Structure and Chemistry (Plart D)

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studies in Natural Products Chemistry Volume 17 Structure and Chemistry (Part D)

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

Vol. 1 Vol. 2 Vol. 3 Vol. 4 Vol. 5 Vol. 6 Vol. 7 Vol. 8 Vol. 9 Vol.10 Vol.11 Vol.12 Vol.13 Vol.14 Vol.15 Vol.16 Vol.17

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)

studies in Natural Products Chemistry Volume 17 structure and Chemistry (Plart D)

Edited by

Atta-ur-Rahman H.EJ. Research Institute of Chemistry, University of Karachi, Karachi 75270, Paicistan

1995 ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon - Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands

ISBN: 0-444-82265-8 © 1995 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science B.V., Copyright & Permissions Department, P.O. Box 521,1000 AM Amsterdam, The Netherlands. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the copyright owner, Elsevier Science B.V., unless otherwise specified. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. This book is printed on acid-free paper. Transferred to digital printing 2005

FOREWORD

The rapid advances in chromatographic procedures, spectroscopic techniques and pharmacological assay methods have resulted in an increasing number of new and interesting natural products being discovered from terrestrial and marine sources. The present volume contains comprehensive reviews on some of the major advances in this field which have taken place in recent years. The reviews include those on novel metabolites from marine gastropods, the chemistry of marine natural products of the halenaquinol family, secondary metabolites from Echinoderms and Bryozoans, triterpenoids and aromatic compounds from medicinal plants, chemistry and activity of sesquiterpenes from the genus Lactarius, the chemistry of bile alcohols, antifungal sesquiterpene dialdehydes, annonaceous acetogenins, nargenicin macrolides, lignans and diarylheptanoids. Tropane alkaloids and phenolies formed by root cultures are also reviewed. Articles on natural Diels-Alder type adducts, the use of computer aided overlay for modelling the substrate binding domain of HLADH, applications of O NMR spectroscopy to natural product chemistry and the use of biological raw materials in synthesis should also be of interest. It is hoped that the present volume will continue to meet the standards set by the earlier ones of this series and provide much material of interest to a large number of natural product chemists. I wish to express my thanks to Dr. M. Saleh Ajaz and Mr. Athar Ata for their assistance in the preparation of the index. I am also grateful to Mr. Wasim Ahmad, Mr. Asif Khan and Mr. Shabbir Ahmad for the typing work and Mr. Mahmood Alam for secretarial assistance.

December 1994

Atta-ur-Rahman Editor

This Page Intentionally Left Blank

Vll

PREFACE Since days immemorial natural products have had a profound impact on humankind. They were our earliest sources of drugs, derived from traditional herbal medicines. They reaped havoc on man in the form of toxins that would kill or maim people, either in natural disasters, like outbreaks of red tide or ergotism, or in incidences inflicted by man, as the executions in old Greece or the poisonings of adversaries that were often a means of settling power struggles throughout history. And they enriched human life in the form of spices and fragrances. Last not least, they have led to the development of the science of organic chemistry, which started out as the chemistry of natural products. Stimulated by important advances in the biological sciences, particularly in the molecular biology of diseases and in the new field of ecology, the last two decades have seen a tremendous renaissance in the field of natural products. We are now accutely aware of the value of the chemical diversity represented by natural products as a source of new leads for bioactive drugs and of the utility of bioactive natural products as tools in dissecting and analyzing life processes at the molecular level. And we are developing an ever keener sense of the importance of natural products in governing the complex relationships of living organisms in our ecosystems. Concomitantly our view of the role of natural products has changed drastically over the years. While at one time they were considered mere waste products of a luxuriating metabolism, the view now prevails that the synthesis of such compounds represents an evolutionary advantage to the producing organism. With the renewed broad interest in natural products it is most appropriate that a continuing series of publications is dedicated to the topic of natural products chemistry. Professor Atta-Ur-Rahman with his worldwide connections to all the leading natural products chemists of our time is the ideal person for the task of editing this series. He has brought this series to life and has done an outstanding job of sustaining it. The present volume again presents an eclectic mix of articles on many different topics ranging from marine natural products, microbial and plant metabolites all the way to topics like molecular modeling, lÔ-NMR spectroscopy or the role of biological raw materials in synthesis. I hope its readers will enjoy this volume as much as I did, and I wish it the same success that its predecessors have enjoyed. Heinz G. Floss University of Washington Seatde, Washington


IX

CONTENTS Foreword Preface Contributors

v vii ix

Novel secondary metabolites of marine gastropods M. ALAM AND K.L. EULER

3

Total synthesis and absolute stereochemistry of novel biologically active marine natural products of Halenaquinol family: Theoretical studies of CD spectra NOBUYUKI HARADA AND TATSUO SUGIOKA

33

Bryozoan secondary metabolites and their chemical ecology A.J. BLACKMAN AND .T.T. WALLS

73

Structure and biological activity of triteipenoids and aromatic compounds from medicinal plants R. AQUINO, F. DE SIMONE, N. DE TOMMASI AND C. PIZZA

113

Sesquiteipenes and other secondary metabolites of genus Lcictarius (Basidiomycetes): Chemistry and biological activity G. VIDARI AND P. VlTA-FINZl

153

Stmcture and biosynthesis of bile alcohols: Disorders of choylesterol side-chain oxidation in cerebrotendinous xanthomatosis BISHAMBAR DAYAI., GERALD SALEN AND SARAH SHEFER

207

Antifungal sesquiteipene dialdehydes from the Warhtirîa plants and their synergists ISAO KUBO

233

Detenuination of relative and absolute configuration in the Annonaceous acetogenins ELIZABETH A. RAMIREZ AND THOMAS R. HO YE

251

The chemistry of the nargenicin macrolides .lAMES KALLMERTEN

283

Some aspects of the chemistry of lignans R.STEVENSON

311

The chemistiy of natural diarylheptanoids G.M. KESERU AND M. NOGRADl

357

Tropane alkaloids in root cuUures of Solanaccous plains M. SAUERWEIN, K. ISHIMARU, K. YOSHIMATSU AND K. SHIMOMURA

395

Phenolics in root cultures of medicinal plants K. ISHIMARU AND K. SHIMOMURA

421

Chemistry and biosynthesis of natural Diels-Alder type adducts from Moraceous plants TARO NOMURA, YOSHIO HANO AND SHINICHI UEDA

451

Modelling the substrate binding domain of horse liver alcohol dehydrogenase, HLADH, by computer aided substrate overlay MAIJA AKSELA AND A.C. OEHLSCHLAGER

479

Applications of 'Ô NMR spectroscopy to natural products chemistry DAVID W.BOYKIN

549

The role of biological raw materials in synthesis JOHN H.P. TYMAN

601

Subject Index

655

XI

CONTRIBUTORS

Maija Aksela

Depaitmcnt ol' Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6

Maktoob Alam

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Houston,, Houston, Texas 77204-5515, U.S.A.

R. Aquino

Dipaitimento di Chimica delle Sostan/e Naturah, Universita Degli Studi di Napoli 'Tederico H", Via D. Montesano 49, 80131 Napoli, Italy

Adrian J. Blackman

Chemistry Department, University of Tasmania, P.O. Box 252C, Hobart, Tasmania-7001, Australia

David W. Boykin

Department of Chemistry, Georgia Slate University, Atlanta, Georgia 30303, U.S.A.

Bishambar Dayal

Department of Medicine, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103-2406, U.S.A.

K.L. Eiiler

Department of Medicinal Chemistry and Pharmacognosy, College of Phannacy, University of Houston,, Houston, Texas 77204-5515, U.S.A.

P. Vita-Finzi

Dipartimento di Chimica Organica, Dell' Universita di Pavia, 27100 Pavia, Italy

Yoshio Hano

Toho University, Faculty of Pharmaceutical Sciences, 2-2-1 Miyama, Funabashi, Chiba 274, Japan

Nobuyiiki Harada

Institute for Chemical Reaction Science, Tohoku University, 2-1-1 Kalahira, Aoba Sendai 980, Japan

Thomas R. Hoye

University of Minnesota, Department of Chemistiy, 207 Pleasant Street, S.E. Minneapolis, MN 55455-0431, U.S.A.

K. Ishimaru

Breeding and Physiology Lab., Tsukuba Medicinal Plant Research Station, National Insliule of Hygienic Sciences, I Hachimandai, Tsukuba, Ibaraki-305, .lapan

James Kallmeiten

Syracuse University, Department of Chemistry, Room 1-041, Center for Science & Technology, Syracuse, New York 13244-4KK), U.S.A.

G.M. Keseru

Research Group for Alkaloid Chemisuy of the Hunganan Academy of Sciences, Technical Univeisily of Budapest, H-1521 Budapest P.O.B. 91, Hungaiy

Isao Kubo

Professor of Natural Products Chemistry, Division of Insect and Microbial Ecology, College of Natural Resources, University of California, Berkeley, CalifomL 94720, U.S.A.

Mihaly Nogradi

Research Group for Alkaloid Chemistry of the Hungarian Academy of Sciences, Technical University of Budapest, H-1521 Budapest P.O.B. 91, Hungary

Taro Nomura


A.C Ochlschlager

Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A IS6

C. Pizza

Dipartimento di Chimica delle Sostan/e Naturali, Universita Degli Studi di Napoli "Federico 11", Via D. Montesano, 49, 8()I31-Napoli, Italy

Elizabeth A. Ramirez

University of Minnesota, Department of Chemistry, 207 Pleasant Street, S.E. Minneapolis, MN 55455-0431, U.S.A.

Gerald Salen

Department ol' Medicine, University ol' Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103-2406, U.S.A.

M. Sauerwein

Breeding and Physiology Lab., Tsukuba Medicinal Plant Research Station, National Instiute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki-305, Japan

Sarah Shefer

Department o\' Medicine, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103-2406, U.S.A.

Koichiro Shimomura

Head of Breeding and Physiology Lab., Tsukuba Medicinal Plant Research Station, National Instiute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki-305, Japan

F. De Simone

Dipartimento di Chimica delle Sostanze Naturali, Universita Degli Studi di Napoli 'Tederico \V\ Via D. Montesano, 49, 80131-Napoli, Italy

R. Stevenson

Department of Chemistry, Brandeis University, P.O. Box 9110, Waltham, MA 02254-9110, U.S.A.

Xlll

Tatsuo Sugioka

Inslitule lor Chemical Reaction Science, Tohoku University, 2-1-1 Katahira, Aoba Sendai 980, Japan

N. De Tommasi

Dipartimento di Chimica delle Soslan/e Naturali, Universita Dcgli Studi di Napoli ^'Federico H",Via D. Monlesano, 49, 8()13l-Napoli, Italy

John H.P. Tyman

Department of Chemistry, Brunei, The University of West London, Uxbridge, Middlesex UB8 3PH, U.K.

Shinichi Ueda


Giovanni Vidaii

Dipartimento di Chimica Organica, Dell' Universita di Pavia, 27100 Pavia, Italy

Justin T. Walls

Zoology Department, University of Tasmania, P.O. Box 252C, Hobart, Tasmania-7001, Australia

K. Yoshimatsu

Breeding and Physiology Lab., Tsukuba Medicinal Plant Research Station, National Insliute of Hygienic Sciences, I Hachimandai, Tsukuba, Ibaraki-305, Japan


structure and Chemistry


Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 17 © 1995 Elsevier Science B.V. All rights reserved.

Novel Secondary Metabolites of Marine Gastropods M . Alam and K.L. Eiiler

Mollusks have attracted the attention of humans since prehistoric times. People historically have associated certain powers with plants and animals that resembled parts of the human anatomy. Mollusks would be a classical example in thatacertain type of power was associated with cowry (anatomical resemblance to female genitalia) and was thought to be transferred to the possessors of cowry. The first written report about mollusks appeared in Aristotle's "History of animals", which contained a detail discussion of Mediterranean mollusks. During the late fourteen and fifteenth centuries the collecting and studying of shells of marine mollusks became hobbies of gentlemen fix)m well-to-do families with interests in natural history. Since the publication of the first book on marine natural products by Professor Scheuer— Chemistry of Marine Natural Products (1) a number of books (2-6) have been published on marine natural products. Similarly, a number of reviews (7-9) dealing with various aspects of the chemistry of marine mollusks have also appeared in the literature. In the present review the authors have attempted to present a summary of the literature dealing with novel compounds from marine mollusks since 1987. For compounds before 1987 the readers are referred to excellent reviews authored by P. Karuso (10) and H. C. Krebs (11). In order to give readers a broader scope of the novel compounds, examples from all three subclasses of the phylum Mollusca—Prosobranchia, Opisthobranchia and Pulmonata have been selected. During the early seventies the occurrence of a variety of compounds from marine mollusks raised serious questions about their origin. Because gastropods are voracious eaters with virtually every type of feeding habit, it was postulated early on that novel compounds from mollusks may have had their origin in the dietary sources of these invertebrates. One of the earliest reports supporting this hypothesis came from the laboratory of Professor Schantz, who showed that saxitoxin [1 ] (one of a group of neurotoxins commonly known as paralytic shellfish poisons), which was originally isolated from the mollusk Saxidomus gigantius, was actually produced by H2NOCO^

HaNOCOs H R-N

^ ^ ^ OH "" 1

' O" OS03H 2

thedinoflagelbitsGonyaulaxcatenella (12). Similarly, saxitoxin derivatives commonly known as

gonyautoxins [2] isolated initially from the clam Mya arenaria were later determined to be secondary metabolites of another species of Gonyaulax— G. tamarensis (=Alexandrium tamarensis) (13). The first report linking a brominated secondary metabolite of the sea hare Aplysia kurodai with its diet, the red alga Laurencia sp„ appeared in 1967 (14). Similarly, earlier work from Professor Moore's laboratory (15) reported the isolation of deromoaplysiatoxin (3) from the blue green alga Lyngbya gracilis, Debromoaplysiatoxin had been isolated previously from the digestive gland of the stSiharQ Stylocheili4S longicauda (16). The presence of 3 in L. gracilis again suggested a direct relationship between the diet and novel metabolites of the sea hare.

Herbivorous marine prosobranchs of the genus Aplysia feed on red, brown, green or bluegreen algae. During the late seventies and early eighties a number of terpcnoidal secondary metabolites were isolated from various species of Aplysia and were assumed to be accumulated by the mollusks from the dietary sources consisting of red algae (17), brown algae (18-22) and green and blue-green alga (23). During the middle eighties research on the secondary metabolites of various species of Aplysia continued to reward researchers with novel compounds. An examination of various species of Aplysia for the presence of aromatic compounds resulted in the isolation of aplysin and related compounds [4-8] (24), which were also found to be present in the red alga of Laiirencia species that was consumed by the sea hare (25, 26). A chemical investigation (27) of

JUC/N^R2

4. 5. 6. 7. 8.

Ri Ri Ri Ri Ri

=r Br, R2 = H = R2 = H = H, R2 = OH = Br, R2 = OH = H, R2= Br

the mid-gut gland of another Aplysia - A. kurodai, collected from Izy-Shimode Beach, Southwest CI

^^4 Br-^^'^^Br 10

CI

J-^^ ^ Jr""! B / ^ > 1 11

Br-^^'^'"'C1 12

Japan, has resulted in the isolation of four isomeric compounds -aplysiapyranoid A - D [9 -12]. The absolute configuration of aplysiapyranoid B was later established by x-ray crystallography (28) Quite often the type of compound isolated from Aplysia species depends on the location from which they were collected, and therefore, on the algae upon which they feed. A. kurodai collected from Mei Prefecture of Japan was found to contain an extended diterpene (with a prenylated eudismane skeleton)-aplysiadiol [13] and its methyl derivative [14] (29) . It could safely be assumed that a brown alga on which this prosobranch feeds was the actual source of aplysiadiol. Recently a biogenetic type synthesis of 14 has been reported (30).

13. R = H 14. R = CH3

An investigation of the more polar fraction of, presumably, the above mentioned collection of A. kurodai, from the Mei Prefecture of Japan resulted in the isolation of three cytotoxic alkaloids — aplaminone [15], neoaplaminone [16] and neoaplaminone sulfate [17] (31). The biogenetic origin

OCH,

(CH3)3N

(CH3)3N

15

16. R = H

17. R = SO3H

of these alkaloids is still open for discussion. However, they could have been derived from tyrosine ortyramine. An examination of another collection of A. kurodai, presumably from Japan, has resulted in the isolation of aplykurodin A [ 18] and aplykurodin B [19] (32), which could have been derived from a steroidal precursor, which was degraded by the sea hare to produce aplykurodin A. However, its presence in the dietary source of the sea hare (such as a brown alga) can not be ruled out at the present time. A bioassay directedfractionationof A. ^wroda/ collected from Mei Prefecture in Japan has

also resulted in the isolation of a novel compound-aplydilactone [2 0] (33) which is an example example of a cyclopropane-ring-containing fatty acid lactone. Cyclopropaneringcontaining fatty acids are quite rare in nature (34,35). Aplydilactone is reported to increase the activity of phospholipase A2— an enzyme that is responsible for the removal of fatty acids from C2 of phospholipids (33).

A subsequent analysis of specimens of A. kurodai collected in 1985 from the Mei Prefecture in Japan yielded a new labdane-type diterpene, 6/7/-aplysin-20 [21], which is a diastereomer of aplysin-20 [2 2](36), together with the enantiomer of isoconcinndiol [2 3](37).

An investigation of the the chemical defense of Aplysia fasciata collected from the Bay of Naples has resulted in the identification of 4-acetylaplykurodin B[24] and aplykurodinine B [25] (38). Recently a collection and chemical examination oi Aplysia Juliana from the intertidal zone of the Karachi, Pakistan coastline yielded a new diterpene lactone-angasiol acetate [26], the

structure of which was established by x-ray crystallography (39).

CH3OCO

24

25

26

Red algae belonging to the genus Laurencia have been reported to to be a rich source of halogenated sesquiterpenes (40). A species of the sea hare- Aplysia dactylomela, which feeds on Laurencia species, has been reported to concentrate about 25 different chlorochamigrene analogues from its diet. In 1986 Sakaietal (41) reported the isolation and chemical structures of new halogenated chamigrenes[27 - 30] from the digestive glands of A. dactylomela, collected from Hisamatsu Miyako, Okinawa. In addition to halogenated chamigrenes two non-terpenoids— brominated diphenylether [31] and maneonene [32] were also isolated. It could be speculated that 3 1 and 3 2 are derived from the dietary sources of the sea hare. However, anisoles are more common in marine invertebrates, such as sponges of the genus Dysidea, as compared to marine algae.

y^-

^f=n

^

Another collection of i4. dactylomela collected from Kohoma Island, Okinawa, on the other hand was found to contain (42) cuparene-related sesquiterpenes - cyclolaurene [33], laurinterol [34], cyclolaurenol [35], cyclolaurenol acetate [36], cupalaurenol [37] and cupalaurenol acetate [3 8]. The authors failed to comment on the presence or absence of chamigrenes in these samples.

33

34

35

CH3OCO

CH3OCO.

36

37

38

Specimens of A. dactylomela from the Caribbean Sea (43) have been reported to contain an uncommon sesquiterpene-dactylol [3 9] with an 8.5 fused ring skeleton. The likely source of dactylol has been speculated to be the red alga Laurencia poiti (43).

39

A new compound from a new class of diterpenes-dactylomelol [40] was also isolated from the specimens of A. dactylomela, presumably collected from the Canary Islands (44). Dactylomelol could be envisioned arising from the cyclization of the two internal double bonds of geranyllinalol to form carbocyclic rings.

40

An examination (45) of juvenile A. dactylomela feeding on the brown alga Stypopodium zonale from Vega Baja on the north coast of Puerto Rico resulted in the isolation of epitaondiol [41] and 3-ketoepitaondiol [42] along with stypodione [4 3] . A comparison of the A. dactylomela

extract with an extract of S, zonale confirmed the dietary nature of the metabolites of juvenile A. dactylomela.

42

41

43

In order to confirm the biotransformation capability of the sea hare -Aplysia punctata^ Quinoa et al. (46) studied a number of marine algae for the presence of halogenated monoterpenes and compared the hplc and gc profiles of the extracts of the hepatopancreas of A. punctata with algal extracts. The results of this study showed a direct correlation between the chromatographic and gc-ms profile of the secondary metabolites of Plocamium coccineum and A. punctata. However, no biotransformation capability was noticed. Nudibranchs are quite often brightly colored, shelUess mollusks which differ from sea hares in that they do not have a mantle cavity and in their dietary habits by feeding on sponges and corals only. In spite of being brightly colored and shell less nudibranchs have few predators. Many nudibranchs employ a chemical defense to deter predators. While a few species are known to synthesize secondary metabolites (47,48), the majority concentrate secondary metabolites from their dietary sources and use them for their defense. Quite often these nudibranchs are capable of concentrating the most repugnant of the minor metabolites of a sponge or coral, which uses these compounds for its own defense against predators. An investigation of the nudibranch Chromodoris funarea from Palau (49), has resulted in the identification of the bromophenylether [4 4], 0-methylfurodysinin [4 5] and hydroperoxide [4 6]

-OOH

10 However, when the animals were stored in acetone for four weeks, the acetone extract gave furodysinin [47], furodysin [4 8], furodysinin lactone [4 9] and epoxylactone [5 0]. Isolation of the artifacts 47 - 50 illustrates the effect of storage and solvent (acetone) on chemical structures of nudibranch*s metabolites.

48

47

49

50

A comparison of the secondary metabolites of C. funerea collected from different locations of Kaibakku Lake in Kaibakku Island, Palau (50), has resulted in the identification of 12-epi-sca\aiin [51], deoxoscalarin [52], luffariellin C [5 3], lufariellin D [54] and ketodeoxoscalarin [55]. 12-E/7/-scalarin [51] and 52 are metabolites of the sponge Spongia officialis and S. nitens respectively (51,52), while 53 and 5 4 are possibly derived from the sponge Liffariella variabilis, which has been reported (53) to contain luffariellin A [56] and luffariellin B [57]. The metabolite profile of C, funerea collected from Kaibakku Lake differs sharply from the profile of C. funerea collected from Iwayama Bay. An examination of the environment revealed that sponges of the genus Dysidea were absent in Kaibakku Lake, possibly

CX:OCH3

53 . R = H 56 . R = OH

H3COCO " ^ \

54. R = H 57. R = OH

11

H3COCO " ^ V _ o

55. X = 3- or 1-kelo because of the vegetation which heavily shades the shallow side of the lake. The resulting shadow quite possibly prevents Dysidea from growing, since Dysidea depends upon either epiphytic or symbiotic cyanobacteria to provide important metabolites required for their growth. These microorganisms in turn require sunlight for their own growth. Chemical studies on the dorid nudibranch Chromodoris macfarlandU collected from Scripp's Canyon, La Jolla, resulted in the isolation of two aromatic norditepenes-macfarlandin A [58] and macfarlandin B [59](54). Both 58 and 5 9 are closely related to aplysulphurin [60] which is a metabolite of the sponge Aplysilla sulphurea (55). A further investigation (56) of the more polar

58

59

60

fractions of the extract resulted in the isolation of three new rearranged diterpcne acetates, which were identified as macfarlandin C [61], macfarlandin D [6 2] and macfarlandin E [6 3]. A comparison of the concentrations of 58 - 63 in seven individual animals led the authors to conclude that that C. macfarlandi must feed on two different aplysillid sponges, one of which contains 6 1 - 6 3 .

OCOCH:.

OCOCH, CH,OCQ

63

12 Another species of Chromodoris -C. norrisi, collected from a mangrove lagoon on the Island of San Jose in the Gulf of California, has been reported to contain norrisolide [6 4] and macfarlandin E [63] along with rearranged diterpene polyrhaphin A [65] and shahamin C [66](57). A comparison of the concentrations of norrisolide, macfarlandin E, polyrhaphin A and shahamin C from C. norrisi with the concentration of rearranged diterpenes from the sponge Aplysilla polyrhaphis collected from the same location confirmed the dietary origin of these metabolites. The data also supported the assertion that this nudibranch does not preferentially concentrate any specific metabolite. Of the nine diterpenes produced by Aplysilla polyrhaphis, only four, 63 - 66, are retained by the nudibranch; this suggests that these metabolites are of importance to the

OCCX:H3

Hjccxro^

H3COCO

Ô

H3COCO—.„

X

.0

nudibranch. Indeed, shahmin C was found to deter predation (feeding) by rainbow wrasse (Thalassoma lucaslu-num ) at a 100 mcg/mg food level. A study of the chemistry of the defense allomones present in C. luteorosea from the Mediterranean Sea (58) resulted in the identification of luteorosin [67], 12-^p/-aplysillin [68], 12epi -12-deacetoxyaplysillin [6 9] and macfarlandin A [58]. Macfarlandin A is a known metabolite of C. macfarlandi (54). Based on a report (59) that C luteorosea feeds on sponges of Spongionella species, the authors speculated that the real source of 67 - 69 is an encrusting species of Spongionella.

OR

67

OCOCH3

68. R = COCH3 69. R = H

The importance of dietary compounds in the determination of the metabolic profile of Chromodoris species is illustrated by the presence of a rearranged diterpene-- chromodorolide A [7 0] from the Indian Ocean's nudibranch Chromodoris cavae (60). A subsequent study of a second

13 collection (61) resulted in the isolation of 7 0 along with chromodorolide B [71]. The biosynthesis of chromodorolides A and B could be envisioned to proceed via the formation of the noirisane skeleton [72], {e.g., the diterpene norrisolide [73](62)) followed by the formation of a new carbon-carbon bond (CI2 -C17). Cyclyzation of the C l l carboxyl group with the bisacetal-oxalane ring would then give the appropriate heterocycles.

CH3OCO..,

CH30C0-«n

100 40

4.4 CATECHINS From the blood-red latex of Croton draconoides, used in Peruvian folk-medicine (53) (+) gallocatechin 68 and (-) epigallocatechin 69 have been isolated. (-) Epicatechin 70, (+) catechin 71 and two galloylesters 72 [(-) epicatechin-3-O-gallate] and 73 [(+)-catechin-7-0-gallate] were isolated from the diuretic and antiinflammatory extracts of Detarium microcarpum , a medicinal plant from Senegal (54). 3'

A //; Table 13. Structures, anti HIV-1 activity and toxicity of flavan 68-73. Position 68 69 70 71 72 73

3 (+)0H (-)0H (-PH (+PH (-)gallate (+PH

5 OH OH OH OH OH OH

6 H H H H H H

7 OH OH OH OH OH Ogallate

3' OH OH OH OH OH OH

4' OH OH OH OH OH OH

5' OH OH H H H H

EC50 5 inactive 2 4 1 10

TC50 >80 >100 >100 >100 >100 >100

4.5 QUINIC ACID DERIVATIVES Two caffeoylquinic acids 95 (3,4,5-tri-O-caffeoylquinic acid) and 96 (4,5-di-O-caffeoyIquinic acid), as well as caffeic acid 97 and synapoic acid 98, have been isolated for the first time from Securidaca longipedunculata

(55), and 3,4,5-tri-O-galloylquinic acid 100 has been isolated from Guiera

senegalensis. (56) (Fig. 11).

145 4.6 ANTIVIRAL ACTIVITY Flavonoids are generally known for their anti-inflammatory, antiallergic and anticarcinogenic activity and some of them have mutagenic properties (57). More recently certain flavonoids have been shown to possess antiviral activity. For example quercetin seems to be effective against herpes simplex virus type 1 (HSV-1) , parainfluenza virus type 3 (Pf-3) and Sindbis virus (SV-1) but it was inactive against poliovirus type 2 and 3 and adenovirus type 3 and 4 infections. Morin was shown to be effective against herpesvirus-suis but rutin did not have this activity. Dihydroquercetin (taxifolin) and dihydrofisetin were virucidal against HSV-1 and herpesvirus-suis but Pf-3 and poliovirus type 2 and 3 were resistant to these two flavonoids. Quercetin, morin, luteolin and fisetin were also active against pseudorabies virus. Subsequent studies confirming earlier observations have shown that naturally occurring flavonoids inhibit infectivity and/or replication of certain RNA (RSV, respiratory syncytial virus, Pf-3, poliovirus ) and DNA (HSV-1) viruses (57). Quercetin and hesperitin affect one or more of the biochemical processes involved in the intracellular replication of each of the viruses studied. Quercetin and catechin were active inhibitors of infectivity, while naringenin totally lacked activity. Thus it is evident that important structure-activity relationships exist between flavonoids and they possess a variety of antiviral activities. More recent studies have shown that 4'hydroxy-3-methoxy-flavones such as 3-methyl quercetin block the replication of poliovirus, apparently by selective inhibition of genomic RNA synthesis (58) and certain flavans such as 4'-6dicloroflavan inhibit human rhinovirus replication by interacting specifically with the VIPI capsid protein to prevent virus uncoating (59). In the past few years the inhibitory effects of flavonoids on the reverse trascriptase (RT) of certain retrovirus including human immunodeficiency virus (HIV) as well as cellular DNA polymerase, have been studied (60-61). As a part of our screening of natural compouds as potential anti -AIDS agents, we isolated flavones, flavans and flavanones as well as quinic acid derivatives from medicinal plants and studied their in vitro anti HIV-1 activity. The bioassays were performed on C8166 cells infected with HI V-III-B strain. Formation of syncitia and gpl20 antigen production were observed. Cell viability of infected cells and cytotoxicity of uninfected cell controls were measured by the MTT-FORMAZAN method (62), and gpl20 antigen production was measiired by ELISA (63). EC50 (the concentration of drug which reduces by 50% the production of gpl20 in infected C86166 cells). TC50 (the concentration which causes 50% of cytotoxicity uninfected C8166 cells) were evaluated. Results are shown in tables 10, 12 and 13. The flavans 68, 70,71,72 and 73 exhibit the most pronounced selective anti HIV-1 activity. In particular the 3-O-galloyl ester derivative of (-) epicatechin, 72, consistenly exhibited the greatest activity (EC5o=l îg/ml, selectivity index >100) followed closely by 70 (-) epicatechin. Differences between isomers were noted in the lower activity of (+) catechin 71 with respect to (-) epicatechin 70, Substitution of the hydroxyl group at C-7 by a gallate moiety in 73 caused a reduction in the antiviral activity and increased the cytotoxicity (Table 13). Of the seventeen flavones (unsaturated pyrone ring ) tested, only two myricetin 57 and kaempferol-3-O-glucoside 61 caused significant inhibition of HIV-1 infection at non toxic concentrations (Table 10). The selective activity of myricetin 57 (selectivity index =20) contrasted with the inactivity of quercetin 56 which differed only in the absence of a 5'-hydroxyl group, indicating that all three hydroxyl groups at 3',4',5' positions

146 of ring B are required for the activity. Compound 58 (3-O-rhamnoside of myricetin ) and compound 59 (3-O-rhamnoside of quercetin) exhibited only very slight selective antiviral activity. In contrast glycosidation at C-3 of kaempferol 54 , which lacks a further 3' hydroxyl group on ring B, elicited selective anti HIV activity in 61 (selective index=10). None of the five flavanones (carbonyl at position 4 of the saturated pyrone ring) tested exhibited activity against HIV-1 infection (Table 12). Ravanones are generally more cytotoxic than the flavans and flavones studied. The active flavans 6873 and flavons 51, 56 and 57 were also tested against HIV-2, SIV and Herpes simplex virus (HSV) infections and elicited comparable activities (Table 14). Compound 72 was in any case the most active compound (41). Table 14. Antiviral activities of flavonols and flavans against HIV-2, SIV and Herpes simplex virus. SIV Herpes simplex Herpes simplex HIV.2 EC50 EC50 TC50 EC50 68 8 10 32 >80 69 >100 >100 10 >50 70 2 2 10 >50 71 5 5 20 >50 72 1 1 1 >100 73 ND ND 10 1 >100 C8166 cells were infected with HI V-2ROD or SIVMAC and Vero cells were infected with Herpes simplex virus type 1. The caffeoylquinic acids 95 and 96, caffeic acid 97, synapoic acid 98 and the structurally related rosmarinic acid 99 and 3,4,5-tri-O-galloylquinic acid 100 whose anti HIV-1 activity has already been reported, were tested for anti HIV-1 activity in the same experimental conditions used to test flavonoids. The results are presented in Table 15. While 97 and 99 were found to be inactive in inhibiting viral replication, 96 and 100 showed similar antiviral activity and 95 showed much higher selective anti HIV-1 activity. EC50 values are quite comparable for 95,96 and 100 but significant Compound

differences are seen, however, in toxicity of 95 (Table 15). Although similar in potency, and having comparable EC5o's the lower toxicity of 3,4,5-tri-O-caffeoylquinic acid 95 gives a higher selectivity index of about 3(X). This compound also exhibits a highly selective inhibition of HSV type 1 replication, comparable to that of ganciclovir. Thus the antiviral action of these compounds is not peculiar to HIV (55). Table 15. Antiviral activity of compounds 95-100.

HlV-lniB 1HIV-2ROD SIVMAC Herpes 1 Compound EC50* TC50 EC50 EC50 TC50 EC50 95 100 0.32 20 2 200 0.08 96 1 40 0.6 8 2 100 0.16 100 0.15 ND ND ND ND 1 15 97 200 200 >200 1 >200 200 >50 98 200 200 ND ND ND ND 99 100 40 100 80 150 20 >1000 0.01 0.016 0.02 ND ND Azr Ganciclovir 1 ND 1 ND ND 1 ND 100 0.08 '''EC50 values are the concentrations of compound in mg/ml which inhibited by 50% the production of gp 120 of HIV or SIV, or herpes simplex virus type 1 surface antigens.

147 Mechanism of action Like a number of polyanionic compounds, including sulphated polysaccharides, polyhydroxycarboxylates and various tannins, the flavonoids that we tested seem to interact with the surface glycoprotein gpl20 to prevent binding of the virus to the sCE)4 receptor (41). Table 16. Inhibition of gpl20/sCD4 interaction by flavan compound 72. Compound

Concentration ue/ml

% Inhibition Washed * Unwashed 72 20 89 97 4 45 53 0.8 35 38 DS500 10 20 81 2 8 76 0.4 4 42 •Compound removed before addition of sCD4 to immobilised gpl20. Like dextran sulphate, compound 72 was more effective when added prior to or at the time of the virus infection; this indicates that it acts at an early stage of infection. But unlike the action of dextran sulphate, which readily reverses on removal of drug, the flavans irreversibly inactivate virus infectivity. Treatment of immobilized gpl20 with the flavans, irreversibly blocked the binding of sCI>4. Some degree of specificity in the interaction of the various tested flavans with gpl20 was apparent from the selectivity inhibition of antibody binding. Whereas flavonoids blocked in a dose dependent manner the interaction of monoclonal antibodies 358 and 380 with the V3 loop and CD4 binding regions of HIV-1 gpl20, they had no effect on the binding of monoclonal antibodies 360 and 323 to the N and C terminal regions of the molecule (Table 17). Thus there is a correlation between the degree of antibody inhibition and sCD4 binding by various flavonoids and their relative effectiveness in inhibiting virus infection. On the other hand, although it has been reported that some flavonoids can inhibit HIV reverse trascriptase in vitro (61), it is apparent that the inhibitory action of flavans is non specific. In fact we observed that they do not inhibit polymerase activity in the presence of serum albumin or detergents such as Triton X-1(X). The fact that human DNA polymerase a, p and y are inhibited to a similar degree by flavans suggests that flavans bind the polymerase without selectivity. From this evidence it is clear that the inhibition of HIV-1 infection by flavans is principally due to a selective interaction with gpl20. In this respect the anti HIV-1 activity of flavans is similar to that of various tannins and .polyanionic compounds. Similar studies (55) on the mechanism of action of quinic acid derivatives 95-100 suggest that they do not inhibit HIV replication by inhibition of HIV-RT as previously reported for 100 (64, 65), but they inactivate virus infectivity by specifically binding to gpl20 which prevents its interaction with CD4 on t-lymphocytes. The inhibition of HIV infection was in fact more pronounced when compounds were present during virus adsorption than when added after infection, as in the case of dextran sulphate. These compounds reduce syncythium formation between chronically infected and uninfected cells while dextrane sulphate inhibits syncytium formation only when added during the mixing of chronically infected and uninfected cells.

148 Table 17. Inhibition of antibody interaction with gpl20 of flavonols and flavans. Compound

I Concentration (jig/ml)

51 56 57 69 70 71 72 DSsoo

10 10 1 10 1 10 50 5 0.5 50 5 10 2 0.4 10 2 0.4

323 0 0 0 0 0 0 0 0

% Inhibition of antI body binding 358 360 0 0 7 0 0 90 0 50 0 0 96 0 44 17 59 0 24 98 0 78 11 92 0 26 0

Table 18. Inhibition of gpl20sCD4 interaction. Compound

Concentration

% Inhibition

(îg/ml)

95 96 97 99 DS500

Azr

50 10 2 20 4 0.8 50 10 2 25 5 1 10 2 0.4 50nM 10

Washed* 87 66 45 77 56 39 14 10 2 41 25 10 20 8 4 5 2

Unwashed 97 88 62 95 63 53 22 12 2 63 29 10 81 76 42 3 3

Compound removed before addition of sCD4 to immobilized gpl20.

380 16 40 48 98 97 0 91 87 36 96 24 99 85 37 93 0 0

149

:ooH R2O'//.

OH

Ri

95

R9

cafl

caffeoyl

caffeoyl

caf

caffeoyl

96

100

galloyl

galloyl

gall

COOH .CCX)H

OH 99

Galloyl=

HO

co-

Fig. 11 Quinic acid and cinnamic acid derivatives from S, Longipedunculata and G. senegalensis.

150 REFERENCES 1. 2.

A. Olayiwola, Fitoterapia 63, (1990), 99. Z.Z. Liang, R.- Aquino, V. De Feo, F. De Simone, C. Pizza, Pianta Medica 56 (1990), 330.

3.

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4.

R. Cerri, R. Aquino, F. De Simone, C. Pizza, J. Nat. Prod. 51 (1988), 257.

5.

R. Aquino, F. De Simone, C. Pizza, R. Cerri, J. F. De Mello, Phvtochemistrv 27 (1988), 2927.

6.

R. Aquino, I. Behar, F. De Simone, M.D' Agostino, C. Pizza, J. Nat. Prod. 49, (1986), 1096.

7.

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8.

P.K. Agrawal, Phvtochemistrv 31 (1992). 3307.

9.

N. De Tommasi, S. Piacente, F. De Simone, C. Pizza, J. Nat. Prod, . in press (1993).

10. R. Aquino, F. De Simone, C. Pizza, C. Conti. M.L. Stein. J. Nat. Prod. 52. (1989), 679. 11. R. Aquino, V. De Feo, F. De Simone, C. Pizza. G. Cirino. J. Nat. Prod. 54. (1991),453. 12. A.M. Yepez, O.L. de Ugaz, CM. Alvarez, R. Aquino, V. De Feo, F. De Simone, C. Pizza, Phvtochemistrv 28 (1991). 1635. 13. R. Aquino, F. De Simone, C. Pizza, J.F. De Mello, Phvtochemistrv 28 (1989), 199. 14. R. Aquino, I. Behar, M.D' Agostino,F. De Simone,0. Schettino, C. Pizza, Biochemical Systematics and Ecologv 15 (1987), 667. 15. A. U. Rahman, M.I. Choudharx, A. Pervil " Principles and Applications of Modem 2D NMR Techniques in Structure elucidation of Complex Method " in A.U. Rahaman Ed. Studies in Natural Product Chemistrv. Elsevier Science Publ. B.V., Amsterdam, 9 (1991), 127. 16. L. Pistelli, A.R. Bilia, A. Marsili, N. De Tommasi, A. Manunta, J. Nat. Prod.55 (1993), 240. 17. M.C. Das, S.B. Mahato. Phvtochemistrv 22 (1983), 1071. 18. G.R. Mallavarapu "Recent Advances in Oleanane Triterpenes" in A.U. Rahman Studies in Natural Product Chemistry. Elsevier Science Publ. B.V., Amsterdam. 7 (1990), 131. 19. R. Aquino, F. De Simone, F.F. Vincieri, C. Pizza, E.Gacs-Bsitz, J. Nat. Prod. 53 (1990), 559. 20. H. Wagner, B. Kreutzkamp, K. Jurcic, Pianta Medica 51 (1985), 419. 21. M.E.O. Matos, M.P. Sousa, M.I.L. Machado, R.B. Filho, Phvtochemistrv 25 (1986), 1419 and references therein cited. 22. A.G. Miana, M.G. Hassan Al-Hazini, Phvtochemistrv 26 (1987), 225. 23. C. Pizza, Z.Z. Liang, N. De Tommasi, J. Nat. Prod. 50 (1987), 927. 24. G. Romussi, S. Cafaggi, C. Pizza, Arch. Pharm. 321 (1988), 753. 25. R. Aquino, I. Behar, F. De Simone, C. Pizza, M. D' Agostino, J. Nat. Prod. 48 (1985), 502. 26. S.B. Singh, R.S. Thakur, H.R. Schulten, Phvtochemistrv 21 (1982), 2079.

151 27. K. Hiller" New results on the structure and biological activity of triterpenoid saponins" in Biologically Active Natural Products Oxford Science Publications. (K. Hostettmann, P. Lea eds.) 12 (1987), 167. 28. R. Pompei, O. Rore, M.A. Marciallis. A. Pani, B. Loddo, Nature 281 (1979), 689. 29. K. Fujisawa. Y. Watanabe, K. Kimura, Asian. Med. J. 23 (1980), 754. 30. M. Baba, S. Shigeta, Antiviral Res. 7 (1980), 99. 31. M. Ito, H. Nakashima, M.Baba. R. Pauwels, E. De Clercq, S. Shigeta, N. Yamamoto Antiviral. Res 7 (1987). 127. 32. R. Aquino, C.Conti, F. De Simone, N. Orsi, C. Pizza, M.L.Stein, Journal of Chemotherapy 3 (1991), 305. 33. N. Abe, T. Ebina, N. Ishida. Microbiol. Immunol. 26 (1982), 535. 34. N. De Tommasi, C. Conti, M. L. Stein, C. Pizza PlantaMedica 57 (1991), 251. 35. M.B. Gupta, T.N.Bhalla, G.P.Gupta, C.R.Nitra, K.P. Bhargave, Eur. J. Pharmacol. 6 (1969), 67. 36. C.A. Winter, E.A. Rislex, G.W. Nuss, Proc. Soc. Exp. Biol. I l l (1962), 544. 37. M. Shimizu, H. Fukumura, H. Tsuji, S. Tanaomi, T. Hayashi, N.Morita, Chem. Pharm. Bull. 34 (1986), 2614. 38. W. Noreen. A. Wadood, H.K. Hydayat, S.A.M. Wahid, Planta Medica 54 (1988), 196. 39. M.D. Ivarra, N. Paya, A. Villar, Planta Medica 54 (1988), 282. 40. N. De Tommasi, F. De Simone, G. Cirino, C. Cicala, C. Pizza, Planta Medica 57 (1991), 414. 41. N. Mahmood, C. Pizza; A. Aquino, N. De Tommasi, S. Piacente, S. Colman, A. Burke, A.J. Hax, Antiviral Research in press (1993). 42. 43. 44. 45. 46. 47. 48. 49. 48. 50. 51. 52.

V. De Feo, M. D' Agostino, F. De Simone. C. Pizza, Fitoterapia 61 (1990), 474. M. D' Agostino, C. Biagi, V. De Feo, F. Zollo, C. Pizza, Fitoterapia 61 (1990), 477. M. D' Agostino. V. De Feo. F. De Simone, C. Pizza. Fitoterapia 61 (1990). 375. V. De Feo. C Delia Valle. F. De Simone. C. Pizza. Annali di Chimica 61 (1990), 474. R.J. Guyglewski, R. Korbut, J. Rodax, J.Jwies. Biochem. Pharm. 36 (1987). 317. M. D' Agostino, F. De Simone, Z.Z. Liang, C. Pizza, Phytochemistry 31 (1992), 4387. G. Romussi, G. Bignardi, C. Pizza, Liebigs Ann. Chem. ( 1988), 989. G. Romussi, G. Bignardi, C. Pizza, N. De Tommasi, Arch. Pharm. 324 (1991), 519. G. Romussi, G. Bignardi, C. Pizza, Liebigs Ann. Chem. ( 1988), 989. R.Aquino, M.L. Ciavatta, F. De Simone, C. Pizza, Phvtochemistrv 29 (1990). 2358. W. Gaffield Tetrahedron 26 (1970). 4093. N.C. Baruah, R.P. Sharma, G.Thyaga Rayani, W. Herz, S. Govidan, Phvtochemistrv 18 (1979). 2003.

53. R.Aquino, M.L. Ciavatta, F. De Simone, Fitoterapia 62 (1991). 454. 54. R.Aquino, M.L. Ciavatta, N. De Tommasi, F. De Simone, C. Pizza. Fitoterapia 62 (1991), 455.

152 55. N. Mahmocxi, P.S. Moore, N. De Tommasi, F. De Simone, C. Pizza, Antiviral Chemistry and Chemotherapy (1993) in press. 56. C. Pizza, personal communication. 57. T.N. Kaul, E. Middleton, RL.Ogra, Journal of Medical Virology 15 (1985), 71 and references therein cited. 58. N. De Meyer, A. Haemers, L. Mishra, H.K. Pandey, L.A.C. Pieters, D.A. Vanden Berghe, A.J. Vlietinck. J. Med. Chemistry 34 (1991), 736. 59. M.A. McKinlay, M.G. Rossmann, Ann. Rev. Pharmacol. Toxicol. 29 (1989), 111. 60. H. Nakane, K.Ono, Biochemistry 29 (1989), 2841. 61. P.S. Moore, C. Pizza, Biochem. J. 288 (1992), 717 and references therein cited. 62. R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter, E. De Clerq J. Virol. Method. 20 (1988), 309. 63. N. Mahmood, A.J. Hay, J. Immunol. Methods 151 (1992), 9. 64. M. Nishizawa, T. Yamagishi, G.E. Dutschman, W. B. Parker, A.J. Bodner, R.E. Kilkuskie, Y.C. Cheng, K.H. Lee. J. Nat. Prod. 52 (1989), 762. 65. W. B. Parker, M. Nishizawa, M.H. Fisher, N. Ye, K.H. Lee. Biochem. Pharmacol. 38 (1989), 3759.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 17 © 1995 Elsevier Science B.V. All rights reserved.

153

Sesquiterpenes and Other Secondary Metabolites of Genus Lactarius (Basidiomycetes): Chemistry and Biological Activity G. Vidari and P. Vita-Finzi

Introduction The genus Lactarius (order Agaricales, family Russulaceae), is one of the largest in the subdivision Basidiomycotina of Whittaker's Kingdom of Fungi (1), and it comprises more than 150 species that grow world-wide in different habitats (2). These mushrooms show several morphological and biological features appealing to the chemists of natural products. For example, the fruiting bodies have various sizes and brilliant colours, and exchange organic materials and salts with several host plants, forming important mycorriza. The flesh of some species is mild and edible, while that of most Lactarius mushrooms tastes pungent or bitter, causing irritation to intestinal walls. The burning sensation develops on the lips and the tongue of an unskilled mycologist within a few seconds up to a few minutes, helping him to recognize inedible and toxic species. Moreover, observing a characteristic milky juice which appears on the surface of damaged fruiting bodies, anyone can easily distinguish a Lactarius species from a congener Russula species or other similar mushrooms. In addition, the colour and taste of this latex can be different from species to species and may change in the air, more or less rapidly, even for the same species, a fact that has a significant taxonomic relevance (2). For instance, the milky juice is permanently white and mild in L. volemus, but it is white and rapidly pungent in L. vellereus; it changes from white to yellow in L. scrobiculatuSy while it becomes bitter and red in L. fuliginosus and violet in L. uvidus. As a rule, only the species with a permanently red-orange juice are surely edible and taste mild. Recently, some aspects of these biochemical processes have been investigated and will be discussed later on in this review. Moreover, the observation of an interesting antibiotic activity for some Lactarius extracts (3) stimulated the search of new biologically active compounds among those isolated from these mushrooms. In fact, simple bioassays (4) led to the identification of new products with antimicrobial, cytotoxic, antifeedant and other interesting activities. No less important was the observation that some species seem to withstand attack from parasites such as snails and insects better than others. In fact, it has been shown that resistant species are armed with a chemical defence system which protects the mushrooms from predators and invaders. In this review we will discuss the chemistry and biological aspects of those secondary metabolites that seem more peculiar to Lactarius than to other mushrooms and have, therefore, a taxonomic relevance. By contrast, other important metabolites, such as triterpenoids, sterols, polyisoprenoids, fatty acids, aminoacids, etc., widely distributed in several species of different

154 genera, will not be considered. Moreover, limitation of space prevents us from including here references of papers describing the total synthesis ofLactarius metabolites, when they are not relevant to structure determination. Since excellent reviews on fungal metabolites have been published in the past (5-7), we will discuss in detail the literature published afterwards until the end of 1993. We have also added some recent yet unpublished results from our laboratory. In Tables 1-23 the structures of isolated and synthetic compounds (the latter in italic) are reported, while Table 24 reports the distribution of secondary metabolites in the investigated Lactarius species, which have been listed according to the subdivision of the genus by M. Bon (2). In Table 24 the metabolites have the same number as in the previous Tables 1-23 and the references are reported in parentheses.

Sesquiterpenes isolated from Lactarius species Sesquiterpenes of several kinds are the characteristic metabolites isolated from Lactarius mushrooms. However, other metabolites such as alkaloids, phenols and derivatives have been found in some species. Except humulene and sterpuranes, sesquiterpenes with the other skeletons shown in Scheme 1 have been isolated from Lactarius species. They have been divided into classes according to their biosynthetic origin from farnesylpyrophosphatc. The small class of farncsanc sesquiterpenes is derived directly from the alicyclic precursor, while drimanes, guaianes and the other classes arise by different cyclizations of farnesylpyrophosphatc. Two different cyclizations of a humulene precursor give rise to the classes of cariophyllanes and protoilludanes. The sesquiterpenes formally deriving from a protoilludane precursor constitute the largest group of Lactarius metabolites. Cyclobutane ring contraction of a protoilludane cation may give rise to the marasmane skeleton, whereas further rearrangements of marasmanes lead to the glutinopallane, lactarane and isolactarane skeletons. In principle, the secolactarane skeleton may arise by bond cleavage of a lactarane, however, the results of some biomimetic-like reactions in vitro (vide infra) seem to indicate their direct origin from marasmanes. In ahemative to the protoilludane-marasmane pathway, isolactaranes may originate from a rearrangement of a suitable sterpurane intermediate, even if this route in the Lactarius species has not been corroborated by the isolation of any sterpurane sesquiterpene. Contraction of the seven membered ring of lactaranes, with loss of the C-8 carbon atom, gives rise to the 8-norlactarane skeleton, whereas loss of the C-13 carbon of marasmanes leads to the 13-normarasmane skeleton. The results of a few biosynthetic investigations, discussed later, are consistent with this general scheme. Moreover, the occurrence of sesquiterpenes with different skeletons in the same species, for instance, marasmane, normarasmane, isolactarane, lactarane, and secolactarane sesquiterpenes in L. vellereus, points out their common biogenesis. Drimane, farnesane, glutinopallane, protoilludane, isolactarane, and guaiane sesquiterpenes have been isolated so far in a few Lactarius species; therefore, they may be considered chemotaxonomic markers. By contrast, large quantities of marasmane, lactarane and secolactarane metabolites occur in almost all Sections, as reported in Table 24. The carbons 5 and 13 of the skeletons of many marasmane, lactarane and secolactarane

155 sesquiterpenes are linked by an oxygen bridge forming an extra ring, either a furan or a y-lactone ring. In the latter the carbonyl group may be located either at C-5 or at C-13. Therefore, it is convenient to subdivide these classes of metabolites into the following groups: simple marasmane and lactarane sesquiterpenes (Tables 6 and 10, respectively), heterocyclic marasmanes (Table 7), 5lactaranolides (Tables 11-12), 8,9-seco-5-lactaranolides (Table 14), 13-lactaranolides (Tables 16 and 17), furanolactaranes (Table 18), and 8,9-secofuranolactaranes (Table 19). Compounds with rearranged structures, obtained by chemical reactions, are reported in Tables 13 and 20. Drimane, guaiane, farnesane and cariophyllane sesquiterpenoids are typical products of the plant metabolism. By contrast, sesquiterpenes with the skeletons derived from a protoilludane precursor have been isolated so far only from Basidiomycetes, but they are not unique to Lactarius species. In fact, marasmanes have been found, for example, also in species of the genera Russula, Lentinellus, Auriscalpium, Bondarzewia, Vararia, Dichostereum, Peniophora, Artomyces, Marasmius, and Fomitopsis; protoilludanes have been isolated from Fomitopsis, Clitocybey Laurilia, and Armillaria species; isolactaranes have been isolated from Stereum and Merulius species, while lactaranes and secolactaranes are also present in Russula, Lentinellus and Fomitopsis species. Anatomical characteristics point to the possibility that several of these genera may form a natural group together with the genera Lactarius and Russula (Russulaceae). Farnesane sesquiterpenes Recendy, the farnesane sesquiterpenes 1.1-1.9 (Table 1) have been isolated for the first time from Lactarius porninsis, the only species of the Section Zonarii investigated so far (8). Pominsal (1.1), porninsol (1.2) and the esters 1.3-1.9 show a high thermal and photochemical lability because of the conjugated tetraene system and readily polymerize when their solutions are taken to dryness. Therefore, special mild conditions are required for their isolation and for recording the spectroscopic data. The composition of the ester mixture 1.3-1.9 was established by capillary GC and GC-MS analysis of the methyl esters obtained by transesterification (8). TABLE 1 - Farnesane sesquiterpenes

>f

Name

Substituents

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9

Porninsal Porninsol Miristoylpominsol Pentadecanoylpominsol Palmitoleylpominsol Palmitoylpominsol Linoleylpominsol Oleylpominsol Stearoylpominsol

13-oxo 13-OH 13-miristoyloxy 13-pentadecanoyloxy 13-palmitoleyloxy 13-palmitoyloxy 13-linoleyloxy 13-oleyloxy 13-stearoyloxy

Ref.

156

P.O.

2

6

8

10

FARNESANES

13 NORLACTARANES

4

8 ' LACTARANES

13SECOLACTARANES

SCHEME 1 - The proposed biogenesis of Lactarius

Cariophyllane

sesquiterpenes

sesquiterpenes

Alcohol 2.1 (Table 2), the only example of this class, was isolated from Lactarius

camphoratus

157 (9), which belongs to the Section Olentes (Table 24). The structure and absolute configuration of this new cariophyllene oxide (2.1) was determined by a combination of spectral data and single-crystal X-ray analysis of the p-bromobenzoate derivative 2.3. TABLE 2 - Cariophyllane sesquiterpenes

N° 2.1 2.2 2.3

Ref.

Substituents No trivial name No trivial name No trivial name

12-OH 12-OAc 12-OBz-p-Br

9 9 9

Drimane sesquiterpenes Drimanes have only been isolated from two Lactarius species of the Section Uvidi: Lactarius uvidus {10-12) 2LndL.flavidus (23). In addition to uvidins A (3.14) and B (3.36) (10), more recently several new fatty acid esters of drimenol (3.2-3.6) and uvidin A (3.16-3.20) (11), and three new sesquiterpenes (12) with the bicyclofarnesane skeleton have been isolated from L. uvidus (Table 3). The stereostructures of the three uvidins C (3.29), D (3.34), and E (3.22) have been established by spectroscopic data and chemical reactions (12). In the interesting synthesis of uvidin E (3.22) from uvidin A (3.14), the Rubottom's procedure was employed for the regiospecific and stereoselective a-hydroxylation at C-5 of the enone 3.10 (12) (Scheme 2). ÔH

ÔTHP

OTHP

ÔH

Scheme 2 Exposure of uvidin A (3.14) or the esters 3.16-3.20 to methanolic KOH led to the new lactone 3.38, arising by a Favorskii rearrangement of the a,p-epoxyketone function (11). The same rearrangement was observed for 11-O-ethoxy ethyl uvidin A; however, in this case the lactone ring involved the tertiary OH group of compound 3.39. Sesquiterpenes 3.38 and 3.39 have a new

158 skeleton named isothapsane (11). TABLE 3 - Drimane sesquiterpenes

14 13 3.1-3.37

3.38

O

3.39

N°

Name

Substituents

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 3.17 3.18 3.19 3.20 3.21 3.22 3.23 3.24 3.25 3.26 3.27 3.28 3.29

Drimenol Palmitoyldiimenol Linoleyldrimenol Oleyldrimenol Stearoyldrimenol 6-Ketostearoyldrimenol No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name Uvidin A No trivial name Palmitoyluvidin A Linoleyluvidin A Oleyluvidin A Stearoyluvidin A 6-Ketostearoyluvidin A No trivial name Uvidin E No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name Uvidin C

ll-OH;7(8)-en;5a-H 1 l-palmitoyloxy;7(8)-en;5a-H 1 l-linoleyloxy;7(8)-en;5a-H I l-oleyloxy;7(8)-en;5a-H II -stearoyloxy ;7(8)-en;5a-H 11 -(6-oxostearoyloxy);7(8)-en;5a-H ll-OH;5a,8a-H 6-OSiMe3;l l-OTHP;5(6),7(8)-dien 1 l-OH;6-oxo;7(8)-en;5a-H 1 l-OTHP;6-oxo;7(8)-en;5a-H ll-OH;6-oxo;5a,8a-H I l-OTHP;6-oxo;5a,8a-H 5a,6a-epoxy;6-OSiMe3;l l-OTHP;7,8-en 7p,8p-epoxy;l l-OH;6-oxo;5a-H 7p,8p-epoxy;l l-OAc;6-oxo;5a-H 7p,8P-epoxy; 11 -palmitoyloxy;6-oxo;5a-H 7p,8p-epoxy; 11 -linoleyloxy;6-oxo;5a-H 7p,8p-epoxy; 11 -oleyloxy;6-oxo;5a-H 7p,8p-epoxy; 1 l-stearoyloxy;6-oxo;5a-H 7p,8P-epoxy; 11 -(6-oxostearoyloxy);6-oxo;5a-H 3p,l l-diOH;6-oxo;7(8)-en;5a-H 5a,l l-diOH;6-oxo;7(8)-en 7,1 l-diOH;6-oxo;7(8)-en;5a-H 5a-OH;l l-OAc;6-oxo;7(8)-en II -OH;7-OAc;6-oxo;7(8)-en;5a-H 7-OH; 1 l-OTHP;6-oxo;7(8)-en;5a-H 3p,l l-diOAc;6-oxo;7(8)-en;5a-H 7-OAc; 11 -OTHP;6-oxo;7(8)-en;5a-H 7p,8p-epoxy;6p,l l-diOH;5a-H

Ref. 10 11 11 11 11 11 10 12 10 12 10 12 12 10 10 11 11 11 11 11 10 12 12 12 12 12 10 12 10,12

159 3.30 3.31 3.32 3.33 3.34 3.35 3.36 3.37 3.38 3.39

No trivial name No trivial name No trivial name No trivial name Uvidin D No trivial name Uvidin B No trivial name No trivial name No trivial name

7p,8p-cpoxy;6p-OH;ll-OAc;5a-H 7p,8p-epoxy;6p,l l-diOAc;5a-H 3p,l l-diOH;6-oxo;5a,8a-H 7a,l l-diOH;6-oxo;5a,8a-H 7p,l l-diOH;6-oxo;5a,8a-H 8p,ll-diOH;6-oxo;5a-H 7p,8p-epoxy;3p,l l-diOH;6-oxo;5a-H 7p,8p-epoxy;3P,l l-diOAc;6-oxo;5a-H (See formula) (See formula)

10,12 12 10 12 12 10 10 10 11 11

Uvidins are attractive chiral starting materials for the synthesis of highly oxidized biologically active drimane-like sesquiterpenes as demonstrated by the syntheses of natural (-)-cinnamodial (115) and (-)-cinnamosmolide (116) from uvidin A (3.14). Guaiane sesquiterpenes Guaiane sesquiterpenes (Table 4) have been isolated so far onlyfromLactarius species of the Section Dapetes (Lactarius deliciosus, L. sanguifluus, etc.) which are characterized by the secretion of a strong coloured milky juice. Usually these mushrooms are edible and of pleasant taste. Each species contains a characteristic mixture of coloured sesquiterpenes responsible for the natural orange, red, green, or even blue colour of the milky juice. The structures of a dozen of guaiane sesquiterpenes were determined by chemical and spectral methods. These compounds are extraordinarily sensitive and could be isolated by employing very mild extraction and purification conditions. For example, two blue pigments of L. indigo were instantaneously converted to an intractable green substance upon addition of MeOH to the acetone solution, or on attempted chromatography (22). Both esters 4.13 and alcohol 4.9 polymerized in air (14). Similarly, delicial (4.5) rapidly polymerized when exposed to light (16). Normal chromatography of delicial was not possible, but small amounts could eventually be obtained by flash chromatography in the dark and with cold solvent, on silica gel prewashed with cold ethyl ether (16). Most isolated guaiane sesquiterpenes show a formyl or free hydroxymethyl group at C-4. However, recent results have shown that they are not present as such in the undamagedfruitingbodies, on the contrary, they are formed enzymatically from fatty acid ester precursors in injured specimens. Interestingly, there are examples of different metabolites produced by the same species collected in different parts of the world. For instance, aldehyde 4.6 has been isolated from Indian (20) but not from European specimens of L. deterrimus, while lactarofulvene (4.1) was isolated from Califomian specimens (13) of L. deliciosuSy but not from European specimens (16). An explanation of these apparent differences between specimens grown in different continents may be the existence of sub-species (16), or a change of the metabolism related to different habitats, or it may be due simply to the formation of artifacts during extraction.

160 TABLE 4 - Guaiane sesquiterpenes 14

N°

Name

Substituents

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 4.14

Lactarofulvene Lactarazulene Lactaroviolin No trivial name Delicial No trivial name DetemDl Sangol No trivial name Stearoyldeterrol Stearoylsangol No trivial name No trivial name Dimers

1(5),2(3),4(15),6(7),9(10),1 l(12)-esaen 13,14 l(10),2(3),4(5),6(7),8(9),ll(12)-esaen 14-16 15-oxo;l(10),2(3),4(5),6(7),8(9),ll(12)-esaen 14,16-18 15-oxo;l(2),3(4),5(6),7(l l),9(10)-pentaen 19 15-oxo;l(2),3(4),5(6),9(10),ll(12)-pentaen 16 15-oxo;l(10),2(3),4(5),6(7),8(9)-pentaen 19,20 15-OH;l(10),2(3),4(5),6(7),8(9),ll(12)-esaen 16 15-OH; 1 (2),3(4),5(6),7( 11 ),9( 10)-pentaen 21 15-OH;1(2),3(4),5(6),9(10),1 l(12)-pentaen 14 15-stearoyloxy;l(10),2(3),4(5),6(7),8(9),ll(12)-esaen 22 15-stearoyloxy; 1 (2),3(4),5(6),7( 11 ),9( 10)-pentaen 21 15-linoleyloxy;l(2),3(4),5(6),9(10),ll(12)-pentaen 16 15-stearoyloxy;l(2),3(4),5(6),9(10),ll(12)-pentaen 14,16 16,22

Ref.

Protoilludane sesquiterpenes Recently, for the first time among the Lactarius sesquiterpenes, the protoilludane skeleton has been assigned to two metabolites oi Lactarius violascens (23) (Table 5). Also this mushroom contains a sesquiterpene alcohol (5.1) and the corresponding 6-oxostearic acid ester 5.2. It is worth noting that this fatty acid (also named lactarinic acid) is peculiar to Lactarius mushrooms where it has been isolated in the form of many sesquiterpenoid esters. The structures of compounds 5.1 and 5.2, particularly the position of the oxygenated group at C-15, have been determined by 2D-NMR spectt-a and NOESY experiments. TABLE 5 - Protoilludane sesquiterpenes O^ 5 J L' ^ 1 4 11^ 13

""^15

N°

Name

Substituents

5.1 5.2

Violascensol 15-(6-Ketostearoyl)violascensol

15-OH 15-(6-oxostearoyloxy)

Ref. 23 23

161 Marasmane and 13-norinarasmane sesquiterpenes Among the not many marasmane sesquiterpenes isolated from Lactarius species (Tables 6 and 7), velutinal esters (7.14, 7.15) deserve a special consideration. Stearoylvelutinal (7.14) was originally isolated by a French group from Lactarius velutinus (39), during the search for the substances that are responsible for the intense blue colour which develops on the gills of a Lactarius specimen by reaction with the sulfo-vanillin reagent. In systematic mycology this reagent is used for identification purposes (40). Independendy, almost at the same time Swedish authors isolated stearoyl- (7.14) and 6-ketostearoylvelutinal (7.15) fromL. vellereus and L. necator (38), in an attempt to clarify the formation of some artifacts. Since then, most Lactarius species have been shown to contain velutinal esters, even if several important exceptions are known (Table 24). Particularly the species of Section Albati, possessing a permanently white milky juice, contain large amounts of velutinal esters. However, these compounds are not unique to Lactarius species, but they have also been found in a number of other genera, for example, in Russula, Lentinellus, Auriscalpium, Artomyces and Peniophora species (40, 113, 117). Catalytic transesterification of esters 7.14 or 7.15 in EtO"/EtOH afforded the free hemiacetal velutinal (7.11) (38), while both velutinal esters gave the methyl acetal 7.13 (38, 40) in HPLC grade methanol. Velutinal (7.11) can be synthesized by selective reduction of isovelleral (6.1) to isovellerol (7.2) using KBH4 in ethanol at r. t , followed by the Sharpless epoxydation of isovellerol (37). Moreover, the treatment of methyl velutinal (7.13) with lithium diisopropylamide afforded, by a p-elimination of the epoxyde, the corresponding 7,13-en-8a-ol derivative 7.9 (36). This product can be easily hydrolysed to isovelleral (6.1) in a THF/H2O mixture containing traces of acid or on prolonged contact with silica gel (36). This conversion may support the biosynthetic pathway proposed for the transformation of velutinals to isovelleral (6.1) in injured mushrooms (36,46). Free velutinal (7.11), its esters 7.14 and 7.15, and methyl acetal 7.13 are labile compounds, and on adsorption on silica gel they yield some of the furanolactarane and secofuranolactarane sesquiterpenes which have been isolated previously from different Basidiomycetes, including Lactarius species. Fast degradation takes place also on dissolving velutinal derivatives in wet acetone or in reagent grade alcohols as under other conditions where traces of acid are probably present (40, 118). Degradation by adsorption on Al203of stearoylvelutinal (7.14) yielded, in addition to the furans, significant amounts of isovellerol (7.2) and lactarol (19.3) (27). The furanoid sesquiterpenes are formed via intermediate dihydrofurans, many of which, in absence of acid, are stable enough to be isolated (85). The formation in vitro of dihydrofurans and furans from velutinal derivatives can be explained by a mechanism via carbocationic rearrangements (Scheme 3) which corroborates the stereochemistry assigned to several Lactarius sesquiterpenes (85, 118). Moreover, this mechanism may be very similar to the enzymatic conversion of velutinal esters to some furanolactaranes and secofuranolactaranes in injured mushrooms (46) {vide infra). One must be aware of the possible formation of artifacts in such conversions and, therefore, strict control must be exerted on any operation where this risk can occur. We must stress that the choice of the solvent for extracting the mushrooms is critical and that preliminary experiments should

162 suggest the best procedure for isolation and chromatographic separation of individual compounds (8, 14, 16, 22, 27, 40, 46). Alcoholic solvents are particularly harmful (119) and instead of them water insoluble solvents like hexane (27), EtOAc (27), ether (21) or CH2CI2 (40) have been recommended. A highly oxygenated marasmane sesquiterpene has been isolated from Lactarius pallidas (29) and named lactaropallidine (6.6). The structure of this compound, including the relative configuration of stereogenic centres, has been elucidated by spectroscopic methods and extensive decoupling experiments. The absolute configuration has been established by the CD measurement for the CO n -> 71* transition.

18.10

18.5

18.17 or 18.19

Scheme 3 - Degradation mechanism of velutinal derivatives Furthermore, reduction of stearoylvelutinal (7.14) with Red-Al (sodium

his{2'

methoxyethoxy)aluminium hydride) in toluene-THF gave directly lactaropallidine in a single step (29). A possible mechanism for this epoxyde-ketone rearrangement is reported in Scheme 4. The absolute configurations of lactaropallidine (6.6) and velutinal esters (7.14, 7.15) were definitively established by an enantioselective synthesis of isovelleral (6.1) (31). This assignment also indicated the absolute configurations of many marasmane, lactarane and secolactarane sesquiterpenes which have been stereochemically correlated to each other and to velutinals or isovelleral (6.1) by chemical reactions. Interestingly, Lactarius sesquiterpenes derived from velutinal esters (7.14, 7.15) have the same configurations as that suggested for the related antibiotic marasmic acid (120), but opposite to that of hirsutic acid, another fungal metabolite (121).

163

Scheme 4 TABLE 6 - Marasmane, isomarasmane and normarasmane sesquiterpenes

6.12-6.22

6.1-6.9

N°

Name

Substituents

6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 6.11 6.12 6.13 6.14 6.15 6.16 6.17 6.18 6.19 6.20 6.21 6.22

Isovelleral [ll'^Hshlsovelleral ^^0-Isovelleral Isovellerdiol No trivial name Lactaropallidine No trivial name No trivial name No trivial name Isoisovelleral No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name

5,13-dioxo;7(8)-en;2a,9a-H 12-2H3;5,13-dioxo;7(8)-en;2a,9a-H i80;5,13-dioxo;7(8)-en;2a,9a-H 5,13-diOH;7(8)-en;2a,9a-H 9a-OH;5,13-dioxo;7(8)-en;2a-H 5,13-diOH;8-oxo;2a,7a,9a-H 5,10a, 13-triOH;7(8)-en;2a,9a-H 5,10a, 13-triOAc;7(8)-en;2a,9a-H 5,7a,8a,13-tetraOH;2a,9a-H 5,13-dioxo;7(8)-en;2a,9a-H 9a-OH;5,13-dioxo;7(8)-en;2a-H 5,7a-diOH;8-oxo;2a,9a-H 5,8a-diOH;7-oxo;2a,9a-H 5,7a-diOAc;8-oxo;2a,9a-H 5,8a-diOAc;7-oxo;2a,9a-H 7a-OH;5,8a-diOAc;2a,9a-H 7a-OH;5,8p-diOAc;2a,9a-H 8a-OH;5,7a-diOAc;2a,9a-H 8P-OH;5,7a-diOAc;2a,9a-H 5,7a,8a-triOAc;2a,9a-H 5,7a,8p-triOAc;2a,9a-H 5,7p,8a-triOAc;2a,9a-H

Ref. 24,25 26 27 25 27,28 29 30 30 25 26,31 28 32 32 32 32 32 32 32 32 32 32 32

164 Other highly oxygenated bicyclic marasmane sesquiterpenes have been isolated from an EtOH extract of L. vellereus, which for many aspects seems an inexhaustible mine of Lactarius sesquiterpenes of any kind (see Table 24). The very unstable 5,10a,13-trihydroxymarasm-7(8)-ene (6.7) is accompanied by the 13-normarasmane isomers 6.12 and 6.13 (32) The latter ketones can derive from lactaropallidine (6.6) by p-elimination and oxidation at C-7. TABLE 7 - Heterocyclic Marasmane sesquiterpenes

NP 7.1 7.2 7.3 lA 7.5 7.6 7.7 7.8

Name No trivial name Isovellerol [n-^Hj]-Isovellerol ^Ô-Isovellerol Rubrocinctal A 6-Ketostearoylrubrocinctal A Rubrocinctal B 6-Ketostearoylrubrocinctal B

7.9 No trivial name 7.10 No trivial name 7.11 Velutinal 7.12 in-^Hsl-Velutinal 7.13 Methylvelutinal 7.14 Stearoyl velutinal 7.15 6-Ketostearoylvelutinal 7.16 No trivial name 7.17 No trivial name 7.18 No trivial name 7.19 No trivial name 7.20 No trivial name 7.21 No trivial name 7.22 Isovellerol dimer

Substituents 5-oxo;7(8)-en;2a,9a-H 5-OH;7(8)-en;2a,9a-H 12-2H3;5-OH;7(8)-en;2a,9a-H i80;5-OH;7(8)-en;2a,9a-H 5-OH;12-oxo:7(8)-en;2a.9a-H 5-(6-oxostearoyloxy); 12-oxo;7(8)-en;2a,9a-H 5-OH;7(8)-en;2a,9a-H;12-acid Me ester 5-(6-oxostearoyloxy);7(8)-en;2a,9a-H; 12-acid Me ester 8a-OH;5a-OMe;7( 13)-en;2a,9a-H 8a-OH;5a-0-stearoyloxy;7( 13)-en;2a,9a-H 7a,8a-epoxy;5a-OH;2a,9a-H 12-2H3;7a,8a-epoxy;5a-OH;2a,9a-H 7a,8a-epoxy;5a-OMe;2a,9a-H 7a,8a-epoxy;5a-stearoyloxy;2a,9a-H 7a,8a-epoxy;5a-(6-oxostearoyloxy);2a,9a-H 9a,10a-diOH;5-oxo:7(8)-en;2a-H 9a,10a-diOAc;5-oxo;7(8)-en;2a-H 7a,8a-diOH;5-oxo;2a,9a-H 7a,8MiOH;5-oxo;2a,9a-H 7a-OH;8a-OAc;5-oxo;2a,9a-H 7a-OH;8p-OAc;5-oxo;2a,9a-H

Ref. 33 27,34 35 27 23 23 23 23 36 36 27,37,38 35 38,39 38,39,40 38 32 32 33 41 33 41 27

The following y-lactone sesquiterpenoids with the parent marasmane skeleton have also been isolated from L. vellereus: 13-OH-7(8)-en (7.1) (33), 7a,8a,13-tri-OH (7.18) (33), 7a,8p,13-triOH (7.19) (41), and 9a,10a,13-tri-OH-7(8)-en (7.16) (32) marasman-5-oic acid y-lactones. Their structures have been established by spectroscopic data of the natural products and of their acetyl

165 derivatives. The value of the coupling constant between the protons H-8 e H-9 in compounds 7.18 and 7.19 indicated the trans and the rather unusual cis configuration, respectively. Furthermore, stereoselective cis dihydroxylation with OSO4 of the C-7,8 double bond of 7.1 occurred from the convex side of the molecule, giving the diol 7.18 in good yield (33). The Polish authors suggested that compound 7.18 is formed in Nature by oxidation of lactone 7.1 and that the biogenesis of lactone 7.19 from velutinal ester (7.14) involves the oxidation of the hemiacetal to the lactone ring, which is then followed by trans diaxial opening of the epoxyde (33). Recently, we have isolated the aldehyde rubrocinctal A (7.5), the carboxymethyl ester rubrocinctal B (7.7), and the corresponding 6-oxostearoyl esters 7.6 and 7.8 from Lactarius ruhrocinctus (Section Ichorati) (23) (Table 7). These compounds are the first examples of 12oxygenated isovellerol derivatives from a Lactarius species. Rearranged marasmane skeletons: glutinopallane and isolactarane sesquiterpenes The only two known natural glutinopallane sesquiterpenes (8.2, 8.3) have been isolated from Lactarius glutinopallens (42) (Table 8). Their structures are strictly related to velutinal esters (7.14, 7.15) for the presence of the cyclopropane ring and the 7a,8a-epoxyde, and to rubrocinctals 7.7 and 7.8 for the carbomethoxy group at C-3. The fascinating lactone isolactarorufin (9.5) (Table 9) is the only example of isolactarane sesquiterpenes isolated from a Lactarius specie (43-45). The structure of isolactarorufin has been elucidated by spectroscopic data and confirmed by X-ray analysis of its p-bromobenzoate 9.8 (45). TABLE 8 - Glutinopallane sesquiterpenes 4

N°

Name

8.1 Methylglutinopallal 8.2 Palmitoylglutinopallal 8.3 Stearoylglutinopallal

12

COOMe ^ H

Substituents 5a-OMe 5a-palmitoyloxy 5a-stearoyloxy

Ref. 42 42 42

Lactarane, lactaranolide, secolactarane and related sesquiterpenes New sesquiterpenes of the lactarane (occasionally named also vellerane) group (Table 10) have been isolated by Swedish authors during their pioneering work on the chemical defence system of Lactarius species. Vellerol (10.8) and vellerdiol (10.20) were first isolated from extracts of Lactarius vellereus made at different times after grinding the mushrooms (27). The reduction of either

166 vellerol (10.8) or velleral (10.5) with KBH4 gave the identical diol 10.20 confirming the stereostructures, particularly the biogenetically important C3-H configuration. TABLE 9 - Isolactarane sesquiterpenes 12

12 1

4

14

^

JL

1

14

9.1-9.8

N°

Name

Substituents

9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 9.10

No trivial name No trivial name No trivial name No trivial name Isolactarorufm (Lactarorufin C) No trivial name No trivial name No trivial name No trivial name No trivial name

8-oxo;2(9)-en 8a-OH;2(3)-en;9a-H 8a-OAc;2(3)-en;9a-H 3p-OH;8-oxo;2a,9a-H 3P,8a-diOH;2a,9a-H 3p,8P-diOH;2a,9a-H 3P-OH;8a-OAc;2a,9a-H 3P-OH;8a-OBz-p-Br;2a,9a-H 4,8a,13-triOH 4,8a,13-triOAc

Ref. A'hAA 43,44 43,44 43,44 43,44,45 43,44 43,44 45 44 44

TABLE 10 - Lactarane sesquiterpenes ,12

13 N°

Name

10.1 Chrysorrhedial 10.2 No trivial name 10.3 No trivial name 10.4 No trivial name 10.5 VeUeral 10.6 ^^0-Velleral 10.7 Chrysorrheal (Scrobicalol) 10.8 Vellerol 10.9 iÔ-Vellerol

^\^ Substituents 5,13-dioxo;2(9),7(8)-dien;3a,6p-H 5,13-dioxo;3( 12),7(8)-dien;2a,6a,9a-H 5,13-dioxo; 3( 12),7(8)-dien;2a,6P,9a-H 5,13-dioxo;4(6),7(8)-dien;2a,3a,9a-H 5,13-dioxo;4(6),7(8)-dien;2a,3P,9a-H i80;5,13-dioxo;4(6),7(8)-dien;2a,3p,9a-H 5-OH; 13-oxo;2(9),7(8)-dien;3a-6p-H 13-OH;5-oxo;4(6),7(8)-dien;2a,3p,9a-H i80;13-OH;5-oxo;4(6),7(8)-dien;2a,3p,9a-H

Ref. 46 26 26 47 24,47,48 27 46,49 27 27

167 10.10 No trivial name 10.11 No trivial name 10.12 No trivial name 10.13 No trivial name 10.14 No trivial name 10.15 No trivial name 10.16 No trivial name 10.17 No trivial name 10.18 No trivial name 10.19 10.20 10.21 10.22 10.23 10.24 10.25 10.26 10.27 10.28 10.29 10.30 10.31 10.32 10.33 10.34 10.35 10.36 10.37 10.38

Chrysorrhediol VeUeidiol Piperdial Epi-piperdial No trivial name No trivial name No trivial name Piperalol Epi-piperalol No trivial name Pipertriol 7-£p/-pipertriol No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name

10.39 No trivial name 10.40 Vellerol dimer

13-OH;5-diOMe;4(6),7(8)-dien;2a,3a,9a-H 13-OH;5-diOMe;4(6),7(8)-dien;2a,3p,9a-H 5-diOMe;4(6)J(8)-dien;2a,3a,9a-H; 13-acid Me ester 5-diOMe;4(6).7(8)-dien;2a,3P,9a-H; 13-acid Me ester 13-diOMe;4(6),7(8)-dien;2a,3p,9a-H; 5-acid Me ester 13-OH;4(6),7(8)-dien;2a,3p,9a-H; 5-acid 5-OAc;4(6),7(8)-dien;2a,3a,9a-H; 13-acid Me ester 5-OAc;4(6),7(8)-dien;2a,3p,9a-H; 13-acid Me ester 13-OAc;4(6) J(8)-dien;2a,3p,9a-H; 5-acid Me ester 5,13-diOH;2(9),7(8)-dien;3a,6P-H 5,13-diOH;4(6),7(8)-dien;2a,3p,9a-H 8a-OH;5,13-dioxo;4(6)en;2a,3p,7a,9a-H 8a-OH;5,13-dioxo;4(6)en;2a,3p,7p,9a-H 9-OH;5,13-dioxo;4(6),7(8)-dien;2a,3p-H 3a,8a-epoxy;5,13-diOH;6(7)-en;2a,9a-H 3a,8a-epoxy;5,13-diOAc; 6(7)-en-2a,9a-H 8a,13-diOH;5-oxo;4(6)en;2a,3p,7a,9a-H 8a,13-diOH;5-oxo;4(6)en;2a,3p,7p,9a-H 5,8a,13-triOH;2(3)-en;9a-H 5,8a,13-triOH;4(6)-en;2a,3p,7a,9a-H 5,8a,13-triOH;4(6)-en;2a,3p,7p,9a-H 5,8a, 13-triOAc;2(3)-en;9a-H 5,8a,13-triOAc;4(6)-en;2a,3p,7p,9a-H 5,8a, 13-triOCONHCCl3;4(6)-en;2a,3p,7p,9a-H 5,8a,13-triOH;2a,3p,6a,7p,9a-H 5,8a, 13-triOH;2a,3p,6p,7p,9a-H 3a,5,8a,13-tetra-OH;6(7)-en;2a,9a-H 3a-OH;5,8a, 13-tri-OAc;6(7)-en;2a,9a-H 3a,5,8a,13-tetra-OH;2a,9a-H 3a-OH;5,8a, 13-tri-OAc;2a,9a-H

47 47 47 47 47 47 47 47 47 46,49 27,50 51 52 27 53 53 51 52 54 51 55 54 55 55 55 55 54 54 54 54 27

Vellerol (10.8), as well as isovellerol (7.2), readily dimerizes when left in reagent grade solvents. Velleral (10.5) was found to be degraded rapidly on attempted preparative chromatography on AI2O3 (27). Moreover, the dialdehyde 10.5 was slowly oxidized to 9-hydroxyvelleral (10.23) in a hexane solution kept at r. t. for two weeks or even at -30° C when a hexane extract of L. vellereus was kept frozen in the air for months (27). In the same conditions isovelleral (6.1) was oxidized to

168 9-hydroxyisovelleral (6.5) (27). This oxidation also occurred when isovelleral was adsorbed on AI2O3 for 5 h in day light. The equally labile and biologically active dialdehydes piperdial (10.21) and ^p/-piperdial (10.22) were isolated from different Lactarius species (Table 24). During chromatography on silica gel ^/7/-piperdial (10.22) was easily converted into piperdial (10.21), which appears to be the more stable epimer, and into velleral (10.5) (52). Like velleral, both piperdial and epi-piperdial rapidly decomposed within few seconds when chromatography on AI2O3 was attempted (113). Piperalol (10.26) was found together with piperdial (10.21) in the same mushrooms (51), while, in addition to compound 10.22, e/?/-piperalol (10.27) (52) and 7-ep/-pipertriol (10.30) (55) were isolated from different extracts of L. necator. In analogy with isovellerol and vellerol (27), piperalol (10.26) and ep/-piperalol (10.27) readily dimerized when exposed to traces of acid (113). NMR data, particularly NOE results, established the position of the formyl groups and the relative configuration of stereocenters of these sesquiterpenes. Moreover, Li AIH4 reduction of lactarorufm N (11.18) gave 7-e/7/-pipertriol (10.30) (55), and both compounds 10.21 and 10.26 were reduced by KBH4 in EtOH to the same triol named pipertriol (10.29) (51). A similar procedure was followed for establishing the stereostructures of two new pungent-bitter aldehydes, named chrysorrhedial (10.1) and chrysorrheal (10.7), that have been isolated from Lactarius scrobiculatus and L. chrysorrheus (46). UV absorption at 313 nm supported the presence of the dienal system in sesquiterpene 10.1, while the cis relationship between H3-I2 and H-6 in compounds 10.1 and 10.7 was firmly established by hydride reduction of both compounds to the same diol 10.19. The latter compound was also obtained by LiAlH4 reduction of the known lactone 16.2 (46). The positive Cotton effect observed for the n -> 7C* transition of the diene system in the diol 10.19 indicated a positive skewness for the chromophore in accordance with the absolute configurations shown in the formulae 10.1, 10.7, 10.19, and 16.2. Interestingly, although one might expect the 1,4-dialdehyde 10.1 and y-hydroxyaldehydes 7.2, 10.7, 10.26, and 10.27 to be in equilibrium with the corresponding hemiacetal forms, only the ^H-NMR spectra of isovellerol (7.2) showed significant amounts of a cyclic product (27). In fact, in common organic solvents isovellerol (7.2) exists as a mixture of approximately equal amounts of the three forms shown below in Scheme 5 (27).

On the other hand, molecular modelling of chrysorrheal (10.7) (46) clearly suggested that ring closure to the hemiacetal form would require a severe conformational rearrangement at a high energetic cost for the loss of the resonance energy deriving from the conjugation of the carbonyl group to the Cy-Cg double bond.

169 The entire group of lactarane sesquiterpenes 10.1, 10.7,10.19, and 16.2 containing the 2(9),7(8)-cycloheptadiene ring, was submitted to conformational analysis by molecular mechanics and ^H-NMR spectroscopy (46). We observed that conformational mobility of each compound is almost restricted to the interconversion of envelope forms of the cyclopentene ring; by contrast, only a single conformation of the seven membered ring is practically populated, owing to the planarity of either the diene (in 10.19) or the diene-carbonyl double bonds (in 10.1,10.7, and 16.2), and the rigid fusion of the y-lactone ring (in 16.2) (46). However, the geometry of the global minima of sesquiterpenes 10.1,10.7, 10.19 is completely different from that of 13-lactaranolide 16.2. In fact, the orientation of the 3-methyl group is pseudoequatorial in the former three compounds, while it is pseudoaxial in 16.2 (Figure 1).

MM2 computed conformations for compound 16.2

MM2 computed conformations for dialdehyde 10.1, hydroxyaldehyde 10.7, and diol 10.19 Figure 1 Recently, several papers have reported the isolation of new lactarane lactones, possessing the methyl group at C-3 either cis or trans to H-2, and the lactone carbonyl group either at C-5 (5lactaranolides, Tables 11-13) or at C-13 (13-lactaranolides, Tables 16-17). Differentiation between these structural alternatives on the basis of spectroscopic data alone has been often risky, especially when only a single isomer is at hand. Therefore, chemical correlations, synthesis of the possible isomers, and molecular mechanics calculations have always been performed in order to corroborate spectroscopic informations. For instance, the correction of the structure of vellerolactone (11.3) (47) led Daniewski and coworkers to revise the structures of lactarorufm N (11.18) and 3-ep/-deoxylactarorufin A (11.20) (58), that had been correlated with compound 11.3 (Scheme 6). Lactone 11.20 was also compared with the C-3 epimer 11.19 (Scheme 6), whose structure was unambiguously proved by synthesis and further chemical transformations (58).

170

H.ISÂH

11.19

H 6 "

11.3 Scheme 6 Lactarorufins D (11.47) and E (11.49), which are the 4a- and 4p-hydroxy derivatives of lactone 11.20, were isolated from L. necator (78). Single-crystal X-ray analysis demonstrated unequivocally the structure and relative stereochemistry of compound 11.47. The ^H-NMR data of lactarorufin E (11.49) were almost identical with those of epimeric lactarorufin D (11.47), except of course the vicinal coupling constant of the proton H-4 with H-3. A preparation of lactarorufins D and E from the more abundant lactarorufin A (11.44) was attempted in order to test their biological activity (77). Oxidative hydroboration of compound 11.13, followed by hydrolysis of intermediate boric esters with cone. HCl in EtOH, yielded lactarorufin A (11.44) (6.7%), S-^pMactarorufin D (11.46) (84.4%), 3-e/7Mactarorufin E (11.48) (1%), and lactarorufin E (11.49) (5.5%) (Scheme 7).

11.48 HO

: H 11.49 HO

Scheme 7 Several representative lactones showing the 8-OH and 3-Me groups cis to H-2 and H-9, as sardonialactone A (11.50), blennins A (11.17) and D, (11.43), 14-hydroxyblennin A (11.51), and lactarorufins D (11.47) and E (11.49) have been submitted to conformational analysis (74, 78, 81). Molecular mechanics showed that, in spite of the different position of the double bond in the

171 cycloheptene ring, a hinge conformation, in which both OH and Me groups are equatorially oriented, is largely preferred by these compounds. By contrast, the strong intramolecular hydrogen bond between the two cis OH groups at C-3 and C-8 of lactarorufms A (11.44) and B (11.71) is the dominant steric factor which affects the overall molecular shape of these two lactones. In fact, in order to form this intramolecular bond, the cycloheptene rings of lactarorufms A (11.44) and B (11,71) must still assume a hinge conformation but folded in an opposite direction to that of 11.47 and 11.49 (Figure 2). For 3-ep/-lactarorufin D (11.46) for which a conformation similar to lactarorufin A (11.44) was expected, dynamic NMR studies indicated the existence of two conformations in solution at r. t. (77).

R = H R' = OH (11.47) R = OH R» = H (11.49)

R = R»=R"=H (11.17) R = R'=H R" = OH (11.43) R'=R" = H R = OH (11.50) R = R"==H R'= OH (11.51)

R = H (11.44) R = OH (11.71)

MM2 computed conformations for compounds 11.17,11.43,11.44,11.47,11.49,11.50,11.51, and 11.71 Figure 2 Additional new identified lactarorufms are 3,12-anhydrolactarorufin A (11.11) from L. necator (63), and 15-hydroxylactarolide A (13-hydroxylactarorufm B) (11.83) from L. mitissimus (83). The structure of the latter compound was confirmed (83) by NaBH4 reduction of the lactol group yielding lactarorufin B (11.71), whose structure had been confirmed by X-ray analysis (72). Furthermore, compound 11.83 was synthesized from lactarorufin B (11.71) (82) by introducing the C-13 hydroxy group in two steps. DIBAL reduction of lactarorufin B gave furantriol (18.27), which was acetylated to the corresponding diacetate 18.28, and this was then oxidized with MCPBA to 11.83. Interestingly, the ^H-NMR spectrum of lactol 11.83 showed only one signal for the proton H-13, owing to a mixture of fast equilibrating epimers. The rate of equilibration was much higher in MeOH than in CHCI3 (82). The 3,8-internal ether of lactarorufin A (11.15) was isolated from L. necator (59), and it was identical with the compound previously obtained from lactarorufin A (11.44) by dehydration with MsCl-Py (53). By comparison with the synthetic isomer 13-oxolactone 16.10 (59), the ^H-NMR spectra of compound 11.15 showed small differences in the chemical shifts, that were attributed to the shorter distance of the protons of 11.15 from the ether oxygen. In addition, the Polish authors found that the acid catalysed dehydration of furandiol (18.14) and 5-deoxylactarolide B (16.15) to the corresponding 3,8-internal ether 18.5 and 16.10, respectively, could be achieved in modest yields by the azeotropic method of removal of water (59) (Scheme 8).

172 The same authors assigned the structure 11.1 to a new polyunsaturated 5-lactaranolide sesquiterpene isolated from an ethanol extract of L. vellereus (56).

p-TsOH CfiHT

Scheme 8

16.10

TABLE 11 - 5-Lactaranolide sesquiterpenes 12

N°

Name

Substituents

11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 11.12 11.13 11.14 11.15 11.16 11.17 11.18 11.19 11.20 11.21 11.22

No trivial name Pyrovellerolactone Vellerolactone No trivial name Lactarotropone No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name Blennin A Lactarorufin N No trivial name No trivial name No trivial name No trivial name

2(3),6(7),8(9)-trien 3(4),6(7)-dien;2a,9a-H 4(6)J(8)-dien;2a,3p,9a-H 8,9-en;2a,3a,6a,7a-H 8-oxo;2(9),3(4),6(7)-trien 3a-OH;6(7).8(9)-dien;2a-H 8a-0H; 1 (2),6(7)-dien;3a,9a-H 8a-OH;l(2),6(7)-dien;3a,9P-H 8a-OH;2(3),6(7)-dien;9a-H 8a-OH;3(4),6(7)-dien;2a,9a-H 8a-OH;3(12),6(7)-dien;2a,9a-H 8a-OAc;2(3),6(7)-dien;9a-H 8a-OAc;3(4),6(7)-dien;2a,9a-H 8a-OAc;3( 12),6(7)-dien;2a,9a-H 3a,8a-epoxy;6(7)-en;2a,9a-H 15-D3;3a,8a-epoxy;6(7)-en;2a,9a-H 8a-OH;4(6)-en;2a,3p,7a,9a-H 8a-OH;4(6)-en;2a,3p,7p,9a-H 8a-OH;6(7)-en;2a,3a,9a-H 8a-OH;6(7)-en;2a,3p,9a-H 8a-OH;6(7)-en;2p,3a,9a-H 8a-OH;6(7)-en;2p,3p,9a-H

Ref. 56 47,57 47,57 58 29 59 60 60 61,62 62 63 61 62,64,65 63 53,59 66 67,68 58,62 58,69 58,69 60 60

173

11.23 11.24 11.25 11.26 11.27 11.28 11.29 11.30 11.31 11.32 11.33 11.34 11.35 n.36 11.37 11,38 11.39 11.40 11.41 11.42 11.43 11.44 11.45 11.46 11.47 11.48 11.49 11.50 11.51 11.52 11.53 11.54 11.55 11.56 11.57 11.58 11.59 11.60 11.61 11.62 11.63 11.64 11.65

No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name Blennin B No trivial name Blennin D Lactaronifin A No trivial name No trivial name Lactarorufin D No trivial name Lactarorufin E Sardonialactone A No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name

8p-OH;6(7)-en;9a-H 29 8a-OAc;3(4)-en;2a,3p,7p,9a-H 62 8a-OAc;4(6)-en;2a,3p,7a,9a-H 67 8a-OAc;6(7)-en;2a,3a,9a-H 58,69 8a-OAc;6(7)-en;2a,3p,9a-H 69 8a-OMs;4(6)-en;2a,3p,7a,9a-H 68 8-oxo;2a,3a,6a,7a,9a-H 58,62 8a-OH;2a,3a,6a,7a,9a-H 58,62 8p-OH;2a,3a,6a,7a,9a-H 58 8a-OAc;2a,3a,6a,7a,9a-H 58,62 2,9-epoxy;8-oxo;3(4),6(7)-dien 70 3a,4a-epoxy;8a-OAc;6(7)-en;2a,9a-H 71 3a,8a-epoxy;15-(OBz-/?-Br);6(7)-en;2a,9a-H 66,72 3a,8a-epoxy;15-OMs;6(7)-en;2a,9a-H 66 3a,8a-epoxy;15-OTs;6(7)-en;2a,9a-H 66 3a-OH;8-oxo;2(9),6(7)-dien 70 3a-OH;8-oxo;6(7)-en;2a,9a-H 53 3a-OEt;8-oxo;6(7)-en;2a,9a-H 73 8a, 13-diOH;2(3),6(7)-dien;9a-H 67 8a,15-diOAc;3(4),6(7)-dien;2a,9a-H 66 2a,8a-diOH;4(6)-en;3p,7a,9a-H 68,74 3a,8a-diOH;6(7)-en;2a,9a-H 53,64,72,75,76 3a,8p-diOH;6(7)-en;2a,9a-H 53 4a,8a-diOH;6(7)-en;2a,3a,9a-H 77 4a,8a-diOH;6(7)-en;2a,3p,9a-H 78 4p,8a-diOH;6(7)-en;2a,3a,9a-H 77 4p,8a-diOH;6(7)-en;2a,3p,9a-H 77,78 7a,8a-diOH;4(6)-en;2a,3p,9a-H 29,78,79 8a,14-diOH;4(6)-en;2a,3p,7a,9a-H 80,81 8a-OH;3a-OEt;6(7)-en;2a,9a-H 54,73 2a-OH;8a-OAc;4(6)-en;3P,7a,9a-H 68 3a-OH,8a-OAc;6(7)-en;2a,9a-H 64,65,82 3a-OH;8p-OAc;6(7)-en;2a,9a-H 53 4a-OH;8a-OAc;6(7)-en;2a,3a,9a-H 77 4P-OH;8a-OAc;6(7)-en;2a,3a,9a-H 77 7a-OH;8a-OAc;4(6)-en;2a,3p,9a-H 79 3a-OH;8a-stearoyloxy;6(7)-en;2a,9a-H 71 3a-OEt;8a-OAc;6(7)-en;2a,9a-H 73 3a,8a-diOAc;6(7)-en;2a,9a-H 64 3a,8a-diO(S02);6(7)-en;2a,9a-H 53 3a-OH;8-oxo;2a,6a,7a,9a-H 53,62 3a,8a-diOH;2a,6a,7a,9a-H 64 3a,8a-diOH;2a,6p,7P,9a-H 64

174 11.66 No trivial name 11.67 No trivial name 11.68 No trivial name 11.69 No trivial name 11.70 Lactarolide A 11.71 Lactarorufin B 11.72 3-Ethyl-lactarolicie A 11.73 No trivial name 11.74 No trivial name 11.75 No trivial name 11.76 No trivial name 11.77 No trivial name 11.78 No trivial name 11.79 No trivial name 11.80 No trivial name 11.81 No trivial name 11.82 No trivial name 11.83 No trivial name 11.84 No trivial name 11.85 No trivial name 11.86 No trivial name

3a-OH;8a-OAc;2a,6,7,9a-H 64 8a-OH;3a-OAc;2a,6,7,9a-H 64,69 3a,8a-diOAc;2a,6,7,9a-H 64 3a,13-diOH;8-oxo;6(7)-en;2a,9a-H 54 3a,8a,13-triOH;6(7)-en;2a,9a-H 54,65,82,83 3a,8a, 15-triOH;6(7)-en;2a,9a-H 64,66,72,84 8a,13-diOH;3a-OEt;6(7)-en;2a,9a-H 54 3a, 13-diOH;8a-OAc;6(7)-en;2a,9a-H 54,65,82 3a,8a-diOH; 15-OBz-/7-Br;6(7)-en;2a,9a-H 66 3a,8a-diOH; 15-OTs;6(7)-en;2a,9a-H 66 3a-OH;8a,13a-diOAc;6(7)-en;2a,9a-H 54,83 3a-OH;8a,13p-diOAc;6(7)-en;2a,9a-H 54,83 3a-OH;8a,15-diOAc;6(7)-en;2a,9a-H 66 3a-OH;8a, 15-diOBz-/?-Br;6(7)-en;2a,9a-H 66 3a-OH;8a,15-diOTs;6(7)-en;2a,9a-H 71 3a-OEt;8a, 13-diOAc;6(7)-en;2a,9a-H 54 3a,8a,15-triOAc;6(7)-en;2a,9a-H 66 3a,8a,13,15-tetraOH;6(7)-en;2a,9a-H 82,83 3a,13-diOH;8a,15-diOAc;6(7)-en;2a,9a-H 82 3a-OH;8a, 13a, 15-triOAc;6(7)-en;2a,9a-H 82 3a-OH;8a, 13p, 15-triOAc;6(7)-en;2a,9a-H 82

TABLE 12 - 5-Lactaranolide derivatives /12

rsp

Name

Substituents

12.1 12.2 12.3 12.4 12.5 12.6 12.7 12.8 12.9

No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name

5-OMe;4(6),7(8)-dien;2a,9a-H 3a,8a-epoxy;5-OH;6(7)-en;2a,9a-H 3a,8a-epoxy;5P-OMe;6(7)-en;2a,9a-H 8a-OH;5p-OMe;2(3),6(7)-dien;9a-H 8a-OH;5p-OMe;3(12),6(7)-dien;2a,9a-H 3a,5,8a-triOH;6(7)-en;2a,9a-H 3a,8a-diOH;5-OMe;6(7)-en;2a,9a-H 8a,5-diOH;3a-OMe;6(7)-en;2a,9a-H 8a-OH;3a,5a-diOMe;6(7)-en;2a,9a-H

Ref. 47 85 85 85 85 85 85 85 85

175 Three new 8-oxolactarane lactones 11.5, 11.33 and 11.38 were isolated from L. scrohiculatus (70) and L. pallidas (29). Ketones 11.5 and 11.38 could also be obtained by PDC oxidation of 2,3-anhydrolactarorufin A (11.9), while the a,P-cpoxyketone 11.33 was considered a biogenetic precursor of the 8-norlactarane sesquiterpene 15.1 (70). Expulsion of the C-8 carbonyl group from the lactarane skeleton of compound 11.33 has been suggested to occur via a benzylic-like rearrangement, followed by a decarboxylative aromatization (70) (Scheme 9).

0

y^-^v

ÔFT

0

0 11.33

Scheme 9 The relative configurations of lactaroscrobiculide A (16.2) and the corresponding 2,9-epoxyde 16.9 have been definitively established by NOE experiments and molecular modelling performed by means of the MM2 program (46). To this purpose, the conformational spaces of the stereostructures 16.2 and 16.9 were explored and compared with the corresponding diastereomers 16.2 A and 16.9 A. It was then found that the experimental ^H-NMR vicinal coupling constants of the two natural sesquiterpenes matched the values calculated for stereoisomers 16.2 and 16.9 instead of those computed for compounds 16.2 A and 16.9 A.

O

1^-^

O

16.9 A

Epoxyde 16.9 is strongly suspected of being an artifact, because it was not found any more in

176 fresh extracts of L. scrobiculatus (46) and it was obtained by oxidation in air of dienelactone 16.2. This oxidation, as well as that with MCPBA, showed a rather surprising diastereoselectivity, since oxidizing agents approached the apparently more hindered face of the C2-C9 double bond of compound 16.2.

TABLE 13 - Rearranged 5-lactaranolide sesquiterpenes

NO

Name

Substituents

13.1 13.2 13.3

No trivial name No trivial name No trivial name

(See formula) 13-OH 13-OAc

Ref. 68 58 58

TABLE 14 - 8,9-Seco-5-lactaranolide sesquiterpenes and derivatives .12

rsp

Name

Substituents

14.1 14.2 14.3 14.4 14.5 14.6

No trivial name No trivial name No trivial name Lactardial Blennin C (Lactaronecatorin A) No trivial name

5-oxo;6(7)-en 5-oxo 5,8-dioxo;2(9),6(7)-dien 5-OH;8-oxo;2(9),6(7)-dien 8-OH;5-oxo;2(9),6(7)-dien 5a-OMe;8-oxo;2(9),6(7)-dien

14.7

No trivial name

8-OAc;5-oxo;2(9),6(7)-dien

Ref. 67 67 49,60 51,85, 61,67,86 85 61

The extremely labile triene-enol-lactone 16.1 was found to be involved in the rapid yellowing of the milky juice and flesh of L. chrysorrheus and L. scrobiculatus, and it could be isolated from the mushrooms under special mild conditions (46). The strong UV absorption of compound 16.1 at

177 370.4 nm was that expected for the cross-conjugated dienone-triene chromophores, while the further unsaturation in the furanone ring was indicated by comparison of the NMR data of compound 16.1 with lactone 16.2. Biosynthetic considerations suggested for lactone 16.1 the same absolute configuration of sesquiterpenes 10.1, 10.7, and 16.2. The new lactone 16.6, one of the few known natural 13-lactaranolides, has recently been isolated from L. vellereus (87). The simulated ^^C-NMR spectra of compound 16.6 suggested that the configuration at C-3 was opposite to that of isomeric lactaroscrobiculide A (16.2). This stereochemistry was established unequivocally by correlation of sesquiterpene 16.6 with 3-deoxy-3^/7/-lactarorufin A (11.20), as shown in Scheme 10 (87). H $.

. > ^ 1)DIBAL / \ 2)AC20/Py ^ ' AcO 1) MCPBA 2)NaBH4 3) Separation

11.20 H 5^

MeOH/H O

: H AcO 11.27 (52.5%)

16.6 Scheme

6

AcO 15.13 (18.9%)

10

For comparison, 3-deoxylactaroscrobiculide B (16.5), the C-3 epimer of natural lactone 16.6, was synthesized from 8-acetyl-5-deoxylactarolide B (16.17), as shown in Scheme 11 (87).

MeOH/H"^ reflux

S c h e m e 11

O

Comparing the ^H-NMR spectra of the epimeric pairs 1 1 . 2 6 , 11.27 and 1 6 . 1 2 , 1 6 . 1 3

178 Daniewski and coworkers noticed that the signal of the proton H-3 cis to the C-8 acetoxy group was shifted to lower field than that of the corresponding trans proton (87). TABLE 15 - 8-Norlactarane sesquiterpenes

O—'12

N°

Name

Substituents

15.1

No trivial name

(See formula)

Ref. 70

TABLE 16 - 13-Lactaranolide sesquiterpenes ,12

N'

Name

Substituents

16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9 16.10 16.11 16.12 16.13 16.14 16.15 16.16

Chrysorrhelactone LactaroscrobicuHde A No trivial name No trivial name No trivial name No trivial name LactaroscrobicuHde B No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name

2(9),5(6),7(8)-trien;3a-H 2(9),7(8)-dien;3a,6p-H 3(4),6(7)-dien;2a,9a-H 4(6).7(8)-dien;2a,3a,9a-H 6(7),8(9)-dien;2a,3a-H 6(7),8(9)-dien;2a,3p-H 3a-OH;6(7),8(9)-dien;2a-H 8a-OAc;3(4),6(7)-dien;2a,9a-H 2p,9p-epoxy;7(8)-en;3a,6p-H 3a,8a-epoxy;6(7)-en;2a,9a-H 3a-OH;6(7)-en;2a,9a-H 8a-OAc;2a,3a,9a-H 8a-OAc;2a,3p,9a-H 3a-OEt;8-oxo;6(7)-en;2a,9a-H 3a,8a-diOH;6(7)-en;2a,9a-H 8a-OH;3a-OEt;6(7)-en;2a,9a-H

Ref. 46 86,46 47 47 87 87 82,88 65 46,89 59 54,88 87 87 73 54,59,65,82 54,73

179 16.17 16.18 16.19 16.20 16.21 16.22 16.23 16.24 16.25 16.26 16.27 16.28 16.29 16.30

No trivial name No trivial name No trivial name LactarolideB 3-Ethyl-lactarolide B No trivial name No trivial name No trivial name No trivial name No trivial name N^ rnv/a/ name No trivial name M? rrzv/fl/ «ame No trivial name

3a-OH;8a-OAc;6(7)-en;2a,9a-H 3a-OEt;8a-OAc;6(7)-en;2a,9a-H 3a,5-diOH;8-oxo;6(7)-en;2a,9a-H 3a,5,8a-triOH;6(7)-en;2a,9a-H 5,8a-diOH;3a-OEt;6(7)-en;2a,9a-H 3a,5-diOH;8a'OAc;6(7)-en;2a,9a-H 3a-OH;5,8a-diOAc;6(7)-en;2a,9a-H 3a-OH;5a,8a-diOAc;6(7)-en;2a,9a-H 3a-OH;5p,8a-diOAc;6(7)-en;2a,9a-H 3a-OH;8a, 15-diOAc;6(7)-en;2a,9a-H 3a-OEt;5,8a-diOAc;6(7)-en;2a,9a-H 3a,5-diOH;8a, 15-diOAc;6(7)-en;2a,9a-H 3a-OH; 8a,5a, 15-triOAc;6(7)-en;2a,9a-H 3a-OH;8a,5p,15-triOAc;6(7)-en;2a,9a-H

54,65,82 73 54 54,65,82 54 54,82 54 82 82 82 54 82 82 82

TABLE 17 - 13-Lactaranolide derivatives ,12

OEt N°

Name

Substituents

17.1

No trivial name

2(9),7(8)-dien;3a,6p-H

Ref. 46

Furanolactarane and secofuranolactarane sesquiterpenes Recently, relatively few new furanolactarane sesquiterpenes have been isolated from Lactarius species (Table 18). Furanodiene (18.1), previously obtained by synthesis (Scheme 12) (60), is also a true metabolite of Lactarius scrobiculatus (46), while the structure of the highly oxidized dioxofuran 18.26, isolated from L. vellereus (56), was confirmed by single crystal X-ray diffraction analysis (95). 3-E/7/-furandiol (18.15) and 4a,8a-dihydroxyfuran (18.16), two isomers of the more widespread 3a,8a-dihydroxyfuran (18.14), have been isolated from L. scrobiculatus (70), and from L. piperatus (94), L. torminosus (94), L. necator (94) andL. circellatus (52), respectively. The relative configuration of furan 18.16 was established by comparing the experimental ^H-NMR coupling constants of sesquiterpene 18.16 with those calculated for all possible stereoisomers (94). The molecular mechanics (MM2) computed conformational mixture of compound 18.16 comprises three conformers, two of which, 18.16 A and 18.16 B, differ mainly in a twist of the cyclopentane ring, whereas the third conformer 18.16 C (40%) shows an entirely different folding

180 of the seven membered ring, which facilitates the formation of an intramolecular hydrogen bond across the ring (94) (Figure 3). The results of molecular modelling (74) also definitively proved the stereostructure 18.13 for furoscrobiculin D, correcting a previous assignment based on the NMR data alone (88). In the preferred conformation 18.13 A of furoscrobiculin D, accounting for more than 95% the entire population, the C-3 methyl and the C-8 hydroxy groups have an equatorial orientation, as in conformers 18.16 A and 18.16 B (Figure 3).

18.16B

18.16C

MM2 computed conformations for compounds 18.13 and 18.16

Figure 3 Furantriol (18.27), isolated from L. mitissimus (84), is one of the few lactarane sesquiterpenes in which one of the gem-methyl groups at C-11 is oxidized and it was chemically correlated (82) with hictarorufin B (11.71), another example of this kind. The Polish authors suggested that lactone 11.71 was enzymatically formed from furan 18.27, and that a C-15 oxidized sesquiterpene of the velutinal type was the common precursor of both compounds in the mushroom (84). Actually, the possibility for the C-15 methyl group to be oxidized at an eariy stage of the lactarane biosynthesis seems to be confirmed by the recent finding of C-15 hydroxylated protoilludane sesquilerpenoids (5.1 and 5.2) in L. violascens (23) (Table 5). Further chemical correlations put stereochemical assignments of most Lactarius sesquiterpenes on a solid basis. Attempted formation of the bromide from the furanosesquiterpene 18.10 with PhaP and CBr4 gave furanether A (18.5) and pyrovellerofuran (18.3), as main products (93). The latter compound had previously been obtained by thermal rearrangement of isovelleral (6.1) (90), while furanol 18.10, isolated from L. vellereus (93), was also formed when velutinal derivatives were decomposed on silica gel (85). This experiment correlated the absolute configuration of isovelleral (6.1) with that of velutinal esters and, indirectly, with the stereostructures of many oihtr Lactarius

181 sesquiterpenes (93). TABLE 18 - Furanolactarane sesquiterpenes ,12

N°

Name

Substituents

18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8 18.9 18.10 18.11 18.12 18.13 18.14 18.15 18.16 18.17 18.18 18.19 18.20 18.21 18.22 18.23 18.24 18.25 18.26 18.27 18.28

No trivial name No trivial name Pyrovellorofuran No trivial name FuranetherA FuranetherB Furoscrobiculin B Furosardonin A Furanol No trivial name No trivial name Furoscrobiculin A Furoscrobiculin D Furandiol 3-£p/-furandiol No trivial name 3-O-Methylfurandiol Furoscobiculin C 3-0-Ethylfurandiol No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name Furantriol No trivial name

l(2),8(9)-dien;3a-H 2(3),8(9>dien 3(12),8(9)-dien;2a-H 3(4)-en;2a,9a.H 3a,8a-epoxy;2a,9a-H 3p,8p-epoxy;2a,9a-H 3a-OH;8(9)-en;2a-H 8a-OH;l(2)-en;3a,9a-H 8a-OH;2(3)-en;9a-H 8a-OH;3(12)-en;2a,9a-H 8a-OAc;2a,3p,9a-H 2,9-epoxy;8-oxo 2p,8a-diOH;3a,9a-H 3a,8a-diOH;2a,9a-H 3p,8a-diOH;2a,9a-H 4a,8a-diOH;2a,3p,9a-H 8a-OH;3a-OMe;2a,9a-H 3a-OH;8a-OEt;2a,9a-H 8a-OH;3a-OEt;2a,9a-H 2P-OH,8a-OAc;3a,9a-H 3a-OH;8a-OAc;2a,9a-H 3p-OH;8a-OAc;2a,9a-H 3a,8a-diOEt;2a,9a-H 3a-OEt;8a-OAc;2a,9a-H 4a,8a-diOAc;2a,3p,9a-H 4,8-dioxo;3P-OH;2(9)-en 3a,8a,15-triOH;2a,9a-H 3a-OH;8a,15-diOAc;2a,9a-H

Ref. 46,60,85 85 90 47 59,79,88 88 88 60,79 91,92 93 87 88 74,88 86,88,91 70 94 85,92 88 54,73,88,92 74,88 65,86 70 88 73 94 56,95 82,84 82,84

The series of reactions shown in Scheme 12 proved the same absolute configuration at C-3 for all natural lactone and furan secolactarane sesquiterpenes (60).

182

18.8

0

.

v^ 14.5

(^

V ^

DIBAL

\^ c c

OH O O^

1 r

14.3

o

11.8

OH

Scheme 12 TABLE 19 - 8,9-SecofuranoIactarane

sesquiterpenes

.12

CHOHCHjCOMe 19.1-19.4

19.5

N°

Name

Substituents

19.1 19.2 19.3 19.4 19.5

Lactaral No trivial name Lactarol No trivial name No trivial name

8-0X0

8-COOMe 8-OH 8-OTHP (See fomiula)

Ref. 96,97 96 51,60,97 97 88

Noteworthy among these conversions are the first successful cyclizations of lactone and furan secolactaranes to the corresponding lactarane sesquiterpenes, which were obtained by a Me2AlCl catalysed ene reaction (60). Under these conditions lactaral (19.1) yielded direcdy the diene 18.1, identical with the dehydration product of furosardonin A (18.8), while smooth cyclization of aldehyde 14.3 gave the lactone 11.8 in which the protons H-8 and H-9 have the "unnatural" cis stereochemistry. This result could be anticipated by examination of the Dreiding models of the two possible transition states 11.8 A and 11.8 B, which showed that unfavourable steric interactions developing between the C-3 methyl group and the bulky >C=0-"A1~ complex are minimized in the

183 transition state 11.8 B leading to lactone 11.8, O CH3

11.8 A

Structure elucidation of many Lactarius sesquiterpenes often requires interconversions of yhydroxybutenolide, butenolide, and furan rings for confirming spectroscopic assignments. Examples of DIBAL reductions of y-lactones to the corresponding furans include the conversions of blennin C (14.5) to lactarol (19.3) (Scheme 12) (60) and of compound 11.20 to 3-deoxy-3-e/7i-furandiol (87) (Scheme 10).

18.21 AcO

O AcO

11.73

HO AcO 29.6 •*" HO _ O _ J ^ H

16.22 25.3 %

Scheme 13

AcO

11-54 22.6 %

184 TABLE 20 - Rearranged furanolactarane sesquiterpenes

Q H N°

Name

Substituents

20.1

No trivial name

(See formula)

Ref. 25

The reverse transformation of a furan to a butenolide ring has been achieved in fair to good yields with NBS in aq. dioxane (Wiesner procedure) (122), as in the following conversions: 18.8 to 11.7 (60), 18.9 to 11.9 (60), and 18.21 to 16.22, 11.54 and 11.73 (82) (Scheme 13). The observed moderate sitoselectivity of the furan ring oxidation at C-5 was attributed to a coordination of the electrophilic Br"^ species with the allylic C-8-OR group, prior to the attack on the aromatic ring (60).

RO 18.19 R = H 18.24 R = Ac

: H RO 11.52 R = H 11.60 R = Ac

o 1 : 3.5 1 :5.7

RO 16.16 R=H 16.18 R=Ac

Scheme 14 MCPBA oxidation of several furanolactarane sesquiterpenes to the corresponding lactarolides (mixture of lactol epimers) has been studied in details by Daniewski and coworkers (59, 65, 73, 82, 84, 87). The sitoselectivity of this oxidation is only moderate and the directing effect of neighbouring oxygenated groups is often unpredictable, so that variable mixtures of C-5 and C-13 lactols are usually obtained. Smooth NaBFit reduction of the separated lactols afforded the corresponding ylactones with the carbonyl group either at C-13 or at C-5. The entire sequence of reactions (MCPBA (or NBS) furan oxidation - NaBH4 lactol reduction) allowed several important correlations of furanolactarane and lactaranolide sesquiterpenes (59, 60, 65, 73, 82, 83, 84, 87), as already reported in Scheme 10 and further illustrated by the examples of Scheme 14.

185

Dibenzonaphthyridinone alkaloids, prenylated phenols, benzofurans and chromenes Sesquiterpenes, as already reported in the previous sections, are the most widespread Lactarius metabolites; however, a few species possess a particular metabolism which leads to secondary metabolites of other classes. Moreover, interesting new compounds with a different biogenesis have been isolated also from species producing large quantities of sesquiterpenes. Interest in the considerable mutagenicity of extracts of Lactarius necator, a mushroom often cited in this review for the occurrence of several lactarane sesquiterpenes, led to the isolation of a highly mutagenic alkaloid named necatorin (4.8 mg from 30 kg of mushroom), for which the structure of 7hydroxycoumaro[5,6-c]cinnoline was originally proposed (101). Necatorin was then shown by direct comparison (100) to be identical with necatorone, isolated almost at the same time by Steglich (99) as one of the pigments of the fruiting bodies of L. necator. Spectroscopic data of this unstable alkaloid established the unusual 5,10-dihydroxydibenzo[de,h][l,6]-naphthyridin-6-one structure (21.2), which was confirmed by total synthesis (100). Necatorone forms red needles which dissolve in DMSO to produce a grass-green solution showing strong green-yellow fluorescence. With aq. ammonia, successive deprotonations of compound 21.2 produce blue and purple anions. Therefore, necatorone is believed to be partially responsible for the change to a deep purple of the dark olivebrown colour of the caps and stalks of L. necator on exposure to ammonia vapours. TABLE 21 - Dibenzonaphthyridinone alkaloids

21.1-21.3

N°

Name

Substituents

21.1 21.2 21.3 21.4 21.5 21.6

10-Deoxynecatorone Necatorone (Necatorin) No trivial name 10,10'-Dideoxy-4,4'-binecatorone 10-Deoxy-4,4'-binecatorone 4,4'-Binecatorone

5-OH 5,10-diOH 5,10-diOMe (See formula) lO'-OH 10,10'-diOH

Ref. 98 99, 100,101 99 98 98 98

186 Necatorone (21.2) was methylated by CH2N2 in methanol/H20 to yield the dimethyl ether 21.3 as the main product. More recendy, other two new necatorone-type alkaloids isolated from L. necator have been identified as 4,4'-binecatorone (21.6) and 10-deoxy-4,4'-binecatorone (21.5) (98). From L. atroviridis, a dark-green North American species, in addition to compounds 21.2, 21.5 and 21.6, 10,10'-dideoxy-4,4'-binecatorone (21.4) was obtained as main alkaloid (98). The structures of all these alkaloids have been established by spectroscopic data and confirmed by synthesis (98). The occurrence of the same alkaloids in L. necator and in L. atroviridis indicates the close taxonomic relationship of both species. Like necatorone, the colour of the DMSO solutions of alkaloids 21.5 and 21.6 changes to purple on addition of alkali, while that of compound 21.4 gives a dove-grey colour with alkali. It is noteworthy that in young, light brown fruiting bodies of L. necator about equal amounts of pigments 21.2 and 21.6 are present, whereas in aged, dark brown specimens the ratio between these compounds becomes 5 : 95. In search for the compounds responsible for the antimicrobial and immunosuppressive activities of L. flavidulus, an edible mushroom in spite of the bitter taste, three geranylphenols have been isolated and named flavidulols A (22.8), B (22.13) and C (22.15) (103, 104). The structure of flavidulol A (22.8) is very similar to that of wigandol isolated from Wiganda kunthii Choisy, the former compound being the methyl ether and the latter the acetate of the same phenol. Flavidulol B (22.13) could be an artifact derived from flavidulol A by a Cope-type rearrangement. The structures of all the flavidulols and their acetyl derivatives (Table 22) could be determined by spectroscopic studies. Particularly, NOE and ^^C-^H-COLOC NMR techniques allowed to establish the configuration of the double bonds in the geranyl moiety of compounds 22.8 and 22.15 as well as the cis stereochemistry at C-2 and C-7 of flavidulol B (22.13) (104). Catalytic hydrogenation of compound 22.8 afforded dihydro and tetrahydro derivatives, 22.11 and 22.12, respectively, while on treatment with 2N HCl in MeOH flavidulol A (22.8) gave two linear tricyclic products 22.17 and 22.18 (104). Recently, geranylgeranylhydroquinone (22.6) and a mixture of fatty acid esters 22.7 have been isolated from L. lignyotus (23). Clearly, these phenols are biogenetically related to flavidulols A-C and to compound 22.1. Compound 22.6 could also be obtained by hydrolysis of the esters 22.7. The acids esterified in 22.7 were identified by GC-MS analysis of the mixture of methyl esters obtained by transesterification (23). Interestingly, the free hydroquinone 22.6 has previously been isolated from the sponge Ircinia muscarum (123) and from plants of the genus Phacelia (124). In a preliminary study on the metabolites of the Lactarius species of the Section Plinthogali the fruiting bodies were extracted by grinding under solvents at r. t.. Unexpectedly, on TLC plates sprayed with the sulfo-vanillin reagent, the metabolites of these species were revealed as green spots, and, therefore, they could easily be differentiated from the metabolites of the ox\\tr Lactarius species. In fact, separation of EtOAc extracts of L. fuliginosus and L. picinus by silica gel column chromatography led to the isolation of benzofuran and chromene derivatives, unprecedented among Basidiomycetes metabolites (102,105) (Tables 22 and 23).

187 TABLE 22 - Prenylated phenols

22.6 and 22.7

OMe

OMe 22.8-22.12

OMe

10

OMe

22.15-22.16

22.13-22.14

OMe 22.17-22.18

N^

Name

Substituents

22.1 22.2 22.3 22.4 22.5 22.6 22.7 22.8 22.9 22.10 22.11 22.12 22.13 22.14 22.15 22.16 21 Al 22.18

No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name No trivial name Flavidulol A No trivial name No trivial name No trivial name No trivial name Flavidulol B No trivial name Flavidulol C No trivial name No trivial name No trivial name

4'-OH;2(3)-en 4'-stearoyloxy;2(3)-en 2,3-epoxy;4'-OH 4'-[2-OH-3-(3-Me-2-butenyl)-5-OMe-phenoxy];2(3)-en 4'-[2-OAc-3-(3-Me-2-butenyl)-5-OMe-phenoxy];2(3)en 2',5'-diOH 2',5'-diOAcyl (*) 4*-OH;Z-2{3),E-6(7)-dien 4'-OMe;Z-2(3),E-6(7)-dien 4'-OAc;Z-2(3),E-6(7)-dien 4'-OH;Z-2(3)-en 4'-0H 4'-0H 4'-0Ac 4',4'-diOH 4',4'-diOAc 6P-0H 6P-0Me

Ref. 102 102 102 102 102 23 23 103,104 103,104 103,104 104 104 103,104 103 103,104 103 104 104

(*) Mixture of esters of the following acids: miristic, pentadecanoic, palmitoleic, palmidc, linoleic, oleic, and stearic acid (23). The structures of the new compounds 22.4, 23.2, 23.5, and 23.8-23.11 have been elucidated by spectroscopic methods; particularly the structures of chromenes 23.9 and 23.11 have

188 been established by NOE experiments and biosynthetic considerations (102). 6-Methoxy-2,2-dimethylchromene (23.5) and benzofuran 23.2 have also been synthesized by alkylation of 4-methoxyphenol to 2-(3-methyl-2-butenyl)-4-methoxyphenol (22.1), followed by acid catalysed cyclization of the corresponding epoxyde 22.3 to 23.3 and 23.6. Dehydration of 23.6 withp-TsOH gave 23.5, while NBS dehydrogenation of 23.3 afforded 23.2 (102). MeO^

MeO,

22.4

OMe

oMe

Scheme 15 - C-C and C-O phenol dimerizations It is worth noting that only one compound, the stearate of 4-methoxy-2-(3-methylbutenyl)phenol (22.2) could be isolated from young intact fruiting bodies of L.fuliginosus and L. picinus extracted in the cold (102). On the other hand, in injured mushrooms the stearate 22.2 was rapidly hydrolysed by lipases to free phenol 22.1. Therefore, the ester 22.2 is the biogenetic precursor not only of compounds 23.2 and 23.5, but also of 22.4 and 23.8-23.11, which can be considered dimerization products of 22.1. Oxidative dimerizations of phenolic compounds occur in Nature by one-electron transfer C-C and C-O couplings which are catalysed by phenol oxidase enzymes. Reactions of the same kind are probably responsible for the reddening of the flesh and milky juice of damaged mushrooms of the Section Plinthogali. In fact, the same change of colour was observed when synthetic phenol 22.1 was added to a mush of Lfuliginosus from which the original metabolites had been washed out with CH2CI2. Moreover, this experiment afforded the same mixture of chromenes and benzofuran as originally isolated from damaged fresh fruiting bodies. The structures of the red pigments are still unknown as they remain irreversibly adsorbed on the top of chromatographic columns.

n The oxidative dimerization of phenol 22.1 was simulated in vitro. Exposure of this compound to the complex Cu(N03)2-pyridine gave rise to dimers 23.8 and 23.9 by a C-C coupling reaction. Compound 23.9 could be cyclodehydrogenated to 23.8 by reaction with DDQ. On the other hand, exposure of phenol 22.1 to K3Fe(CN)6 gave the product 22.4 of a C-O coupling, which was then transformed into 23.10 by DDQ cyclodehydrogenation (102) (Scheme 15). In addition to dimers, a natural trimer 23.12 of phenol 22.1 has been isolated from L. fuliginosus and its structure has been elucidated by accurate and extensive NOEDS experiments (102). Furthermore, a qualitative evaluation of the contents of different extracts of the same or different Lactarius species of the Section Plinthogali has been carried out by GC and GC-MS analysis, using a Dexsil 300 column (125). By this method, the simple 2,2-dimethylchromene (23.4) has been identified in an extract of L. picinus (102). TABLE 23 - Benzofurans and Chromenes MeO

«X3 Pd/C

'

2. KOH. CH^H

b

b b

OH 24

[24

HO

Figure 4. Two Acetogenin Derivatives, 9 and 10, with Single Crystal, X-ray Structures.

With an ever-increasing number of Annonaceous acetogenins being reported, there have been many efforts to develop methods for elucidation of configuration that are more generally applicable. Table 1 shows a time-line chronicling the major contributions that address various stereochemical issues in these molecules. Beginning with our report in 1986 describing the relative configuration of the bis-THF portion of uvaricin,i3 research has yielded many complementary methods for

255

determining configuration within these natural products. This chapter presents an overview of these methods (through 1993). The classification/labeling scheme for the structural subunits of the Annonaceous acetogenins proposed^ and later expanded^ by McLaughlin will be used in this chapter. 1"* The terminal lactone moiety is labeled "A", and this subunit is designated A1, A2, A3, A4, or A5 depending on Its structure (Figure 5). For example, the a,punsaturated lactone without other functionality on the carbon chain, such as found in uvaricin (1), is known as A1. The letter "B" refers to the THF subunit, labeled B1, 82, 83, or 84 ; for example, 83 refers to the non adjacent bis-tetrahydrofuranyl subunit. Other, miscellaneous functionality is represented by the letter "C": hydroxyl (C1), carbonyl (C2), acetate ester (C3), vicinal diol (C4), epoxide (C5), and olefin (C6). This scheme for organization and labeling of the acetogenins simplifies reference to molecules possessing common structural features; for example, instead of referring to "a monoTHF subunit bearing only one adjacent hydroxyl group", one can simply say "a 84 subunit." o Vo Rx.--V..>**,>/^

o OH V - o R^sX^s.^'SV^

i^ o—f o "vX^'ÂsX^

o V-o n^^X-Ss..A^

o V-o ^^.y^-^^y'^Kf^

OH

A1

OH

A2

^—'

B1

A3

OH

OH

OH

A4

OH

OH

B2

B3

^—'

A5

OH

84

OH

Y

^r >" v^

Figure 5- Lactone (A#), Tetrahydrofuran (8#), and Miscellaneous (C#) Substructural Units Found in the Annonaceous Acetogenins.^

The strategy we have used to develop general methods for the determination of both relative and absolute configuration within various substructural units of the acetogenins consists of four parts, i) An appropriate set of model compounds is identified and synthesized so as to provide a complete set of diastereomers of unambiguously known

256 Table 1 . Time-line of Important Events, Including Major Advances in Determination 1982

First Annonaceous acetogenin, uvaricin, reported.

Cole et al. J. Org. Chem. 47,3151.

1985

C(36) of uvaricin determined to be S by degradation to lactic acid.

Cole et al. J. Nat. Prod. 48, 644.

1986

Model compounds of bis-THF structure prepared for NMR correlation studies; observation that ^H NMR shifts of acetate methyl groups correlate with relative configuration of C(15)/C(16) and C(23)/C(24) in uvaricin (later proven to be correct).

Hoye et al. Tetrahedron 42, 2855.

1987

""H NMR correlation studies suggest that bis-THF portion of uvaricin possesses threo/trans/threo/trans/erythro relative configuration among C{15)-C(24). Establishes general method for determining relative configuration.

Hoye et al. J. Am. Chem. Soc.

1987

Rolliniastatin I reported, and successful X-ray studies on p-bromophenylurethane derivative establish complete relative configuration.

Pettit et al. Can. J. Chem. 65, 1433.

1988

''H NMR correlation method further validated by comparison of rolliniastatin I NMR data with the now known relative configuration. Method made more quantitative, relying less on visual inspection. Relative configuration of bis-THF moiety of aslmicin verified.

Hoye et al. J. Org. Chem. 53, 5578.

1989

Rollinicin reported, containing a vicinal diol along Sneden et al. one carbon chain; relative configuration assigned as J. Nat. Prod. 52, 822. erythro based on cims fragmentation pattern.

1989

Bullatacin and bullatacinone reported; absolute configuration at C(4) assigned as S based on ORD spectral data (later proven to be incorrect); bullatacin successfully converted to bullatacinone, proving that they possess the same relative configurations along the adjacent bis-THF backbone.

McLaughlin et al. J. Nat. Prod 52, 463.

1990

Annonin I (squamocin) reported; successfully studied by X-ray analysis of a derivative. Previously described NMR correlation method gives results in accordance with structure determined by X-ray. Complementary 1H as well as i^C NMR correlation method developed to determine the configurational relationship between a THF ring and an adjacent hydroxylated carbon.

Born et al. Planta Med. 56,312.

109, 4402.

257

of Configuration, in the Development of Annonaceous Acetogenin Chemistry. 1991

"^H NMR-based method for assigning relative configuration (i.e., cis vs. trans) of 2-acetonyl-4alkylbutanolides.

Hoye et al. J. Org. Chem. 56, 5092.

1991

Synthesis of 15,16,19,20,23,24-êxep/-uvaricin (a diastereomer of the natural product) confirms relative configuration and establishes absolute configuration (via Mosher esters) of the first acetogenin, uvaricin.

Hoye et al. J. Am. Chem. 113,9369.

1992

Gigantetronenin and gigantrionenin reported, first Annonaceous acetogenins found to contain a double bond along one of the aliphatic chains. Configuration in both determined to be cis from ^H NMR coupling constants.

McLaughlin et al. J. Nat Prod. 55, 1655.

1992

Relative configuration of some mono-THF acetogenins confirmed by correlation of ^H and "^^C NMR chemical shifts with two mono-THF model compounds of known configuration.

Figaddre et al. Tetrahedron Lett. 33, 5749.

1992

Absolute configuration of C(4) in C(4)-hydroxylated compounds determined by NMR analysis of Mosher ester derivatives and comparison to model compounds. All configurations studied determined to be R at C(4). Method also applicable for determining the relative configuration between C(4) and C(36), as well as the absolute configurations of carbinol centers adjacent to THF rings.

McLaughlin, Hoye, et al. J. Am. Chem. Soc. f 74, 10203.

1993

General method for determining relative configuration of mono-THF acetogenins by correlation of 1H chemical shifts with mesitoylated model compounds.

Cassady et ai. Tetrahedron Lett. 34, 5847/5851

1993

Total synthesis of enf-bullatacin, the enantiomer of the bis-THF natural product, confirms absolute configuration of bullatacin.

Hoye et al. Tetrahedron Lett. 34, 5043.

1993

Total synthesis of solamin and reticulatacin, two mono-THF acetogenins, confirms their absolute configuration.

Keinan et al. J. Am. Chem. Soc.

115,4891.

Soc.

258 relative and/or absolute configuration, ii) An appropriate battery of spectral data from this set is carefully collected, interpreted, and tabulated, iii) Trends In these data are observed, iv) Relevant data from the natural products themselves, or appropriate derivatives, are collected and compared with those from the set of model compounds to deduce the relevant configurational relationships. Many of the general methods described herein make use of comparisons of NMR chemical shift data between a molecule with an intact natural product skeleton and another, skeletally simpler, model compound. It is more convenient to draw parallels between the two if the numbering scheme used to refer to the atoms involved are the same in both stmctures. Therefore, wherever possible, atoms on the carbon skeleton of the model compound(s) will be numbered corresponding to the natural product(s) they are intended to mimic, regardless of the "proper" numbering for the model structure.

II. THF BACKBONE 1. Adjacent bis-THF Structures (B1) In 1986, this laboratory''^ described the synthesis of a series of twelve acetylated model compounds 11a-l (Figure 6) for the bis-THF structure of uvaricin (1), one of only a handful of Annonaceous acetogenins known at the time. Among other things, we noted that the ''H NMR chemical shifts of the acetate methyl groups on the models showed a clear correlation with the relative configuration (either three or erythro) between the carbon bearing the acetoxy group and the adjacent carbon in the THF ring. Specifically, an erythro relationship between C(15)/C(16) or C(23)/C(24) in the model compounds led to a 5 of 2.051 ± 0.007 ppm, while a three relationship placed the methyl group at 2.075 ± 0.008 ppm. Since the acetate methyl group in uvaricin (1) resonates at 2.049 ppm, while the acetate derivative of uvaricin (the diacetate 12) showed acetate signals at 2.049 and 2.074 ppm, we concluded that the relationship between C(23)/C(24) in uvaricin is erythro, while the C(15)/C(16) relationship is three (Figure 7). Notice that very small differences in chemical shift were meaningful in the trend just described--the acetate methyl groups for each set of six diastereomeric compounds having either both erythro or both three terminal diastereomeric relationships all fell within a range of just over one one-hundredth of a ppm. Moreover, the two different diastereomeric environments led to a difference of only slightly more than two onehundredths of a ppm (i.e., A5 = 0.024). This requires a certain degree of care in measuring and reporting chemical shift data. To ensure reproducibility and confidence in our measured 8 values, we always include TMS as an internal standard in our samples, and we always set the TMS resonance to 5 = 0.00 ppm before printing spectral peak positions. Although this may seem obvious, it Is apparent to us that many

259

< ^Me

AcO,.,^ n-CsH,^

AcO,,,^ n-CgH^^ AcO,

fl5 ^16 O

erythrocis

n-CsH,, AcO,,.^ n-CsHn

P

I P

AcO,,.^n-C5Hii

I P

"MQ

threo-

J 20

cis

>

123 l24

erythro-

"^Mo

AcO**'^n-C5Hii

AcO^^^n-CsHn AoO^''^n-CsHn AcO^^nCgHt,

AcO*'*^n-CgHi,

\AB

11a

lib

11c

erythro/cis/threo/cis/erythro « er/c/lh/c/er

erlVth/der

er/t/th/t/er

lid er/c/er/c/er

lie erA/er/c/er

111 erfV&rfVer

AcO^^n-CsHii

AcO^^n-CsHii AcO^^n-CsHn

AcO^^n-Crf-lii

AcO^^n-CsHn

AcO^^n-CsHn

AcO*

AcO^^n-CgHt^ AcO*'

AcO

AcO**

AoO

n-CgHii

n-CsHn

n-C^n

iig

11h

111

Ih/c/th/c/th

th/VtfVc/th

iy

tlWtfWth

th/c/er/cmi

n-CsHn

tt/t th/Ver/c/th

n-CsHn

111 fhn/erfm

Figure 6- Twelve (of Twenty Possible) Diastereomeric, Synthetic, Model Compounds for the bis-THF Core of B1 Acetogenins. researchers are in the habit of referencing spectral resonances to some standard value of the solvent peak (e.g., residual CUCb in the CDCI3 to 8 = 7.26). This is dangerous because the solvent chemical shift is solute-dependent (e.g., CHCI3 is a weak hydrogen-bond donor); the inert TMS standard much less so.

or both down (i.e.. threo)

r^]"^

O

HgC^ O S 2.049

5 2.049

Figure 7. Acetate CJbis Chemical Shifts of Uvaricin Acetate (12) Define the C(15)/C(16) and C(23)/C(24) Relative Configurations for Uvaricin (1).

260 Further comparison of the model compounds H a - P ^ with uvaricin acetate (12) supported the assignment of C(23)/C(24)-erythro and C(15)/C(16)-threo. This analysis contributed additional information about the three-dimensional structure of uvaricin (1). Each stereorelationship along the THF backbone (three and erythro for pairs of adjacent oxygenated carbons, or cis and trans across THF rings) was correlated with a distinctive set of chemical shifts for the protons along the bis-tetrahydrofuranyl structure. The differences observed were in some cases small, but still significant enough to impart confidence In deducing the relative configuration along the B1 subunit. The chemical shift patterns of the twelve model compounds and of uvaricin and uvaricin acetate are recorded in Table 2 and shown graphically in Figure 8. (Due to the symmetry of the model compounds, and to simplify the graphic, we here revert to the numbering scheme used in the original paper.) In addition to the previously noted acetate methyl shifts, key observations were: i) H(5) and H(2) each appeared 0.04 - 0.08 ppm downfield in the trans/trans models compared to the cis/cis Isomers (where cis and trans refer to the substitution pattern on each THF ring); ii) if the configuration of the model is either cis/cis or trans/trans, H(2) is shifted farther downfield for the relationship C(2)/C(2') = three than for C(2)/C(2') = erythro; and iii) in the unsymmetrical (cis/trans) models, the resonances for H(2) and H(2') are nearly superimposed for C(2)/C(2') = erythro but significantly separated when C(2)/C(2') = three. Visual comparison of the ''H NMR spectrum of uvaricin acetate (12) to the model compounds yielded the closest match with the .../trans/threo/trans/... models; this, coupled with the previous information about the C(15)/C(16) and C(24)/C(25) relationships led to the conclusion that uvaricin has a three/trans/threo/trans/erythro configuration along the THF backbone, proceeding from carbon 15 to carbon 25 (see Figure 8). Table 2.

^H NMR Chemical Shift Values (in ppm) for the Methine Protons Associated with Oxygenated Carbons In the Model Compounds l l a - l .

1 ^ Configuration

H(2)

H(2')

H(5)

H(5')

H(6)

H(6')

Ac

Ac'

11a

er/c/th/c/er

3.81

3.81

3.94

3.94

4.90

4.90

2.045

2.045

lib

er/t/th/c/er

3.76

3.88

3.93

4.01

4.91

4.91

2.053

2.048

11c

er/tAh/t/er

3.88

3.88

3.98

3.98

4.91

4.91

2.045

2.045

lid

er/c/er/c/er

3.71

3.71

3.91

4.95

er/t/er/c/er

3.80 3.84

3.91

3.91 3.97

4.95

lie

4.91

4.96

2.045 2.058

2.053

3.99

3.99

4.92

4.92

2.050

2.050

3.86

3.93

3.93

4.94

4.94

2.069

2.069 2.077

2.045

lit

er/t/er/t/er

3.80 3.84

iig 11h

th/c/th/c/th

3.86

th/t/th/c/th

3.84

3.93

3.91

4.08

4.88

4.88

2.077

111

th/t/thAAh

3.90

3.97

3.97

th/c/er/c/th

3.77

3.93

3.93

4.85 4.84

4.85 4.84

2.074 2.074

11| 11k

3.90 3.77

th/t/er/c/th

3.82

3.82

4.850

th/t/er/t/th

3.84

3.84

3.93 3.97

3.97

111

3.97

4.84

2.073

2.073

4.852

2.080

2.075

4.84

2.071

2^07;^J

261

er/c/tfi/c/er er/t/th/c/er

o^Ms

er/t/th/t/er

nc f/ef

er/t/er/c/er er/t/er/t/er

-J5-J ;

th/t/th/c/th .n-CsHii t h / t / t h / l / t h

15

Figure 8.

th/t/th/l/th

I

A

2

A_..

I..5. 11k 111

: 5.0

.5._5lL...J2 5 i 2

6

4.9

r 4.8

f2 74

A

5 !

... i.. 6 !-«•

....lij... 2i^15

H(24)

A

•.A...

2415

I

...A.

i.

.A;A_.

4.1

-4-

th/c/th/c/er

.A:A.

I

5(ppm)

th/t/th/t/er

.-_.A.

J5-...J.?....:.

* __..5

..6..;. 6 f^ ....6!..

;.

i..2i.;.

^

._.j5l

..e..i :«--^

Ih/c/th/c/th

Ih/c/er/c/th th/t/er/c/th th/t/er/l/lh

t d .....^-.

rs 24/15;

.;

4.0

3.9

-4—

-4—

"T" 3.8

u.-Ji 23/16 20/19

.; ;

3.7

-I 1-—

i ...i

J.J.J AcO

'1623 i 20/19' 23/16201/19

\

AcO

•

i.Ul..LJ.L -.:-l-—1-

2.08 2.06 2.04

i

-^-

AcO

AcO

AcO's!

Graphical Comparison of Proton Chemical Shift Data for Model bisAcetates 11a-l and the Peracetate Derivatives of Uvaricin (12), Rolliniastatin I (14), and Asimicin (15).

In that early work w e relied much more on proton than carbon chemical shift trends. This was, in part, driven by the limited quantities of some of the twelve pure, synthetic, model compounds. However, the proton shift trends were also more meaningful than the carbon for this particular set of model compounds. It is our contention that proton chemical shift data should be used more frequently for this purpose and that this underutillzation Is largely a bias of technological origin. From the advent of '•^c NMR spectroscopy chemical shift trends were recognized to be of primary importance. Relatively large field dispersion and the routine lack of coupling data predisposed

262 researchers to rely heavily on "^^c chemical shifts. In the case of protons, only in the last decade has the routine availability of spectra recorded at Increasingly higher magnetic fields provided relatively complete assignments of the majority of resonances in spectra of complex molecules. Thus, proton chemical shift trends in complex molecules now warrant very careful attention. The 1987 report of the X-ray structure of a derivative of rolllniastatin I (8)''2 permitted us to further validate this ''H NMR correlation method."'^ The relative configuration along the THF rings in rolllniastatin I (8) (I.e., threo/cis/threo/cis/erythro) as determined by chemical shift correlation (Figure 8) matched exactly with the crystallographically determined structure. In the course of this analysis, some refinements to the method were made. Unlike uvaricin acetate (12), the triacetylated rolllniastatin I (14) did not exhibit a clear correlation with a single set of model compounds; two possibilities for the relative configuration were identified by simple visual inspection. Therefore, it was necessary to make the method more quantitative to arrive at an unambiguous conclusion. This was accomplished by comparing each of the eight measured "'H chemical shifts [H(15), H(16), H(19), H(20), H(23), H(24), and the two acetate methyl groups] for the natural product derivative with the analogous resonances for each of the model compounds, and taking the sum of the observed chemical shift differences. (For the six, unsymmetrical diastereomers of the model compounds that were JTJQI made, the expected chemical shifts were extrapolated from the relevant symmetrical model compounds.) The model having the smallest sum of the absolute values of chemical shift differences (I|A5's|) compared with the natural product represents the most likely relative configuration. The results of this comparison both for rolllniastatin I (8) and for aslmicin (13), another recently (at the time) discovered Annonaceous acetogenin,''^ are summarized In Table 3. Rolllniastatin I triacetate (14) shows the best match with a hypothetical erythro/cis/threo/cis/threo model, which corresponds exactly with the relative configuration determined by X-ray crystallography on the derivative of the natural product. Aslmicin triacetate (15) was determined to be threo/trans/threo/trans/threo, which is the same conclusion reached by visual inspection. A comment must be made at this point about the limitations of this method. It leaves open the question of directionality of the stereochemical relationships. For example, were the complete relative configuration of rolllniastatin I (8) not known from the x-ray crystallographic study, it would not be possible to tell whether the order of relative configuratlonal relationships proceeding from C(15) to C(24) was threo/cis/threo/cis/erythro or erythro/cis/threo/cisAhreo. We refer to this as the "endedness" problem, and it is an issue of structural ambiguity that has been overlooked In a number of instances.

263

Table 3.

Quantitative Comparison of ''H NMR Chemical Shifts for the Peracetates of Uvaricin (12), Rolliniastatin I (14), and Asimicin (15) with each of the Twelve Model Compounds lla-l as Well as with Eight Additional Extrapolated Unsymmetrical Isomers. 2:|A5's| model

12

14

15

er/c/th/c/er er/lAh/c/er er/l/th/t/er

tra lib 11c

0.11 0.17 0.21

0.36 0.52

er/c/er/c/er

lid

0.30 0.28 0.08 0.62

5 6 7

er/l/er/c/er er/t/er/t/er th/c/th/c/th

lie 11f

0.34

8 9 10 11

thMh/cAh th/lAh/tAh th/c/er/c/th

0.44 0.28 0.36 0.32 0.02

th/l/er/c/th

111 11k

0.15 0.19 0.19 0.23 0.29 0.27

th/l/er/t/lh

111

0.22

0.15 0.21

0.22

12 13

er/c/th/c/th

11a/11g

0.26

0.09

0.36

entry

descriptor

1 2 3 4

119 11h 111

0.18 0.26 0.32 0.12 0.44 0.28

0.41

0.16 0.72

0.38 0.16

14

er/t/th/c/th

11b/11h

0.19

0.15

0.27

15

th/t/th/c/er

11h/11b

0.35

0.26

0.41

16 17

er/t/th/l/lh

0.05 0.47

0.09 0.54

18 19

er/l/er/c/lh th/t/er/c/er

20

er/l/er/t/lh

11C/11I 11d/111 lle/11k 11k/lie 11f/111

0.22

er/c/er/c/th

0.26 0.26 0.14

0.30 0.19 0.16 0.17

0.34 0.32 0.22

Born et. al.^ have reported a complementary technique to determine the relative configuration between a carbon in a THF ring and an adjacent carbinol center. This approach is applicable to all B1, 82, B3, or B4 substructures. The model compounds 16-ef and 16-f/i (Figure 9) were synthesized as a mixture of diastereomers and separated chromatographically as their acetate derivatives. These acetates were assigned as three or erythro by the observed ^H NMR coupling constant between H(15) and H(16). The isomer with the smaller coupling constant (J15/16 = 5.2 Hz) was assigned the three relative configuration while that with the larger (J15/16 = 6.0 Hz) was assigned as erythro. Such a small difference in J's suggests that this assignment was somewhat tenuous. However, it has since been confirmed by subsequent stereospecific synthesis and correlation of model mono-THF compounds."^^ô jhe

264

i^

O I

i^-'CioHai

(^

7\

63.84(82.29)"

^\1^15^^10^21

O ""5 3.84(71.83)

6 3.79(82.47)'

^ ^ , ^ " " 5 3.40 (73.87)

Figure 9. Diagnostic Proton (and Carbon) Chemical Shift Data for Simple erythroand threo-a-HydroxyalkyI Tetrahydrofurans. acetates were then reconverted to the free alcohols 16, which were studied by ''H and "•^c NMR spectroscopy. A correlation was found between the three or erythro configuration of the models and the chemical shifts of nearby ''H and ""^c nuclei, particularly C(15) and H(16). These results are summarized in Table 4 along with the relevant data for annonin I (4), the discovery of which was reported in the same paper. It is clear from these data that annonin I possesses one three and one erythro relationship between C(15)/C(16) and C(23)/C(24). The question of which was which (I.e., the endedness), however, was resolved only through X-ray crystallographic structural analysis of the derivative 10. This work provided another verification of our original approach to assignment of bis-THF relative configuration. Table 4.

Correlation of ''H and "•^c NMR Chemical Shift Values Between Annonin I (4) and the Diastereomeric Pair of Model Compounds 16-er and 16-th. 16-er

4 (Annonin 1)

16-th

H(15)

3.84

3.40

H(15), H(24)

3.40, 3.87

H(16) C(15)

3.84 71.83

3.79 73.87

H(16), H(23) C(15). C(24)

3.88, 3.87 71.7,74.1

1 C(16)

82.29

82.47

C(16), C(23)

83.4, 82.9

All of the methods discussed so far, however useful, still leave open the question of absolute configuration. In 1992, we described a study carried out in collaboration with the McLaughlin group^"' detailing our studies of Mosher ester (i.e., methoxytrifluoromethylphenylacetate) derivatives22-24 of various carbinol centers in the acetogenins. Since all THF-contalning acetogenins have at least one hydroxyl group in the B subunit, Mosher derivatization of these groups provides an opportunity to draw conclusions about the absolute configuration in this portion of the molecule. The principle behind the Mosher ester technique is illustrated in Figure 10. The two enantiomers of the Mosher acid chloride, (f?)-MTPA-CI and (S)-MTPA-CI, are used to derivatize a stereo-

265 genie carbinol center to the (S)- and (f?)-MTPA esters, 17-Sand 17-/?, respectively.25 Assuming that the preferred conformation is as shown, with the trifiuoromethyl group eclipsed with the carbonyt, conclusions can be drawn regarding the absolute configuration of the carbinol center based on "^H and ''^F NMR spectroscopic data.26 Since the phenyl group will tend to have a shielding effect on nearby atoms, protons in the L3 portion of the ester should appear farther upfield in the "• H NMR spectrum of 17Sthan in 17-/?, while those in the L2 substituent should display the opposite trend. more highly shielded

less highly shielded OMe

^m a Streptomyces strain; details of the structure have not yet been reported. ^^ 2. Biological Activity The nargenicins exhibit activity against gram-positive bacteria, especially Staphylococcus aureus (Table 1). A comparison of activities indicate that nargenicin Ai 1 is considerably more active in vitro than the C9-hydroxy congener nodusmicin, 2 J 18-Deoxynargenicin Ai is less active than 1 against staphyloccocus strains but shows pronounced activity against streptococci. Table 1. Agar-diffusion Antimicrobial Spectrum of 1, 2 and 3J Organism Bacillus subtilis Staphylococcus aureus Micrococcus luteus Klesiella psewnoniae Mycobacterium avium Penicillum oxalicum Saccharomyces pastorianus Bacteroidesfragilis Clostridium perfringens

Nodusmicin 0 — 35 27 26 0 0 33 33

INHIBrnON ZONE SIZE* Nargenicin Ai 18-Deoxynargenicin Ai 0 36 42 32 30 0 0 37 48

15 27 31 18 26 0 0 21 36

* In mm from 12.6 mm disc dosed with 0.08 ml of 1mg/ml solution. The activities of nargenicin Ai and Bi against S, aureus strains are comparable to that of erythromycin. Studies by the Pfizer group have indicated that both nargenicins retain activity against multiple-drug resistant Staphylococcus strains (Table 2). 10 Table 2, Comparative In Vitro Spectrum and Potency of 1 and 5.^® Organism Staphylococcus aureus Staphylococcus aureus^ Staphylococcus epidermis Staphylococcus epidermis*"^ Neisseria sicca Escherichia coli Pasteurella multocida

ANTIBIOTIC and MIC (Hg/ml) Nargenicin Ai Nargenicin Bi Erythromycin 0.1 0.2 0.8 0.8 25 2.0 25

0.8 1.6 1.6 6.3 0.8 12.5 200

*Multiple-dmg resistant strain. **Methicillin/erythromycin resistant strain.

0.1 >200 0.1 >200 1.6 1.6 0.4

286 Studies by the Upjohn group confumed the potent activity of nargenicin Ai against multiple-drug resistant strains of S. aureus and demonstrated the in vivo activity of the antibiotic in mice (Table 3)7 Table 3. Activity of 1, 2 and 3 Against Drug-Resistant S. aureus Strains^ In VitrO'MlC (îg/ml) Sa Resistant Strain UC76 (control) UC6685 (P.T,C,N,K,E)* UC6686 (G,K)* UC6687 (P,K)* UC6686 (P,T,C,Ch,E)* UC76 (subcutaneous) UC76 (oral)

Nodusmicin >100 >100 >100 >100 >100

>320

Nargenicin Ai 18-Deoxynargenicin Ai 0.2 0.2 0.2 0.2 0.2

1.0 1.5 3.1 1.5 3.1

In Vivo CD50 (mg/kg) 17.4 MOO 50

* Resistant by Kirby-Bauer disc diffusion method to: penicillin G (P), Tetracycline (T), Clindamycin (C), Novobiocin (N), Kanamycin (K), Chloroamphenicol (Ch), Erythromycin (E). Originally isolated from a screen for antibiotic activity against anaerobic bacteria, coloradocin, 8, exhibits pronounced and selective activity against pathogenic anaerobes and microaerophiles, displaying only limited activity against aerophilic microorganisms. ^^ The biological spectrum of 8 contrasts those of the structurally-related nargenicins, which are primarily active against aerobic targets. In vitro studies have demonstrated that coloradocin has activity comparable to vancomycin against selected anaerobes, including Clostridium difficile, the causative organism for pseudomembranous colitis.^ 1 Coloradocin is particularly active against the niicroaerophilic organisms Neisseria gonorrhoeae, Haemophilus influenzae and Legionella strains, and is effective against ampicillin-resistant strains ofN. gonorrhoeae and H, influenzae. Acute toxicity of coloradocin is low (LD50 in mice injected intra-peritoneally is >500 mg/kg). 3. Biosynthesis The polyketide biosynthetic origin of the nargenicins has been independentiy confirmed by Cane 15 and Rinehardt.l^. Feeding experiments with l^C-labeled sodium acetate indicate that carbon pairs C1-C2, C3-C4, C5-C6, C7-C8 and C11-C12 of nargenicin Ai are acetate-derived; analogous studies with 13C-labeled propionate have shown that the C17-C18-C19, C15-C16C20, C13-C14-C21 and C9-C10-C22 triads of 1 derive from propionate (Figure 3). The C23 methyl group of nargenicin Ai originatesfromL-methionine; the pyrrole 2-carboxylic acid moiety of 1 derivesfrompropionate/acetate via the succinate-a-ketoglutarate-dehydroproline pathway. Cane has demonstrated that advanced di-, tri- and tetraketide fatty acid precursors are incorporated directiy into the nargenicins, and has noted that an early intermediate in the nargenicin pathway is common to the biosynthetic scheme leading to erythromycin A and

287 Figure 3

=

A Me"

CH0. r S ^ W ' S " "

^ y ^

=

'O-

M e ^ ^ ^ .

o isl ,^^^v^ HO

20 Incorporation of Acetate and Propionate Subunits Into Nargenicin A

19

methymycm.l7 A three stage sequence has been proposed for the overall biosynthetic pathway leading to the nargenicin macrolides, consisting of: (1) elaboration of an extended, hranched-chain fatty acid 9 from propionate-acetate condensations, (2) cyclizations to establish the cis-fused octalin and macrolide systems of 11, and (3) final oxidations and introduction of the C23 methyl and C9-0-acyl groups of 1 (Figure 4). Labeling studies with 180-Iabelled propionate-acetate precursors reveal that oxygen atoms at CI, C9, C l l and C17 are derived from propionate and acetate, while fermentation studies conducted in an ^^02 atmosphere indicate that the C2 and C18 oxygen substituents and the C8-C13 ether bridge of nargenicin Ai originate from molecular oxygen, an event that presumably occurs late in the biosynthetic pathway. While mechanistic details of the biosynthetic elaboration of tiie octalin and macrolide systems have yet to be defined, Cane has suggested that the nargenicin octalin nucleus may be generated by the intramolecular Diels-Alder cyclization of an oxygenated tetraene intermediate 10.^ Figure 4 COS-Enz

S-Enz

10 .OH

O2

11

1; nargenicin A^

288 4. Chemistry of the Naturally-Occurring Nargenicins The novel structural features of the nargenicins impart a unique chemistry to the macrolides. Not unexpectedly, the decenolide system is relatively sensitive and readily undergoes acid or base-catalyzed ring cleavage (Figure 5). Whaley and coworkers have reported that hydrolysis of nodusmicin by methanolic sodium hydroxide affords the corresponding seco acid 12; further degradation with sodium periodate affords acetaldehyde and aldehydic acid 13.3 Solvolysis of nargenicin Ai 1 in basic ethanol initially affords a ring-expanded lactone 14; at extended reaction times 14 undergoes lactone cleavage to yield ester 15.^ Alternatively, treatment of nargenicin Ai with acidic ethanol affords seco-tsitx 15 directiy. Figure 5

MeO.

2; nodusmicin

12

13 .O-CP

1; nargenicin A^ CP« 2-carboxypyrrole

Reagents: (a) IH NaOH, 1:1 H20:MeOH; (b) aq NaI04; (c) NaOEt, EtOH; (d) HCl, EtOH. The sensitivity of the nargenicin decenolide system to cleavage is fortunately not reflective of the ease with which the macrolide can be regenerated from seco acid derivatives. The presence of three sp^-hybridized carbons and the fusion to the rigid 1 l-oxatricyclo[4.4.llA0^''7]undecene nucleus impart considerable rigidity to the lactone system and reduces the number of degrees of rotational freedom available to seco derivatives. Steliou has demonstrated a facile, tin-mediated closure of the tetrahydropyranyl-protected nodusmicin seco acid 16 to lactone 17 (Figure 6)}^ Early synthetic studies by Magerlein and co-workers established that the rate of reactivity of the nargenicin hydroxyl substituents with a variety of electrophilic reagents follows the general trend C 1 8 > C 9 » C 1 1 . ^ The structural relationship between nodusmicin, 2, and nargenicin Ai, 1, was confirmed by silylation of the CIS hydroxyl group of 2, followed by selective acylation of the C9 hydroxyl of 18 and deprotection (Figure 7).^^ This strategy has been used to prepare a series of 9-C)-acyl analogs of nargenicin Ai from the C18-silylated intermediate 18; in general.

289 Figure 6 ,OTHP

a.b MeO.

Reagents: (a) 10 eq. dihydropyran, CSA, CH2Q2; (b) 1.1 cq KOH, 1:1 HiOiMeOH, reflux; (c) 1 eq Me2SnO, mesitylene, reflux. diminished antibiotic activity is observed for this series (Table 4). Finally, nodusmicin has been transformed to 18-deoxynargenicin Ai, 3, by thioacylation and reductive deoxygenation of thioimidazole 19 via the Barton protocol^ Figure 7 ,o-cp

CPa 2-carboxypynrolel

3; 18-deoxynargenicin A

Reagents: (a) rBuMe2Sia, imidazole, CH2CI2; (b) DCC, pym>le-2-carboxylic acid, pyridine; (c) aq BU4NF; (d) l,r-tiiiocarbonyldiimidazole, THF; (e) nBusSnH, THF. A notable aspect of the chemistry of the nargenicins is the remarkably inert C14-C15 trisubstituted olefin. In their original isolation smdies, Whaley and coworkersreportedthat while the C5-C6 olefin of 2 undergoes rapid hydrogenation, the C14-C15 olefin was uneffected by hydrogenating conditions, even at extended reaction times.3 Subsequent studies on the seco derivative 20 and tetraacetate 21 have revealed that the C14-C15 olefin of these compounds is similarly unreactive towards bromination and oxidation with a variety of reagents, including osmium and ruthenium tctroxides and ozone (Figure 8).^^ The lack of reactivity of the C14-C15 olefin is presumably a consequence of the extreme steric environment of this group, which is

290

Table 4.

Activity of 9-0-Acyl Esters of Nodusmicin Against S.

9-0-Sub^tituent

MIC (^g/ml)

H (nodusmicin, 2) Pyrrole-2'-carbonyl (nargenicin Al, 1) Pyrrole-3'-carbonyl Benzoyl Thiophene-2'-carbonyl Thiophene-3'-carbonyl Furan-2'-carbonyl Furan- 3 '-carbonyl Nicotinoyl Isonicotinoyl 4-MethylpyrTole-2'-carbonyl N-Methylpynole-2'-carbonyl L-Prolyl A.3'-L-Prolyl Pyrrole-2'-acetyl PyiTole-2'-acryloyl

125 0,125 0,39 >250 3,9 0,5 7,8 0.5 250 >250 0,78 >250 62,5 12.5 >100 >50

AureusA^

blocked from approach by external reagents by both the C4 and CI 1 substituents. In an attempt to mitigate the steric environment at C14-C15, iodolactone 24 was prepared from 21; however, the lack of reactivity of 24 towards oxidizing and other reagents parallels that of 20 and 21.^1 Figure 8

MeO

23a; R» H 23b; R« Ac

Reagents: (a) K2CO3, MeOH; (b) AciO, DMAP; (c) Br2, CCI4; (d) OSO4, THF; (e) I2, MeCN. As part of an effort to prepare compounds for correlation with advanced synthetic intermediates, Plata examined the stepwise degradation of the nargenicin C19-C14 macrolide subunit.î Following the earlier work of Whaley,^ ester 20 was subjected to periodate oxidation and the remaining C9 and C l l hydroxyls protected as methoxymethyl ethers (Figure 9),

291 Hydrogenation of the C5-C6 olefin of 25 and oxidative decarbonylation afforded enone 26. Attempts to oxidize the C14-C15 olefin of 26 using a variety of reagents (O3, OSO4, MCPBA) were unsuccessful, as were efforts to generate a p-alkoxy ketone for an anticipated retro-aldol reaction by conjugate addition of oxygen nucleophiles to 26. Exposure of 26 to aqueous acid resulted in the selective cleavage of the Cll ether and intramolecular addition to the enone, yielding tetracyclic 27 as a single isomer of undetermined stereochemistry. Figure 9 PMOM

25 .OMOM

Reagents: (a) NaI04, THF, H2O; (b) MeOCHaCl, (iPr)2NEt; (c) H2,5% Pd/C, MeOH; (d) DABCO, O2, Cu(0Ac)2,2,2'-bipyridyl; (e) HQ, H2O. n. SYNTHETIC STUDIES OF THE NARGENICIN MACROLIDES The unique structural features of the nargenicin macrolides pose an intriguing synthetic challenge and several groups have recorded efforts directed at the total synthesis of these compounds.^2-24 Critical structural and stereochemical elements which must be addressed in an effective synthetic route to the nargenicins include: (1) the novel, highly-functionalized 11-oxatricyclo[4.4.1.l»^»'7]undecene nucleus, (2) the remote stereogenic centers at C2, CI6, C17 and CI 8 of the macrolide system, and (3) the presence of an acid/base sensitive decenolide ring. Steliou's lactonization of a protected 5ec

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