolates strongly supporting the documented ethno-medical uses and reported pharmacological activity. The most common activity exhibited by extracts and pure isolates was mostly microbial related. It was also interesting to note that some compounds showed high efficacy against resistant organisms, a very important aspect, since most used drugs especially antibiotics tend to produce resistance to certain strains of organisms. The survey showed furthermore that certain structural features were essential for certain activities and also that compounds that were found active were usually effective against not just one but several disease functions. One cannot help but surmise that more activities are yet to be reported for these same isolates. The challenge remains for researchers in the various pertinent disciplines to carry out more work on these to fill in the knowledge gaps that still exist. I. INTRODUCTION The genus Erythrina comprises around one hundred and ten species of orange or red flowered trees, shrubs and herbaceous plants. It is divided into five sub-genera and twenty-six sections, largely on the basis of morphology, especially the colour and shapes of the flowers and fruits and of inflorescence characteristics. They are found throughout the tropical and semi-tropical

822 regions of the world. In the southern Africa, it occurs in scrub forest, dry woodland and coastal bush throughout the four northern provinces of South Africa, and also in Mozambique, Botswana, Zimbabwe, Angola, Zambia and Malawi. The genus Erythrina is part of the Papillionaceae sub-family of the Leguminoseae (Fabaceae). There are twenty-eight species and three sub-species in Mexico, twenty-six species in Central America, twenty-two species in South America, five species and two varieties in West Indies, thirty species and sub-species in tropical Africa, six species in South Africa seven species in continental Asia and seven species in Malaysia and the Pacific [1-2]. II. SOME ETHNOBOTANICAL AND PHARMACOLOGICAL INFORMATION ON ERYTHRINA Traditionally, Erythrina species have found use amongst different communities for various ailments. The bark of E. fusca and E. indica, for example, has been used for the treatment of fever, malaria, rheumatism, toothache, boils and fractures. Boiled roots of the same were taken internally as a remedy for beriberi. In Kenya, a concoction of dried bark of E. abyssinica has been used for the treatment of trachoma (oral) and elephantiasis (external) and its roots against malaria and syphilis, while the pounded green stem bark is taken as an antihelminthic [3]. The water extract of flowers of E. americana have been used in Mexico for the treatment of insomnia. The rest of other documented traditional medicinal uses are listed in Table 1. The seeds of Erythrina species are known to contain toxic alkaloids, as well as blood clotting substances, which may be of value in the treatment of thrombosis. The seeds, stem and root bark of some Erythrina species have been used also to stupefy fish, as narcotic, purgative, and diuretic [1,4], while crushed seeds of some have been used as rat poison. All the Erythrina species examined have yielded alkaloids, mostly of the erythrinaline type, having the curare like poisoning action [1,4]. Several of the more common species are used for decorative purposes in gardens and city streets. A number of biological activity tests have been carried out on some species of this genus, which corroborate the reported ethnomedicinal values. Table 2 [5] gives a summary of some of the results obtained. Table 2 also includes a very recent report of antitrypanosomal and antiplasmodial activities of a 90% ethanolic extract of E. senegalensis from Cote d'lvoire. A noteworthy observation from this report is that antitrypanosomal activity is reported for the first time from the genus [5b]. III. PHYTOCHEMICAL WORK ON NON-ALKALOIDAL CONSTITUENTS OF ERYTHRINA Extracts of the leaves, bark and roots of the genus Erythrina have a significant history of medicinal use for the treatment of diseases such as female infertility, stomach pain and gonorrhoea [6] and others shown in Table 1, and biological tests on Erythrina plant extracts [5] showed that these have various pharmacological activities (Table 2). In spite of this, earlier work on Erythrina tended to concentrate on the alkaloidal content of the plants. It was perceived, then, that alkaloids could be the only active constituents in these plants. It was for this reason perhaps that there was initially very little interest in the neutral non-alkaloidal constituents of the genus. It was not until the late seventies and early eighties that serious work started on the non-alkaloidal constituents of some Erythrina species [7-9]. Subsequently more reports started to appear on non-alkaloidal Erythrina content [10-12]. The research on these neutral constituents has since intensified and a number of studies have been conducted on the biological activity of these constituents. Nakanishi and co-workers [2] did some work

823 which could be the first to mark the beginning to serious biological activities of the neutral constituents of Erythrina. The following sections give an account of the biologically active neutral metabolites of the genus Erythrina. The major class of neutral metabolites that the genus Erythrina elaborates is the flavonoids in a very broad sense and these are divided into flavonoids sensu strictu [i.e. in a narrow sense], Fig. (1) and isoflavonoids, Fig. (2) depending on their biogenetic origin. Table 1: Ethnomedicinal Application of Some Erythrina Species (NAPRALERT, 2004) Plant

Part

Remedy

E. flabelliformis

seed

Toothache contraceptive

E. folkersii

bark

inflammation of womb (oral) diuretic

seed E. fusca

bark seeds

infested sores (washed externally) fungal dermatosis anti-inflammation agent skin infections

E. glauca

not specified

malaria (oral decoction) narcotic and purgative

E. humeana

bark

tuberculosis (oral decoction)

E. indica

bark leaf leaf

antihelminthic (oral) expectorant(oral) aphrodisiac (oral) menstrual troubles

E. mulungu

bark

fevers (oral)

E. sacleuxii

leaf bark

malaria (oral) malaria (oral) serious wounds

E. senegalenses

bark bark + leaf entire plant

oracular infections (oral) bronchial affections (oral) jaundice (oral) female infertility (oral) dysentery (oral) venereal disease (oral)

E. strictu

bark

epilepsy (oral)

E. subumbrans

leaf

menorrhagia (oral)

E. variegata

bark + coconut oil flowers leaf

swellings and inflammations (external application) skin ulcers antiasthmatic (oral) augmenting milk flow

E. zeyheri

bark

tuberculosis

824 Table 2: Reported Bioactivities for Some Erythrina Species (NAPRALERT, 2004) Plant species E. abyssinicci E. berteronna

Part bark root root bark root back

E. caffra

Bark

E. crisla-galli E. flnbelliformis

Leaf Seed

E. fusco E. latissima E. lysistemon

Seed Bark root

E. sacleuxii E. senegalensis E. suberosn

leaf flowers stem bark leaf

E. vnriegaln

bark

Activity antibacterial antifungal smooth muscle relaxation antifungal anticoagulant antibacterial cyclooxygenase inhibition antibacterial larvicidal diuretic CNS depressant antibacterial antiviral cytotoxicity antimalarial Molluscicidal Antiplasmodial, antitrypanosomal antitumour hypotensive antiulcer plant germination inhibition Smooth muscle relaxation Antifeedant

p-Hydroxycinnamoyl-CoAl 3x malonyl-CoAli

Figure 1: Biosynthetic relationships amoung different classes of tlavonoids

Extract CHC13 Ethanol 60% Methanol CH2C12 Acetone EtOAc

H2O EtOAc MeOH:CH2Cl2 1:1 EtOAc. H2O Ethanol 90% [5b] EtOAc/H2O 1:1 EtOAc. MeOH Acetone

825 •COOH

\

H

f T L* ^J T V°!l ^ 0^ 5 ^) Pterocarpans

6 a-hydroxypterocarpans

1 1 Pterocarpenes

Figure 2: Major isoflavonoid classes and their biosynthetic relationship [ adapted fromTahara and Ibrahim (13)]

IV. BIOACTIVE COMPOUNDS FROM ERYTHRINA A survey of non-alkaloidal secondary metabolites from Erythrina revealed the presence of flavanones, flavonols, chalcones, cinnamoyl phenols, stilbenoids, isoflavones, isoflavans, isoflavanones, pterocarpans, isoflav-3-enes, 3-phenoxychromones, coumastans, 3phenylcoumarins, lignans, cinnamate esters, simple phenolics, triterpenes, sesquiterpenes, squalene, C-18 unsaturated hydroxycarboxylic acid, and «-octacosanol. The documented bioactivity of some of the isolated metabolites includes antimicrobial activity [9,14-32], antiinflammatory [33-35], inhibition of platelet aggregation [9,15,32], tyrosinase inhibition [32], phospholipase A2 (PLA2) inhibitors [34,36], cyclooxygenase inhibitors [23], antioxidant [13,33,35,37-40], inhibitors of NA+/H+ exchange system [41], phospholipase C inhibitors [42], behavioural depression [15], muscle relaxation [15], P-adrenergic inhibition [15], diuretic [15], anticancer [13,43], cytotoxic [44-45], DNA-repair properties [44], oestrogenic [13] or proestrogenic activities [13], antiplasmodial [5b,46], and anti-HIV [47, 47b] activities. V. BIOACTIVE FLAVONOIDS FROM ERYTHRINA SPECIES In this section, flavonoids will be referred loosely to as compounds whose biogenetic origin is depicted in Fig. (1). Thus, in this section, chalcones, 1,3-diphenylpropanes (cinnamoylphenols) and stilbenoids (not generally considered as flavonoids) will be included as well as other 'conventional' flavonoid classes. Bioactive compounds will therefore be discussed according to class rather than common activity, and as such compounds that exhibit the same activity but belong to different classes will be discussed together with those from the same class albeit displaying different bioactivities. The same reasoning will be used in

826 discussing isoflavonoids. Bioactive flavonoids have been reported in this genus and their breakdown, first according to class (or subclass) and then type of activity, is given below. 1. Bioactive Flavanones: Liquiritigenin Derivatives Liquiritigenin [40] (5-deoxynaringinin) 1 and its derivatives seem to be restricted to very few Erythrina species and are found predominantly in E. abyssinica [9,46,48] and E. latissima [24,38-39] with reports of one or two metabolites of the class each in E. lysistemon [40], E. sigmoidea [49-50], and very recently E. fusca [32b]. In a screen for phytoestrogens, based on the binding affinity for the bovine uterine receptor, 1 was found to have affinity similar to that of the common dietary phytoestrogens genistein and daidzein [13b]. Nakanishi and coworkers [9] did some work that led the isolation and biological activity assessment of the novel neutral metabolites from E. abyssinica [9], viz abyssinones I 2, II 3, III 4, IV 5. The driving force for this work came probably from an ethno-medical observation that the roots of the plant were used to treat malaria and syphilis [3] and from an earlier finding by the same group that 60% aqueous methanolic extract of these roots was found to possess strong activity against fungi and Gram-positive bacteria [51]. Abyssinone II 3 was found to be active against Gram-positive bacteria Staphylococcus aureus & Bacillus subtilis (MIC 50 ug/ml each), Micrococcus lysodeikticus (25 ug/ml); against yeasts Saccharomyces cerevesiae & Candida utilis (100 ug/ml each) and against fungi Sclerotinia libertiana (12.5 ug/ml), Mucor mucedo (50 ug/ml), and Rhizophus chinensis (100 ug/ml). Abyssinone II also inhibited rabbit platelet aggregation induced by 38 ug/ml of collagen (IC50 3.5 ug/ml), and 0.57 uM sodium arachidonate (IC50 74 ug/ml, 40 % inhibition), but no inhibition against that induced by 10 uM ADP. Abyssinone I 2 was active against S. aureus & B. subtilis (MIC 50 ug/ml each); S. cerevesiae & C. albicans (100 ug/ml each), S. libertiana (12.5 ug/ml) & M. mucedo (50 ug/ml) but inactive against R. chinensis (>100 ug/ml). Abyssinone III 4 was not active against any bacteria, yeasts or fungi up to 100 ug/ml and was not tested for inhibition of platelet aggregation, while abyssinone IV 5 showed activity against both S. aureus (25 ug/ml) and B. subtilis (12.5 ug/ml) but was inactive (>100 ug/ml) against yeasts and fungi. Abyssinone IV further showed some antiplasmodial activity against the chloroquine-resistant W2 (IC50 7.7 fig/ml; chloroquine 0.093 ug/ml, quinine 0.21 ug/ml) and chloroquine-sensitive D6 (9.0 ug/ml) strains of Plasmodium falciparum [46]. Abyssinone VII 6 and erylatissin C 7 are new metabolites from E. abyssinica [49] and E. latissima [38,39] respectively, which have displayed free radical scavenging properties (IC50 27.7 and 710 ug/ml respectively) against 2,2-diphenyl-l-picrylhdrazyl (DPPH) radical. These compounds showed some weak preliminary antibacterial (0.1-0.5 ug) and strong antifungal (0.01-0.lug) activity using TLC bioautography technique. Table 3: Liquiritigenin Derivatives from Erythrina Species Compound Liquiritigenin 1 Abyssinone I 2 Abyssinone II 3 Abyssinone III 4 Abyssinone IV 5

Abyssinone VII 6 Erylatissin C 7

Erythrina source E. lysistemon [40] E. abyssinica [9] E. abyssinica [9] E. latissima [38,39] E. abyssinica [9] E. abyssinica [9,48,46] E. latissima [24] E. sigmoidea [27,30] E. abyssinica [48] E. latissima [38,39]

Activity Phytoestrogenic activity Antibacterial, antifungal Antibacterial, antifungal, antiyeast, inhibits platelet aggregation Antibacterial; antiplasmodial W2 (chloroquine resistant strain-7.7|j.g/ml), D6 (chloroquine sensitive strain -9.0 ug/ml) DPPH radical scavenging properties Weak antioxidant(DPPH), antibacterial (Gram-positive), antifungal.

827

No

R1

R2

R3

Name

1

H

OH

H

Liquiritigenin

2

3 ,4 (2,2-dimethylchromeno)

H

Abyssinone I

3

3,3-DMA

H

Abyssinone II

4

3,3-DMA

5

3,3-DMA

6 7

OH

4 ,5 (2,2-dimethylchromeno)

Abyssinone III

OH

3,3-DMA

Abyssinone IV

OH

OH

3,3-DMA

Abyssinone VII

OH

OMe

3,3-DMA

Erylatissin C

2. Bioactive Flavanones: - Naringenin Derivatives Naringenin [38-39] 8 and its derivatives seem to be more widely distributed among Erythrina species than liquiritigenin derivatives. Abyssinone V 9, first reported by Nakanishi and coworkers from E. abyssinica [9], showed antimicrobial activity against Gram-positive bacteria, S. aureus (50 ug/ml), B. subtilis (25 ug/mi) & M. lysodeikticus (12.5 ug/ml) and was non active (>100 ug/ml) against yeasts and fungi. It also showed a weak to moderate phospholipase A2 (PLA2) inhibition activity with IC50 value of 6 uM (trifluoropirazine at 20 uM showed >80% inhibition under assay conditions). The report of a PLA2 inhibitor, 6prenylabyssinone 10, first isolated from the methanolic extract of the stem bark of the Samoan medicinal plant E. variegata [36] and which has demonstrated strong PLA2 activity, is perhaps a good example of how ethno-medical data can provide clues or leads to certain putative pharmacological activities. The Samoan healers prepare extracts of the bark in coconut oil and apply the extract externally for swellings and inflammations. The extract is also used to treat skin ulcers that appear as boils or burns which burst and suppurate. It thus appeared, from this data, that E. variegata extractives in Samoa could be effective against inflammation or inflammation-like processes. Compound 10 was isolated from this bark extract and found to inhibit PLA2 with IC50 of 10 uM. Burttinone 11, first isolated from the stem bark of E. burttii [53] and later from E. lysistemon [43], was evaluated by the National Cancer Institute (NCI) as an anticancer agent through the 60 cell panel screen. The IC50 values for 11 were less than 50 uM against 43 cell lines. Burttinone further showed maximum toxicity against colon cancer cell line, HCC 2998 (IC50 = 20 uM), while the IC50 were higher than 50 uM in all the five tested leukaemia cell lines. It was, however, not selective in its action against any tested panel, indicating that it is a general cytotoxic agent which may probably have no clinical value.

828

Sigmoidins A 12 and B 13, first isolated from the stem bark of E. sigmoidea [11-12,32], exhibit weak but significant in vitro activity against both Gram-positive and Gram-negative bacteria. These two compounds were very recently [33] analysed for their ability to inhibit the stable l,l-diphenyl-2-picrylhydrazyl (DPPH) free radical and arachidonic acid metabolism. These were further studied in two experimental models of inflammation induced in mouse ears by 12-O-tetradecanoylphorbol 13-acetate (TPA) and the phospholipase A2-induced rat paw oedema. Both compounds proved to be potent scavengers of DPPH, while arachidonic acid metabolism inhibition studies showed them to be selective inhibitors of 5-lipoxygenase with no effect on cyclooxygenase-1 activity. Sigmoidin A, 12 gave IC50 of 31 uM. In the phospholipase A2-induced mouse paw oedema assay, only sigmoidin B 13 inhibited oedema formation at 60 minutes, showing a percentage inhibition below that obtained with cyproheptadine (59% vs 74%). In the TPA test, Sigmoidins A and B decreased the induced oedema by 89% and 83% respectively. The data for the two compounds seem to suggest that they have different mechanisms of action and this depending on whether the compound has one or two prenyl (or y,y-dimethylallyl or 3,3-dimethylallyl) units in the B-ring. Sigmoidins A and B also display oestrogen-like activity and thus displayed low but significant binding affinities on bovine uterine oestrogen receptor [60], albeit at much lower activity level than for 17P-ostradiol, a putative oestrogen. Compounds 14-17, recently isolated from the stem bark of E. fusca, displayed weak to moderate antibacterial activity (Table 5) against S. aureus, B. subtilis and E.faecalis [32b]. Table 4: Bioactive Naringenin Derivatives from Erythrina Species Compound Naringenin 8 Abyssinone V 9

6-pvenylabyssinoneV 10

Erythrina source E. latissima [38,39] E. abyssinica [9,48,52] E. burttii [53] E. eriotriocha [54] E. sigmoidea [55] E. variegata [36] E. variegata [36]

Activity Antibacterial; Phospholipase A2 inhibitor IC50 6 u-M

Phospholipase A2 (PLA2) inhibitor 1C5O 10 U.M

Burttinone 11

E. burttii [53] E. lysistemon [43]

Sigmoidin A 12

Sigmoidin B 13

Erysenegalone

14

Citflavanone Lonchocarpol A Lupinifolin

15 16 17

Anticancer-against 60 cell panel lines but not selective therefore not useful therapeutically. Antibacterial, anti-inflammatory.

E. abyssinica [52] E. eriotriocha [21 ] E. sigmoidea [11,12] E. latissima [24,56] E. abyssinica [48,52] E. berteroana [ 16] E. latissima [24,56] E. sigmoidea [11,12,57,58] E. suberosa [59] E, senegalensis [15] E. fusca [32b] E. fusca [32b] E. fusca [32b] E. fusca [32b]

Antibacterial, anti-inflammatory

Antibacterial Antibacterial Antibacterial Antibacterial

829

OH

O

No

R1

R2

R3

R4

R5

R6

Name

8

H

OH

H

H

OH

H

Naringenin

9

H

OH

H

3,3-DMA

OH

3,3-DMA

Abyssinone V

10

3,3-DMA

OH

H

3,3-DMA

OH

3,3-DMA

6-prenyl-

11

H

OH

H

a

OMe

3,3-DMA

Burttinone

12

H

OH

3,3-DMA

OH

OH

3,3-DMA

Sigmoidin A

13

H

OH

H

OH

OH

3,3-DMA

Sigmoidin B

abyssinone V

OH

O No

R1

R3

R2

R4

14

OH

3,3-DMA

7,8(2,2dimethylchromeno)

15

OH

H

7,8(2,2dimethylchromeno)

16

OH

3,3-DMA

17

OH

6,7(2,2dimethylchromeno)

OH

Name Erysenegalone Citflavanone

3,3-DMA

Lonchocarpol A

3,3-DMA

Lupinifolin

830 Table 5: In vitro Antibacterial Activity of Compounds from E.fusca [32b] Compound

Staphylococcus aureus

Bacillus subtilis

Enterococcus faecalis

MIC"

MBC"

MIC

MBC

MIC

>100

>100

50

50

100

100

12.5

>100

12.5

12.5

>100

>100

Lonchocarpol A

6.25

>100

3.13

3.13

6.25

6.25

Lupinifolin

12.5

>100

6.25

6.25

50

50

Tetracycline

(Isosenegalensin) Senegalensin

32

Table 9: Bioactive Isoflavone Derivatives from Erythrina Species

Compound

Erythrina source

Auriculatin 37

E. eriotriocha [84]

Activity Antibacterial, diuretic, PLA2 inhibitor,

E. senegalensis [15,78,83,85,86] cytotoxic E. variegata [87] Warangalone 39

E. addisonae [34]

(Scandenone)

E. arborescens [77]

Anti-inflammatory

E.eriotriocha [88] E. sigmoidea [21,50] E. variegata [18] 6,8-diprenylgenistein 40

E. senegalensis [78,79]

Antibacterial, diuretic, p-adrenergic

E. sigmoidea [21,50]

stimulation, platelet aggregation inhibitor

E. variegata [18,90,91] E. vogelii [92] 8-prenylluteone 38

E. eriotriocha [88]

PLA2 inhibitor, cytotoxic

E. lysistemon [40] E. senegalensis [42,78,79]

837

OH

No

R1

37 38 39 40

R2

O

R3

R4

Name

6,7(2,2-dimethylchromeno)

3,3-dimethylallyl

OH

Auriculatin

3,3-dimethylallyl

3,3-dimethylallyl

OH

8-prenylluteone

6,7(2,2-dimethylchromeno)

3,3-dimethylallyl

H

Warangalone (scandenone)

3,3-dimethylallyl

3,3-dimethylallyl

H

6,8-diprenylgenistein

OH

OH

Auriculatin 37, first isolated from Milletttia auriculata [93], was subsequently reported in several Erythrina species (Table 9). A detailed pharmacological screen on some isolates from E. senegalensis revealed the compounds auriculatin 37 and 6,8-diprenylgenistein 40, to be non-toxic, exhibiting antimicrobial activity against Gram-positive bacteria and having diuretic properties [15]. In addition, 40 also exhibited some P-adrenergic stimulation and platelet aggregation properties [15]. Auriculatin 37, along with 8-prenylluteone 38, inhibited phospholipase C (PLC) in vitro and the formation of total inositol phosphates (IPj) in plateletderived growth factor (PDGF)-stimulated NIH3T3yl cells. These compounds exhibited potent activity on phosphoinositides (PI)- turnover in response to PDGF stimulation [42]. These two compounds, both with at least one prenyl group, were shown to have an inhibitory effect on the Pi-turnover in the NIH3T3yl cells and the enzymatic activity of PLC with an IC50 of 20 (J.M. When the anti-PLC of 37 and 38 was compared with that of common flavonoids (luteolin, quercetin, hesperetin and genistein), the common flavonoids were found to exhibit very weak inhibitory activity with IC50 values of around 250 uM against PLC [42]. It appears that the presence of a prenyl group at position 8 was related to the inhibitory action against PLC-yl. These compounds further showed cytotoxicity against several human tumour cell lines viz PC-3 (prostate), NCI-H226 (lung), CRL1579 (melanoma) in vitro (Table 10), with moderate cytotoxicity (IC50 range 9.0-20 uM), with 37 showing more potent inhibition than 38 on PC-3 and CRL1579 cell lines [42] (Table 10). Since PLC plays an important role in intracellular signal transduction and cell proliferation, 37 and 38, as PLC inhibitors, maybe cell growth inhibitors and could find application in cancer treatment or as tools for exploring the mechanism of PLC-mediated signal transduction. Table 10: Cytotoxicity of 37 and 38 Against Some Human Tumour Cell Lines

Compound

Human Cell Line (ICJO values in \M) PC-3 (prostate)

NC1-H226 (lung)

Auriculatin 37

9.4

12.1

CRL1579 (melanoma) 9.0

8-prenylluteone 38

20.0

15.1

16.4

838 Warangalone or scandenone 39, first described by Pelter and Stainton [94], has been reported in a number of Erythrina species [18,21,34,50,77,88]. Its anti-inflammatory activity was discovered through a bioassay-guided fractionation of an EtOAc extract of the bark of E. addisonae, which showed activity on a model of ovalbumin hind paw oedema [34]. The antiinflammatory activity of the extract and the warangalone (the main active constituent of the extract) was assayed on phospholipase A2 (PLA2) and carrageenin-induced mouse paw oedema, 12-O-tetradecanoylphorbol 13 acetate (TPA)-induced mouse ear oedema, and a model of chronic dermatitis caused by repeated administration of TPA. Warangalone showed marked effectiveness as an anti-inflammatory agent on the PLA2-induced paw oedema (Table 11) and on the TPA-induced ear oedema (Table 12) in mice after systemic and local administration respectively [34]. Table 11: Effect of Test Drugs on PLA2-Induced Mouse Paw Oedema" Test drug

Time 30min

%I

60 min

%I

Control

6.50

E. addisonae EtOAc extract

3.50

46

3.80

39

Warangalone 39

5.16

21

2.00

69

Cyproheptadine

2.16

66

2.32

63

6.30

"E. addisonae EtOAc extract (50 mg/kg), warangalone (5 mg/kg), and cyproheptadine (5 mg/kg) were intraperitoneally administered 30 min before subcutaneous plantar injection of PLA2 and oedema was measured 30 and 60 min later. Table 12: Effect of Test Drugs on TPA-induced Ear Oedema"

Test drug

Control E. addisonae EtOAc extract Warangalone 39 Indomethacin

Ear swelling

%1

MPO activity

14.25 1.80 3.07 2.57

87 78 82

155 n.d 91 99

41 36

"E. addisonae EtOAc extract, warangalone, and indomethacin were administered at 1, 0.25, and 0.5 mg/ear respectively, on the ear surface together with TPA. b

MPO = myeloperoxidase activity.

(b). Bioactive Daidzein Derivatives from Erythrina Genus It is interesting to note that most common activity that daidzein and its derivatives exhibit seems to be predominantly antimicrobial (Table 13). Daidzein 41, a possible precursor for 5deoxyisoflavones, has been reported in several Erythrina species [38-40,76,94-96] and is a well-known dietary phytoestrogen [13b]. It has been reported also to exhibit weak to moderate antibacterial activity and weak radical scavenging properties on the DPPH radical [38-39]. 8prenyldaidzein 42, so far reported only in two Erythrina species [21,95], was found to be active against S. aureus (MIC 10.5 ug/ml, tetracycline 0.1 ug/ml, erythromycin 0.2 ug/ml), B. subtilis (10.5 ug/ml, tetracycline 0.2 ug/ml) and Mycobacterium smegmatis (12.5 ug/ml,

839

erythromycin 0.78 ng/ml). Neobavaisoflavone 43, first isolated from the seeds of Psoralea corylifolia [100], and later in a few Erythrina species (Table 13), exhibits weak radical scavenging properties on the DPH radical [38-39]. It also showed antifungal properties against Cryptococcus neoformans (MIC 50 ug/ml) and Aspergillus fumigatus (50 ug/ml), was found inactive against Candida albicans (> 100 ug/ml) and showed antibacterial activity against Gram-positive S. aureus (2.5 ug/ml) [50,97]. It may be worth noting that a comparison between 41, 42 43 and 44 seems to point to the fact that the presence of a 3,3-dimethylallyl (y,Y-dimethylallyl or loosely referred to as prenyl) enhances activity towards bacteria and fungi and that the presence of a prenyl unit at 3 - position (as opposed to 8-position) enhances activity against bacteria and also broadens the spectrum wide enough to make the compound fungitoxic. When a prenyl unit is cyclised as in corylin 44, the activity against fungi (C. neoformans, A. fumigatus and C. albicans) is lost completely [97]. Erylatissin A 45 was only weakly antibacterial and radical scavenging [38-39], while erysubin F 47 was only very weakly anti-MRSA with MIC range of 100 ug/ml and with all 13 MRSA resistant strains [75]. Bidwillon C 46 exhibited activity against Streptococcus mutants (IC50 25 ug/ml), Prevotella intermedia (50 ug/ml) and Porphyromonas gingivalis (50 ug/ml) [19]. Ulexone A 48, first reported from gorse (Ulex europeaeus [101]), later in Ulex jussiaei [102] and so far reported only in E. vogelii [99], showed insect feeding deterrent activity against Costelytra zealandica (Coleopteran) larvae [101] with an FD50 (concentration which reduces feeding by 50%) of 17 ppm. Table 13: Bioactive Isoflavones (Daidzein Derivatives) from Erythrina Species Compound

Erythrina source

Daidzein 41

E. arborescens [76 ]

Estrogenic,antibacterial, weakly DPPH

E. indica [95]

radical scavenging

Activity

E. latissima [38,39] E. lysistemon [40] E. orientalis [94,96] 8-prenyldaidzein 42

E. eriotriocha [21 ]

Antibacterial

E. indica [95]

Neobavaisoflavone 43

E. abyssinica [48] E. latissima [24,56,65] E. sigmoidea [50,97]

Antibacterial, antifungal, DPPH radical scavenging

Corylin

E. sigmoidea [97]

Antifungal

44

Erylatissin A 45

E. latissima [38,39]

Bidwillon C 46

E. x bidwillii [19]

Antibacterial, antifungal

E. variegata [30] E. poeppigiana [29] Erysubin F 47

Antibacterial (anti-MRSA)

E. suberosa var glabrescenses [75] E. variegata [26,98]

E. vogelii [99] Ulexone A* 48 *genistein derivative but included here for convenience

Insect feeding deterrent

840

R1

No

R1

R2

R3

R4

R5

41

H

OH

H

H

OH

42

H

OH

3,3-DMA

H

OH

H

8-prenyldaidzein

43

H

OH

H

3,3-DMA

OH

H

Neobavaisoflavone

44

H

OH

H

3 ,4 (2,2-dimethylchromeno)

H

Corylin

45

H

OH

H

OH

OCH3

3,3-DMA

Erylatissin A

46

H

7,8(2,2dimethyl

H

OH

H

Bidwillon C

OH

H

Erysubin F

H

Ulexone A

R6 H

Name Daidzein

chromeno) 47

H

OH

3,3-DMA

3,3-DMA

48

OH

OH

H

3 ,4 (2,2-dimethylchromeno)

Bioactive Pterocarpans from Erythrina Species Pterocarpans form the second largest class of metabolites reported from Erythrina species after isoflavones but represent the largest number of bioactive compounds in any given subclass (Tables 14-19). A large number of pterocarpans are phytoalexins, that is, antibiotic chemicals that are produced in response to microbial challenge [103]. The activity profile spans "normal" pterocarpans, through 6a-hydroxypterocarpans to 6a, 11 a-dehydropterocarpenes, Fig. (2). The major activity exhibited is antimicrobial (Tables 14-16), but other activities such as inhibition to rabbit platelet aggregation [9] (Table 17), cytotoxicity and potential antitumour [44] (Table 18), antiplasmodial [46] (Table 19), phospholipase A2 inhibitors [36], and antiHIV [47]are shown by this class. The antimicrobial profile (Tables 14-16) can be divided into general antibacterial and antifungal properties (Table 14), antibacterial activity against resistant strains thus giving anti-MRSA properties (Table 15) and activity against cariogenic oral bacterial strains (Table 16). What seems to emerge (though more structure activity work needs to be done) is that for general activity against bacteria, the presence of a 3 or 9- hydroxy (preferably both) and of both 2- and 10-prenyl in a pterocarpans nucleus is essential for the activity (erycristagallin 53, erycristin 54, and erythrabyssin II 56), while for general fungitoxicity, the non-availability of a 9-hydroxy either through a cyclisation process with 10prenyl (phaseollidin 58 vs phaseollin 59) or through methyl ether formation (erythrabyssin I or cristacarpin 55 vs erythrabyssin II 56). It appears that for anti-MRSA, (Tablel5) a general pterocarpans nucleus (erycristagallin 53, orientanol B 65, orientanol C 66 and 2-prenyl-6ahydroxyphaseollidin/demethylerystagallin A 67, sandwicensin [27] 69), with either 3/9-

841 dihydroxy, or 2/10-diprenyl units (53 and 67) or presence of at least one of 3/9 hydroxy and at least one of 2/10 prenyl units (65, 66 and 69 [27]) is essential. The activity against various mutants streptococci strains of cariogenic bacteria [31] (Table 16) reveals the essence of a pterocarpans nucleus. Erycristagallin 53 and sigmoidin K 68 are structurally very similar except that the presence of a 6-oxo functionality makes the latter a coumastan as opposed to a pterocarpan. Sigmoidin K was found to be totally inactive (>100 ug/ml) against nine strains of mutants streptococci and one strain of Lactobacillus casei and showed a very wide variability in activity (6.25->100 ug/ml) against the seven strains of other streptococci but gave good activity range (1.56-6.25 ug/ml) against Actinomyces. Erycristagallin conversely was very active against all organisms with very narrow activity range (mutant streptococci 6.25 ug/ml, other streptococci 3.13-6.25 ug/ml, Actinomyces 1.56 ug/ml, Lactobacillus casei 6.25 ug/ml). The other more active compounds are erystagallin A 60, demethylerystagallin A 67 and orientanol B 65 in that order. The presence of either one or both 3/9 hydroxyls and one or both of 2/10 prenyl units seems important for activity. It appears that activity is greatly enhanced by the presence groups (2,10-diprenyl, 3,9-dihydroxy) (53, 67), attenuated by the presence of both a single 2-prenyl and a 9-hydroxy (65, 60), while the absence of both of these groups reduces or removes the activity completely (55, 61). The antibacterial effect of erycristagallin to mutant streptococci was based on bactericidal action. Erycristagallin (6.25 ug/ml: MIC) also completely inhibited radio-labelled incorporation of thymidine into Streptococcus mutants cells, as well as the incorporation of radio-labelled glucose into bacterial cells. This points to the potential that erycristagallin has as a phytochemical agent for prevention of dental caries by inhibiting the growth of cariogenic bacteria and by interfering with the incorporation of glucose responsible for the production of organic acids. It is important to note that according to criteria for resistance of MRSA to methicillin and oxacillin strains are defined as resistant if they are not inhibited at a concentration of above 12.5 ug/ml, and as sensitive if they are inhibited at/or below this concentration [26]. Cristacarpin 55, erythrabyssin II 56, phaseollidin 58 and phaseollin 59, were tested for their inhibitory effect on rabbit platelet aggregation [9] (Table 17) and only erythrabyssin II showed significant activity. An important feature of many tumour cells is that they have defects in their ability to repair damage to DNA as compared to normal cells. Therefore, agents with selective toxicity towards repair-deficient cells might be potential antitumour agents. In this respect, DNA repair-deficient and repair-proficient yeast mutants can be used to screen for potential antitumour agents. The chloroform extract of the bark of E. burana showed selective activity against the repair-deficient rad 52 yeast strain. Activity-guided fractionation of the chloroform extract [44] yielded compounds cristacarpin 55 and phaseollidin 58. Both compounds showed selective activities in rad 52 and rad 321 assays compared to with the wild-type RAD+ strain. Cytotoxicity activities of cristacarpin and phaseollidin were determined in three cell lines (Table 16). Phaseollidin was found to be moderately active in the wild-type CHOC cytotoxicity assay, while cristacarpin, interestingly, was inactive against wild-type CHOC but showed activity against a P-glycoprotein overproducing cell line [44], showing unusual reversal of activity. It is also interesting to note that the roots of £. abyssinica have long been used in ethno-medicine to treat various ailments such as malaria and syphilis [3], Recent work by Yenesew and co-workers [46] on the acetone extract of the roots of E. abyssinica showed potent activity against the chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum with IC50 values of 0.49 and 0.64 ug/ml respectively, explaining its wide traditional use as an antimalarial [3]. This extract yielded, among others, pterocarpans isolates [46], erythrabyssin II 56, erycristagallin 53 and eryvarin D (we found it to be identical to 3-hydroxy-9-methoxyl0-prenylpterocarpene) 62, with antiplasmodial activity against W2 and D6 strains of Plasmodium falciparum (Table 19). Erythrabyssin II was found to be the

842 most active of the pterocarpans tested [46]. Erycristagallin was also reported as a pterocarpans isolate from a Samoan E. variegata whose bark extract exhibited strong PLA2 inhibition activity [36]. Three isolates were obtained from this plant and erycristagallin gave the strongest inhibition of the three with an IC50 value of 3 uM (trifluoropirazine at 20 \xM showed >80% inhibition under these assay conditions) [36]. The ethyl acetate extract from E. mildbraedii, in the carrageenin induced mouse paw oedema, showed anti-inflammatory activity [35], and erycristagallin was isolated as the active ingredient. The erycristagallin from this extract, in vivo, significantly inhibited the PLA2-induced mouse paw oedema, as well as the mouse ear oedema induced by TPA (ID50 < 10 ug/ear). It further reduced significantly the chronic inflammation and leukocyte infiltration induced by repeated application of TPA. In vitro, erycristagallin inhibited the arachidonic acid metabolism via the 5-lipoxygenase pathway in rat polymorphonuclear leukocytes (IC50 23.4 (xM), but had no effect on cyclooxygenase-1 metabolism in human platelets, while showing antioxidant activity in the DPPH test. The anti-inflammatory activity of erycristagallin, as with other phenolics, may be based on its capacity to inhibit arachidonic acid metabolism via the 5-lipoxygenase pathway [35]. HIV inhibitory pterocarpans 3-0- methylcalopocarpin (we found to be identical to orientanol B) 65 and sandwicensin 69 were isolated from an organic bark extract of a Guatemalan E. glauca that showed preliminary anti-HIV activity [47]. The two compounds inhibited the cytopathic effects of in vitro HIV-1 infection in human T-lymphoblastoid cell line (CEM-SS). Orientanol B (3-O-methylcalopocarpin) was cytoprotective over a modest concentration range (EC50 0.2 ng/ml; IC50 3.0 ug/ml) with a maximum of 80-95% protection, while sandwicensin was less effective (EC50 2 ug/ml; IC50 7 ug/ml), with a maximum protection of only 50-60%. Compound 69 was also very recently found to be weakly anitbacterial against S. aureus, B. subtilis and E. faecalis with MIC values of 50, 100 and 50 ug/ml respectively [32b], Table 14: Erythrina Pterocarpans with Antibiotic Activity Bacteria (MIC in ug/ml)

Yeast

Compound

Fungus

(Ug/ml) S.a

Erybraedin A 49

B.s

Ms

12.5

6.25

Erybraedin C 50

12.8

12.5

Erybraedin D 51

78.3

Erybraedin E 52

22.1

Erycristagallin 53

1.56

Erycristin

54

6.25

6.25

1.56

Eryabyssin I 55

12.5

6.25

3.13

3.13

S.m

Pg

A.a

6.3

6.3

6.3

M.I

(H-g/ml)

S.c

C.u

S.I

M.m

R.c

50

50

6.25

25

*

*

*

*

(cristacarpin) Erythrabyssin 56

II

0.78

3.13

*

Isoneorautenol 57

25.0

Phaseollidin 58

50

25

25.0 *

*

#

*

*

Phaseollin 59

12.5

6.25

25

50

12.5

12.5

12.5

S.a = Staphylococcus aureus, B.s = Bacillus subtilis, M.s ~Mycobacterium smegmatis, S.m = Streptococcus mutant, P.g.= Porphyromonasgingivalis,

A.a = Actinobacillus actinomycetencomitans, M.I = Micrococcus lysodeikticus, S.I =

Saccharomyces cerevesiae, C.u = Candida utilis, S.I = Sclerolinia libertiana, M.I = Mucor mucedo, R.c = Rhizopus chinensis. * = > 100, # = >50

843 Table 15: Anti-MRSA Activity of Pterocarpans from the Genus Erythrina

Antibiotic parameter (in ng/ml)

Proportion

Compound

Anti-MRSA range

MIC50

MIC,o

of resistant strains

Cristacarpin SS (Erythrabyssin 1)

100

100

100

13/13

Erycristagallin

53

3.13-6.25

6.25

6.25

0/13

Erystagallin A

60

12.5-25

25

25

9/13

Eryvarin A

61

>100

>100

>100

13/13

Eryvarin D

62

12.2-25

25

25

9/13

Eryvarin E

63

25-M00

>100

>100

13/13

Folitenol

64

6.25->100

12.5

50

6/13

Orientanol B 65 (3-O-methylcalocarpin)

3.13-6.25

6.25

6.25

0/13

Orientanol C

66

6.25-25

12.5

12.5

2/13

Phaseollidin

58

25-50

50

50

13/13

Phaseollin

59

25

25

25

13/13

Demethylerystagallin A 67 (2-prenyl-6a-hydroxyphaseollidin)

6.25-12.5

12.5

12.5

0/13

Methicillin

12.5->100

>100

>100

13/13

Oxacillin

12.5->100

>100

>100

13/13

IC50 and IC90 cone, needed to inhibit 50% (seven strains) and 90% (12 strains) of strains tested Table 16: Activity of Erythrina Pterocarpans Against Cariogenic Oral Bacteria [31].

MIC range in ng/ml

Compound Mutants streptococci (9 strains)

Other streptoc

Actinomyces

Lactobncillus

occi (7 strains)

(3 strains)

casei (1 strain)

Erycristagallin 53

6.25

3.13-6.25

1.56

6.25

Cristacarpin

100

50-100

25

100

Demethylerystagallin A 67

12.5-25

6.25-12.5

3.13

12.5

Erystagallin A

60

6.25-12.5

6.25-12.5

3.13

12.5

Eryvarin A

61

>100

>100

100->100

>100

Orientanol B

65

6.25-12.5

6.25-12.5

6.25

6.25

>100

6.25->100

1.56-6.25

>100

55

(3-O-methylcalocarpin) Sigmoidin K. 68

844 Table 17: Inhibition of Rabbit Platelet Aggregation (IC50 in (xg/ml) by Pterocarpans from E. abyssinica [9|.

Compound

Inhibition induced by ADP (10 uM

Collagen (38 ng/ml)

"

74 (slight inhibition)

74

Erythrabyssin II 56

23

9

34

Phaseollidin

58

30

23

34

Phaseollin

59

30

36

80

Arachidonic acid (0.57 u,M)

Erythrabyssin I 55 (cristacarpin)

Table 18: Bioactivity Data for Pterocarpans 55 and 58 Isolated from E. burana |44|

Compound

Organism (IC]2 in )xg/ml RS322YK

Cristacarpin 55

RS167N

RS321

Cell line (IC50 in jalVI) RS188N

P-388

CHOC

CHOC-

+

rad52

rad 6

rad 321

RAD

80

540

108

330

>10.0

>20.0

4.0

500

>1000

400

5500

>10.0

4.0

7.6

PGO

erythrabyssin 1 Phaseollidin 58

P-388= wild type P-388 murine leukaemia cells; CHOC = wild type Chinese hamster ovary cells; CHOC-PGO = Pglycoprotein overproducing Chinese hamster ovary cells. Table 19: In vitro Antiplasmodial Activity of Pterocarpans from Root Bark of E. abyssinica [46] Against Strains W2 and D6of Plasmodium falciparum.

IC50 in uM

Compound W2 (chloroquine-resistant) Erycristagallin

D6 (chloroquine-sensitive) 19.0

53

20.1

Erythrabyssin II 56

6.5

8.1

Eryvarin D

20.6

21.9

Chloroquine

0.093

0.008

Quinine

0.21

0.042

62

845

No

R1

R2

R3

R4

R5

49

H

OH

3,3-DMA

H

OH

50

H

OH

3,3-DMA

3,3-DMA

OH

H

Erybraedin C

51

H

OH

3,3-DMA

8,9(2,2 -dimethyl-

H

Erybraedin D

R' 3,3-DMA

Name Erybraedin A

chromeno) 52

2,3-furano

H

H

OH

3,3-DMA

Erybraedin E

54

3,3-DMA

OH

H

H

OCH3

3,3-DMA

Erycristin

56

3,3-DMA

OH

H

H

OH

3,3-DMA

Erythrabyssin II

57

H

OH

H

8,9(2,2-dimethyl

H

Isoneorautenol

3,3-DMA

Phaseollidin

chromeno) 58

H

OH

H

H

OH

59

H

OH

H

H

9,10(2,2dimethyl-

Phaseollin

chromeno) 64

3,3-DMA

OH

H

H

9,10(2,2dimethyl-

Folitenol

chromeno) 65

3,3-DMA

66

2,3(2,2-dimethylchromeno)

69

H

OCH3

H

H

OH

H

Orientanol B (3-O-

H

H

OH

3,3-DMA

Orientanol C

H

H

OCH3

3,3-DMA

Sandwicensin

methylcalopocarpin)

OH

846

No

R1

R!

R3

R4

R5

55

H

OH

H

H

OCH3

60

3,3-DMA

OH

H

H

OCH3

61

H

OCH3

H

H

67

3,3-DMA

OH

H

H

R« 3,3-DMA

Name Cristacarpin (Erythrabyssin I)

3,3-DMA a

OH

Erystagallin A Eryvarin A

3,3-DMA

Demethylerystagallin A 2-prenyl-6a-hydroxyphaseollidin

a =

o

R1

No

R1

R2

R3

R4

R5

53

3,3-DMA

OH

H

H

OH

62

H

OH

H

H

OCH3

R6 3,3-DMA 3,3-DMA

Name Erycristagallin Eryvarin D (3-hydroxy-9-methoxy-l 0pren ylpterocarpan)

63

3,3-DMA

OH

H

H

OCH3

3,3-DMA

Eryvarin E

68

3,3-DMA

OH

H

H

OH

3,3-DMA

Sigmoidin K

3. Bioactive Isoflavanones from Erythrina Species Eriotrichin B 70 (also called Bidwillon A) was found to be antibacterial against S. aureus with MIC50 of 8.3 ug/ml [20], while 2,3-dihydropratensein (5,7,3 -trihydroxy-4 methoxyisoflavanone) 71 showed antifungal activity against Cladosporium cucumerinum (2 jj.g) in a TLC bioautography preliminary assay [12]. Orientanol F 72 showed Anti-MRSA

847 against 13 strains with a range of 3.13-12.5 ug/ml, MIC50 of 6.25 ug/ml and MIC90 of 12.5 Hg/ml [26], while eryzerin A 76 showed sensitivity to 3 out of 13 strains (MIC50 = MIC90 =25 ug/ml) and eryzerin B 77 was inactive with all 13 tested strains resistant (MIC50 = MIC90 =>50 ug/ml) [28]. The organic extract from the roots of a Tanzanian strain E. lysistemon was found to be active in the NCI's primary anti-HIV screen [47], and the activity-guided isolation gave modestly active 5-deoxyglasperin F 73 (EC50 11.5 ug/ml) and 2,3- -dihydro-2hydroxyneobavaisoflavone 75 (EC50 7.6 ug/ml) and barely active 5-deoxylicoisoflavanone 74 together with three other inactive isoflavonoids. Table 20: Bioactive Isoflavanones from Erythrina Species

Compound

Erythrina source

Eviotrichin B 70

E. eriotriocha [20]

(Bidwillon A)

E. orientalis [105]

2,3-dihydropratensein 71

E. berteroana [12]

Orientanol F 72

Activity Antibacterial (S. aureus 8.3 |ag/ml)

Antifungal (Clndosporium cucumerinum 2

E. latissima [24]

Hg)

E. orientalis [105]

Anti-MRSA (MICso 6-25 fig/ml)

E. variegata [26] E. lysistemon [47 ]

Anti-HIV (IC5o 11.5 ug/ml)

flavanones 74

E. lysistemon [47]

Anti-HIV (weak)

2,3-dihydro-2 -hydroxy

E. lysistemon [47]

Anti-HIV (lC5o7.6|ig/ml)

5-deoxyglasperin F 73 5-deoxylicoiso-

-neobavaisoflavone 75 Eryzerin A 76

E. zeyheri [28]

Anti-MRSA (M1C5O 25 ug/ml)

Eryzerin B

E. zeyheri [28]

Anti-MRSA (M1C50>50 ug/ml)

77

848

No

R1

R2

R3

R4

70

H

3,3DMA

OH

3,3DMA

OH

71

OH

H

OH

H

H

72

H

3,3DMA

7,8(2,2-dimethylchromeno)

H

73

H

H

OH

H

74

H

H

OH

H

R5

R' H

R7

R8

Name

OH

H

Eriotrichin B

OH

OCH3

3,3DMA

2,3-dihydropratensein

OH

OH

H

Orientanol F

2,3 (2,2-dime

OH

H

5-deoxyglaperin F

3 ,4 (2,2-dime-

H

5-deoxylicoisoflavanone 2,3-dihydro-2-hydroxy

thylchromeno) OH

thylchromeno) 75

H

H

OH

H

OH

76

H

3,3DMA

OH

H

77

H

3,3DMA

OH

3,3DMA

3,3DMA

OH

H

OH

3,3DMA

OH

H

Eryzerin A

OCH3

H

OH

H

Eryzerin B

neobavaisoflavone

4. Bioactive Isoflavans from Erythrina Eight isoflavans have (i.e erythribidin A, phaseollinisoflavan, methoxyphaseollinisoflavan, 2 O-methylphaseollidinisoflavan, eryzerins D and E, eryvarin C, and 4 -O-methylglabridin), to date, been reported in Erythrina genus and only three viz eryzerin C 78, eryzerin D 79 (both isolated from E. zeyheri [28] ) and eryvarin C 80 (from E. variegata [26,98]) have been reported to have some anti-MRSA activity. Eryzerin C gave MIC50 and MIC 90 values of 6.26 ug/ml each, inhibiting all 13 strains tested, while eryzerin D was slightly less active with MIC50 and MIC90 values of 12.5 ng/ml each, again inhibiting all 13 strains tested.

OH R1

No

R1

R2

R3

Name

78

3,3-dimethylallyl

OH

3,3-dimethylallyl

Eryzerin C

79

6,7(2,2-dimethylchromeno)

3,3-dimethylallyl

Eryzerin D

80

6,7(2,2-dimethylchromeno)

H

Eryvarin C

849 5. Bioactive isoflav-3-enes from Erythrina Seven isoflav-3-enes viz eryepogin A [27,72] (burttinol B [64]), eryepogin B [72] (burttinol C [64]), burttinol A [64], 7,4 -dihydroxy-2,5 -dimethoxyisoflav-3-ene [46], bidwillol A [19,106], eryvarins H and I [30], have to date been reported in the genus, and of these only two have been reported to be biologically active. 7,4 -dihydroxy-2 ,5 -dimethoxyisoflav-3-ene 81 isolated from E. abyssinica [46] was reported to have antiplasmodial activity against chloroquine-resistant (W2: IC50 27.2 uM) and chloroquine-sensitive (D6: IC50 18.2 uM) strains of Plasmodium falciparum. Bidwillol A 82, reported from E. x bidwillii [19] and E. orientalis [106], was found to be weakly active against Lactobacillus fermentum (IC50 25 ug/ml) and Actinomyces naeslundii (IC50 25 ug/ml [19]. OMe

81: R1 = H, R2 = OCH3

7,4 -dihydroxy-2 ,5 -dimethoxyisoflav-3-ene

82: R' = 3,3-dimethylallyl, R2 = H

Bidwillol A

6. Bioactive Coumastans from Erythrina Five coumastans (4-hydroxycoumasterol [50], Indicamines A [95] and B [22], robustic acid [95] and sigmoidin K [26,104]) have been reported to date in the genus, and only two of these have been reported to have biological activity. Indicamine B 83 was isolated from the root bark of E. indica [22] and was found moderately active against S. aureus (MIC50 9.7 (ig/ml, streptomycin 5.5 ug/ml) and M. smegmatis (18.5 ug/ml, streptomycin 1.7 ug/ml) but inactive against E. coli (>1000 ug/ml, streptomycin 5.0 p.g/ml). Sigmoidin K 68 has been discussed under bioactive pterocarpans. ^0. 6

1^ OMe

l

OH

J3 \ ^

L "OH

83: Indicamine B

VII.

BIOACTIVE CINNAMATE ESTERS FROM ERYTHRINA

Cinnamate esters are the only non-flavonoid (word used in a very general sense) and nonalkaloidal, bioactive, identifiable secondary metabolites reported in the genus. Cinnamate esters have been reported in several Erythrina species [24,48,56,90,107-108]. Several cinnamate esters have been reported in the genus and these include erythrinasinate (octacosanyl E isoferulate), erythrinasinate B (octacosanyl E ferulate), erythrinasinate C (tetradecanyl E ferulate), erythrinasinate D (hexacosanyl E isoferulate), hexacosanyl E

850 ferulate, and triacontanyl 4-hydroxycinnamate. The three esters 84-86, isolated from E. senegalensis and E. excelsa, were thoroughly screened for pharmacological activity [107], involving over fifty tests. The two esters, erythrinasinate B 84 and erythrinasinate 86 were isolated from E. senegalensis [21,108] while 85 hexacosanyl E ferulate was reported from E. excelsa [107]. The three esters exhibited activity that could be broadly described as having CNS, cardiovascular and metabolic. Compound 84 was completely non toxic when administered orally or peritoneally. Its CNS activity was manifested by reflex depression, behavioural depression, muscle relaxant and antielectric shock properties. It showed antiarrhythmic effects (cardiovascular agent) as well as aquaretic properties (metabolic agent). Compound 85 was non toxic when administered orally or peritoneally. It exhibited reflex depression, behavioural depression, muscle relaxation, cholinergic activation, antiarrhythmic and aquaretic properties. Compound 86 was completely non toxic regardless of whether it was administered orally or peritoneally and it showed reflex depression, behavioural depression, muscle relaxant, cholinergic activation, anti-electric shock, antiarrhythmic and aquaretic properties.

O

No

R1

R2

R3

Name

84

H

OCHj

-(CH 2 ) 27 CH 3

Erythrinasinate B

85

H

OCH 3

-(CH 2 ) 25 CH 3

Hexacosanyl E-ferulate

86

CH3

OH

-(CH 2 ) 27 CH 3

Erythrinasinate

VIII.

CONCLUSION

The survey revealed that the genus Erythrina was very rich in secondary metabolites particularly of the flavonoids class. The bioactivity profile represented the various classes fairly reasonably but it became apparent that a number of these isolates have not yet been tested for biological activity. The challenge remains for researchers to carry more work on these to fill in the knowledge gaps that still exist. The survey revealed very close agreement between ethno-medical use of the various Erythrina extract preparations (Table 1) and the results of biological activity (Table 2). The reported activities of pure isolates strongly support the documented ethno-medical uses and reported pharmacological activity on the various extracts which are, by and large, mostly microbial related. It was also interesting to note that some compounds showed high efficacy against resistant organisms - a very important aspect - since most used drugs especially antibiotics tend to produce resistance to certain strains of organisms. It also emerged from the survey that certain structural features were essential for certain activities. Compounds that were found active were usually effective against not just one but several disease functions. One cannot help but surmise that more activities are yet to be reported for these isolates.

851 ACKNOWLEDGEMENTS RRTM gratefully acknowledges grants from University of Botswana Research and Publication Committee (UBRPC-R475) and IFS (F/2698-2). CCWW and BFJ acknowledge DAADANSTI and DAAD-NAPRECA respectively for scholarships. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10]

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

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CHEMICAL CONSTITUENTS AND PHARMACOLOGY OF ARISTOLOCHIA SPECIES TIAN-SHUNG WU*, AMOORU G. DAMU, CHUNG-REN SU, AND PING-CHUNG KUO Department of Chemistry, National Cheng Kung University, Tainan, Taiwan; Tel: 886-6-2747538. Fax: 886-6-2740552 E-mail: [email protected] ABSTRACT: Aristolochia species have long been known for their wide use in traditional medicine and have attracted intense research interest because of their numerous biological activity reports and unique constituents, aristolochic acids. Aristolochia species are sources of a number of physiologically active compounds of different classes. Aristolochic acid derivatives with various carbon skeletons, aporphines, benzyliso- quinolines, isoquinolines, protoberberines, protopines, amides, chlorophylls, mono-, sesqui-, and diterpenoids, lignans, biphenyl ethers, flavonoids, tetralones, benzenoids, and steroids have been identified from different Aristolochia species. The major focus of recent research is on the negative aspects of aristolochic acids because of the nephro- toxicity of some aristolochic acids. This report addresses the complex array of the biologically active and chemically diverse metabolites identified from Aristolochia species during the past seventy years, in addition to biosynthetic studies, ecological adaptation and chemotaxonomy to show the rapid development in the phytochemistry and pharmacology of the Aristolochia species.

INTRODUCTION The family Aristolochiaceae with seven genera: Apama, Aristolochia, Asarum, Euglypha, Holostylis, Saruma and Thottea is nested with in the magnoliid (or eumagnoliid) clade of primitive angiosperms, which include Piparales, Magnolidales, Laurales, Wintarales, Chloranthaceae and monocotyledons.There are about 600 to 700 species, most of them in the genus Aristolochia (ca. 500 species) and most of the rest in Asarum (ca. 100 species). The majority of the Aristolochiales are tropical, though a number of them range as far north as Canada, Scandinavia, and Northern Japan. They may grow as climbing vines, as short creeping herbs and a few are shrub-like [1-3]. Aristolochia species are herbaceous perennials, under shrubs or shrubs, often scandent, scrambling, twining, sometimes lianas, usually with prostrate or tuberous rhizomes or rootstocks, and alternate, pinnate, polymorphic or lobed leaves bearing essential oils. The

856 flowers are axillary, bilaterally symmetric, tubular and calyx usually mixture of purple, brown, green or red with fetid odors. Fruits are dry capsules with flattened or rounded seeds. Species of Aristolochia were widely distributed in tropical, subtropical and temperate regions of the world. They are known to occur in Asia, Africa, North and South America and Australia but there is a wide distribution across tropical Asia. Members of Aristolochia have been cultivated in gardens as ornamentals due to their attractive leaves and flowers, often with bizarre colors or patterns [4-7]. Some oddly shaped flowers have been given names like "bird's head" and "Dutchman's pipe". Many species of Aristolochia have been used in the folk and traditional medicines as medicaments and tonics (Table 1) [8-14]. Especially, the use of Aristolochia species in Chinese popular medicine has a long tradition [15-19]. Some species have been used in the form of crude drugs as anodynes, antiphlogistics and detoxicants in Mainland China. The mature fruits of A. debilis, known in China by the name, Madouling are still used for the treatment of snakebite, tuberculosis and as antihypertension [20]. The dry roots of A. indica are reputedly used in Indian folk medicine as an emmenagogue or abortifacient [21-24]. A. albida, native of West Africa has been used in traditional medicine for various ailments including skin diseases, dysentery, gastrointestmalcolics and snakebites [25-27]. The roots of A. argentina, popularly known as Charrua or Charruga are used in the Argentinean folk medicine [28,29] as an emmenagogue and for treating arthritis, poisoning and pruritus. A. bracteata, an Indian medicinal plant [30] is reputed for its efficacy as anthelmintic, purgative, emmenagogue, and for expelling round worms. The rhizomes of A. brevipes, commonly known as guaco are used by the local Tarasc people [31] to treat arthritis and diarrhea and also applied to cure wounds from snakebites. A. chilensis is known by the vernacular names ' Oreja de zorro' (fox ear) and hierba de lavirgen maria (virgin mary's herb) and a decoction of its roots [32,33] was drunk at least well in to the second half of last century to reduce abundant lochia (puerperal secretions). The roots of A. cinnabarina are used as painkiller in Chinese folk medicine [34]. The aerial parts of A. constricta, a medicinal plant forward in Equador and South America are empirically used in folk medicine [35-37] as antispasmodic, anthelmintic, emmenagogue and against snakebites. The aerial parts of A. grandiflora are said to have antimicrobial, uterotonic, and cytotoxic properties and also used for treating snakebites [38,39]. The fruits and roots of A. zollingeriana, an endemic species [15] of Taiwan have been used as an alternative for the famous Chinese medicine 'Madouling', as an analgesic,

857

Table 1. Traditional/folklore medicinal uses of Aristolochia species Botanical name A. acutifdia

Trivial name Jiquiro, Jarrinha

A. albida

A. argentina

Charrua, Charruga

A. baetica A. birostris

A. bracleala

A. brevipes

A. chamissonis A. chilensis

Part used whole plant

Papular use treatment of erysipelas

References 47

rizhomes

ailment for skin diseases; treatment of dysentery, gastrointestinal colics and snakebites; as an adjuvant as emmenagogue, antiseptic, diaphoretic and diuretic; treatment of arthritis, poisoning, and pruritus as emmenagogue; in cancer in cancer

25,26,27

as emmenagogue, anthelmintic, purgative, mosquitoes repellent, antidote, anodyne and insecticide treatment of arthritis, wounds, snakebites and toothache in debility treatment of abundant lochia (Puerperal secretions); as emmenagogue as analgesic and pain killer treatment of cancer, menstrual troubles, legulcer, wound and tumor; as depurative and insect repellent in cancer

30

aerial parts

roots Capivara, Angilico, Jarrinha, Ukulwe, Bracteated birthwort

Guaco

Oreja de zorro, Hierba de la virgin maria

roots

whole plant

rhizomes

whole plant roots

Sichuan Zhusalian Upright birthwort

roots

roots

A. constricia

Pipevine, Ma dou ling, Tian xian teng Saragez

A. cucurbitifolia

Qing muxiang

A. cinnabarina A. clematits

A. contorta

roots

aerial parts

fruits, roots

as antispasmodic, analgesic, anticancer, antimalarial and antiinflammatory; as emmenagogue; treatment of snakebites as anodynes, antiphlogistics, expectorant, antitussive

28,29

48,49 50,51

31,52 53

32,33

34 54

55

35,36,37

56

858

A. debilis

A. gibertii

Ma dou ling, Qing muxiang, Seimokkou, Birthwort long, Chinese fairly vine, Birthwort Mil hombres hembra, Contrayerba, Patito

roots

and antiasthmatic; treatment of snakebites and lung inflammation as bronchiectatic; to decrease high blood pressure

20

whole plant

treatment of swelling; in stomachpain; as ailments

57

A. gigantea

whole plant

58,59

A. grandiflora

aerial parts

as emmenagogue, abortive and antiseptic; treatment of wounds and skin diseases as uterotonic, cytotoxic and antimicrobial; treatment of snakebites; as flies and maggots repellent as expectorant, antitussive, antiasthmatic and analgesic; treatment of snakebites as emmenagogue, cardiotonic, diuretic, antiinflammatory, abotifacient and mild sedative; against intestinal worms poisonous bites and stings treatment of asthma, cough and piles; as antivenomous, antibacterial, antipruritic, hypotensive, expectorant, and emetic as expectorant and antitussive, analgesic and antiasthmatic as antiphlogistic, detoxicant and anodyne treatment of cancer, sclerosis, uterus tumor and nose cancer treatment of snakebites and rheumatism; as circulatory stimulant, antiinflammatory, antiseptic and abortive as bronchiectatic;

A. helerophylla

Yellowmouth, Dutchman's pipe

fruits, roots

A. indica

Indian birthwort

roots

A. kaempferi

Yellowmouth, Dutchman's pipe

whole plant

A. kankauensis

fruits, roots

A. liukiuensis

roots

A. longa

Tian xian teng

whole plant

A. macroura

Mil hombers, Patito Coludo, Jarrinha, Isipomilhombres

whole plant

A. manshuriensis

Manchurian

whole plant

15

15

21,22,23,24

60

15

61 62,63

64

65,66,67,68,

859

A. maurorum

birthwort, Manchurian Duchman's pipe, Guan mu tong, K.an-mokutsu, Mokuboi, Kwangbanggi Zarand

A. mollissima

Xun gu feng

fruits, roots

A. paucinervis

Barraztam

whole plant

roots

whole plant

A. rotunda A. rodriguesii

Sangue-de-Cristo

roots, aerial parts

A. triangularis

Mil hombres

bark

roots

A. tuberosa

A. yunnanensis

Yunnan ma douling, Nan mu xiang

roots

A. zollingerinnn

Ma douling

fruits, roots

to decrease high blood pressure

69,70

as antiseptic; in wound healing; for scab of sheep as analgesic, anticancer, antimalarial, antiinfiammatory, antirheumatic and bronchiectatic; to decrease high blood pressure; treatment of stomach ache and abdominal pain treatment of skin infections, gas gangrene, abdominal pain and infections of the upper respiratory tract in cancer; as depurative as abortifacient and antiinfiammatory; treatment of snakebites as antirheumatic, antifertility, diaphoretic, diuretic, antiseptic, emmenagogue, antidote, and abortive; treatment of wounds and skin diseases treatment of sore throat, venomous snakebites, and tuberculosis treatment of gastrointestinal diseases, tricomoniasis, and various pains as expectorant, painkiller, antitussive, antiasthmatic, and analgesic; Treatment of snakebites

71,72

40

73, 74, 75

76 77

41,42,43,44

78

45,46

79

expectorant, antitussive, antiasthmatic and also for the treatment of snakebite and lung inflammation. The roots and fruits of A. mollissima [40] ("Xun Gu Feng" in Chinese) are employed as analgesic, anticancer, antimalarial and anti-inflammatory agents and also for the treatment of

860 stomach ache, abdominal pain and rheumatism. A. triangularis, a medicinal plant [41-44] found in South America was used in the treatment of wounds and skin diseases, as emmenagogue, antidote, abortive, antirheumatic, antiseptic and also as tonic agent by local people. A. yunnanensis [45,46] (Yunnan Ma Do Ling) is a Chinese crude drug, Nan Mu Xiang recommended for gastrointestinal diseases, trichomoniasis, and various pain conditions in traditional medicine. ETHNOPHARMACOLOGY Several bioactivity studies have been reported to assess the traditional uses of Aristolochia species. Table 2 summarizes the biological activities of the members of Aristolochia. Lee et al. reported that the extract of A. debilis [80] showed potent inhibition of COX-2 activity and iNOS activity in lipopolysaccharide (LPs)- induced mouse macrophages RAW 264.7 cells. Extracts of the whole plant of A. grandiflora [81] showed moderate neutralization ability against the heamorrhagic effect of Bothrops atrox venom. Some of Aristolochia species have been reported to possess insecticidal and repellent activity. For example, A. clematitis used as insect repellent, A. grandiflora used against flies and maggots [82] and A. bracteata extracts showed clear activity against mosquitoes [83]. The essential oil of A. indica [84] found to show antibacterial activity. An extract of A. indica showed reproducible tumor inhibitory activity [85] against the adenocarcinoma 755 test system. The crude petroleum ether, chloroform, and alcoholic extracts of the roots of A. indica [89-92] were found to exert 100 % abortifacient activity in mature female mice. Methanolic extract of A. macroura [93] showed cytotoxicity against a human hepatocelluar carcinoma cell line, HepG2. The deffated chloroform fraction obtained from the rhizomes of A. paucinervis [94] has a high bacteriostatic activity against bacterial strains like Clostridium perfringens ATCC13124 and Enterococcus faecalis ATCC 29212. Another study indicated that the defatted chloroformic rhizome fraction of A. paucinervis [95] was most active against Clostridium perfringens, Clostridium difficile, Enterococcus faecalis, Micrococcus lutens and Bacillus subtilis. The defatted chloroform fraction of leaves of this plant also possessed a high bacteriostatic activity against both anaerobic and aerobic strains. Results of this study support the use of A. paucinervis in Moroccan traditional medicine to treat skin and soft-tissue infections, especially gas gangreen and intestinal diseases. Bio-screening study reported by Coussio et al. [96] indicated that A. triangularis contained

861

Table 2. Ethnopharmacological reports on Aristolochia species Botanical name A. albida

Plant material rhizomes

A. argentina

aerial parts

A. bracteata

seeds

A. bracteolate A. constricta A. debilis

whole plant whole plant whole plant

A. elegans A.fructus A. grandiflora A. indica

A. macroura A. manshuriensis

A. mollissima A. (iff. orbicularis A. papillaris A. paucinervis

A. taliscana A. triangularis

A. trilobata

whole plant

Bioactivity insecticidal antifeedant an ti spasmodic insecticidal, antibacterial, antifungal antiinflammatory, antibacterial, analgesic; toxicity to goat antiplasmodial anti spasmodic inhibition of testosterone-5ccreductase; inhibition of COX-2 activity, inhibition of iNOs activity; inhibition of lipid peroxide formation; inhibition of melanin formation

antimitotic; antiviral whole plant leukotriene B4 receptor antagonist activity whole plant neutralizing ability against the haemorrhagic effect aerial parts antibacterial; roots tumor inhibitory; abortifacient activity; interceptive activity; antispermatogenic effect cytotoxicity leaves cell suspension culture cardiotonic, whole plant antihypoxic activity; synergistic effect for pesticides; inhibition mutagenicity of Trp-P-1 whole plant antirheumatic arthritis; conctraceptive roots repellent effect against the corn borer Sitophilus zeamais smooth muscle relaxant activity whole plant bacteriostatic, bactericidal; rhizomes, leaves antibacterial; smti-Helicobacter pyroli acitivity; antidermatophytic, antifungal trypanocidal roots cytotoxicity, whole plant inhibition of growth of crown gall tumors; typical c-mitotic action antibacterial; leaves, bark antiphlogistic, antiinflammatory

Ref. 100 99 101 102

104

105 106 107 108 80

109, 110 111 112 113 81 84 85 86,92 89,91 90 93 114 115 116 117 118 119 120 94 95 95 121 122 96

123 97 98

862 cytotoxicity against KB cells and inhibited the growth of crown gall tumors. The hexane extracts of A. trilobata leaves [97] and bark were most active against Staphylococcus aureus and validated its traditional use to heal the deep and surface wounds. The chloroform extract of A. trilobata leaves [98] showed anti-inflammatory effect with an ID50 value 108 )ug/cm2 comparable to that of indomethacin (93 ug /cm2). The phytochemistry and pharmacology of Aristolochia has evoked a great deal of interest because of their numerous traditional uses and number of enthopharmacological reports on their crude extracts. Consequently, Aristolochia has become one of the most intensely investigated genera and a large number of papers have been published on the production of physiologically important metabolites by Aristolochia. Thus the primary aim of this chapter is to bring up-to-date, in graphical form, those data with the metabolites of Aristolochia which have appeared in the literature up to June 2004, concerning the isolation, structural elucidation, biological activity and literature references. CHEMICAL CONSTITUENTS Over the last seventy years over sixty species of Aristolochia have been exploited for chemical examination by research groups throughout the world and a variety of compounds have been isolated. The spectrum of physiologically-active metabolites from Aristolochia species covers 14 major groups based on structure: aristolochic acid derivatives, aporphines, amides, benzylisoquinolines, isoquinolones, chlorophylls, terpenoids, lignans, biphenyl ethers, flavonoids, tetralones, benzenoids, steroids, and miscellaneous. The aristolochic acid derivatives, host of phenanthrene derived metabolites were further classified into aristolochic acids, sodium salts of aristolochic acids, aristolochic acid alkyl esters, sesqui- and diterpenoid esters of aristolochic acids, aristolactams, denitroaristolochic acids, and aristolactones. The terpenoids can further be subdivided into 4 groups: mono-, sesqui-, di- and tetraterpenoids. Aristolochic acids

The constituents of the Aristolochia have became the active subject of a phytochemical and pharmaceutical work since the discovery of compounds belonging to the aristolochic acids group. The naturally occurring aristolochic acids, with 3,4-methylenedioxy-10-nitrophenanthrenic-1-acid skeleton are typical constituents of the Aristolochia, as well

863 as the butterflies that feed on such plants and are claimed to be responsible for the biological activity of Aristolochia species. The first paper on the aristolochic acids from the genus Aristolochia which appeared in 1943 by Rosenmund and Reichstein concerned A. clematitis, a plant used in China and Taiwan for the treatment of cancer, menstrual troubles, leg ulcer and tumors [124]. It was named aristolochic acid A by Tomita and Sasagawa and its structure was elucidated by Pailer et al. in 1956 by means of chemical reactions [125,126]. Table 3 lists the sixteen aristolochic acids that have been isolated so far from Aristolochia. Aristolochic acid I (5) is the abundant aristolochic acid found in almost all species of Aristolochia studied with few exceptions and has been reported to be the major constituent responsible for the high antitumor activity of Aristolochia samples [99,127-131]. Since its first isolation in 1943, the aristolochic acid I (5) is without a doubt the most extensively studied of the hundreds of known secondary Aristolochia metabolites due to its potent antitumor activity and have recently attracted intense research interest because of their nephrotoxicity. The major focus of much recent research has been on the negative aspects of these compounds because of the Chinese Herb Nephropathy [224]. Recently health food supplements containing aristolochic acid have been prohibited for use in weight reduction, with full scientific support [225, 226].The aristolochic acid II (1) is a simple and second abundant aristolochic acid derivative devoid of oxygenation in ring-C [227]. A general structural feature of these aristolochic acids which exhibited simple substitutions by hydroxyl, methoxyl, ethoxyl and/or methylenedioxy groups is that oxygenated substituents are usually present at the C-6 or C-8 position of the lower aromatic ring. When two substituents were present, both C-6 and C-8 were substituted. However, aristolochic acids V (13) [130,230] and Va (9), and Vila (11), E (12) and 7-methoxy aristolochic acid I (15) have been reported to be oxygenated at C-6 & 7 and C-7 & 8, respectively. Aristoloside (16) [65] and aristolochic acid IIIa-6-0glucoside (7) are the only two glucosylated aristolochic acids in nature. Aristoloside (16) was first isolated from A. manshuriensis and was then also found in A argentina [130], A cinnabarina [142,143] and A elegans [168]. Peter and Muzaffer reported 9-methoxy aristolochic acid II (6), as only aristolochic acid with oxygenation at C-9 from A. ponticum [207]. Aristolochic acid Via (8) found in A. argentina is the only example to be oxygenated at C-2 position [130]. Tseng and Ku isolated an unusual aristolochic acid namely debilic acid (17) with a nitrophenanthrenyl acetic

864 acid skeleton from A. debilis [242,243]. Later it was also found in the A. mashuriensis [193] and A. tubflora [220]. Table 3. Aristolochic Acids Isolated from Aristolochia Species Compound

Name

Source

Ref.

A. argentine! A. auricularia A. baetica A. cinnabarina A. clematitis

127,130 132 48,49 142, 143 127, 144, 146, 147, 149, 153, 227 157 158 127, 163, 166 127 228 229 62, 127 127, 193, 195,241 144 203 206 207 127,211 127 220 223

0

c -V

OH NO2

R5 R

R4

2 R,

Ri

H

R2 H

H

R. H

R5 H

Aristolochic acid 11(1)

A. contorta A. cucurbitifolia A. debilis A. esperanzae A. heterophylla A. kaempferi A. longa A. manshuriensis A. mollissima A. moupinensis A. pallida A. ponticum A. rotunda A. sipho A. tubflora A. zollingeriana

H

OH

H

H

H

Aristolochic acid Ilia (Aristolochic acid C) (2)

A. argentina A. cinnabarina A. clematitis A. contorta A. cucurbitifolia A. debilis

130 142, 143 146 230 159 127, 162,

865 A. fangchi A. heterophylla A. indica A. kaempferi A. longa A. manshuriensis A. rotunda A. tagala A. tuberosa A. tubflora A. watsonii A. zollingeriana

H

H

H

OH

H

Aristolochic acid la (3)

H

OCH3

H

H

H

Aristolochic acid III (4)

H

H

H

OCH3

H

Aristolochic acid I (Aristolochic acid A; Aristolochic acid) (5)

166 127, 172, 230 228, 232 180 223,233 62 195 127,211 216 218,219 220 127 223 129,130 A. argentina 127, 140 A. chilensis 158 A. cucurbitifolia 231 A. fangchi 127, 130 A. argentina 132 A. auricularia 52 A. brevipes 127,146 A. clemalitis 163, 166, A. debilis 234 A. esperanzae 127 A. longa 62 A. versicolar 222 A. zollingeriana 223 A. acuminata 127, 128 A. albida 99 A. argentina 127, 129, 130, 131 A. auricularia 132 A. austrozechuanica 133 A. badamae 127 A. baecita 48, 49, A. bracteata 127, 134 A. brevipes 127,135A. championii 137 A. chilensis 52 A. cinnabarina 138, 139 A. clematitis 127, 140 A. contorta 141-143 A. cucurbitifolia 127,144A. debilis 153 A. elegans 154-157 A. esperanzae 56, 158, A. fangchi 159 A.fimbriata 127,160A. foveolata 166 A. griffithii 127, 167A. heterophylla 171 A. indica 127 127, 172, 173 A. kaempferi

866

A. ponticum

127 174 175 176,177 92, 127, 178-185 61, 127, 156,158, 164, 186 187 127, 188190 62, 127, 183,191, 192 65, 127, 193-196, 241 127, 197 127 127, 198204 176,205 127,128 127 206 127 207 208 127,209 210 127,211, 212 127, 183 127,213215 216 217 218,219 220 221,222 127 223 207

A. baetica A. cinnabarina

49 142,235

A. argentina

130

A. argentina A. debilis A. fangchi

130 127, 165 127, 172

A. kunmingensis A. kwnngsiensis A. longa A. manshuriensis A. maurorum A. maxima A. mollissima A. moupinensis A. multiflora A. ornithocephala A. pallida A. pandurata A. ponticum A. pubescens A. reticulata A. rodriguesii A. rotunda A. serpentaria A. sipho A. tagala A. triangularis A. luberosa A. tubflora A. versicolar A. westlandii A. zollingeriana

H

H

H

H

OCH3

H

Ogle

H

H

H

OH

H

H

OCH3

H

H

OH

OCH3

H

H

9Methoxyaristol ochic acid-II (6) Aristolochic acid-IIIa-6-OP-glucoside (7) Aristolochic acid-Vla (8) Aristolochi acid-Va (Aristolochic

867

H

OH

H

OCH3

H

acid-B) (9) Aristolochic acid IVa (Aristolochic acid D) (10)

A. acuminata A. albida A. argenlina A. clematitis A. cucurbitifolia A. debilis A. elegans A. esperanzae A. helerophylla A. indica A. kaempferi A. longa A. manshuriensis A. mollissima A. moupinensis A. multiflora A. rigida A. triangularis A. zollingeriana A. argenlina A. contorta A. cucurbitifolia A. debilis A. foveolata', A. heterophylla A. longa A. pubescens A. tagala A. tubflora

H

H

OH

OCH3

H

Aristolochic acid Vila (11)

H

H

OCH,

OH

H

H

OCH3

OCH3

H

H

H

OCH3

H

OCH,

H

Aristolochic acid-E(12) Aristolochic acid-V (13) Aristolochic acid IV (14)

A. contorta A. debilis A. argentina A. pallida A. argentina A. auricularia A. clematitis A. cucurbitifolia; A. debilis A. esperanzae A. kwangsiensis A. longa A. manshuriensis A. moupinensis A. pallida A. rigida A. versicolar A. zollingeriana

H

H

OCH3

OCH3

H

7-

A. contorta

127,128 99 127 127, 146 56, 158 127,166 168, 169 127 92, 177, 178,179 92, 127, 178, 179 229,233 62 65, 127, 194-195 199,202204,237 205 127, 128 238 239 223 130 230 56 127, 162, 163 174 232 62 208 216 220 230 166 130 206 127,130 132 127,146 56, 158 163 127 190,240 62 127, 194, 196 205 206 238 222 223 230

868

H

Ogle

H

c

OCH3

H

Methoxyaristol ochic acid-1 (15) Aristoloside (16)

Debilic acid (17) N0 2

A. debilis A. longa A. argentine! A. cinnabarina A. elegans A. manshuriensis A. debilis A. manshuriensis A. tubflora

127, 162, 163 62 130 142,143 168 65, 127, 194,241 127,242, 243 127,193 220

cr

[

"OCH,

Sodium Salts of Aristolochic Acids It is interesting to note that Formosan Aristolochia species including A. cucurbitifolia, A. foveolata, A. heterophylla, A. kaempferi and A. zollingeriana are the only species [159,228,244-246] from which sodium salts of aristolochic acids were isolated. However, very recently Lopes and co workers have also isolated sodium aristolochate I (21), II (18), Ilia (19) and IVa (22) from Brazilian species, A. pubescens [208]. Isolates of this class from various Aristolochia species were listed in Table 4. Aristolochic Acid Alkyl Esters Table 5 summarizes the occurrence of fourteen alkyl esters of aristolochic acids in Aristolochia species. It is noted that all the reported aristolochic acid alkyl esters are methyl esters except 32 which is an ethyl ester of aristolochic acid la with an ethoxy group at C-8 [129]. Compounds 25-31 are methyl esters of reported aristolochic acids whereas compounds 33-38 are methyl esters of non reported aristolochic acids. Among alkyl esters of aristolochic acids reported so far aristolochic acid I methylester (28) and aristolochic acid IV methylester (30) are frequently encountered in Aristolochia species. Formosan Aristolochia species are known to be rich source for alkyl esters of aristolochic acids, particularly A. zollingeriana [171,223] and A cucurbitifolia [56,159]. Sesqui- and Diterpenoid Esters of Aristolochic Acids Table 6 shows the eleven sesquiterpene esters of aristolochic acids isolated from Aristolochia species. Isolation of the aristoloterpenate I (40) from the radix of A. mollissima is the first report of such compounds in

869 Table 4. Sodium Salts of Aristolochic Acids Isolated from Aristolochia Species Compound

Name

Source

Ref.

A. cucurbitifolia A.foveolata A. heterophylla A. kaempferi A. pubescens A. zollingeriana A. foveolata A. heterophylla A. kaempferi A. pubescens A. zollingeriana A. cucurbitifolia A. cucurbitifolia A. foveolata A. heterophylla A. kaempferi A. manshuriensis A. pubescens A. zollingeriana

159 174 228 233, 244 208 245 174 228,232 244 208 245 159 56, 159 174,246 228,232 229,233, 244 241 208 245 159 174 228 244 208 245 56 174 228

0

cXT

•^^I^^O-Na*

i

R. R?

R, H

R, H

R3 H

Sodium aristolochate II (18)

OH

H

H

Sodium aristolochate Ilia (19)

OCH3 H

H H

H OCH3

Sodium aristolochate III (20) Sodium aristolochate I (21)

OH

H

OCH3

Sodium aristolochate IVa (22)

H

OH

OCH3

H

OCH3

OCH3

Sodium aristolochate Vila (23) Sodium aristolochate VII (24)

A. cucurbitifolia A. foveolata A. heterophylla A. kaempferi A. pubescens A. zollingeriana A. cucurbitifolia A. foveolata A. heterophylla

which the carboxylic acid group of aristolochic acid forms ester linkage with a sesqui- or diterpene [204,250]. It is interesting to note that, among eleven compounds of this type from Aristolochia species, we discovered eight sesquiterpene esters of aristolochic acids from the roots and stems of

870

Table 5. Aristolochic Acid Alkyl Esters Isolated from Aristolochia Species Compound

Name

c Ri

Source

Ref.

0 II OR NO2

A R?

R CH3

R, H

R2 H

R3 H

CH3

H

H

OH

CH3

OCHj

H

H

CH3

H

H

OCH3

CH3

OH

H

OCH3

Aristolochic acid IVa methyl ester (29)

A. elegans A. indica A. kwangsiensis A. manshuriensis A. moupinensis A. versicolar

CH3

OCH3

H

OCH3

Aristolochic acid IV methyl ester (30)

A. brevipes A. championii A. cucurbitifolia A. elegans

Aristolochic acid 11 methyl ester (25)

Aristolochic acid la methyl ester (26) Aristolochic acid III methyl ester (27) Aristolochic acid I methyl ester (28)

A. argentina A. cucurbitifolia A. heterophylla A. longa A. manshuriensis A. zollingeriana A. kaempferi

129 159 228 62, 192 241 223 244

A. cucurbitifolia A. kaempferi A. argentina A. cucurbitifolia A. debilis A. elegans A. heterophylla A. indica A. kaempferi A. longa A. manshuriensis A. mollissima A. sipho A. versicolar

159 244 129 159 163, 165 168 228 127, 179 244 62, 192 65,241 203 209, 215 221 168 127, 178 247 241 205 222 52 138, 248 56, 159

871 A. kaempferi A. kwangsiensis A. longa A. manshuriensis A. moupinensis A. versicolar A. zollingeriana

CH3 CH2CH3

H

OCH3

OCH3

H

H

OCH2CH3

Aristolochic acid VII methyl ester (31) Aristolochic acid-la ethyl ester ethyl ether (32) O

A. cucurbitifolia A. argentina

168 244 127, 188, 190, 240 62, 192 65 205 221, 222 223 56 129

OCH3 R2O^~

.NO 2

5c

)

R4

CH3

R3 H

R4 H

CH3

CH3

H

H

H CH3 H CH3

CH3 CH3 CH3 CH3

OH OH H H

H H OCH3 OCH3

Ri

R2

H

Aristolochic acid All methyl ester (33)

A. auricularia A. kaempferi A. zollingeriana

Ariskanin-A (Aristolochic acid BII methyl ester) (34) Ariskanin-B (35) Anskanin-C (36) Ariskanin-D (37) Ariskanin-E (38)

A. manshuriensis A. zollingeriana A. A. A. A.

zollingeriana zollingeriana zollingeriana zollingeriana

132 61, 158, 244 171, 223 195 223 223 223 223 223

A. heterophylla [249,251] and a diterpene ester of aristolochic acid from the roots and stems of A. elegans [252] and thus, A. heterophylla is considered to be a rich source of sesquiterpene esters of aristolochic acids. Aristoloterpenate I (40) and III (42) are esters of aristolochic acid I (5) with the sesquiterpenes, madolin M (305) and L (306), respectively, whereas aristoloterpenate II (39) and IV (41) are esters of aristolochic acid II (1) [249]. In these four esters C-ll of the aristolochic acid and C-4' of sesquiterpene were involved in the ester linkage. The stereochemistry at

872 Table 6. Sesqui- and Diterpenoid Esters of Aristolochic Acids Isolated from Aristolochia Species Compound Aristoloterpenate II (39) Aristoloterpenate I (40)

Aristoloterpenate IV (41) Aristoloterpenate III (42)

Aristophyllide C (43) Aristophyllide A (44) Aristophyllide D (45) Aristophyllide B (46) Aristolin (47) Aristoloin I (48) Aristoloin II (49)

Occurrence A. heterophylla A. heterophylla A. kaempferi A. mollissima A. heterophylla A. cucurbitifolia, A. heterophylla; A. kaempferi A. mollissima A. heterophylla A. heterophylla A. mollissima A. heterophylla A. heterophylla A. elegans A. pubescens A. pubescens A. pubescens

Ref. 228,249 228, 249 244 204,250 228,249 159 228, 249 244 204 228,251 228,251 204 228,251 228,251 252 208 208 208

C-4' of aristoloterpenate I-IV (39-42) was determined as R by circular dichroic studies. Aristophyllide A (44) and B (46) are aristolochic acid I (5) esters of the rearranged e«/-elemane type sesquiterpenes, aristophyllene and its stereoisomer, respectively [251]. Aristophyllide C (43) and D (45) are aristolochic acid II (1) esters of aristophyllene and its stereoisomer, respectively [251]. In these esters aristolochic acid and sesquiterpene were bound by an ester linkage between C-ll and C-12'. The absolute configuration of C-5' and C-12' of these four esters were determined by application of the circular dichroic exciton chirality method. Aristophyllide A (44) and C (43) have R and S configurations at C-5' and 12' whereas aristophyllide B (46) and D (45) possessed opposite stereochemistry S and R at C-5' and 12' centers. Aristolin (47) is the first example of an ester composed of aristolochic acid and a diterpenoid, in which C-16 hydroxy group of e«f-kauran-16(3, 17-diol involves in the ester linkage with C-ll carboxylic acid group of aristolochic acid [252]. Very recently, Isabele et al have reported the isolation of two more diterpene esters of aristolochic acids, aristoloin I (48) and II (49) together with aristolin (47) from the tubercula of A. pubescens [208]. In aristoloin I (48) and II (49), aristolochic acid I (5) and II (1) were bound to a kaurane diterpene, 16a,17-en?-kauranediol by an ester linkage through C-ll and C-17' centers. Thus, from these reports it appears that a general theme running through the chemistry of Aristolochia is the ability to form esters

873 .CHj

R 43 H 44OCH,

R 45 H 46 OCH3

Fig. (1). The Structures of Sesqui- and Diterpenoid Esters of Aristolochic Acids

of aristolochic acids with a number of different types of secondary metabolites. Denitroaristolochic Acids The twelve denitroaristolochic acids isolated from Aristolochia species are presented in Table 7 and endemic Formosan Aristolochia species, A. cucurbitifolia was noted for profuse production of denitroaristolochic acids [56,159,254]. Research work by our group revealed that majority of

874 Table 7. Denitroaristolochic Acids Isolated from Aristolochia Species Compound

Name

Source

Ref.

0 OR

] T

R3

R, R H

R> H

R2

H

R3 H

H

H

H

CH3

H

H

A. manshuriensis

241,253

OCH3

Demethylaristofolin E (Aristolic acid II) (50) Aristolic acid I (51)

A. albida A. cucurbilifolia A. helerophylla A. indica A. kaempferi A. reticulata A. versicolar

H

H

Aristofolin E (52)

H

OH

OCH3

Aristofolin B (53)

H CH3

OH H

H H

OCH3 OCH3

Aristofolin D (54) Aristolic acid methyl ester (55)

H

OCH3

H

OCH3

H

Ogle

H

OCH3

6-Methoxy aristolic acid (56) Aristofolin A (57)

CH3

OCH3

H

OCH3

6-Methoxy aristolic acid methyl ester (58)

Na

Ogle

H

OCH3

Sodium aristofolin A (59)

CH3 Na

H H

OCH3 OH

OCH3 OCH3

Aristofolin C (60) Sodium aristofolin B (61)

A. kaempferi A. manshuriensis A. cucurbilifolia A. helerophylla A. kaempferi A. cucurbitifolia A. cucurbitifolia A. heterophylla A. indica A. kaempferi A. kwangsiensis A. versicolar A. cucurbitifolia A. kaempferi A. kwangsiensis A. versicolar A. championii A. cucurbitifolia A. heterophylla A. cucurbitifolia A. cucurbitifolia

99 56, 159, 254 228,232 179,182 229, 244 209 222 244 241 56, 254 228,232 229 56, 254 56, 254 232 179, 182 244 247 222 56, 254 229,233 188, 190 222 138,248 56, 254 232 56, 254 159

denitroaristolochic acids were the constituents of Formosan Aristolochia species [254]. Aristolic acid I (51) is the abundantly reported denitroaristolochic acid in Aristolochia species and has been shown to be

875 extensively studied compound after aristolochic acid I (5) and II (1). The effect of aristolic acid I (51) on a diverse array of physiological process has been examined, as it is a denitro derivative of an active ingredient aristolochic acid I (5) to understand the role of nitro group in the bioactivity. Aristofolin A (57), B (53), C (60), D (54), and E (52) [244,254] reported as metabolites of Aristolochia species of Taiwan origin were denitro derivatives of aristoloside (16), aristolochic acid Vila (11), 7-methoxy aristolochic acid I (15), aristolochic acid FVa (10) and aristolochic acid II methyl lester (25), respectively. Compounds 52, 55, 58 and 60 were isolated as methyl esters of denitroaristolochic acids. Aristofolin A (57) and sodium aristofolin A (59) are the only examples of denitroaristolochic acid containing glucosyl units [233,254]. Compounds 59 and 61 were isolated as sodium salts of denitroaristolochic acids from A. cucurbitifolia [254] and A. heterophylla [232], and A. cucurbitifolia [159], respectively. Aristolactams Aristolactams are reduction products of aristolochic acids and regarded as biogenetic intermediates in the biosynthetic pathway of aristolochic acids. They are supposed to originate in the plants by oxidation of aporphines. From Tables 8 and 9, it is evident that thirty six aristolactams have been reported so far from Aristolochia species. Aristolactams are by no means confined to Aristolochiaceae; at least species of Annonaceae, Menispermaceae and Monimiaceae have been reported to contain one or more of the over sixty known aristolactams [255]. Occurrence of aristolactams in these four families is of taxonomic significance and supports the view that the Aristolochiales is related to Magnoniales and Ranunculales [256]. The majority of aristolactams also possess oxygenated substituents at C-3 and C-4, C-6 and/or C-8, as in aristolochic acids. Aristolactams 62-82 showed similar substitution pattern to that of the accompanying aristolochic acids. However, aristolactams 83-97 differ in that they lack methylenedioxy substituents, instead they possess methoxyl or hydroxyl groups at C-3 and C-4. The seven aristolactam glycosides that have been described to date in Aristolochia include compounds 68-73, and 85. Compound 73 was found to possess a bioside on C-5, whereas the remaining all are iV-glycosides of aristolactams [239]. Compound 72 is the only representative of acylated glycoside of aristolactam, with ?ra«s-/?-coumaroyl moiety as acyl group on C-6 of glucose unit [260]. A. argentina was found to be rich in aristolactams

876

Table 8. Aristolactams Isolated from Aristolochia Species Compound

R H

Ri

c

Name O

Source

Ref.

257 132 56, 158, 159 158 174 158 158,229, 244 158,241 210 158 239 220 158 223 257 48,49 158, 159 158 158,228, 232 158,229, 244 158 158 218 158 257 191 210 239 257 132 49 207 223 99 127,257

\ NR

it

R3

H

R2 H

H

Aristolactam II (Cepharanone A) (62)

A. argentina A. auricularia A. cucurbitifolia A. debilis A. foveolata A. heterophylla A. kaempferi A. manshuriensis A. rodriguesii A. tagala A. triangularis A. tubftora A. westlandii A. zollingeriana

H

OH

H

H

Aristolactam Ilia (Aristolactam C) (63)

A. argentina A. baetica A. cucurbitifolia A. debilis A. heterophylla A. kaempferi A. manshuriensis A. tagala A. tuberosa A. westlandii

H

H

OH

H

Aristolactam-Ia (64)

H

OCH3

H

H

Aristolactam III (65)

H

H

OCH3

H

Aristolactam I (Aristolactam A)

A. argentina A. longa A. rodriguesii A. triangularis A. argentina A. auricularia A. baetica A. ponlicum A. zollingeriana A. albida A. argentina

877 (66)

A. auricularia A. baecita A. bractenta A. brevipes A. chilensis A. contorta A. cucurbitifolia A. debilis A. elegans A.fangchi A.foveolata A. heterophylla A. indica A. kaempferi A. manshuriensis A. mollissima A. ponticum A. reliculata A. rotunda A. tagala A. tubflora A. westlandii A. zollingeriana

H

H

H

OCH3

glc

H

H

H

glc

OH

H

H

Aristolactam C W-p-D-gluco- side (69)

glc

H

OH

H

glc

H

OCH3

H

Aristolactam la NP-D-gluco- side (70) Aristolactam I A^P-D-gluco- side (71)

9-Methoxyaristolactam-II (67) Cepharanone A N(5-D-gluco- side (68)

A. auricularia

A. cinnabarina A. contorta A. foveolata A. heterophylla A.mollissima A. cinnabarina A. contorta A. cucurbitifolia A. elegans A.foveolata A. heterophylla A. indica A. kaempferi A. mollissima A. pubescens A. rotunda A. tuberosa A. zollingeriana

132 48 258 52 259 260, 262 59, 158, 159 127, 158, 234 168, 174 127,172 246 158,228 127, 178, 181,182 158 241 202 207 209 127 158 220 158 223,245 132

A. foveolata A. pubescens

142,261 262 246 228,232 203 142, 143, 261 262 56 168, 169 246 228 127, 180 244 204 208 263 218 223 174 208

A. cinnabarina A. clematitis A. contorta A. cucurbitifolia

142, 143 153 230, 260 158

878 A. debilis A. foveolata A. helerophylla A. indica A. knempferi A. manshuriensis A. mollissima A. pubescens A. rotunda A. tagala A. tuberosa A. wesllnndii A. zollingeriana 6'-pcoumarylglc

H

OCH3

H

c H

H

glc-glc

H

OH

H

OCH3

H

H

OCH3

OH

H

H

H

OCH3

OH

Aristolactam-/V(6'-trans-pcoumaroyl)-|3-Dglucoside (72)

158, 163 174 158,228, 232 92, 127, 178, 180 158,244 158 203, 204 208 263 158 218 158 223,245

A. conlorta

260

A. triangularis

239

A. cinnabarina

142,261

A. cucurbitifolia

159

A. brevipes A. foveolata

52 174

0 NH

(

c

R4 R3

R2 Triangularine-B (73) 2-Hydroxy-8methoxycepharanone-A (74) Cepharanone B (75) 9Hydroxyaristolact am-I (76) 0 NH

R4 Ri

R3 R2

879 OCH3

OCH3

H

H

OCH3

H

0CH3

H

5,6-Dimethoxyaristolactam (77) Aristolactam-IV (78)

A. mollissimn

202

A. argentina

127, 130 178 202

A. indica A. mollissima H OCH3 9OCH3 A. auricularia Methoxyaristolactam A. cucurbitifolia 1(79) A. elegans A. heterophylla A. kaempferi A. manshuriensis A. mollissima A. ponticum A. zollingeriana H OCH3 OCH 2 CH 3 9A. mollissima Ethoxyaristolactam-I (80) H OCH3 9OCH3 A. auricularia Methoxyaristolactam A. cucurbitifolia IV (81) OCH3 Aristored (82) A. bracteata O A. reticulata

H

H

OCH3

y

CT

H3CO

NH

132 56, 159 169 228 244 241 204 207 223 202

132 159 127 127, 183, 209 183

A. serpentaria

1

^N)CH,

followed by A. heterophylla and A. cucurbitifolia. Aristolactam I (66) followed by aristolactam All (83), aristolactaml-A^- P-glucoside (71) and aristolactam II (62) were the frequently encountered aristolactams in the Aristolochia species. Aristolactam II (62) found in several species of Aristolochia is a simple aristolactam without substitution on ring B and C. Unusual aristolactams which have C-2 substitution, namely 2-hydroxy-8methoxycepharanone A (74), aristored (82), aristolactam DII (92), Dili (96), CII (93), CIII (97), CIV (95) and aristoliukine A (94) were described from Aristolochia species. Among these, compounds 92 and 96 reported from A argentina [130,266] contain -COOH group on C-2, where as 93, 95, 97 from A. argentina [228,266] and 94 from A. kaempferi [229,244] possess hydroxymethyl group at C-2. The presence of a carboxyl group in 92 and 96 suggest that they might be biogenetically derived from 3carboxyphenylalanine or 3-carboxy-4-hydroxyphenylalanine [266]. On the other hand, aristolactams 93 and 97 might either arise from

880 Table 9. Aristolactams Isolated from Aristolochia Species Compound

Name

Source

Ref.

A. aculifolia A. argentina A. baetica A. contorta A. cucurbitifolia A. debilis A. elegans A. foveolata A. heterophylla A. indica A. kaempferi A. manshuriensis A. mollissima A. rodriguesii A. tagala A. triangularis A. westlandii A. zollingeriana A. cucurbitifolia A. elegans A. heterophylla A. kaempferi A. cucurbitifolia

210 127,257,264 49 262 56, 158, 159 158 168, 169, 171 246 158,228,232 92, 127, 181 61, 158,229,244 158,241 204 210 158 239 158 223 159 168, 169 228,232 244 159

A. argentina A. baetica A. cucurbitifolia A. debilis A. heterophylla A. kaempferi A. manshuriensis A. rodriguesii A. tagala A. triangularis A. westlandii A. argentina A. elegans A. foveolata

127,257,264 49 56, 158, 159 158, 163 158,228 61,244 158,265 210 158 239 158 257 168, 169 174

0 RO

/ \ \ NH

X R, H

R3 H

R4

CH3

H

Aristolactam All (83)

CH3

H

H

H

Piperolactam A (84)

CH3

glc

H

H

CH3

CH3

H

H

Piperolactam AO-P-D- glucoside (85) Aristolactam BII (86)

H

CH3

OH

H

R2

Aristolactam AHIa (87)

881

H

CH3

H

OH

H

CH3

OCH3

H

CH3

CH3

OCH3

H

CH3

CH3

H

Aristolactam- Ala (88) Aristolactam AIII (89)

Aristolactam Bill (90)

OCH3 Aristolactam-Bl (taliscanine) (91)

A. heterophylla A. kaempferi A. mollissima A. triangularis A. argentina A. triangularis A. argentina A. baetica A. cucurbitifolia A. debilis A. heterophylla A. kaempferi A. manshuriensis A. tagala A. westlandii A. zollingeriana A. acutifolia A. argentina A. zollingeriana A. argentina A. taliscana

228, 232 158,229,244 204 239 257 239 127,257,264 49 158 158 158,232 61, 158,244 158 158 158 223 210 127,257,264 223 264 127

0 R2O——. NH

CO2H

OH

OCH3

H

CH2OH

OCH3

OCH3

H

CH2OH

OH

OCH3

OH

CH2OH

OH

OCH3

OCHj

CO2H

OH

OCH3

OCH,

CH2OH

OCH3

OCH3

OCH3

R4 Aristolactam DO (92) Aristolactam- CO (93) Aristoliukine A (94) Aristolactam CIV (95) Aristolactam DIN (96) Aristolactam- CIII (97)

A. argentina A. foveolata A. manshuriensis A. argentina A. triangularis A. kaempferi A. mollissima A. heterophylla

130,266 174 265 266 239 229, 244 204 228

A. argentina

130,266

A. argentina

266

aristolactams DII (92) and Dili (96) by reduction and methylation reactions or directly originate from the ammo acids 3hydroxymethylphenylalanine and 3-hydroxymethyl-4-hydroxyphenylalanine [266]. The 9-oxygenated aristolactams are rare in Aristolochia with compounds 67 [132], 76 [52], 79 [132], 80 [202] and 81 [132] being the examples with hydroxy or methoxy or ethoxy group at C-9.

882 Compound 80 is the only example of 9-ethoxy aristolactam found in A. mollissima [202]. Aporphines Twenty aporphine alkaloids have been isolated so far from Aristolochia species (Table 10). Glaziovine (98) was detected as the sole proaporphine component from Chilean species, A. chilensis [259]. Aporphines, magnoflorine (105) followed by cepharadione A (107) and 4,5-dioxo-dehydroasimilobine (110) are found in several species of Aristolochia. Aporphines with iV-formyl substitution, ./V-formylnornantenine (102), and its 6ot,7-dehydro derivative, 6a,7-dehydro-./Vformylnornantenine (103) were described by Touche et al. from A. brevipes [273]. The latter aporphine (103) was reported to exist in a 19:1 ratio of E/Z conformers due to stereoisomerism of the jV-formyl group. An 7V-acetyl aporphine derivative, ./V-acetylnornuciferine (99) besides magnoflorine (105) has been encountered in A. bracteata, an Indian medicinal plant by Pakrashi et al. [258]. Magnoflorine (105) and N,Ndimethylindcarpine (106) were polar quaternary aporphines with N,Ndimethyl group found in Aristolochia species. Aporphines 107-114 were representatives of 4,5-dioxoaporphines known to Aristolochia. The 4,5dioxoaporphines are a small group of aporphinoids found mostly among members of the botanical families Menispermaceae, Berberidaceae, Annonaceae, Fumariaceae and Aristolochiaceae [276]. Since aporphine alkaloids have been postulated as precursors of aristolactams and aristolochic acid in plants, 4,5-dioxoaporphines may be considered as possible intermediates. Tuberosinone iV-glucoside (109) is the only Nglucoside of aporphine found in Aristolochia species, A. tuberosa [218] and A. cinnabarina [141]. Lysicamine (115) from A. contorta [272] and isomoschatoline (116) and oxonuciforine (117) from A. elegans [169] were fully aromatic aporphines with a rare 9-oxo function. An unusual aporphine, dimethylsonodione (118) with 5,8-dioxo function was also identified from A. manshuriensis [241]. Most of the aporphines found in Aristolochia species possess 4,5-tetrahydro basic skeleton. Protoberberines Sixteen protoberberine type alkaloids reported from Aristolochia species were listed in the table 11. Occurrence of this type alkaloids were rare in Aristolochia, they were found only in four species, A. arcuata

883

Table 10. Aporphines Isolated from Aristolochia Species Compound

Name (-)-Glaziovine (98)

Source A. chilensis

Ref. 259

(-HV-Acetylnornuciferine (99)

A. bracteatn

258

Isoboldine (100)

A. papillaris

120

(-)-Isocorydine (101)

A. pubescens

208

H,CO

H,CO

884

I \

/V-Formylnomantenine (102)

A. brevipes

273

6a,7-Dehydro-JVformylnornantenin

A. brevipes

273

Asimilobine (104)

A. cucurbitifolia

159

Magloflorine(105)

A. argentina A. austrozechuanica A. baetica A. bracteata A. clemntitis A. conlorta A. cymbifera A. debilis A. elegans

131 133 49, 134 135 153 154, 155 267 268,269 167, 168, 169, 170 231 176, 177,236 92 164 270 194 176,205 239

\

r

0

\—o

e(103) |/

CHC

H3CO'"'

I

105 106

R, CH 3 H

TH

R2 H CH 3

R3 H H

CH3

R4 CH 3 CH 3

W.W-Dimethyllindcarpine (106)

A. fangchi A. helerophylla A. indica A. kaempferi A. macedonica A. manshuriensis A. moupinensis A. triangularis A. triangularis

239

885 0 o

JK.

\

/ ~~—

x

f/

.0

Cepharadione A (107)

J

1\

1

Tuberosinone (108) R CH3 H glc

107 108 109

Tuberosinone-A'-pD-glucoside (109)

R, H OH OH

4,5Dioxodehydroasim ilobine (110)

A. chilensis A. cucurbitifolia A. foveolala A. heterophylla A. indica A. kaempferi A. mollissima A. tagala A. Iriangularis A. zollingeriana A. cinnabarina A. kaempferi A. tuberosa A. cinnabarina A. tuberosa

A. chilensis A. contorta A. cucurbitifolia; A. elegans; A. heterophylla; A. indica A. kaempferi

0 /

^ N

s.

^ ^

1 T

R2O

Aristoliukine B (112)

r

/

J

1 3

R2 R. H CH3 CH 3 H CH3 H CH3 H H CH3

R

110 111 111 113 114

Aristolodione (111)

H H CH 3 H H

Triangularine-A (113) Aristoliukine C (114)

Lysicamine (115) Isomoschatoline (116)

I

H3CO"

r

J

1 R 115 H 116 OH

R2 H H

246

228, 232 92, 181 229,244 204 158 239

223,245 141,142,143 244

219,274,275 141,142,143 218,219,220

271 272

56, 159 169

228,232 181

61, 158,229,244 204 239 271 232 244 232 204

229, 244 239

A. kaempferi

244

A. contorta A. elegans

168, 169

R3 H OH H OH OCH

1H3CO^

A. mollissima A. iriangularis A. chilensis A. heterophylla; A. kaempferi A. heterophylla A. mollissima A. kaempferi A. triangularis

271

56, 159

R3 H H

272

886 Oxonuciforine (117) Demethylsonodion

A. elegans

168,169

A. manshuriensis

241

H3aX^>''x)

R 120 H 121 glc

H,CO.

Ogle

122

123

R,O.

H,CO' H3CO

R2 R

124 125 126 127 128 129

i

CH 3 H H H Ac Ac

R

2

H OH OH OH H OAc

R3 H OH Ogle Oxyl OAc OAc

R4 H H H H glc-Ac4 H

R2 Rs H H

130

H H Ac Ac

131 132 133 134

Fig. (2).The structures of Proto berberines

Ri H H H Ac Ac

R2 OH H H OAc H

R3 OH Ogle Oxyl Oglc-Ac4 OAc

R4 H OH

Rs CH 3

OH H

H Ac

OAc

glc-Ac

H

887 Table 11. Protoberberines Isolated from Aristolochia Species Compound Cyclanoline (119) 2,10-Dihydroxy-13-oxidodibenzo[a,g]quinoiizinium (120) 2-Hydroxy-10-O-[glucopyranosyl]-13oxidodibenzofa,gl-quinolizinium (121) 8-Benzylberbine-A (122) jV-Oxide 8-benzylberbine-B (123) (-)-8p-(4'-Hydroxybenzyl)-2,3-dimethoxyberbin10-ol(124) (-)-8p-[4'-Hydroxybenzyl]-2-methoxyberbin3,10,11-triol (125) (+)-10-O-[p-Glucopyranosyl]-8|3-[4'hydroxybenzyl]-2-methoxyberbin-3,ll-diol (126) (+)-8p-[4'-Hydroxybenzyl]-2-methoxy-10-O-[Pxylopyranosyllberbin-3,11 -diol (127) 3,10-Diacetoxy-8p-[4'-O-(a-glucopyranosyltetraacetate)-benzyll-2-methoxyberbine (128) 3,10,11 -Triacetoxy-8p-[4'-acetoxybenzyl]-2methoxyberbine (129) (-)-8a-[4'-Methoxybenzyll-2-methoxyberbin3,10,11-triol (130) (+)-10-O-[-p-Glucopyranosyl]-8a-[4'hydroxybenzyl]-2-methoxyberbin-3,9-diol (131) (+)-8a-[4'-Methoxybenzyl]-10-O-[Pxylopyranosyl]berbin-3,9-diol (132) (-)-3,ll-Diacetoxy-8a-[4'-acetoxybenzyl]-10-O-[pglucopyranosyltetraacetate]-2-methoxyberbine (133) 3,9,10-Triacetoxy-8a-[4'-O-(p-glucopyranosyltetraacetate)benzyll-2-methoxyberbine (134)

Source A. debilis A. nrcuala

Ref. 268 277

A. arcuata

277

A. gigantean A. gigantean A. constricta

278, 279 278,279 280

A. gigantea

279

A. gigantea

279

A. gigantea

279

A. gigantea

279

A. gigantea

279

A. gigantea

279

A. gigantea

279

A. gigantea

279

A. gigantea

279

A. gigantea

279

[277], A. constricta [280], A. debilis [268] and A. gigantea [278,279]. Among them, A. gigantea is rich in this type alkaloids. Out of sixteen protoberberine type alkaloids found in Aristolochia, ten were from A. gigantea [278,279]. Seven of them were occurred as glycosides. Compounds 122-134 are 8-benzyltetrahydroprotoberberine type alkaloids [278,279]. Their unusual carbon skeleton had been obtained by introduction of a benzyl group at C-8 of berberine. On the basis of proposed biosynthetic pathway, where the berberine bridge carbon is considered to be derived from formate, Lopes suggested that the CHCH2Ar unit of 8-benzyltetrahydroprotoberberine alkaloids of Aristolochia comes from p-hydroxybenzaldehyde through a biosynthetic step involving two molecules of ^-hydroxybenzaldehyde condensing with one of L-tyrosine [279]. It is reported that rings B and C of the

888 tetrahydroprotoberberine alkaloids adopt a half-chair conformation and B/C cis configuration [278]. The 8-benzyl group was in P-orientation in alkaloids 124-129, whereas in alkaloids 130-134 it is in a-orientation. Compound 123 is the only N-oxide among these alkaloids. Compounds 119-121 were quaternary alkaloids. Compounds 120 and 121 reported from A. arcuata [277] are the 13-oxidodibenzo[a,y]-quinolizinium alkaloids. It is known that oxyberberinium and oxyprotoberberinium salts were generally obtained from natural products, which were deoxygenated in both A and D rings, having the L-tyrosine and p-hydroxybenzaldehyde as common biogenetic precursors. The largest difference between 13oxidodibenzo[a,y]-quinolizinium and oxyberberinium and oxyprotoberberinium alkaloids is the presence of only one oxygenated function at A and D rings. Since, 13-oxidodibenzo[a,y]-quinolizinium alkaloids from A. arcuata [277] are presumably biogenetically related to 8-benzylberberine and benzylisoquinoline alkaloids isolated from Aristolochia species, Ltyrosine and p-hydroxybenzaldehyde are considered to be their biogenetic precursors. Protopines Protopine alkaloids are apparently not very common in Aristolochia and however, constrictosine (135), 3-0-methylconstrictosine (136), 3,5di-O-methyl-constrictosine (137), 5,6-dihydroconstrictosine (138) and 5,6-dihydro-3,5-di-0-methylconstrictosine (139) are the only examples of the protopine alkaloids [280]. Their occurrence in Aristolochia was also strictly confined to A. constricta [280]. Protopine alkaloids are usually reported as C-2 substituted on the basis of biogenetic considerations. The unusual absence of C-2 substitution was noticed in the protopines of Aristolochia. R,O

R| 135 H 136 CH3 137 CH3

R,O.

R2 H H CH 3

Fig. (3). The Structures of Protopines

R. 138 H 139 CH,

R2 H CH,

889 Benzylisoquinolines Fourteen benzylisoquinoline type alkaloids have been identified in Aristolochia species (Table 12). Coclaurine (140), oblongine (141) and reticuline (142), respectively found in A. papillaris [120], A. triangularis [239] and A. reticulate [281], were simple benzylisoquinoline alkaloids [169]. Aristoquinoline A (143), B (145), C (144) isolated from A. elegans were novel benzoyl benzyltetrahydroisoquinoline ether iV-oxides. Compounds 143 and 145 were conformational isomers which differ in the conformation of C-ring and configuration of iV-oxide. Alkaloids 146 and 147 from A. elegans [167,282] and 148-150 from A gigantea [283] were bis-l-benzyltetrahydroisoquinoline alkaloids with one diphenyl ether link between rings C and C , usually through C-ll and C-12' centers. These alkaloids usually have \R,VR absolute configuration, however, 146 reported to possesses IS,\'R configuration. Compounds 151 and 152 from A. fangchi [231] and 153 and 154 from A. debilis [161,284] were bisbenzyltetrahydroisoquinoline alkaloids with two diphenyl ether links. These ether linkages were usually between rings C and C , and B and B', except in aristolochine which contained two ether links between rings B and C , and C and B'. Benzylisoquinolines and benzoyl benzyltetrahydroisoquinoline ether JV-oxides were assumed to be biogenetic intermediates in the catabolic process of bisbenzyltetrahydroisoquinoline alkaloids. Table 12. Benzylisoquinolines Isolated from Aristolochia Species Compound Coclaurine (140) Oblongine (141) Reticuline (142) (-)-(-ft)-Aristoquinoline A (143) (-Hfl)-Aristoquinoline C (144) (-)-(/?)-Aristoquinoline B (145) (-)-Temuconine (146) (-)-(«,«)-7'-O-Methylcuspidaline(147) (-)-Pedroamine (148) (-)-Pampulhamine (149) (-)-Geraldoamine (150) Fangchinoline (151) (-)-Obamegine(152) Tetrandrine(153) Aristolochine (154)

Source A. papillaris A. triangularis A. reticulata A. elegans A. elegans A. elegans A. elegans A. elegans A. gigantea A. gigantea A. gigantea A. fangchi A. fangchi A. debilis A. debilis

Ref. 120 239 281 169 169 169 282 167 283 283 283 231 231 284 161

890 H,CO.

R,0'

140 141

RT R3 OCH, H H H CH, OH

R4 H H

142

OCH, H

OH OCH, H

H

Rj OH H

H OH

~CH,

H,C

147 148 149 150

R H CH, CH, CH,

R, CH, H CH, CH,

R2 CH, H H CH,

^.OCH, H,CO. .OCH,

H,CO 151 152 153

R, H H CH,

R, H CH, CH 3

Fig. (4). The Structures of Benzylisoquinolines

Isoquinolines The occurrence of isoquinoline alkaloids in the genus Aristolochia is limited to three species, A. arcuata, A. elegans and A. gehrtii. Isolates of this calss were listed in the Table 13. Most of these alkaloids reported from Aristolochia were tetrahydroisoquinoline type, except 168 which is fully aromatic. It is interesting to note that all the alkaloids isolated from A. arcuata [285] were isoquinolines whereas all the alkaloids from A. elegans were isoquinolones with a carbonyl function at C-l. The

891 isoquinolines (156-160) isolated from A. arcuata [285] were reported to contain rare substituents, like TV-ethyl, TV-fructosyl and TV-glyceryl. Iwasa et al. [286] proposed that salsolinol (155), a simple isoquinoline alkaloid is formed by the condensation of dopamine with acetaldehyde, and it has been suggested that the O-methylating enzymes of salsolinol may be different from those of dopamine. On the basis of this proposal, Francisco et al. [285] suggested that the alkaloids 157-160 come from acetoacetate condensation with a dopamine derivative followed by decarboxylation. Isoquinoline alkaloids were also considered as biogenetic intermediates in the catabolic process of bisbenzyltetrahydroisoquinoline alkaloids. Amides The amides are another class of compounds isolated from several Aristolochia species (Table 14). Most of the amides from Aristolochia species, on structural investigation were found to contain a tyramine unit connected to phenolic acids like coumaric, ferulic, cinnamic acids, and rarely with acetic acid through an amide linkage. 7V-/?-/ra«.s-Coumaroyltyramine (178) and N-p-trans-iQuxXoyXtyrdsmne (179) are more frequently encountered amides in Aristolochia species. Their cis isomers, N-p-ciscoumaroyltyramine (174) and TV-cw-feruloyltyramine (175) were also isolated from Aristolochia species. Aurantiamide acetate (172), a rarely occurring modified phenylalanine dipeptide alkaloid was reported from only one species, A. tubflora [288]. A^raws-Cinnamyltyramine (177) was described only from A. cucurbitifolia [159] and A. elegans [169]. Aristomanoside (181) isolated from A. manshuriensis [241] is a 4,4'diglucoside of /V-feruloyl-3-methoxytyramine. Its aglycone, N-transferuloyl-3-methoxytyramine (180) was also found in A. manshuriensis. The last mentioned amide (180) and its cis isomer (176) were also reported from A. gehrtii [241]. The co-occurrence of biosynthetic derivatives, isoquinolones and alkamides in Aristolochia spcies, which could be formed by at least one unit C6-C2 is remarkable. Nacetyltyramide (171) from A. cucurbitifolia is the only example of an aliphatic acid amide of tyramine [56]. In addition, three simple amides, namely pyridine 3,5-dicarbozamide (169), 3,4-dimethoxy-Af./V-dimethylbenzamide (170) and cinnamamide (173) were also described from A kaempferi [61,244]. The occurrence of the tyramine amides and the modified dipeptides in these plants is of considerable biogenetic and chemotaxonomic significance.

892 Table 13. Isoquinolines Isolated from Aristolochia Species Compound

Name

Sourece

Ref.

Salsolinol (155) 6,7-Dihydroxy-l -methyl-A'-(6'fructopyranosyl)-l ,2,3,4tetrahydroisoquinoline (156)

A. arcuata A. arcuata

285 285

6,7-Dihydroxy-l, l-dimethyl-1,2,3,4tetrahydroisoquinoline (157) 6,7-Dihydroxy-l ,1 -dimethyl-/V-ethyll,2,3,4-tetrahydroisoquinoline(158) 6,7-Dihydroxy-l,l-dimethyl-A'-(2'glyceryl)-l,2,3,4-tetrahydroisoquinoline (159) 6,7-Dihydroxy-l ,1 -dimethyl-A'-(6'fructopyranosyl)-l ,2,3,4tetrahydroisoquinoline (160)

A. arcuata

285

A. arcuata

285

A. arcuata

285

A. arcuata

285

CH3 R 155 H 156 6'-Frc

H3C

CH3

R 157 H 158 CH2CH3 159 CH(CH2OH)2 160 6'-Frc

K

JL '

2

O

161 162 163 164 165 166 167

R F1 FI FI FI CH3 CH 3 C H3

K.]

IV2

OH OH OCH3 OCH3 OH OCH3 OCH3

OH OCH3 OH OCH3 OCH3 OH OCH3

Pericampylinone-B (161) Pericampylinone-A (162) Northalipholine (163) Corydaldine (164) 3,4-Dihydro-6-hydroxy-7-methoxy-2methylisoquinolin-1-one (165) Thalipholine (166) yV-Methyl corydaldine (167)

yV-Methyl-6,7-dimethoxyisoquinolone (168)

A. A. A. A. A.

elegans elegans elegans elegans elegans

168 169,206 169 169 168

A. gehrtii A. elegans A. elegans

287 169 169

A. elegans

168

0

Amino Acids Fifteen amino acids, alanine (182), glycine (183), leucine (184), proline (185), vallme (186), asparagine (187), glutamine (188), senne (189), threonine (190), aspartic acid (191), glutamic acid (192), tyrosine.

893 Table 14. Amides Isolated from Aristolochia Species Compound Pyridine-3,5-dicarbozamide (169) 3,4-Dimethoxy-A',A'-dimethylbenzamide(170) yV-Acetyltyramide (171) Aurantiamide acetate (172) Cinnamamide (173) yV-p-m-Coumaroyltyramine (174)

7V-/>-c/s-Feruloyltyramine (175)

c/s-TV-Feruloyl-3 -O-methyldopamine (176) yV-Jrans-Cinnamyltyramine (177) Af-/7-(r««i-Coumaroyltyramine (178)

,¥-/ra/j.s-Feruloyltyramine (179) (Moupinamide)

(ra/ts-Ar-Feruloyl-3-O-methyldopamine (N-transFeruloylmethoxytyramine) (180) Aristomanoside (181)

Source A. kaempferi A. kaempferi A. cucurbitifolia A. tubflora A. kaempferi A. gehrtii A. mollissima A. moupinensis A.Zollingeriana A. elegans A. gehrtii A. heterophylla A. zollingeriana A. gehrtii A. elegans A. cucurbitifolia A. elegans A. grandiflora A. heterophylla A. gehrtii A. kaempferi A. manshuriensis A. mollissima A. moupinensis A. zollingeriana A. acutifolia A. elegans A. gehrtii A. heterophylla A. manshuriensis A. moupinensis A. papillaris A. pubescens A. zollingeriana A. gehrtii A. manshuriensis A. manshuriensis

Ref. 244,245 244 56 288 61,229 287 204 205 223 168, 169 287 228 223 287 168, 169 159 168, 169 289 232 287 229 241 204 205 223 210 168, 169 287 228 241 205 120 208 223 287 241 241

(193), histidine (194), lysine (195), and y-aminobutyric acid(196) were found in A. clematitis which is known by its vernacular name Upright birthwort growing in Mainland China [297,298]. Asparagine (187) is also isolated from A kaempferi [233] and A. rotunda [212]. Chlorophylls The naturally occurring chlorophylls with a porphyrin skeleton are another group of constituents present in Aristolochia species. Aristophyll

894

OCH3

N 169

174 175 176

R, H OCH3 OCHj

H OCHj

R, 177 H 178 H 179 OCH, 180 OCH, 181 OCH,

R, R3 H H OH H OH H OH OCH, Ogle OCH3

R4 H H H H gle

Fig. (5). The Structures of Amides

A (197) and B (198) were reported as new chlorophyll derivatives from A. elegans [168,292] and aristophyll C (199) was also found as a new chlorophyll in A. heterophylla [232]. Methyl pheophorbide-a (200) and methyl 21-hydroxy-(21i?)-pheophorbide-a (201) were isolated from A. cucurbitifolia [56,159]. A known chlorophyll, pheophytin-a (202), was isolated from A. kaempferi [229]. Another new pheophytin-a derivative described from A. heterophylla is 132-hydroxy-(1325)-pheophytin-a (203) [232]. Incidentally, all these chlorophylls were isolated by our group from Aristolochia species collected in Taiwan.

Table 15. Phenanthrenes Isolated from Aristolochia Species Compound 2-(Phenanthro[3,4-d]-l,3-dioxole-6-nitro-5carboxamido)propanoicacid (204) 9-Ethoxyaristolactone (205) Aristolophenanlactone 1 (206) Aristolide-B (207)

Aristolide-A (208) Aristolide-C (209) Aristolamide (210) Argentinine (211) Aristololide (212)

Source A. longa

Ref. 62

A. tnollissimn A. tubflora A. cucurbitifolia A. heterophylla A. manshuriensis A. cucurbitifolia A. heterophylla A. cucurbitifolia A. heterophylla A. indica A. argentina A. indica

201 220 56, 159 293 241 56, 159 293 56, 159 293 179 295 294

895

O

COOCH3

O

COOCHj O—Phylyl

H3COOC

R

200 201

H OH

202 203

R H OH

Fig. (6) The Structures of Chlorophylls

Phenanthrenes Aristolide A-C (207-209) are the examples of novel dihydrophenanthrenelactones known to Aristolochia species. Aristolide A (208) and C (209) were reported from A. cucurbitifolia [56,159] and A. heterophylla [293] have R configuration at C-10, whereas aristolide B (208) found in the same plants as well as in A. manshuriensis [241] possesses S configuration at C-10. Aristolophenanlactone I (206) and 9ethoxyaristolactone (205) occur in A. tubflora [220] and A. mollissima [201], respectively are also aristolactone derivatives with 8,9-oxygenation at 8 and 9 positions. Aristolamide (210) isolated from A. indica [179] contain -CONH 2 group at C-l. Teresa et al. isolated compound 204, as a new companion of aristolochic acid with -CONH-CH(CH3)-COOCH3 group at C-l and its optical rotation suggests the possibility of L-(+)alanine derivative [62]. Aristololide (212), a phenanthroid lactone is the first 10-oxygenated aristolic acid derivative encountered in nature [294].

896 Argentinine (211), isolated from A. argentina is the only -CH2CH2N(CH3)2 containing phenanthroid amine reported so far [295].

OR, 206 204 205

R, H OCH3

R2 CH3 CH3

NH 2

R. 207 CH3 208 CH3 209 CH3

NHCH(CH3)COOH N(CH3)2

NO,

OCH3 210

R2 H OH OCH2CH3

OCH, 211

212

Fig. (7). The Structures of Phenanthrenes

Monoterpenoids Monoterpenoids were widely distributed in essential oils of Aristolochia species being mostly present as acyclic monoterpenoids, menthanes, pinanes and camphanes (Table 16). In addition to these types, thujanes, caranes, fenchanes and tricyclic monoterpenes were also identified as minor components. A total of forty-three compounds were identified in the essential oils from leaves, aerial stems and underground organs of A. argentina, constituting 84-98 % of total essential oils [296]. The essential oils of A. argentina are characterized by the unusual presence of argentilactone (223) and isomers of undecatriene (219-222). Argentilactone (223), a volatile compound was identified as the lactone of the (-)-(5/?)-5-hydroxydodeca-Z,Z-2,6-dienoic acid [297]. Its presence and quantitative prevalence over any other component in the volatile oils is the

897

Table 16. Monoterpenoids Isolated from Aristolochia Species Structure Acyclic monoterpenoids CH, CH3

JL.

H,C

.-

Source

Ref.

Geraniol (213)

A. brevipes A. gigantea A. ovalifolia A. acutifolia

304 301 306 299

A. argentina A. asclepiadifolia A. gigantea A. indica A. longa A. elegans A. debilis A. longa

296 305 301 307 311 308 298 311

;ra«i-Ocimene (217)

A. argentina A. gibertii

296 300

Myrcene(218)

A. argentina A. debilis A. gibertii A. argentina

296 298 300 296

(3E,5Z)1,3,5-Undecatriene (220)

A. argentina

296

(3E,5E)1,3,5-Undecatriene (221)

A. argentina

296

(2Z,4Z,6E)2,4,6Undecatriene (222)

A. argentina

296

Argentilactone (223)

A. argentina

296, 297

Limonene (224)

A. argentina A. asclepiadifolia A. debilis A. elegans A. gibertii A. gigantea A. ringens

296 305 298 308 300 301 309

JL. CH2 OH

^ ^ ^ ^ ^ ^ CH,

CH, CH3

Compound

H ,C

o

pH

Geranyl acetone (214)

(SVLinalool (215)

2 H,C ^W^ ^ - ^/ \^ •^C/CH ^

CH,

CH3

JL

J^ c H

CH 3

^L

ri,L

(3-0cimene (216)

CH 2

^^^ 11 ^ C H 2

^^^^CH,

CH2

r^^cH, /

(3Z,5E)1,3,5-Undecatriene (219)

\

^W^^CH,

1 1 Men thanes r. \

H3c—V \

/

)

CH 2

'/ y,CH

3

898

H( =

v \_/ C H 3

HiC

A. brevipes

304

a-Teqiineol (226)

A. acutifolia A. asclepiadifolia

299 305

a-Terpenyl acetate (227)

A. argentina

296

a-Terpinolene (228)

A. indica

307

y-Terpinene (229)

A. ovalifolia

306

a-Phellandrene (230)

A. argentina A. ringens

296 309

P-Phellandrene(231)

A. argentina A. gibertii

296 300

Perillyl aldehyde (232)

A. brevipes

304

Perillyl acetate (233)

A. brevipes

304

Cuminyl alcohol (234)

A. heterophylla

228

p-Cymen-8-ol (235)

A. brevipes A. ringens

304 309

1,8-Cineole(236)

Carvacrol (237)

A. argentina A. brevipes A. debilis A. elegans A. gibertii A. brevipes

296 304 298 308 300 304

Carvone (238)

A. gibertii

300

\/~foH N

'

H3C—V ^ H

m-/7-Menth-2-en-l-ol (225)

CH3 >

f-OAc CH,

'

3C

/

^

/

CH3

'

CH,

'

H 2 C=^

CHj

>

( CH,

'

^

0—v

/

\

CH, CH3 /

/ H,C HOH2C—(/ y \—' H 3 C—^ \

^ ' H3C

CH3 ( CH, ^OH CH,

^ ^ " 3

0

».^_/H CH] HO O

V

/

CH,

899

6t:

HjC^XH/

(3-Cyclocitral (239)

A. argentina

296

Borneol (240)

A. asclepiadifolia A. brevipes A. debilis A. elegans A. helerophylla A. mollissima A. ovalifolia A. reticulata A. zenkeri

Bornyl acetate (241)

A. longa A .debilis

305 304 298 308 228 203, 204 306 281 310 311 298

Bornanol (242)

A. debilis A. elegans A. longa

298 308 311

Isobornyl formate (243)

A. argentina A. gibertii

296 300

Camphor (244)

A. brevipes A. debilis A. ovalifolia

304 298 306

Camphene (245)

A. argentina A. brevipes A. debilis A. elegans A. gibertii A. zenkeri

296 304 298 308 300 310

ercrfo-Fenchyl acetate (246)

A. brevipes

304

Bicyclic monoterpenoids Camphanes

CHO

H,C^/-

Fenchanes OAc

900 Pinanes CH,

CH3

CH2

CH3

CH2OH

a-Pinene (247)

A. argentina A. debilis A. elegans A. gibertii

296 298 308 300

Pin-2-en-8-ol (248)

A. longa

311

8-Acetoxy-pin-2-ene (249)

A. longa

311

(5-Pinene (250)

Pinocarvone (251)

A. argentina A. brevipes A. debilis A. elegans A. gibertii A. brevipes

296 304 298 308 300 304

Verbenone(252)

A. brevipes

304

Myrtenol (253)

A. brevipes

304

A-2-Carene (254)

A. gibertii

300

A-3-Carene (255)

A. gibertii A. reticulata

300 281

a-Thujene (256)

A. brevipes

304

Sabinene (257)

A. gibertii

300

cw-Sabinol (258)

A. brevipes

304

Caranes

1*^'

1

Thujanes

H3CH(3 ^ 2

\ HO

C

/

CH3

/

CH,

/—\ F^ \^J

CH,

901 Tncyclic monoterpenoids H3C^/CH3

Tricyciene (259)

A. gibertii

300

most relevant chemical characteristic of A. argentina. Four isomers of undecatriene are reported to have very interesting olfactive properties. The strong earthy odor that the essential oils of A. argentina display can be also largely assigned to the presence of E, Z (220) and E, E (221) undecatriene isomers. The essential oils of A. debilis were analyzed by Hayashi et al. [298] and identified eleven monoterpenoids. Myrcene (218) and limonene (224); and camphor (244) and camphene (245) were the major components of aerial parts and roots of A. debilis, respectively. Two monoterpenoids, geranylacetone (214) and a-terpineol (226) were identified as minor constituents in the hexane extract of the aerial parts of A. acutifolia [299]. Nineteen monoterpenes were identified in the essential oils of leaves and stems of A. gibertii [300]. The essential oils isolated from stems were dominated by limonene (224) content. A comparative study of the essential oils of the stems and leaves of A. gigantea also showed a significant increase in the percentage of monoterpenes in the stem oil, although in this case such increase is probably due to a higher content of linalool (215) and a-terpineol (226), rather than limonene (224) [301]. The essential oil of A. brevipes was characterized by the presence of a high percentage of monoterpenes by Nieves et al. and 1,8-cineole (236), camphor (244), and P-pinene (250) were the main constituents [304]. Linalool (215) and borneol (240) were the major and limonene (224) and terpineol (226) were the minor monoterpenoids found in the freshly prepared essential oil of roots of A. asclepiadifolia which has a characteristic pleasant aroma and smells like many absolutes [305]. The essential oil of leaves of^. ovalifolia [306] was reported to contain thirty two compounds corresponding to 80.43 % of the oil. The major components were geraniol (213), ^-terpinene (229), borneol (240) and camphor (244). Four monoterpenes, linalool (215), bornyl acetate (241), pin-2-en-8-ol (248) and its acetyl derivative (249) were isolated from the essential oil of the aerial parts of A. longa [311]. The later compounds are first cases to be described in the literature of pinanes, functionalized at C-

902 Sesquiterpenoids The sesquiterpenoids with various types of CI5 carbon skeletons constitute the largest group of metabolites in Aristolochia. Twenty four types of sesquiterpenoids were reported so far from the genus Aristolochia (Table 17). However, all these types of sesquiterpenoid frame work arise from the common precursor, farnesyl pyrophosphate, by various modes of cyclizations followed, in many cases, by skeletal rearrangement. The major types of sesquiterpenoids that have been found in the Aristolochia species include cadinanes, aristolanes, germacranes and bicyclogermacranes. Palmeira et al. identified fourty-seven components in three nonpolar fractions from the hexane extract of A, acutifolia [299]. In all the fractions, sesquiterpenes, P-bisabolene (264), oc-selinene (319), aeudesmol (325), spathulenol (372) and trans a-bergamotene (386) were the most abundant. Urzua et al. separated sesquiterpenoids, P-farnesene (260), (-)-P-bisabolene (264), (+)- sesquiphyllandrene (266), p-elemene (268), p-caryophyllene (313), l(10)-aristolene (331), y-cadinene (345), guaiol (360), P-aromadendrene (369), a-aromadendrene (370), ochimachalene (380), a-trans- bergamotene (384), and cc-cedrene (384) from a terpenoid mixture with germination inhibiting property obtained from a nonpolar fraction of the roots of A. chilensis [312]. Volatile oils from roots, stems and leaves of A. elegans [308] were found to be rich in sesquiterpenoids. Sesquiterpene hydrocarbons, in particular bicyclogermacrene (288), P-caryophyllene (313), and isocaryophyllene (314) were the predominated components in the oils from the leaves, whereas the oxygenated sesquiterpenes, mainly is-nerolidol (263), were the main constituents of the oils from stems and roots. Extensive research on Aristolochia species by our group has revealed that A. mollissima, A. heterophylla and A. cucurbitifolia were rich sources of sesquiterpenoids [313,314]. Moreover, sesquiterpenoids were widely distributed in the stems and roots of these species rather than in leaves. Seven sesquiterpenoids, madolin A-E (300, 299, 296, 295, 271) as new and aristolactone (278) and manshurolide (307) as known,were isolated from the stems and roots of A. cucurbitifolia, an endemic species of Taiwan [159,315]. The only difference in the structures of madolins A (300), B (299) and C (296), all possessing 1,10-epoxybicyclogermacrane skeleton was the functionality of C-14: an aldehyde group in the former, with carboxylic acid group in the second and with simple hydrogen in the third. Madolin-D (295), with bicyclogermacrane skeleton is an acetyl derivative

903

Table 17. Sesquiterpenoids Isolated from Aristolochia Species Compound Farnesanes CH3

CH3

Name

Occurrence

Ref.

P-Farnesene (260)

A. argentina A. elegan A. macroura

296 308 301

Faraesol (261)

A. elegans A. peltato-delloidea

308 352

Z-(ft)-Nerolidol (262)

A. elegans

308

£-(/?)-Nerolidol (263)

A. brevipes A. elegans A. gibertii A. gigantea A. macroura A. triangularis

304 308 300 301 301 301,30 2, 303

Bisabolanes CH,

P-Bisabolene (264)

A. acutifolia A. chilensis

299 312

CH 3

ot-Bisabolene (265)

A. elegans A. asclepiadifolia A. impudica

308 305 353

CH 3

P-Sesqui- phellandrene (266)

A. acutifolia A. chilensis

299 312

CH,

CH,

CH3 HC

CH2

CH,

CH3 - ^ ^ - ^ ^ i H3C^ JOH

c c c

H c'

J

j

H

H

904 CH,

a-Curcumene (267)

A. elegans

308

(3-Elemene (268)

A. acutifolia A. argentina A. brevipes A. chilensis A. debilis A. elegans A. gibertii A. rodriguesii A. triangularis

Y-Elemene (269)

A. elegans A. giganiea A. macroura A. triangularis

299 296 304 312 298 308 300 301 301, 303 308 301 301 301, 303

8-Elemene (270)

A. argentina A. elegans A. gibertii A. macroura

296 308 300 301

Madolin E (271)

A. cucurbitifolia A. heterophylla

159, 315 228

Madolin R (272)

A. mollissima

204

Madolin S (273)

A. mollissima

204

Elemanes

CH2

CH,

1

CH,

Germacranes

905 Germacrene A (274)

A. brevipes A. birostris A. impudica

304 301 353

Germacrene B (275)

A. elegans

308

Germacrene D (276)

A. argentina A. brevipes A. elegans A. gibertii A. odoarlissima

296 304 308 300 354

Hedycaryoi (277)

A. elegans A. peliato-deltoidea

308 352

Aristolactone (278)

A. conlorta A. cucurbitifolia A. gibertii A. helerophylla A. kaempferi A. kunmingensis A. liukiuensis A. mollissima

230 159, 315 300 228 244 187 328 200, 203, 204, 329 281, 318 281, 318 222

.CH, CH-,

CH,

CH,

CH,

H,C

CH,

CH,

•O

CH3

A. reticulata A. serpentaria A. versicolar

Isoaristolactone (279)

A. versicolar

222

Madolin J (280)

A. heterophylla

228

906 Versicolactone B (281)

O

CH3

A. heterophylla A. mollissima A. versicolar

203,

Madolin X (282)

A. mollissima

204

Madolin U (283)

A. mollissima

204

Madolin I (284)

A. heterophylla

228

Costunolide (285)

4. yunnanensis

333

Mollislactone (286)

A. elegans A. mollissima

308 200

Melompolide (287)

A. yunnanensis

333

Bicyclogermacrene (288)

A. argentina A. elegans A. gibertii

2% 308 300

228 204 322

CH,

O

H,C

Bicyclogermacranes

907

OHC

(+)-Isobicyclogermacrenal (289)

A. heterophylla A. kaempferi A. manshuriensis A. mollissima

232 244 325 203, 204

Madolin P (290)

A. kaempferi

244

Madolin K (291)

A. heterophylla A. mollissima

238 204

Madolin T (292)

A. mollissima

203, 204

Madolin Y (293)

A. mollissima

317

Madolin N (294)

A. heterophylla

232

Madolin D (295)

A. cucurbitifolia

159, 315

Madolin C (296)

A. cucurbitifolia

159, 315

H3C^CH

I^CH3

CH,

C

°°H

A/ OHC

HjC^Y^

0H

H3C

\Jr\\.,

OHC

OHC

\jr\\^,

ri3^-

CH OAc

H3C^^CH H2C

H3C H2C

n3^

OH

^tH OH

CH OAc CH3

908 CH 3

\

Madolin 0 (297)

A. heterophylla A. kaempferi

232 244

Madolin V (298)

A. mollissima

203, 204

CH3

Madolin B (299)

A. cucurbitifolia A. heterophylla A. kaempferi A. mollissima

159, 315 228 244 204

\ H CH3

Madolin A (300)

A. cucurbitifolia A. heterophylla A. kaempferi A. mollissima

(-)-Lepidozenal (301)

A. heterophylla A. kaempferi A. mollissima

159, 315 228, 232 244 203, 204 228 244 204

1,10-Epoxylepidozenal (302)

A. heterophylla A. mollissima

228 204

a-Humulene (Caryphyllene) (303)

A. argentina A. birostris A. elegans A. gibertii A. indica A. macroura A. papillaris

296 301 308 300 307 301 301

"'•£-)

OHC

H3C H3C

\ H

Tj

''',

nnp

C^

3

HOOC

OHC

CH O Ac

H3C

H3C H,C

OHC

OH

H,C

\

H

\ H

3

CH3

OHC

H3C^^CHi Humulanes CH,

H3C-J H,C

1

909 H,C.

Humulene epoxide II (304)

A. acutifolia

299

Madolin M (305)

A. heterophylla A. mollissima

228 204

Madolin L (306)

A. heterophylla

228

Neoaristolactone (manshurolide; versicolactone A) (307)

A. cucurbitifolia A. heterophylla A. manshuriensis A. mollissima A. versicolar

159, 315 228 327 204 320, 321

Versicolactone D (308)

A. heterophylla A. kunmingensis A. versicolar

228 187 324

Madolin W (309)

A. mollissima

204

CH3

H3CO*" CHO

HO

Madolins

v/

CHO

910 AcO,,

Madolin H (310)

A. heterophylla A. mollissima

228, 316 204

MadolinG(311)

A. heterophylla

228, 316

Madolin Z (312)

A. mollissima

317

(3-Caryophyllene(313)

A. acutifolia A. argentina A. brevipes A. chilensis A. debilis A. elegans A. gibertii A. indica A. longa A. melanoglossa A. ringens A. argentina A. elegans

299 296 304 312 298 308 300 84, 307 311 355 309

A. acutifolia A. elegans A. indica A. longa A. melanoglossa A. peltato-delloidea A. chamissonis A. pubescens

299 308 307 311 355 352

CHO

AcO/,,,

CHO

OH

CHO Caryophyllanes H3C

H

Isocaryophyllene (314)

H3C^ ' V HC I

\, % *CH, ^_V

Caryophyllene oxide (315)

8/?,9«-Oxide-pcaryophyllene(316)

H,C

/

296 308

356 357

911

H,C

H ^ )

A. pubescens

357

Caryophyllenol 1(318)

A. elegans

308

a-Selinene (319)

A. acutifolia

299

Selina-3,7(ll)-diene (320)

A. argentina

296

8-Selinene(321)

A. peltato-deltoidea

352

(3-Selinene (322)

A. acutifolia A. brevipes A. gibertii

299 304 300

5PH, 7|3, lOa-Selina4,ll-diene(323)

A. indica

335

Aristolochene (324)

A. indica

334

a-Eudesmol (325)

A. acutifolia A. peltato-deltoidea

299 352

'OH

CH Eudesmanes CH,

c:H,

Kobusone(317)

CH,

CH,

H ( • H, CH,

C

\ C^H, CH,

(:H,

do T

YP

TCH,

L/CH,

Ti CH, ,,lfCH,

i CH, CH, CH,

CH,

CH, CH,

CH,

912 CH

P-Eudesmol (326)

A. brevipes A. odorntissimn

304 354

a-Cyclocostunolide (327)

A. yunnanensis

333

Isoalantolactone (328)

A. debilis

337

Dihydroisoalantolacto ne(329)

A. debilis

337

(5-Gurjunene (330)

A. argentina

296

A1(10)-Aristolene (calarene)(331)

A. brevipes A. arcuata A. chilensis A. debilis A. elegans A. longa A. papillaris A. contorta

304 188 312 344 308 311 301

Aristolanes

i CH3 CH 33

CH3

"K CH3 CH,

I CH3 CH3 A-Aristolene (332)

CH3

I CH3 CH,

CH,

9-Aristolen-la-ol (333)

A. debilis

230 344

. peltato-deltoidea

352

913

i CH3 CH3

^f,,, 1 'CH3 CHO

^ CH, CH3

l(10)-Aristolen-13-al (334)

A. debilis

A'"0)-Aristolone (2oxocalarene) (335)

A arcuata A. clematitis A. debilis

I CH3 CH3

3

346

A -Aristolenone (336)

/(. debilis

188 153 338, 339, 344 311 ~344~

A1(10)'8-Aristolodien-2one (337)

A. debilis

345

A'uu)-Debilone (338)

A. debilis

344

9a-Hydroperoxy1(10)- aristolenone (339)

A. debilis

345

A9-Aristolone(-)aristolone) (340)

A. albida A. debilis

99 344

1 a-Hydroxy-9aristolenone (341)

A.debilis

345

A. longa

I CH3 CH3

CH3

345,

C CH, OH

OOH

= CH3 CH3

I CH3 CH,

CH,

914 CH,

HO* 1 H CH,

Maaliol (342)

A. longa

Madolin F (343)

A. heterophylla

228, 316

5-Cadinene (344)

A. acutifolia A. debilis A. elegans A. odoratissima A. papillaris

299 298 308 354 301

Y-Cadinene (345)

A. acutifolia A. debilis A. elegans A. gibertii A. papillaris

299 298 308 300 301

y-Muurolene (346)

A. acutifolia A. elegans

299 308

T-Muurolol (347)

A. elegans

308

8-Cadinol (348)

A. ovalifolia

306

""K 1 CH, CH3 CH

Cadinanes

915 HO

T-Cadinol (349)

A. elegans

308

a-Cadinol (350)

A. acutifolia A. brevipes A. elegans

299 304 308

Cubeno1(351)

A. elegans

308

e/?/-Cubenol (352)

A. elegans

308

Calamenene (353)

A. brevipes A. debilis A. elegans

304 298 308

CH2

p-Calacorene (354)

A. giberlii

300

CH3

a-Calacorene (355)

A. brevipes A. elegans A. giberlii

304 308 300

CH

HJC^^CHJ

II

1 1

CH3 OH? '

0

H

3

C ^

916 CH3

Cadalene (356)

A. brevipes

304

0

CH3

Mansonone G (357)

A. liukiuensis

328

o

X

Dehydrooxoperezinon e (358)

A. manshuriensis A. liukiuensis

325 328

a-Guaiene (359)

A. brevipes A. debilis

304 298

Guaiol (360)

A. chilensis A. elegans A. asclepiadifolia

312 308 305

8-Guaiene(361)

A. debilis

298

Bubnesol (362)

A. brevipes

304

H,C Guaianes H

VM

H3C

^-CH

3

CH3

H,C^H" 3 H3C

H3C

H

>-CH3 H,C

H3C

C; H3C

H

A~CH3 H3C

OH

917 H3C

3

'

6,9-Guaiadiene (363)

A. elegans

308

Guaiazulene (364)

A. elegans

308

y-Gurjunene (365)

A. aculifolia

299

DehydrocostuslactoneA(366)

A. yunnanensis

333

Madolin Q (367)

A. heterophylla

314

Versicolactone C (368)

A. heterophylla A. versicolar

228 324

P-Aromadendrene (369)

A. chilensis

312

H,C H3C

H3C

^ C H

3

H,C H3C H ':

vC)

H3C

U~CHl

H7C H2C H U

O

CH

H C

n~

2

°

O Aromadendranes H2C J_T

H3C

H

\\

'""f^

918 H2C

H

HP

>L H3C

L

H3^SC H3C H2C

"3 1 "

H

A. chilensis A. debilis A. gibertii

312 298 300

fl//o-Aromadendrene (371)

A. acutifolia A. brevipes A. gibertii

299 304 300

Spathulenol (372)

A. acutifolia A. argentina A. chamissonis A. elegans A. gibertii A. melanoglossa A. mollissima A. peltato-deltoidea A. brevipes A. indica A. ovalifolia A. asclepiadifolia

299 296 356 308 300 355 204 352 304 351 306 305

Globulol (374)

A. elegans

308

Viridiflorol (375)

A. elegans

308

Aromadendrane4p,10p-diol (376)

A. heterophylla

228

"3

LHi

1H HO

^J AN-u H3C C " 3 HQ CH3

C

5

C

"3

7^ H,C C H 3 H3C OH

H^C

H3C

7^ H,C C " 3 HO H

H0

H

(+)-Ledol (373)

^

H,C H3C pU

H3
ara-quinone function belongs to a novel skeleton, normaaliane type [316]. Madolin G (311) and H (310) were bicyclic sesquiterpenes with novel skeleton named as madolin type which consists of a three- and an eleven membered rings [316]. Madolin G (311) is a 10,11-epoxide of madolin H (310). Madolin I (284) and J (280) were very similar to aristolactone (278), but first one having ketone and a terminal methylene groups at C-l and C-10, respectively instead of a double bond, whilst the second contains (3,y-unsaturation in the lactone ring [228]. Madolin-K (291) is a bicyclogermacrane identical with isobicyclogermacrenal (289) and possessing cis geometry at C-4 and -5 and an exo 12-hydroxymethyl group [228]. Madolin L (306) has a C-l2 membered ring with three E- configurated double bonds, an aldehyde at C-l and a

922 methoxyl at C-4 with R stereochemistry. The only difference between madolin M (305) and L (306) is the Z-configuration of A2'3 double bond in the former instead of ^-configuration in the latter [228]. Madolin-Q (367) considered to be an artifact formed in the course of extraction and separation with CHC13. The CC13 radical obtained from CHC13 was substituted on C-l position. Madolin Q (367) has a rearranged guaiane skeleton with a 4,6-y-lactone [314]. The spectral data of aromadendrane4p,10P-diol (376) was found to be similar to aromadendrane-4a,10p-diol, isolated from Brasilia sickii, however, a single crystal X-ray analysis and NOESY experiments indicated that the hydroxyl groups at C-4 and -10 have P- relative stereochemistry [228].137Madolin N (294) and O (297) along with madolin A (300), (-)-lepidozenal (301) and isobicyclogermacrenal (289) have been isolated from the leaves of A. heterophylla [232]. Madolin N (294) has a bicyclogermacrane skeleton with a ketone function at C-l, an aldehyde at C-4 and a terminal methylene group at C-10. Madolin-0 (297) is very close to madolin-N (294), but having an epoxide and a methyl group on C-l and -10 instead of ketone and terminal methylene groups. A bicyclogermacrane type sesquiterpenoid with a C-l4 carboxylic acid, madolin P (290) was isolated from stems and roots of A. kaempferi [244]. It is very similar to madolin B (299) but having a C-l/10 double bond instead of an epoxide. Sesquiterpenoid metabolites, madolin R-Z (272,273,292,283,298,309, 282,293,312) along with several other known sesquiterpenoids have been reported from A. mollissima [203,204,317] growing in Mainland China. Two interesting products, madolin R (272) and S (273) are the only examples, having rearranged elemane frame work, with 2-substituted propyl side chain and an aldehyde group attached to C-6 and C-4, respectively of the elemane skeleton. Madolin S (273) is, essentially, the methyl derivative of C-l 2 hydroxy group of madolin R (272) [204]. Madolin T (292), V (298) and Y (293) are bicyclogermacranes with a conjugated formyl group. Madolin T (292) and V (298) were acetyl derivatives of madolin K (291) and O (297), respectively [203]. Moreover, madolin V (298) is an epoxide of madolin T (292) [203]. Madolin Y (293) is very close to madolin N (294), the only difference is the presence of a hydroxyl group at C-l instead of keto function [317]. Thus, biogenetically, it may obtained by the reduction of C-l keto group ofmadolinN[287].

923 Madolin U (283), W (309), X (282) and Z (312) were discovered as minor sesquiterpenoid metabolites from roots and stems of A. mollissima [204,317]. Madolin W (309) and Z (312) are 4,6-cyclohumulane derivatives belongs to madolin type sesquiterpenoids. The only difference between madolin W (309) and Z (312) were the presence of a C10/11 vinyl methyl group in the former instead of a terminal methylene and an additional hydroxyl group in the second compound. Madolin U (283) and X (282) were 4,6-y-lactones of germacrane type sesquiterpenoids. Madolin U (283) is quite similar to madolin I (284), but with a hyrodxyl group at carbon C-l instead of a carbonyl group. Madolin X (282) was structurally closely related to versicolactone B (281), but having an opposite geometry (Z) at A910 double bond. A sesquiterpenoid lactone, aristolactone (278) has first been isolated from A. reticulata, then from A. serpentaria and was shown to be a germacrene type sesquiterpene with P,y-unsaturated lactone by Williams and co workers [281,318]. Later, Smith et al. [319] revised the structure of aristolactone (278) as a,P-unsaturated lactone. Isoaristolactone (279), isolated from A. versicolar was quite similar to aristolactone (278), but just differ in the position of double bond [222]. Zhang et al. [320-322] have isolated versicolactone A (307), B (281) and C (368) from the roots of A. versicolar collected in China. Versicolactone A (307) is an humulane type sesquiterpenoid with 12-membered ring and an ct,p-unsaturated-y-lactone ring. It was also named as manshurolide and neoaristolactone [320,321]. Versicolactone B (281) is a germacrane type sesquiterpenoid with an a-oriented hydroxyl group at C-l position. Versicolactone C (368) was neither a psuedoguaianolide nor guaianolide. From the X-ray analysis it was found that, it has a novel hydroazulene skeleton with a- and P- oriented hydroxyl groups at C-l and C-5 positions, respectively. The biogenesis of versicolactone C (368) from the versicolactone B (281) was proposed through addition of H2O and cyclization [322,323], Scheme (1). A structurally interesting sesquiterpenoid lactone, versicolactone D (308) was also obtained from the roots of A. versicolar [324], for which the structure was characterized with the aid of X-ray analysis and found to be possessed a 12-membered ring, 10-membered ring with a y-lactone and a 6-membered ring. Finally, it was assumed that this sesquiterpene lactone was formed by the condensation of aristolactone (278) and versicolactone A (307). These two molecules connected at 4, 5 and 6 , 1 5 , respectively, which resulted in the formation of a new 6-membered ring. Rucker et al. [325] isolated

924 OH

rr

OH

Scheme (1). The proposed biogenesis of versicolactone C

(+)-isobicyclogermacrenal (280) from the stems of A. manshuriensis of Korean origin. It was identical in all respects with (-)-isobicyclogermacrenal, isolated from the liverwort, Lepidozia vitrea [326], but having opposite optical rotation. It is a (+)-(6S,7/?)-enantiomer of (-)(6i?,7.S)-isobicylcogermacrenal. A sesquiterpene lactone, manshurolide (307) was reported from the stems of this species [327]. It has a 12membered ring with an a,p-unsaturated-y- lactone ring. This is the first report of the occurrence of a 12-membered ring among the sesquiterpenes. Recently, P. L. Wu et al. [241] reported a tricyclic sesquiterpene, dehydrooxoperezinone (358) from the stems of A. manshuriensis of Chinese origin. Its structure with (9-naphthoquinone basic skeleton is very similar to mansonone G (357), isolated from A. liukiuensis [328], but having an additional five membered ring which is formed by cyclization between C-2 and C-8a through oxygen. Three sesquiterpene lactones [200,201,203,204,329-332], aristolactone (278), neoaristolactone (307) and mollislactone (286) were isolated from radix of A. mollissima. Mollislactone (286) has a novel skeleton with 10- membered ring, it may be formed from the germacrene skeleton via rearrangement [200,201]. Versicolactone D (308) and aristolactone (278) were also separated from the aerial parts of A. kunmingensis [187]. Sesquiterpene lactones, costunolide (285), dehydrocostuslactone A (366), a-cyclocostunolide (327) and melampolide (287) were characterized from the underground parts of A. yunnanensis [333] growing in Yunnan province of China. Among them, costunolide (285) and dehydro- costuslactone A (366) were identified as germacranolide and guaianolide, respectively, whereas occyclocostunolide (327) as eudesmanolide. The last compound, melampolide (287) was characterized as l(10)-czs- costunolide by X-ray analysis and it possessed non-oxygenated melampolide skeleton. Nine eudesmane derived sesquiterpenes were isolated from Aristolochia species. Among them, a- and P- eudesmols (325), (326), a-, p- and 5- selinenes (319), (322), (321) and selina-3,7(ll)-diene (320) were found in the essential oils of various Aristolochia members. Govindachari et al. [334] isolated aristolochene (324), a sesquiterpene

925 hydrocarbon from the roots of A. indica. It has an eremophilane type carbon frame work. Aristolochene (324) represents structurally the simplest member of the biogenetically interesting and steadily growing group of eremophilane type sesquiterpenoids. Another sesquiterpene hydrocarbon belonging to the eudesmane group, 5p//,7p,10a-selina4(14),ll-diene (323) has also been isolated from A. indica by Govindachari et al. [335] Catalytic hydrogenation over PtO2 in ethanol gave two stereoisomeric saturated hydrocarbons one of which was enantiomeric with P-selinene (322). Formation of this product during hydrogenation was due to epimerization at C-7, possibly through the migration of double bond of the isopropenyl group, which is well documented in the sesquiterpenoids bearing an axial isopropenyl group [336]. From the biogenetic point of view, the co-occurrence of aristolochene (324) and 5p//,7p,10a-selma-4(14), 11-diene (323) in the same plant suggests that both are derived from a common precursor. Wall et al. separated two lactones, isoalantolactone (328) and dihydroisoalantolactone (329) from the antimutagenic fractions from A. debilis [337]. Fourteen aristolane derived sesquiterpenes were reported so far from the Aristolochia species and this is one of most abundant sesquiterpenoid groups of Aristolochia. Moreover, aristolanes are particularly abundant in the essential oils obtained from the roots of A. debilis. The aristolone (335), an aristolane type sesquiterpenoid component of the essential oil of A. debilis [338-341] was reported to contain a double bond on one side and cyclopropane ring on the other side, conjugated with the ketone group on the basis of the analyses of its reduction products: dihydroaristolone, deoxoaristolone and aristolol. Aristolone (335) has a hydronaphthalene skeleton with ketone group at C-2 position and a methyl group at C-5 position. The underground parts of A. debilis also gave aristolane sesquiterpenoids, A1(10>-331 and A9- aristolenes (332), A1eo-kolavelool-3-oicacid (397), (-)-13-epr- 2oxokolavelool (399), (-)-2p-hydroxykolavelool (401), (-)-2p-hydroperoxykolavelool (402), (+)-13-epz'-2o>hydroxykolavelool (400), (-)kolavelool (398) and (-)-3a,4P-dihydroxykolavelool (403) were isolated

928

Table 18. Diterpenoids Isolated from Aristolochia Species Structure Phytanes

Compound

Source

Ref.

(£)-Phytol (396)

A. elegans A. odoratissima A. peltato-deltoidea

308 354 352

2)-o6eo-kolavelool3-oic acid (397)

A. chamissonis

356

Kolavelool (398)

A. chamissonis A. cymbifera A. galeata

356 267 363

13-ep/-2-oxokolavelool (399)

A. chamissonis

356

(+)-13-epi-2a-hydroxykolavelool (400)

A. chamissonis

356

(-)-2[5-hydroxykolavelool (401)

A. chamissonis

356

("*H

H C

HOH C^^V^^^^^^T CH, Clerodanes

CH, OH

(+)-(4

J "CH3

HO 2 C—4,

OH

TH? 3

OH

1 CH CH3 3 OH

H | HO"""

] ^

f""CH3 #

|' 'CH 3 C H 2

TH™ 3 OH

CH3

3

929

A. chamissonis

356

(-)-3a,4p-dihydroxykolavelool (403)

A. chamissonis

356

A1314-Kolavenic acid (404)

A. brasiliensis A. galeala

360 363

Kolavenic acid methyl ester (405)

A. esperanzae A. galeata

360 363

(2S, 5R, 8 R, 9S, \0R)-2Hydroperoxy-en/-3cleroden-3,13 -diene-15-oic acid methyl ester (406)

A. esperanzae

360

A1-Kolavenol (407)

A. galeata

363

rel-(5S, &R, 9S, ]0R)-entClerod-3,13- diene-15-oic

A. brasiliensis

360

hydroperoxykolavelool (402) CH,

CH,

CH, rcHCOOCH3

CO,H

CH,

acid (408)

930 CH,

A 1314 -2-Oxokolavenic acid

A. brasiliensis

360

2-Oxokolavenicacid methyl ester (410)

A. esperanzae

360

Populifolic acid (411)

A. brasiliensis A. cymbifera A. galeata

360 267 363

Populifolic acid methyl ester (412)

A. esperanzae A. galeata

360 363

Dihydrokoiavenol (413)

A. galeata

363

(2S,5R$R,9S,\0R)-2Hydroperoxy-enf-3cleroden-15-oic acid methyl ether (414)

. esperanzae

360

2-Oxopopulifolic acid (415)

A. brasiliensis A. cymbifera A. galeata

360 267 363

(409) ,,CHjCO2H

I""CH3COOCH

CO 2 H

-,, C H CO 2 H

931

CH,

2-Oxopopulifolic acid methyl ester (416)

A. esperanzae

360

rel-{5S, %R, 95, 10/?)-2-oxo • ent-3- cleroden-15-oic acid (417)

4. brasiliensis

360

e/?('-Populifolic acid (418)

A. cymbifera

267

Columbin(419)

A. albida

364

e«/-Labd-8P-ol-14-ene (420)

A. cymbifera

363

A13l4-e«r-Labdan-8|3-ol-15oic acid (e«(-labd-13-ene8P-ol-15-oic acid) (421)

A. galeata

363

-,CHCO2CH3

I CH3C°2H

COOH

Labdanes

H,C

CH

.„„ CO22H "OH

1

H,C

CH,

932 Copalic acid (422)

A. cymbifera A. esperanzae A. galeala

363 363 363

en/-Labd-8|3-ol-15-oic acid (423)

A. galeata

363

en/-Labd-6p-ol-8(17),13dien-15-oic acid (424)

A. esperanzae

363

e«r-16P(H)-Kaurane (425)

A. Iriangularis A. elegans

270 366

Kauranal (426)

A. elegans

366

Kauranoic acid (427)

A. elegans

366

(-)-Kaur-16-en(428)

A. acutifolia A. argenlina A. chilensis A. Iriangularis

299

Kauranes

v

AH H,C

CH,

AH H,C

CHO

H,C

t\ H COOH

296 312 302,303

933

H,C

H,C

H,C

H,C

303 366

ent-\ 6(3,17-Epoxykauran (430)

A. triangularis A. elegans

270 169

(-)-Kaur-16-ene-18-ol(431)

A. triangularis

269

ent-3f>, 19-Dihydroxy- kaur16-ene(432)

A. rodrigueisii

365

(-)-Kaur-16-en-18-al(433)

A. triangularis

269

(-)-Kaur-16-en-18-oic acid (434)

A. triangularis A. rodrigueisii

269 365

(-)-Kaur-16a-ol-18-a1(435)

A. triangularis

269

CH?OH •CH

H,C

A. triangularis A. elegans

CH,

- _ C CH,

H,C

16a, 17-Epoxykauran (429)

CH,OH

CHO

COOH

CHO

934

H,C

16o-Hydroxy-(-)-kauran-l 9al (436)

A. rodrigueisii

365

(-)-Kauranol (437)

A. rodrigueisii

365

e«M 60,19Dihydroxykaurane(438)

A. rodrigueisii

365

e«2)-afeeo-clerodane skeleton. It has been suggested that this abeo clerodane diterpene could be formed from kolavenic acid via oxidative cleavage followed by Aldol conden- sation pathway, since the yielded rearranged aldehyde could be oxidized into the corresponding carboxylic acid [359]. It is interesting to notice that (+)-13epz'-2a-hydroxykolavelool (400) is unstable, being rapidly transformed into (-)-13-e/>/-2-oxokolavelool (399) under storage. The diterpene, (-)2p-hydroperoxykolavelool (402) having p-hydroperoxy group at C-2 is also not stable and is easily transformed into its 2-oxo derivative. Twelve clerodane diterpenes 404-406, 408-412, 414-417 have been reported from the stems of A. brasiliensis and roots of A. esperanzae, collected in Brazil

936 [360]. It constitutes the first report of the occurrence of clerodane diterpenes in Aristolochiaceae. Treatment of 412 with m-chloroperbenzoic acid resulted in the oxidation of C-2, the reaction product was identified as 416. The formation of this carbonyl compound was explained by an acid promoted 1,2-nucleophilic rearrangement of the epoxide initially formed in the reaction [361]. The presence of peroxide function on C-2 in 414 and 406 was confirmed by dehydration of 414 with acetic anhydride and pyridine to 416 [362]. The compounds 414 and 406 differ only by the saturation 414 or unsaturation 406 at C-13. These two compounds undergo rapid decomposition. On the basis of rapid conversion of the peroxy derivatives 414 and 406 to their corresponding carbonyl derivatives 416 and 410, it was suggested that the 2-oxo-3- clerodene diterpenoids could be artifacts. Among these diterpenes, 415, 416, 404, 412 and 414 possessed a saturated side chain, whereas 409, 410, 417, 408, 405 and 406 contain a double bond between carbons C-13 and -14. It is of interest to note that diterpenes 415, and 417 are the only diterpenes possessing cis stereochemistry at the junction of A and B rings. Lopes et al. [363] examined three Brazilian Aristolochia species A. cymbifera, A. esperanzae and A. galeata and reported labdane and clerodane diterpenoids. Extraction of the leaves of A. cymbifera gave two labdanes, copalic acid 422 and e«f-labd-8P-ol-14-ene 423. Compound 423 has a hydroxyl group at C-8 in the axial orientation. Two labdane diterpenes were isolated from the leaves of A. esperanzae. The structures of these compounds 422 and 424 were elucidated through their corresponding methyl esters. The diterpene 424 contains a side chain analogous to that of 422 and a secondary alcohol group in the axial orientation on the C-6. The roots of A. galeata contain six clerodane diterpenoids, populifolic acid (411), kolavenic acid (404), kolavenol (407), dihydrokolavenol (413), kolavelool (398) and 2-oxopopulifolic acid (415). Populifolic acid methyl ester (412), kolavenic acid methyl ester (405), acetyl derivatives of 406 and 413 were also identified [370]. The conversions 411 to 415 and acetyl derivative of 413 to corresponding 2-oxo derivative reinforce the previous suggestion that the 2-oxokola- venic compounds are artifacts. The clerodane diterpenes, populifolic acid 411 and its new C-5 epimer, epipopulifolic acid (418) together with other diterpenoids, 2-oxopopulifolic acid (415) and kolavelool (398) were isolated from the roots of A. cymbifera [267]. This result suggested that clerodane diterpenes predominate in the roots whereas labdane diterpenes predominate in the leaves of the species. A furanoid diterpene lactone belongs to clerodane type was isolated from the rhizomes of A. albida [268] and identified as

937 columbin (419). Rucker et al. [269] have reported seven e«^-kaurane diterpenes 428, 430, 431, 433, 434, 435, and 442 from the roots and stems of A. triangularis collected in Rio Grande do sul. Further investigation on A. triangularis [270] collected in Parama by Lopes and co workers led to the isolation of seven en?-kaurane type diterpenes 425, 428, 430, 439, 442, 444, and 446. Their studies provide evidence that the main difference in the chemical composition between A. triangularis collected in Rio Grande do sul and in Parama is the occurrence in the species from the first region of considerable amounts of kaurane diterpenes oxidized at C-19. These two reports revealed that A. triangularis is a rich source of e«Z-kaurane diterpenoids. A diterpene (-)-kaur-16-ene (428) was also found in the essential oils of A. acutifolia [299], A. argentina [296], and A. chilensis [312]. Isabele et al. [208,357] reported the e«r-kaurane-16a,17-diol (439) from A. pubescens, which was previously isolated from A. triangularis. Eight e«/-kaurane diterpenoids 425-427, 429, 430, 439, 442, and 444 were discovered by Tsai and co-workers [168], and Luiz et al. [366] in A. elegans. It is of interest to note that the Aristolochia species of Brazilian origin were the only rich sources of diterpenoids. Triterpenoids The triterpenoids are apparently rare in Aristolochia. The foliar epicuticular waxes of leaves of A. esperanzae from Cerrado was analyzed by Oloveira et al. and triterpenoids lupeol (447), P-amyrin (448), epifriedelinol (449), and ursolic acid (451) were identified as major constituents [367]. These triterpenoids clearly predominate over alkanes in the waxes from the Cerrado species. Another triterpenoid friedelin (450) was found in A. indica [368] and A cucurbitifolia [166]. Tetraterpenoids Loliolide (249), an apocarotinoid was isolated as the sole tetraterpenoid from Aristolochia species, A. gehrtii [287]. Apocarotenoids are carotenoids in which the carbon skeleton has been shortened by the formal removal of fragments from one or both ends. Lignans Lignans were another important class of metabolites found in several species of Aristolochia. There are six types of neolignans and lignans with

938 H,C

,CH

r

CH 3

452

Fig. (8). Structures of Tri- and Tetraterpenoids

structural diversity reported to date from Aristolochia genus (Table 19). Compounds 453 and 454 from A. manshuriensis (374) and 455-458 from A. birostris [370] were acyclic neolignans found in Aristolochia. Ligans 459-468 were examples of 2-aryl-3-methyl-2,3-dihydrobenzofuran type neolignans of Aristolochia. They are also termed as eupomatenoids. Eupomatenoids are 3-methyl-2-phenyl-5i?-propenylbenzofuran derivatives and they owe their name to the family of Eupomatiaceae, which is a rich source of these compounds. Among the ten lignans of this type from Aristolochia, 461, 462, and 464-468 were reported from A. pubescens [357]. Licarinediol A (465) and B (467), and O-mehyllicarinediol A (466) and B (468) related to lacarin A (464) were separated as two lildiastereoisomeric mixtures, in which A represents the (25r,35r,85,9/?)

939

Table 19. Lignans Isolated from Aristolochia species Compound &ypropen-3-ol)-phenoxy)-propan-l,3-diol (453) Erythro-1 -(4-hydroxy-3-methoxyphenyl)-2-(2,6-dimethoxy4-(l-(£)-propen-3-ol)-phenoxy)-propan-l,3-diol (454) re/-(8fl)-A-3,4-Methylenedioxy-3',5'-dimethoxy- 8.0.4'neolignan (455) re/-(8/?)-A8'-3,4-Methylenedioxy-5,3',5'-trimethoxy-8.0.4'neolignan (456) re/-(7«,8i?)-A-3,4-Methylenedioxy-3' ,5,5 '-trimethoxy-7hydroxy-8.0.4'-neolignan (457) re/-(8R)-A-3,3',4,5,5'-pentamethoxy-8.0.4'-neolignan (458) Eupomatenoid-1 (Eupomatene) (459) Eupomatenoid-7 (460)

(2R,3«)-2,3-Dihydro-2-(4-hydroxy-3-methoxyphenyl)-7methoxy-3-methylbenzofuran-5-aldehyde (461) (2«,3ft)-2,3-Dihydro-2-(4-hydroxy-3-methoxyphenyl)-7methoxy-3-methylbenzofuran-5-carboxylic acid (462) Eupomatenoid-8 (licarin-B) (463) (+)-(ra«s-Dehydrodiisoeugenol (licarin-A) (464) Licarinediol A (465) O-Methyllicarinediol A (466) Licarinediol B (467) O-Methyllicarinediol B (468) Zuihonin-B (469) 3-e/j/-Austrobailignan-7 (fragransin Ei) (470) Nectandrin-B (471) re/-(7S>,8S,7'«,8'«)-3,3\4,4',5'5'-Hexamethoxy7.0.7',8.8'-lignan (472) (+)-Austrobailignan-7 (473) (+)-Calopptin (474) (+)-Aristolignan (475) (-)-Galbacin (476) Zuonin-A (477) re/-(8R,8'fi)-3,4;3',4'-Dimethylenedioxy-9(3-hydroxy8.0.4'-ligan (478) (-)-Cubebin (479)

Source A. manshuriensis

Ref. 369

A. manshuriensis

369

A. birostris

370

A. birostris

370

A. birostris

370

A. birostris A. laliscana A. arcuatn A. peltato-deltoidea A. taliscana A. tubflosa A. pubescens

370 122,371 277 352 122,371 288 357

A. pubescens

357

A. taliscana

122,371

A. pubescens A. laliscana A. pubescens A. pubescens A. pubescens A. pubescens A. arcuata A. chilensis A. taliscana A. chilensis A. ponticum A. birostris

357 122,371,372 357 357 357 357 277 373 122 374 207 370

A. chilensis A. taliscana A. chilensis A. chilensis A. arcuata A. triangularis A. chilensis A. birostris

374 122 374 374 277 303 259, 374 370

A. birostris A. chamissonis A. cymbifera A. elegans A. esperanzae A. galeala

370 356 267 169 363 363

940

3',4'-Dimethoxy-3,4-desmethylenedioxycubebin (480) 3,4-Dimethoxy-3',4'-desmethylenedioxycubebin (481) p-Methylcubebin (482) a-Methylcubebin (483) rel-(%R,%'S,9R)-3,4-Dimethoxy-3',4'-methylenedioxy- 9aethoxy-8.8',9.O.9'-ligan (484) re/-(8«,8'5,95)-3,4-Dimethoxy-3',4'-methylenedioxy-9pethoxy-8.8',9.O.9'-ligan (485) Aristelegin-C (486) Aristelegin-B (487) (-)-Hinokinin (488)

Kusunokinin (489)

Pluviatolid (490) Bursehernin (491) (-)-5"-Methylhinokinin (492) Aristelegin-A (493) /•e/-(8/?,8'/?)-3,4-Dimethoxy-3',4'-methylenedioxy-9-oxo8.8',9.O.9'-ligan (494) Savinin (495) (-)-Dihydro cubebin (496) Piperitol (497) (+)-Methylpiperitol (498) (-)-Eudesmin (499) Asarinin (500) Fargesin (501)

Sesamin (502) (+)-Eudesmin (503) (+)-Medioresinol (504) (-)-Kobusin (505) (-)-Pinoresinol (506) (-)-Aristotetralol (507) (-)-Aristotetralone (508) (-)-2-Hydroxyaristotetralone(509) (-)-2-Acetoxyaristotetralone(510) (-)-Aristochilone(511)

A. gehrtii A. indica A. pubescens A. triangularis A. triangularis A. triangularis A. elegans A. elegans A. peltato-deltoidea

287 294 208, 357 302,303, 375 303, 375 303,375 169 169 352

A. peltato-deltoidea

352

A. elegans A. elegans A. birostris A. chamissonis A. cucurbitifolia A. cymbifera A. elegans A. gehrtii A. indica A. pubescens A. triangularis A. galeata A. pubescens A. triangularis A. triangularis A. peltato-deltoidea A. pubescens A. elegans A. elegans A. peltato-deltoidea

169,206 169,206 370 356 159 267 169 287 294 208 302 363 208 302 302 352 208 169 169,206 352

A. indica A. pubescens A. gehrtii A. gehrtii A. gehrtii A. galeata A. albida A. cymbifera A. galeata A. pubescens A. tagala A. pubescens A. elegans A. elegans A. elegans A. chilensis A. chilensis A. chilensis A. chilensis A. chilensis

92 208 287 287 287 363 376 267 363 357 158 357 169 169 168 377 377,378 377 377 377

941 A. chilensis A. chilensis

(-)-Aristoligone (512) Aristosynone (513)

311 377

HjCO

R 453 H 454 OCH,

455 456 457 458

-CH,-CH,-CH,CH 3 CH 3

H OCH3 OCH 3 OCH3

R, R, 459 -CH,460 H CH,

H H OH OCH,

OR, 4

469 470 471 472

-CH,H -CH,H CH 3 H H CH 3 CH 3 CHj

*^-s

-CH,CH 3 H CH 3 CH 3 CH3 CH,

*^fi

H H H CH 3

473 474 475

R, R, -CH,CH,CH, H

Rj CH 3 CH 3 CH 3

R, H CH 3 CH,

isomer and B the (2S,3S,8R,9R) isomer. Lignans 461 and 462 were the corresponding bisnor-neolignan aldehyde and acid of licarin A (464). Considering that the configurations of C-2 and C-3 do not change during transformation one could suggest that the enantiomer of licarinediol A (465) might be the key biosynthetic intermediate from licarin A to its bisnorneolignan aldehyde. Thus this later one by oxidative processes could yield bisnorneolignan acid. Eupomatenoid-1 (459), -7 (460), -8 (463), and dehydrodiisoeugenol (464) were isolated from A. taliscana [122,371,372]. The lignans, 469-477 were representatives of 2,5-diaryl3,4-dimethyltetrahydrofuranoid lignans, in Aristolochia species. Zuihonin B (469) from A. arcuata [277], 3-e/?/-austrobailignan-7 (470), zuonin A

942 OH l

R,0

H,CO.

HjCO'

'OR,

478 479 480 481 482 483

OH H H H OCHj H

H -CH,OH -CH," OH -CH,OH CH, CH, H -CH2OCH, - C H r

-CH,-CH;CH, CH3 -CH,-CHr -CH2-

484

H

485

0 C H

,CH

H,CO

R,0

R 4 0'

Rr,

R, R, R4 R5 Ri -CH H -CH,CH, CH, H -CH,-CH ,H CH, H -CH H CH, CH, -CH H -CH,OCH , -CH,-CH

488 489 490 491 492 493

xx

orientalinol -» stephanine -> aristolochic acid I, which involves a phenol coupling step and a dienol-benzene rearrangement [396]. From [2-14C]DL-noradrenaline incorporation experiments, they speculated that the aristolochic acids are related to 4-hydroxynorlaudanosoline, rather than to norlaudanosoline, and that it is the presence of a benzylic hydroxyl group which predisposes an aporphine intermediate to oxidative conversion into an aristolochic acid. Later, this hypothesis was confirmed by the feeding experiments of Schuette et al. [397] In their experiments, they found that [4-14C]- tetrahydropapavarine-HCl feeding to A. sipho gave no radioactive aristolochic acid I, whereas feeding of [4-14C]- norlaudanosolineHCl yielded acid with the carboxylic group containing 69% of the radioactivity. Further labeling studies in A. sipho by Comer et al. proved that tyrosine, dopa, dopamine and noradrenaline can be served as specific precursors in the biosythesis of aristolochic acid. On the basis of the feeding experiment with doubly labeled [p-14C,15N] tyrosine they predicted that the nitro group of aristolochic acid originated from the amino group of tyrosine [398]. The incorporation of tyrosine, (3,4-dihydroxyphenyl)alanine, nororientaline, prestephanine, and stephanine into aristolochic acid in A. bracteata by Sharma et al. demostrated specific utilization of nororientaline [399]. This strongly supported the hypothesis that the oxidative coupling of orientaline gives prestephanine, which is converted to stephanine; oxidative cleavage of stephanine then furnished aristolochic acid. An experiment with doubly labeled nororientaline showed its incorporation intact into the product and confirmed the view that the methylenedioxy group in aristolochic acid originates from an Omethoxyphenol precursor. Parallel feedings of (-)- and (+)-orientaline confirmed that the stereospecificity is maintained in the biosynthesis of aristolochic acid from the 1-benzyltetrahydroquinoline precursors. These feeding experiments strongly supported the following sequence for the

960 biosynthesis of aristolochic acid: norlaudanosoline -» nororientaline -> orientaline -> prestephanine -> stephanine -> aristolochic acid, Scheme (3).

H,CO.

Scheme (3). Biosynthetic pathway of aristolochic acid (5)

Concurrent isolation of the aristolochic acids, aphorphines, 4,5-dioxoaporphines, 7-oxoaporphines, aristolactams and aristolactam Af-glycosides in Aristolochia species is of interesting point in view of biogenesis [260]. Several aristolactams showed a similar substitution pattern to that of the accompanying aristolochic acids [257]. The structural relationship between both these two groups suggested that aristolochic acids are derived from aristolactams rather than directly from quarternery aporphine alkaloids as proposed [400]. Also, Castedo et al. suggested that aporphine alkaloids be postulated as precursors of aristolactams in plants. Thus, the biosynthetic pathway can be enlarged with the introduction of the oxoapophinoids and aristolactams as possible intermediates of aristolochic acids [401]. The 4,5-dioxoaporphine generated from the oxidation of aporphines can function as intermediate for the biosynthesis of aristolactam, while aristolochic acids were derived from aristolactam, Scheme (4). Indeed, conversion of pontevedrine, a 4,5-dioxoaporphine, into an aristolactam takes place in vitro and can be regarded as a benzilic acid rearrangement followed by loss of carbon.

961

HO

Scheme (4). Biosynthetic pathway of aristolactam

Aristolactones, the 10-oxygenated denirtoaristolochic acid derivatives can be viewed as a by-product of the biosynthesis of aristolactam from aristo- lochic acid [294]. Thus the intermediate amino acid might tautomerize to the corresponding imine which could then be hydrolyzed to the lactone via hydroxyacid, Scheme (5).

'OCH 3

'OCH,

Scheme (5). Biosynthesis of aristolactones

OCHj

OCHj •OCH,

962 9-Methoxytariacuripyrone and 7, 9-dimethoxytariacuripyrone with 5nitro-2//-benzo[h]chromen-2-one skeleton, for which Achenbach et al. suggested the trivial name, tariacuripyrone, might originate from a corresponding aristolochic acid: oxidative cleavage of ring A between C-l and C-2 and subsequent decarboxylation and oxidative decarboxylation steps directly produce the tariacuripyrones [52], Scheme (6). This hypothetical biosynthetic pathway is corroborated by the identical substitution pattern observed in aristolochic acid III (4) and 9-methoxytariacuripyrone (629) as well as in aristolochic acid IV (14) and 7, 9-dimethoxytariacuripyrone (630). Lou et al. proposed the biogenetic path way for the neoaristolactone (manshurolide) a typical sesquiterpenoid of Aristolochia with C-l2 membered ring skeleton from the farnesylpyrophosphate [167], Scheme (7). This compound was formed by the end to end coupling of three isoprene molecules. The isolation of a series of manshurolide type sesquiterpenoids with slight structural variations allowed us to propose possible biosynthetic conversions of these sesquiterpenoids from each other, Scheme (8) [314].

Scheme (6). Hypothetical biosynthetic pathway to tariacuripyrones.

Bisbenzylisoquinoline alkaloids can be biogenetically formed by the dimerization of two enantiomeric benzylisoquinoline units through phenolic oxidative pathway. In contrast, oxidative cleavage of bisbenzyltetrahydroisoquinolines through TV-oxide to produce tetrahydroisoquinolone-benzyltetrahydroisoquinoline dimers as well as that of a simple monomeric benzyltetrahydroquinoline, which in turn lead to tetrahydroisoquinolones and biphenyl ethers is an intrinsic part of the general alkaloid catabolic process. It has also been adumbrated that the tetrahydroisoquinolone alkaloids originate in plants from the oxidation of simple benzyltetrahydroisoquinolines [402,403]. These assumptions were corroborated by the co-occurrence of bisbenzyltetrahydroisoquinolines, TVoxide benzoyl benzyltetrahydroisoquinolines, tetrahydroisoquinolines and biphenyl ethers in A. elegans. The structural kinship of these metabolites allowed us to consider a definite possibility that these metabolites were derived biogenetically from bisbenzyltetrahydro- isoquinolines in general alkaloid catabolic process [169]. Thus we proposed a possible biogenetic transformation pathway of these metabolites, Scheme (9).

963

Double bond tranformation OPP

Farnesylpyrophosphate

Cyclization Oxidation Hydroxylation

-H.O COOH

Cyclization

Scheme (7). Biosynthetic pathway for neo-aristolactone

SPECTRAL PROPERTIES Spectroscopy methods are widely used for the structural determination of aristolochic acids. These compounds all show characteristic UV absorption bands of a substituted phenanthrene chromophore at 223, 250, 318 and 390 nm whereas aristolactams absorb at 241, 250, 259, 291 and 300 nm [125,404]. The phenolic derivatives of this series generally display considerable bathochromic shifts on addition of alkali [129,140]. The IR spectrum is useful for detecting functional groups of aristolochic acids [405]. These compounds show two characteristic bands at 1550 and 1350 cm"1 due to the nitro group, and the carboxyl OH group appears at 3000-2500 cm"1 as a broad continuous absorption. Hydroxy derivatives of aristolochic acids or aristolactams show OH and NH absorptions at 33003500 and 3200-3400 cm"1. The carboxylic or lactamic carbonyl is present at 1710-1690 cm"1, whereas the carboxylic group absorption of the sodium salts of aristolochic acids appears at 1540-1580 cm"1 [159,171, 174,246]. In general, the aromatic ring system shows stretches at 1625-1575 and

964 ,CH,

An A

41 R= Anstolochic acid I 42 R= Anstolochic acid II

312

CHO

SAM- S-Adenosyl-L-methionine H COO~

CHO

CHO 311

NH 3 + Ad

Scheme (8). Biogenetic sequences of madolin type sesquiterpenoids

1525-1475 cm"1 as usual, and observation of the bands in the range of 900700 cm"1 is based on the substitutions in the aromatic ring. 1 H-NMR is the most commonly used technique in the structural elucidations of aristolochic acids. The aristolochic acids and aristolactams display a C-2 aromatic proton at 5 7.50-7.90 and C-3, C-4 hydroxy at 8 10.00, or methoxy at 8 3.90-4.10, or methylenedioxy at 8 6.35-6.55 of

965

H3C

R, = H, R, = b-H, R, = CH3, R4 = a-H, (•)-(R, R)-7'-0-metliylcuspidaline (147) R, = CH3, R, = b-H, R3 = H, R4 = b-H, (-)-temuconine (146)

(143) R,= -CH3, R, = -H,R, = C (145) R, - -CH3, R, « -H, R3 = (144) R,= -CHj, R2= -H,R3 =

(162) R, =H,R 5 = R,, = OH

'oxidative cleavage

(166) R, = CH3, R5 = OH, R(, = OCH3 (163) R, = H, R, = OH, R6 = OCH3 (167) R, = CH3, R5 = R6 - OCH3 R7

(543) R6 = CH,OH, R7 = COOH, R8 = OCH3 (546) R,, = R7 = COOH, R, = OCH3 HjCN

A'-methyl-6, 7-dimethoxyisoquinolone (168)

(541) R6 = COOCH 3 , R7 - CHO, R, = OCH 3

(542) R6 = CHO, R7 = COOCHj, R, = OCH3 (545) R6 = R, = COOCH3, R, = OCH, (540) R6 = R7 = COOCH3, R, = OH (544) R6 = CH,OH, R7 = COOCH3, R, = OCH3

Scheme (9). Possible biotransformation pathway from bisbenzyltetrahydroisoquinolines

ring A. The presence of a strongly deshielding nitro group results in a downfield shift about 0.9 ppm of the H-9 of ring B to form a chemical shift at 5 8.40-8.70 in aristolochic acids, but in aristolactams H-9 appears

966 rather upfield at 8 7.05-7.40. The H-9 of the sodium aristolochates shifts upfield and resonate usually from 8 8.15-8.35. The chemical shift of H-5 of ring C can be recognized by its downfield position at 5 8.10-9.12. Most of the aristolochic acids and aristolactams have one or two substitutions on the ring C; therefore, interpretation of coupling constants is useful to determine the ortho or meta H-H splitting and thus the positions of substituents [62,65,127,178,257]. The 13C NMR spectrum is an elegant method to distinguish aristolactams and sodium salts of aristolochic acids from aristolochic acids. The carbonyl carbon of aristolochic acids usually resonates around 8 170-175, whereas it shows an upfield shift of 5-10 ppm in aristolactams and downfield shift of 10-15 ppm in sodium salts of aristolochic acids [246,262]. The mass spectra of aristolochic acids were first reported by Pailer et al. [146], who found that the nitro radical is very easily split off from the molecular ion, giving the base peak [M-46]+, and then the CH3, CO, etc. were removed. Eckhardt et al. [406] found that primary cleavage of aristolochic acid occurs in the condensed aromatic system having a carboxy and a nitro functions in the peri positions, with elimination of NO2, and then loss of small units such as H, CH3, CO, and CHO. In 1987, Priestap reported the mass fragmentation pattern of aristolochic acids [130], Scheme (10). Rucker et al. described the mass spectral fragmentation of methyl ester of debilic acid. It displayed strong molecular ion peak followed by fragments [M-31]+, [M-46]+, [M-46-15]"1" and [M-46-15-15]+ [193]. Priestap explained the principal cleavages observed in the mass spectrum of aristolactam CII and DII [266], Scheme (11, 12). The mass spectrum typical of aristolactams exhibits very strong [M]+ peaks, usually the base peak, and the principal ions were associated with loss of methyl and carbonyl derived from initial cleavages around the methoxy functions. Aristolactam showed a preferential loss of a hydrogen atom to give the base peak followed by the elimination of a carboxy group. Expulsion of a hydrogen atom from the CH2OH group is more pronounced in aristolactam CII, presumably because the resultant ion was stabilized by the two adjacent methoxy groups. Initial expulsion of methyl may represent an equally likely process. The degradation path [M-2Me4CO-HCN]+ can be formulated in which fission of the CH2OH group proceeds with migration of hydrogen atoms on the aromatic ring system. In contrast to typical aristolactams, the [M]+ peak of aristolactam CII is not recognizable, possibly because of preferred stabilization through loss of one hydrogen atom and also by a more facile methyl elimination.

967

Scheme (10). Mass spectral fragmentation of aristolochic acid OCH,

Scheme (11). Mass spectral fragmentation of aristolactam CII (93)

Cleavage of one methoxy group with expulsion of a methyl radical may be favoured by a hydride transfer from the adjacent CH2OH group to the ether oxygen. In mass fragmentation pattern aristolactam DII behaves like typical O-hydroxybenzoic acids. It showed a preferential loss of water to give the most abundant fragment species. The primary cleavage of the

968

Scheme (12). Mass spectral fragmentation of aristolactam DII (92)

methoxy group with loss of methyl observed in aristolactams, is noted only to a very small extent in aristolactam DII. An alternative mode of breakdown in aristolactam DII occurs through dehydration between the carboxyl and methoxy groups with cyclization to a new ring. The resulting fragment then suffers the successive losses of hydrogen, three carbonyls, and hydrogencyanide. The mass spectra of 4,5-dioxoaporphine alkaloids were characterized by a direct loss of CO ([M-28]+) from the molecular ion leading to a prominent peak and subsequent loss of a methyl group accounted for other significant peak [181]. Teresa et al. have explained the mass spectral fragmentation of 2-(phenanthro[3,4-GT]-l,3-dioxole-6-nitro-5- carboxamido)propanoic acid methyl ester isolated from A. longa [62], Scheme (13). It has amino acid side chain joined to the phenanthrene structure by means of an amido linkage. The main fragmentation is due to the loss of the nitro group to give the base peak, and/or cleavage of the amino acid side chain. Priestap et al. reported the mass fragmentation of argentilactone, an active constituent of the rhizomes of A. argentina [297], Scheme (14). We have applied the circular dichroic (CD) exciton chirality method to determine the absolute configuration of sesquiterpene esters of aristolochic acids [407,408], Scheme (15,16). A positive Cotton effect at 221 nm was indicative of the ^-configuration at C-5' of aristophyllide A (27) and C (29) which is due to a clockwise configuration between the double bond and unsaturated aldehyde of the sesquiterpene, aristophyllene. A negative Cotton effect at 262 nm caused by the exciton interaction between the 3,4-methylenedioxybenzoate and the ring double bond inferred the ^-configuration at C-12' of 27 and 29 [251]. The opposite Cotton effects, i.e., a negaive Cotton effect at 227 nm and a positive Cotton effect at 261 nm concluded C-5'5 and C-12'/? stereochemistry for

969

o ,

-NO,

Scheme (13). Mass spectral fragmentation of compound (211)

m/z 150 /-C 2 H 3

C7H13 m/z 97 \ C7H,, mh 95

^\~^ 0 m/z 65 m/z 117

nk 91

Scheme (14). Mass spectral fragmentation of argentilactone (223)

970

JOOOO-

Scheme (15). CD spectra of 39-42

Scheme (16). CD spectra of 27-30

971 aristophyllide B (46) and D (45) [251]. A negative Cotton effect at 250 nm due to the arylcarboxylate chromophore proved the ^-configuration at C-4' for aristoloterpenate I (40), II (39), III (42) and IV (41) [249,409]. TOTAL SYNTHESIS Kupchan et al. achieved the first total synthesis of aristolochic acid involving photocyclization of substituted 2-iodostilbenes [410]. Piperonal (689) provided a suitable skeleton to build ring A of aristolochic acid. It was reduced to the piperonoyl alcohol (690) with lithium aluminium hydride. Bromination of 690 afforded 6-bromopiperonoyl bromide (691). It was then hydrogenated to the corresponding 2-bromo-4,5methylenedioxytoluene by «-butyllithium carbonation method. The toluic acid (693) was converted to acid chloride by oxalyl chloride, and bromination of acid chloride and by radiation with a 200-W tungsten lamp followed by methanolysis afforded the ester 694, which on treatment with silver nitrate produced methyl ester (695). In the synthetic sequence of ring C of aristolochic acid, 2-nitro-6-methoxytoluene (696) was oxidized to the nitroaldehyde (697), by Kronhke reaction. It was converted to the oxime 698 and then to iodaldehyde 700 via Sandmeyer reaction. The synthetic precursor of ring A on condensation with the Schiff s base of 695 in glacial acetic acid gave the 2-carbomethoxy-4,5-methylenedioxy-2'-iodomethoxy-a-nitro-c/5-stilbene (702). Photolysis of this product afforded aristolochic acid I methyl ester. Aristolochic acid (5) was prepared by hydrolysis of its methyl ester using Pailer and Schleppnik method [411], Scheme (17). PHARMACOLOGY To provide scientific support for the wide use of Aristolochia species in folk/traditional medicines, number of scientific groups world wide studied the pharmacological properties of both crude extracts and constituents of Aristolochia species. Many worthy achievements in the pharmacology of Aristolochia have been published. The aristolochic acids have been considered to be the most potent fraction of the Aristolochia constituents. Aristolochic acid I, the most active constituent of Aristolochia has been used for medicinal purposes since the GraecoRoman period [412]. The pharmacopeia of the People's Republic of China indicated that aristolo- chic acid can be used to relieve pain by subdueing hyperactivity of the liver, counteract toxicity, and cause subsidence of

972

HO Ac 68%

THF ] 00%

])n-BuLi CO, 2)H' AgNOj 3)Me0H 46%

ccc

CH, COOH

Scheme (17). Total synthesis of Aristolochic Acid I (5)

swelling [413]. On top of that it was also reported to relieve pain and induce diuresis. Following the observations that the compound was mutagenic and carcinogenic, it was removed from pharmaceutical products since a decade [412]. Antitumor Activity/Cytotoxicity Kupchan and Doskotsch found that an alcoholic extract of A. indica possessed reproducible activity against the adenocarcinoma 755 test system and isolated aristolochic acid I (5) as an active principle [414].

973

5 A A 1 R=H I AA-II R=OCH,

66 AA-I R=H 62 AA-II R=OCH,

+ DNA

dG-AA I dG-AA II

Scheme (18). Metabolic activation and DNA adduct formation of AAI and AAII

Kamatsh and co-workers reported that growth of mouse sarcoma-37 cells incubated with aristolochic acid (5) at concentration of 100-200 u.g/ml for 3 hours was completely inhibited. Treatment of mice with aristolochic acid (5) (1.25-5 mg/kg ip per day) for 3 days, after subcutaneous implantation of sarcoma-37 cells inhibited tumor growth by 40-50 %. The cytotoxicity on HeLa cells in culture was observed at concentration of 25 ug/ml [415]. The acute toxic effects of aristolochic acid I (5) were observed in rats and mice, oral or intravenous administration of high doses was followed by death from acute renal failure within 15 days [416]. The biological activities of aristolochic acid I (5), aristolactam I

974 (66), and aristolactam-/V-P-D-glucoside (71) have been evaluated towards Erlich ascities carcinoma cells in Swiss albino mice [417]. These studies showed that the cytotoxic activity is in the order aristolochic acid I aristolactam-A^-P-D-glucoside aristolactam I. Aristolactam III (65) has been reported to exhibit cytotoxic activity against three kinds of human cancer cell lines (A-549, SK-ME L2 and SK-OV-3) [418]. It is interesting that the cytotoxicity of aristolochic acid I (5) was not only obsereved in animal cells, has also been confirmed for plant cells [419]. Thus Aristolochia plants seem to have developed the aristolochic acid I (5) as chemical barriers against herbivores. Aristoloside (16), a glucoside, was also reported to possess antitumor activity [420]. Aristoloside (16) was given in the drinking water for 30 days to 4 months old SHN mice, a strain with a high incidence of mammary tumors. It inhibited preneoplastic mammary gland growth and elongated the estrous cycle. Aristoloside (16) had little effect on normal mammary gland growth. Since these findings were consistent with the antitumor effect of Guanmu-tong (Gmt, radix of A. manshuriensis), aristoloside was considered to be one of the active component of Gmt [421]. It was developed by the Otsuka Pharmaceutical Co. group as an antitumor agent [420]. In cytostatic potential screening of aristolochic acids and aristolactams against cultured KB and P388 cells by Pezzuto et al, aristolochic acid I (5) showed to be most potential with ED50 value 0.58 uM and aristolactam I (66) and aristolactam iV-(3-D-glucopyranoside (71) also demonstrated appreciable activity with ED50 vlaues 4.2 uM and 6.0 uM, respectively against P-388 cells [413]. In our cytotoxicity screening program on the isolates from Formosan Aristolochia species, most of the compounds exhibited some degree of cytotoxicity against KB, P-388, A-549, HT-29 and HL-60 cell lines with significant ED 50 values [159,223,244], Table 24. Aristolochic acid I (5) displayed excellent inhibition on the growth of HT-29 cell line with the ED50 value of 8.3xlO"4 ug/ml. Incidentally, ariskaninB (35) and cepharanone A (62) are equally potent towards P-388, A-549 and HT-29 cell lines. Ariskanin A (34), aristolochic acid All methyl ester (33) and aristolactam III (65) were cytotoxic against only P388 cell line, whereas ariskanin C (36) was active against only A-549 cell line. Interestingly, aristolactam I (66) and isorhamnetin-3-O-rutinoside (531) well inhibited the growth of all cell lines tested, KB, P-388, A-549, HT-29 and HL-60 cell lines, however the latter compound showed greater extent of inhibition with ED50 values of 1.3, 1.9, 0.8, 1.2 and 0.5 u.g/ml, respectively. Ariskanin E (38), aristolochic acid II methyl ester (25) and

975 Table 24. Cytotoxicity of compounds Isolated from Aristolochia ED50 Cone (u.g/ml) Compound ariskanin A (34) ariskanin B (35) ariskanin C (36) ariskanin D (37) ariskanin E (38) aristolochic acid A II methyl ester (33) aristolochic acid 11 methyl ester (25) aristolochic acid C (2) aristolochic acid I (5) aristolochic acid II (1) aristolactam (66) aristolactam III (65) aristolactam All (83) cepharanone A (62) aristolactam A III (89) aristolactam-A'-P-D-glucoside (71) aristolactam-C-jV-P-D-glucoside(69) isorhamnetin-3-O-rutinoside (531) KB : human epidermoid carcinoma P-388 : mouse lymphocytic leukemia A-549 : lung adenocarcinoma HT-29 : colon adenocarcinoma HL-60 : human leukemia

KB >50 4.1 6.2 11.4 >50 >50 >50 >50 4.0 >50 3.3 13.5 >50 4.1 15.8 >50 >50 1.3

P-388 1.5 2.3 5.1 0.5 0.5 1.5 0.5 8.8 0.7 10.9 1.0 3.7 1.3 2.3 0.8 N 2.4 1.9

Cell lines A-549 >40 1.7 2.3 3.8 1.2 17.4 1.2 >20 5.0 >50 3.2 19.1 2.7 1.7 1.7 N 4.6 0.8

HT-29 8.0 3.3 7.3 2.2 4.9 14.8 4.9 17.1 0.00083 8.4 2.6 18.6 13.2 3.3 4.5 N 12.9 1.2

HL-60 9.3 3.9 5.8 1.3 >50 "1 11.9 >50 40 3.4 >50 2.4 6.8 12.9 3.9 1.8 N 11.4 0.5

aristolactam All (83) were active against P-388, and A-549 cell lines, whereas ariskanin D (37) and aristolactam AIII (89) were active against P388, HT-29 and HL-60 cell lines and P-388, A-549, HL-60 cell lines, respectively. In addition, the novel constituents from our research, sesquiterpene esters of aristolochic acids (39-46) have also showed moderate cytotoxicity against hepatoma G2,2,2,15 cell line [249,251]. Aristolactam All (83) snowed cytotoxicity against PS and KB cells in culture at ED50 3.2, and 2.1 g/ml, respectively [422]. In vitro cytotoxicity of aristolactam la (64) and aristolochic acid I (5) isolated from the roots of A. longa against P-388 lymphocytic leukaemia and NSC LCN6 (brainchial epidermoid carcinoma of human origin) was observed [191]. Zhang et al. reported that the versicolactone A (307) showed growth inhibition against human liver cancer cell line QQy-7703 [320]. An amide, aurantiamide acetate (172) found in A. tubflora showed cytotoxicity against A-549, MCF-7, and HT-29 cells in culture with ED50 values of 2.15x10"', 7.73xlO"2 and 2.60 |ig/ml, respectively [288]. NCoumaroyl- tyramine (178) suppressed the growth of human tumor cells such as U937 and Jurkat cells. In addition, the suppression of the growth

976 of the cells was strongly associated with an increased percentage of cells in the S phase of the cell cycle progression. Furthermore, Ncoumaroyltyramine (178) was able to inhibit the protein tyrosine kinases including epiderma growth factor receptor (EGFR) [423]. Matsuda et al. demonstrated the antimitotic activity of argentilactone (223) isolated from A. argentina [424]. However, it bears a close structural relation with parasorbic acid. Parasorbic acid and a series of ot,p-unsaturated lactones have been reported to induce cancer in rats [425-427]. Argentilactone has irritative effects on the skin and mucosa and also may cause allergic reactions on the skin. Thus Priestap et al. warned the health risk of ingesting the tinctures of A. argentina as it contain 0.05 mg/ml of argentilactone, a possible carcinogenic agent [296]. Mutagenic Activity Pezzuto et al. found that aristolochic acid I (5) serves as a direct-acting mutagen in Salmonella typhimurium strains TA100, TA102, TA1537 and TM677, but was not active in the nitroreductase defficient strains TA98NR and TA100NR [412]. However, aristolic acid (51) was also found to be a direct-acting mutagen in Salmonella strains TA98, TA100, TA102, TA1537, and TM677 as well as strains TA98NR and TA100NR. Both compounds were active mutagens in cultured Chinese hamster overy cells. Thus nitro group at position 10 is not required to induce a mutagenic response. A series of structural relatives, aristolochic acid I methyl ester (28), aristolic acid methyl ester (55), aristolochic acid D (10), aristolactam I (66), aristolactam All (83) and aristolactam-jV-P-D- glucoside (71) were found to be inactive with S. typhimurium strain TM677. But compound 28 and 55 were found to be active mutagens with strains TA98, TA100, TA102 and TA1537. Pistelli et al. isolated aristolochic acid IV (14) from A. rigida and evaluated the mutagenic activity using the plate incorporation assay for S. typhimurium strain TA100 [238,428]. Aristolochic acid IV (14) showed dose dependent activity on mutation of the TA100 strain. The assay with S9, rat liver enzyme system which is added to stimulate metabolic activation process showed no significant dose response relationship, only variations of revertants per plate with in spontaneous levels were noted. On the contrary, in the absence of S9, a significant increase of revertants per plate took place. These results indicated that aristolochic acid IV (14) is endowed with weak direct mutagenic properties, and this effect seemed to be inhibited at least in part, by metabolic reactions.

977 9-Methoxytariacuripyrone (629), a nitro phenanthrene compound isolated from A. brevipes showed strong mutagenic activity in strain TA98, TA100 and some YG strains of S. typhimurium with and without S9 addition [429]. Incubation with cytosol resulted in a heavy increase in mutagenecity. When incubated with microsomes the activity was dramatically decreased. Schimmer et al. viewed that enzymes may possibly involved in activation and detoxification of the compound. The role of the basic structure on the mutagenecity mediated through the nitro group was also considered. Antifertility Activity Aristolochic acid (5) and its methyl ester (28) were found to possess significant antifertility activity in mice [87-89]. Wang and Zheng reported that aristolochic acid (5) showed significant antiimplantation and early pregnancy-interrupting effects when administered orally to mice at a dose of 3-4 mg/kg [430]. This acid showed neither estrogenic nor antiestrogenic actions. Treatment with exogenous progesterone failed to prevent its pregnancy interrupting action. In addition, intra-amniotic injection of aristolochic acid I (5) in mid-term pregnant dogs and rats led to termination of pregnancy. Later, Che et al. reported that aristolochic acid (5) and its methyl ester (28), aristolic acid (51) and (125)-7,12-secoishwaran-12-ol (390) were ineffective in antifertility tests, when given to mice, hamsters and rats [92]. Anti-Oestrogenic and Anti-Implantation Activity Studies on endocrine and contraceptive property of aristolic acid (51) in the pre-implantation stage of pregnancy in mice by Pakrashi et al. elicited anti-oestrogenic nature as it prevented oestrogen induced weight increase and epithelial growth of mouse uterus and prevented implantation in the early stage of pregnancy in mice [431]. Finding of antioestrogenicity of aristolic acid was corroborated by the depletion of alkaline phosphatase activity, glycogen content and mitotic counts of oestrogen treated uterus. Ganguly et al. reported that aristolic acid disrupted nidation in mice when administered on day 1 of pregnancy [432]. The compound did not affect the tubal transport of eggs, but the uterine blue reaction caused by extravasation of the dye, pontamine blue at future implantation sites was inhibited significantly in treated mice.

978 This acid induced impairment of development and reconciled with decreases found in uterine weight and its total protein contents. It prevented specific uterine alkaline phosphatase activity. On the otherhand, specific uterine acid phosphatase activity remained low on days 4 and 5 and increased significantly thereafter. It indicated that aristolic acid interferes with steroidal conditioning of the uterus and renders it hostile to ovum implantation. A sequiterpene, (12S)-7,12-secoishwaran-12-ol (390) isolated from the root of A. indica which is reputed for its emmenagogic and abortifacient properties was found to exert 100% interceptive activity and 91.7 % antiimplantation activity in adult female mice at a single oral dose of 100 mg/kg body weight on day 1 of pregnancy [89]. Subsequent lower doses showed lower percentages of activity. Laparotomy indicated abortion to occur between day 8 and 10 of pregnancy. No toxic effect was found at the dose levels used. It also showed anti-estrogenic potency when administered to immature female mice along with estrogen [433]. When administered alone, sesquiterpene did not showed any uterotropic activity but when given together with estrogen, it inhibited the uterotropic action of estrogen. Since estrogen is necessary for implantation, and the compound is having anti-implantation and abortifacient activities, this action may be due to inhibition of estrogen by the compound during the pre- and post implantation period. Interceptive Activity A phenolic acid compound, /7-coumaric acid (585) isolated from A. indica has been investigated for its antifertility effect at different stages of pregnancy in mice [434,435]. It showed 100% interceptive activity in mice when administered at 50 mg/kg dose. The therapeutic value of the 100 % active dose is investigated by acute toxicity and teratogenic studies. In acute toxicity study, it had high margin of safety (>1000 mg/kg). The compound did not show teratogenic effect. Abortifacient Activity Methyl ester of aristolic acid (55) isolated from A. indica exerted 100%, 25% and 20 % abortifacient activity in mice when administered at the dose level of 60 mg/kg body weight on days 6 or 7, 10 and 12 post coitum, respectively [88].

979 A sequiterpene, (12S)-7,12-secoishwaran-12-ol (390) exerted 100% abortifacient activity in mice when administered at a single oral dose of 100 mg/kg body weight on 6th or 7th day of pregnancy [89]. Short Term Toxicity Study As methyl ester of aristolic acid (55) showed potent abortifacient activity, Pakrashi et al. designed a short term toxicity study to predict reasonable safety and to identify undesirable drug effects [436]. Chronic adminis- tration of methyl ester of aristolic acid (55) at the dose level of 60 mg/kg/day for 5 days per week over a period of 4 weeks increased liver alkaline phosphatase activity, depleted liver glycogen and decreased kidney alkaline phosphatase activity in the treated group showing damage to liver and kidney. There was also an increase in the treated uterine weight. The treated animals which later became mothers were indifferent towards their letters and exhibited cannibalism. These results provide indications of undesirable drug effects one should look for in human, during clinical trials. Since a possible damage to liver and kidney is indicated here, a special attention should be paid to these organs in experiments with higher dose in same or higher animals. Immunomodulating Activity Experiments performed in rabbits and guinea pigs showed marked stimulation of leukocyte phagocytosis following application of Aristolochia extracts, and aristolochic acid I (5) was characterized as the active principle. It was capable of offsetting chloramphenicol- and prednisolone- impaired phagocytic activity without influencing the number of leukocytes. When phagocytosis was impaired by prednisolone, acid showed the effect depending on the dose of prednisolone: from 2.5 mg/kg prednisolone upwards its lesional activity could not be influenced, but for doses under 1 mg/kg values were clearly normalized by treatment with aristolochic acid I (5). Offsetting of damage by cyclophosphamide to phagocytosis was seen even after higher drug doses. Further investigation proved that 5 has a protection activating effect on the phagocytosis of leukocytes. This effect was also observed in cold blooded animals including carps, Aesculopius snake, and Elaphe longssima [437]. It acts not on the invading pathogens but on the naturally existing endogenous defense of the diseased organism, via vigorous stimulation of the phagocytic activity of host leukocytes [437]. Aristolochic acid I (5)

980 stimulated the reticuloendothelial system (RES) and abolished the RESdepressing effect of chloramphenicol in mice and rats. Treatment with aristolochic acid I (5) increased the oxygen consumption and thus the metabolic activity in mice liver cells and splenocytes [438]. Antibacterial Activity Aristolochic acid I (5) was also reported to exhibit antibacterial action against Staphylococcus aureus, Diphococcus pneumoniae and Streptococcus pyogenes in infected mice at 50 ug/kg ip [415]. When, rats with wounds infected with S. aureus were treated intraperitoneally or orally with aristolochic acid I (5), they recovered much faster than control. In mice with Pneumococci infections were influenced very well by aristolochic acid I (5). Rabits after intravenous application of aristolochic acid I (5) showed an increased antibacterial action of serum. Aristolactam la (64) and aristolochic acid I (5) showed antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, S. faecalis, S. aureus and S. epidermides [191]. Neurological disorders, especially Parkinson's diseases have been treated by the administration of the aristolactam taliscanine (91) to the affected patient [439]. Cepharadione A (107) exhibited antimicrobial activities [440]. An aristolactam, aristolactam ./V-p-D-glucopyranoside (71) characterized from the roots of A. contorta showed significant antibacterial activity against Gram-positive bacteria [260]. The essential oil of A. indica containing P-caryophyllene (313), a-humulene (303), caryophyllene oxide (315), and linalool (215) as major constituents was found to be moderately active against Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, B. shaericus, and Salmonella typhimurium [84]. A sequiterpenoid, l(10)-aristolen-13-al (334) isolated from A. debilis showed moderate antimicrobial activity against Staphylococcus aureus [346]. Anti HIV Activity In anti HIV evaluation by Wu et al, dehydrooxoperezinone (358) isolated from A. manshuriensis showed moderate anti HIV activity in acutely infected H-9 lymphocyte cells with IC50 and EC50 values of 25.1 and 17.5 |^g/ml, respectively and therapeutic index is 1.43 [241].

981 Antiplatelet Aggregation Xu and Sun reported that moupinamide (179), an amide found in several Aristolochia species, inhibited rat platelet aggregation and MDA formation in platelets in vitro [205]. In our antiplatelet aggregation activity evaluation studies, aristolochic acid All methyl ester (33), ariskanin A-E (34-38), aristolactam All (83), aristolactam AIII (89), piperolactam A (84), aristolide A (208), aristolactone (278) and manshurolide (307) showed different degree of inhibition of rabbit platelet aggregation based on inducers, arachidonic acid (AA), collagen (Col), platelet activation factor (PAF) and thrombin (Thr) [159,223,244], Table 25. Among screened, no compound inhibited significantly the rabbit platelet aggregation induced by thrombin (Thr) at even high concentrations (100 u.g/ml). Aristolochic acid All methyl ester (33), ariskanin A-E (34-38), aristolide A (208), manshurolide (307), aristolactam All (83), and piperolactam A (84) displayed 100% inhibition of platelet aggregation induced by AA at 100 ug/ml and however, aristolactam AIII (89) and aristolactone (278) also showed little less inhibition at the same concentration. Among these inhibitors, aristolochic acid All methyl ester (33), piperolactam A (84), aristolactam All (83), and ariskanin-E (38) exhibited 100% even at 50 ug/ml whereas ariskanin B-D (35-37), aristolide A (208), and manshurolide (307) showed little less inhibition at this concentration. Aristolochic acid All methyl ester (33), ariskanin B-E (35-38) and piperolactam A (84) were most potent inhibitors (100%) of platelet aggregation induced by Col and ariskanin A (34) and aristolactam AIII (89) also inhibited significantly. Only ariskanin C (36) showed 100% inhibition of platelet aggregation induced by PAF at 100 |ug/ml, but ariskanin A (34), B (35), aristolactam AIII (89) and aristolactone (278) showed significant inhibition. Wang et al. also reported that aristolactone (278) showed inhibition [187] of platelet aggregation induced by PAP with an IC50 = 2.63 x 10"5 M. Smooth Muscle Relaxant Activity Lemos et al. studied the nonspecific and reversible smooth muscle relaxant activities of the ethanol extract of A. papillaris (EE), a fraction containing tertiary alkaloids (TAF) and three alkaloids isolated from it [120]. In non pregnant rat uterus, EE and TAF inhibited both the oxytocin-induced contractions and the amplitude of rhythmic spontaneous contractions with the IC50 values of 0.91 and 0.22 (ig/ml in the first experiment and 1.0 and 0.17 u.g/ml in the second, respectively. The rythmic contractions of the uterus obtained from 21-day pregnant rats

982 Table 25. Antiplatelet aggregation activity of compounds Isolated from Aristolochia Inducer Compounds Cone, (nig/ml Inhibition (%) Compounds

Tlir(0.1U/ml)

Col(10nig/ii]l)

AA(100nM)

PAF(2 g/ml)

100

50

100

50

20

100

50

20

100

50

iclliyl plicophorbidc-ii (21

A

A

A

A

0.9 ± 1.1

N

N

0.1 ± 4.1

N

N

45.2 ±15.1

15.5 ±6.4

0.5 ±3.8

22.5 ±4.8+

N

13.5 ±4.5*

N

N

48.3 ±21.7*

N

arisiohde-A (208)

-0.6 ± 1.8

N

100.0 iO.Of. 89.6 ±8.6*

aristolactonc(278)

-1.5 ±0.8

N

9I.7±6.8+.

11.2 ±7.3

N

maiishurolidc (307)

-3.2 ± 2.6

N

100.0 ±0.0$ 77.3±11.5f

18.2 ±6.7* 21.9 ±5.9+

N

N

51 1±12.3+

N

arsto!aciam-All (83)

0.8 ±3.9

N

100.0 ±0.0J 100.0 ±O.0J 95.3 ± 3.7J J6.8± IO.2t

N

N

10.6 ± 4 1 +

N

pipcroliiciiini-A (84)

4.0 ± 3.4

N

!00.0±0.0j 100 0±0.0f

38.8 ± 13.8+

N

anskaiiin A (34)

7.5 ± 1.7

N

100.0± 1 3

N

N

76.2 ± 5 8

N

N

61.2 ±9.0

N

iiriskiiniii B (35)

4.7 ± 1.2

N

100.0 ± 1.3

80.4 ± 4.1

3.1 ± 2.5

100 0±0.6

36.8 ±6.8

12.6 ± 1.4

60 6 ±8.9

ariskanmC(36)

3I.9±7.3

N

100.0 ± 1.3

51.5 ± 8.0

5.2 ±2.7

100 0 ±0.6

80.9 ± 7.5

13.5 ± I.I

100.0 ± 1.2

19.0 ± 3 1

ariskanin D (37)

4.1 ± 1.9

N

100.0 ± 1.3

93.4±3.8

47.8 ±9.3

100,0 ±0.6

IOO.O±O.6

8.1 ±0.7

90.0 ± 4.9

15.3±2.9

anskanin E (38}

6.9 ± 1.9

N

100.0 ±0.4

100.0±0.4

4.0 ± 1.7

100.0 ±0.5

100.0 ±0.5

12.6 ±4.5

45.3 ± 8.9

N

10.3 ± 1.6J 100.0 ±0 0; 60.6 ± 2 ) 1 * 22 3 ± 2.4

N " "

N

100.0 ±0.4

100.0 ±0.4

6.6 ± 1.3

100.0 ±0.5

I7.8±4.2

2.5± t.6

100.0 ± 1.2

14.2±4.0

arsiolaciam-AH] (89)

3.7 ± 1.2

N

77.9 ±9 1

N

N

67.9 ±5.1

N

N

58.9 ±5.5

N

aspirin

2.5 ± 1.2

N

!00.0± 1.4 51.0 ± 12.5

3.6 ± 1.3

7.3 ±3.9

N

N

2.4 ± 1 2

N

rislolocliic acid A II metli 16.3 ± 10.6 esler (33)

N = not tested. A = platelet aggregation promoted. Thr = thrombin, AA = arachitonic acid, Col = collagen, PAF = platelet activation factor.

were also reduced by EE and TAP with IC50 values of 25.5 and 11.2 )ag/ml, respectively. The relaxation of isolated guinea pig trachea produced by EE and TAP were also observed with the compounds isolated from TAF, moupinamide (179), coclaurine (140) and isoboldine (100) with IC50 values of 1.58xlO"4 M, 3.98xlO"4 M and 7.10xl(T4 M, respectively. Propranolol significantly antagonized the effects of coclaurine (140) and isoboldine (100) but failed to inhibit the responses to moupinamide (179) which suggests that these compounds produce muscle relaxation by P-adrenoceptor-dependent and -independent mechanisms. Antispasmodic Activity The methanol extract of the aerial parts of A. constricta actively inhibited the electrical induced contractions of the isolated guinea-pig ileum with IC50 value of 196.3 ug/ml, Table 26. Five protopine alkaloids,

983 Table 26. IC50 Values and Confidence Limits of Extracts, Fraction VI, and Pure compounds from A. constricta on ECI and Ach- and Histinduced Contractions of Guinea-pig Ileum IC50 values

CHCl 3 -MeOH extract MeOH extract fraction VI

137 139 138 135 136 137 139 138 135 136 137 139 138 135 136

confidence limits (lower-upper) effect on ECI 515.7 711.3 M 681.7 ng/mL 160.3 272.1 M 196.3 ng/mL 180.3 265.4 M 220.3 u.g/mL 6.6 x 1 0 ' M 4.8 x 10"' M 8.9 x 10"'M 1.9 x 10"'M 1.4 x 1 0 ' M 2.5 x 10"'M 1.6 x 10"'M 2.6 x 10"'M 2.1 x 1O"5M 4.6 x 10"' M 9.4 x 10"'M 6.6 x 10"'M 8.6 x 10"'M 5.8 x 10-'M 1.3 x 10"'M effect on Ach-Induced Contractions 4.3 x 10"'M 7.8 x 10-'M 5.8 x 10"' M 2.0 x 10"'M 1.5 x 10"'M 2.6 x 10-'M 2.6 x 10"'M 1.4 x 10"'M 2.8 x 10"'M 5.1 x 1 0 ' M 1.1 x 10"'M 7.5 x 10"'M 8.6 x 10"'M 6.0 x 10"'M 1.2 x 10""'M effect on Hist-Induced Contractions 4.9 x 1 0 ' M 9.3 x 1 0 ' M 6.8 x 10"'M 2.2 x 10"'M 1.5 x 1 0 ' M 3.1 x 10"'M 1.8 x 10"'M 3.9 x 10"'M 2.6 x 10"'M 3.7 x 10"'M 6.9 x 10"'M 5.1 x 10-'M 5.9 x 10"' M 4.2 x 10"'M 8.2 x 10"'M

3,5-di-O-methylconstrictosine (137), 5,6-dihydro-3,5-di-O- methylconstrictosine (139), 5,6-dihydroconstrictosine (138), constrictosine (135) and 3-0-methylconstrictosine (136) were isolated as compounds responsible for the observed antispasmodic activity of the extract. These compounds exerted a significant activity on the ECI, at concentrations of 2.5xlO"5, 5*10~5, 10"4 M. The relative order of potency was: 137 R2=OH 83 R,=R2=OH

80

81R=H 82 R=OH

O

84R=H 87 R=OH

85 R,=OH, R2=H 86 R,=C1, R2=H 89 R,=C1, R2=OH

88

Comparison of the structure of jaborosalactone 2 (85) with that of trechonolide A (64) indicated that both compounds may have closely related biosynthetic precursors. In the former withanolide, ring closure on C-12 has occurred with a C-22 hydroxyl as shown in Fig. (4) pathway a. Oxidation of this intermediate or a related compound to the 22-ketone would allow cyclization between C-23 and the C-12 ketone to give a spiranoid withanolide (Fig. (4) pathway b). The co-occurrence of trechonolide A (64) and jaborosalactone 2 (85) in J. araucana supports this hypothesis.

1034

.0

OH

o

OH

t 77

64

85

Fig. (4). Proposed biosynthetic routes to trechonolides (pathway a) and spiranoid withanolides (pathway b) via a common precursor. The spiranoid intermediate could render both types of spiranoid withanolides either by reduction at C-22 or dehydration of the 20-hydroxyl.

Aromatic ring-D withanolides Withanolides with a six-membered aromatic ring D constitute nowadays an important group. A small number of these compounds, the nicandrenoids, were isolated from the Peruvian "shoofly" plant Nicandra physaloides (e.g. Nic-1, 90) [1,2] and remained as a curiosity within the withanolides, for many years. A family of these type of withanolides and related ergostane derivatives (termed salpichrolides) were isolated from Salpichroa origanifolia (Lam.) Thell in the last decade. The major components, in plants collected in Cordoba and Buenos Aires provinces (Argentina), were salpichrolides A (91) and G (92), salpichrolides B (93) and C (94) being isolated as minor compounds [45-47]. Compound 91 was the first withanolide having a 5,6-epoxide with a-stereochemistry, a feature found afterwards in several other salpichrolides.

o

o

= O

;o OH ""6 90

91R=H 92 R=0H

1035

O

OH

= O

= O

93

From plants collected in Buenos Aires in winter, two ergostane derivatives, salpichrolides E (95) and F (96) were isolated [48]. The stereochemistry of the C-24 methyl could not be determined. It is noteworthy that this type of side chain has been found only in N. physaloides, the other plant known to contain ring D withanolides [1,2].

o

A group of withanolides hydroxylated in the side chain, was isolated as minor components from S. origanifolia collected in Buenos Aires province in winter and in Salta province in summer. They were named salpichrolides H (97), I (98), J (99), K (100) and M (101) [47,49] Salpichrolides H and M correspond to the two possible products of hydrolytic {trans) cleavage of the side chain epoxide. Salpichrolides H (97) and I (98) could be intermediates in a degradative pathway, leading from salpichrolide A (91) to salpichrolide E (95), by oxidative cleavage of the C-25-C-26 bond, Fig. (5). C-26 would give rise to the formyloxy group. Salpichrolide F (96) may be derived analogously from the corresponding 5a,6(3-diol (salpichrolide C), or by hydroytic cleavage of the 5,6-epoxide of 95.

1036

0

97 26S/26S 2.5:1

9S26S/26R 1:3.5

0

o

I 99

100 R,=OH, R2=H 101 R!=H, R2=OH (26S/26R 1.3:1

A common feature of most withanolides, is the oxidation level of C-22 and C-26, C-26 being oxidized in most instances to the carboxylic acid level, thus allowing the formation of a 22,26-lactone. In some withanolides (e.g. in most salpichrolides) it is at the aldehyde level, allowing the formation of a 22,26-lactol. Salpichrolide J (99) and K (100) are the first withanolides with a side chain in which oxidation levels at C22 and C-26 are reversed. Salpichrolide K (100) slowly cyclized to salpichrolide J (99) in solution. A possible biosynthetic pathway for these compounds is presented in Fig. (6).

Fig. (5). Proposed degradative pathway for the formation of the side chain in salpichrolides E (95) and F (96). Starting from salpichrolide A (91) the first two intermediates correspond to compounds 97 and 98.

1037

Fig. (6). Proposed biosynthetic pathway for the formation of the side chain in normal salpichrolides and in salpichrolides J (99) and K (100).

Besides withanolides with an aromatic D ring, three withanolides with a normal (5-membered) D ring were isolated from S. origanifolia, salpichrolides D (102), L (103) and N (104) [46,49]. All of them retained the characteristic 5a,6a-epoxide moiety, unique to S. origanifolia. ,0

OH

102

103

104

The structure of salpichrolide N (104) is particularly interesting regarding the biosynthesis of withanolides with an aromatic D ring. Whiting has proposed as a possible pathway to ring D aromatization, the oxidation of C-18 followed by a 1,2-shift of C-17 to form a new six-

1038

membrered ring via a cyclopropyl fused intermediate; this would lead to salpichrolide A and related compounds upon cleavage of the C-13-C-17 bond as shown in Fig. (7) pathway a. The cleavage of the C-13-C-18 bond of the cyclopropyl intermediate would result in migration of the angular methyl (C-18 to C-17 via a 13,15-diene intermediate) to yield salpichrolide N (104) (Fig. (7) pathway b). Salpichrolide L (103) may be the precursor of a putative 14,16-diene intermediate.

103

Fig. (7). Proposed biosynthetic pathways for the formation of withanolides with an aromatic D ring (e.g. 91, pathway a) and for the rearranged skeleton in salpichrolide N (104, pathway b).

Acnistins and withajardins The acnistins are withanolides with a bicyclic side chain at C-17 similar to that of the withametelins [1-3], but with C-21 directly bonded to the lactone ring (C-24) via a C-C bond instead of an ether bond. Formation of the new 21,24 bond is considered to take place by a SN2-type reaction in withanolides having a good leaving group at C-21 [1]. The first acnistins, acnistin A (105) and E (106) were isolated from plants of Acnistus ramiflorus Miers (one of the synonyms of A. arborescens) collected in Merida (Venezuela) [50,51]. Luis et al. isolated acnistins B (107), C (108), D (109), F (110), G (111) and H (112) from Dunalia solanacea Kunth. collected in Medellin (Colombia) [52-54] This type of bicyclic side chain was also found in the withasteroid glycosides tubocapside A and tubocapside B isolated from Tubocapsicum anomalum Makino [1].

1039

O

o

O

""OAc 107

105 R!=H, R2=H 106 R,=OH, R2=H 108 R,=H, R2=OAc

109

O

110

111

112

Withajardins are closely related to acnistins, in this case C-21 is directly bonded to C-25 giving rise to a bicyclic lactone side chain with a six-membered homocycle. Withajardins A-E (113-117) were isolated from plants of Deprea orinocensis (Kunth) Raf. collected in El Jardin, Colombia. A common precursor has been proposed in the biogenetic routes to acnistins, withajardins and withametelins [55-57].

O

o

113 R=H 116R=Ac

114 R=H 115R=Ac

117

1040

BIOLOGICAL ACTIVITY Antifeedant and insecticidal properties Insecticidal properties of withanolides were first noticed on components isolated from the Peruvian plant Nicandra physaloides. Nicandrenone (Nic-1) (90), the major component isolated from this plant, was known by its bitter taste and its insecticidal properties. [58-61] During an infestation by larvae of the Egyptian cotton leafworm Spodoptera littoralis (Boisd) in the summer of 1978, it was noticed that shrubs of Physalis peruviana L. (cape gooseberry) were not attacked, whereas other Physalis and Nicandra spp. suffered heavy damage. Asher and co-workers demonstrated that withanolide E (118) and 4(3hydroxywithanolide E (119), isolated from P. peruviana, as well as several related steroids, had insect antifeedant properties. Further studies on other withanolides showed antifeedant effects and species-specific activity on three insects, S. littoralis (Boisd.) (Lepidoptera), the Mexican bean beetle, Epilachna varivestis Muls. (Coleoptera) and the red flour beetle, Tribolium castaneum (Herbst) [62]. R,0

,COOR

o

118 R=H 119R=OH

R2O' 120 R|=glc-(l-»2)-glc-6'-Ac, R2=R3=H, R4=OH 121 R,=glc-(l-»4)-glc-(l-»2)-glc-6'-Ac, R2=glc, R3=R4=H 122 R,=glc-(l-»4)-glc-6"-Ac-(l-»2)-glc-6'-Ac, R2=glc, R3=R4=H 123 R,=glc-(l-»4)-glc-(l->2)-glc-6'-Ac, R2=glc, R3=Ac, R ^ H 124 R,=glc-(l-»4)-glc-6"-Ac-(l->2)-glc-6'-Ac, R2=glc, R3=Ac, R4=H 125 R,=R2=glc, R3=R,=H 126 R,=glc-(l->2)-glc, R2=glc, R3=R4=H 127 R,=glc-(l->2)-glc-6'-Ac, R2=glc, R3=R4=H 128 R,=glc, R2=R3=H, R4=OH

Waiss and co-workers examined P. peruviana as a possible source of insect resistance in intergenetic hybridization and found that its foliage is highly inhibitory to growth and development of Helicoverpa zea, an insect that is an economic pest of numerous crops including the solanaceous plants tobacco and tomato. Bioassay directed extraction and

1041

fractionation of leaf material led to the isolation of several steroidal glycoside esters (120-128) that reduced the growth of H. zea. These compounds are structurally related to withanolides, with the 5-lactone side chain open and the carboxyl group esterified by mono- di- or trisacharides [63,64]. Artificial diets containing the test compounds at several levels were presented to larval H. zea, and their growth was determined after a 10-day period. The most active substance was the 11-hydroxy diglucoside ester 120, which reduced the weight of larvae to 50% of control values (ED5o) at a dietary concentration of 5.4 ppm. The triglucoside esters with 3-0glucosyl substitution, 121 and 122, had ED5o's of 15 and 50 ppm respectively. This may be compared to the 35 ppm value for both 123 and 124 that are the corresponding analogues with position 22 acetylated. Monoglucoside ester 125, had an ED5o of 85 ppm, the corresponding diglucoside esters 126 and 127 had ED5o's of 64 and 22 ppm respectively. The least active compound was the 11-hydroxy monoglucoside ester 128, at 110 ppm. By comparison, 4p-hydroxywithanolide E (119), which was found in P. peruviana at concentrations of over 2000 mg/kg (dry basis), had an ED50 of about 250 ppm. No clear structure-activity relationship could be established for compounds 120-128, the most striking difference was that between 128 and 120, that showed a ca. 27-fold change in activity. Their structures differ only by a single acetoxy glucose unit, and their polarities -as estimated by chromatographic partitioning between the stationary and mobile phases- were very similar, thus in this case the differences in insect inhibitory effects appear to be governed by very subtle factors. The above compounds were not lethal over the concentration range studied; for example, 120 was tested at 10 times the ED50, and all animals lived. This was consistent with the behavior of H. zea on fresh P. peruviana leaves where the larvae search and sample without settling down to feed. Moreover, the leaves showed a fine pattern of "shotgun" holes instead of the usual serrated feeding zones on preferred hosts where a large amount of plant material had been ingested. On the basis of these data, the authors suggested that growth inhibition was a consequence of feeding deterrence, leading to semi-starvation of animals. Baumann and co-workers studied the variation in the concentration of withanolide E (118) and 4(3-hydroxywithanolide E (119) in the berry as well as in the surrounding calyx during fruit development in Physalis

1042

peruviana [65]. On a fresh weight basis, they all decreased except for 4phydroxywithanolide E (119) that remained almost unchanged in the calyx. However, when related to the tissue water to obtain a measure for chemical defense, there was a decrease in the berry but a strong increase in the calyx during maturation, for both withanolides. When the withanolides content was compared with the ppm-concentration reported for antifeedant effect of those compounds, data suggested that the berry itself could be protected by intrinsic withanolides only when young. It appears that chemical defense is later taken over by the calyx abundantly equipped with 119. These authors also determined the concentration of 118 and 119 in leaves (related to water), 640 ppm for 118 and 1140 ppm for 119, which is high enough to explain the full protection against predation. The antifeedant effect of several withanolides isolated from Salpichroa origanifolia were investigated on larvae of the sanitary pest Musca domestica [66], the stored grain pest Tribolium castaneum [67] and the Mediterranean fly Ceratitis capitata [68]. The time needed to pupate 50% of the surviving M. domestica larvae (PT50) exposed to salpichrolides A (91), C (94) and G (92) is summarized in Table 2. On the basis of the intermediate dose (500 ppm), compound 91 showed the greatest development delay. The 2000 ppm concentration produced in all cases 100% mortality before pupation occurred, not allowing the calculation of the PT50. The concentration needed to inhibit complete development in 50% of the larvae (EC50) was calculated from the dose response curves in each experiment with the three natural withanolides, salpichrolide G (92) being the most toxic (ED50 203 ppm). With salpichrolides A (91) and G (92), adults failed to enclose from puparia. Development delays similar to those obtained with salpichrolide A (91) were observed when medium and low nutrition diets -without withanolides- were offered as food, supporting the idea that these compounds act as feeding deterrents. In the case of Tribolium castaneum, significant developments delays from larva to adult were also observed in treatments with salpichrolide C (94) at 2000 ppm and with salpichrolides A (91) and G (92) at 500 ppm and higher concentrations (Table 2). The results paralleled those obtained previously with M. domestica larvae, salpichrolide A showing the greatest development delay. On the other hand, no development delay was observed with salpichrolide C (94) in T. castaneum at 500 ppm. The

1043

different responses may be explained by species-specific detoxification mechanisms. Comparison of adult size data in treatments that produced development delays showed that control adults were significantly bigger (3.60 ± 0.10mm) than individuals treated at 500 ppm with compounds 91 (3.22 ±0.10 mm) and 94 (3.27 ±0.15 ppm), suggesting feeding inhibition by these compounds [67]. Table 2. Pupation time in Musca domestica larvae and development time for T. castaneum larvae exposed to natural salpichrolides A (91), G (92) and C (94) [66,67]. Musca domestica

Tribolium castaneum

Treatment

Cone (ppm)

PT50 (days)

DT50 (days)

control

-

7.7 (7.5-7.9)

57.3 (52.5-60.8)

91

500

10.3(10.1-10.6)

70.7 (68.3-73.0)

91

2000

ID

85.9(81.5-90.4)

92

500

8.0 (7.8-8.2)

69.1(61.9-74.6)

92

2000

ID

106.6(101.8-112.6)

94

500

8.1 (7.8-8.4)

54.6(49.1-58.1)

94

2000

ID

87.0 (82.0-92.2)

ID: Incomplete development

A group of synthetic analogues of natural salpichrolides was assayed on M. domestica and T. castaneum to assess structure-activity relationships. Results indicated that oxidation of the hemiacetal side chain to the lactone (compound 129) eliminated the biological activity on both species. Acetylation of the hemiketal on the side chain (compound 130) resulted in a nonsignificant decrease of the activity in M. domestica and drastically reduced the observed effect in T. castaneum. Reduction of the 2,3-double bond (compound 131) had a small negative effect on the feeding deterrent activity compared to salpichrolide A (91) [66,67]. These results prompted a study of the influence of modifications in rings A and B of the steroid nucleus on the antifeedant activity. Lethal and sublethal effects of natural salpichrolides and synthetic analogues were evaluated on the Mediterranean fly Ceratitis capitata [68]. The analogues selected for testing involved two major modifications of the A and B ring functionalities. On one hand three analogues with varying degrees of

1044

reduction of the ring A enone system were synthesized (compounds 93, 131 and 132). Although salpichrolide B (93) occurs naturally in S. origanifolia, it is a very minor component and cannot be isolated in sufficient amounts for biological testing; it was prepared from salpichrolide A (91). The second modification involved cleavage of the 5,6-epoxide, followed by oxidation (compound 133) or dehydration (compound 134). Significant development delays from larvae to puparia were observed in treatments with the three natural salpichrolides, A (91), C (94) and G (92); these results were similar to those previously obtained with M. domestica and T. castaneum larvae in which salpichrolide A (91) showed the greatest development delay (Table 3).

o =o

E

129 R=O 130 R=a-H, p-OAc

O

131 R=O 132 R=a-0H, p-H (X

JDH

O

= O

-- o OH 134

Table 3. Pupation time (PT50) and mortality of Ceratitis capitata larvae exposed to natural and synthetic salpichrolides (500 ppm). Treatment

a

PT50 (days)

Mortality (%)

91

10.54(10.18-10.83)

47.5

92

8.47 (8.00-9.00)

37.5

94

8.44(8.1-8.75)

5.0

131

6.84(6.53-7.12)

20.0

93

_•

95.0

132

11.64(10.42-15.02)

77.5

133

5.79(5.61-5.97)

7.5

134

6.15(5.87-6.41)

22.5

Control

5.36(5.14-5.58)

10.0

The high mortality produced by salpichrolide B (93) did not allow PT50 calculation

1045

Oxidation of the 6-hydroxy group in salpichrolide C (compound 133) or cleavage of the 5,6-epoxide in salpichrolide A followed by dehydration (compound 134), resulted in loss of the inhibitory effect. Although reduction of the 2,3-double bond (compound 131) had a smaller effect, the ring A reduced analogue 132 showed the greatest delay among synthetic analogues. Salpichrolide B (93) produced a high mortality before pupation, not allowing the PT50 calculation. The resulting EC50 of salpichrolide B (93) was 83 ppm, being this value lower than those informed for salpichrolide A (91) and G (92) against M. domestica. Exposure of adults of Ceratitis capitata to drinking water containing natural salpichrolides A (91), G (92), B (93) and C (94) produced mortality in all cases, with salpichrolide B producing the highest effect. The fact that the reduction of the 2-en-l-one system increased toxicity is in agreement with the inhibition observed by Waiss and co-workers on Helicoverpa zea larvae exposed to withanolides and related esters isolated from Physalis peruviana [63,64]. In that case compounds with a reduced 2-en-l-one system exhibited higher activity in comparison with 4(3hydroxywithanolide E (119). The content of the salpichrolides in S. origanifolia was monitored by HPLC during plant development, reaching a maximum during summer (Dec 21 st to march 21 st in the southern hemisphere) when insect populations are higher [66]. These results in conjuction with the observed toxic and feeding deterrent activities suggest that these compounds may provide protection against predation by certain phytophagous insects acting as chemical defense. Feeding deterrant activity of the major components of J. odonelliana, jaborosalactone P (77) and jaborosalactone 10 (78), was studied against the stored grain pest Tribolium castaneum [44]. In this case, only jaborosalactone P (77) produced a significant delay in the development of neonatae larvae. Dinan and coworkers studied withanolides as potent ecdysteroid agonists and antagonists to assist in the further elucidation of the mode of action of ecdysteroids and, possibly, as novel invertebrate pest control agents [69]. Sixteen withanolides which had been isolated from Iochroma gesneriodes (Kunth) Miers (Solanaceae) were assessed for agonistic/antagonistic activity using the Drosophila melanogaster B l l cell line bioassay. Those possessing an oxygen-containing function at C-3 (hydroxy or methoxy) and an a,(3-unsaturated ketone in the side chain

1046

ring showed antagonistic activity, with 2,3-dihydro-3Phydroxywithacnistine (135) being the most active (ED50 2.5 x 10~6M versus 5 x 10~8 M for 20-hydroxyecdysone (20E)). Oxygen-containing functions at C-3 are rare among natural withanolides and in many cases they are artifacts of the isolation procedure (especially, methoxy groups), thus it is not clear if the antagonistic activity of the above mentioned withanolides is serendipitous or whether withanolides could be activated upon ingestion by insects [70]. Recently Dinan and coworkers surveyed 128 species of solanaceous plants for the presence of ecdysteroid agonist and antagonist activities. Only weak antagonist activity was associated with a few of the methanolic extracts, including those from species known to contain high levels of withanolides [71].

AcO

o

135

Cancer chemopreventive activity of withanolides The induction of the phase II drug-metabolizing enzyme quinone reductase (QR), using Hepa Iclc7 hepatoma cells, has been currently used to determine the potential cancer chemopreventive activity of withanolides [72]. Induction of QR activity was calculated from the ratio of specific enzyme activities of compound-treated cells in comparison with a solvent control. The concentrations required to double and quadruple QR activities in the cells, CD and CQ, respectively, were generated. To observe only the induction on QR and to avoid cytotoxic effects, the half-maximal inhibitory concentration of cell viability, IC50, was also determined. From the ratio between the IC50 and CD or CQ values, chemopreventive indices (CI) were calculated. Such measurements not only predicted anticarcinogenic activity but also provided a reasonable index of potency and toxicity.

1047

The first studies on cancer chemopreventive activity were performed by Kennelly and co-workers on withanolides isolated from Physalis philadelphica. The most potent compounds were found to be ixocarpalactone A (34), philadelphicalactone A (36), 4p\7p,20Rtrihydroxy-l-oxowitha-2,5-dien-22,26-olide (42), and ixocarpalactone B (38), all of which contained a 4p-hydroxy-2-en-l-one structural unit [73]. Lately, thirty-seven naturally occurring withanolides isolated from southamerican Solanaceae plants were evaluated for their potential to induce quinone reductase [74]. Jaborosalactone 1 (84), jaborosalactone O (71), jaborosalactone P (77), trechonolide A (64) and withaphysalin J (17), were demonstrated to be significant inducers with CD values in the range of 0.27-1.52 mM. In each subgroup of withanolides analyzed, it was found that some substituents lead to changes in quinone reductase activity. These results indicated that a functionalized methyl-18 plays an important role in improving QR activity. On the other hand, the presence of 5a-substituents resulted in lower activities. In general, spiranoid and trechonolide type withanolides exhibited good QR induction. In terms of CI values, some of the compounds described compared favorably with sulphoraphane, a known chemopreventive agent. Among these compounds, the spiranoid jaborosalactone P (77) was one of the most promising in terms of inducing potency and low toxicity. To further evaluate the potential of jaborosalactone P (77) a preliminary study was performed to test the capacity of this agent to induce steady-state levels of quinone reductase in multiple organ sites of BALB/c mice. Sulforaphane was used for comparison in this in vivo study. With Jaborosalactone Ptreated mice, a significant induction was observed in liver and colon, but not in lung, stomach, or mammary gland. The in vivo study confirmed the in vitro results, indicating that withanolides may function as potent phase II enzyme inducers. Activity-monitored fractionation of a chloroform-soluble extract of Deprea subtriflora using a quinone reductase induction assay led to the C-18 norwithanolides mentioned previously. Six of the active compounds obtained from this plant (44, 46, 47, 49, 52 and 53), presentes an a,Punsaturated ketone unit in ring A. Compound 55 -with a doubly unsaturated ring A ketone- was found to be inactive in the QR assay, while compound 54 was active [72]. It has been suggested that the presence of an a,P-unsaturated ketone unit in ring A of withanolides is important for inducing activity in the cell-based QR induction assay,

1048

however other structural features may compensate the lack of this functionality or block its beneficial effects. Withanolides 26 and 27 isolated by Minguzzi and co-workers from A. arborescens were very potent as monofunctional inducers of quinone reductase (CD value), but their selectivity (CI value) was marginal [22]. Phytotoxic activity Recently withanolides isolated from Iochroma australe and the norbornane-type withanolide jaborosalactol 18 (3) isolated from Jaborosa bergii, showed phytotoxic activity on monocotyledoneous and dicotyledoneous species. Iochroma australe extract and the major constituent (17S,20R,22R)-4p,7P,20-trihydroxy-l-oxowitha-2,5,24trienolide (24,25-dehydro-42) reduced growth of the radicle of the weeds Sorghum halepense (Monoct.) and Chenopodium album (Dicot.) [75]. Jaborosalactol 18 (3) showed significant inhibition of radicle growth at 2 x 1(T3 M on the dicotyledoneous species Chenopodium album, Ipomea purpurea and Lactuca sativa (phytogrowth inhibitory activity > 49%) [13]. On the other hand, in the monocotiledoneous species tested {Zea mays and Sorghum halepense) the phytogrowth effect of compound 3 was stimulatory. Thus, the authors suggest that compound 3 could act as a selective phytogrowth controller, stimulating radicle growth of monocotyledoneous species. Trypanocidal leishmanicidal and bactericidal activities. In the course of screening extracts from Bolivian plants against Trypanosoma cruzi, Leishmania spp., Bacillus subtilis and Staphylococcus aureus, Dunalia brachyacantha (Griseb.) Sleumer was found to be active. The bioassay-guided purification of the leaf extract led to the isolation of two known acetoxywithanolides (136 and 137), which displayed antiparasitic and antimicrobial activity (Table 4) [25]. This constitutes the first report of antileishmanial and antitrypanosomial (Chagas'disease) activities for steroidal lactones.

1049

AcO.

OH 136

137

Table 4. Antiparasitic activities of 18-acetoxywithanolide D (136) and 18-acetoxy-5,6-deoxy-5-withenolide D (137) [25].a Cone, (ng/tnl) 1JO

m

JU

Tc TT

La

TTT

TTT

+++

+++

J_L_L.

4-4—U

4-4-4-

TTT

TTT

TTT

+++

+++

25

0

^C

ZJ

10

Lb

+

Ld TTT

+++ 4-4-4-

TTT

+++

1

0 ++ + + a Tc= Trypanosoma cruzi, ii=Leishmania braziliensis, La= Leishmania amazonensis, Ld= Leishmania donovani.. 0; number of epimastigotes or promastigotes identical to control; +: 75% epimastigotes or promastigotes, with few degenerative forms; ++: 50% epimastigotes or promastigotes, with few degenerative forms; +++: total lysis of parasites.

ACKNOWLEDGEMENTS Financial support by CONICET (Argentina), Universidad de Buenos Aires, SeCyT-UNC, Agencia Cordoba Ciencia and FONCYT is gratefully acknowledged

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1051 [32] Cirigliano, A.M.; Veleiro, A.S.; Oberti, J.C; Burton, G.; Phytochemistry, 1995, 40, 611-613, [33] Veleiro, A.S.; Cirigliano, A.M.; Oberti, J.C.; Burton, G.; J. Nat. Prod., 1999, 62, 1010-1012. [34] Carcamo, C ; Fajardo, V.; Tojo, E.; Heterocycles, 1993, 36, 1771-1774. [35] Bonetto, G.M.; Gil, R.R.; Oberti, J.C.; Veleiro, A.S.; Burton, G.; J. Nat. Prod., 1995,55,705-711. [36] Lavie, D.; Besalle, R.; Pestchanker, M.J.; Gottlieb, H.E.; Frolow, F.; Giordano, O.S.; Phytochemistry, 1987, 26, 1791-1795. [37] Parvaez, M.; Fajardo, V.; Shamma, M.; Ada Cryst., 1988, 44, 553-555. [38] Fajardo, V.; Podesta, F.; Shamma, M.; Freyer, A.J.; J. Nat. Prod., 1991, 54, 554563. [39] Cirigliano, A. M.; Veleiro, A. S.; Bonetto, G. M.; Oberti, J. C ; Burton, G.; J. Nat. Prod., 1996, 59,717-721. [40] Monteagudo, E.S.; Burton, G.; Gros, E.G.; Gonzalez, CM.; Oberti, J.C. Phytochemistry, 1989, 28, 2514-2517. [41] Misico, R.I.; Oberti, J.C; Veleiro, A.S.; Burton, G.; J. Nat. Prod, 1996, 59, 66-68. [42] Veleiro, A.S.; Trocca, C.E.; Burton, G.; Oberti, J.C; Phytochemistry, 1992, 31, 2550-2551. [43] Monteagudo, E.S.; Oberti, J.C; Gros, E.G.; Burton, G.; Phytochemistry, 1990, 29, 933-935. [44] Cirigliano, A.M.; Veleiro, A.S.; Oberti, J.C; Burton, G.; J. Nat. Prod, 2002, 65, 1049-1051. [45] Veleiro, A.S.; Oberti, J.C; Burton, G.; Phytochemistry, 1992, 31, 935-937. [46] Veleiro, A.S.; Burton, G.; Bonetto, G.M.; Gil, R.R.; Oberti, J.C; J. Nat. Prod., 1994,57, 1741-1745. [47] Tettamanzi, M.C.; Veleiro, A.S.; Oberti, J.C; Burton, G.; J. Nat. Prod., 1998, 61, 338-342. [48] Tettamanzi, M.C.; Veleiro, A.S.; Oberti, J.C; Burton, G.; Phytochemistry, 1996, 45,461-463. [49] Tettamanzi, M. C ; Veleiro, A. S.; de la Fuente, J. R.; Burton, G.; J. Nat. Prod., 2001, 64, 783-786. [50] Usubillaga, A.; Castellano, G.; Zabel, V.; Watson, W.H.; J. Chem. Soc. Chem. Commun., 1980, 854-858. [51] Luis, J.G.; Echeverri, F.; Quinones, W.; Gonzalez, A.G.; Torres, F.; Cardona, G.; Archbold, R.; Rojas, M.; Perales, A.; Steroids, 1994, 59, 299-304. [52] Luis, J.G.; Echeverri, F.; Garcia, F.; Rojas, M.; Planta Med., 1994, 60; 348-350. [53] Luis, J.G.; Echeverri, F.; Gonzalez, A.G.; Phytochemistry, 1994, 36, 769-772. [54] Luis, J.G.; Echeverri, F.; Gonzalez, A.G.; Phytochemistry, 1994, 36, 1297-1301. [55] Echeverri, F.; Torres, F.; Cardona, G.; Lopez, J.; Quinones, W.; Gallego, L.H.; Pelaez, C ; Rojas, M.; Garcia, F.; Restrepo, L.M.; Rev. Boliv. Quim., 1991, 10, 2123. [56] Luis, J.G.; Echeverri, F.; Quinones, W.; Gonzalez, A.G.; Torres, F.; Cardona, G.; Archbold, R.; Perales, A.; Tetrahedron, 1994, 50, 1217-1226. [57] Echeverri, F.; Quinones, W.; Torres, F.; Cardona, G.; Archbold, R.; Luis, J.G.; Gonzalez, A.G.; Phytochemistry, 1995, 40, 923-925.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

1053

BIO ACTIVE SECONDARY METABOLITES RELATED TO LIFE-CYCLE DEVELOPMENT OF OOMYCETE PHYTOPATHOGENS MD. TOFAZZAL ISLAM AND SATOSHITAHARA Laboratory of Ecological Chemistry, Graduate School of Agriculture, Hokkaido University, Kita-Ku, Sapporo 060-8589, Japan

ABSTRACT: Members of the oomycete genera, e.g., Phytophthora, Pythium and Aphanomyces, which are phylogenetically distinct from fungi, are the most devastating pathogens of plants, animals, fishes and humans. The zoospores of phytopathogenic oomycetes are believed to locate their host plants by chemotaxis, after which they undergo a series of morphological changes before penetrating the host tissues to establish the diseases. Bioassay-guided chromatographic separation visualized some host-specific plant signals which are not only responsible for chemotaxis but also trigger developmental transitions (encystment and germination) of zoospores on host surface to initiate infection. In contrast, nonhost plants possess diverse secondary metabolites that can directly affect motility and viability of the phytopathogenic zoospores, indicating their involvement in plant resistance against oomycetes. This review summarizes isolation, identification and bioactivities of diverse secondary metabolites identified in host and nonhost plants toward the most infamous phytopathogenic oomycete zoospores namely, Phytophthora, Pythium, and Aphanomyces. The possible ecochemical role and the mode of action of these active compounds are discussed in relation to the biorational control of oomycete phytopathogens. The importance of bioassay methods in isolating novel zoospore regulating molecules from the host and nonhost plants is discussed. In addition, a brief discussion on the chemical basis of host-specificity in animal, fish and human pathogenic oomycetes are also included.

INTRODUCTION Oomycetes (Peronosporomycetes in new classification [1]) are phylogenetic relatives of brown algae that cause many destructive diseases of plants, as well as several animal and human diseases [2,3]. They are mostly water or soil inhabiting organisms [4]. The members of oomycetes genera Phytophthora (Ph.), Pythium (Py.) and Aphanomyces are known as the most devastating pathogens of dicot plants [5]. For example, Ph. infestans, which causes the late-blight disease of potato, destroyed the Irish potato crop in 1845 and 1846, resulting in the historic Irish potato famine [6]. This is still a damaging disease, annually costing over $ 5 billion worldwide in crop losses and control measures [7]. Some members of

1054

oomycetes are pathogenic on species other than plants include: Py. insidiosum, which infects animals (swamp cancer); Lagenidium giganteum, which parasitizes the larval stage of mosquitoes is being used as potential control agent; Aphanomyces invadans, A. astaci and Saprolegnia spp. are the devastating pathogens of several fish species [1,2,8,9]. Phytopathogenic oomycetes give a special stage of motile spore with two dissimilar flagella called zoospore, in their life cycle (Fig. 1). Both flagella are ornamented with complex hairy structures probably for swimming, sensing and precisely docking on the host surface [1,5,10,11]. Zoospores of those plant pathogens are liberated from a mycelium or a sporangium within an hour; disease caused by these oomycetes can be multi-cyclic, resulting in severe epidemics that can destroy whole crops within a single season [12]. Zoospores show interesting responses to their host and nonhost roots as well as their secondary metabolites in addition to environmental physicochemical factors. However, very restricted chemical factors regulating the life cycle of such organisms have been known so far, even though understanding well about such chemical factors seems to be very significant to establish new techniques for control of those soil-borne oomycete phytopathogens still problematic in agriculture. Our knowledge of their biology is limited, but their physiology differs from that of fungi, and many fungicides are ineffective against oomycetes [7,14]. New approaches are needed to find novel targets and to develop the 'oomicides' for a sustainable and biorational management of those notorious phytopathogens [14]. •

a

PF

b

\' ^x^v\/)///

C

Fig. (1). Scanning (a) and transmission (b & c) electron micrographs showing the morphological features of an Aphanomyces cochlioides zoospore and its flagella. a. A reniform-ovate secondary zoospore with a shorter anterior (AF) and a longer posterior (PF) flagella. b. Terminal part of an anterior flagellum possessing tripartite tubular hairs, c. Fine tubular hairs on the tapered tip and surface of a posterior flagellum shaft. Circular objects in the background of micrograph (a) are pores (size: 0.6 |am) of SEMpore membrane.

Zoospores of the phytopathogenic oomycetes accumulate at the potential infection sites of host roots by chemotaxis [11,15-17]. Root released electrical currents (electrotaxis) are also found to participate with

1055

chemotaxis in homing responses of zoospores toward roots [18]. A few host-derived chemical signals have been identified as the chemotactic factors of phytopathogenic zoospores (Fig. 2) [19-23]. For example, indole3-carbaldehyde (1) from cabbage seedlings [19], and prunetin (2) from pea seedlings [20], is specific attractants for A. raphani and A. euteiches, respectively. Similarly, cochliophlin A (3) in roots and root exudates of spinach, and daidzein (4) and genistein (5) in soybean root exudates, are the specific attractants for A. cochlioides [21] and Ph. sojae zoospores [23], respectively. The other host-derived attractant so far reported is N-transferuloyM-O-methyldopamine (6) from the roots of Chenopodiwn album as a potent attractant of A. cochlioides zoospores [22]. All these compounds showed their attractant activity at concentrations as low as micromolar to nanomolar levels. Host-specificity in chemotaxis in oomycete zoospores has been described in some earlier reviews [13,17,25, 26]. Recently, some of the host-specific plant attractants were reported to trigger differentiation of zoospores as shown by the host roots suggesting the chemical basis of the host-specificity in phytopathogenic oomycetes [27]. Little is known about the signal transduction mechanism of zoospore chemotaxis and differentiation by host signals, whilst some stimuli related to general signal transduction have been reported. In recent reports, it was claimed that zoospore perceive host signal by a G-protein-coupled receptor and translate into responses via phosphatidic acid and/ or phosphoinositide signaling cascades [13,27,28]. Among the oomycetes, Aphanomyces species cause some of the destructive plant and fish diseases in the world [8, 29-32]. Species of the phytopathogenic Aphanomyces exhibit high degree of specialization and can infect a limited number of plant species [32, 33]. For example, most of the plants are resistant to the strains of A. cochlioides that infect sugar beet, spinach and a few other members of Chenopodiaceae and Amaranthaceae [32-34]. This phenomenon of nonhost resistance, the ability of a pathogen to cause a disease in particular species but not in others, has always intrigued plant pathologists but remains poorly understood especially in oomycetes [3, 35]. Survey of non-susceptible plants using A. cochlioides zoospore bioassay revealed that they possess diverse chemical weapons to defend themselves from the attack of oomycetes [36]. Here we review our research results concerning zoospore attractants, repellents, cytotoxins and inhibitors of zoospore motility together with bioassay systems to survey chemical regulators toward zoospores. The first part of this review described some new findings on the effects of hostspecific plant signals on chemotaxis and differentiation of oomycete zoospores. The potential role and mode of action of nonhost secondary

1056

metabolites in plant-pathogen compatibility is also discussed in the second part of this review in relation to the biorational control of oomycete phytopathogens on the basis of natural product chemistry. This updates our earlier reviews on chemotaxis of oomycete zoospores [13, 37] and homing responses of Phytophthora and Pythium by Deacon and Donaldson (1993) [17], and complements a recent review on molecular basis of recognition between oomycete plant pathogens and their hosts [26].

1: indole-3-carbaldehyde Host plant: Brassica campestris var. capitata Oomycete: Aphanomyces raphani

3: cochliophilin A Host plant: Spinacia oleracea Oomycete: Aphanomyces cochlioides

2: prunetin Host plant: Pisum sativum Oomycete: Aphanomyces euteiches

4: R =• H : daidzein 5: R * OH : genistein Host plant: Glycine max Oomycete: Phytophthora sojae

6: N-frans-feruloyl-4-O-methyldopamine Host plant: Chenopodium album Oomycete: Aphanomyces cochlioides Figure (2). Structure of some host-specific attractants for oomycete zoospores

HOST-SPECIFIC PLANT SIGNALS REGULATING THE LIFECYCLE DEVELOPMENT OF OOMYCETES Motile zoospores are an important means of initiating infection by oomycetes. Zoospores do not divide, however, but have high-affinity

1057

receptor-based recognition systems for locating hosts by chemotaxis [13,25,26]. The accumulated zoospores differentiate into adhesive cysts (a process called encystment), which in turn germinate to produce hyphae that are actually responsible for invading the host tissue [11]. Recent reports suggest that all these key pre-infection events of soil-borne zoosporic phytopathogens are triggered by chemical signals released from the host roots [24,26]. Zoospores Use Host^specific Chemical Signals to Target Roots Preferential chemotaxis toward hosts

Preferential chemotaxis followed by encystment of zoospores toward compatible hosts has been reported in many cases for plant pathogenic oomycetes. Encystment in vivo involves recognition of a host or substrate surface. Held (1973) was among the first who established this because zoospores of Rozella allomyces were attracted to both host (Allomyces) and nonhost (Blastocladiella) thalli; but encyst only on the host [38]. A scanning electron microscopic observation showed that significantly higher numbers of zoospores of Ph. cinnamomi encysted on the surface of a susceptible cultivar of avocado compared to that of the tolerant cultivar [39]. Fewer cysts were germinated on the roots of the tolerant cultivar than on the susceptible cultivar. Mitchell and Deacon (1986) observed that zoospores from Pythium graminicola and Py. arrhenomanes, which characteristically infect graminaceous hosts, preferentially accumulated behind root tips of grasses compared to dicots, whereas zoospores of the broad host range species Py. aphanidermatum and Py. ultimum did not show preference for grass roots [40]. Similarly, zoospores of a cotton pathogen, Py. dissotocum were attracted to cotton roots but zoospores of Py. catenulatum which is not compatible with cotton were not. The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola [41], and those of the nematode parasite Catenaria anguillulae accumulate at the mouth of the host [42]. All these interesting phenomena suggest that some host-specific chemical signals might play a vital role in locating host and differentiation of zoospores on host surface before penetration. On the other hand, saprophytic species selectively colonize of the host depending on their food sources [43,44,45].

1058

100 wm

Fig. (3). Response of Aphanomyces cochlioides zoospores toward spinach roots. A. Photomicrograph (dark field) of aggregated zoospores (dots close to root surface) just behind root cap of spinach root tip. B. A mass of cystospores (arrow) on spinach root [11].

Evidence of host-specific signals for zoospore chemotaxis

The question of specificity in chemotaxis is an important one as it relates to the contribution of chemotaxis, and subsequent steps of infection by zoospores, to host selection and host specificity [26]. Several host-specific attractant signals have already been identified for phytopathogenic oomycete zoospores. For example, indole-3-carbaldehyde (1) isolated from cabbage seedlings is a chemoattractant down to concentration of 1 nM for A. raphani zoospes [19]. Prunetin (2) isolated from the roots and root exudates of pea seedlings [20] is a potent attractant (down to 10 nM) for zoospores of the A. euteiches, while the zoospores of A. cochlioides are strongly attracted to host metabolites, cochliophilin A (3) and N-trans-femloyl-4-Omethyldopamine (6) at concentrations down to 0.1 nM and 10 nM, respectively [21,22]. Zoospores of Ph. sojae are attracted to daidzein (4) and genistein (5) from the roots and root exudates of its host, soybean, at a concentration down to 0.1 nM [23,24]. In contrast, some nonhost secondary metabolites including flavonoids displayed potent repellent activity toward pathogenic zoospores [13,36,46]. All these reports are interesting because they parallel the behavior of host-specific Rhizobium species, which also are attracted to the flavonoids of their hosts and can be repelled by nonhost flavonoids, the ability to recognize flavonoids released by their hosts appear to be also critical for first recognition step in the Rhizobium4eg\ime interaction [47]. So far, there are two best documented examples of specificity in chemotaxis and subsequent differentiation of oomycete zoospores by host plant signals. The first one is the attraction, encystment and germination of A. cochlioides zoospores to a rare flavone, cochliophilin A (3), and the other one is Ph. sojae zoospores to isoflavones daidzein (4) and genistein (5)

1059

[21,23,24,27]. In both cases, these compounds are present in the host seeds, roots and exuded by the roots. The details of each interaction between host signal and the respective pathogenic zoospores are described in the following section. Host-specific attractants for Aphanomyces spp. zoospores

The genus Aphanomyces consists of both saprophytic species as well as specialized parasites on plants and fishes [8,36]. In crops, they generally cause damping-off and root rot diseases, for example, A. euteiches on peas (Leguminosae), A. cochlioides on spinach, sugar beet and few other members of Chenopodiaceae and Amaranthaceae, and A. raphani on radishes (Cruciferae). Studies on the ecological chemistry of phytopathogenic Aphanomyces spp. revealed that the zoospores of those specialized pathogens locate their host plants guided by specific host signals released from the roots. The zoospores of phytopathogenic Aphanomyces attracted only to the potential infection sites of host roots but not to those of nonhost [36]. In a detailed study, Ui and Nakamura (1963) observed that the A. cochlioides was highly compatible with most of the members of Chenopodiaceae and a few members of Amaranthaceae (Table 1) [32]. Members of other plant families were less compatible or incompatible with A. cochlioides. When root extracts of 15 plant species belonging 10 families were subjected to A. cochlioides zoospore bioassay, interestingly, the crude extracts from the plants belonging to Chenopodiaceae and Amaranthaceae showed strong attractant activity indicating a direct correlation between host-pathogen compatibility and zoospore attractant activity toward plant root extracts (Table 1) [36]. At least three host-specific attractant signals (1-3) have been isolated for Aphanomyces zoospores by detailed bioassay-guided fractionation procedures [19-21]. Another potent attractant, JV-frans-feruloyM-0-mehyldopamine (6) for A. cochlioides zoospores was isolated from the roots of a host plant, Chenopodium album. These compounds show attractant activity only toward the respective pathogenic zoospores down to the nanomolar level. Other non-pathogenic oomycetes do not respond to these compounds. The third attractant for A. cochlioides zoospores was identified from the leaves of spinach as 5,4'-dihydoxy-6,7-methylenedioxyflavone (7) which is less active than the root attractant cochliophilin A (3). Although this compound has already been found as a glucuronide derivative in spinach, the ecological significance of 7 in host-pathogen relationship is yet to be clarified [49].

1060 Table 1: Responses of zoospores toward the extracts of plants belonging to different families and varying levels of compatibility with Aphanomyces cochlioides*

Plant

Degree of compatibility

Minimum active cone. (ppm)b

Chenopodiaceae

Spinacia oleracea L.*

attractant (30)

Chenopodium album L.*

attractant (30)

Beta vulgaris L. var. saccharifera*

attractant (100)

B. vulgaris L. var. cicla*

attractant (30)

Papaveraceae Papaver rheos L.

attractant (30)

Amaranthaceae Celosia cristata L.*

attractant (30)

Amaranthus retroflexus L.*

attractant and weak halting (100)

A. gangeticus L. Potulacaceae

attractant and halting (100)

Portulaca olercea L. Cruciferae

halting (100)

Raphanus sativa L. Graminae

repellent (500)

Zea mays L. Leguminosae

NA (1000)

Trifolium repens L. Solanaceae

NA(1000)

Lycopersicon esculentum L. Compositae

NA (1000)

Taraxacum officinalae L. Anacardiaceae

NA (1000)

Lannea coromandelica L. halting and lytic (200) a

Adapted from the reference of [8,32,36,37,48].

* Reported hosts.'+' and '-' signs indicate the degree of compatibility. and incompatibility of pathogen to the respective plant species, respectively. Acetone extractives.

b

1061 Host-specific plant signal, cochliophilin A

The specific signaling compound, cochliophilin A (3) for A. cochlioides zoospores has been found in the seeds, roots and root exudates of spinach [37]. Cochliophilin A (3) is also contained in the roots of two other hosts of A. cochlioides, C. album and sugar beet [50]. The detailed isolation and quantification procedures of compound 3 from the fresh spinach roots and root exudates were elaborately discussed in an earlier review [37]. It was estimated that fresh spinach roots contained approximately 1.9 x 10~5 mol/kg cochliophilin A (3) and exude sufficient amount (34 ng/plant/day) of this compound for attracting the zoospores of A. cochlioides [37]. So far, the distribution of 3 is completely restricted in the hosts (Chenopodiaceae) of A. cochlioides. Therefore, Tahara and Ingham (2000) suggested that the sensitive attraction of A. cochlioides zoospores to this rare flavone may account for a part of the mechanism that determines the host range of this phytopathogen [37]. This hypothesis is now strengthening when cochliophilin A (3) was found to trigger developmental transitions of zoospores at a physiologically relevant concentration [27]. Host-specific plant signals involved in host recognition as well as germination of pest propagules have been reported in many cases including soil-borne fungi [51], parasitic plants [52] and nematodes [53]. Structure-activity relationships

The specificity of A. cochlioides for a flavone, cochliophilin A (3) has been defined using a number of synthetic compounds with various levels of structural similarity to the flavone [37,54]. It revealed that A^ing oxygenation at C-5 and C-7 positions in the flavones with unsubstituted firing were the most important determinants of attractiveness. The 5-hydroxy group in flavones is known to form a strong hydrogen bond with certain metal ions but it is not known at present if this property is linked with the ability of 5 to attract zoospores. Although a limited number of compounds were investigated, the 6,7-methylenedioxy-substituted flavone, cochliophilin A (3) and its 6,7-dimethoxy derivative (8) showed almost equivalent stronger activity than those of other compounds tested. The presence of small alkoxy groups at C-6 and C-7 would seem to be effective in enhancing the zoospore attractant properties. Flavones with a 5-hydroxy7^nethoxylated A^ing, and an unsubstituted B^ing, exhibited stronger activity than chromone which lacked the B^ing, or apigenin-7,4'-di-Omethyl ether with a /?-methoxylated B^ing [37,54]. These results suggest that amongst flavones, strong activity may be related to the presence of an

1062

unsubstituted B^ing. In contrast, many other flavones and isoflavones showed little or no attraction [36,37]. For example, genistein (4) and prunetin (5) have respectively been reported as zoospore attractants of Ph. sojae and A. euteiches, respectively; these isoflavones were essentially inactive toward A. cochlioides at a concentration of 10"6 M [37]. An important finding of these studies was that A. cochlioides zoospores could respond to a wide range of phenolic compounds, albeit at significantly higher concentration than that for the flavones. Furthermore, in some cases the response observed was repulsion rather than attraction, for example, 8prenylated naringenin (9) and medicarpin (10) [36].

OCH, OCH, OH

OH

O

O

OH

OCH3

OH O 10

Recently, we tested some synthetic flavonoids those of which showed potent repellent activity toward Ph. sojae zoospores. Surprisingly, most of the compounds tested, showed strong attractant activity toward A. cochlioides zoospores, where some of them were more powerful attractants than the host-specific cochliophilin A (3) [36]. Although, the attractant property of those synthetic flavonoids were similar to cochliophilin A (3), however, very less number of attracted zoospores were encysted and germinated by those synthetic attractants than that of the natural plant signal (3). Structural requirements for attractant activity of synthetic flavonoids revealed that hydrophobic B-ring plus alkylated (methylated) A^ing in the flavonoid skeleton is responsible for strong activity. For example, 7hydroxy-5^nethylflavone (11), displayed 100 folds higher attractant activity than that of cochliophilin A (3). Similarly, a methoxylated B^ing at 4'position with unsubstituted A^ing (e.g. 4'-methoxyflavonol, 12, active at 1011 M) also showed potent attractant activity. Therefore, hydrophobicity in

1063

A^ing also increases attractant property. These observations raise the possibility that A. cochlioides zoospores can integrate a large amount of information about their chemical environment, over and above their attraction to host flavone (3). Although the detailed structure-activity studies of compounds related to the ferulamide attractant (6) have not been carried out. However, among the four regio-isomers tested, the natural ferulamide (6) displayed the strongest attractant activity. The substitution patterns of 4-OH or 3-OCH3 in amides are popular in nature, but they are less active than 4-O-methyldopamine. Compound 6 is the first naturally occurring amide possessing a rare substitution pattern (4-O-methyldopamine moiety) in the dopamine part may be indicative of the presence of a specific relationship between the host material and the mobile pathogen [54]. However, this compound (6) seemed to operate through a different receptor from cochliophilin A (3) because a background of one of them did not affect the response to the other. Similarly, Sekizaki et al. (1993) studied the specificity of A. euteiches zoospores for prunetin (2) using wide variety of isoflavones (non-, mono-, di-, and tri-substituted) [55]. They observed that a hydroxy group (but not a methoxy) at the C-5 position of an isoflavone is necessary to strongly attract A. euteiches zoospores. In addition, the presence of an additional hydroxy group at C-7 or C-4' enhanced the attractant activity, which was further increased by 7-O^nethylation, but slightly decreased by 4'-O^ethylation. The strongest activity amongst 26 isoflavones was associated with the natural attractant prunetin (2). The structural requirement for another host-specific attractant, indole-3 carbaldehyde (1) has been studied by using 16 compounds (namely, indole, indole-3-ethanol, indole-3-propionic acid, indole-3-butyric acid, indole-3acrylic acid, indole-3-acetic acid, indole-2-carboxylic acid, indole-3carboxylic acid, indole-5-carboxylic acid, acetoaldehyde, vanilline, nbutyraldehyde, cinnamaldehyde, citrol, citronellal and tryptamine hydrochloride) structurally related to compound 1 [19]. Only few compounds, namely, indole-3-carboxylic acid, indole-3-acrylic acid, indole3-acetic acid, and indole-2-carboxylic acid showed weak attractant activity toward A. raphani zoospores only at very high concentrations (>10"6 M). Tryptamine hydrochloride exhibited attractant activity at 10 |ag/ml however; at 100 |ug/ml it immobilized zoospores. Obviously, none of the compounds displayed any attracting activity at concentrations lower than 10"6 M suggesting that compound 1 is a host-specific plant signal which is an attractant at 10"9 M for A raphani zoospores. Indole-3-carbaldehyde (1) did not attract zoospores of A. cochlioides and A. euteiches which further

1064 strengthen the assumption that compound 1 is a host-specific one for A. raphani. Attractant for Phytophthora and other oomycete zoospores

Among the oomycete genera, Phytophthora and Pythium are known as the most devastating pathogen of dicot plants. Most of them have wide host range. Zoospores of those Phytophthora and Pythium species show a relatively nonspecific attraction to amino acids (particularly aspartate, glutamate, asparagine, glutamine, arginine and methionine), sugars (e.g. glucose) or volatile compounds (e.g. alcohols, isovaleraldehyde, valeraldehyde), all of which are common components of root exudates [17,26]. Attraction to these compounds may account for the nonspecific attraction of many Phytophthora and Pythium zoospores because, the threshold level for chemotaxis is usually higher than the micromolar order which may not practically exist in the rhizosphere. Many researchers have been studied on the preferential chemotaxis of Phytophthora and Pythium zoospores; however, convincing information available on the molecular mechanism of host recognition in those phytopathogens is limited. Species of Phytophthora and Pythium, especially those with restricted host ranges, appear to exhibit more specificity in their attraction toward root exudates. The most characterized example of specificity is the attraction of Ph. sojae zoospores to the isoflavones, daidzein and genistein [23]. The zoospores of Ph. sojae, which are chemotactically attracted to the isoflavones, daidzein (4) and genistein (4), released from the soybean roots [23,46] were used as the test organisms to check the response to a wide range of compounds possessing some structural similarity to genistein, including isoflavones, flavones, chalcones, stilbenes, benzoins, benzoates, benzophenones, acetophenones, and coumarins [46]. Of 59 compounds examined, 43 elicited some responses. A comparison of the chemotactic responses elicited by the various compounds revealed a primary role for the phenolic 4'- and 7-hydroxy groups on the isoflavone structure. An important finding of this study was that Ph. sojae zoospores could respond to a very wide variety of phenolic compounds, albeit at significantly higher concentration (1 uM) than for the isoflavones [26,46]. Tyler and coworkers (1996) observed substantial levels of genetic variation in the attraction of zoospores of different Ph. sojae genotypes to isoflavones [46]. Genetic crosses between the isolates showed that a single gene was responsible for the difference in attraction to genistein and other isoflavones [26]. The genetic differences of zoospores in response to nonisoflavone phenolics were determined by at least six additional

1065

independently segregating genes, supporting the notion that Ph. sojae has an extensive array of receptors capable of sensing the phenolic environment of the zoospores. Based on these findings, Tyler (2002) hoped that detailed mapping of these genes may provide a route to cloning the Ph. sojae receptors responsible for detecting isoflavones and other phenolic compounds [26]. Similar extensive array of receptors may exist in other oomycetes for sensing chemical environment in the rhizosphere. During the study on structure-activity relationships, Tyler et al. (1996) also surprisingly observed that a few flavonoidal compounds were acted as good repellents, notably methylated flavones (7-hydroxy-5-methylflavone 11, 7-hydroxy-3-methylflavone 13, 5,7-dimethoxyflavone 14, and 4',5,7trimethoxyflavone, 15) with mainly a hydrophobic B^ing [46]. Interestingly, one of the most potent repellents for Ph. sojae zoospores, 7hydroxy-5-methylflavone (15), was also reported to be the most potent inhibitor of the nodulation response of several genotypes of Bradyrhizobium japonicum [56]. However, all those alkylated synthetic flavonoids showed potent attractant activity toward A. cochlioides zoospores (particle method). In organisms that use klinokinesis as a chemotaxis strategy, such as Phytophthora species and many bacteria, attraction and repulsion are parts of a continuum of responses: attraction results from a lower-than-average frequency of turning, while repulsion results from a higher-than-average frequency of turning [16,46,57]. In Escherichia coli, the same receptors and the same signal transduction pathways mediate both attraction and repulsion [57]. OCH 3

OH

12

OCH3 H,CO H3CO H,CO H,CO

O

O 14

15

1066

Specific sensitivity of zoospores to the host-derived compounds has also been found in other pathogenic oomycetes. Kerwin et al. (1992) observed that Py. marinum exhibited encystment on the surfaces of red algae (its hosts) but not on green or brown algae (non-host) [58]. Galactose or anhydrogalactose contents in the surface of red algae were found to be responsible for such a specific response. Similarly, fish and mosquito larval parasites showed positive chemotaxis toward their host surface chemical constituents [59-62]. Developmental Transitions of Zoospores Triggered by Host-Specific Plant Signals A common characteristic among most of the oomycete and fungal plant pathogens is that each specialized on a narrow range of specific plants as hosts [51]. One adaptation to a specific host plant is the recognition of the host's chemicals which can be used to trigger genes or developmental pathways needed for pathogenesis. The production of characteristic secondary metabolites (e.g. flavonoids) by plants, particularly those exuded from roots (e.g. legumes), appears to be used as signals for various microbes, including symbionts as well as pathogens. Although zoospores of oomycetes rapidly differentiate after docking on the potential infection sites of host, but it has long been unknown whether some host-specific plant signal are involved in this developmental pathway. Differentiation of zoospore involves encystment of a zoospore and germination of a cystospore to a hypha. Recently, a few host-specific attractants were found to trigger differentiation of zoospores, essential for penetration into host roots (Table 2). Developmental transitions by cochliophilin A

Despite of the discovery of some host-specific chemoattractants of zoospores, it was a big question whether the same signaling molecule induces subsequent encystment and germination on host surface or these following events are regulated by different host signals. Moreover, it was unknown whether the stages of pre-infection are necessarily under separate control or a part of the signaling cascade. This is important because a success of infection depends on the completion of sequential events. Deacon (1996) suggested that zoospores might be induced to encyst by the effect of specific root surface components [25]. Evidence supporting the involvement of any host-specific plant signal in differentiation of

1067

pathogenic zoospores has been lacking for long. However, in vitro studies revealed that zoospores are encysted by root surface mucilage, fucosyl residues, pectin, alginate or specific polysaccharides, lectin or monoclonal antibodies specific for flagella [17]. However, in all those cases the threshold concentrations were high. Recently, investigation of the host factors triggering encystment and germination of A. cochlioides zoospores on spinach roots revealed that a gradient of the host-specific attractant, cochliophilin A (3) triggers encystment and germination of zoospores at a concentration approximately ten times higher than that observed to elicit chemotaxis (Fig. 4) [27]. The effects of three host-derived attractants identified for A. cochlioides zoospores, cochliophilin A (3), N-trans-feruloyM-O- methyldopamine (6) and 5,4'-dihydroxy-3,3'-dimethoxy-6,7-methylenedioxy-flavone (7) have been assayed by particle bioassay method. Only, the cochliophilin A (3) induced encystment of the attracted zoospores at a range of 10"8 to 10"6 M concentration in a dose dependent manner and formed a mass of cystospores on and around the Chromosorb W AW particles treated with 3. Initially the attracted zoospores became sluggish, moved in a circular fashion, halted and rapidly changed into round-shaped cystospores. Interestingly, the attracted zoospores landed and encysted on the surface of Chromosorb particle coated with 10~7 to 10"6 M solution of cochliophilin A (3). All encysted zoospores germinated (100%) on and around the particles within 30^4-0 min. The cystospores germinated adjacent to the particles coated with host-specific attractants showed germ-4ubes tropism toward the particles. The particles coated with lower than 10"8 M concentration of cochliophilin A exhibited only attractant activity but not induced encystment of zoospores. On the other hand, the control particles treated with solvent alone neither affected the normal motility of zoospores in the aquatic medium nor resulted in encystment of any zoospore. The other two host-specific attractants (6 and 7) did not induce encystment and germination up to 10"6 M concentrations.

Zoospore

Immature cystospore

Mature cystospore

Germinating cystospore

Fig. (4). Morphological changes (developmental transitions) of Aphanomyces cochlioides zoospores triggered by a host-specific plant signal, cochliophilin A (3). White bars = 1 |JM.

1068

The effect of cochliophilin A (3) on the encystment and germination of zoospore was evaluated by the direct application of 3 suspended in water at a range of 10"12 to 1CT6 M concentration [27]. The direct application of 3 into the zoospore suspension as a homogeneous solution at a range of 1CT12 to 10"8 M just stimulated the motility of zoospores for 10-15 minutes without resulting any encystment and germination. However, at higher concentrations (10~7 to 10"6 M) of cochliophilin A (3) in the above conditions, it showed no effect on the motility of zoospores. Interestingly, very slow release of 5xl0~12 to 5xl0~10 M cochliophilin A (3) solution to the zoospore suspension by a microsyringe showed strong stimulation of the motility of zoospores followed by encystment and germination. In most cases, the stimulated zoospores formed the clumps of aggregated cells scattered at the bottom of glass petri dish, and then encysted and germinated. It clearly indicates that a gradient of host signal is necessary for taxis and differentiation of zoospores which seems to reflect exactly the natural event at rhizosphere. Thus, our particle bioassay appeared to be a suitable method for studying chemotaxis and subsequent differentiation of zoospores where a gradient of chemical is essential for the response of cells [63]. The germ tubes of the cystospores germinated adjacent to the aggregate exhibited tropism toward the aggregate center. Tropic responses of hyphal germlings to host-specific signals have also been observed in Ph. sojae [24], and autoaggregation of zoospores in the absence of a host seems to be characteristic of many other oomycetes [64]. It is feasible that the hostspecific compounds might also induce the similar aggregation phenomenon. Aggregation of inoculated zoospores on a certain point of the host root might increase the vigor of the inoculums for successful infection. To understand the mechanism of halting response of the zoospores to cochliophilin A (3) followed by encystment and germination, we undertook a time-course observation of changes by scanning electron microscopy (SEM). SEM observation revealed that zoospores stimulated by 3 or spinach root tip underwent a similar sequence of morphological changes up to germination of cystospores. In both cases, the stimulated zoospores became almost round shape by shedding their flagella within 20 min of stimulation and soon became the enlarged cystospores (8.5-10.5 jam i.d.) bounded by a smooth cyst-coat [11]. The flagella of the zoospore were found to lose their fine structures immediately after detachment. The initial cystospore coated with a smooth cyst-coat rapidly changed into a mature cystospore (5.7-7.1 |im i.d.) coated with rough cell wall within 20-30 min and finally germinated within 40-60 min. Interestingly, the sequence of morphological changes of zoospores by cochliophilin A (3) was identical to those occurred during interaction with spinach roots.

1069

Therefore, the behavior of zoospores on and around Chromosorb particles coated with cochliophilin A (3) was identical to that of zoospores toward spinach roots. The amount of 3 in the spinach root (ca, 1.9 x 10"5 mol/kg fresh root) seems to be enough to initiate encystment of zoospores followed by germination which are regenerated by Chromosorb particles coated with a 10"8 M solution of 3 as a dummy of the spinach root [27]. These observations suggest that cochliophilin A (3) is indeed a host-specific plant signal which may play essential roles in both locating host roots and initiating encystment and germination. Interestingly, an almost similar phenomenon was observed in bacteria. As a signal for chemotaxis of rhizobia a concentration as low as 10"9 M luteolin is sufficient, and at 10"6 M concentration luteolin stimulates nod gene expression [65,66]. Furthermore, the growth and sporulation of A. cochlioides on a corn meal agar medium were unaffected up to 10 M concentration of cochliophilin A (3). This information supports that this oomycete can grow well and produces zoospores for further dissemination of pathogens to spread the disease through surrounding healthy plants. All these interesting features of host-pathogen interactions might have ecological significance, and may find useful application in the investigation of biochemical and molecular mechanism in pathogenicity where it is definitely desired to synchronize the development of pathogen with that of the host. Differentiation of zoospores by soybean isoflavonoids

The specific attractants of soybean pathogen, Ph. sojae also trigger encystment followed by germination of zoospores at the higher concentration than that needed for attracting activity. This phenomenon was confirmed by capillary bioassay methods. When a capillary tube containing 20 |jM of daidzein (4) or genistein (5) was introduced to the chemotaxis chamber and left undisturbed, zoospores rapidly plugged the capillary tube and other encysted around the mouth of the tube and germinated [23,24]. This phenomenon was identical to the responses of zoospores toward a soybean root tip. Addition of the solution of soybean isoflavones at a low concentration directly to the zoospore suspension before vortexing also significantly increased the germination ratio of cystospores than that of control indicating that host signals are effective in germination of pathogenic spores [67]. Flavonoids have also been reported to germinate many pest propagules of soil-borne fungi, parasitic plants and mycorrhizal spores [51].

1070 Table (2). Triggering Che mot axis and differentiation (encystment and germination) of Some Oomycete Zoospore by Host-specific Plant Signals Host-specific

Host plant

plant signal

Threshold concentration (n M) Oomycete

Taxis

Encystment

Germination

3-indolecarbaldehyde (1)' Cabbage

A. raphani

1

nt

nt

Prunetin (2)a

Pea

A. euteiched

10

nt*

nt

[20]

Cochliophilin A (3)b

Spinach

A. cochlioides

0.1

10

10

[21,27]

Daidzein (4)*

Soybean

Ph. sojae

1

100

100

[23, 24]

Genistein (5)a

Soybean

Ph. sojae

1

100

100

[23, 24]

Feruloyldopamine (6)b

C. albumm

A. cochlioides

10

na

na

[22, 27]

[19]

* nt = not tested; na = non-active; Jcapillary method; particle method.

4: R = H : daidzein 5: R = OH : genistein

Chemotropism of hyphal germlings

Factors that influence the direction of hyphal growth after germination of a cystospore are less explored than chemotaxis. Autoaggregation of zoospores in the absence of an available host is characteristic of some if not all oomycetes [64]. Zentmyer (1961) demonstrated that Ph. cinnamomi cysts that were adjacent to the root of their host germinated rapidly and grew in the direction of the root, but host-specific chemical signals regulating this behavior has been discovered very recently [24,27]. However, tropic responses of growing hyphae to nutrient sources have also been demonstrated in several saprophytic and parasitic oomyetes [68,69]. Recently, Morris et al. have investigated the role of the host-specific isoflavones, daidzein (4) and genistein (5) on chemotropic behavior of germinating cysts of Ph. sojae [24]. They demonstrated that hyphal tips respond chemotropically to 4 and 5, suggesting that hyphal tips from the zoospore cysts that have encysted adjacent to the root may use specific host isoflavones to locate their host. Thus chemotropic responses of oomycetes hyphae might also contribute to their effectiveness as plant pathogen.

1071 Receptors in zoospores

It is assumed that the responses of zoospores to different host-specific plant signals or environmental chemical stimuli are mediated by chemoreceptors in the zoospore of flagellar membrane. But no receptor has yet been purified and characterized for a zoosporogenic oomycetes. In a biochemical study, Sakasai analyzed a putative cochliophilin A (3) receptor protein in the membrane of A. cochlioides zoospores [70]. He designed a cochliophilin A analog, AF-bio (16) according to the results of structure-activity relationships analyzed by Kikuchi et al. [54] and Takayama [71]. The analog (16) consists of the required part structures as an attractant, a biotin part to be trapped by a horseradish peroxidase-avidin conjugate, and an azido group, which is for photoaffinity labelling of the zoospore protein(s). AF-bio (16) showed attractant activity toward A. cochlioides zoospores and competition against cochliophilin A (3) itself in the zoospore chemotaxis.

H

OH O OH O biotin part

attractant part 16

photoaffinity ligand

17

According to his preliminary experiments, a fresh zoospore suspension containing 16 was treated by UV-light, and then the membrane proteins were fractionated and subjected to SDS PAGE. The proteins in the gel were transferred to a polyvinylidine difluoride (PVDF) membrane and treated with a horseradish peroxidase-avidin conjugate. Peroxidase active region on the PVDF membrane was detected by ECL™ (enhanced chemi luminescence) method. Finally, he found AF-bio (16) binding protein at ca 70 kDa, presumably a reputed receptor protein for cochliophilin A (3), because the band disappeared completely when the zoospores treated with 3 for photoaffinity labelling in the presence of excess amounts of AF (17) lacking a biotin part structure. Further progress of characterization of this AF-bio binding protein is eagerly waited.

1072 Signal transduction pathways in zoospores

G-proteins are believed to be key components of signal transduction pathways in chemotaxis of many other motile cells [72]. Mastoparan is commonly used as a diagnostic agent for the participation of G-proteins in both animal and plant signal transduction pathways [73,74]. Interestingly, the heterotrimeric G-protein activator, mastoparan showed encystment of both A. cochlioides and Ph. infestans zoospores at a micromolar concentration [13,27,28]. The synthetic peptide analog Mas 17, predicted not to form an amphipathic helix at lipid interface because of the replacement of Leu-6 by Lys, is totally devoid of agonist activity. The concomitant application of mastoparan and the host-specific attractant cochliophilin A (3) appeared to further enhance encystment of zoospores and rapid germination of A. cochlioides cystospores. In addition, chemicals interfering with phospholipase C activity (neomycin) and Ca2+ influx/release (EGTA and loperamide) suppressed cochliophilin A and mastoparan induced encystment and germination. Changes of Ca2+ fluxes during differentiation of zoospores have been observed by early investigators [67,75]. By an X^ay microanalysis of individual encysted zoospores, Connolly and co-worker (1999) also demonstrated that the hostspecific plant signals, daidzein (4) and genistein (5) trigger a calcium influx in Ph. sojae [67]. These results suggest that the zoospore differentiation by host-specific cochliophilin A (3) might be mediated by G^irotein-coupled receptors to activate both phosphoinositide and Ca2+ second messengers pathways. Genes encoding a and |3 subunits of heterotrimeric G-proteins have already been characterized in Ph. infestans [76]. However, in a recent study, Latijnhouwers et al. demonstrated that differentiation of zoospores of late blight pathogen Ph. infestans is triggered not only by mastoparan, but also by di-octanoyl phosphatidic acid (DOPA), n-and sec-butanol but not terr-butanol [28]. Likewise, mechanical agitation of zoospores, which also induced encystment of zoospores, resulted in increased levels of phosphatidic acid (PA) as well as its phosphorylation product diacylglycerolphosphate (DGPP). They also found that the accumulation of PA during encystment by mastoparan and rc-butanol is caused by the stimulation of PLD but not PLC activity. They concluded that PLD is involved in zoospore encystment by mastoparan. It is known that encystment of phytopathogenic oomycete zoospores by mechanical agitation regenerate into next generation of zoospores instead of germination. We also observed that both n- and 5ec-butanol also induced encystment of A. cochlioides zoospores where the cystospores did not advanced to germination rather regenerated into zoospores. Whereas,

1073

induction of encystment by a host-specific plant signal synchronously germinate into hyphae within a few minutes. These raises a question whether encystment of zoospores by mechanical agitation or PA or nbutanol follow a different signal transduction pathway than encystment induced by a host-specific plant signal. Additionally, PA has been linked to a variety of plant treatments and responses, most of these involve biotic or abiotic stresses, suggesting a role for PA as a general stress-signalling molecule [77]. A further comparative study on lipid metabolism in zoospore after induction of encystment by host-specific plant signals and other chemicals (e.g., n-butanol) would give suggestive information of the signal transduction pathways in zoospores. Since the components of the pathway represent attractive targets for developing alternative disease control methods, agricultural practice may benefit from such kind of research results in the long term. Concluding Notes Much progress has been made in the past decade in understanding the signaling and interactions between root-infecting oomycetes and their hosts, and the findings indicate several points of potential general significance. A common characteristic among oomycetes and fungal pathogens of plants is that each specializes on a narrow range of specific plants as hosts. One adaptation to a specific host plant is the recognition of the specific host's chemicals which can be triggers for specific gene expression or developmental pathways needed for pathogenesis [51]. The production of characteristic plant secondary metabolites by plants, for example, flavonoids particularly those exuded from the roots, appear to be used as signals for various microbes, including symbionts as well as pathogens. The phenomenon of host recognition through host-specific plant secondary metabolites which function as chemical signals directing several key steps in the early stages of the infection response of oomycete phytopathogens is summarized as follows: a)

They mediate chemotaxis of swimming zoospores toward the root tips [11,23], where most of the signaling compounds are exuded by the root [37,78].

b)

Exposure of zoospores to elevated levels of host-specific attractant signals cause encystment followed by 100% germination of cystospores on host roots as well as on

1074

artificial surfaces such as Chromosorb particle, capillary tube or plastic membrane [24,27]. c)

Host-specific attractants also induce chemotropic growth of germlings toward the roots [11,24,27].

d)

Zoospore perceives host-signal by a G-protein-coupled receptor and then translate into responses (chemotaxis and differentiation) via phosphoinositide/ Ca2+ or phosphatidic acid second messengers pathways [27,28].

^ Fig. (5). Chemotaxis and subsequent differentiation of Aphanomyces cochlioides zoospores by host-specific plant signal cochliophilin A and host (spinach) roots, a. Zoospores aggregated, encysted, and germinated on and around a Chromosorb W AW particle coated with lxlO"6 M cochliophilin A (3). Cystospores germinated adjacent to the particle showed germ tube tropism toward the cochliophilin source; b. A SEM micrograph showing cystospored adhered and germinated on the surface of a cochliophilin A coated particle; c. Cystospores germinated on a host (spinach) root. White bars = 100 |im.

DIVERSE NONHOST SECONDARY METABOLITES AFFECTING MOTILITY AND VIABILITY OF ZOOSPORES In contrast to susceptible plants, non-susceptible plants may possess some chemical weapons to defend themselves from the attack of zoosporogenic oomycetes [79]. This hypothesis has been tested by surveying physiologically active secondary metabolites in more than 200 nonhost plant extracts using A. cochlioides zoospores by particle bioassay method [80]. The crude extracts showed a wide range of biological activities toward zoospores, and the active principles of several plant extracts have been identified by bioassay-guided chromatographic techniques. This section summarizes our research findings along with current knowledge on nonhost plant secondary metabolites in relation to their resistance against oomycete phytopathogens.

1075

Survey of Physiologically Active Secondary Metabolites in Nonhost Plant Extracts In recent years, there has been renewed interest in examining interactions between nonhost plants and oomycetes [3]. The molecular basis of nonhost resistance remains one of the major unknowns in the study of plant-microbe interactions. Plant disease resistance can be conferred by constitutive features such as structural barriers or performed antimicrobial secondary metabolites. Performed barriers and compounds such as saponins are ubiquitous in plants and play important roles in nonhost resistance against filamentous fungi [35,81]. Studies concerning nonhost resistance against oomycetes by plant secondary metabolites are very few. Screening extracts of nonhost plants revealed that nearly half of the extracts had direct effects on motility and viability of A. cochlioides zoospores (Table 3). Although, some of the nonhost plant extracts exhibited attractant activity, however, none of them showed attractant and subsequent differentiation of zoospores as shown earlier by cochliophilin A (5) [27,36]. In addition nonhost extracts exhibited some deleterious activities for example, repellent, stimulant, halting, lysis etc. against the zoospores. Some unusual activities like sudden inhibition of motility by Portulaca oleracea (Portulacaceae), attraction and halting by Amaranthus gangeticus (Amaranthaceae), motility inhibition and subsequent lysis of zoospores by Lannea coromandelica (Anacardiaceae) and Ginkgo biloba (Ginkgoaceae), and negative chemotaxis by Dalbergia odorifera (Leguminosae) and Magnolia kobus (Magnoliaceae) extracts were noticeable (Table 3) [36,82]. These interesting effects of nonhost plant extracts toward phytopathogenic oomycete zoospores prompted us to investigate the active principles by detailed chemical studies. Screening results thus indicated that many nonhost plants might use secondary metabolites to directly defend themselves from the attack of oomycete phytopathogens. Isolation of various nonhost defense factors (chemical weapons) against oomycetes may give some new interesting targets for controlling oomycete phytopathogens. Based on the screening results, we identified the active principles in nonhost plant extracts by detail bioassay-guided chemical fractionations, and the results are reviewed in the following sections. The modes of actions of the isolated compounds on zoospores are also illustrated.

1076 Table 3. Activities of Some Nonhost Plant Extracts3 toward Aphanomyces cochlioides Zoospores Plant name

Family

Achyranthes sp. Amaranthus gangeticus A. caudatus A. tricolor Lannea coromandelica Mangifera indica Catharanthus roseus Basella alba Terminalia arjuna T. chebula Attractylodes lancea Aucklandia lappa Cyperus rotundus Phyllanthus emblica Ricinus communis Ginkgo biloba Leucas zeylanica Leonurus heterophyllus Akebia quinata Pueraria lobata var. chinensis Dalbergia odorifera Allium chinensis A. cepa A. sativum Magnolia kobus Hibiscus rosa sinensis Azadirachta indica Sinomenium acutum Papaver somniferum Ampelygonum chinense Portulaca oleracea Nigella sativa Paeonia suffruticosa Aegle marmelos Capsicum annuum Abroma augusta Cuminum cyminum Foeniculum vulgare Vitex negundo Curcuma longa Zingiber officinale Elettaria cardamomum

Amaranthaceae

Anacardi aceae Apocynaceae Basell aceae Combretaceae Compositae Cyperaceae Euphorbiaceae Ginkgoaceae Labiatae Lardi zabal aceae Leguminosae Li li aceae

Magnoliaceae Malvaceae Meliaceae Menispermaceae Papaveraceae Polygonaceae Portulacaceae Ranunculaceae Rutaceae Solanaceae Sterculiaceae Umbelliferae Verbenaceaee Zingiberaceae

Plant organ

Types of activity* (MAC Mg/ml)

stem bark leaves whole plant aerial part rhizome root rhizome aerial part unripe fruit whole plant aerial part root heartwood bulb

fruits aerial part stem whole plant root seed root bark aerial part whole plant aerial part seed ripe fruit aerial part rhizome

+ = attractant; -= repellent; s = stimulant; h = halting motiliity; b = bursting zoospores (cell lysis). MAC - minimum active concentration. *Particle bioassay method was used to test the activity of extracts. a Plant materials were ground or cut into small pieces and extracted with 70% acetone.

+ & h (200) + & h (30) + & h (50) + & h (30) h & b (200) -(200) s(100) + (30) -(500) -(500) -(1000) (1000) -(1000) -(200) - & h (200) h & b (200) + & s (500) + (500) + (200) + (500) -(200) + (500) (1000) + (1000) -(200) + (500) -(100) + (200) + (200) -(200) + & h (200) s (500) -(200) + (200) + (30) s (500) + (500) -(500) s (500) -(100) s (500) -(500)

1077

Zoospore Motility Inhibitors in Portulaca oleracea Isolation of active principles

To identify the zoospores motility inhibitory principles from the common purslane, Portulaca oleracea, 1.15 kg of fresh roots were first extracted with MeOH and then subjected to chemical fractionation using n-hexane, diethyl ether and EtOAc (Scheme 1) [83]. The diethyl ether extract (1.72 g) Fresh roots of Portulaca oleracea (1.15 kg) extraction with MeOH Coned. MeOH extracts (50% MeOH)

Hexane layer

MeOH-H2O layer MeOH evaporation Ether extraction

Aqueous layer (780 mg)

Ether extract (1.72 g)

EtOAc extraction

SiO2 C.C. in EtOAc^vleOH-H 2 Oconc.NH4OH = 60:15:5:1

Aqueous layer Repellent fraction (small amount)

EtOAc extract SiO 2 C.C. (CHCl3-MeOH = 2:l)

Stimulant fraction (321 mg)

Repellent fraction (42.2 mg)

SiO2 C.C. (CHCl 3 -MeOH=20:l) Stimulating fraction (30.2 mg)

CHC13 layer

HPLC (CHCl 3 -MeOH=10:l) Stimulant (19, 12.5 mg)

Partition between CHC13 and aq. 2N HC1 Aqueous layer

SiO 2 C.C. (CHCl3-MeOH-H2O = 65:25:4)

Repellent (20, 8.5 mg)

Scheme 1. Isolation procedure for compounds in Portulaca oleracea roots exhibiting stimulant and repellent activity on zoospore of A. cochlioides. SiO2 C.C. : silica gel column chromatography.

1078

was applied to silica gel (200 g) column chromatography using EtOAcMeOH-H 2 O-conc. NH4OH 60:15:5:1 to give 12 fractions (100 ml each) in which two active components, a stimulant (fractions 2-4) and a small amount of repellent (fraction 6) were detected. Fractions 2-4 were rechromatographed and the stimulant was finally purified by HPLC using an Inertsil column (6.0x250 mm) in CHCl3-MeOH 20:1, flow rate 1 ml/min, to yield 12.5 mg of the stimulant (?R ca 16.3 min). As the amount of repellent in the ether extract was insufficient for further purification, the EtOAc extract (Scheme 1) was used as an alternative source. This extract was initially applied to a silica gel (60 g) column and the repellent was eluted with CHCl3-MeOH 2:1. Three repellent fractions (6-8, 40 ml each) were combined, the volume reduced to near dryness in vacuo, and the residue redissolved in EtOAc and washed with 2N HC1. The EtOAc-soluble constituents were further purified by passing through a silica gel Sep-Pak column (3 cc) with CHCl3-MeOH-H2O 65:25:4 as eluting solvent to give the repellent (8.5 mg). Structure elucidation ofmotility inhibitors

HR-EI-MS indicated the empirical formula C18H19O4N for the stimulant, whilst the 'H-NMR spectrum revealed two hydroxy groups [5 7.88 (1H, s) and 5 8.09 (1H, s)], seven aromatic protons [5 6.7-7.2 (7H)], one methoxy group [5 3.88 (3H, s)], two methylene groups [5 2.74 (2H, t, 7=7.3 Hz) and 5 3.48 (2H, q, .7=7.3 Hz)], and two olefmic protons [5 6.47 (1H, d, 7=15.5 Hz) and 5 7.42 (1H, d, 7=15.5 Hz)]. The coupling constant of 15.5 Hz indicated the presence of a frans-distributed olefinic bond. The detection of protons assignable to a methoxy, and a hydroxy group, an olefinic group and a l,2,4^risubstituted benzene, as well as an EI-MS fragment at m/z 177, strongly indicated the presence of a feruloyl part structure. The remaining four aromatic protons [5 6.75 and 7.06 (both 2H, d, J=8.4 Hz)] and four aliphatic protons attributable to two methylene groups were assigned to those of tyramine. Most of these signals were very similar to those of Nfran.s-feruloyl-3-O-methyldopamine (18) and iV-frans-feruloyM-O-methyldopamine (6) previously isolated from Spinacia oleracea [84] and Chenopodium album [22]. The stimulant was thus considered to be N-transferuloyltyramine (19). The identification was confirmed by acylation of commercially available tyramine with ferulic acid in the presence of N,N'dicyclohexylcarbodimide to yield Af-frans-feruloyltyramine (19) which possessed physicochemical properties indistinguishable from those of the natural stimulant. The stimulant activity of natural iV-?rans-feruloyltyramine

1079

(19) towards the zoospores of A. cochlioides was also identical to that observed for the synthetic compound (19). The purified repellent, containing minute amounts of a homologue (or homologues), gave a positive response to the Dittmer test which indicated the presence of a phosphate group in the molecule [85]. In the 31P-NMR spectrum, a phosphorus atom resonated at 4.54 ppm (internal standard: triphenylphosphine). Alkaline methanolysis of the repellent was yielded methyl linoleate indistinguishable from an authentic sample. In the ! HNMR spectrum, signals assignable to two double bonds via one methylene (-CH=CH-CH2-CH=CH-), and a methyl doublet coupled with 31P via an oxygen atom [5 3.66 (3H, d, 7=10.9 Hz)] were detected. The molecular formula of the repellent was found to be C22H41O7P by HR-FAB-MS (negative ion mode: [M-H+]~, m/z 447). From an analysis of all the available physicochemical data, the repellent was considered to have structure 20 [83]. This structure was confirmed as follows. Commercially available 1oleoyl-24ysophosphatidic acid (21) was methylated with diazomethane in ether to yield the monomethyl and dimethyl esters, which were respectively positive and negative in the Dittmer test. Chromatographic and spectroscopic properties of synthesized monomethyl ester (22) were in good agreement with those of the natural repellent except for minor features reflecting differences in the fatty acid part of each molecule. Biological activity and the possible function of the motility inhibitors

Compounds 19 and 20 along with some acylated phosphatidic derivatives in addition to 1,2-dioleoylphosphatidic acid, were tested using the particle bioassay [83]. Commercially available l-oleoyl-2lysophosphatidic acid (21) possessed repellent activity which was enhanced by monomethylation. When A. cochlioides zoospores were pre-treated with an excess of the natural stimulant N-trans-feruloyltyramine (19), and then exposed to Chromosorb W AW particles coated with various test compounds, it was found that l-oleoyl-24ysophosphatidic (21, 100 ppm) and its monomethyl ester (22) (10 ppm), as well as the natural repellent 1linoleoyl-2-lysophosphatidic acid monomethyl ester (20, 30 ppm), effectively inhibited zoospore motility [83]. However, l-oleoyl-2lysophosphatidic acid dimethyl ester (23) and 1,2-dioleoylphosphatidic acid tested with and without the stimulant (19) showed neither repellent nor motility inhibitory activity. The bioassay revealed that compounds possessing repellent activity are monoacylated phosphatidic acid derivatives containing at least one hydroxy group on the phosphoryl unit [83].

1080

HO OCHi 19 (natural stimulant)

H

°

H-C-O' H-C-OH o H-C-O-P-OH H 20 (natural repellent) O H H-C-O' H-C-OH H-C-O-R H O 21: R=—P-OH OH

O 22: R = —P-OH OCH3

23: R

O P-OCH3 OCH3

The characteristic behavior of Aphanomyces zoospore movement is shown in Fig. 6, where zoospore movement is inhibited in the area close to a Chromosorb W AW particle treated with a mixture of the stimulant and repellent factors from roots of P. oleracea, whilst in area remote from the

1081

particle, they are still swimming quite actively. Based on the bioassay results, we conclude that a mixture of the stimulant, N-transferuloyltyramine (19), and the repellent, l-linoleoyl-24ysophosphatidic acid monomethyl ester (20), in Portulaca root is responsible for inhibiting the motility of zoospores of A cochlioides.

Fig. (6). Photomicrograph of zoospores of A. cochlioides after exposure to a mixture of stimulant, N-transferuloyltyramine (19) and the repellent, Hinoloyel-24ysophosphatidic acid monommethhyl ester (20) released from Chromosorb particle treated with a 1000 ppm and a 100 ppm solution of 19 and 20. Photograph was taken through a microscope with an exposure time of 0.5 s. Dots close to the particle: inhibited zoospores. Lines in the area remote from the particle: traces of swimming zoospores.

The behavior of zoospores treated with a mixture of these pure compounds was very similar to the effect observed when zoospores were exposed to segments of fresh roots of P. oleracea suggesting that both compounds exude from the roots. Nevertheless, natural compounds possessing important in vivo functions, which remain to be fully elucidated. When bioassayed using the particle method, it was found that cochliophilin A (3), like the stimulant iV-frans-feruloyltyramine (19), could act together with the natural repellent 14inoleoyl-24ysophosphatidic acid monomethyl ester (20) to completely inhibit zoospore motility. Under the microscope, the treated zoospores were first seen to become stationary, and then settle at the bottom of the petri dish where they encysted to give cystospores. These cystospores germinated within 1-2 h, although germination would not normally be expected in the absence of a host plant. This is the first report on the inhibition of zoospore motility as a result of

1082

the interaction of a zoospore stimulant (iV-frans-feruloyltyramine, 19) and a repellent (14inoleoyl-24ysophosphatidic acid monomethyl ester, 20). The data also indicate a new biological action for lysophosphatidic acid, derivatives of which are already known to exhibit chemoattractant effects on the amoeba, Dictyostelium discoideum [86]. The biochemical properties of lysophosphatidic acids are described in detail in the latest review [87]. Recent identification and cloning of lysophosphatidic acid-specific receptor has led to the elucidation of G-protein and signaling pathways through which lysophosphatidic acid functions [88]. Recently, di-octanoyl phosphatidic acid (DOPA) was found to cause encystment of 100% of Ph. infestans zoospores at 15 (ig/ml within 10 min [28]. Hydroxylated iVcinnamoyl-/?-phenylethylamine derivatives including JV-rrans-feruloyltyramine (19) are relatively widespread in higher plants [89]. Their physiological functions are of general interest because their biosynthesis from the corresponding acyl-CoA and amine derivatives, under the influence of enzymes such as tyramine feruloyl transferase, is stimulated in response to pathological infection [90,91]. Isoflavonoidal Repellents from Dalbergia odorifera The extracts of a famous Chinese herbal medicine, the heartwood of Dalbergia odorifera displayed potent repellent activity toward A. cochlioides zoospores. Three isoflavonoids were isolated as the active factors from the extract by a series of column chromatography followed by preparative TLC [36,82]. Their chemical structures were assigned on the basis of physicochemical data including 2D NMR. Structure elucidation

The first isolate gave an intense molecular ion peak at m/z 270 ([M]+, 100%) in the FD-MS spectrum and analysis of HR-EI-MS established the molecular formula of 10 as C16Hi404. The UV, EI-MS, *H- and 13C-NMR data were found to be reasonably matched with those reported for medicarpin [92,93]. Thus the structure of 10 was confirmed as (±)medicarpin (3-hydoxy-9-methoxypterocarpan, 10) ([oc]29D 0° in MeOH, c = 0.045). The HR-EI-MS of the compound 24 exhibited the exact molecular mass (calcd., 286.0841, obsd., 286.0834) corresponding to the molecular formula C16Hi405. The UV, EI-MS, ! H and 13C-NMR data agreed with those of the reported (-)-claussequinone (24) [94]. The optical data recorded for 24 was ([a] 28D -31.5° in MeOH, c = 0.0069). The HR-EI-MS and the

1083 !

H-NMR spectra of compound 25 estimated its molecular formula as C16H12O4. The ] H- and 13C-NMR assignments were compared with those of the literature and all chemical shifts and coupling patterns were found to be identical with those of formononetin [92,95]. Thus the 25 was confirmed as formononetin (7-hydoxy^T^nethoxyisoflavone, 25). The compound 25 was acetylated and the bioactivity of acetylated formononetin (26) was also evaluated.

10. (±)-medicarpin

24. (-)-claussequinone

25. R = OH, formononetin 26. R = OAc

Biological activity The bioactivity of compounds 10 and 24-26 were evaluated by particle method. The possible combinations of these four compounds were also tested. All these four compounds (10 and 24-26) showed different activities to the motility of the zoospores. (i)-Medicarpin (10) showed repellent activity at 150 |ug/ml, while (-)-claussequinone (24) and formononetin (25) showed stimulating and attracting activities at 100 and 50 |Jg/ml, respectively. Mixture of these three isoflavonoids (1:1:1, w/w) exhibited repellent activity at 50 |ug/ml. The repellent activity of (i)-medicarpin (10) was enhanced in the presence of 24 and 25. Compounds 10, 24 and 25 are known to be antimicrobial as well as bioregulating in human physiology [96-98]. The negative chemotaxis of isoflavonoids may be interesting because a plant flavone, cochliophilin A (3), is a host-specific plant signal for A. cochlioides. Medicarpin has been found as phytoalexin in many legumes [99]. Antimicrobial activities of these three isolates (10, 24 and 25) have been reported but the repellent activity of those isoflavonoids toward zoospores has not been claimed. Negative chemotaxis of zoospores from isoflavonoids those were found in many plants, raises questions on the occurrence of this phenomenon particularly during plant/parasite interactions.

1084

Motility Inhibitory and Zoospore Lytic Factors from Unripe Ginkgo Fruits The maidenhair tree, or Ginkgo, is a gymnosperm that has been described as a 'living fossil' because it is known to have existed early in the Jurassic period 170 million years (Myr) ago [100]. It is one of the most important medicinal plants that received a considerable interest [101-103]. During the survey of physiologically active secondary metabolites in traditional medicinal plants toward zoospores of A. cochlioides, we observed that the EtOAc soluble extracts of unripe Ginkgo biloba fruits induced potent motility inhibition followed by lysis of the zoospores. Anacardic acids in the Ginkgo extracts were found to responsible for such a characteristic biological activity [104]. Isolation of the active principles along with their biological activities toward zoospores is discussed here. Isolation of anacardic acids, cardol and cardanol

The whole unripe Ginkgo fruits (15 kg) were extracted with MeOH (10 1) and the MeOH extract was concentrated in vacuo to remove the solvent. The resulting aqueous solution was diluted with deionized water to 4 1 and extracted successively with n-hexane (4 1) and then EtOAc (4 1). The EtOAc solubles (ca. 70 g) were chromatographed on a silica gel (900 g) column and the constituents were eluted with a mixture of n-hexane and EtOAc (15:1 v/v) to yield 40 g of an anacardic acid mixture. A part of the anacardic acid mixture (1.5 g) was refluxed in cone. H2SO4-MeOH (1:20 v/v) followed by silica gel column chromatography to yield a mixture of methyl esters (975 mg) and a slowly eluting non-derivatizable constituent (99 mg). The mixture of methyl anacardates was applied to medium-pressure column chromatography using an ODS column eluted with 3% H2O/MeOH to give two major peaks (fr. 1 and fr. 2, 478 and 360 mg, respectively). The major component in fr. 1 was further purified by HPLC (Prep-ODS column, 1% H2O/MeOH, flow rate 5 ml/min, l i?-66.6 min) to yield 281 mg of first methylated product. From the latter fraction, two major peaks were separable by HPLC (Prep-ODS column, 3% H2O/MeOH) to give 280 mg of crude second and 60 mg of crude third methylated products, respectively as oils. The second and third methylated products were purified from each HPLC fraction by repeated HPLC (100% MeOH, flow rate 5 ml/min) as colorless oils (180 and 24 mg, respectively). Methylated products (1st, 2nd and 3rd) thus purified were separately hydrolyzed in MeOH-aq. 2 M KOH (1:1) and the resulting products purified by preparative TLC (n-

1085

hexane-EtOAc-HCOOH, 14:2:1 v/v/v) to yield 27, 28 and 29, respectively in good yields. 21:l-io -Cardol (30) containing fraction (66 mg) eluted from the first silica gel column followed after the anacardic acid mixture was purified by preparative TLC in CHCl3-EtOAc-HCOOH (60:10:3, v/v/v, Rf 0.27, 35 mg). 21:l-w7-Cardanol (31) found as the unchanged constituent (99 mg) in the methylation reaction mixture from 1.5 g of the crude anacardic acid mixture was separated by preparative TLC (Rf 0.65 in CHCl3-EtOAc-HCOOH = 60:10:3), and finally purified by HPLC (Prep-ODS column, 3% H2O/MeOH, '/J-53.3 min) to give 40 mg of a colorless syrup. The structures of all derivatives were confirmed by spectroscopic methods [104].

27: R r

, R2=R3=H, (22:lco7-anacardic acid)

32: R r

, R2=H, R3=Me

28: Rj=

, R2=R3=H, (24: Ito9-anacardic acid)

29: Rj=

, R2=R3=H, (22:0-anacardic acid)

R

CF,

30:R 1 =OH(21:lco 7 -cardol) NO 2 7

31: R r H (21:lco -cardanol)

33: fluazinam

Biological activities of anacardic acids and related compounds

In homogeneous solution method [105], the active EtOAc solubles of unripe Ginkgo fruits exhibited lytic activity at a range of 0.1-1 |ag/ml

1086

toward A. cochlioides zoospores. After adding the sample solution into the zoospore suspension, cells quickly became immobile or moved in an unusual circular fashion for a few minutes and then halted. Initially the halted zoospores became round-shaped spores by losing their flagella and part of them was burst gradually. Relatively high motility inhibition and lysis-inducing activities against A. cochlioides zoospores were expectedly observed in 22:l-oo72-O-methylanacardic acid (32) and 22:l-w7-anacardic acid (27) as with a reputed fungicide fluazinam (33) [106]. 21:l-o:7-Cardol (30) and 24:l-co9-anacardic acid (28, a homologue of 27) were a little less active. Among the studied compounds, the halting and lytic activities were observed in decreasing order of 33 > 32 > 27 > 30 > 28 > 29. Compound 27 at 10~6 M caused 96% motility inhibition in 20 min and 67% lysis of the zoospores within 3 h. To achieve similar lytic activity, relatively longer time (6 h) was required for 30 and 28. In contrast, 22:0-anacardic acid (29 with a saturated 27 at the aliphatic side chain) exhibited relatively a weak activity and 21:1-w7-cardanol (31, a decarboxylative product of 27) was thoroughly inactive up to 10"4 M. Table 4. Motility Inhibitory and Zoospore Lytic Activities Induced by Ancardic Acids, Related Compounds and a Synthetic Pesticide Tested compounds*

Motility inhibition (%)

Lysis (%)

10 min

20 min

30 min

60 min

120 min

27

75

98

100

31

47

68

28

55

72

99

24

34

43

29

36

54

99

4

14

28

30

56

82

99

28

39

54

32

80

100

100

36

51

72

40

58

79

0

0

0

33

84

100

100

Control

2

4

7

ISO min

Final concentration of each compound in zoospore suspension was 10 M. Adapted from reference [104]. *The number of tested compounds is corresponding to that in the text.

To understand the mode of action, we examined the morphological changes of zoospores interacting with anacardic acid (27) (Fig. 7). Time-course scanning electron microscopic observation revealed that anacardic acid (homogeneous solution method, 5xlO"5 M) first damaged fine hairs of flagella and thus halted the zoospores within few min. The affected zoospores immediately turned into a nearly

1087

round-shaped spores (10 min after treatment) leaving or after complete destruction of one or both flagella. The immobile round spores appeared to be dehydrated and squeezed within 20 min after treatment (Fig. 7a). The membranes of dehydrated spores ruptured at a single point through which the cellular materials gradually released into water (Fig. 7b, c). Finally, all inner materials of the affected cells were came out and dispersed into water within 60 min (Fig. 7d).

a

b

r

Q

liun

Fig. (7). SEM micrographs showing lytic activities of 22:l-co7-anacardic acid (27) at 5xl0' 5 M [104]. Circular objects in the background are pores (size: 0.6 |jm) of SEMpore membrane.

Structural requirements for motility inhibition and lysis activities of anacardic acids and related compounds were briefly studied by testing several derivatives of anacardic acids and related compounds [104]. In respect of structure-activity correlation, active compound possessed common part structures, an aliphatic side chain with one olefinic bond and a carboxy group on the aromatic ring, which are likely to be necessary to show the activity. Structural modification of 27, for example 2-O-methylation (32) could improve their activities. Cardol (30) having no carboxyl group, but two hydroxyl groups on the aromatic ring also exhibited noticeable antizoosporic and antibacterial activities, whilst the content of 30 in the crude extract of Ginkgo fruits was very small (750 ft) of Berkeley Pit Lake [9]. Their structures were deduced by spectroscopic analysis and confirmed by single crystal x-ray analysis on berkeleydione (13). Both compounds inhibited signal transduction enzymes caspase-1 and matrix metalloproteinase-3. Berkeleydione (13) was also active against non-small cell lung cancer in NCI's 60 cell line anti-tumor screen. o .OH

14 Fig. (4). Structures of berkeleydione and berkeleytrione

The crude organic extracts of a Penicillium sp. isolated from a depth of 885 ft. were active against Staphylococcus aureus and in the brine shrimp lethality screea These extracts were further tested using enzyme inhibition assays for two different signal transduction enzymes - matrix metalloproteinase-3 (MMP-3) and caspase-1 (Casp1). Compounds 13 and 14 were isolated from the chloroform extracts of the broth filtrate of & Penicillium sp. found growing in Berkeley Pit Lake. Berkeleydione (13, 5.5 mg/L) was isolated as a crystalline

1168

solid. High-resolution CIMS (chemical ionization mass spectrometry) established the molecular formula of C26H33O7 (M++H) with 11 units of unsaturation. Extensive ID and 2D NMR data generated three possible structures, but there were inconsistencies with each structure proposed. The quantity and proximity of the many quaternary carbons made an unambiguous structural determination impossible. A single crystal was submitted for X-ray crystallographic analysis (Fig. 5).

Fig, 5

ORTEP drawing of berkeleydione

With the structure in hand we could make the spectral assignments which were largely straightforward based on extensive ID and 2D NMR experiments. Berkeleytrione (14, 3.4 mg/L) was isolated as an amorphorus solid. High-resolution EIMS established the molecular formula of C26H34O7 with 10 units of unsaturation. The structure was determined through spectroscopic methods. Several hybrid sesquiterpene-dimethyl orsellinate metabolites are known from Aspergillus sp [111-114]. All of these are highly oxygenated and have undergone rearrangements. Biosynthetic studies have demonstrated that the precursor of the terpenoid portion is farnesyl pyrophosphate and of the nonterpenoid portion is a bis-Cmethylated polyketide [115]. Berkeleydione (13) and berkeleytrione (14) effectively inhibited both MMP-3 and caspase-1 in the micromolar range. Berkeleydione

1169

(13) was tested in NCI's anti-tumor screen against 60 human cell lines. It showed selective activity towards non-small cell lung cancer NCI-H460 with a Logio GI50 of -6.40. This extreme selectivity is noteworthy in a natural product that has not been derivatized or tailored towards a particular cancer type. Although we are still at an early stage in this overall research endeavor we have already found the microbes of the Berkeley Pit Lake to be a source of new and interesting secondary metabolites. It is not often that scientists have the opportunity to explore such a unique environment and we are fortunate to have easy access this dynamic ecosystem. Based on preliminary data, we expect to find much interesting chemistry in this project. Of equal importance, however, is the learning environment that this research has provided not only our undergraduate students, but also other students in related Pit research projects. Our combined efforts should afford new insights into the acid mine waste phenomenon and the organisms that live in these inhospitable waters. As to the secondary metabolites and their microbial producers - they could be the richest products ever mined from "the richest hill on earth". ABBREVIATIONS DNA PCR Taq pH HBOI P-388 HCT8 A549 HSV-1 CaN CPP32 ICE2 ICP PDB

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

deoxyribonucleic acid polymerase chain reaction Thermus aquaticus -log(H+) Harbor Branch Oceanographic Institution murine (mouse) leukemia human colon cancer human alveolar cancer Herpes Simplex Virus-1 calcineurin caspase-3 interleukin-2 converting enzymes inductively coupled plasma potato dextrose broth

1170

PDBH+ TSB TSBH+ myc act Pit II PCP TMP-SMX casp-1 MMP-3 TIMPs 5-HT CHO FDX FDR NMR

potato dextrose broth acidified to pH 2.7 tryptic soy broth tryptic soy broth acidified to pH 2.7 mycological broth actinomyces broth Pit Lake water broth Pneumocystis carinii pneumonia trimethoprim-sulfamethoxazole caspase-1 matrix metalloproteinase-3 tissue inhibitors of metalloproteinases 5 -hydroxytryptamine Chinese hamster ovary freeze-dried extract freeze-dried residue nuclear magnetic resonance

ACKNOWLEDGEMENTS We thank our colleagues from the Department of Chemistry, Montana State University: S. Busse for assistance with NMR spectroscopy and L.J. Sears for mass spectral data and J.Madison, Montana Bureau of Mines and Geology, for Pit water samples. We thank the National Science Foundation grant # 9506620 for providing funding for NMR upgrades at the MSU facility and grant #CHE-9977213 for acquisition of an NMR spectrometer; NIH grants GM/OD 54302-02 and NCRR Grant # P20 RR15583 to the NIH-COBRE Center for Structural and Functional Neuroscience for funding the neurotransmitter bioassay work; NIH Grant P20 RR-16455-02 (BRIN Program of the National Center for Research Resources); USGS grant 02HQGR0121, and NIH grant CA24487 (JC) for financial support of this research. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of NIH or the U.S. Government.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

ISOFLAVONESAS COMPONENTS

FUNCTIONAL

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*F.R. MARIN1, J.A. PEREZ-ALVAREZ2, C. SOLER-RIVAS1. Departamento de Quimica-Fisica Aplicada (Area de Tecnologia de Alimentos), Facultad de Ciencias, Universidad Autonoma de Madrid, 2804,. Madrid, Spain. 'Departamento de Tecnologia Agroalimentaria (Division de Tecnologia de Alimentos), Escuela Politecnica Superior de Orihuela, Universidad Miguel Hernandez de Elche, 03312, Orihuela (Alicante), Spain. * Author to whom correspondence should be addressed: Francisco R. Mar in (e-mail: [email protected]; Phon.: 3414973585; Fax.: 34914973579) ABSTRACT: Isoflavones constitute a characteristic and very important subclass of flavonoids. Their structures are based on the 3-phenylcromen skeleton that is chemically derived from the 2-phenylchromen skeleton, by an aryl-migration mechanism. Structurally, isoflavonoids can be classified according to the oxidation of the Ci5 skeleton, their complexity and the internal formation of the heterocyclic rings. Isoflavones are mainly found in legumes and particularly in soy, although their presence has also been reported in black beans, green split peas, chickpeas, lima beans, split peas, alfalfa sprouts, sunflower seeds and clover sprouts. Moreover their natural distribution in raw materials, their presence as an ingredient in the composition of several foods, soy products in infant foods, vegetarian formulations, etc. lead to their ubiquitous presence in foodstuffs. Due to their ubiquitous distribution in food and the claimed beneficial health effects of foods containing isoflavones, their distribution in foods and healthy properties have been reviewed. Isoflavones have been proposed to have estrogenic activity and play a putative role in the prevention of climacteric syndrome. Other healthy properties as a possible role in renal disease protection, learning memory behaviour during aging, prevention of some types of cancer, bone metabolism, and thrombogenicity, among others, have been reported. Related to their biological activities, studies at molecular level involving interaction between different types of isoflavones and estrogen receptors, and others, show hierarchies correlating biological activities and chemical structures. Key Words: Isoflavones, nutraceuticals, functional foods.

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INTRODUCTION Flavonoids are a type of compounds omnipresent in the plant kingdom and, unavoidably, form part of our diet, because they constitute up to 2% of the total photosynthesised carbon. Flavonoids are plant secondary metabolites (they have not been reported as naturally-occurring in animals), aromatics and belong to the group of plant phenols [1]. From a purely chemical point of view, flavonoids are characterised by a skeleton of three units, C6-C3-C6, that forms a cyclic structure in most cases [2], In this skeleton two aromatic rings, referred to as A and B (in chalcones), can be distinguished, with an additional third ring (C) in the rest of the flavonoids. This last ring appears as a cyclation of chalcones with a hydroxyl in 6' position (Fig. (1)), while the A and B rings have a different metabolic source. The B ring is formed in the shikimate pathway, while the A ring comes from the condensation of three units of malonyl Co-A [3, 4]. Flavonoids can be classified according to the degree of oxidation of the three-carbon central segment. From lower to higher degree of oxidation, flavonoids are usually classified as catechins, chalcones, flavanones, isoflavones, flavan-3, 4-diols, flavones, aurones, flavonols and anthocyanins (Fig. (2)) [5], Isoflavonoids are a characteristic and very important subclass of flavonoids. Their structures are based on the 3-phenylchromen skeleton that is chemically derived from the 2-phenylchromen skeleton, by an arylmigration mechanism. Structurally, isoflavonoids can be classified according to the oxidation of the C15 skeleton, their complexity and the internal formation of heterocyclic rings [6]. Isoflavonoids are metabolically derived from the flavanones. The central step is the migration from the C2 to the C3 of the aryl block, which constitutes the B ring of the flavanone intermediate. This reaction is catalysed by 2-hydroxyflavone synthase, a cytochrome P450. At the same time, the isoflavones are precursors of a substantial number of compounds involved in the biosynthesis of phytoalexins and pterocarpanes. Epidemiological data report that isoflavones have multi-biological and pharmacological effects in humans, including estrogen agonist and antagonist activity, cell signalling conduction, as well as cell growth and death promoting. The mechanism through which isoflavones exert the above-mentioned functions is not only based on their estrogenic properties, but also on their roles as protein tyrosine kinase inhibitors,

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regulators of gene transcription, modulators of transcription factors and as antioxidants. They may also alter some enzymatic activities [7].

Fig. (1). Basic skeleton of flavonoids: (i) Chalcones, (ii) Phenylbenzopiran-4-one.

HO.

HO.

OH OH

Anthocyanldin

OH 0H

Catechin

Fig. (2). Chemical structures of the various classes of flavonoid.

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CHEMICAL FEATURES OF ISOFLAVONES Isoflavones constitute one of the largest groups of natural flavonoids, with about 364 aglycones (unconjugated forms) reported yet [6]. The most thoroughly investigated and perhaps most yet interesting phytoestrogens are the isoflavones, i.e. daidzein, genistein, formononetin and biochanin A, together with the coumestan coumestrol -a coumarine-like compoundmetabolically derived from the isoflavone daidzein. The isoflavones (daidzein and genistein) exist in four related chemical structures, namely aglycones (daidzein and genistein), the 7-O-glucosides (daidzin and genistin), the 6'-O-acetylglucosides (6'-O-acetyldaidzin and 6'-O-acetylgenistin) and the 6'-O-malonylglucosides (6'-Omalonyldaidzin and 6'-O-malonylgenistin). Other naturally occurring isoflavones are metabolically derived from these. Thus, daidzein is the metabolic precursor of formononetin and genistein of biochanin A. On the other hand, both formononetin and biochanin A are metabolised, after ingestion, to daidzein and genistein, respectively, and subsequently to equol and p-ethylphenol, respectively, as we will see below. In addition, glycitein, an isoflavone similar in structure to daidzein and genistein, has been isolated from plants, but only one report on its estrogenic activity has been found when preparing this review. Similarly to daidzein and genistein, there are four different chemical structures for glycitein: the aglycone glycitein, 7-O-glucoside glycitin, 6'-Oacetylglycitin and 6'-O-malonylglycitin. Other compounds, such as coumestrol, cannot be classified as isoflavones. However, coumestrol is a natural metabolic product of daidzein, together with other chemicals, such as the phytoalexins, phaseollin, dalbergioidin and kievitone. From a structural point of view, the coumestran coumestrol has been described as a free form and as an aglycone, although no reports on glucosylated and/or other natural forms of conjugation have been found. In Fig. (3), which shows some of the above-mentioned chemicals, the structural similarity with natural estrogens can be seen. Distribution of Isoflavones Fransworth and co-workers [8] have reported 94 plants as exhibiting estrogenic activity; not all these plants are edible. Isoflavones are mainly found in legumes and particularly in soy. Soybeans and soy products are a

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particularly a rich source of isoflavones, which may range in dry weight, from 0.2 to 1.6 mg/g. The principal isoflavones found in soy foods are genistein, daidzein and glycitein. Their presence has also been reported in black beans, green split peas, clover sprouts, chickpeas and other legumes [4]. Other isoflavones, such as brochamin and formononetin, have been found in green beans, chickpeas, lima beans, split peas, alfalfa sprouts, sunflower seeds and, particularly, in red clover [9, 10]. The widespread use of soy products in infant foods, vegetarian formulations and as an ingredient in the composition of several foods contributes to its ubiquitous presence in foodstuffs. Soy isoflavones are found in four chemical forms: the unconjugated form, as glucoside (daidzein, genistein and glycitein), acetylglucoside and malonylglucoside, Fig. (3) [10]. Moreover, processing and fermentation of the soybean has been reported to influence the forms isoflavones take. Similarly, after ingestion, isoflavones are modified during gut transit and/or in the liver to give a batch of metabolic by-products, including glucouronic conjugates, such as p-ethylphenol [11]. Reinli & Block [10] reviewed more than 36 research papers in 1996 with the aim of compiling and standardising all the previous information. Due to the variability of the research results, partially due to analytical procedures, isoflavone levels were recalculated on a wet weight basis. Table 1 shows some of the most relevant results reported by Reinli & Block -together with others from different sources- on the ratio of isoflavones present in different foods. However, it has to be borne in mind that substantial variations in the phytoestrogen content may occur as a consequence of the genetic differences of various varieties, environmental conditions, such as location and harvesting year and degree of maturity. BIO AVAILABILITY AND METABOLISM Isoflavones, a special kind of polyphenols, show similar behaviour regarding absorption and metabolism to this family of compounds. Thus, Scalbert et al [12] put forward two primary sources of evidence concerning the absorption of polyphenols: first, indirect evidence for their absorption through the gut barrier as can be seen from the increase in the antioxidant capacity of the plasma following the consumption of polyphenol-rich foods, and second, their recovery in urine after the ingestion of given amounts of a particular polyphenol.

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OH

0

Genistein

OH

Daidzein

0 OCH3 OCH3

Formononetin

Biochanln A

OH

0

Glycitein

OCH3

O-DMA

Coumestrol

OH

Estradiol Fig. (3). Chemical structures of major isoflavones, metabolic derivatives, coumestrol and estradiol.

1183

Since polyphenols are retained until they, or their metabolic products, are excreted by the urine or faeces, they undergo a number of complex microbial and chemical transformations that may include chemical conjugation, hydrolysis and biliary excretion [12]. The few existing studies in humans show that the quantities of polyphenols found intact in urine vary from one phenolic compound to another. Among flavonoids, recovery is low for some flavonols, such as quercetin and rutin (0.31.4%), but high for other flavonoids, such as catechins, anthocyanins, flavanones and soy isoflavones (3-27%) [13]. Absorption It has been suggested that the polyphenol structure may have a major impact on intestinal absorption. The most widely discussed structural parameters are molecular weight, glycosylation and esterification. As mentioned above, single flavonoids, such as isoflavones, have higher recovery values than polymeric flavonoids, such as tea theaflavins (0.0006%) and other kinds of flavonoids, such as flavanols. However, the recovery values of isoflavones are lower than those reported for phenolic acids [14]. On the other hand, a different pattern of urine recovery has been described for the soy isoflavone structure. For genistein, for example, a urine recovery of approximately 8% has been reported while for daidzein the rate was approximately 3%. This difference may be explained by structural differences. Thus, genistein has a hydroxyl moiety in C-5, while daidzein does not have it, Fig. (4). This slight difference in the hydroxyl substitution patter influences their water solubility. The Merck Index describes that genistein is slightly soluble in hot water, while daidzein is not at all soluble in water, which goes a long way to explaining their differential behaviour in urine.

OH Genistein

0H

Daidzein

Fig. (4). Differences between the isoflavones, genistein and daidzein, which might influence their recovery in urine.

1184

The glucosylation pattern seems to influence absorption through the gut barrier. Thus, the absorption of quercetin, a flavonol, has been measured in ileostomised volunteers. The absorption of the quercetin glycosides contained in onions was higher (52%) than that of quercetin aglycone (24%) [15]. In contrast, rhamnosides of quercetin are more poorly absorbed than the former glycoside and aglycone structures. It is proposed that the gut absorption of these rhamnosides would require deglycosylation by the colonic microflora, as suggested by their delayed absorption compared with the glycosides [16]. On the other hand, the ready absorption of quercetin glucosides might be due to their hydrolyses either by the lactase phlorizin hydrolase or the cytosolic P-glycosidase in the enterocyte, although this explanation cannot be extended to all classes of flavonoids [11]. The main soy isoflavones (genistein and daidzein) are 7-O-P-Dglucosides, while the aglycone forms (genistein and daizdein) are minor compounds compared with the glycosylated forms. Some authors suggest that after ingestion, the conjugated form of isoflavones is hydrolysed by intestinal (3-glucosidases -lactose phlorizin hydrolase, a membrane-bound enzyme found in the brush-border of the small intestine, which releases the principal bioactive aglycones, daidzein and genistein. These compounds may be absorbed or further metabolised in the distal intestine with the formation of specific metabolites, such as equol, O-DMA and pethylphenol [11]. Regarding the formation of these isoflavone derivatives, it is extremely interesting that some people are unable to produce equol or that they produce it in very low amounts. In fact, studies report that a third part of the general population cannot form equol. This demonstrates that the breakdown of isoflavones by the microflora in the gut determines the recovery of the compounds, and that the excretion to the urine of equol and other isoflavones derivatives, such as O-DMA, is dependent on the different composition of intestinal microflora. To confuse the issue even more, some researchers report that the production of isoflavone derivatives, such as equol, also depends on diet and gender: a high fat/meat content diet increases equol production in women but not in men, which is explained by promotion of the growth or the activity of the bacterial populations responsible for equol production. On the other hand, no age-related differences for isoflavone metabolism have been reported [12, 13]. There are contradictory findings as regards the need for the intestinal modification of isoflavones before they can be absorbed and

1185 Table 1. Food sources and content in major isoflavones. Where concentrations are available they are quoted as extreme values [8,9]. ND: Not Detected. Trace Daidzein Genistein Formononetin Biochanin A Food product Soybeans Black soybeans Green soybeans Soy nuts Tofu Tofu, fermented Soy flour Soy meal, defatted Soy meal, whole Soy flakes Soy flakes, defatted Soy flakes, whole Soy granule Soy protein, textured Soy concentrate Soy milk Alfalfa (buds) Alfalfa sprouts Clover sprouts Mung bean sprouts Soy bean sprouts Tempeh Soy sauce Miso Soy hot dog Soy bacon Tofu yoghurt Soy mozzarella Soy fibre Green beans Large lima beans Red beans Chick-pea Kidney beans Pink beans

(mg/kg)

(mg/kg)

(mg/kg)

(mg/kg)

676-1007 270-699 546 575 29-146 36-117 226-655 575-706

200-1382 277-612 729 729 50-166 40-218 478-1125 683-1000

ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND

706 221-880 419-1166

1000 280-1561 1411-1951

ND ND ND

ND ND ND

744

1320

ND

ND

549 83-568

748 568-707

ND ND

ND ND

43-107 37-74 ND ND ND ND

58-211 41-103 ND ND ND ND

ND ND T 3.4 22.8 T

ND ND T ND 4.4 ND

138-211 137-273 8-14 71-366 34 28 57 11 171 ND ND T ND ND ND

112-230 235-398 5-9 260-524 82 69 94 36 210 ND ND 3 ND ND ND

ND ND ND ND ND ND ND ND ND 1.5 14.8 ND ND 10.5 T

ND ND ND ND ND ND ND ND ND ND ND ND 15.2 ND ND

become available to the organism. Virtually all circulating polyphenols are glucuronidated and/or sulphated and no free aglycones are found in plasma [12, 17, 18], except for particular flavonoids, such as phloretin,

1186

which is present in rat plasma both in conjugated and non-conjugated forms [19] and for diosmetin, which is the circulating form in plasma of its glycosylated forms [20], In general terms and according to quercetin model, O-glucouronides are formed in the gut barrier, as mentioned above and secreted back either to the gut lumen or to the serosal side. These conjugates then reach the liver, where they are further metabolised [21]. However, free flavonoid aglycones are found when pharmacological doses are administered, indicating a possible saturation of the conjugation pathways [22]. Notwithstanding, other authors do not agree with this point of view. Andlauer et al [23], for example, reported that genistein was partly absorbed without a previous cleavage. Consistent with this suggestion is the finding that both aglycones and their glycosides are absorbed very rapidly [24]. Other authors even report that isoflavone aglycones are absorbed faster and in greater amounts than their corresponding glycosides in humans [25], which of course contradicts the above assumption. A different behaviour has been described in non-monogastric animals. Thus, in ruminants, isoflavones are mainly absorbed in the rumen, where the gastrointestinal epithelium is the major site of metabolism and the liver contributing very little to total isoflavones metabolism in these animals [26]. Metabolism The aglycones, along with any other bacterial metabolites derived from them, are absorbed from the intestinal tract and transported via the portal venous system to the liver, where the isoflavones and their metabolites are metabolically modified [7]. In general terms, all polyphenols are extensively metabolised either in tissues after absorption through the gut barrier, or, in the case of the nonabsorbed fraction and the fraction re-excreted in the bile, by the colonic microflora. All polyphenols, including isoflavones, are conjugated to form O-glucouronides, sulphate esters and O-methyl ethers. Lundth [26] proposed that isoflavones and their metabolites are conjugated mainly (95%) with glucuronic acid and, to a lesser extent, as sulphate conjugates. The formation of anionic isoflavone derivatives by conjugation with glucouronides and sulphate groups facilitates their urinary and biliary excretion and explains their rapid elimination. Thus, when the oral intake of soybean products is studied in human, genistein

1187

and daidzein are found in plasma two hours after feeding and reach their highest concentration six hours later. The half-life of genistein and diadzein is estimated to be 8.3 and 5.8 hours, respectively, both showing a high degree of recovery in faeces [12]. These results indicate that isoflavones persist more in the body than other classes of fiavonoids, which are quickly eliminated both in urine and bile after ingestion. In human, a post-prandial peak is observed 1-2 h after ingestion of various flavonols and flavanols. In the case of most fiavonoids absorbed in the small intestine, the plasma concentration then rapidly decreases, the repeated ingestion of polyphenols over time being necessary for the maintenance of high concentrations in plasma [11, 27]. Isoflavones can be detected in many animal tissues. A tissue distribution of daidzein of 40 mg/kg body weight has been reported in rats 15 min after intravenous injection. Daidzein concentration was found to be high in plasma, liver, lung and kidney (about 30 ug/g wet weight) to be moderate in skeletal muscle, spleen and heart (about 15-20 ug/g wet weight) and to be low in brain and testis. After feeding rats with 5, 100 and 500 ug/g genistein, other researchers reported a dose-dependent increase in total genistein concentration in brain, liver, mammary, ovary, prostate, testis, thyroid and uterus, with the highest content being observed in the liver and kidney where it is three to five times more concentrated than in plasma [28, 29, 30]. Polyphenol conjugates may also exert biological activity after deconjugation at the cellular level. Deglucouronidation and demethylation of phenolic estrogen conjugates have been described in the presence of lysosomes isolated from hamster kidney and liver. Similarly, the hydrolysis of estrone sulphates by mammary cancer cell lines has been described [31, 32]. The extent of polyphenol methylation would also affect the biological properties of polyphenols, although this is not important, as major food isoflavones do not have a cathecol pattern, Fig. (3), unlike other bioactive polyphenols such as catechin, quercetin or caffeic acid which are O-methylated "in vivo" [11]. While the last part of the small intestine is where fiavonoids glycosides are hydrolysed, conjugated with glucouronides and absorbed. Those not absorbed, such as aglycones and other fiavonoids, undergo metabolic change in the colon. In general terms, polyphenols, once they reach the colon, are extensively metabolised by the microfiora. The flavonoid glycosides, such as rutin, which do not absorb in the upper part of the intestinal tract can be hydrolysed and the aglycone absorbed. The flavonoid glucouronides excreted in the bile can also be hydrolysed by the

1188

microflora and the resulting aglycone reabsorbed, thus entering an enterohepatic cycle [11, 33, 34]. Further, aglycones are also metabolised to a wide array of low molecular weight aromatic acids, such as phenylvaleric, phenylpropionic, phenylacetic and benzoic acids, that are well absorbed through the colonic barrier. Several authors report that the lowest degree of polyphenols absorption takes place in the small intestine the highest amount reaching the colon, where the tissue is exposed to microbial flavonoids metabolites and the non-absorbed flavonoids from the small intestine [35, 36]. Most studies on the biological properties of polyphenols have been carried out on flavonoids in their native form. However, many of their biological effects observed in animal or clinical studies may be equally explained by their microbial metabolites. As regards isoflavones, little is known so far. On the one hand, as we see below, some isoflavone activities may be explained by their similarity with estrogenic hormones and therefore molecular integrity or minimal modification of the structure must occur to preserve activity. For example, equol (a metabolite of daidzein isoflavones) showed higher affinity for estrogen receptors than did daidzein itself, while daidzein and genistein glucouronides had, respectively, a 10- and 40-fold lower affinity for estrogenic receptors as compared to their aglycones [37, 38]. On the other hand, many other isoflavone activities, see below, are not related with their structural similarity with estrogens and therefore their physiological activity may depend directly on their own structure, or conjugates, or putative products of low molecular weight produced by microflora metabolism, which have not been described yet. Whatever the case, our knowledge on polyphenol bioactivity is reaching a point whereof the results used to explain their activity must be reconsidered. FUNCTIONAL PROPERTIES During recent years, isoflavones have received increasing interest due to their estrogenic and antiestrogenic effects, and have been associated with the reduced incidence of breast and prostate cancer, cardiovascular diseases or osteoporosis, although they also exhibit other favourable effects through an array of different mechanisms. These mechanisms are not only based on their estrogenic properties, but also on their properties as antioxidants, gene transcription modulators and enzyme inhibitors. Thus, we will review their hormonal effects, the influence on cell signalling, on cell proliferation and some of their pharmacological and

1189

therapeutic effects, their putative anti-cancer properties, their claimed ability to prevent cardiovascular diseases and their effect on the immune system being the most relevant. Hormonal Effects The classical definition of phytoestrogens encompasses compounds that exert estrogenic effects on the CNS (Central Nervous System), induce estrus and stimulate growth of the genital tract of female animals [39]. Broadly, the term phytoestrogens may also refer to chemicals that show effects suggestive of estrogenicity, such as binding to estrogen receptors, the induction of specific estrogen-responsive gene products and the stimulation of those receptors, which could encourage breast cell cancer growth. Although, it has been known since 1931 that soybeans contain relatively high concentrations of isoflavones, genistein, being the first isoflavones isolated from soybean by Walter, there was no knowledge that these compounds could show some biological activity in animals [7]. It was approximately 15 years later when these were recognised in playing a role in the infertility syndrome in sheep, in which they were correlated with the animal's hormonal potency [40]. Isoflavones and some metabolic derivatives, such as the coumestran coumestrol, Fig (3), are structurally similar to mammalian endogenous estrogens and may act as estrogen agonists or antagonists, depending on their concentration or the tissue in which they act [41]. In this respect, crucial in demonstrating that phytoestrogens share a common mechanism of action were studies in experimental systems, in which phytoestrogens competed with radiolabelled estradiol to bind to the estrogen receptor and elicited estrogenic responses in estrogen-responsive tissues and cells [42]. Further, it was found that they act mainly by binding to the ERJ3, which was found to be expressed in many tissues, including the hypothalamus, pituitary gland, lung and thymus [43, 44]. Genistein (4',5,7-trihydroxyisoflavone) is the principal, most active isoflavone and shows the highest binding affinity for the estrogen receptor [45]. Its methoxy derivative, biochanin A, does not bind to the estrogenic receptor but is estrogenic in vivo [46]. This curious paradox may be explained, as described previously, by the fact that biochanin A, after ingestion, is metabolised in its chemical precursor, the isoflavone genistein. On the other hand, daidzein (4\7-dihydroxyisoflavone) has a

1190

higher binding affinity for the estrogen receptor than its methoxy derivative, formononetin, although both are weak estrogens in vivo. In 1961, Bickoff proposed that methylation could be the mechanism by which the estrogenic potency of isoflavones is reduced. In addition, the different potency of genistein and daidzein could be due to the presence of the hydroxyl group of genistein [7]. Zava & Duve [47] reported that genistein has estrogenic and ERindependent cell growth inhibitory actions. Over a physiologically relevant concentration range, genistein could serve both as a surrogate estrogen agonist and as a growth regulator. Some researchers have suggested that an optimal pattern of hydroxylation seems to be necessary for a flavonoid to have estrogenic activity. According to this, it has been reported that those flavonoids with hydroxyl moieties at C-4' and C-7 were invariably estrogenic and that an additional hydroxyl group at the C5, such as genistein, increased estrogen activity. Obviously, these structural criteria do not differentiate between the different classes of flavonoid, and include some flavanones, such as naringenin, and/or flavonols, such as quercetin, which obviously are not isoflavones [48, 49], Notwithstanding, the above authors indicated that isoflavones were better ligands, for ER, than flavones; and rings A and C of isoflavones were thought to mimic rings A and B of estrogens [50, 51]. Therefore, two kinds of criteria may be highlighted when putative estrogenic activity is based on its chemical nature. The first criterion is based on the hydroxyl moieties at positions 4' and 7', plus an activity enhancer when C-5 is hydroxyl substituted; and a second criterion as regards B-ring location at C-2 or C-3, this last position having an enhancing effect through its simulation of a natural oestrogen structure. On the other hand, criteria to explain structures that probably do not have estrogenic activity have also been proposed. Thus, more than four hydroxyl substituents, as in the case of the flavonol quercetin, or a 4' methoxylated substituent, such as the flavone diosmin, appear to abolish the estrogenic activity of flavonoids, Fig (5) [49]. The structural dependence of the estrogenic activity on the hydroxylation pattern has been reported by several research teams [48, 49, 50, 51]. Thus, Collins et al [52] examined the agonist/antagonist activity of various flavones and isoflavones, using a yeast estrogen system in which yeast cells were cotransformed with the human ER and two copies of an estrogen response element linked to the lacZ gene. The IC50 (the concentration of the ligand competitor at which the binding of radiolabelled 17p-estradiol to the human ER was reduced to 50%) for

1191

coumestrol, genistein, biochanin A, chrysin (a flavone) and naringenin (a flavanones) were determined to be 0.01, 2.0, 6.0, 33 and 45 uM, respectively, indicating that such isoflavones as genistein and biochanin A bind to the ER up to 5-10 times more strongly than flavones such as chrysin and naringenin.

OH

OH

m

Fig. (5). Structural criteria that enhance the estrogenic activity of flavonoids. I, II: Criteria based on the hydroxyl moiety pattern [47, 48, 49]. Ill, IV: Criteria based on the simulation of a natural estrogen structure [50, 51].

Coumestans, an isoflavone-^netabolic derivative, represents a fully oxidised version of the flavonoid pterocarpans and share the same systematic numbering. The most prominent and potent representative, coumestrol, has higher binding affinity for the estrogen receptor than genistein [53]. The hydroxyl moiety of coumestrol at position 12 corresponds to the hydroxyl moiety at C-4' proposed above as a structural criterion [48, 49]. This higher binding affinity suggests that the internal cyclation corresponding to the isoflavone structure at carbons 6 and 4, and/or the presence of an extra keto group in the coumestrol may improve the affinity for the ER and, therefore, of the estrogenic activity. One last additional criterion may be mentioned: The glycosylation pattern, which has until recently been ignored by researchers, should be taken into consideration. Recent studies have shown, as we will see below, that the aglycone forms show higher affinity for ER than the glycosylated ones.

1192

Besides the affinity of genistein, daidzein, biochanin A, formononetin, coumestrol and some other flavonoids, there is little information regarding other isoflavones, of which more than 300 have been reported to exist [6]. One such example is glycitein, whose estrogenic activity has been reported in one study. The data indicated that glycitein, when fed to female mice at 3 mg/day for 4 days, produced weak estrogenic activity comparable to that of other isoflavones, but much lower than that produced by diethylstilbestrol and 17(3-estradiol [54]. As regards these and those previously reported, a hierarchy of estrogenic activity may be proposed, in which flavone 7, 4'-dihydroxylate has the lowest activity and coumestrol with its internal cyclation the highest, Fig. (6). Table 2 shows the relative potencies of different isoflavones and coumestrol in different models for evaluating estrogenic activity.

Fig. (6). Estrogenic hierarchy, in increasing order, of flavonoid structures. A: Flavone 7, 4' di-OH. Rp H or OH, B: Flavanone 7, 4' di-OH, R,: H or OH. C: Isoflavone 7-OH, R^ H or OH, R2: H or CH3. D: Isoflavanone 7,4' di-OH. E: Isoflavone 5, 7, 4' tri-OH. F: Coumestrol.

When isoflavones are compared to estradiol, their estrogenic effects are seen to be weaker. In the mouse uterine growth assay, genistein and daidzein are roughly 100,000 times less effective than estradiol [43]. Moreover, the circulating forms of isoflavones, such as the glucouronides, showed between 10 and 40-fold lower activity for estrogenic receptors compared with their aglycones, but still showed weakly estrogenic activity at physiological concentration [11, 38].

1193 Table 2. Relative potency of some phytoestrogens in human adenocarcinoma cells [9].

Estradiol Coumestrol Geni stein Equol Daidzein Biochanin A Formononetin

In vitro 1,186 2.40 1.00 0.72 0.16 0.08 0.01

endometrial

In vivo 100,000 35 1.00 0.75 0.46 0.26

Apart from epidemiological evidence then, how is it possible to explain the physiological effect of isoflavones? Although, their estrogenic potency is extremely low compared with animal sterols, their blood concentrations may reach 50-800 ng/mL (0.2-3.2 ug/mL) in adults consuming modest quantities of soy food containing approx. 50 mg/d of isoflavones. Similarly, in response to the consumption of soy foods, blood isoflavone concentrations may be doubled. When soy is consumed on a regular basis, plasma isoflavone levels far exceed normal estradiol concentrations, which both in men and women generally range between 40 and 80 pg/mL [12, 55]. These observations lead to the hypothesis that isoflavones may be biologically active, conferring health benefits that could explain the relatively low incidence of hormone-dependent diseases in countries where soy is a dietary staple. However, the effects of soy consumption on hormonal metabolism have been inconsistent, probably as a result of methodological differences in the studies as regards the characteristics of the subjects, study design, isoflavone form and other factors [7], Soy isoflavones appear to affect the menstrual cycle and the concentrations of reproductive hormones in premenopausal women, by increasing the length of their follicular phase [56]. Moreover, it has been reported that dietary genistein exerts estrogenic effects upon the hypothalamic-pituitary axis in rats, and increases plasma prolactine. Also, genistein and daidzein may suppress glucocorticoid and stimulate androgen production in cultured human adrenal cortical cells [56, 57]. In brief, it is generally accepted that isoflavone consumption exerts various hormonal effects. However, the resulting health benefits are of uncertain clinical significance and further research is necessary to determine

1194

whether the responsible constituents are the isoflavones contained in food or other food constituents. Besides the estrogen agonist effect of isoflavones, an antagonist effect has also been described for them. Thus, isoflavones at concentrations 100-1000 times higher than that of estradiol have been considered capable to compete effectively with endogenous mammalian estrogens and prevent estrogen-stimulated growth in mammals [43]. This estrogen antagonist pattern of isoflavones, by competing with estradiol for the ER, may end up by interfering with the release of gonadotropins and interrupting the feedback-regulating system of the hypothalamuspituitary-gonadal axis. Kuiper et al [43] and Cassidy [58] reported the identification of a novel rat ERp. A subsequent study of the ligand selectivities and the tissue distribution of both ER subtypes a and P have thrown new light on the estrogenic activity of phytoestrogens. Both coumestrol and genistein exhibit a significantly higher affinity for ERP protein than for ERa. On the other hand, ERP is expressed prominently, in testicular tissue, secretory epithelial cells of the prostate, in the vascular system and, apparently, in breast tumour cells. ERp is also found in brain, bone, bladder and vascular epithelia, which have been seen responsive to classical hormone replacement therapy [59, 60, 61]. Although the regulation of the sex hormone receptors at the transcriptional level may not be considered as a hormone-like activity, it is one of the ways to control the hormonal signal. Thus, it has been reported that genistein, at concentrations of 500 ug/g body weight, decreased ERa mRNA expression [62]. In a similar way, it has been described that daidzein can decrease ERp mRNA levels in the hypothalamus of newborn piglets [63]. On the other hand, some authors find that soy isoflavones increase nerve growth factor mRNA and brain derived neurotrophic factor mRNA in rats [64, 65]. Since the discovery of a second estrogen receptor, ERP, it is necessary to re-evaluate the molecular basis for the action of estrogen and its agonists. Structurally ERP is highly homologous to ERa in the DNA binding domain, with more than 95% amino acid identity but only 55% homology in the ligand binding domain [66]. The structural differences lead to different relative binding affinities in ligand binding assays. Compared with ERa, isoflavones have a greater relative binding affinity to ERP, while estradiol binds to ERa and ERP with an equal affinity. Thus, structural biology shows that genistein is completely buried within the hydrophobic core of the protein and binds in a manner similar to 17p-

1195

estradiol. However, in the genistein complex, the activation-function does not adopt the distinctive agonist position but instead lies in a similar orientation to that induced by estrogen antagonist, which is consistent with genistein's partial agonist character in ERp [67, 68, 69]. Isoflavones in Hormone Replacement Therapy Estrogen deficiency in peri- and postmenopausal women results in a variety of neurovegetative, physical and somatic symptoms and may contribute to serious diseases within the aged female population. Such estrogen deficiency symptoms and the resulting diseases can be relieved, or their progression slowed down by conventional hormone replacement therapy (HRT) involving 17P-estradiol esters or conjugated estrogens [70]. However, estrogen has been demonstrated to be associated with the increased incidence of breast and endometrial cancer after prolonged treatment. In addition, during HRT, venous thrombo-embolic complications are encountered more frequently than in women not undergoing HRT [71]. Therefore, there is a growing interest in using isoflavones as a potential alternative to the estrogens in hormone replacement therapy. However, in this respect, there is no clear evidence that isoflavones are superior to a placebo when used to alleviate climacteric complaints such as hot flushes [71, 72], although some studies suggest the opposite [73]. Similarly, there is no evidence that isoflavones have a positive effect on the urogenital tract as stabilizers of the acidity of the vaginal milieu and thus preventing ascending infections [74]. On the other hand, isoflavones have been shown to have mild osteoprotective effects on the bones of ovarictemised rats, as well as on the bones of postmenopausal women [75, 76]. Also, a protective effect against cardiovascular risk has been described, as we shall see below [77, 78], whereas any protective effect on mammary cancer and endometrial cancer may be linked to the dosage [79].

Antiproliferative Effects and Regulation of Signalling Most of the support for the anticancer effects of isoflavones comes from epidemiological studies, for which data suggest that a diet rich in

1196

isoflavones provides protection against several forms of cancer, particularly those that are hormone-dependent, such as breast, prostate and lung cancer [7, 80, 81]. In vitro data lend support to the view that isoflavones inhibit cancer cell growth, prostate cancer and MCF-7 human breast cancer cells being the most relevant [82, 83]. Moreover, over 100 in vitro studies have shown that genistein inhibits the growth of a wide range both of hormone-dependent and hormone-independent cancer cell lines, with IC50 values ranging between 5 and 100 umol/L [84]. Some authors suggest that isoflavones may exert cancer-preventive effects by decreasing estrogen synthesis and altering metabolism away from genotoxic metabolites towards inactive metabolites. Thus, it has been found that daily consumption of a soya diet (providing 113-207 mg/day of total isoflavones) reduces circulating levels of 17(3-estradiol by 25%, and of progesterone by 45% compared with the levels recorded for a controlled diet in healthy and regularly menstruating women [85, 86]. It has become apparent that the anticancer mechanisms of isoflavones do not operate exclusively via the estrogen receptor. The intervention of isoflavones in tyrosine kinase activity is one of the ways of interfering cancer development. Enhanced protein tyrosine kinase activity due to the overexpression of receptor and/or protein tyrosine kinases leads to a continuous signalling that results in uncontrolled cell proliferation, which produces cancer growth. Many of the transduction pathways of peptide growth factor signals that have been implicated in certain cancers involve the action of tyrosine kinases. Therefore, a circulating tyrosine kinase inhibitor, such as genistein, may have beneficial effects on the prevention and treatment of cancers [87]. It has been reported that genistein can downregulate the intrinsic protein tyrosine kinase involved in neuroblastoma development [88]. It has also been described that in androgen-independent, human prostate carcinoma DU145 cells, genistein inhibits the transforming growth factor (TGF)-a-caused activation of membrane receptor erBl (a component of RTK family, Receptors of Tyrosine Kinase), before inhibiting downstream cytoplasmic signalling [89]. Indeed, more than 2000 papers supporting the regulation of tyrosine kinase by genistein are available in the scientific literature [90]. Most isoflavone studies on cell proliferation were performed using estrogen-dependent human breast carcinoma MCF-7 cells. The results pointed to a biphasic effect: stimulation of growth at low concentrations and inhibition at high concentrations. Thus, it has been reported that cell growth is stimulated by daidzein and genistein at low concentrations of < 0.25 ug/mL and < 10 ug/mL, respectively, while at higher concentrations

1197

(over 25 ug/mL and 20 ug/mL) cell growth is significantly inhibited in a dosage-dependent fashion [91. 92]. It has also been reported that isoflavones may inhibit cell proliferation through the inhibition of angiogenesis, which is a well-regulated and limited process intimately bound to vessel growth and carcinomas. The generation of new capillaries from pre-existing vessels does not occur in the healthy adult organism except in very few cases. However, pathological angiogenesis occurs during the development of some diseases and particularly in tumours. Well-vascularised tumours expand both locally and by metastasis, whereas avascular tumours do not grow beyond a diameter of 1-2 mm [93, 94]. Genistein is a well-known and potent inhibitor of cell proliferation and in vitro angiogenesis [95, 96], which may suggest that this property is related to its isoflavone structure. However, some studies have shown that some flavonoids, but not isoflavones, are even better antiangiogenic agents than genistein. Fotsis et al [97] found that several flavonoids such as 3',4'-dihidroxiflavone, luteolin and 3-hidroxyflavone were stronger inhibitors of angiogenesis that genistein. Apigenin, eriodyctiol and quercetin showed a similar effect, while luteolin glycoside, fisetin, myricetin, hesperetin and catechins had a less pronounced effect. These results suggest that a nonhydroxylated C ring with oxo function at position 4 and a double bond between C2 and C3 are required for maximal biological activity. Moreover, their glycosylation pattern seems to imply the lack of these properties. Based on these findings, Fotsis et al [97] suggested that this behaviour might be correlated with early events and some enzymatic inhibitors, such as tyrosine kinases [98] and protein kinases [99, 100]. The structural properties highlighted previously would make isoflavones competitive inhibitors with respect to the ATP binding site in a variety of enzymes, a region of considerable homology among kinases [101]. On the other hand, the concentration of genistein required to inhibit angiogenesis in vitro was reported to be higher than the genistein concentration likely to be achieved in vivo. However, it has been found that isoflavones in vitro may also be active at physiological concentrations (< 5-6 umol/L) [38, 102, 103]. In vitro studies have revealed that numerous mechanisms may be involved apart from the tyrosine kinase mechanism. In several cell lines, genistein did not alter tyrosine phosphorylation of the EGF receptor or other tyrosine kinase substrates. On the other hand, some authors have repotted that genistein inhibited the expression of the EGF receptor in the

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rat dorsolateral prostate, suggesting that the effect of genistein is via transcriptional processes rather than directly on tyrosine kinase activity [104, 105]. Thus, it has been suggested that the variable effects of isoflavones in estrogen-sensitive tissues may depend on the production of paracrine and autocrine growth factors that cause proliferation of cells not expressing ERa or ER.p [106]. Another anti-cancer mechanism of isoflavones may involve inhibition of key enzymes of estrogen metabolism, such as 3 P-hydroxy steroid dehydrogenase, 17P-hydroxysteroid dehydrogenase, 5a-reductase and aromatase, as a consequence of which the level of active steroid hormones is affected [81, 106, 107]. Isoflavones may also exert their activity either directly on DNA expression, or by protecting it. Thus, isoflavones may either inhibit DNA topoisomerase I and II activity, which is thought to cause DNA damage [108, 109]. The transcription factor p53 has become one of the most important tumour suppressors, and genistein has been shown to induce the upregulation of this protein [110, 111]. Genistein may also inhibit cell growth both by increasing the expression and production of the transforming growth factor (TGF) pi [112]. Some of the anti-cancer effects of isoflavones may result from their modulation of apoptosis. Thus, genistein inhibits the proliferation and differentiation of N2A, JC, SKNSH, MSN and Lan5 neuroblastoma cell lines and induced apoptosis in one (N2A line) [113]. Davis et al [114] reported that genistein induced apoptosis by inactivating NF-KB, in this way promoting cell death. Traganos et al [115] demonstrated that genistein at 5-20 ug/mL produced cell cycle arrest in both the Gl/S and G2/M phases of the human myelogenous leukaemia HL-60 line and the lymphocytic leukaemia MOLT-4 cell line. Further studies reported that genistein up to 60 uM arrested the development of human gastric cells at G2/M. Also, studies conducted in a non-small-cell lung cancer cell line demonstrated that genistein, at 30 uM, induced G2/M arrest through the p21 upregulation and the induction of apoptosis [116, 117]. Cell cycle arrest and the induction of apoptosis could be functionally related to the activation of p53 and/or the inhibition of cell cycle kinase activity, and research results suggest that flavonoids may be more effective in controlling the growth of tumours with certain mutational spectra [118]. A non-cancer related influence of isoflavones on cell signalling was reported by Liu et al. [119], who found that when pregnant sows where fed with daidzein, fetal growth was promoted, sow milk production was

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improved and postnatal growth was positively affected. Further, it was demonstrated that when sows were fed with daidzein the expression of IGF-1R gene in the longissimus muscle of new born piglets was markedly enhanced, suggesting that daidzein may influence fetal growth via the upregulation of IGF-1R expression in skeletal muscle [44, 120, 121]. Effect of Isoflavones on Cardiovascular Diseases Many research papers have demonstrated that soy protein inhibits cardiovascular diseases and reduces the risk of atherosclerosis in animals and humans. Indeed, in October 1999, the USFDA acknowledged the health claims regarding the beneficial effects of soy-based foods in a heart-healthy diet, after reviewing research from 27 studies that showed soy protein's value in lowering levels of total cholesterol and low-density lipoprotein [7, 79, 122]. Most authors support the view that the beneficial effects of soy are primarily due to isoflavones and are mediated by many mechanisms [123]. On the other hand, the mechanisms associated with soy's beneficial effects on cardiovascular health are not fully understood and, it remains unclear which components of soy protein contribute to its protective effects. It is possible that soy substances other than isoflavones, such as saponins, phytic acid, or a protein-isoflavone interaction, among others, may be involved in the multifarious process. However, most researchers consider that these positive effects are due to a reduction of plasma LDL (low density lipoprotein) cholesterol and triglyceride concentrations [124, 125], although other mechanisms, such as lowering diastolic blood pressure in women and improving the vascular and endothelial function, have also been proposed [126]. On the other hand, other authors suggest that the benefits of soy protein on cholesterol levels may be mediated through the upregulation of LDLreceptor activity, thus providing a novel mechanism of plasma cholesterol reduction different from currently available diets and hypolipidaemic drugs [127, 128]. Other proposed mechanisms for the effects of isoflavones include the prevention of atheroma formation through their antioxidant ability, and recently, the oxidative theory of atherogenesis has provided another avenue of therapy involving antioxidants [129]. According to this theory, antioxidants should protect lipoproteins against oxidative modification and reduce the biological consequences. Thus, Tikkanen et al [130] showed that the intake of soy protein containing 60 mg of isoflavones per

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day might provide protection against the oxidative modification of LDL. The oxidative modification of LDL particles is considered to be a prerequisite for the uptake of LDL by macrophages in the artery wall, which is an initial step in the formation of atheroma. Other Effects Most autoimmune diseases are more common in women than in men and quite frequently occur when estrogen levels change dramatically. The gastrointestinal tract and the immune system have often been overlooked and not considered as targets of estrogen. However, ERp has been found highly expressed in the human thymus and the gastrointestinal tract. Therefore, some of the immuno-modulatory effects of estrogens might be mediated via ERp. In this respect, daidzein has been proven to increase the activation of murine lymphocytes and activate natural T killer cells [38, 102]. Similarly, in vivo, it has been shown that daidzein, at a high dosage, enhances several immunological functions [131]. Theoretically, phytoestrogens could be expected to improve the cognitive function, particularly verbal functioning, as suggested for conventional hormone replacement therapy. As regards cognitive decline and soy isoflavone intake, the scientific data are varied. Thus, some studies suggest that increased tofu in the diet is associated with cognitive decline. On the other hand, animal data indicate the potentially beneficial effects of soy isoflavones in neuroanatomy and simple memory tasks [79]. Finally, there is growing evidence that the dietary intake of phytoestrogens has a beneficial role in chronic renal disease. Data suggest that the consumption of soy-based products rich in isoflavones and flaxseed rich in lignans retards the development and progression of chronic renal disease. In several animal models of renal disease, both soy protein and flaxseed have been shown to limit, or reduce, proteinuria and the renal pathological lesions associated with progressive renal failure. Isoflavones appear to act through various mechanisms that modulate cell growth and proliferation, extracellular matrix synthesis, inflammation, and oxidative stress. However, further investigation is needed to evaluate their long-term effects on the progression of renal disease in patients with chronic renal failure [132],

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ARE ISOFLAVONES TOTALLY INNOCUOUS? The excessive consumption of soybean and its products has been considered goitrogenic in humans and animals. Several researchers have reported the induction of goitre in iodine-deficient rats maintained on a soybean diet [133, 134, 135]. In some cases, the extreme intake of soybean has been correlated with cancer. Thus, Kimura et al reported an increase of up to 40% in thyroid carcinoma in rats fed on iodine-deficient defatted soybean [136], Recent studies have highlighted an explanation for this undesirable effect of soy flavonoids. The function of the thyroid is to synthetise thyroid hormones and TPO (Thyroid Peroxidase), which catalyses the iodination of thyrosyl residues on thyroglobulin and the subsequent coupling of iodotyrosyl residues required for iodothyronine hormone formation. In the presence of iodine ions, genistein and daidzein (the major soy flavonoids) block TPO-catalysed tyrosine iodination by acting as alternate substrates, yielding mono-, di- and triodoisoflavones. Genistein can also inhibit the synthesis of thyrosine by using iodinated casein or human goitre thyroglobulin as substrates for the coupling reaction [137], Genistein and genistin have also been known to be strong cytotoxic agents in vitro. This characteristic can be an advantage when target cells are malignant but can be disadvantageous when they are normal cells. Recent experiences carried out with rat myogenic cells (L8) showed that genistein and genistin strongly inhibited in vitro myoblast proliferation and fusion in a dose-dependent manner. Genistein also inhibited protein accretion in myotubes. Decreased protein accretion is largely a result of the cell (myofibrillar) protein synthesis rate, while no adverse effect on protein degradation has been observed. The results suggest that if sufficient circulating concentrations are reached in tissues of animals consuming soy products, genistein potentially affects normal muscle growth and development [138]. On the other hand, it is widely accepted that isoflavones can freely pass the placenta barrier and in humans isoflavone concentrations in the neonate are similar to those in the maternal plasma. It has been reported that isoflavones at concentrations found in a standard, natural-ingredient diet may affect the sexual differentiation of female rats in the uterus [7, 44]. Finally, the healthy effects of isoflavones on some individuals may be negative on others. Thus, as we have seen above, genistein may induce

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the upregulation of p53 protein. The Arg form at codon 72, instead of the Pro, has been associated with an increased risk of human papiloma virus related cancer in humans [139]. This finding could imply an extra risk associated with higher intakes of isoflavones. However, the former theory is not fully accepted, as other researchers argue that there is no relation between the kind of codon and an extra risk [140]. When soy-based products are promoted as healthy foods possessing putative beneficial estrogenic and anticarcinogenic activity, some of these properties due to their isoflavones, the findings mentioned in the previous paragraph should be highlighted, due to the widespread use of these products in infant food formulas and the consumption of soy products by people with vegetarian diets. ABBREVIATIONS CNS EGF ER HRT LDL 0-DMA RTK TGF USFDA UV/V

= Central Nervous System = Epidermal Growth Factor = Estrogen Receptor = Hormone Replacement Therapy = Low Density Lipoprotein = O-Desmethylangolensin - Receptors of Tyrosine Kinase = Transforming Growth Factor = U.S. Food and Drug Administration = Ultraviolet/Visible

ACKNOWLEDGEMENTS The authors would like to thank Prof. Victor Kuri, University of Plymouth, United Kingdom, for his helpful assistance in preparing this manuscript.

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1209 SUBJECT INDEX Abortifacient acivity 978 ofaristolicacid 977,978 Abyssinone II 836 against Mucor mucedo 826 against Staphylococcus aureus 826 Acanthosicyos 430 Acetoxyverruculogen 580 structure of 580 8-Acetoxywithanolide D 1049 antiparasitic activity of 1049 Acetylcholinesterase inhibition 143 by suvanine 143 12-O-Acetyl-16-O-deacetyl-12,16episcalarolbutenolide 151 cytotoxic activity of 151 Acetylcholine esterase 989 inhibition of 989 Achlya bisexual is 1102 Acid mine waste extremophiles 1123 bioactive metabolites from 1123 Acid mine waste lakes 1138 Acidophiles 1126 in acidic environment 1126 Acnistins 1022,1038,1039 structures of 1039 Acremonium luzulae 512 Actones 671 Acylation 576 Acylsaponins 223 from Silene fortunei 223 Adaptogens 456 cucurbitacins as 429,430-435,438, 439-446,447,456 Adenosine biosynthesis 4 Adjuvant activity 239 and HLB value 239 AF-bio binding protein 1071 Aframomum stipulatum 804 against malaria 371 against remota 371 Aging 705 role of oxygen species in 705 Ajuga decumbens 371 in cough 371 in inflammation 371 in respiratory disease 371

Akaloid phthalide 619 sources of 619 Akebia quinata 1103 Alcholic esters 248 Aldehydes 248 Aldol reaction 10,11,13 ofKetoaldehyde 4,10,13 Aldose reductase 1137 inhibition of 137 Alkaliphiles 1126 in an alkaline environment 1126 Alkaloids 182,248,808-814 actinidine 248 against Plasmodium falciparum 182 ibogaine 808 iboxygaine 808 noribogaine 808 tabernanthine 808 Alkaloid phthalide subtype 630 structures of 630 Allanblackia stuhlmannii 705 HIV-inhibitory activity of 705 Allelopathic 835 5-hydroxyisoflavonoids as 835 y4//o-Cerdran-type sesquiterpenes 409-412 sources of 409-412 structures of 410 Altohyrtin A 71 total synthesis of 71 Altohyrtin C 71 first total synthesis of 71 Alzheimer's disease 386,795 Genipine against 384,386 Amaranthus gangeticus metabolites 1095 regulation of development transitions of zoospores by 1095 Amarogentin 261 from Swertia chirata 261 Amaroswerin 261 Amauromine 571 as vasodilator 571 structure of 571 Amides 891,894 from Aristolochia species 891 structures of 894 Amines 85,86 structures of 86 Aminophenol 20 oxidative cyclization of 20

1210 Amtimicrobial property 463 of cucurbitacins 463 Anacardic acids 1084-1086 and related compounds 1084 biological activities of 1085 isolation of 1084 motility inhibitory activity of 1086 Analgesic 446 cucurbitacins as 429,430-435,438, 439-446,447,456 Analgesic actions 654 ofcuanxiong 654 Analgesic activity 156,209,382,444,816 ofiridoids 248,251,252,291, 305-333,340,352,353,365,381 ofglutinol 816 of Kageneckia oblonga A21,AAA ofsaponins 209 of triacetyl disidein 156 Angolesin 834 anti-MRSA activity of 834 Angolesin (oc-methyldeoxybenzion) 834 structure of 834 Anisatin 419 neurotoxic activity of 419 Anisatin-subtype sesquiterpenes 397 from Illicium floridanum 397 from Illicium minwimense 397 Anislactone B 414 chemical conversion of 414 transformation of 416 Anislactone-type sesquiterpenes 395,412419 biosynthetic route of 418 chemistry of 395 from Illicium species 395 neurotoxic activity of 419 neurotrophic activity of 395,419 sources of 412-419 structures of 413 Anti HIV activity 356 of olive leaves 356 Antiangiongenic agents 1197 isoflavones 1197 Antibacterial activity 125,156,185,209, 482,704,805 of azaphilones 481 of hyperibones A-D 704 of Lippia multiflora 805 of manzamines 185

of neomangicol B 156 ofsaponins 209 Antibiotic activity 842 of.£>y//w7«apterocarpans 842 Antibiotic aspirochlorine 519 from Aspergillus flavus 519 from Aspergillus oryzae 519 from Aspergillus tamarii 519 Antibiotics 1107 and conventional 1107 Anticancer activity 825,859 of Aristolochia mollissima 859 of bioactive compounds 825 Anticancer agents 149,827,835 burttinone as 827 isosenegalensin as 835 search for 1149 Anticancer drugs 1134 development of 1134 Anticarcinogenic activity 1202 of soy-based products 1202 Anticarcinogenic effects 214 of Panax ginseng 214 Anticholinergic property 262 of swertiamarin 262 ofsweroside 262 Anti-convulsant 638 3#-Butyphthalide as 638 3.S-Butyphthalide as 638 Anti-dementia 774 huperzine A as 774 Antidote 266 Genticma lufea as 265,266,270 Antifeedant activity 130,140,144,986, 988 against Carassius aurantus 144 of Aristolochia albido 986 of Aristolochia albida metabolites 988 of Cacospongia linteiformis 130, 140,144 of cyclolinteinone 130 of 12-deacetoxyscalaradial 149 of 3-deoxy derivative 130 Antifertility activity 979 of aristolochic acid 979 Antifertility effects 462 of Cucumis sativus 461 of cucurbitacins 429,430-435,438, 439-446,447,456

1211 Anti-free radical activity 354 of 3,4-dihydroxyphenylethyl-4formy lmethy 1-1 -4-hexenoate (3,4-DHPEA) 354 Antifungal activity 128,179,185,198, 209,477,482,524 against Aspergillus fumigatus 524 against Candida albicans 524 against Cryptococcus neoformans 524 of azaphilones 481 of(S)-cucurphenol 198 of forronychomycosis 477 ofhyrtiolide 128 of itraconazole 477 of Kalihinane diterpenoids 179, 180 of manzamines 185 ofsaponins 209 ofterbinafine 477 Anti-glycation activity 764 in fructose-BSA assay 764 Antihelmintic effects 1095 of condensed tannins 1095 Antihepathotoxic activity 356 ofiridoids 356 of secoiridoids 356 Antihepatitis drugs 262 used as tonic in chronic diarrhoea 262 use in asthma 262 use in bronchitis 262 use in dropsy 262 use in dry cough 262 use in gonorrhoea 262 use in phthisis 262 Antihepatotoxic effects 463 of cucurbitacins 463 Anti-HIV bioassay-guided fractionation 676 Anti-inflammatory activity 110,123,130, 139,150,152,185,209,252 of cavernolide 139 of cyclointeinone 130 of horrosalaonenes 152 ofiridoids 252 ofmanoalide 123 of manzamines 185 of marine metabolite 110 ofsaponins 209 on ear oedema in mice 150

Anti-inflammatory agent 135,156,442 cacospongionolides 135 cucurbitacin R 442 23,24-dihydrocucurbitacin B 442 indomethacine 156 Anti-inflammatory compound 139 luffolide 139 Anti-inflammatory effects 775 ofTWG 775 Antiinflammatory properties 129,354 ofluffariellin A 129 of luffariellin B 129 ofoleuropein 354 Luffariellin A 129,354 antiinflammatory properties of 129,354 Luffariellin B 129,354 antiinflammatory properties of 129,354 Oleuropein 354 antiinflammatory properties of 129,354 Antileishmanial activity 357,1048 against Leishmania donovani 317 in vitro 357 in vivo 357 ofiridoids 357 of secoiridoids 357 of steroidal lactones 1048 Antimalarial 774 artemisinin as 774 Antimalarial activity 145,178,180,181, 188,195,196,989 in vitro 178,181,989 of 3-alkoxy-l,2-dioxane 195 of chondrillin 196 of halerosellinic acid 145 of heptyl prodigiosin 188 of marine isonitriles 180 ofmuqubilone 196 Antimalarial agent 139 deoxy-diacarnoate B 139 Antimalarial derivatives 179 isonitrile-containing 179 Antimalarial diterpenoids 178 from Cymbastela hooperi 178 Antimalarial drugs 128,170,171 against Plasmodium falciparum 128 chloroquine 170,171 mefloquine 170,171

1212 pyrimethamine 170,171 Antimalarial lead compounds 169 from marine organisms 169 Antimicrobial activity 112,115,116,131, 135,1147 against Sarcina lutea 112 against Staphylococcus aureus 112 of cacospongionolides 135 of hippospongin A 116 of palauolol 131 specific assays for 1147 Antimicrobial agents 1146 search for 1146 Antimutagenic activity 210,211 in mammalian cells 210 ofaflatoxinBl 211 of kalopanaxsaponin A 211 ofsaponins 210 Anti-OVA antibody levels 238 Antioxidant activity 354,705 in lipid peroxidation 705 ofiridoids 354 of secoiridoids 354 Antiparasitic activity 187 against Plasmodium falciparum 187 Antiplasmodial activity 177,179,181, 841,844 against Plasmodium falciparum 841 by preventing heme detoxification 181 of axisothiocyanate-3 177 of isonitrile derivatives 179 Antiproliferative effects 1195 of isoflavones 1195 Antirrhinoside 250 Antispasmodic activity 129 of hippospongin 129 Anti-spermatogenesis actions 7776 ofTWG 776 Antithrombin 143 suvanine sodium salt as 143 Antitrypanosomial activity 1048 of steroidal lactones 1048 Antitrypsin activity 143 of suvanine sodium salt 143 Antitubercular activity 147 of heteronemin 147 Antitumor activity 147,214

in vivo 147 ofsaponins 214 on sarcoma-180-implanted mice 147 Antitumor compound 175,535 743 (ET-743) 175 Antitumor promoting activity 212 ofsaponins 212 Antiviral (HSV and PVl)/cytotoxic activities 119 of furanoterpene acid 119 Antiviral activity 113,138,209,355 against Herpes simplex (HSV) 113 against Herpes simplex virus type-1 138 against Polio vaccine (PV1) viruses 113 against vesicular stomatitis virus 138 in vitro 113,355 ofiridoids 355 ofsaponins 209 of secoiridoids 355 of variabilin 113 Antiviral drug 157 arabinofuranosy ladenine 15 7 Anthraquinones 498-502 Apama 855 Aphanomyces 1053,1059 root rot diseases caused by 1059 Aphanomyces astaci 1054 Aphanomyces cochlioides 1055,1058 zoospores of 1058 Aphanomyces euteiches 1055 Aphanomyces invadans 1054 Aphanomyces westlandii 1002 Aphanomyces zollingeriana 856,868 as analgesic 856 Apocynin 253 effects on neurophil oxidative burst 253 Apoptosis inducing activity 224 ofsaponins 224 Apoptotic effects 226 of saikosaponin D 226 of Bupleururm falcatum 226 Aporphines 810,882-886 from Aristolochia species 883-886 Aquilegiosides C-F 231 from Aquilegia vulgaris 231

1213 Aranochlors 529 inhibitory activities of 529 against microorganisms 529 Aranochor A 528 from Pseudoarachniotus roseus 528 structure of 529 Arbortristoside A 355,356 against Candida albicans 356 semilko forest 355 Arcinol 710 in vivo 710 cancer chemopreventive activity of 710 Ardeemin 571 structure of 571 Argentilactone 969 mass spectral fragmentation of 969 Aridanin 812,816 against Biomphalaria glabrata 816 from Tetrapleura tetraptera 816 molluscicidal activity of 816 structure of 812 AristofolinA 875 Aristofolin B 875 AristofolinC 875 Aristofolin D 875 Aristolactam 875,876-879,961 biosynthetic pathway of 961 from Aristolochia species 876-879 Aristolactam III 974 Aristolactam taliscanine 980 in Parkinson's diseases 980 Aristolactaml-A/-P-glucoside 879 Aristolactones 961 biosynthesis of 961 Aristolanes 912 from Aristolochia longa 912 Aristolic acid 977,978 abortifacient acivity of 978 anti-estrogenic activity of 977 anti-implantation activity of 977 from Aristolochia indica 978 immunomodualting activity of 979 short term toxicity study of 979 Aristolin 872 Aristolochia 1002 chemotaxonomy of 1002

Aristolochia sp. 855,857-859,862 chemical constituents of 855,862 ethanopharmacology of 860 medicinal uses of 857-859 pharmacology of 855,971-992 Aristolochic acids 864-868,869,959,967 antitumor activity of 972 biosynthesis of 959 diterpenoid esters of 868 mass spectral fragmentation of 967 sesquiester of 868,869 sodium salts of 868 sources of 864-868 Aristolochic acid alkyl esters 868,870, 871 from aristolochia species 870,871 Aristolochic acid I 971 pharmacology of 971 total synthesis of 972 Aristolochia albido 986 antifeedant activity of 986 aristolic acid from 986 Aristorochia alcinous 1000 Aristorochia arcuata 888 13 -oxidodibenzo [oc,y] -quinolizinium alkaloids from 888 Aristolochia argentina 856,875 in arthritis 856 in poisoning 856 in pruritus 856 Aristolochia bracteata 856 as antihelmintic 856 Aristolochia chilensis 856 to cure wounds 856 to treat arthritis and diarrhea 856 Aristolochia clematitis 863 in cancer 863 in leg ulcer 863 in menstrual troubles 863 in tumors 863 Aristolochia constricta 982 antispasmodic activity of 982 Aristolochia contoria 1004 Aristolochia curcubitifolia 868,879 Aristolochia cymbifera 1004 Aristolochia debilis 860 inhibitor of iNOS activity 860 Aristolochia euteiches 1059 Aristolochia euteiches zoospores 1063 Aristolochia fangchi root 998

1214 Aristolochia foveolata 868 Aristolochia galeata 1004 Aristolochia gigantea 1003 Aristolochia heterophylla 868 Aristolochia kaempferi 868 Aristolochia longa 975 against 1-388 lymphocytic leukaemia 975 Aristolochia manshuriensis 1002 Aristolochia mollissima 859 anticancer activity of 859 antimalarial activity of 859 as anti-inflammatory agent 859 as analgesic 859 in abdominal pain 860 in rheumatism 860 in stomach ache 860 Aristolochia ochraceus 570 Aristolochia onoei 1003 Aristolochia papillaris 981 smooth muscle relaxant activity of 981 Aristolochia paucinervis 860 bacteriostatic activity of 860 in skin and soft-tissue infections 860 Aphanomyces raphani 105 5,1059 Aristolochia ringens 1004 Aristolochia rotunda 1003 Aristolochia shimadai 1002 Aristolochia triangularis 860 in skin diseases 860 in wounds 860 uses of 860 Aristophyllide A 872 Aristophyllide B 872 Aristophyllide C 872 Aromadendranes 917 from Aristolochia chilensis 917 Aromatase 1198 Aromatic compounds 515-521 structures of 516-521 Aromatic ring-A withanolides 1032 Aromatic ring-D withanolides 1034-103 8 Arteflene 192 structure of 192 Artemether 189 structure of 189 Artemisia annua 171,189 antimalarial activity of 189 in fever 189

in folk Chinese medicine 171 Artemisinin 189,190 mechanism of action of 190 structure of 189 Artesunate 189 structure of 189 Asalina bioassay 117 activity of cacospongionolide D in 117 Asari herba 996 Asarum 855 Ascochlorin analogs 534 cytotoxic activities of 534 Asmolactones 352 formation of 352 Aspergilloxide 156 from genus Aspergillus 156 Aspergillus amstelodami 572 Aspergillus rubber 573 Aspergillus ustus 481,570 Asperparalines 594-604 bioactivity of 596 biosynthesis study of 596-598 structure of 595-596 synthetic study of 596-600 Aspochracin 550 from Streptomyces sp. 550 Astragalosie I 230 Astragalus sponins 231 Astrophaneura alcinous 999 Aswagandha 1020 from Withania somnifera 1020 medicinal properties of 1020 Atheroma 1200 Atherosclerosis 136,705 role of oxygen species in 705 Aucubajaponica 370 Aucubigenin 386 Aucubin 249,250,279,383,386,387 as antidote 279 as chemopreventive 383 astringent property of 279 effects on collagen synthesis 387 in dysentery 279 in vivo assay 386 liver protective effects of 279 stimulant effects of 279 Auriculatin 837 antimicrobial activity of 837 from Millettia auriculata 837 Aurones 1178

1215 Austocystins 499 from Aspergillus ustus 499 structure of 499 Autoimmune diseases 1200,1150 therapies for 1150 Auxarconjugatins A and B 529 from Auxarthron conjugation 529 structures of 530 Avicins 213 effects on chemically induced mouse skin carcinogenesis 213 Avicins D 213,226 apoptosis inducing activity of 226 Axinella verrucosa 578 Axinella cannabina 176,177 axisonitrile-1 from 176 axisothiocyanate-1 from 176 isonitrile terpenoids from 177 Axisonitrile-3 177 from Acanthella klethra 111 Azaphilone 485,487 from Penicillium sclerotiorum 485 inhibitory activities of 487 structure of 486,487 Azaphilone metabolites 482 production of 482 Azaphilones 481 antibacterial activity of 482 antifungal activity of 482 l-Azaspiro[4.5]decan-8-ones 15 Sorensen and Ciufolini synthesis of 15 Azaspirodecanediones 17 from ./V-methoxyphenylamides 17 synthesis of 17 Azatricyclic compound 34 Wardrop's alternative route to 34 Bacillus subtilis 123,125,1087 activity to oomycete zoospores 1087 Badrinal 256 Battus philenor 1000 Bee venom PLA2 124,143 against Escherichia coli 125 cytotoxic activity against 143 dehydromanoalide effects on 124 inhibitor of 143 Benicasa 430

Benzenoids 945,949,950,952 from Aristolochia species 945 Benzophenone synthase 722 from Centaurium erythraea 722 Benzophenones 721-725,748-761 biological activities of 721 from Clusia 722 from Garcinia 722 from Hypericum 722 isolation of 721,724,748-752 sources of 725-746 structures of 721,725-746 synthesis of 722-724 Benzylisoquinolines 889,890 from Aristolochia species 889 structures of 890 Benzyltetrahydroisoquinolines 962 Berkeleydione 1167 isolation of 1167 structure of 1167 Berkelytrione 1167,1168 formula of 1168 isolation of 1167 Beticolins 1 500 structure of 500 Beticolins 2 500 structure of 500 Beticolins 3 500 structure of 500 Beticolins 4 500 structure of 500 Beticolins 6 500 structure of 500 Beticolins 8 500 structure of 500 Bicarbocyclic sesterpenoids 131 antimicrobial activity of 131 antiproliferative activity of 131 cytotoxic activity of 131 sources of 131 Bicyclo-(3.3.1)-nonane derivatives 693 conformers in 693 tautomers in 693 Bicyclo-[3.3.1 ]-nonane derivatives 682-697 sources of 683 structures of 684 Bicyclogermacranes 906 from Aristolochia elegans 906 BidwillolA 849 structure of 849

1216 Bioactive cinnamate esters 849 from Erythrina 849 Bioactive cinnamoylphenols 833 from Erythrina 833 Bioactive compounds 825 anticancer activity of 825 anti-inflammatory activity of 825 antimicrobial activity of 825 DNA-repair properties of 825 muscle relaxation properties of 825 Bioactive coumastans 849 from Erythrina 849 Bioactive daidzein derivatives 838 from erythrina genus 838 Bioactive flavanones 826,827 naringenin derivatives 827 Bioactive flavonoids 825 from Erythrina species 825 Bioactive isoflav-3-enes 849 from Erythrina species 849 Bioactive isoflavanones 846,847 activity of 847 sources of 846,847 Bioactive isoflavone derivatives 836 activity of 836 from Erythrina species 836 Bioactive isoflavones 839 activity of 839 from Erythrina species 839 Bioactive marine secondary metabolites 158 by aquaculture 158 by cell culture 158,159 by chemical synthesis 158 by cultivation of marine organisms 158,159 Bioactive metabolites 74,95,97,100, 1134,1143 flaccidoxides 95 from deep-sea sponges 1134 from microorganisms 1143 hamiltonins 100 latrunculins 100 nakafurans 97 pyrroloiminoquinones 74 sarcoglane 95 search for 1143 spongiane diterpenes 100 variabilins 97 Bioactive pterocarpans 840

as antiplasmodial compounds 840 as antibacterial compounds 840 as antimicrobal compounds 840 as phospholipase A3 inhibitors 840 from Erythrina species 840 Bioactive saponins 209 cancer related 209 developments in 209 immunomodulatory activity of 209 Bioactive secondary metabolies 1133 extremophiles source of 1133 Bioactive stilbenoids 831 from Erythrina 831 Bioactivemetabolites 81 halichlorensin 81 halitulin 81 Bioactivity 429,591,596 of asperparalines 594-604 of brasiliamides 584-594 of cucurbitacins 429,430-435,438, 439-446,447,456 of dihydrobrasiliamide B 588, 591-592 Bioactivity isoflavans 848 sources of 848 Biosynthesis 251,411,434-435,475, 591-592,958,959,961 of aristolactones 961 of aristolochic acids 864-868, 869,958, 959,967 of brasiliamides 584-594 of dihydrobrasiliamide B 588, 591-592 of griseoflavin 471-479 of illicinolides 411 ofiridoids 248,251,252,291, 305-333,340,352,353,365,381 Biosynthetic origin 250 ofiridoid 250 Biphenyl ether 948 structure of 948 Bisabolanes 903 from Aristolochia acutifolia 903 Bisbenzyltetrahydroisoquinolines 963 biotransformation pathway from 963 Bishomosesterterpenoids 153 from Phyllospongia foliascens 153

1217 Bisindole 569 Sw-pyrroloiminoquinone alkaloids 74 tsitsikammammine A and B 74 Bit is artetans 984 Bobolstemma paniculatum 212 Bonjoch's synthesis 37-40 of ketone enolates 37-40 via palladium-catalyzation of aminotethered vinyl bromides 37-40 Bothrops atrox 860 as insect repellent 860 hermorrhagic effect of 860 Boucheafluminensis 381 as regulator of digestive function 381 anti-inflammatory activity of 381 Bourbonanes 921 from Aristolochia gibertii 921 Bovine endothelial GM7373 140 Brasiliamides 584-594 bioactivity of 591 biosynthesis of 591-592 conformational analysis of 589 discovery of 584 related compound 593 Brasiliamide A 586 structure of 585 Brasiliamide B 586 structure of 586 Brasilianum Batista JV-379 585 convulsive acivity of 585 Brevianamide E 571 structure of 571 Bridgehead azabicyclic iminium ions 35-39 Kibayashi's approach from 35-39 Brine shrimp lethality assay 95 6-Bromoaplysisnopsin 188 structure of 188 5-Bromoochrephilone 486 from Penicillium multicolor 486 6-Bromopenitrem E 582 'H-NMR spectrum of 583 structure of 582 Bronianone 672 structure of 672 Brummond's synthesis 28-32 ofFR.901483 28-32 via tandem cationic aza-Cope

rearrangement-Mannich cyclization reaction 28-32 Bryonia 430 Bursting activities 1114 homogenous solution method for 1114 Burttinone 827 as anticancer agent 827 Butylidencphthalide 638 as anti-angina 638 as anti-platelet / anti-thrombosis 638 in cardiac function modulation 638 in inhibition of learning and memory impairment 638 in sedation and sleep enhancement 638 in smooth muscle relaxation 638 Butyliphthalide 638,648 against cerebral injuries 648 against cerebral ischemia 638 in blood viscosity reduction 638 protective effects of 648 37?-Butyphthalide 638 as anti-convulsant 638 3S-Butyphthalide 638 as anti-convulsant 638 in inhibition of learning and memory impairment 638 protection against cerebral ischemia 638

Ca2+influx-efflux 1097 blockade of 1097 in zoospores 1097 Cacospongia 139 Cacospongia linteiformis 130,140,144 antifeedant activity of 140 cyclolinteinone from 130 lintenone from 144 Cacospongia scalaris 113,146 ircinin from 113 scalarin from 146 Cacospongionolides 135 effects on panel of secretory PLA2 135

1218 Cadinanes 914 from Aristolochia heterophylla 914 Cancer 134,370,371,705,863 Aristolochia clematitis in 863 Catalpa ovata against 370 drugs against 134 Kigelia pinnata in 371 oxygen species role in 705 Cancer cell lines 66,82,94,111,155 cephalostatins against 64-66 eleutherobin against 94 halitulin against 82 idiadione against 111 neomangicols A and B against 155 Cancer cell proliferation 136 Cancer chemopreventive activity 713-716 of polyisoprenylated benzophenone 713-716 Candida albicans 136,198,356,704 arbortristoside A against 355,356 (S)-cucurphenol against 198 in Candida infections 704 sulfircin activity against 136 Candida gilchristi 65 Candida hamiltoni 101 spongiane diterpene lactones from 101 Candida lucanusianus 805 Candida maritimaum 273 Candida nemorosa 680 Candida ovata 371 Candida pulchellum 273 Candida quintense 273 Candida scalaris 147,150 deacetylscalaradial from 150 Candida spicatum 273 Candida tenuiflorum 273 Cantauhum erythrea 272 as stomachic 272 in blood purification 272 in jaundice 272 in sores 272 in wounds 272 Capillary zone electrophoresis 995 Cardanol 1084 isolation of 1084 Cardol 1084 isolation of 1084

Carnosoflogein A 458 from Hems ley a carnosiflora 458 Carrageenan-induced inflammation 775 in vivo 775 Carvacrol 810,815 structure of 810 Caryophyllaceae saponins 224 Caryophyllanes 910 from Aristolochia argentina 910 Caspase-1 1155 Catalpa ovata 370 against inflammation 370 against cancer 370 Catalpol 249,386 Catechins 1178 Catenaria anguillulae 1057 Catnip tea 289 in chicken pox 289 in colic fevers 289 in headache 289 in insomnia 289 in measles 289 in nervousness 289 Cavernosolide 133 structure of 133 Cayaponsides B 451 strucure of 451 Celastrol 795 antioxidant activity of 795 anti-inflammatory activity of 795 Celastrol type compounds 782 immunosuppressive activity of 782 anti-inflammatory activity of 782 Celosia cristata 1096 Centaurium erythrea 269,273 for languid digestion with heart burn 273 for muscular rheumatism 273 Cephalostatins 64-66 against cancer cell lines 66 as cell growth inhibitors 64 cytostatic properties of 66 from Cephalodiscus gilchristi 65 from Ritterela tokioka 65 trisdecacyclic pyrazine structure of 65 Cephalostatin 1 65 structure of 65 Cephalostatin 2 65 structure of 65

1219 Cephalostatin 3 65 structure of 65 Cephalostatin 4 65 structure of 65 Cepholostatins 5 66 structure of 66 Cephalostatin 6 65 structure of 65 Cephalostatin 7 67 structure of 67 Cephalostatin 8 67 structure of 67 Cephalostatin 9 67 structure of 67 Cephalostatin 10 68 structure of 68 Cephalostatin 11 68 structure of 68 Cephalostatin 12 68 structure of 68 Cephalostatin 13 68 structure of 68 Cephalostatin 14 68 in vitro 68 Cephalostatin 15 68 in vitro 68 Cephalostatin 16 69 structure of 69 Cephalostatin 17 69 structure of 69 Cepharadione A 980 antimicrobial activity of 980 CepharanoneB 989 antimalarial activity of 989 in vitro 989 Chaetoviridines A 491 as monoaminooxidase inhibitor 491 effect on growth of Penicillium oryzae 491 inhibitory effect on monoaminooxidase 491 structure of 491 Chaetoviridine B 491 structure of 491 Chaetoviridine C 491 structure of 491 Chaetoviridine D 491 structure of 491

Chaga's disease 988 eupomatenoid-1 988 hearinAin 988 Chalcones 830 activity of 830 from Erythrina sp. 830 Chamigranes 919 from Aristolochia debilis 919 Chamone I 765 Chemical components 791 bioactivity of 791 from TW plant 791 Chemoreceptors 1071 of flagellar membrane 1071 Chemotaxis 1065 Chemotropism 1070 of hyphal germlings 1070 Chicken ovalbumin (OVA) 239 in mice 239 Chinese hamster ovary (CHO) cells 210 DNA damage in 210 Chinese-herbs nephropathy 990-993 Chlorflavonin 523 structure of 523 Chlorinated anthraquinones 501 structures of 501 Chlorinated benzophenone antibiotic 538 pestalone 538 Chlorine containing polyketides 527 chlorocarolide A and B 527 Chloroform extract 442 anti-inflammatory activity of 442 from Cayaponia tayuya 442 Chlorofusin 493 structure of 493 Chloroorselinic acid A 489 from Emehcella falconensis 489 structure of 4889 Chloroorselinic acid B 489 from Emericella falconensis 489 structure of 489 Chloroorselinic acid C 489 from Emericella falconensis 489 structure of 489 Chlorophylls 893,895 structures of 895 Chlovalicin 530 structure of 530 Chondrillin 197 structure of 197 Chramaloside A 217

1220 Chromatographic techniques 1088 Chromeno-coumarin calanolide A 702 from Calophyllum langiferum var. Austrocoriaceum 702 inhibitor of HIV-1 reverse transcriptase 702 Chromodoris inornata 143 Chronic inflammatory conditions 134 Chronic inflammatory disorders 134 drugs against 134 Ciclopentane derivatives 323 origin of 323 structures of 323 Ciona intestinalis 85 C/s-jasminoside 350 Citrullus colocyhthis 431 as purgative 431 Ciufolini synthesis 22-25 via oxidative cyclization 22-25 of oxazolinetethered phenol 22-25 Classical hormone replacement therapy 1194 (-)-Claussequinon 1082,1083 13 C-NMR data of 1082 structure of 1083 Clavelina lepadiformis 85,185 Cnidilide 638 in blood viscosity reduction 638 in inhibition of learning and memory impairment 638 Cochliobolus lunata 481 Cochliophilin A 1056,1061 from Chenopodium album 1061 from Aphanomyces cochlioides zoospores 1061 structure of 1056 Colon adenocarcinoma 975 Columbin 984 anti-snake venom activity of 984 Communesins 574-579 biosynthetic pathway of 576,577 discovery of 574 identification of 574 insecticidal activity of 576 molecular formula of 574,575 related compounds 577-579 structures of 574,575 Conopharyngine 809 structure of 809 Convulsive activity 550 against silkworms 550

Convulsive compounds 579-593 Coscinoderma mathewsi 143 suvanine from 143 Cacospongionolide B 132 from Fasciospongia cavernosa 132,142 Cacospongionolides E inhibition 135 of human synovial PLA2 135 Cytoxicity 134 of cacospongionolide B 134 Coumarin 521-526,813,817 from Tabernanthe tetraptera 817 Coumestrol 1191 affinity for estrogen receptor 1191 COX-2 expression 139 cavernolide effects on 139 COX-2 inhibitors 447 cucurbitacins as 447 Cuanxiong 654 analgesic actions of 654 for asthma 654 for stroke 654 Cucumis sativus 461 antifertility effects of 462 antimicrobial properties of 463 toxicity of 462 Cucurbita ficifolia 432-434 chemistry of 432-434 hypoglycemic effects of 432 Cucurbitacin 7?-diglucoside 456 against stress-induced alterations 456 Cucurbitacin-glycosides 452 structure of 452 Cucurbitacin glyscosides 254 structure of 254 Cucurbitacins 429,430-435,437,438, 439-446,447,456,462 agonist activity of 459 analytical separation of 435,437 anticancer effects of 447 antifertility effects of 462 as adaptogens 456 as analgesic 446 as anti-inflammatory agents 439-446 as ecdysteroid antagonists 431 bioactivity of 429 biological property of 439 biosynthesis of 434-435 chemistry of 432-434

1221 cytotoxic effects of 447 detection of 435 effects on immune system 456 effects on insects 457 effects on plant parasites 457 extraction of 436 from protostane 435 gibberellin-antagonistic activity of 430 identification of 435 isolation of 435 occurrence of 429 pharmacological activity of 431 purification of 437 structural elucidation of 438 toxicity of 462 Cucurbitacins E 447 from Conobea scoparoides 448 Cucurbitacins WG, 430 from Wilbrandia 430 Cucurbitacins WG2 430 biological significance of 430 gibberellin-antagonistic activity of 430 from Wilbrandia 430 Cucurbitane 433 structure of 433 (S)-Cucurphenol 198 against Candida albicans 198 antifungal activity of 198 Cudraphenones A-D 711 against HGF 711 against HSC-2 711 against HSC-2 cells 711 Curcurbitacins B 440 structure of 440 Curcurbitacins E 440 structure of 440 Cyclic peroxides 81 from Plakortisaff simplex 81 structures of 81 Cyclization 566,723 Cycloartane type triterpene saponins 231 from Astragalus peregrinus 231 Cyclochlorotine 502 from Penicillium islandicum 502 Cycloechinulin 570 structure of 570 Cycloparvifloralone-type sesquiterpenes 407-409 structures of 407-409

Cycloperoxide-containing antimalarial agents 191 Cycloperoxides 189 CyclosporinA 232 Cyclotryprostatin A 581 structure of 581 Cyclotryprostatin B 581 structure of 581 Cyclowithanolides 1023 Cymbastela hooperi 180 marine isonitriles from 180 Cystodytes delechiajei 85 Cystospores 1068,1069 germination of 1068,1069 Cytosolic PLA2 113 variabilin inhibitor of 113 Cytotoxicity 121,143 against human leukaemia MOLT4 cells 121 against human myeloid K562 cells 121 in Artemia salina bioassay 1443 of rhopaloic acid A 121 Cytotoxicity activities 115,120,125,131, 136,138,145 against central nervous system carcinoma XF49 8 116 against human tumor cell lines 131 against human tumor cell lines 138 against NSLC-N6 cells 145 against ovarian carcinoma SKOV-3 116 against skin carcinoma SK-MEL-2 116 in Artemia salina bioassay 120 of 1,2-dioxanes 138 of hipposulfates B 136 ofircinin-2 116 ofluffariolides 125 of mycaperoxides A/B 138 of petrosaspongiolide L 145 ofsarcotinG 116 ofsarcotinH 116 on KB cells 145 Cytotoxic depsipeptides 82 geodiamoli de TA 82 hemiasterlin de TA 82 jaspamide de TA 82

1222 Daidzein 1056,1183 structure of 1056,1183 Dalbergia odorifera 1082 isoflavonoidal from 1082 repellent activity of 1082 structure elucidation of 1082 Danggui 613,654 in treatment of female irregular menstruation 613 use in pain 654 De novo DNA synthesis 231 De novo purine nucleotide biosynthetic pathway 4 2-Deacetoxy-21 -acetoxy scalarin 148 cytotoxic activity of 148 from Japanese H. erecta 148 12-O-Deacetyl-12-ep/-scalarin 146 12-Deacetylhyrtial 151 from H. erecta 151 Deacylsaponins 236 Debilicacid 868 from Aristolochia debilis 868 from Aristolochia longa 868 Debenzylation 11 of diphosphate ester 11 Decarboxylation 959 Decatromicins A 506 against Staphylococcus aureus 506 structure of 506 Decatromicins B 506 structure of 506 Deglucouronidation 1187 of phenolic estrogen conjugates 1187 Dehydration 936 6,7-Dehydrofevucordin A 434-435 biosynthesis of 434-435 structure of 434 (-)-Dehydrogriseofulvin 475 microbial transformation of 475 reduction of 475 Dehydrogriseofulvin 479 from Peltaspermum martinsii 479 Dehydro-luffariellolide diacid 127 structure of 127 Dehydrooxoperezinone 980 from Aristolochia manshuriensis 980 Deinococcus radiodurans 1130

(+)-DemethylaminoFR901483 32-35 Wardrop's formal synthesis of 32-55 Denitroaristolochic acids 873-875 from Aristolochia species 873-875 Densitometric method 996 25-Deoxycacospongionolide B 133 5-Deoxyisoflavones 838 daidzein precursor for 838 Deoxyloganic acid 340 esterification of 340 oxidation of 340 22-Deoxy-variabilin 114 against Bacillus subtilis 114 against Candida albicans 114 from Thorecta sp. 114 Depsides 507-510 structures of 508-510 Depsidones 507-510 Depurative 288 Lamium labiatae as 288 Desaturation 566 DesmethylaminoFR901483 32-35 formal synthesis of 32-35 (±)-Desmethylamino FR901483 9-12,33 synthesis of 9-12,33 Detection 993-999 of aristolochic acids 993-999 Deutzioside 249 Diacamus levii 128 e«Mnuqubilin from 128 Diastereoselective spirocyclization 18 TV-acylnitrenium ion-promoted 18 Dibenzylphosphate 21 hydrogenolysis of 21 Didemmaones B 73 from ectynonanchora flabellate 73 Didemnum listerianum 85 Didemnum sp. 185 20,24-Dihomoscalaranes 154 against KB cells 154 Dihydrobrasiliamide B 588,591-592 bioactivity of 591 biosynthesis of 591-592 (-)-Dihydrocubebin 943 from Aristolochia pubescens 943 Dihydrooxyresveratrol 831 structure of 831 Dihydroplakortin 193 from Plasm odium falciparum 193 structure of 193

1223 Dilemmaones A-C 73 from Cram be chelastra 73 from Ectynonanchoraflabellate 73 Dimeric secoiridoid glucosides 317 2,2-Dimethyl-l,3-dioxan-5-one 26 condensation of 26 7,12-Dimethylbenz[a]anthracene 212 1,2-Dioleoylphosphatidic acid 1079 Dioscin 225 from Polygonatum zanlanscianenens 225 Dioxin 26 retro Diels-Alder cycloaddition of 26 Diphosphate ester 11 debenzylation of 11 Disidein 156 from Dysdidea pallescens 156 stereochemistry of 156 Diterpenoids 777-781,811,927-937 from Aristolochia species 928-935 fromTW 777-781 structures of 780 D-limonene 1110 mycelial growth inhibition by 1110 DNA topoisomerase 1 1198 DNA-damaging agent 188 lissoclinotoxin A 188 £>-Pinitol 1001 defensive role of 1001 against herbivores 1001 Dragmacidon 593 structure of 593 Dragmacidon A 593 structure of 593 Dragmacidon B 593 structure of 593 Drugs 1144 industrial production of 1144 Dunalia brachyacantha 1048 Dysentery 279 aucubin in 249,250,279,383,386, 387 Dysidea avara 158 against skin disorder 158 Dysidea sp. 114 against protein phosphatase enzyme 114 isopalinurin from 114

Dysideapalaunic acid 137 effects on aldose reductase 137 stereochemistry of 137 Ecdysteroid antagonists 459 structures of 459 ECE inhibition 77 of Pachastrella extract 77 Elemanes 904 from Agrostophyllum brevipes 904 Eleutherobin 94 against cancer cell lines 94 Eleuthosides 92-94 from Eleutherobia aurea 92-94 structures of 92-94 Emericellafalconensis 481 Encystment activity 1113 of zoospores 113 Enterohepatic cycle 1188 £«/-kurospongin 120 cytotoxicity of 120 Enzymatic inhibitors 1197 Enzymatic reduction 137 of glucose 137 EPA-Superfund site 1139 bioprospecting in 1139 £f>/-acetylscalarolide 147 cytotoxic activity of 147 Epikingiside 342 biosynthetic pathway of 342 £p/-kingiside derivatives 329 origin of 329 structures of 329 Epikingiside derivatives 344 routes to 344 7-Epinemorosone 689 structure of 689 12-ep;-Scalarin 146 Epoxidation 576 28,29-Epoxyplukenetione A 699 structure of 699 2,3-Epoxysqualene 434 formation of 434 from squalene 434 Epstien-Barr virus early antigen (EBVEA) 451 inhibitory effects on 451

1224 Erdin 479 from Aspergillus terreus 479 from Penicillium sp. 479 Erycristagallin 842 anti-inflammatory activity of 842 Eryepogin F 832 as anti-methicillin 832 Erypostyrene 833,834 against 13 MRSA strains 833 anti-candidal activity of 833 antimicrobial activity of 833 anti-MRSA activity of 833 inhibitory effect of 834 structure of 833 Erythrina abyssinica 822 against malaria 822 against syphilis 822 Erythrina americana 822 in insomnia 822 Erythrina fusca 822 in fever 822 in malaria 822 Erythrina glauca 842 anti-HIV activity of 842 Erythrina indica 822,835 against human KB cells 835 in fever 822 in malaria 822 in vitro 835 Erythrina pterocarpans 842 antibiotic activity of 842 Erythrina species 822-824 as narcrotic 822 as purgative 822 ethnomedical application of 823-824 role in thrombosis 822 Erythrina variegata 827 against inflammation 827 Eryvariestyrene 833,834 against Staphylococcus aureus 833 against Salmonella gallinarium 833 from Erythrina variegata 833 inhibitory effect of 834 structure of 833 Escherichia coli 125 Bee venom PLA2 against 124 Esterification 348 with iridane 348

Estradiol 1182 structure of 1182 Estrogen deficiency 1195 symptoms of 1195 Estrogenic 1202 soy-based products 1202 Estrogenic activity 1190,1191 offlavonoids 1190 Etherification 712 9-Ethoxy aristolactam 882 from Aristolochia mollissima 882 9-Ethoxyaristolactone 895 Eudesmanes 911 from Aristolochia acutifolia 911 Euglena mutabilis 1141 Euglypha 855 Excdeconolides A 1026 from Exodecosus maritimus 1026 Exodeconolides B 1026 from Exodecosus maritimus 1026 Exodeconolides C 1026 from Exodecosus maritimus 1026 Exremophilic microorganisms 1127 Extremophiles 1130 from inland environments 1130 Extremophilic microbes 1128 of yellowstone national park 1128 Extremophilic organisms 1125 Extremozymes 1131 source of 1131 Falconensine E 490 from Emericella falconensis 490 structure of 490 Falconensine K 490 from Emericella falconensis 490 structure of 490 Falconensine L 490 from Emericella falconensis 490 structure of 490 Falconensine M 490 from Emericella falconensis 490 structure of 490 Farnesanes 903 from A. argentina 903 Fascaplysinopis reticulate 127 iso-dehydroluffariellolide from 127 Fasciospongia 1139

1225 Fasciospongia cavernosa 132,139,142 cacospongionolide B from 132 cavemosolide from 142 F a s c i o s p o n g i a sp. I l l Feeding activity 1045 against Tribolium castaneum 1045 of jaborosalactone P 1045 FellutanineD 571 structure of 571 t-Ferulloyl 254 structure of 254 Fever 374,381,822 Erythrina fusca in 822 Erythrina indica in 822,835 Gardenia jasminoides in 374 Logotis brevituba in 381 Fevillea cordifolia 438 cucurbitacins from 439 Fibrosing interstitial nephropathy 991 Flaccidoxides 95,96 structure of 95,96 Flavones 813,817,1179 structure of 1179 Flavonoids 671,944,945,947,1062,1178 from Aristolochia species 945,946 repellent activity of 1062 structures of 947 Fluorescence microscopy 1094 Fluroindolocarbazoles 498 structure of 498 Foliaspongin 153 antiinflammatory activity of 153 Fontanesia secoiridoids 341 biosynthetic pathway to 341 Fontoanesia 340,345 secoiridoids from 340 Formamide-containing sesquiterpenoids 176 axamide-1 176 axamide-2 176 axamide-3 176 axisothiocyanate-3 176 Formononetin 1185 sources of 1185 Forsythia iridoids 340 biosynthetic pathway to 340 (-)-FR901483 8,12-15,21,24 Ciufolini's synthesis of 24 Snider's synthesis of 14

Sorensens synthesis of 21 synthesis of 12-15 total synthesis of 8 (±)-FR901483 25 via amidoacrolein cycloaddition 25 FR901483 3,4,6,7,8-15,19,22,25,29,31, 35-40 biosynthesis of 4 Bonjoch's synthesis of 37-40 Brummond's formal synthesis of 31 Brummond's retrosynthetic analysis of 29 Ciufolini synthesis of 15,22 construction of 6 framework of 6 from Cladobotrym sp. 3 Funk's retrosynthetic analysis of 25 immunosuppressive activity of 3 in vitro 3 Kibayashi's synthesis of 35-39 Snider's synthesis of 8-15 Sorensen synthesis of 15 structure of 3 FR901483 skeleton 32 synthetic approaches to 32 FR901483 synthesis 5-8 main features of 6 Fraxinus excelsior 342 Fromonoetin 1083 structure of 1083 biological activity of 1083 Fumitremorgin B 580 structure of 580 Fungi 471,481,483 Halogen containing compounds from 471,481 isochromophilones II against 483 Fungal antibiotics 518 from Pterula species 518 Fungal metabolite 512 from Fusarium sp. 512 Fungal origin 549 of bioactive alkaloids 549 Fungicides 1107 Funk's synthesis 25-28 via amidoacrolein cycloaddition

1226 25 of(±)-FR901483 25-28 Furnish diketopiperazine 569 cyclization to 569 Furospinosulin-1 110 from Ircinia spinosula 110 Fusarium heterosporum 155 neomangicols A-C from 155 Galium aparine 290,291 as antiscorbutic 291 as apeient 291 as diuretic 291 as refrigerant 291 iridoid glycosides from 291 Galium atuntsiensis 269 Galium cambogia 765 against gastric ulcers 765 Galium campestris 270 Galium depressa 270 Galium gelida 270 Galium hombroniana 672 Galium karroa 266,267 in improving appetite 267 in leucoderma yunani 267 in stimulating gastric secretion 267 in syphilis 267 Galium kurroa 270 Galium linearis 268,270 iridoids from 268 Galium lufea 265,266,270 against snake bites 266 amarogentin from 265 as antidote 266 as antirabies agent 266 gentianine from 265 gentiopicroside from 265 in dyspepsia 266 in gastric inflammation 266 in hepatic/gall bladder disease 266 in liver disorders 266 in stomach ailments 266 sweroside from 265 swertiamarin from 265 Galium macrophylla 270 Galium manshurica 269 Galium punctata 270 Galium purpurea 270

Galium pyrenaica 270 Galium rigescens 269,270 Galium rubiaceae 290 Galium septemfida 270 Galium tessmanii 804 Galium tibetica 267,270 in bacterial infections 267 in constipation 267 in hepatitis 267 secoiridoid glycosides from 267 Gallium triflora 269 Galtonia 220 fom Candicans geltonia 220 Gangliosides GM2 235 Garcinol 704-708,764 anti-MRSA activity of 704 antioxidant mechanism of 708 antioxidative activity of 705 chelating activity of 706 in Fenton reaction system 706 radical scavenging activity of 705 Garciosaphenone A 705 anti-HIV activity of 705 antimicrobial activity of 705 Gardeniajasminoides 374 in inflammation 374 in hypertension 374 in fever 374 Gardenoside 250,386 against Alzheimer's disease 386 Gastric inflammation 266 Galium lufea in 265,266,270 Gastric ulcer 707 oxygen species role in 707 ge/w-Methyls 689 13 C-chemical shifts of 689 Genipine 384,386 against Alzheimer's disease 386 chemopreventive activity of 384 effects on glutathion-transferase 386 hepatotoxic activity of 386 Geniposide 384,386,387 anti-asthamatic property of 387 antioxidant activity of 384 antitumoral property of 384 chemopreventive property of 384 effects on collagen synthesis 387 effects on mieloperoxidase (MPO) 384

1227 Genistein 834,1056,1182 derivatives of 834 structure of 1056,1182 Gentian 270 iridoids from 270 secoiridoid glycosides from 270 Gentiana chyrayta 261 Gentianae radix 265 used in folk medicine 265 Gentiopicrin 261 Gentiopicroside 248,261,262,266 anti-inflammatory activity of 266 fungicidal activity of 266 Genus didiscus 198 Genus erythrina 821,822,825 bioactive non-alkaloidal constituents from 821 bioactive compounds from 825 in female infertility 822 in gonorrhoea 822 in stomach pain 822 pharmacological information of 822 Genus Illicium 395 Genus iridomirmex 247 Genus Sarcotragus 119 Genus Spongia 139 Geodiamolide 83 against P-388 cancer cells 83 structure of 83 Geodin 479 from Penicillium sp. 479 Geranylfarnesol 110 from cochliobolus 110 Germination 1070 hyphal growth after 1070 Germination inhibiting activity 988 of(-)-(3-bisabolene 988 Giant redwoods 1124 endosymbionts of 1124 Gibberellin-antagonistic activity 430 of cucurbitacins 429,430-435,438, 439-446,447,456 Gillusdin 479 from Aspergillus terreus 479 Gilmaniella humicola 531 Gilmaniellin 532 from Gilmaniella humicola 532 Ginkgo fruits 1084 motility inhibitory factor from

1084 zoospore lytic factors from 1084 Ginkgo metabolites 1087 motility inhibitory/zoospore lytic properties of 1087 Ginsenoside Rg3 224 vinblastine efflux inhibition 224 Ginsenosides Rbl 232 Gintiana lutea 265 uses in folk medicine 265 Gisan-depsidone biosynthetic pathway 510 Glaziovine 882 from Aristolochia chilensis 882 Gleditsioside E 227 against MCF-7 cell lines 227 against HL-60 cell lines 227 Glucanase inhibitor proteins (GIPs) 1106 Gluconeogenesis inhibitor 540 Glucosidase-catalysed hydrolysis 350 Glucosylation 1184 (3-Glucuronidase inhibitors 817 8-hydroxytricetin 7-glucuronide 817 isovitexin 817 Glutinol 816 analgesic activity of 816 anti-inflammatory effects of 816 Glycosides 226 apoptosis inducing activity of 226 Glycyrrhizin 230 antitumor properties of 230 characterization of 1071 effects on macrophage-derived NO production 230 in signal transduction pathways 1072 structure of 230 Gratiola officinalis 430,432-435 Griseoflavin 471-479 anti-inflammatory properties of 477 as fungistatic 477 biosynthesis of 475 biological activity of 476 chlorine-containing antibiotic 471 determination of 476 fermentation conditions of 471,472 for onychomycosis 477

1228 for treatment of dermatophytoses 477 from Penicillium griseofulvum 471 from Penicillium janczewskii 474 from microorganisms 472 from Penicillium urticae 474 isolation of 471,475 large-scale prodution of 473 purification of 475 side effects of 478 spatial arrangement of 478 to Treat tinea capitis 477 used in dermatophyte onychomycosis 477 vasodilatory effects of 477 Gonorrhoea 262,822 antihepatitis drugs in 262 Genus Erythrina in 821,822,825 Guaianes 916 from Aristolochia linkiuensis 916 Guibourtia tessmanii 804 Guttiferones A-E 702,712 cytopathic effects of 702 in A2780 human ovarian cell 712 in vitro 702 Gypenosides 226 apoptosis inducing activity of 226 Gypsetin 571 structure of 571 Halichondria sp. 131 palauolide from 131 Haliclona tulearensis 81 Haliotis rufescens 147 Halistanol disulfate B 76,77 effect on ECE 77 structure of 77 Halisulfates 9 136 Halisulfates 2 131 antimicrobial activity of 131 from Halichondria sp. 131 Halitulin 82 against cancer cell lines 82 cytoxicity of 82 structure of 82 Halogen containing compounds 471,481 from fungi 471,481 Halophiles 1127 salty environment for 1127

Hamiltonins A-D 100 from Chromodoris hamiltoni 100 structures of 100 Hansenula anomala (Pichia anomala) 1159 Harpagoside 249 Harpagophytum erecta 151 Hyrtialfrom 151 Harpagophytum procubens 374 a-Hederin 223 HeLa cells 225 proliferation of 225 HelicusinA 488 from Talaromyces helicus 488 HelicusinB 488 from Talaromyces helicus 488 HelicusinC 488 from Talaromyces helicus 488 HelicusinD 488 from Talaromyces helicus 488 Hemiasterline 83 against P-388 cancer cells 83 structure of 83 Hepatitis 267,381 Galium tibetica in 267,270 Logotis brevituba in 381 Hepatoprotective activity 262 of gentiopicroside 262 ofsweroside 262 Heptylprodigiosin 188 in vitro 188 structure of 188 Heteronema erecta 147 heteronemin from 147 Hexanorcucurbitacin I 434 structure of 434 High blood pressure 3 81 Logotis brevituba in 381 Hippospongia sp. 116,117 hippospongins A-C from 116 inhibitory effects on human Rasconverting enzyme 117 sesterterpenoid acid from 117 HIV-1 replication 833 inhibitors of 833 15-HLO 135 halisulfate 1 inhibitor of 135 Homobaldrinal 256 Homofascaplysin A 187 from Hyrtios erecta 187

1229 from Plasmodium falciparum strains 187 Homofascaplysin A 187 structure of 187 Hormonal effects 1189 of isoflavones 1177,1180,1181, 1185 Hormone replacement therapy 1195 isoflavones role in 1195 Host-specific attractants 1056,1059 structures of 1056 Host-specific chemical signals 1057 Host-specific chemoattractants 1066 Host-specific plant signal 1061-1064 in host recognition 1061 in germination of pest propagules 1061 Host-specific signals 1058 evidence of 1058 for zoospore chemotaxis 1058 5-HT2A receptor ligand 1156,1164 from Pithomyces sp. 1164 Human 122-lipoxygenase(12-HLO) 135 inhibitor of 135 Human bronchopulmonary non-small-celllung carcinoma cell line 141 petrosaspongiolides A-J against 141 Human cdc25A protein phosphatase 137 dysidiolide inhibitor of 137 Human colon tumor (HCT-116) cytotoxicity 76 of pyrroloiminoquinones 76 Human KB cells 836 cytotoxicity against 836 Human synovial PLA2 135,139,141,142 cacospongionolides E inhibition of 135 cavernolide effects on 139 effects on 142 Human tumor cell lines 136 Humualnes 908 from Aristolochia birostris 908 Huntington's disease 1150 therapies for 1150 Hydrogenolysis 21 of dibenzylphosphate 21 10-Hydroxyoleoside 249 8-Hydroxygeraniol 250 5-Hydroxyisoflavonoids 835 as allelopathic 835

Hydroxylation 347,566 ofoleosides 347 8-Hydroxymanzamine A 182 structure of 182 5P-Hydroxynicandrin B 1029 structure of 1029 8-Hydroxytricetin 7-glucuronide 813 structure of 813 28-Hydroxywithanolide E 1024 structure of 1025 20-Hydroxywithanolide glycosides 1027 from Dunalia brachyacantha 1027 19-Hydroxywithanolides 1032 Hymenoxin 813,817 against human tumour tissues 817 cytotoxic effects of 817 in vitro 817 structure of 813 Hymonymic Chinese crude drugs 998 Hyperibones A-D 704 antibacterial activity of 704 Hypertension 374 Gardenia jasminoides in 374 Hypertensive stroke-prone rat 652 Hyphal growth 1070 after germination 1070 Hypoglycemic activity 262,356 of gentiopicroside 262 ofiridoids 356 of secoiridoids 356 ofsweroside 262 of swertiamarin 262 Hypoglycemic effects 540 in vivo 540 Hypolipidemic active metabolites 512 ascofuranone 512 ascofuranol 512 Hyrtios erecta 127,146,147 hyrtiolide from 127 Hyrtios erectus 143 hyrtiosal from 143 Hyrtios sponge 71 Hyrtiosal 154 from Hyrtios erecta 154

Iberis amara 461 Iberis umbellate 431 antagonist activity of 431

1230 Ibogaine 814 anti-addictive properties of 814 Ichthyotoxic effects 153,154,157 against Gambussia affinis 153 Ichthyotoxicity 117,143,144 in Artemia salina assay 144 in fish lethality assay 117 to Gambussia affinis 117,143 Idiadione 111 in Artemia salina bioassay 111 in cancer cell lines 111 IFN-Y

238

in vitro production of 238 IL-2 238 in vitro production of 238 IlicicolinD 512 from Cylindrocladium ilicicola 512 Illicinolides 411 biosynthesis of 411 Illicium anisatum 396 toxic substance from 396 Illicum vernum 396 fruits of 396 Immune system 456 cucurbitacins effects on 429, 430-435,438,439-446,447,456 Immunoadjuvant activity 209 ofiridoids 356 ofsaponins 209 of secoiridoids 356 saponins as 233 Immunomodualting activity 209,979 of aristolic acid 977,978 of bioactive saponins 209 Immunomodulatory activity 229,979 of arisolochic acid I 979 ofsaponins 229 Immunomodulatory constituents 573 from Microascus tardifaciens 573 Immunomodulatory effects 233 of Albizzia adianthifolia 233 Immunosuppressants 1136 Immunosuppressive activity 3,774,782 of celastrol type compounds 782 ofFR901483 3,4,6,7,8-15,19,22, 25,29,31,35-40 of Tripterigyum wilfordii 114 IndicamineB 849 strucutre of 849

Indoles 493-498 from Penicillium crustosum 493 Indole alkaloids 809 structures of 809 Indole-3-carbaldehyde 1056 structure of 1056 Inflammatory activity 116 of Thorecta horridus 116 in vivo 116 Influence type A virus 355 Inorolides A 155 cytotoxicity of 155 Insecticidal activity 262,550 against silkworms 550 ofchirata 262 Insecticidal compounds 550 Insecticidal okaramines 582 discovery of 582 Integrin-mediated cell adhesion 441 inhibition 441 Interleukin (IL-2) transcriptional activation 793 inhibitor of 793 Iochroma coccineum 1025 Ipecoside 249 Ircinia 139 Ircinia fasciculate 115 fasciculatin sulphates from 115 palinurin from 115 Ircinia oros 119 Ircinia variabilis 115 fasciculatin sulphates from 115 palinurin from 115 Ircinin 112 anti-inflammatory activity of 112 Ircinin-1 111 from Ircinia oros 111 Ircinin-2 111 in mouse ear edema 111 Iridodial 365 structure of 365 Iridodialogentiobioside 249 Iridoid bearing plants 247 chemical and biological aspects of 247 of temperate region 247 Iridoids 248,251,252,291,305-333,340, 352,353,365,381 analgesic activity of 3 82 antiinflammatory activity of 353,382

1231 antitumoral activity of 383 biological activities of 252,352 biosynthesis of 251,365 cardiovascular activity of 352 characterisation of 251 chemopreventive activity of 383 extraction of 251 from Oleaceae 305 hypothetical biosynthetic pathway to 340 in myxopyreae 340 pharmacological activities of 252,365 structure of 248,291,305-333 Iridoid containing drugs 252 Iridolactones 366 from actinidia polygama 366 from Nepeta cataria 366 Iridomyrmecin 247 defensive mechanisms of 247 Insect repellent 860 Bothrops atrox as 860 Insecticidal activity 576 of communesins 574-579 Insomnia 289,822 catnip tea in 289 Erythrina Americana in 822 Intramolecular aldol reaction 4 ofketoaldehyde 4,10,13 Iridoids 268,355-357 antileishmanial activity of 357, 1048 antiviral activity of 113,138,209, 355 from Grevillea linearis 268,270 hypoglycemic activity of 262,356 Iridomyrmex ants 365 Ishwaranes 920 from Aristolochia argentina 920 Islanditoxin 502 from Penicillium islandicum 502 Isoaristolactone 923 from Aristolochia versicolar 923 Isochromophilone IX 485 from Penicillium sp. 485 GABA-containing metabolite 485 Isochromophilones I 482 anti-HIV activity of 483 120CD4 binding inhibitors 482 from Penicillium multicolor 482 Isochromophilones II 483

against Aspergillus niger 483 against Bacillus subtilis 483 against Candida albicans 483 against Escherichia coli 483 against fungi 483 against Micrococcus luteus 483 against Piricularia oryzae 483 Isochromophilones III-VI 483 against Acholeplasma taidlawii 484 against Aspergillus niger 484 against Bacillus subtilis 484 against Bacteroides fragillis 484 against Candida albicans 484 against Escherichia coli 484 against Micrococcus luteus 484 against Mucor racemosus 484 against Myeobacterium smegmatis 484 against Pseudomonas aeruginosa 484 against Pyricularia oryzae 484 against Saccharomyces sake 484 against Staphylococcus aureus 484 against Xanthomonas oryzae 484 from Penicillium multicolor FO-3216 483 inhibitors of ACAT 484 Isochromophilones VII-VIII 44 against Bacillus subtilis 485 against Micrococcus luteus 485 against Myeobacterium smegmatis 485 against Pyricularia oryzae 485 antimicrobial activity of 485 from Penicillium sp. 484 in vitro 484 Isocoumarins 521-526 Isoflavones 1177,1180,1181,1185 absorption of 1183 antagonist effects of 1194 antiproliferative effects of 1195 as functional food components 1177 bioavalability of 1181 chemical features of 1180 distribution of 1180 effect on cardiovascular diseases 1199

1232 estrogen agonist effect of 1194 food sources of 1185 functional properties of 1188 hormonal effects of 1189 metabolism of 1181,1186 osteoprotective effects of 1195 structures of 1187 Msoferulloyl 254 structure of 254 Isoflavonoids 834,1178,1083 chemotaxis of 1083 from Erythrina species 834 Isogarcinol 714 effects on human leukemia cell lines 714 Isogarcinol-xanthochymol mixture 710 apoptosis-inducing effects of 710 against human leukemia 710 Isonitriles 176 and analogues 176 Isoprenylated tyrosine derivatives 1164 structures of 1164 Isoquinolines 890,892 from Aristolochia arcuata 890 from Aristolochia elegans 890 from Aristolochia gehrtii 890 from Aristolochia species 892 Isosenegalensin 835 as anticancer agent 835 Isoxanthochymol 705 anti-HIV activity of 705 Ixocarpalactone A 1027 from Physalis philadelphica 1027 (-)-Jaboromagellonine 1029 from Jaborosa magellanica 1029 structure of 1030 Jaborosalactol 1023 Jaborosalactone 1033 from Jaborosa araucana 1033 Jaborosalactone 8 1024 from Jaborosa leucotricha 1024 Jaborosalactone O 1032 from Jaborosa leucotricha 1032 Jaborosalactone P 1045 feeding activity of 1045 Jaborosalactone R 1030 from Jaborosa sativa 1030 structure of 1030

Jasmineae 346 biosynthetic pathway in 346 Jasminum lanceolarium 350 Jasminum hemsleyi 309 jashemsloside A from 309 jashemsloside C from 309 jashemsloside D from 309 Jasminoside 350 esterification of 350 Jasmoaldehydes 352 formation of 352 Jasmolactones 332 origin of 332 structures of 333 Jaspamide 83 against P-388 cancer cells 83 structure of 83 Jaspamide TA 82 cytotoxic depsipeptides 82 from Hemiastrella minor 82 Jaspiferals E-F 145 against L1210 145 cytotoxicity of 145 Jaspis johnstoni 520 Jaspolinaloside 347 from Jasminum. polyanthum 2>A1 Jenisseenssosides C,D 227,,232 apoptosis inducing activity of 227 jurkat cells proliferation effects of 232 Jesterone 1109 as mycelial growth inhibitor 1109 Jurkat cells proliferation 232 effects of Jenisseenssosides C,D 227,232 Jujuboside A 238 structure of 238 Jujuboside B 238 structure of 238 Jujuboside Bl 238 structure of 238 Jujuboside C 238 structure of 238 Jujubosides 238 immunological adjuvant activity of 238 Kageneckia oblonga A31,AAA analgesic activity of 444 anti-inflammatory activity of 444

1233 antipyretic activity of 444 as antioxidant 444 cucurbitacins from 437 Kaikosaponin III 210 from Pueraria thunbergiana 210 antimutagenicity activity of 211 Kaitocephalin 527 structure of 527 from Eupenicillium shearii 527 Kalihinane diterpenoids 179,180 antifungal activity of 179 antihelmintic activity of 179 from A canthella sp. 180 KalihinolA 179 against mouse mammary tumor 179 cytotoxicity activity of 179 KalihinolA 180 against Plasmodium falciparum 180 Kalopanaxsaponin A 222 Kampo medicines 998 Ketoaldehyde 4,10,13 aldol reaction of 13 intramolecular aldol reaction of 4 preparation of 14 synthesis of 10 Ketone enolates 38 cyclization of 38 Kibayashi's synthesis 35-39 ofFR901483 3,4,6,7,8-15,19,22, 25,29,31,35-40 Kigelia pinnata 371 in cancer 371 in skin infections 371 Kohamaic acids A and B 137 against P388 cells 137 Kolanone 705,765 against Bacillus subtilis 705 against Staphylococcus aureus 705 antimicrobial activity of 705 Lactonization 350 Lagenidium giganteum 1054 Lamiosie 250 Lamium labiatae 288 against hemorrhage 288 antiinflammatory properties of 288

as antispasmodic 288 as astringent 288 as depurative 288 as haemostatic 288 in bowel movment 288 in menorrhagia 288 Lannea 1088 bioassay-guided fractionation of 1088 Lannea coromandelica 1088,1089 characterization of 1089 MALDI-TOF-MS of 1089 motility inhibitory activity of 1088 zoosporicidal polyflavonoids from 1088 Lannea extracts 1088 physicochemical properties of 1088 Lannea tannins 1094 lysis of zoospores by 1094 Lamium amplaxicaule linn 288 Latrunculin A and B 101 from Chromodoris hamiltoni 101 LC-MS methods 476 LC-MS-MS methods 476 LC-PDA detection method 997 Learning and memory impairment 638 butylidencphthalide in inhibition of 638 35-butyphthalide in inhibition of 638 cnidilide in inhibition of 638 Leg ulcer 863 Aristolochia clematitis in 863 Leishamania spp. 1048 LepadinD 185 structure of 185 Lepadin E 185 structure of 185 Lepiochlorin 180 526 an antibacterial lactol 526 Leukemia 710 against human isogarcinolxanthochymol mixture 710 Levistolide B 638 in blood viscosity reduction 638 Lewis lung carcinoma cells 223 Life-cycle development 1056 ofoomycetes 1056

1234 Lignans 937,939-944 from Aristolochia species 939-944 structures of 941 Ligstral-type secoiridoids 350 Ligusticum acuminatum 613 Ligusticum acutilobum 613 Ligusticum chuanxiong 613 Ligusticum frondosa 152 homoscalarens from 152 Ligusticum jeholense 613 Ligusticum jeholense var. tenuisectum 613 Ligusticum mutellina 613 Ligusticum offwinalc 613 L igusticum porteri 613 Ligusticum sinense 613 Ligusticum sinense cv. chaxiong 613 Ligusticum tenuissimum 613 Ligusticum variabilis 124 dehydromanoalide from 124 Ligusticum wallichii 613 Liguistilide 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Ligustroside 356 against Escherichia coli 356 Limestone formations 1138 Linear sesterterpenoids 110 Lintenolide 141 cytotoxicity of 141 against a tumor cells 141 Lippia multiflora 805 antibacterial activity of 805 pediculocidal activity of 805 5-Lipoxygenase 112 ircinin inhibitor of 112 Liquiritigenin derivatives 826,828 activity of 826 sources of 828 Lissoclin disulfoxide 87 structure of 87 Lissoclinotoxin A 188 against Plasmodium falciparum 188 structure of 188 Liver disorders 266 Gentiana lufea in 265,266,270 Liver protective 279 effect of aucubin 249,250,279, 383,386,387

7-ep/-Loganic acid 341 oxidation of 341 Loganin 356 hepatotoxic activity of 356 Logotis brevituba 381 in fever 381 in hepatitis 381 in high blood pressure 381 Lonicera (caprifoleaceae) 273 antitussive activity of 274 as antipyretic 274 as sedative 274 bis-iridoids from 273 coumarin glycosides from 274 hypotensive activity of 274 iridoids from 273 sulfur containing monoterpenoids from 273 triterpeoids saponins from 274 Lonicera angustifolia 275 use in gastric troubles 275 Lonicera caerulea 277 caeruloside A from 277 caeruloside B from 277 epivogeloside from 277 ketologanin from 277 loganin from 277 secologanin from 277 sweroside from 277 Lonicera japonica 21A caffeoylquinates (CQs) from 274 effects on HIV 274 for rashes 274 for skin ailments 274 Lonicera implexa 371 Lonicerapericlymenum 275 as antispasmodic 275 as diuretic 275 biosidic ester iridoid glucoside from 275 secoiridoid glucosides from 275 use in respiratory tract 275 /-Triptolide 787 synthesis of 787 via /-dehydroabietic acid 787 Luff a operculata 430 Luffalactone 132 from Luffariella variabilis 132 Luffariella geometric 133 Luffariella variabilis 133 Luffariolide F 125

1235 Luffariolide G 125 Lung adenocarcinoma 975 Luteusin C 488 from Talaromyces luteus 488 structure of 488 Luteusin D 488 from Talaromyces luteus 488 structure of 488 Luteusin E 488 from Talaromyces luteus 488 structure of 488 Lymphocyte proliferation 231 action on 231 Lymphoproliferative activity 233 Maclurin 672 structure of 672 Macrocycles 502-506 Macrocystic pyrifera 147 Macrophage activation 230 role in host defense mechanisms 230 Madolis 909 from Aristolochia mollissima 909 Magnoflorine 882 from Aristolochia 882 Majonoside-R2 212 from panax vietnamiensis 212 Majucin-subtype sesquiterpenes 401-404 Malaria 169,170,822 by Plasmodium falciparum 169 by Plasmodium ovale 169 by Plasmodium malariae 169 Erythrina abyssinica against 822 Erythrina fusca in 822 Erythrina indica in 822,835 symptoms of 170 Malaria infection 170 quinine for 170 Mammalian sex hormones 1099,1100 activities of 1100 repellent activity of 1099 Mammary tumor cell line MCF7 140 spongianolides A-E inhibitory effects on 140 Manoalide 123,141 isolation of 123 Manzamine A 182 against Plasmodium falciparum 183

in vitro activity of 183 structure of 182 Manzamine alkaloids 185 against malarial parasite 185 Manzamine F 183 structure of 183 Marcfortine B 604 structure of 604 Marine natural products research 63 in southern Africa 63 Marupone 672 structure of 672 Measles 289 catnip tea in 289 Medicarpin 1083 as phytoalexin 1083 (i)-Medicarpin 1083 structure of 1083 Medico-magic plants 803,806,707 bioactive natural compounds from 803 of Bantu area 803 use of 806,807 Melanin synthesis inhibitor 463 cucurbitacins 463 MelledonalC 511 MelledonalD 511 from Armillaria spp. 511 Menodora robust a 350 Menorrhagia 288 Lamium labiatae in 288 Merrilactone A 417 synthesis of 417 Methanococcus jannachii 1130 7-Methoxyaristolochic acid I 863 6-Methoxybenzoxazolinone 818 from Scoparia dulcis 818 pharmacological activity of 818 Methyl protoneodioscin 214,218 antitumor activity of 214 Methyl-3-epinuapapuanoate 198 against Plasmodium berghei 198 in vivo 198 Methyl-3-epinuapapuanoate 198 structure of 198 24-Methylscalaranes 152 from Dictyoceratida sp. 152 from Halichondria sp. 152 8-O-Methylsclerotiorinamine 487 from Penicillium multicolor 487 structure of 488

1236 Microbial fermentation schemes 1157 Microbial products 549 as antibiotics 549 as enzyme-inhibitors 549 as fungicides 549 as herbicides 549 Micrococcus luteus 125 Mikrolin 532 structure of 532 Millecrols 98 structure of 98 Millecrones 98 against bacillus subtilus 98 against staphylococcus aureas 98 structures of 98 Mimosa 1093 zoosporicidal activity of 1093 Mitochondrial ATP synthase inhibitor 118 oligomycin 118 Miwanensin-type sesquiterpenes 400 MMP inhibitors 1154 therapeutic use of 1154 Modolin type sesquiterpenoids 964 biogenetic sequences of 964 Molluscicidal activity 123 ofmanoalide 123 against Biomphalaria glabrata 123 Monocarbocyclic sesterterpenoids 123 from genus Luffariella 123 Monodora myristica 807,815 antibacterial activity of 807 in vitro 807 Monomeric secoirided glucosides 310 origin of 311 structures of 311 Monordens C-E 539 from Humicola sp. 539 Monoterpenoids 810,896-901,897 from Aristolochia species 897-901 Monotropein 250 Morinda citrifolia 376 Morphine withdrawal 142,983 effect of constrictosine in 983 role of petrosapongiolide M in 142 Motility inhibitors 1078,1079 biological activity of 1079 function of 1079 structure elucidation of 1078

Motility inhibitory activity 1086 of anacardic acids 1084-1086 Motility inhibitory factor 1084 from Ginkgo fruits 1084 Mouse lymphocytic leukemia 975 MPP+ induced apoptosis 990 inhibition of 990 Multiple sclerosis 1151 Muqubilone 197 Muqubilone (or aikupikoxide A) 123 against herpes simplex virus type 123 antiviral activity of 123 from Diacarnus erythraeanus 123 in vitro 123 Murine allogenic bone marrow transplantation 775 Musanga cecropiodes 805,807 analgesic effects of 807 Mycobacterium smegmatis 838 Mycobacterium tuberculosis 263 activity against 263 Mycorrhizinol 532 structure of 532 Mycorrhizins 523 biosynthesis of 523 Myxopyreae 345 biosynthetic pathway in 345 N. arbortristis 305 arborside A from 305 arborside B from 305 arborside C from 305 arbortristoside A from 305 arbortristoside B from 305 arbortristoside C from 305 arbortristoside D from 305 arbortristoside E from 305 nyctanthoside from 305 Narcotic 822 Erythrina species as 822-824 Naja naja PLA2 enzymes 141 Naja nigricollis 984 Nakafurans 97 structures of 97 Nannizzia gypsea var. incurvata 571 Naringenin derivatives 828 activity of 828 sources of 828

1237 Natural phthalides 624-637 biological activities of 637 classification of 624-637 Natural products 1103 bioactivites of 1103 Natural semi-synthetic quillajasaponins 235 Nectandrin-B 942 from Aristolochia chilensis 942 Nematocidal halogenated dihydoisocoumarins 522 from Lachnum papyraceum 522 Nemorosone 688,704,705,716 against human cervix crcinoma 716 against human larynx carcinoma 716 antimicrobial activity of 705 from Clusia grandiflora 704 from Clusia rosea 688 Nemorosone II 765 Neoanistatin 419 neurotoxic activity of 419 Neobavaisoflavone 839 antifungal properties of 839 from Aspergillus fumigatus 839 from Psoralea corylifolia 839 Neoechinulins D and E 572 «eo-Kauluamine 184 structure of 184 (6Z)-Neomanoalide 123 (6£)-Neomanolide 123 Nepatalactone 247 form Nepta cataria 247 Nepeta (labiateae) 289 as diaphoretic 289 as refrigerant 289 as soporific 289 Nepetariaside 249 Neurodegenerative disorder 1152 Neurological disorders 354 Neurotrophic acivity 396 of Illicium jiadifengpi 396 of Illicium merrillianum 396 of Illicium minwanense 396 of Illicium tashiroi 396 Nicotinamide 1095,1096 halting activity of 1096 motility-inhibiting activity of 1096 structure of 1095

Nigella sativa 223 antitumor activity of 223 Nigerazine A 593 structure of 593 Nigerazine B 593 structure of 593 Nigragillin 593 structure of 593 Nitric oxide (NO) 131 Nitrogen-tethered phenol derivatives 15-19 iodine oxidation of 15-19 Nitrone rac-9 9 1,3-dipolar cycloaddition of 9 Nitrongen-tethered phenols 16 spirocyclization of 16 yV-methoxyamide 17 cyclization of 17 NO synthase 650 Nomofungin 578,579 structure of 579 Non-glucosidic secoiridoids 332 origin of 332 structures of 333 Non-alkaloid phthalide 613-619 sources of 613-619 Non-alkaloid phthalide subtype 630 structures of 630 Non-alkaloidal constitutents 822 of Erythrina 822 Non-competitive GAB A antagonist 419 Non-glycosidic secoiridoids 350 Nonhost extracts 1075 Nonsteroidal anti-inflammatory drugs 713 in colonic tumor 713 in colon cancer 713 «or-cucurbitacins WGi 446 chemical structures of 446 «or-cucurbitacins WG2 446 chemical structures of 446 Norscalarals A-C 151 against tumor cell lines 151 from Cacospongia scalaris 151 23-Norscalarane petrosaspongiolide K 152 against NSCLC-N6 cells 152 18-Norwithanolides 1025 Novel bis-anthrones 501 from Anaptychia obscurata 501

1238 Novel sesquiterpenoid MMP-3 inhibitors 1165 from acid mine waste extremophile 1165 isolation of 1165 NPK fertilizers 260 application of 260 iV-fr-ara-Feruloyltyramine 1079 as natural stimulant 1079 iV-/rara-ferulyl-4-0-methyl-dopamine 1059 from Chenopodium album 1059 attractant activity of 1059 Nuclear DNA 1094 fragmentation of 1094 Ochrocarpinones A-C 711 against A2780 ovarian cancer cells 711 from Ochrocarpos punctatus 711 Oil-soluble artemether 190 in treatment of severe malaria 190 Okaramine A 551-573 absolute confirguration of 562 biosynthetic pathway of 564-567 discovery of 551 'H-NMR spectrum of 551 insecticidal activity of 563-564 molecular formula of 551 related compounds 570-573 Okaramines N and J 567-570 synthetic study of 567-570 Oleaceae 303 iridoids from 303 secoiridoids from 303 Oleacin inhibitor 352 of converting enzyme 352 Oleae 346 biosynthetic pathways in 346 Oleanolic acid 263 Olefinic bond 677 epoxidation of 677 Oleosides 347 hydroxylation of 347,566 Oleoside derivatives 343 biosynthetic pathway to 343 Oleoside biosynthesis 343 role of 7-ketologanic acid role in 343

Oleoside-type glucosides 346 biosynthesis of 346 Oleuropein 354-356 against RSV 356 antioxidant activity of 355 hypoglyscemic activity of 356 scavenging effects of 354 Olive oils 355 against reactive oxygen species 355 Oliverine 810,814 from Pimelea suaveolens 814 microfilaricidal activity of 814 structure of 810 Onjisaponins A 239 mucosal adjuvant activities of 239 Onjisaponins E 239 mucosal adjuvant activities of 239 Onjisaponins F 239 mucosal adjuvant activities of 239 Onjisaponins G 239 mucosal adjuvant activities of 239 OocydinA 1109 as mycelial growth inhibitor 1109 structure of 1109 Oomycetes 1070,1075 chemotropic responses of 1070 resistance against 1075 Oomycete phytopathogens 1053 bioactive secondary metabolites related to 1053 life-cycle development of 1053 Oomycete species 1111 host-specifity of 1111 Oomycete zoospores 1064 attractant for 1064 Ophelia chirata Grisebach 261 Organic acids 671 Ornoside 356 against Escherichia coli 356 against Staphylococcus aureus 356 1,2,34-Oxamanzamine A 184 structure of 184 Oxidation 341,936,1025 of 7-epMoganic acid 341 Oxidation products 710 ofgarcinol 719 mechanism of formation of 710 Oxidative decarboxylation 962 Oxidative ozonolysis 87

1239 19-Oxygenatedscalaranes 148 12-Oxygenated withanolides 1029 from Datura ferox 1029 Oxyresveratrol 831 depigmenting effects of 832 in tyrosinase inhibition 831 structure of 831 P-388 lymphocyte leukemia cell line screen 65 Pacchastrella sp. 77 effects on endothelin enzyme (ECE) 77 Paederia scandends 376 Palauolide 131 from Halichondria sp. 131 Palinurin 115 from Ircinia fasciculate 115 Panax saponins 215-219 structures of 215-219 Pancreatic cancer 1135 Papilio xuthus 1000 Para influence type 3 virus 356 Paraherquamide family 602 structures of 602 Paralytic activity 550 against silkworms 550 Paralytic compounds 594-604 Parkinson's diseases 980 aristolactam taliscanine in 980 Particle bioassay 1112 p-Coumaric acid 978 interceptive activity of 978 PDEs phosphodiesterases 650 Penicillium chrysogenum 1158 Penicillium expansion 550 insecticidal activity of 550 Penicillium fellutanum 571 Penicillium multicolor 486 5-bromoochrephilone from 486 Penicillium roqueforti 1166 Penicillium simplicissimum 550 insecticidal activity of 550 Penicillium urticae A1A transformation of 474 Penitrems 494 from Aspergillus species 494 from Penicillium 494 structure of 494 PenitremA 494,582-584

against Bombyx mori 494 against Heliothis zea 494 against Spodoptera frugiperda 494 convulsive effect induced by 584 from Penicillium crustosum 494 identification of 582 insecticidal activities of 494 related compound 583 structure of 495,582 Penitrem C 494 against Bombyx mori 494 against Heliothis zea 494 against Spodoptera frugiperda 494 as convulsive 494 from Penicillium crustosum 494 insecticidal activities of 494 structure of 494 Penitrem F 494 against Bombyx mori 494 against Heliothis zea 494 against Spodoptera frugiperda 494 as convulsant 494 from Penicillium crustosum 494 insecticidal activities of 494 structure of 495 Pentacarbocyclic sesterpenoids 156 Pentaglycoside 232 immunostimulant activity of 232 Pentobarbital-induced sleep 640 Pericosines A 537 from Periconia byssoides 537 Perophoramidine 578,579 from Perophora namei 578 structure of 579 Peroxyplakoric acids A3 195 structure of 195 Peroxyplakoric acids methyl esters 196 synthetic analogues of 196 Petrosaspongia 13 9 Petrosaspongiolide K 145 Petrosaspongiolide P 141,142 effects of 141 Petrosaspongiolides M-R 141 PG27 775 immunosuppressive activity of 775 Phychium cinnamomi 1057

1240 Phychium infestcms 1053 late-blight disease by 1053 Phychium sojae 1055,1058 zoospores of 1058 Pharmacology 855,971-993 of Aristolochia sp. 855,857-859, 862,971-993 Phenanthrenes 894,895,896 from Aristolochia species 894 structures of 895,896 Phenol 20 synthesis of 20 Phenolic amides 16 oxidative O-spirocyclization of 16 Phenolic oxazolines 17 oxidative spirocyclization of 17 Phenolic sulfonamides 19 oxidative spirocyclization of 19 Phleodictynes 186 structure of 186 Phomalactone 1108 by Nigrospora sphaerica 1108 Phomopsin A 505 from Phomopsis leptostromiformis 506 structure of 506 Phorbazoles 80 immunomodulatory activity of 80 structure of 80 PhorbazoleC 80 first total synthesis of 80 Phospholipase C activity 1072 Phospholipase injection 985 in edema 985 Phthalide 612, 611,613-624,640,642, 647,649,652,702 actions on central nervous system 611 against cerebral ischemia 646-647 against seizure 640 anti-spasmodic potencies of 651 as anti-angina 611,642 as anti-hypertensive agents 651 as anti-platelet 611,641 as anti-smooth muscle proliferation 611 as anti-thrombosis 611,641 biological activities of 611,702 chemical structure of 612 chemistry of 611 classification of 612

for food flavorings 611 in cardiac function 611 in cardiac function modulation 642 in dietary supplements 611 in fungi 611 in herbal remedies 611 in inhibition of smooth muscle cell proliferation 643 in smooth muscle relaxation 649 naturally occurring 611 sources of 612-624 vasodilatory actions of 651 vasorelaxing actions of 652 Phthalide derivatives 643 anti-proliferative activity of 643 Phthalide dimer type 620-622,634 sources of 620-622 structures of 634 Phthalide isoquinoline type 622-624,637 sources of 622-624 structures of 637 Phyllactones A 154 against KB cells 154 Phyllogenone B 157 from Phyllospongia foliascens 157 against P388 cells 157 Phylum coelentrata 88-96 bioactive metabolites from 88-96 Phylum hemichordata 64-78 bioactive metabolites from 64-70 Phylum mollusca 96-102 bioactive metabolites from 96-102 Phylum porifera 70-85 bioactive metabolites from 70-85 Physalins 1022 Physalis peruviana 1024 Phythium 1053 Phythium graminicola 1057 zoospores from 1057 Phytoestrogens 1099,1193 in human endometrial adenocarcinoma cells 1193 in vitro 1193 in vivo 1193 relative potency of 1193 repellent activity of 1099 Phytomedicines 996 used as slimming regimens 996

1241 Phytopathogenic oomycete zoospores 1058 Phytophthora 1053,1064 attractant for 1064 Phytophthora spp. 1105 Picfelterracin VI 443 structure of 443 Picracin 450 from Picrorhiza scrophulariaeflora 450 Picroliv 252,385,387 anti-allergic property of 387 anticholestatic activity of 252 antioxidant activity of 385 as chemopreventive agent 385 cholerectic activity of 252 hepatoprotective activity of 252, 385 injury protective activity of 385 Picrorhizia kurroa 252,253,378 in liver disorders 252 picroside-IV from 253 Picrorhizia scrophulariflora 252 in liver disorders 252 Picrosides I 252 hepatoprotective effects of 252 Piericidins 550 from Sreptomyces sp. 550 Pinicoloform 527 antibiotic activity of 527 cytotoxic activity of 527 Piperazinomycin 593 structure of 593 Piperolactam 981 antiplatelet aggregation activity of 981 PIT lake microbes 1140 initial discovery of 1140 PIT microbes 1159 biological activity profiles of 1159 PLA2 134 cacospongionolide B inhibitor of 134 PLA2 activity 986 in vitro inhibition of 986 PLA2 enzymes 986 TFVPL-lb 986 TFV-PL-la 986 PLA2-induced mouse paw oedema 838 effects of E. addisonae EtOAc

extract on 838 effects of warangalone on 838 PlakortideE 193 structure of 193 PlakortideF 194 antimalarial activity of 194 structure of 194 PlakortideK 194 antimalarial activity of 194 structure of 194 PlakortideL 194 structure of 194 PlakortideO 194,195 against Plasmodium falciparum 194 Plakortide P 194 against Plasmodium falciparum 194 Plakortin 193,194 against human colon carcinoma 194 against mouse lymphoma cells 194 structure of 193 Plakortis halichondraides 192 plakortin from 192 Plakortis sp. 196 against Plasmodium falciparum 196 Plantago altissima 280 Plantago asiatica 280 Plantago atrata 280 Plantago berghei infection 196 Plantago cornuti 280 Plantago lanceolata 280 in asthma 280 in cough 280 in inflammed surfaces 280 in pulmonary diseases 280 iridoids from 280 Plantago lundborgi 280 Plantago major 277,279 3,4-dihydroxyaucubin from 278 as diuretic 279 gardoside from 278 geniposidic acid from 278 in dysentery 279 in griping pain 279 in wound healing 279 majorside from 278 use in diarrhea 279

1242 use in eye wash 279 Plant ago major 374 in cancer 374 in skin/respiratory ailments 374 Plantago media 280 Plantago ovata 280 Plantago patagonica 280 Plantago renformis 280 Plasmodium falciparum 128,171 cytotoxicity against 128 Plasmopara viticola 1057 Plukenetione A 699 structure of 699 Plukenetione B 699 structure of 699 Plukenetione C 699 structure of 699 Plycitone 88 effects on retroviral reverse transcriptase enzymes 88 Pneumocystisjaroveci 1148 Polyalthie suaveolens 804,808 Polycitrins 87-88 structure of 88 Polyflavonoid tannins 1090,1092 against zoospores 1092 lytic activities of 1092 motility inhibition by 1092 structural units (A-D) of 1090 Polygalaceae saponins 228 Polyisoprenylated benzophenone derivatives 671 -673,702,703,705-716 against HIV-l 672 anti-cancer activity of 713-716 anti-HIV activity of 702-703 antimicrobial activity of 672, 703-705 antioxidant activity of 705-710 biological activity of 671,702 chemistry of 671 cytotoxicity of 710-713 from Allanblackia 672,673 from Clusia 672 from Garcinia 672 from Hypericum genera 672,673 from Moronobea 672 from Ochrocarpus 672 from Symphonia genera 672,673 from Tovomiptosis 672 from Tovomita genera 672,673

from Vismia genera 672,673 occurrence of 673 Polyketide derivatives 192 Polymerization 1089 Polyphenols 1186 metabolism of 1186 Polyprenylated benzophenones 671 Polyvinylidine difluoride (PVDF) 1071 Pomadasys commersonni 102 diterpenes from 102 Portulaca oleracea 1077 zoospore motility inhibitors from 1077 Preferential chemotaxis 1057,1058 toward hosts 1057,1058 Prenylation 723 Prenyldaidzein 838 against Staphylococcus aureus 838 seco-Prezizaane 395,411,419 biosynthesis of 411 chemistry of 395 from Illicium species 395 neurotoxic activity of 419 neurotrophic activity of 395 neurotrophic activity of 420 seco-Prezizaane-type sesquiterpenes 397 Prianos sp. 128 Promomonilicin 510 antimicrobial spectrum of 510 Propolone A 705 antimicrobial activity of 705 Prostaglandin (PGE2) 131 Prostaglandins 90 biosynthesis of 90 Protein kinase C (PKC) 140 spongianolides A-E inhibitory effects on 140 Protein synthesis inhibitors 113 Protoberberines 882,886,887 structues of 886 from Aristolochia species 887 Protodioscin 225 inhibitory effects on human leukemia HL-60 cells 225 Protojujuboside 238 Protoneodioscin 218 Protopanaxadiol saponin M1 229 antimetastatic property of 229 Protopines 888 structures of 888

1243 Prunetin 1056,1058 from pea seedlings 1058 structure of 1056 Pruritus 856 A. argenuna in 856 PS45A-4 1163 against caspase-1 1163 Pseudoanisating 420 insecticidal activity of 420 neurotrophic activity of 420-425 Pseudoarachniotus roseus 528 aranochor A from 528 Pseudoanisatin-type sesquiterpenes 397,398 structures of 398 Pseudomajucin-subtype sesquiterpenes 404-407 Pseudomajucin-type sesquiterpenes 404-407 structures of 405 Psoriasis 136 12-HLOrolein 135 Pterocarpans 843,844 anti-MRSA activity of 843 effect on rabbit platelet aggregation 844 from the Genus Erythrina 843 Purgative 822,431 Citrullus colocynthis as 431 Erythrina species as 822-824 Pythium aphanidermatum 1057 zoospores from 1057 Pythium arrhenomanes 1057 Pythium insidiosum 1054 Pyrite 1138 oxidation of 1138 Pyrroloinenoquinones 75 structure of 75 QS-21 234 adjuvant activity of 234 Quebracho 1093 zoosporicidal activity of 1093 Quebracho tannin 1089 Quillaja saponaria 234,236 aduvant activity of 234 Quillajasaponins 234 aduvant activity of 234

Racemichydroxylaminerac-10 9 condensation of 9 synthesis of 9 Racemic triptolide 787,788 total synthesis of 787,788 Radix (Angelicae sinensis) 611,655 estrogenic effects of 655 for treatment of cerebroand cardio-vascular disease 611 in female irregular menstruation 611 use in obstructive pulmonary diseases 655 Ram semmal vesicle (RSV) microsome 992 Rebeccamycin 497 as indolocarbazole antitumor agent 497 from Saccharothrix aerocolonigenes 497 Receptor independent apoptosis 69 of leukaemia cells 69 Recombinant human synovial PLA2 133 inhibitory effect on 133 Regeneration activity 1113 of zoospores encyst 1113 Rehmannia glutinosa 379 Respiratory disease 371 Ajuga decumbens in 371 Respiratory syncytial virus 355 Rhabdastrella (jaspis) stellifera 144 jaspiiferals C-F from 144 Rhabdastrella globostellata 144 aurorals from 144 Rhizobium-legume interaction 1058 Rhizoma chauanxiong 611 in female irregular menstruation 611 for treatment of cerebroand cardio-vascular disease 611 Rhopaloic acids A-C 121 from Rhopaloeides 121 minimum inhibitory concentration of 121 Rhopaloic acids A-E 121 from Hippospongia sp. 121 Rietone 90 from Alcyonium fauri 90 in NCI's CEM-SS cell line screen 90 structure of 90

1244 Ring-A aromtatic withanolides 1022 Ring-D aromatic withanolides 1022 RNA synthesis inhibitors 113 Rozella allomyces 1057 zoospores of 1057 Rubrorotiorin 492 in Pyrenula hirayamae 492 structure of 492 Saophularia buergeniana 379 Saikosaponin-D 226 apoptotic effects of 226 structure of 226 Salmahyrtisol A 154 against cancer cells 154 Salmahyrtisol B 147 against human gastric carcinoma 147 Salpichrolides E 1036 degradative pathway for 1036 Salpichrolides J 1036 biosynthetic pathway for 1037 Sampsoniones A-J 700 from Clusia plukenetii 700 structures of 701 Saponification 568 SaponinQS-7 236 Saponins 209,214,230,231,817,1075, 1104 as immunostimulants 230 against microbial pathogens 1104 antitumor activity of 214 antiviral activity of 113,138,209, 355 cyototoxic activity of 215 effects on luteinising hormonereleasing hormone 817 effects on (LHRH)-induced LH release 817 immunostimulant activity of 231 in vitro 214 in vivo 214 macrophage activation by 230 Saprolegnia spp. 1054 Saprosma scortechinii 376 Sarcoglane 95,96 structure of 95,96 SarcotinsN 120 against human tumour cell lines 120

cytotoxicity of 120 from sarcotragus 120 Sarcotins O 120 against human tumour cell lines 120 cytotoxicity of 120 Sativolides 1022,1030 18-epz-Scalaradial 150 cytotoxicity by 150 Scalaradial 150 effects on human neutrophils 150 Sclerotiorin 492 from Pyrenula japonica 492 from Penicillium sclerotiorum 492 structure of 492 Scopadulcic acid B 811,815 in vitro 815 structure of 811 Scopadulciol 811 structure of 811 Scopadulin 812 structure of 812 Scoparia dulcis 804 Scoparic acid A 811 structure of 811 Scopoletin 813 structure of 813 Staphyloccus aureus 123,126,128 luffariolides H against 126 luffariolides J against 126 Scrophularia auriculata 379 Scrophularia deserti 380 as cardiotonic 380 as diuretic 380 in cancer 380 in fever 380 in hypoglycemia 380 Scrophulariafrutescens 380 in inflammation 380 Scrophularia ningpoensis 380 Scrophularia scordonia 3 80 in inflammation 380 Scrophularia punicea 264 swertiapunimarin from 264 Secoiridoids 248,305-333,335-353, 355-357 antiinflammatory activity of 353 antileishmanial activity of 357, 1048

1245 antiviral activity of 113,138,209, 355 biological activities of 352 biosynthesis of 335-352 cardiovascular activity of 352 from Oleaceae 305 hypoglycemic activity of 262,356 structures of 305-333 Secoiridoid 5-hydroxy derivatives 261 Secoiridoid glucosides 317 Secologanin 249 condensation of 249 Secologanoside 342 biosynthetic pathway of 342 Secologanoside derivatives 331 origin of 332 structures of 333 Secomanoalide 124 isomer of manoalide 124 Secoxyloganin 344 routes to 344 Securiosides A 227 apoptosis inducing activity of 227 Sedanenolide 638 as anti-convulsant 638 as anti-platelet 638 anti-thrombosis activity of 638 Senkyunolide A 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Senkyunolide H 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Senkyunolide I 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Senkyunolide J 638 in blood viscosity reduction 638 in inhibition of learning/ memory impairment 638 Senkyunolide P 638 in blood viscosity reduction 638 Sensitized tumor necrosis factor 794 Sequoyitol 1000 Serotonin 5-HT2c 188 Sertiamarin 262 anticholinergic properties of 262

Sesquiterpenes 98 structure of 98 Sesquiterpenoids 176,784,902-927 from Aristolochia species 903-927 from Axinnella cannabina 176 from Triterygium wilfordii plant 784 structures of 784 Sesterstatins 1-3 148 against P388 cells 148 cytotoxic activity of 148 from Hyrtios erecta 148 Sesterterpenes 511 against CACO-2 (human colon carcinoma) 512 from genus Fusarium 511 neomangicols A and B 511 structure of 512 Sesterterpenoids 109 from marine 109 Sialytransferase inhibitor 210 of soyasaponin I 210 Sigmoidins A 828 against gram-positive bacteria 828 against gram-negative bacteria 828 from Erythrina sigmoidea 828 Sigmosceptrellin A 197 against Plasmodium falciparum 197 structure of 197 Sigmosceptrellin B 197 structure of 197 Signal transduction enzymes 1153 in drug discovery 1153 use of 1153 Signal transduction pathways 1072 glycyrrhizin in 230 Silyloxydiene 26 Diels-Alder cycloaddition of 26 Simple Benzophenone derivatives (SBDs) 673 Simple polysoprenylated benzophenones 674 from Clusiaceae 674 sources of 674 structures of 676,679,681 Simplex herpes type 1 virus (HSV-1) 355 Skin inflammation 442 subchronic model of 442

1246 Smooth muscle relaxation 650 mechanisms of action of 650 effects of butylidenephthalide on 650 Snider's synthesis 8-15 ofFR901483 8-15 via 1,3-dipolar cycloaddition 8-15 via intramolecular aldol reaction 8-15 Sodwanones 78-79 from Axinella weltneri 78-79 structures of 78-79 Sodwanones G-I 80 cytotoxicity of 80 against cancer cell lines 80 Soil-borne zoosporogenic phytopathogens 1087 Soluble guanylate cyclase 650 Songarosaponin C 232 from Vaerbascum songahcum 232 immunosuppressive activity of 232 Sorbitol cycle 137 Sorensen's synthesis 19-22 of amino-teterahedphenol 19-22 via oxidative cyclization 19-22 Southern African coastline 62 bio-geography of 62 Southern African marine ascidians 85-88 bioactive metabolites from 85-88 Southern African marine invertebrates 61 bioactive natural products from 61 Southern African marine mollusks 96-102 bioactive metabolites from 96-102 Southern African marine soft corals 88-96 bioactive metabolites from 88-96 Southern African marine sponges 70-85 bioactive metabolites from 70-85 Southern African marine worms 64-78 bioactive metabolites from 64-70 Soy isoflavones 1194 effects on nerve growth factor mRNA 1194 Soyasaponins 239 adjuvant activity of 239 Soyasaponin A1 239 adjuvant activity of 239 Soyasaponin A2 239 adjuvant activity of 239

Soybeans 1189 source of isoflavones 1189 Spectrofluorimetric methods 476 Spermicidal activity 795 in vitro 795 Spiranoid 1034 Spiranoid withanolides 1032 from Jaborosa odonelliana 1032 from Jaborosa runcinata 1032 structures of 1033 Spirostanol saponins 225 apoptosis inducing activities of 225 Spiroxins 536 mechanism of action of 536 against ovarian carcinoma 536 Spongia idia 111 Spongia sp. 139 spongianolides A-F from 139 Spongiane diterpenes 100 from Chromodoris hamiltoni 100 Spongidines A-D 146 against human synovial PLA2 146 Spongionolide A 140 total synthesis of 140 stereochemistry of 140 Spongiostatins 70-85 antineoplastic mechanism of action of 72 from Spirastrella spinispirulifer 70 Spongiostatin 1 71,72 effect on glutamate-induced polymerization of tubulin 72 total synthesis of 71 Spongiostatin 1-9 73 cytoxicity in NCI's 60 cell line screen 73 inhibitor of tubulin polymerization 73 Sponins 210 chemopreventive activities of 210 as cytotoxic agents 210 as antitumor agents 210 Sporidesmins 495 structure of 496 Stachytarpheta cayennensis 381 Staphylococcus aureus 1048 STAT6 activation inhibitor 525 TMC-264 525

1247 Stephacidins 603 structures of 603 Stephania tetrandra 990 Steroids 949,953,954 from Aristolochia species 953,954 structures of 953 Steroid saponins 220-221 Stroke 654 cuanxiong for 654 Store-operated calcium channel 650 Streat-plate method 1142 Streptococcus erythaeanus 128 Streptomyces pyogenes 123 Strictosidin 249 Strobilurus renacellus 512 Structure-activity relationships of 1061 Suberitenones A 154 from Suberites sp. 154 Subtrifloralactones A-L 1028 Subtriflora-y-lactones 1022 Swamp cancer 1054 Swern oxidation 26 Sweroside 248,262 hepatoprotective activity of 262 hypoglycemic activity of 262,356 Swertia 261 iridoids from 261 secoiridoids from 261 Swertia alata 263 belidifolia from 263 oleanolic acid from 263 Swertia chronic 262 use in chronic fever 262 Swertia japonica 264 swertiaside from 264 sunburiside-II from 264 Swertia nervosa 263 augustiamarin from 263 sweroside from 263 swertiamarin from 263 Swertiamarin 261,266 Swinholide 83 Swinholide A 84 against cancer cell lines 84 structure of 84 Synovial phospholipase A2 112 Synthesis 10,12-15,722-724 of benzophenones 721-725, 748-761 of(-)-FR901483 8,12-15,21,24 ofketoaldehyde 4,10,13

Synthetic isonitrile derivatives 181 Syphilis 267,822 Erythrina abyssinica against 822 Gentiana karroa in 266,267 Syringa jasminum 346 oleoside 11-methyl ester from 346 Tabernanthe iboga 804 aphrodisiac property of 804 hallucinogenic property of 804 Tabernanthe litoralis 1132 L-aminoacylase of 1132 Tabernanthe tetraptera 807 antibacterial activity of 807 Tabernanthine 814 bradycardisant activity of 814 Talaromyces sp. 481 TAN1251 alkaloids 3,5,40,44 Snider's synthesis of 44 structures of 5 synthesis of 3,40 TAN1251A 4,5,42,46 as muscarinic antagonists 5 Kawahara-Nagumo retrosynthetic analysis of 42 synthesis of 46 (+)-TAN1251A 41-43 Kawahara-Nagumo synthesis of 43 (-)-TAN1251A 46,49-50,52-55 from proline derivative 54-55 Honda's approach to 52-53 Honda's synthesis of 53 Kawahara-Nagumo approach to 54-55 Proline-based approach to 54-55 Snider's synthesis of 46 Wardrop's synthesis of 49-50 TAN1251A-D 5 from Penicillium thomii 89 TAN1251B 5,48 as muscarinic antagonists 5 synthesis of 48 (+)-TAN1251B 48 Snider's synthesis of 48 TAN1251C 4 (±)-TAN1251C 45,51-52 Ciufolini's synthesis of 51-52 Snider's synthesis of 45 TAN1251D 4

1248 (±)-TAN1251D 47 Snider's synthesis of 47 TAN1251A 18 synthesis of 18 Tashironins 411 biosynthesis of 411 Tasnemoxides A-C 129 against cancer cell lines 129 Taxol 794 anti-tumor activity of 794 Taxol-producing microbes 1124 TCM herbs 653 rhizoma chuanxiong 653 rhizoma ligustici 653 sources of 653 Terpenes 510-515 from Armillaria spp. 510-515 Terpene derivatives 197 antimalarial activity of 197 Terpenoids 810,815-816 diterpenoids 815 from Scoparia dulcis 815 in blennorhagia 815 in hypertension 815 in stomach disorders 815 Tetracarbocyclic sesterterpenoids 146 Tetracycline 704 for Staphylococcus aureus 704 Tetrahered vinyl halides 38 cyclization of 38 Tetralones 948 structures of 948 Tetraprenyltoluquinols 94-95 structures of 94-95 Tetraterpenoids 937 from Aristolochia species 937 structures of 938 Thl immune response 233 in production of cytotoxic lymphocytes 233 Thl response 234 against intracellular infection agents 234 Thelpin 479 from Thelepus setosus 479 Theonella swinhoei 83 Theopalauamide 83 from Theonella swinhoei 83 Theopalauamide 84,85 in standard paper disk assay 84

inhibitory effect of 84 structure of 85 Thermophiles 1129 from deep-sea vents 1129 Thermus aquations 1127 Thomitrem 493 Thorecta sp. I l l Thorectandra excavatus

133

Thorectandrol E 132 Thorectandrols A-D 132 Thrombin receptor antagonist 77 from Halichondria cf. moorei 11 Thyroid peroxidase 1201 H-Thymidine incorporation 232 in jurkat T cells 232 TNF-oc production 139 cavernolide effects on 139 Tohottea 855 Topoisomerase I inhibitors 498 topopyrones A 498 topopyrones B 498 Topopyrones 498 against gram-positive bacteria 499 against herpes virus 498 in vitro 498 structure of 499 Tosylamide 23 jV-acylation of 23 Total synthesis 8,71,971,972 of altohyrtin A 71 of aristolochic acid I 971 of(-)-FR901483 8,12-15,21,24 Toxocara canis 1095 Toxoplasma gondii 128 Traditional Chinese medicinal herbs 652 phthalide-containing 652 rraw-geranylnerolidol 110 biosynthetic routes to 1034 from Cochliobolus 110 from Ceroplastse albolineatu 110 Trechnolides 1030,1034 from Jaborosa laciniata 1030 from Jaborosa magellanica 1030 Trechonolide A 1033 Tricarbocyclic sesterpenoids 139 Tricyclic intermediate 55 synthesis of 55 Tricyclic skeleton 36 Kibayashi's synthesis of 36

1249 Trim eric secoiridoid glucosides 317-319 origin of 318 structures of 319 Trimusculus costatus 102 16,19,20-Trioxygenetated withanolides 1028 Tripartite tubular hairs (TTHs) 1093 Triprenylhydroquinones 98 structure of 98 Triprenylquinones 98 structure of 98 Triptolide 784,787-791,796 characterization of 784 enantioselective total synthesis of 789-790 isolation of 784 source of 796 structural modification of 784 synthesis of 787-791 Triterpene saponins 214,232 cytotoxic activity of 214 from caryophyllaceae 232 in vitro of 214 Triterpenoids 671,783,816,937 from Triterygium wilfordii 783 structures of 783 Triterpenoid tripterine 795 antiinflammatory activity of 795 immunosupprressive activity of 795

from Xenia macrospiculata 89 structures of 89-90 Tubemioside II 212 in cancer prevention 212 Tubular necrosis 991 Tumors 863 Aristolochia clematitis in 863 Tumor necrosis factor-oc (TNF-a) 134 Tyrosine kinase inhibitor 1196 genistcin 1196 Unsaturated amino alcohols 85-87 against A549 (non-small cell lung) 87 against LOX (melanoma) 87 against OVCAR-3 (ovarion) human tumor cell lines 87 against SNB-19 (CNS) 87 structures of 86 Untenospongin A 120 from hippospongia 120 Untenospongins A and O 120 coronary vasodilating activity of 120 from Hippospongia 120 Urothelial carcinoma 991 Ustilago violaceae 128 hytiolide against 128

Triterygium wilfordii lTi,ll^,116-lM bioactivities of 774 bioactive compounds from 773 chemical components of 776-784 clinical use of 776 diterpenoids from 776 extracts of 774 side effects of 776 Triterygium wilfordii plant 781 alkaloids from 781 Trypanosoma cruzi 1048 Tryprostatin A 581 structure of 581 Tryprostatin B 581 structure of 581 Tryptamine hydrochloride 1063 attractant activity of 1063 Tsitsikamma favus 74 Tsitsixenicins 89-90 from Capnella thyrsoidea 89

Valdivones 91,92 anti-inflammatory properties of 91 from Alcyonium valdivae 91 structures of 91,92 Valepotriates 256,366 Valerenic acid 255,258 by thin-layer chromatography 258 spasmolytic effects of 255 Valerian 259 film-coated tablets of 259 Valeriana edulis 257,258 acevaltrate from 257 isovaleroxyhydroxydidrovaltrate from 257 in vitro 258 mutagenic properties of 258 valtrate from 257 Valerianajatamansi 255,257-259,260 acevaltrate from 257

1250 essential oils from 258 isovaltrate from 257 lanarin isovalerate from 259 4-methoxy-8-pentyl-1 -naphtholic acid from 259 use in ayurvedic system of medicine 255 volatile oil of 260 Valeriana offwinalis 255,257-259 acetoxyvalerenic acid from 257 actinidine from 259 essential oils from 258 extraction of 259 hyroxyvalerenic acid from 257 isoferulic acid g-aminobutyric acid from 259 kanakoside C from 257 kanakoside A from 257 kanokoside D from 257 tinctures prepared from 259 valerenic acid from 257 use as mild sedative 255 Valtrate 249 Variabilin 97,112,113 anti-inflammatory activity of 97,113 anti-microbial properties of 97 anti-tumor properties of 97 antiviral activity of 113,138,209, 355 from Isotericola variabilis 112 icthyotoxic properties of 97 structures of 97 Variabilin inhibitor 113 of cytosolic PLA2 113 Vasodilatory effects 477 ofgriseoflavin 471-479 Vassobia lorentzii 1025 Verbascum 286,287 against influenza 286 antifungal activity of 287 anti-inflammatory property of 286 antitussive activity of 286 antiviral activity of 286 as astringent 286 as diuretic 286 as emollient 286 as heart stimulant 286 as sedative 286 in allergies 286 in asthma 287

in bronchitis 287 in chicken embryos 286 in chronic hard cough 287 in congestion 286 in fever 286 in migraine 286 in pulmonary complaints 287 in tuberculosis 287 in tumor formation 286 in whopping cough 286 Verbena 284,285 as laxative 285 as rubefavient 284 as tonic 284 hypotensive effects of 285 in asthma 284 in dysmenorrhea 284 in gallstones 285 in healing of wounds 284 in insomnia 284 in nervous coughing 284 in rheumatism 284 Verbena officinalis 284 as antispasmodic 284 as diaphoretic 284 as nerve tonic 284 as relaxant 284 as sedative 284 Veronica (scrophulariaceae) 287 Veronica anagallis -aquatica 287 anti-scorbutic properties of 287 use in bladder troubles 287 Veronica arvensis 287 as diaphoretic 287 as diuretic 287 expectorant properties of 287 Veronica beccubunga 287 iridoids from 287 Verbascum thapus 286 coumarin from 286 flavonoids form 286 iridoid glycosides from 286 oligosaccharides from 286 polysaccharides from 286 Verruculogen 579,580 identification of 579 related compounds 580 structure of 579 Verticillium hemipterigenum 534 Vertihemipterin A 534

1251 Vinorelbine 794 anti-tumor activity of 794 Vipera russelli 985 Vismiaguianones A-E 711 against cancer cell line 711 Vismiaguianone B 711 moderate DNA strand-scission activity of 711 Vitex 281 effects on the pituitary gland 281 in amenorrhea 281 in dysmenorrhea 281 in endometriosis 281 in menorrhagia 281 in menstrual complaints 281 in premenstrual syndrome 281 in treatment of menopause 281 Vitex agnus castus 283,284 against anxiety earlybirth 284 antifungal activity of 284 as diuretic 284 in digestive problems 284 in hyperprolactinemia 284 iridoids from 283 treatment of premenstrual problems by 284 Vitex negunda 281-283 analgesic activity of 283 antibacterial activity of 283 antifungal activity of 283 antihistamine activity of 283 anti-inflammatory activity of 283 antioxidant activity of 283 as antihelmintic 283 as expectorant 283 diuretic properties of 283 hepatoprotective activity of 283 in catarrhal fever 282 in headache 282 in rheumatism 283 in skin infections 283 in swelling of joints 282 pain suppressing activity of 283 use in dyspepsia 283 Vitex trifoliate 283 Vitex verbenaceae 280 Voacangine 809 structure of 809 Volatile organohalogen 526 l-chloro-5-heptadecyne 526 Voltage-operated calcium channel 650

Wardrop's formal synthesis of 32-55 Ct)-demethylamino FR901483 32-35 Water-soluble artesunate 190 in treatment of severe malaria 190 WithaferinA 1019,1020 from Acnistus arborescens 1020 from Withania somnifera 1019 structure of 1020 Withajardins 1022,1038,1039 structures of 1039 Withametelins 1022 Withanolides 1019-1023,1046-1045, 1048 antifeedant properties of 1040-1045 as anticancer compounds 1020 as anti-feedant compounds 1020 as anti-inflammatory compounds 1020 as antitumor compounds 1020 as cytotoxic compounds 1020 bactericidal activities of 1048 biological activity of 1019,1040 cancer chemopreventive activity 1046-1048 chemistry of 1019,1023 classification of 1021-1023 from South American solanaceae 1019 immunomodulating activity of 1020

in Ajuga parviflora 1020 in Cassia siamea 1020 insecticidal properties of 1040-1045 occurrence of 1020 phytotoxic activity of 1048 trypanocidal/leishmanicidal activity of 1048 Withaphysalins 1022 Xanthobaccin A 1107 structure of 1107 Xanthochymol 704,755,760,761-764 antibacterial activity of 761,762 antifungal activity of 761,762 anti-MRSA activity of 704

1252 antioxidant activity of 761,762 antiviral activity of 761,762 biological activities of 761 I3 C-NMR data for 761 COSY correlations for 760 cytotoxic activity of 761,763 DEPT data for 761 'H-NMR data for 761 'H-spectrum of 755 HMBC data for 761 molluscicidal activity of 761,763 trypanocidal activity of 761,764 Xanthones 671 Xanthone-O-glucosides 263 as anti-convulsant 263 as cardiovascular stimulant 263 as CNS depressant 263 Xenicane diterpenes 93 against A-549 human lung carcinoma 93 against HT-29 human colon carcinoma cell lines 93 against MEL-28 human melanoma 93 against P-388 mouse leukaemia 93 Xerophenone A 699 structure of 699 Xerophenone B 699 structure of 699 Yingzhaosu A 192 structure of 192 Z- Butylidenephthalide 638 as anti-angina 638 Zahavins 92-94 Zerynthia polyxena 1000 Z-Ligustilide 638 as anti-platelet aggregation/ anti-thrombosis 638 Zoospores 1060,1066,1071,1074 bioassay-guided chromatographic techniques for 1074 developmental transitions of 1066 differentiation of 1066 metabolites affecting motility of 1074 receptors in 1071

responses of 1060 viability of 1074 Zoospore regulation 1112 bioassay methods for 1112 Zoospore lytic factors 1084 from Ginkgo fruits 1084 Zoosporicidial activity 1093 of polyflavonoid tannins 1093 Zuihonin B 941 from Aristolochine arcuata 941