Alkaloids

THE ALKALOIDS: Chemistry and Pharmacology VOLUME 50

THE ALKALOIDS Chemistry and Biology

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THE ALKALOIDS: Chemistry and Pharmacology VOLUME 50

THE ALKALOIDS Chemistry and Biology Edited by

Geoffrey A. Cordell College of Pharmacy University of Illinois at Chicago Chicago, Illinois

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

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Copyright 0 1998 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center. Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0099-9598/98 $25.00

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I

CONTENTS

CONTRIBUTORS. ........................................................................... PREFACE .................................................................................

xi ...

XI11

R. H. F. Manske: Fifty Years of Alkaloid Chemistry

D. B. MACLEAN A N D V. SNIECKUS 1. Introduction ................................................................. .................................... 11. Childhood and Formative Years.. 111. Higher Education and Early Empl .......... ................

IV. V. VI. VII. VIII.

Scientific Career and Research ............................................ Editorship.. ......... ................................................. ............... The Scientist and SOC Naturalist. Orchidist, Concluding Remarks ................................................. Publications of R. H. ...............

3 7 8 18 40 42 45 47 51

Chemistry and Biology of Steroidal Alkaloids A N D M. IQBALCHOUDHARY ATTA-UR-RAHMAN

I. Introduction. ......................................... ....... .... 11. Isolation and Structure Elucidation ........................................... 111. Physical Properties . .... ..................................... IV. Biogenesis.. .................... V. Some Synthetic Studies and Chemical Transformations.. .................... VI. Pharmacology.. ................ References ...........................................

61 63 75 90 92 98 103

Biological Activity of Unnatural Alkaloid Enantiomers ARNOLD BROSSIA N D XUE-FENG PEI Introduction ............... ....................... Analytical Criteria. ....... ........................... Unnatural Alkaloid Enan (+)-Morphine.. ............................................................. (+)-Physostigmine.. .. ................................................. VI. (+)-Colchicine ............................. VII. (+)-Nicotine ................................................................ 1. 11. 111. IV. V.

V

109 110

112 118

123 128 133

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CONTENTS

VIII. Conclusions . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .. . . . . . .. . . . . . .. . .. . .. .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .

135 136

The Nature and Origin of Amphibian Alkaloids JOHNW. DALY Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. .. . .. . .. .. ... .. .. 1. Introduction.. 11. Sarnandarines . . . . . . . . . . . ................................................... .. ... .. ... . .. .. . .. ... .. ... .. ... .. . 111. Batrachotoxins., .. ... ... .. .. . . . . .. . .. .. , ................... . . . .. . .. . _. _ _. _... .. .. . .. .. . . . .. . .. ...

IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

The Purniliotoxin Class. ...................... Histrionicotoxins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histrionicotoxins.. ....................................... Gephyrotoxins . . . . . . . . . . Gephyrotoxins Decahydroquinolines.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Decahydroquinolines.. Cyclopenta[b]quinolizidines.... .. . .. ...... .. .. ..... ... .. ... . .. . . . .. ... . . .. ... .. . ....................................... ...................... Epibatidine.. . . . . . . . . . . . . Epibatidine.. ...................... Pseudophrynamines . . ,. . Pseudophrynamines Pyrrolizidine Oximes Pyrrolizidine Oxirnes . . . . . . . . . . . . . . . . . . . . .................................. . . . .. . . . . . . . . . . . . Coccinellines Coccinellines.. . . . . . . . . . . ........................................................ .................................. .... Bicyclic “Izidine” Alkal Monocyclic Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. . . . .. . . . . . . . . . ....................................... Summary and Prospects Prospects References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Biochemistry of Ergot Alkaloids-Achievements DETLEF GROCERA N D HEINZG. I. 11. 111. IV. V. VI. VII. VIII.

141 142 142 145 149 151 1.51 152 154 1.54 155 156 157 157 158 159 164 165 16.5 167

and Challenges

FLOSS

Introduction.. . . ........................................................ Historical Background.. ...................... The Natural Ergot Alka Producing Organisms.. . ...................... ...................... Biosynthesis.. . . . . . . .. . .. Biotechnologica Pharmacologica ...................... Future Challenges . . . . . . .. . .. .. . . . . . . . . . . . . References . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

172 172 173 182 183 20 1 204 208 212

Natural Polyamine Derivatives-New Aspects of Their Isolation, Structure Elucidation, and Synthesis HESSE ARMIN GUGGISBERC A N D MANFRED

....................................... I. Introduction.. . . . . . . . . . . . ........... 11. Alkaloids with the Sper 111. Spermine Alkaloids. . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. 3-Phenylpropenoyl Derivatives of Sperrnine and Spermidine . . . . . . . . . . .. . . . . V. Polyamines from Spiders, Wasps, and Marine Sponges References . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

219 22 1 243 247 249 254

CONTENTS

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Molecular Genetics of Plant Alkaloid Biosynthesis

TONIM. KUTCHAN I. Introduction.. .......................................................... 11. Monoterpenoid lndole Alkaloids.. ............................................ 111. TetrahydrobcnzylisoquinolineAlkaloids ...................................... IV. Bisbenzylisoquinoline Alkaloids ........................... V. Tropane and Nicotine Alkaloids.. ............................................. VI. Acridone Alkaloids ................................ VII. Conclusions and Fu .................................. References ......................................................................

258 259 272 290 295 304 309 311

Pseudodistomins: Structure, Synthesis, and Pharmacology

ICHIYA NINOMIYA. TOSHIKO KIGUCHI. A N D TAKEAKI NAITO I. Introduction. .................................................................... ................. 111. Synthesis ........................................................................ IV. Biogenesis.. ..................................................................... V. Pharmacology References ...................................................................... 11. Isolation and Structure.. . . .

317 318 322 338 340 341

Synthesis of the Aspidosperma Alkaloids

J. EDWIN SAXTON I. 11. 111. IV. V. VI. VII. VIII.

Introduction.. .................................................. The Aspidospermine Group ................................................... Vindorosine and Vindoline ... ................................... ..... The Vincadifformine Group ................................. The Vindolinine Group ........................................................ .................................. The Meloscine Group . . . . . . . . . . The Aspidofractinine Group.. .............................. The Kopsine Group ............................................................ References ...................... .................................

343 344 346 355 361 366 366 369 374

Synthetic Studies in Alkaloid Chemistry CSABASZANTAY 1. Introduction. ....................................................................

11. Synthesis of Ipecacuanha Alkaloids ........................................... 111. Synthesis of Yohimbine Alkaloids. ........................................

IV. V. VI. VII. VIII.

Synthesis Synthesis Synthesis Synthesis Synthesis

of of of of of

Corynantheidine Alkaloids.. ..................................... Rauwolfia Alkaloids.. ............................................ Berbane Vincamine and Structurally Related Alkaloids ................. Aspicfospenna Alkaloids .........................................

377 379 380 383 384 385 386 399

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IX. Synthesis of Alkaloids from Catharanthus roseus.. ........................... X. Synthesis of Morphine.. ........................................................ XI. Synthesis of Epibatidine.. ...................................................... References ......................................................................

400 405 407 41 1

Monoterpenoid Indole Alkaloid Syntheses Utilizing Biomimetic Reactions HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAl 1. Introduction.. .........................

...........................

11. Biomimetic Syntheses of Corynanthe aloids from Secologanin. Strictosidine. and Their Analogs. .............................................. 111. Biomimetic Syntheses of Aspidospernia and fboga Alkaloids ............... IV. Biomimetic Skeletal Rearrangements and Fragmentations ......... V. Biomimetic Synthesis in the Sarpagine Family.. .............................. ............................ VI. Biomimetic Bisindole Alkaloid Syntheses ...... VII. Conclusions ......................................... References ................................................

415 416 419 428 436 444 447 448

Plant Biotechnology and the Production of Alkaloids: Prospects of Metabolic Engineering I. 11. 111. IV. V. VI.

V A N DER HEIJDEN. A N D J. MEMELINK RoeERr VERPOORTE. ROBERT Introduction ...................... Plant Cell Cultures for the Production of Alkaloids ......................... Metabolic Engineering ............................ Transcriptional Regulati ansduction Pathways .............. Conclusions ..................... ........ ................ Future Prospects.. ................................. ................ References .............. .......................................

453 455 462 491 496 497 499

History and Future Prospects of Camptothecin and Taxol

c. W A N 1 MONROE E. WALL A N D MANSUKH I. Camptothecin ................................................................... 11. Taxol ............................................................................ References ......................................................................

509 521 531

Alkaloid Chemosystematics PETERG. WATERMAN

I. Introduction.. ................................................................... 11. Alkaloids in Chemical Systematics: Laying Down the Rules ................ 111. The Evolution of Alkaloids.. .................................................. IV. Handling Alkaloid Data in Systematic Studies ...............................

537 539 540 544

CONTENTS

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V. Systematically Significant Distributions of Alkaloids in Higher Plant Taxa ........................................................... VI. Concluding Comments ......................................................... References ......................................................................

548 563 564

....................................................... CUMULATIVE INDEX OF TITLES.. INDEX ..................................................................................

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

AITA-UR-RAHMAN (61), H. E. J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan ARNOLD BROSSI (109), School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599 M. IQBALCHOUDHARY (61), H. E. J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan JOHNW. DALY(141), Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 (171), Department of Chemistry, University of WashingHEINZG. FLOSS ton, Seattle, Washington 98195 DETLEFGROCER(171), Institute for Plant Biochemistry, Halle (Saale), Germany ARMINGUCCISBERG (219), Organisch-chemisches Institut der Universitat Zurich, 8057 Zurich, Switzerland MANFRED HESSE(219), Organisch-chemisches Institut der Universitat Zurich, 8057 Zurich, Switzerland TOSHIKO KIGUCHI (317), Kobe Pharmaceutical University, Higashinada, Kobe 658, Japan

TONIM. KUTCHAN (257), Laboratorium fur Molekulare Biologie, Universitat Munich, 80333 Munchen, Germany

D. B. MACLEAN (3), Department of Chemistry, McMaster University, Hamilton, Ontario, Canada L8S 4M1 J. MEMELINK (453), Institute of Molecular Plant Sciences, Leiden University, 2300RA Leiden, The Netherlands TAKEAKI NAITO(317), Kobe Pharmaceutical University, Higashinada, Kobe 658, Japan xi

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CONTRIBUTORS

ICHIYA NINOMIYA (317), Kobe Pharmaceutical University, Higashinada, Kobe 658, Japan XUE-FENG PEI(109), Laboratory of Bioorganic Chemistry, National Institutes of Health, Bethesda, Maryland 20892 SHIN-ICHIRO SAKAI (415), Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263, Japan J. EDWINSAXTON (343), Department of Chemistry, The University of Leeds, Leeds LS2 9JT, United Kingdom V. SNIECKUS (3), Guelph-Waterloo Center for Graduate Work in Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 CSABA SZANTAY (377), Institute of Organic Chemistry, Technical University, and Central Research Institute for Chemistry, H-1525 Budapest, Hungary HIROMITSU TAKAYAMA (415), Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263, Japan ROBERT VAN DER HEIJDEN (453), Division of Pharmacognosy, Leiden/ Amsterdam Center for Drug Research, Leiden University, 2300RA Leiden, The Netherlands ROBERT VERPOORTE (453), Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300RA Leiden, The Netherlands MONROE E. WALL(509), Research Triangle Institute, Research Triangle Park, North Carolina 27709 MANSUKH C. WANI(509), Research Triangle Institute, Research Triangle Park, North Carolina 27709 PETERG. WATERMAN (537), Phytochemistry Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow G1 lXW, Scotland, United Kingdom

For many younger chemists and biologists, for whom this volume may be the initial foraging into the mystical, marvelous world of alkaloid chemistry and biology, the name “Manske” has an indescribable aura attached to it. Perhaps advised by a more senior colleague or faculty member to “look it up in Manske,” the younger scientist’s prototypical response is the question “What’s ‘Manske?’ Is it some acronym for a computerized database on alkaloids?” (“Many Alkaloids, New and Structurally Korrect, ‘Ere” comes to mind, and, incidentally, reflects my Cockney upbringing.) “Oh, it’s that book series on alkaloids. Can’t recall who’s the editor now. Used to be Manske in the old days. Don’t really know who he was though,” comes back the response from the learned professor. Thousands of alkaloid chemists and biologists, as well as many natural product scientists, know this series only as “Manske” or “Manske’s Alkaloids.’’ Only when they have to write a citation reference do these chemists and biologists discover that the last volume edited by Manske was published in 1977, the year of his death, and that the title of the series began as The Alkaloids: Chemistry and Physiology and was changed, with the publication of Volume 21 in 1983, to The Alkaloids: Chemistry and Pharmacology. This volume marks a transition in the title of the series, which will be changed again as of Volume 51 to The Alkaloids: Chemistry and Biology. I believe that this reflects the transition that is being made to cover not only the biological and pharmacological effects of alkaloids once isolated, but also their role in their host organism or secondary site, as well as the substantial advances in the biotechnological aspects of alkaloid formation and production. The period following the death of Manske benefited from the expertise of two other editors. Russell Rodrigo, a colleague of Manske, served as editor for Volumes 17-20, and then Arnold Brossi took over as very energetic editor for Volumes 21-40. Brossi and I coedited Volumes 41 and 45. Why then isn’t it called “Brossi’s Alkaloids?” Chapter 1 in this celebratory volume may provide an answer, as well as a response to some of the other issues raised above. When I first decided to put together a special volume of the series in celebration of the publication of Volume 50, I had the idea to ask a select group of alkaloid chemists to prepare a chapter on their own areas of

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PREFACE

interest, indicating some of the recent progress and speculating on where their area of the field would be moving in the years ahead. I was extremely fortunate to persuade many outstanding scientists to contribute to this volume. Then I received a letter from Victor Snieckus indicating that he and D. B. MacLean were preparing a biography on Manske. They were asking if I could help them publish this article in the series or recommend another site for publication. The synchronicity was perfect. Their outline was exciting; it reflected a very personal view of an exceptional human being, and thus it was an easy decision that this biography would be the first chapter in the celebratory volume. Embellished with Manske’s own autobiographical and laboratory notes and some wonderful anecdotes and photographs, the completed chapter shows Manske as an outstanding alkaloid chemist and as a person who was committed to the role of scientist as a contributor to society (“If we leave the decisions to politicians and theologians we will inherit a society which scientists will not like and we will only have ourselves to blame,” p. 44). In addition, it shows his love of cooking, of growing orchids, and of ecology. Suddenly, this is not merely the name on the spine of some musty old volumes-not just the name in colloquial use for a book series. This is a real person, someone who has almost been brought back to life. There is no longer an excuse when asked “Who was Manske?” or “Why is the series still called Manske’s Alkaloids?” In addition to bringing out the human qualities of the founder of this series, this chapter reveals another astonishing fact: that the chemistry that Manske and his colleagues accomplished was done, for the most part, without the benefit of either chromatography or spectroscopy. Current graduate students and postdocs should stand in awe of these achievements, and those of the other legends of alkaloid chemistry, for that matter. We are truly standing on the shoulders of giants, yet their presence is rarely acknowledged as we rush to run the next gradient-enhanced HMBC spectrum. As a result, this unique perspective of alkaloid chemistry offers a wonderful historical overview of life as an alkaloid chemist in the mid1920s to the mid-1970s. The remaining chapters in this volume are written by a selection of the leading scientists working in the field of alkaloid chemistry and biology today and are arranged alphabetically by author. Atta-ur-Rahman and Chaudhary describe some of the prominent recent chemical and biological work, much of it conducted in their own laboratories, on the steroidal alkaloids from terrestrial plants and animals and from marine organisms. Since most physiologically active alkaloids are pure enantiomers, it is intriguing chemically and biologically to prepare and evaluate the unnatural enantiomers of important alkaloids. Brossi and Pei describe some of the recent work in this area. Amphibians are also recognized as being a source

PREFACE

xv

of chemically and biologically significant alkaloids, and Daly updates the recent studies that have led to the isolation of epibatidine and several other interesting metabolites. The critical issue of the future sourcing of these alkaloids is also discussed. Groger and Floss are recognized as leaders in the field of ergot alkaloid chemistry and biosynthesis, and for the first time in many years they bring this area up-to-date and clearly indicate the opportunities for future research development. The natural polyamine derivatives derived from spermine and spermidine are under rapid development currently from both an isolation and a synthetic perspective, and Guggisberg and Hesse describe these recent results based substantially on their own studies. The tremendous impact that .enzyme isolation and molecular genetics are having, and will continue to have, in the future strategies for understanding the formation and availability of important alkaloids is reviewed in detail by Kutchan. Tunicates of the genus Pseudostoma have yielded a number of novel metabolites whose structure elucidation and synthesis have been engaging several Japanese research groups. Ninomiya, Kiguchi, and Naito clarify the confusion that has surrounded the structures of these particular alkaloids. The past 18 years have seen some remarkable developments in the efficient formation of various members of the Aspidosperma group of alkaloids, and Saxton provides an authoritative review of this area. Paralleling the history of The Alkaloids series have been the tremendous synthetic efforts in alkaloid chemistry conducted at the Central Research Institute for Chemistry in Budapest in the past 40 years, principally under the leadership of Szantay, who here reviews some of the highly directed work on various indole and other alkaloid groups that has led to the enhanced commercial availability of several alkaloids. The structural diversity of the monoterpenoid indole alkaloids has led to numerous biogenetic ideas as to the formation of these structure types, very few of which have been tested in vivo. However, many of them have been evaluated, successfully, through chemical incitement, and these efforts are reviewed by Takayama and Sakai. Substantial drama in the past 20 years has surrounded the impact of biotechnology on plant secondary metabolism. The chapter by Verpoorte, van der Heiden, and Memelink nicely complements that of Kutchan in focusing on the experimental issues that have come to light with the use of cell cultures for the production of alkaloids and on how metabolic engineering still faces numerous challenges. Together these chapters define well the need for more concerted studies on how and where alkaloids are actually produced in plant cells and indicate the mountainous pathway ahead which must be traversed for the commercial production of medicinally important alkaloids in vitro. Two plant alkaloids, taxol and camptothecin, have recently been approved for marketing for the treatment of various cancerous states after

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many years of dedicated effort by researchers following their isolation by Wall and Wani. This saga is described by these discoverers, and the future developments in these important fields of alkaloid research are outlined. Finally, the chemosystematics of alkaloids, such as it is known at present, is discussed by Waterman, and some pertinent questions are asked. Have we progressed since the early work by Hegnauer? What is the significance for chemosystematics (and for alkaloid chemistry and biology) that “dormant” genes for alkaloid production can be turned on? It is a stimulating thought indeed that many plants may already have the genes for the production of diverse alkaloids and that in our isolation studies we are merely looking at those genes in operation today. Is the common genetic pool for alkaloid production more widely distributed than we have imagined? What are the signal transducers and transcription factors for these genes to be turned on and off? With the revolution underway in plant biotechnology these questions will surely be answered in the next few years, and the challenges of generating medicinally valuable agents within new, fast-growing host systems in large bioreactors will be surmounted. The holy grail of a continuous-flow operation for the production of an alkaloid through stabilized enzymatic synthesis will undoubtedly be achieved, and the field identification of the individual components of complex alkaloid mixtures will become a reality through global communications technology. Alkaloid synthesis will continue to improve as higher yield, more steroselective, more compact, and more economical procedures become available. And, as our understanding of human biology and the diseases with which we are afflicted improves, so more and more significant alkaloids will be detected from the terrestrial and marine environments. I have no doubt that the vibrancy of this field of alkaloid chemistry and biology will contribute even more substantially in the next 50 years to the health and welfare of humankind than it has in the past. Thus, while we celebrate this volume of The Alkaloids: Chemistry and Pharmacology as a milestone of continued scientific achievement, I conclude that with dedication, intuition, and an appropriate level of investment, it will be shown that our present state of knowledge is merely a beginning to an even greater level of understanding and awareness of our world and its potential for sustainable development. Geoffrey A. Cordell University of Illinois at Chicago

THE ALKALOIDS Chemistry and Biology

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R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY D. B. MACLEAN Department of Chemistry McMaster University Hamilton, Ontario, Canada L8S 4M1

V. SNIECKUS Guelph- Waterloo Center for Graduate Work in Chemistry University of Waterloo Waterloo, Ontario Canada N2L 3G1

I. Introduction ............................................ 11. Childhood and Formative Years .................. 111. Higher Education and Early Employment ............................................... A. Queen's University (1919-1924) ................................................. B. Manchester University (1924-1926) ................................................... C. General Motors Corporation (1926-1927) and Yale University (1927-1929) ................................................................. IV. Scientific Career and Research .................. A. Calycanthine ...

V. VI. VII. VIII.

C. The Isoquinoline Alkaloids ............................................................ D. The Lycopodiurn Alkaloids ... E. Miscellany .................................................................................. F. Heterocyclic Chemistry ......... Editorship ............................................... The Scientist and Society ........... Naturalist, Orchidist, Musician, and Cuisinier .......................................... Concluding Remarks ..................................................................... Publications of R. H. F. Manske ...........................................................

8 9

20 36

45 51

I. Introduction My mother discovered that tincture of laudanum relieved my insomnia. . . . I slept long and peacefully and became a model child. -R. H. F. Manske commented on his first acquaintance with alkaloids at the age of 18 months. [2] THE ALKALOIDS, VOL. 50 0099-Y5Y8/YX $25.00

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Copyright 8 IYYX hy Academic Press All rights of reproduction in any form reserved.

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MACLEAN AND SNIECKUS

Richard H. F. Manske was an outstanding Canadian chemist who will be remembered for his many contributions to the isolation and structural elucidation of alkaloids, particularly those of the isoquinoline family. As a leading authority on alkaloids, he was chosen to become the founding editor of The Alkaloids in 1950 and continued as editor until his untimely death in 1977. We were fortunate to have known him as a boss and collaborator (D. B. M. from 1946) and as a colleague (V. S. from 1966) and we, and many others, benefited from his broad knowledge and his enthusiasm for research. Outside his office and laboratory, he found time to be an avid gardener and orchid grower; also, he enjoyed music, played the violin, watched birds and stars, made an excellent martini, was keen to discuss science, religion, and philosophy, and even wrote a book on cooking. A truly remarkable man! The celebration of the fiftieth volume of The Alkaloids is an opportune occasion to honor his eminent contributions to alkaloid chemistry. All of these studies were accomplished by what may now be known as the classical methods-reactions carried out in glass with the usual inorganic reagents . . . , with reagents for the detection of functional groups, but without electronic gadgetry. There were no crooked lines to interpret because there were no machines to make them. [ 1 )

When Manske began his research, alkaloids were separated by fractional crystallization [3] of the bases or their salts and purified to constant melting point by repeated crystallization. Thus by trial and error, infinite patience, and superb experimental skill, separation of complicated mixtures was achieved. Compositions were established by elemental analysis and molecular weight determinations of the alkaloids and their derivatives and functional group analyses were used extensively to gain initial structural insight. Complex structures were elucidated by degradation to smaller fragments and these, after identification (usually by synthesis), were intuitively reassembled to arrive at a tentative structure in accord with the molecular composition. The ultimate proof of structure was the synthesis, by unambiguous methods, of the proposed structure and the establishment of its identity with the natural product [4]. The chemists of the day were limited to the determination of the skeletal arrangement of the atoms in the molecule since, without NMR spectroscopic and X-ray techniques, degradation and synthesis often provided little stereochemical information. Although enantiomeric relationships were readily resolved, the establishment of absolute stereochemistry was not possible. Diastereomeric relationships were recognizable, e.g., in the phthalideisoquinoline alkaloids, but the determination of relative stereochemistry was seldom realized. Morphine, the Proteus of organic compounds, succumbed to the assaults upon it and strychnine was just beginning to give up some of its mysteries. [l]

1.

R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

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These are some of the classical problems to whose solution I was a spectator. Perhaps the most spectacular was that of strychnine because more skilled chemists had been concerned with it than with any other substance . . . [ l ] It is taken for granted that almost any compound can be synthesized if enough manpower is available for it. Even so, organic chemistry has not yet reached the stage when a synthesis can be achieved by merely pushing buttons. 111

Despite the above limitations, complex structural problems were being tackled with great promise. Morphine, strychnine, thyroxine, Vitamin A, cholesterol, and the bile acids were among the significant molecules which revealed their structures using classical methods; UV spectroscopy was available but its use for structural work began only in the late 1920s. In the arena of complex synthesis, strychnine, sucrose, and, in the later years of Manske’s life, chlorophyll and Vitamin BI2were conquered and retrosynthetic analysis became common practice [5]. Richard Manske’s introduction to research was oriented toward physical organic chemistry, first under the direction of J. A. McRae at Queen’s University, Kingston, Ontario, and subsequently with A. Lapworth at ManChester, England. His first experience with alkaloids was gained also in Manchester where, as part of his Ph.D. thesis under the supervision of Robert Robinson, he accomplished the total synthesis of harmaline. As Eli Lilly Research Fellow and Sterling Fellow at Yale University, he continued work on alkaloids and, in 1931, shortly after joining the National Research Council (NRC) of Canada as Associate Research Chemist, he published his first paper on the degradation of calycanthine, an alkaloid that he had isolated at Yale. This paper was followed by the first of several papers on the Senecio alkaloids and, in 1932, the first of a flood of publications, initially from NRC and later from the Dominion Rubber Co., on alkaloids of the Fumariaceous plants. This work greatly expanded the number of isoquinoline alkaloids and resulted in the discovery of several new ring systems. Through his outstanding research on the Fumariaceous plants, he gained early recognition and became an internationally renowned alkaloid chemist. Beginning in 1942, Manske, in collaboration with Leo Marion, examined the Lycopodiaceae for alkaloid content, an investigation which led to the isolation of some 30 alkaloids, and opened up a completely new field of alkaloid research [6]. As Head of the Organic Chemistry Section at NRC, Manske championed the pursuit of fundamental research and, by example, did much to improve the quality of research in Canada. Leo Marion, who succeeded him at NRC, followed similar objectives with equal vigor [6]. Manske regarded Marion as an excellent chemist, and from Marion’s account [7], the admiration was reciprocated. It was in Ottawa that his two daughters, Barbara and Cory, were born. At the time of Cory’s birth he was reaping a great harvest of

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MACLEAN AND SNIECKUS

alkaloids from Corydulis species, hence the name [8]. Also during this period he was made a Fellow of the Royal Society of Canada (1935) and was awarded the D.Sc. degree from Manchester University (1937). Staff and equipment were difficultly accessible in 1943 but we lit our first Bunsen burner on June first of that year. It has burned ever since. [l]

In 1943, given carte blanche by the then President, Paul C. Jones, Manske assumed the challenging position of Director of Research, Dominion Rubber Co., in Guelph, Ontario, and saw the research laboratories develop into a leading industrial research center in Canada. Although understandably relegated to a secondary position, alkaloids, were not neglected. Thus, it was here that he resolved the structure of the cularine alkaloids by exploiting a key reductive cleavage reaction of diary1 ethers. Furthermore, he continued work initiated at NRC on the synthesis of quinolines and the isomeric pyridocarbazoles in collaboration with M. Kulka and A. E. Ledingham whose contributions he warmly acknowledged. On Marshall Kulka, he remarked, “I regard him as one of the more skillful experimentalists that I know,” and on Archie Ledingham, he commented, “. . .a superb operator in the organic laboratory. We performed many experiments which required the use of four hands. His pair were as efficient as mine and I often marveled at the synchronism that we achieved.” Whenever time was available from his diverse duties, the Director was found at the bench. He encouraged his younger colleagues to collaborate in alkaloid work on a part-time basis thereby stimulating some into academic careers. It was also here that, without Xerox or Chemdraw, the maiden volume of The Alkaloids was compiled and saw publication in 1950. It . . . is my opinion that a group of scientists whose sole objective is practical application will soon degenerate into mere technicians. Consequently, I laid special emphasis on pursuing basic research problems, not so much to find whole products or processes, but to maintain an active esprit de corps and to develop ever more competent scientists. I am proud to record that the results bear out my contention although I do not entirely overlook the smile of lady luck. I do maintain however that fortune would not have been our reward without a staff of highly competent scientists. I further maintain that the solution of major problems seldom lies in a pointed attack. It is the by-products, those observations that had not and in general could not have been anticipated, that generate new attacks and new solutions. Models on a much grander scale are the research laboratories of the General Electric Co. and of the Bell Telephone co. [l]

True to these principles, Manske hired the best chemists available, some of whom established their careers under his guidance and others, such as A. N. Bourns, R. Y.Moir, and J. M. Pepper, left the company and became excellent teachers, researchers, and administrators at Canadian universities. In the ensuing years, his contributions to science were recognized by several

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

7

institutions. He was named Centenary Lecturer, The Chemical Society, London (1954), received the Chemical Institute of Canada Medal (1959), was awarded an honourary D.Sc. from McMaster University (1960), was named a Canadian representative to the NATO Conference on Taxonomy and Natural Products in Paris (1962), and became President of the Chemical Institute of Canada (1964). Some years before his retirement from the Dominion Rubber Co., his wife Jean, succumbed to a chronic illness. Later, he married Doris Williams who survived him. In 1966, Manske retired and joined the Department of Chemistry at the University of Waterloo as Adjunct Professor. Having regained full freedom for alkaloid research, he lost little time and no enthusiasm; an alkaloid isolated 25 years ago revealed its structure; another, the most complicated, fell to X-ray analysis some 40 years after isolation. And, of course, The Alkaloids continued. Furthermore, his Waterloo colleagues were enriched by his extraordinary grasp of practical organic chemistry. To recall an incident, one of us (V. S.) directly learned how to prepare a rare oxygenated benzoic acid-it was easy, Perkin had done it before the turn of the century! He regularly gave guest lectures on his beloved benzylisoquinoline alkaloids to the enjoyment of undergraduate and graduate students. These special treats were rich in chemistry, spiced with anecdotes about Robinson and other famous organic chemists, and sprinkled with lessons in scientific writing and the work ethic. One of us (V. S.) observed on numerous occasions the amazement of students accustomed to spectroscopic methods, when they realized that structures had once been elucidated using elemental analysis, degradation, and, in large part, chemical intuition. Judging from one of his last lectures [9], the rapidly advancing field of molecular biology did not escape his attention.

11. Childhood and Formative Years

. . . I should make a correction. My first contact with alkaloids was just before age zero. In order to expedite the count down prior to my birth the attending doctor resorted to the use of tincture of ergot. [2]

Richard Helmuth Fred Manske was born in Berlin, Germany, on September 14,1901, and emigrated to Canada in November 1906. His father, John, a factory worker, and his uncle Gustav preceded the family in order to select a homestead. Bertha Manske, Richard, and his brother Hans, 3 years his senior, sailed (third-class) to Quebec City and traveled by train to Battleford, Saskatchewan, at that time, “the frontier town at the end of

8

MACLEAN A N D SNIECKUS

the steel.” Reunion of the family was not immediate since “nature has ways of interfering with the plans of men, particularly if the affected men are not wise in the ways of nature,” and occurred only on Christmas Eve in blizzard conditions. In the Spring, after a survey of arable land by ox cart (“. . . necessarily slow. . . . The process o f . . . remastication . . . for contented oxen must be done deliberately”), the Manske family built a sod-house (“. . . with materials abundantly available, . . . essentially fire proof, and above all. . .very warm”) near the Alberta border, nurtured the homestead with meager resources, and eventually flourished by hard and honest work, available in large part owing to the expansion of the Canadian Pacific and other railroads in Western Canada. It was this environment of extreme bleakness (“There are few scenes as awe-inspiring as endless miles of snow at 40 degrees below zero Fahrenheit”) and immense beauty (“. . . the entire prairie assumed a blue hue from the profusion of the . . . crocus”) which profoundly influenced his early years. With no books, save a German bible, as reading material, the young immigrant turned to the myriad of mysteries of his surroundings, discovering the infinity of birds and plants and the vastness of nature which, by his later admission, “urged me to study her even if not to explain.” From this prairie homestead 110 miles from the nearest post office (Battleford), which was to be the home of his parents for half a century, and his brother much longer, Manske took an enormous step: “. . . from an agrarian existence . . . to one of the seats of learning at the forefront of science and the humanities.” Observing the development of a bright mind (he was awarded a Governor General’s bronze medal in an Alberta school), his parents offered strong encouragement and Manske found himself on the road to Queen’s University.

111. Higher Education and Early Employment (1919-1924) A. QUEEN’SUNIVERSITY It was a cold and clammy evening early in September of 1919 when 1 said goodbye to my mother. . . . I rode our pony down the lane that led to the road and the railway station. . . . When I dismounted and sent my obedient pony homeward I still had a peculiar sensation in the visceral region. . . . Not until I was firmly ensconced on a dusty leather seat and speeding eastward did I believe that I was really going to Kingston in Ontario. [l]

At Queen’s University, Kingston, Ontario, Manske “abruptly learned that facts alone d o not constitute an education,” adapted, and obtained

1. R.

H. F.

MANSKE:

FIFTY YEARS OF ALKALOID CHEMISTRY

9

B.Sc. (1923) and M.Sc. (1924) degrees “under instruction that was generally good and often excellent.” He especially acknowledged the impact of Professors K. L. Clark, J. A. McRae (see below), and w. C. (Billy) Baker, the latter providing two lessons in the first lecture: “avoid haste in passing judgment on your fellow man” (a reference to the fact that there was another Baker, a janitor, at Queen’s) and “facts are only important when they can be related.” Manske’s M.Sc. thesis (Fig. 1) “The Mechanism of Condensation of Aldehydes and Ketones with Compounds Containing an Active Methylene Group,” was eventually published in part [6]. The thesis addressed a controversy of that period, with respect to the position of the double bond, a, p, or 0, y , in the condensation products. The results of his research, consistent with the modern viewpoint, favored the former and showed that alkylation of the initial condensation products led to &y-unsaturated products. During his M.Sc. studies, Manske held a NRC of Canada Bursary. Encouraged by his M.Sc. supervisor, J. A. McRae, Manske sailed to Manchester for Ph.D. work. McRae had also studied at Manchester and was “the major cause of my winning the 1851 Exhibition Scholarship” to support his studies. It was at Queen’s University that he met his future wife, Jean Gray, whom he married before moving to Manchester for his doctoral studies. B. MANCHESTER UNIVERSITY (1924-1926) I was about to study chemistry under two of the world’s most famous men. . . . Not only this, but I was to meet the greatest that England had to offer in more than a casual way. W. H. Perkin, jun, a co-author on the harmaline story, . . . often discussed my work with me. [l] After I had determined the equilibrium constants of some twenty ketones and aldehydes I did that of cyclohexanone. To my surprise and to that of my professor it proved to be extraordinarily reactive. That being so, cyclohexanone cyanohydrin should form a reasonably stable potassium salt and therefore the ketone should dissolve . . . in a solution of potassium cyanide. At his request I prepared a strong solution of the latter and to this he added a liberal amount of cyclohexanone. On gentle shaking the cyclcohexanone quickly dissolved and almost instantly the solid potassium salt of the cyanohydrin separated in a mass of crystals. As he handed me the test tube he thanked me and went on his way to reappear several days later with a specimen of cycloheptanone. [l] (See also Fig. 2.)

Richard Manske entered the Ph.D. program at Manchester where “the smog . . . did nothing to lessen” his enthusiasm for learning. In the first year, he carried out research under the supervision of Arthur Lapworth while his second year was spent on a problem set by Robert Robinson although “In actual fact it was my second year with Robinson. He was also interested in mechanism and had paid me frequent visits, . . . and donated many rare carbonyl compounds.”

10

MACLEAN AND SNIECKUS

FIG.1. First page of the M.Sc. Thesis of R. H. F. Manske submitted to Queen’s University, 1924.

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

11

FIG.2. A page from the Ph.D. Thesis of R. H. F. Manske submitted to Manchester University, 1926, depicting an aldol condensation and describing the equilibrium constants for cyanohydrin formation for carbonyl compounds which laid the foundations of modem mechanistic organic chemistry.

12

MACLEAN AND SNIECKUS

Manske’s Ph.D. thesis is comprised of three parts. Part I, under the supervision of Lapworth, was entitled “The Influence of Groups on the Reactivity of Organic Compounds. Cyanohydrin Formation,” and led to three publications (5,7,23) (Fig. 2). The first paper (5) showed that the previously proposed structures of menthone cyanohydrin and camphor cyanohydrin were untenable. This conclusion was based on the results reported in the second and third publications (7,23) in which the dissociation constants of a large number of ketone cyanohydrins were measured. Furthermore, an examination of 0-, m-, and p- substituent effects on the dissociation constants of cyanohydrins of aromatic aldehydes was recorded and the results, interpreted in terms of contemporary theory of electronic and steric effects (7) (Fig. 3), may be considered to be the forerunner of the modern Hammett free-energy relationship treatment [lo]. It took me six months to synthesize harmaline-an achievement which I now would expect a third-year student to complete in six afternoons. [2] The sequence of reactions was scribbled in a hurry. He (Robinson) was vaguely aware that diazonium salts can be made to react with acetoacetic esters. . . . [ l ]

Part I1 of Manske’s thesis, “The Synthesis of Harmaline and Some of its Derivatives,” supervised by Professor Robert Robinson, triggered his interest in alkaloids which became the focus of his research career [2]. Herein is described the synthesis of harmaline (2) (Fig. 4) by ingenious application of the Japp-Klingemann reaction and, by accident, of rutaecarpine (4). The former was a benchmark achievement of synthetic confirmation of structure; the latter work, not included in his Ph.D. thesis, came about while attempting to convert P-3-indolepropionic acid (1) (Scheme l),obtained by sequential Japp-Klingemann and Fischer reactions (3), to tryptamine using the Curtius method; instead, he obtained the 0-carboline 2. From meager available structural evidence on rutaecarpine (3), Manske reasoned that 2 might be converted into the alkaloid by reaction with methyl anthranilate. This reaction in fact yielded a compound with the right m.p. However, since it was nonbasic, it violated the classical definition of an alkaloid and the crystals from this accidental total synthesis lay dormant for a year before the puzzle was clarified and the results were published. The authors called it “an unexpectedly simple synthesis” (4) [2].

. . . it was desirable to have the hydrazide of benzylphthalamic acid and this was to be prepared by heating the acid with hydrazine. Unexpectedly at that time but later perfectly obvious the result was benzylamine and phthalylhydrazide. [l] Part I11 of his thesis, “A Modification of the Gabriel Synthesis of Primary Amines,” also stemmed from an accidental discovery [2]. In an attempt to prepare the hydrazide (5) of benzylphthalamic acid (4) by treatment with hydrazine, followed by acid hydrolysis, Manske observed the formation

1.

R. H. F. MANSKE: FIFIY YEARS OF ALKALOID CHEMISTRY

13

FIG.3. A page from the Ph.D.Thesis of R. H. F. Manske which shows, in part, rationalization of the effect of benzaldehyde substituents on cyanohydrin formation.

14

MACLEAN A N D SNIECKUS

FIG.4. A page from the Ph.D. Thesis of R. H. F. Manske delineating the biogenesis of harman from tryptophan as suggested by Perkin and Robinson.

1. R.

15

H. F. MANSKE: FIFIY YEARS OF ALKALOID CHEMISTRY

2

1

3

SCHEME 1. The synthesis of rutaecarpine (3) from P-3-indolyl propionic acid (1).

of phthalyl hydrazide (6) and benzylamine (7) (Scheme 2) (Fig. 5). This prompted him to use hydrazine in the Gabriel synthesis and led to the modification, now deservedly known as the Manske-Ing reaction [111which represented a major improvement over prior practice because the traditional hydrolysis, in acidic or basic media, was often slow and incomplete. In fact, Manske used this procedure in his harmaline synthesis (2). It was the previous observation that prompted Manske to use hydrazine in the Gabriel synthesis. I had cleared my benches and received my degree but I had incurred an overdraft of thirty-five pound sterling at the storeroom. I did not possess such an astronomic sum and went to the treasurer hoping to make arrangements to pay my debt at a later time. There was no debt. Prof. Lapworth, unknown to me, had paid it. [l]

C. GENERAL MOTORS CORPORATION (1926-1927) A N D YALEUNIVERSITY (1927-1929) I felt like one who had received exhaustive swimming instructions but had never been in water. The assigned problem was to develop a better synthesis of ephedrine. [l] Inspiration came indirectly from Prof. Lapworth. He had sent me the draft of a paper in which the reactivities of a number of ketonic compounds were compared. CONHCH2Ph

HPNNH~

TI-

a

CONHCHpPh CONHNH,

5

Ct

4Co2H

1
+

H2NCH2Ph

0 6

7

SCHEME 2. Experiment which led to the Manske-Ing modification of the Gabriel synthesis of primary amines.

16

MACLEAN A N D SNIECKUS

FIG.5. A page from the Ph.D. thesis of R. H. F. Manske describing the experiments which led to the development of the Manske-Ing modification of the Gabriel synthesis of amines.

1. R.

H. F. MANSKE: F I F W YEARS OF ALKALOID CHEMISTRY

17

The carbonyl of propiophenone was less reactive than that of phenylacetone by several orders of magnitude. 1 inferred therefore that the carbonyl adjacent to the methyl phenyl diketone would react with methylamine to yield a monoketimine with the other carbonyl intact. [ I ]

In 1926, having completed his Ph.D. and with dubious prospects for a job, Manske returned to North America and was fortunate to obtain a position as a research chemist with General Motors Corp. in Detroit. His tenure as an industrial chemist was brief for, in the following year, he was offered an Eli Lilly Research Fellowship by Professor T. B. Johnson at Yale University, having been recommended by Dr. Elizabeth Gatewood, a former student of Johnson and Manske's former laboratory partner at Manchester [l].As a Lilly Fellow (1927-1929), he developed a new synthesis of ephedrine, a then important drug for the treatment of asthma and hay fever whose natural source (Chinese plant, Ephedru vulgaris) was scarce. Inferring from the work of his former mentor, Lapworth, that the C-2 carbonyl of l-phenyl-1,2-propanedione(8) (Scheme 3) would react preferentially with methyl amine [l],Manske reduced a mixture of the two components in the presence of hydrogen and Pt and obtained d,l-ephedrine (9) as the major product, along with small amounts of $-ephedrine. In collaboration with Johnson, this synthesis was published (8)and was applied to the preparation of a series of ephedrine analogs ( 9 ) ;the resolution of d,l-ephedrine using optically pure mandelic acid was also described (9) . In related work, a new synthesis of l-phenyl-l,2-propanedioneand other adiketones was reported (10). Following observations made at Manchester, Manske showed that urethanes and ureas, by-products formed in the original Curtius procedure, upon successive treatment with phthalic anhydride and hydrazine may be readily converted to primary amines (11). Thus, another connection to the Manske-Ing reaction was established. During his tenure at Yale, he also reported the isolation of the alkaloid, calycanthine, from Merufiu pruecox (12). In work apparently not linked with the Lilly Fellowship, Manske collaborated with the biochemist R. W. Jackson on the synthesis of 3-indolyl butyric

(48%)

8

9

SCHEME 3. The original synthesis of d,l-ephedrine (9) by Manske.

18

MACLEAN AND SNlECKUS

acid derivatives using the Japp-Klingemann and Fischer indole reactions (15). His interest in indole chemistry, originating in Manchester, was thus revitalized and occupied his attention for some years. This research was linked to the known involvement of 3-indolyl derivatives in plant metabolism (vide infra). In this period at Yale, Manske also reported on the occurrence of D-mannose in Fucus vesicufosus and its separation from fucose (24) and, as a Sterling Research Fellow (1929-1930) in work sponsored in part by the Rockefeller Foundation, on the attempted synthesis of the partially reduced phenanthrene system present in morphine (16).

IV. Scientific Career and Research . . . new alkaloids were discovered at a rate that would make the discoverer of islands in the St. Lawrence envious. [2]

In 1930, Manske returned to Canada to assume the position of Associate Research Chemist at the NRC Laboratories in Ottawa. Shortly thereafter (1934), Leo Marion joined the NRC and he and Manske collaborated on several researches in alkaloid chemistry.

A. CALYCANTHINE That alkaloid was one of my first loves and indeed Leo Marion and I succeeded in writing a completely satisfying and quite elegant structure for it. Actually there was only one thing wrong with it, namely, the structure. [Z]

Encouraged by Professor G. Barger, a former colleague at Manchester [l], Manske attempted to elucidate the structure of calycanthine (10) (Scheme 4) and, in his first papers from NRC, described the degradation of benzoylated calycanthine to Nb-methyl-Nb-benzoyltryptamine (11) whose structure was confirmed by synthesis (17J9). Further degradation studies, jointly with L. Marion and M. Kulka (45,54,90),some of which were carried out at Dominion Rubber Co., led to the isolation of substituted indoles, quinolines, and P-carbolines. They were probably misled by the prevalence of indoles and P-carbolines and failed to deduce the correct structure of the alkaloid [e.g., see (U)] or of a key degradation product, calycanine (13). It remained for the groups of Woodward at Harvard and Harley-Mason at Cambridge to propose the correct structure in 1 9 0 , which was verified, in an accompanying paper by Robertson and co-workers at Glasgow, through an X-ray analysis [12].

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

19

10 (Kalycanthine)

' 12 (Manske's partial structure of calycanthine) SCHEME

N'

13 (Calycanine)

4. Structure of I-calycanthine: Manske's ncmesis.

B. THESENECIOALKALOIDS In 1931, Manske published the first of several papers on the alkaloids of Senecio species (20). From S. rerrorsus, he isolated a new alkaloid retrorsine (C18H2s06N), and demonstrated that, on basic hydrolysis, it was converted into a basic and an acidic fraction, retronecine and retronecic acid, respectively. Other Senecio alkaloids behaved similarly and he proposed that, as a group, the bases be called necines, and the acids, necic acids; these terms are still used today. In the course of several years, he examined some 16 Senecio species from which he isolated several new alkaloids, necines and necic acids (33,47), four of which still carry names assigned to them by Manske. In the final paper in this series, he reported, for the first time, the presence of Senecio-type alkaloids in a related genus, Erechtites (48). From E. heirucifoliu, he isolated a crystalline base (later shown to be a mixture) which he named heiracifoline and showed that it was comprised of necine and necic acid components. For reasons, not apparent now, he abandoned the Senecio alkaloids and devoted his energies to the Fumuriu alkaloids, an area in which he had already made notable contributions, and to the Lycopodium alkaloids, a field ripe for investigation at that time. The structures of the Senecio alkaloids were eventually determined by Roger Adams and others [13].

20

MACLEAN A N D SNIECKUS

C . THEISOQUINOLINE ALKALOIDS 1. Introduction At an early date I involved myself with plants belonging to the Fumariaceae family and to my surprise I found one or more new alkaloids in virtually all of the thirty species that I examined. Only about four of these species were native to eastern Canada. Several of the others were obtained from collectors but most of them were grown in my own garden. [l]

The first of the series of papers, “Alkaloids of Fumariaceous Plants,” (21), appeared in 1932; the last, the fifty-seventh communication, in 1969

(239) [14]. Systematically and without spectral information, Manske made his major mark in science through these contributions. Manske developed a method of separation based on the solubility properties of hydrochlorides in chloroform (24). This procedure simplified the separation process and it was not uncommon for him to isolate and characterize eight or more alkaloids from a single plant extract by fractional crystallization without the modern-day benefit of chromatography. By application of this technique to new and previously examined species, many new members of established ring systems, as well as many alkaloids which defied structural categorization at that time, were discovered. The latter were carefully preserved for further study in Manske’s celebrated little brown bottles (Fig. 6) which, in turn,

FIG.6. Manske’s little brown bottles: left: 3-indolyl-propionic acid (see Section 1V.F); right: bicuculline (Dicentru cuculluriu).

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

21

were stored in pipe tobacco cans at least until 1952 when he quit smoking in protest to an increase in the federal tax on tobacco [15]. Among these were alkaloids of the cularine, spirobenzylisoquinoline, and cancentrine families, whose structures, representing new ring systems, were resolved many years after their isolation. In the beginning, Manske designated each new or uncharacterized alkaloid by a Greek letter; soon, however, he exhausted the Greek alphabet and devised a new open-ended system (42), e.g., bicuculline, originally Alkaloid-a, became F1; the last alkaloid of the series, F64, is now known as fumariline. The accumulation of aporphine and protoberberine alkaloid types led Manske to speculate on their biogenesis. These speculations were based primarily on structural relationships among the various types and are summarized in two reviews (R5,MZ). Manske also had a strong commitment and interest to use alkaloid content in plant taxonomy, especially in those cases where plant morphology was unable to provide a definitive classification (Fig. 7). The ensuing account of Manske’s contribution to the isoquinoline alkaloids will be organized in relation to the various classes of the alkaloids. We begin with the phthalide isoquinolines and continue with ring systems that were known when he began his researches. This section will conclude with the new ring systems discovered by him. 2. New Alkaloids of Established Ring Systems The structures of many of the alkaloids which were all isoquinolines, were for the greater part easily determined. Frequently it was only necessary to determine the position of a hydroxyl or of a methylenedioxy group. [l]

Although the prevalence of alkaloids of the phthalideisoquinoline, protoberberine, and protopine families in Fumaria species was already recognized at the time, Manske’s research greatly expanded their number (Tables I and 11, Schemes 5 through 8). His first success in this area was the isolation and structural elucidation of bicuculline. From Dicentra cucullaria (22),he isolated several known alkaloids and two unidentified bases, Alkaloid-a and Alkaloid+. The structure of the former was soon established as a phthalideisoquinoline; it was later named bicuculline (23).The latter was shown subsequently to be the hydroxy acid derived from bicuculline by opening of the lactone ring (28);it was named bicucine. Bicuculline holds the distinction of being the first new alkaloid whose structure Manske determined. The discovery of other members in this group followed at a fast pace. Thus, adlumine and adlumidine were isolated from Adlumina fingosa (24,25)and the structure of the former established by oxidative degradation (25).Capnoidine, a new alkaloid isolated from Corydalis sempervirens (26) proved to be the enantiomer of adlumidine and diastereomeric

22

MACLEAN AND SNIECKUS

FIG.7. Lecture delivered by R. H. F. Manske on the subject of alkaloids as an aid to plant taxonomy.

with bicuculline (202). Corlumine (34),found in C. scouleri (35),C. sibiricu (36),C. nobifis (60),and D.cucuffariu (34),was shown to be a diastereomer of adlumine (34).Corlumidine, found only in C. scouleri (35),was converted

1. R.

23

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

TABLE I PHTHALIDEISOQUINOLINES A N D

SECOPHTHALIDEISOQUINOLINES

Alkaloid

Source"

Bicuculline Bicucine" (+)-Adlumine (-)-Adlumine Adlumidine Capnoidine Corlumine Corlumidine Cordrastine Fumarimined Bicucullinine"

Dicentra cucullaria D. cucullaria Adlumina fungosa Corydalis sempervirens A . fungosa C. sempervirens C. scouleri C. scouleri C. aurea C. ochroleuca C. ochroleuca

Referencesh

" Plant source in which it was first reported. References given for isolation and structural elucidation ' Hydrolysis product of bicuculline. " Secophthalideisoquinolines.

into corlumine upon treatment with diazomethane (34);later, it was shown that the phenolic OH was located at C-7 of the isoquinoline nucleus (38). Cordrastine, apparently cordrastine I, was found in C. u r e a and I-adlumine in C. sempervirens (42). TABLE I1 TETRAHYDROPROTOBERBERINES Substitution Pattern Alkaloid

Source

Capaurine Capauridine Capaurimine Coreximine Aurotensine (? )-Tetrahydropalmatine Caseamine Caseadine Ophiocarpine Cheilanthifoline Caseanidine

Corydalis aurea C. aurea C. pallida Dicentra eximia C. aurea C. aurea C. caseana C. caseana C. ophiocarpa C. cheilantheifolia C. caseana

References

OH

OMe

1 1 1,10 2.1 1 29

2,3,9,10 2,3,9,10 2,3,9

1.11 1 13R 2 1

3,10 3,10 2,3,9,10 2,10 2,10,11 9,10 3 2.9.10

OCH,O

2.3 9.10

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MACLEAN AND SNIECKUS

(+)-Bicuculline (1 s) (1 'R) (+)-Adlumidine (1s) (1 's) (-)-Capnoidine (1 R) (1 'R) (- )-Bicucine (Lactone hydrolysis product of Bicuculline)

Me (+)-Corlumine

Cordrastine-1

R = Me

Bicucullinine

(1s) (1 'R)

Fumaramine

SCHEME 5. Phthalideisoquinoline alkaloids isolated by Manske (see Table I).

C. ochroleuca yielded two secophthalideisoquinolines, F45 and F46 (53). F45 found itself in one of Manske's little brown bottles until 1976 when it was shown to be identical with fumarimine (163).In the same communication (163),the structure of F46, given the trivial name bicucullinine, was established. It was suggested that both alkaloids may be derived in the plant from bicuculline by N-methylation, opening of the heterocyclic ring,

Hunnemanine Allocryptopine

R=H R = Me

SCHEME 6. Protopine alkaloids.

1. R.

25

H. F. MANSKE: F I R Y YEARS OF ALKALOID CHEMISTRY

Me0 MeO OMe

Capaurine R = Me Capauridine = (t)-Capaurine Capaurimine R = H

Caseamine Caseadine

R’ = R4 = Me; R2= R3 = H R’ = R3 = R4 = Me; R2= H

OH

Caseanidine

Coreximine

OMe

OMe

(+)-TetrahydropalmitineR’ = R2 = R3 = R4 = Me Cheilanthifoline R’ = Me; R2= H; R3 + R4 = CH2 (3)-Aurotensine R‘ = R3= Me; R2 = R 4 = H

Ophiocarpine

SCHEME7. Tetrahydroprotoberberine alkaloids isolated by Manske (see Table 11).

and oxidative degradation. The alkaloids are listed in Table I and their currently accepted structures are shown in Scheme 5. Their structures were determined largely by intuition while making up for liquid and salt losses by temperate imbibition. In one unfortunate case this process failed and resort to experiment was necessary. The structure arrived at unfortunately was compounded of a series of errors and the alkaloid turned out to be a specially pure sample of cryptopine. Be it remembered though in extenuation that we had no IR machine and no lithium aluminum hydride. [2]

26

MACLEAN AND SNIECKUS

(+)-Thalictrifoline (2 )-Cavidine (t)-/\pocavidine

% ‘ OR’

‘

R’ = R2 = R3 = Me; R4 = H R’ = R2 = R 4 = Me; R 3 = H R‘ = R4 = Me; R2 = R3 = H

Me0 OMe

OR2

OMe (+)-Thalictricavine

R’ = R2 = R4 = Me; R3 = H

Epiapavidine

R’ = R4 = Me; R2 = R3 = H

Solidaline

SCHEME 8. 13-Methyltetrahydroprotoberberinealkaloids isolated by Manske.

Protopine, whose structure had already been established, was a common constituent of the extracts from Fumariu plants. However, only one new alkaloid of this group, namely hunnemanine, derived from Hunnemannia fumuriaefolia, was discovered (71).The skeletal structure of the alkaloid was established by its conversion into allocryptopine by methylation (Scheme 6), and the position of the phenolic group was determined by degradation of its 0-ethyl ether. In contrast with the protopines, many new protoberberine alkaloids were discovered in the Fumariaceae (Table 11, Scheme 7). Corydalis aurea (28) was the first of the plants in this series to afford new protoberberines, namely capaurine and capauridine. The latter was later shown to be racemic capaurine. Capaurine is a 1,2,3,9,1O-pentasubstitutedtetrahydroprotoberberine with four groups and a single O H at C-1 (84). Capaurimine (F50), isolated from C. pallida, has three OMe and two phenolic O H groups. Treatment with diazomethane afforded 0-methyl capaurine. The location of the phenolic groups was established later (91) [16]. Coreximine, from D. exirnia (42) proved to be, according to his own admission [l],a surprise in that it carried C-2 and C-11 hydroxyls and C3 and C-10 methoxy groups (104,109), a “wrong” substitution pattern. It

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

27

was the first example of a norcoralydine in Nature. Aurotensine (F18), a constituent of C. aurea (42), was later shown to be comprised mainly of (?)-scoulerine (62). d,f-Tetrahydropalmitine was also isolated from C. aurea (42). Corydafiscaseana (43) afforded two new tetrahydroprotoberberine alkaloids, F33 and F35, which were given the trivial names, caseamine and caseadine, respectively (136).Caseadine was monophenolic and caseamine diphenolic; methylation with diazomethane furnished the same tetramethoxy compound. They were established to be tetrahydroprotoberberines with a novel 1,2,10,11-substitution pattern (136). Although the single O H of caseadine was assigned to C-1, it was impossible to assign the OH groups of caseamine other than that it had one OH in each of rings A and D. In the interim, the structure of caseadine has been confirmed by synthesis [17] and the structure of caseamine resolved by NMR methods and confirmed by synthesis [MI. The investigation of C. ophiocarpa afforded the new alkaloid, ophiocarpine (F39), a tetrahydroprotoberberine substituted with an alcoholic O H group (50). The OH group was assigned, correctly, to C-13 because other positions were considered untenable on the basis of the chemical behavior of the alkaloid (67). This alkaloid has been considered a link between the phthalide and protoberberine alkaloids. Cheilanthifoline (F13), present in small quantity in C. scouleri (35) and in C. sibirica (36),was found in larger amounts in C. cheifantheifofia(59), hence the derivation of its name. Its structure was established by degradative methods and by its conversion into sinactine by methylation with diazomethane (59). Caseanidine, from C. caseana (147), was monophenolic and contained three OMe groups. It also was a protoberberine and had a 1,2,9,10-substitution pattern with the OH group situated at C-1. Several new 13-methyltetrahydroprotoberberineswere isolated and characterized (Scheme 8). Examination of C. thafictrifolia (76) afforded four new bases, namely thalictrifoline, a quaternary base from which (+)-thalictrifoline was obtained upon Zn/HCl reduction, and two other new bases designated F59 and F60. The basic carbon framework of thalictrifoline and its oxygenation pattern were established by its conversion into ( 2 ) mesocorydaline, of known structure, and by its oxidation to rn-hemipinic acid (4,5-dimethoxyphthalic acid). Alkaloid F59 was shown later to be the C-13 epimer of thalictrifoline; it was named cavidine (146). Apocavidine, derived from C. tuberosa, afforded cavidine on 0-methylation; the phenolic group is situated at C-2 (146). Thalictricavine, isolated from C. tuberosa (116)is an isomer of thalictrifoline in which the positions of the substituents are transposed. Epiapocavidine, also found in C. tuberosa, is des-0-methylthalictricavine carrying the phenolic group at C-10 (153).

28

MACLEAN A N D SNIECKUS

Based on spectroscopic examination of solidaline, a minor alkaloid of C. solida (166),it has been proposed that the alkaloid is a protoberberine with methoxyl groups at C-2, C-3, C-9, and C-10, a methyl and an OH group at C-13, and an intriguing C-8-C-14 methylenedioxy bridge. Corypalline (Scheme 9), was isolated from C. pallida and C. aurea (38). Its 0-ethyl ether was identical with a synthetic sample of 7-ethoxy-6methoxy-2-methyl-l,2,3,4-tetrahydroisoquinolinethereby establishing its structure. The bisbenzylisoquinoline, dauricine, was isolated from Menispermum canadense (74,124)and a new aporphine alkaloid, analobine, from Asimina triloba (41). Manske also examined a number of papaveraceous plants, some of which were nurtured in his garden and hence required the use of his considerable persuasive tactics with the Royal Canadian Mounted Police to avoid their confiscation [15]. From Bocconia arborea, he obtained, in addition to several alkaloids of known constitution, four new substances designated P61, A, B, and C (78) which were subsequently identified (140) as 1,3-bis(llhydrochelerythriny1)acetone (A), a previously unknown compound, dihydrosanguinarine (B), oxysanguinarine (C), and 11-0-methylsanguinarine (P61) (Scheme 9). In 1964, Manske and Shin reexamined (126) Eschscholtzia californica and isolated six alkaloids of established structure, including N-methyllaurotetanine for which they suggested the trivial name, lauroscholtzine. Two apparently new alkaloids (Scheme lo), eschscholtzine, and a small amount

Corypalline

Dihydrochelerythrine

bH Analobine

1 I-Oxosanguinarine

SCHEME 9. Miscellaneous alkaloids isolated by Manske.

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

0

OMe

0

(-)-Eschscholtzine

29

(-)-Eschscholtzidine

Me0

M e o F i M e Lauroscholtrine N-Methyllaurotetanine =

Me0

SCHEME 10. Eschscholrzia alkaloids isolated by Manske.

of a phenolic base, m.p. 254"C,were also reported. The latter was ultimately shown to be bisnorargemonine (131). Eschscholtzine proved to be a new member of the pavine (argemonine) group of alkaloids (127) containing two methylenedioxy groups. A related alkaloid, eschscholtzidine, was reported several years later (232). Subsequently, the absolute configuration of this group of alkaloids was examined in an ORD study (135). In his untiring search for new alkaloids, Manske examined other fumariaceous plants of the genera Dicentra (29,30,39), Corydalis (51,65, 73,89,101,119),Dactylicapnos (77) as well as other papaveraceous plants (55,66,98,117).All afforded alkaloids, but none of novel structure. Those interested in natural product structure will be intrigued to know that there are several F-designated substances stored in the Manske little brown bottles which appear not to have been investigated further. These include F53, F54, and F55 from C. nobilis (60),F56 and F57 from C. montana (65),F58 from H . fumariaefolia (71), and F60 from C. thalictrifolia (76). 3. Alkaloids with New Ring Systems Cularine Alkaloids I had worked on cularine for a number of years and though my intuition had given me a satisfactory structure I was not able to confirm it experimentally. One day I was looking up a paper on the vapor phase methylation of aniline but what really got my attention was the following one in which the authors said that metallic sodium dissolved in ammonia will quantitively hydrogenolyze diary1 ethers. After reading it a second time and re-checking with my secretary, who also reads English, I confirmed the structure of cularine while my assistant made some dimethylaniline. [2]

30

MACLEAN AND SNIECKUS

In 1938, Manske reported the isolation of cularine (Scheme 11) and cularidine from D. cuculluriu and of cularine and cularimine from D. eximiu (42). The fourth member of this group, cularicine (125) was separated subsequently as a minor component of a mixture of phenolic bases obtained from D.cluviculutu (58), of which the major component was cularidine. It was only in 1950, after Manske had become Director of Research at Dominion Rubber Co., that the structures of cularine and cularimine (des-Nmethylcularine) were resolved by a series of degradation experiments (ZOO) (14, Scheme 12). It was known that cularine had three methoxyl groups, an N-methyl group, and an ether oxygen indifferent to attack by standard ether-cleaving reagents. Hofmann degradation (2 stage) afforded a dimethine (15) containing two double bonds which, upon oxidation, yielded a tricarboxylic acid (16) with loss of a single carbon atom but with retention of the three methoxyl groups and the ether oxygen. Also, a monocarboxylic acid was isolated containing one less carbon atom than the tricarboxylic acid; Manske inferred that it was a xanthone (17). (Phenanthroquinone undergoes an analogous series of reactions on oxidation in alkaline media to afford fluorenone.) From these data it was concluded that the Hofmann product was probably a substituted dibenz[b,f]oxepin (15). The absence of reference compounds prompted Manske to devise a synthesis of this heterocyclic ring system (see Section 1V.F). He recognized that cleavage of the diphenyl ether linkage of cularine a substance more might yield a 1-benzyl-1,2,3,4-tetrahydroisoquinoline, amenable to structural study than cularine itself. The cleavage of diphenyl ethers with sodium in liquid ammonia had been reported earlier and when this reaction was applied to cularine, it afforded a single ring-opened product in which the ether oxygen was retained as a phenolic group on the aromatic nucleus of the benzyl group. The 0 - M e derivative of the cleavage product afforded a dimethine on Hofmann degradation, which was oxidized as before to give 4-methoxyphthalic acid and asaronic acid (2,4,5-trimethoxybenzoic acid). These degradation experiments defined the structure of

R

’

O

-

T R4

\ / R~O

Cularine Cularimine Cularidine Cularicine

R’ = R2 = R3= R4= Me R’ = R2 = R3 = Me; R4 = H R’ = H; R2 = R3 = R4 = Me R‘ = H; R2+ R3= CH2; R4 = Me

OR^ SCHEME 11. Manske’s cularine alkaloids.

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

31

Two-stage Hofmann degradation

14 (Cularine)

15 (Dimethine)

16 (Tricarboxylic acid)

17 (Xanthone)

SCHEME 12. The structure of cularine. Hofmann degradation and oxidation of the resulting dimethine.

the alkaloid. The terminus of the ether linkage was located at C-8 of the isoquinoline nucleus on the basis of steric considerations and the observation that, in the isoquinoline alkaloids, oxygen substituents on aromatic rings are normally adjacent. The configuration of the cularine alkaloids was established by others, by chemical and X-ray methods; it was shown that they had the (1s) configuration (Scheme 13). The structure of cularicine was resolved through its conversion to cularine (125). The base was 0-methylated with diazomethane, the methylenedioxy group cleaved by heating with phloroglucinol in sulfuric acid, and the resulting diphenolic compound 0-methylated with diazomethane to afford cularine. Cularidine underwent 0-methylation to afford cularine, thereby establishing its skeletal structure and its substitution pattern. The position of the phenolic group was ascertained by way of degradation of its 0-ethyl ether. 4-Ethoxyphthalic acid was obtained when 0-ethylcularidine was subjected to ether cleavage with Na/NH3 and the product subsequently oxidized with permanganate (130).

32

MACLEAN AND SNIECKUS

Me0W

N.Me Na I liq NH3

Meo? Me0\ Me0

-

/OMeMe OMe 18

14 (Gularine) 1. OMethylation 2. Two-stage Hofmann

OMe

\ 2 H Me0a C 0 COpH

OMe

OMe 19

20

%

. Me0

+ HOpC J+ )

I

OH-I MnO;

/

OMe

OMe

21

SCHEME 13. The structure of cularine. Reductive ether cleavage and degradation of the cleavage product.

The Spirobenzylisoquinoline Alkaloids Manske was the first to isolate alkaloids of this group (Scheme 14) and was intimately involved in their structural elucidation. As early as 1936, ochotensine was found as Alkaloidi in C. sibirica (36) and shortly thereafter as F17 in D. cucullaria (42). It was given its present name when it was discovered in relatively abundant amounts in C. ochotensis (56) where it is accompanied with ochotensimine (0-methylochotensine). Other alkaloids isolated by Manske which subsequently proved to be of the spirobenzylisoquinoline type include ochrobirine (F14), initially obtained from C. sibirica (36) and subsequently from C. lutea (52) and C. ochroleuca (53),fumaricine (44),and the related fumaritine and fumariline (139), from F. oficinalis, and sibiricine from C. sibirica (139). Thirty years after its detection, the brown bottles of ochotensine and ochotensimine were reopened and their structures were elucidated in collaboration with S. McLean (132) [20]. The tool of the 1960s, NMR spectroscopy, rather than chemical degradation, played the key role in the structural elucidation and verification was achieved by X-ray crystallographic analysis

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

Ochotensine R' = H; R2= Me = ~e Ochotensimine R' =

33

Fumaricine R = Me Fumaritine R = H

Fumariline

Sibiricine

Ochrobirine

Fumarofine

SCHEME14. Spirobenzylisoquinoline alkaloids isolated by Manske.

of ochotensine methiodide [20]. Once the structures of ochotensine and ochotensimine were established, it was soon discovered that the other alkaloids noted above were spirobenzylisoquinolines, but lacked the exomethylene group. Instead, they were oxygenated in the five-membered ring either with one oxygen atom at C-8, fumaricine, fumaritine, and fumariline (137,138,143),or with an oxygen atom at each of C-8 and C-13, ochrobirine (142) and sibiricine (141). The structures of these alkaloids were deduced by application of NMR techniques. Alkaloid F38, fumarofine (44,148), was incorrectly placed into the spirobenzylisoquinoline class and was later shown to have an interesting benzazepine structure by Shamma and co-workers [21].

34

MACLEAN AND SNIECKUS

The Cancentrine Alkaloids Then there was M2 later called cancentrine. 111

From Dicentra canadensis, a species which holds the distinction of being the first of the fumariaceous plants which he examined (21), Manske isolated a base, as orange needles, which was not named at that time but later designated F22 (42). This alkaloid was named cancentrine in 1970, some 30 years after its isolation, when it yielded its secrets (22, Scheme 15) to X-ray crystallographic analysis of a degradation product (145). A single stage Hofmann followed by hydrogenation and treatment with diazomethane gave the dihydromethine O-methyl ether (23) whose golden-yellow hydrobromide provided suitable crystals for X-ray determination. From this X-ray structure, and NMR examination of cancentrine, its O-methylether, its 0-acetate, and its methine, the structure of cancentrine was deduced. Two dehydro derivatives of cancentrine were subsequently characterized (150) and its reactions were studied (149,156). The dimeric structure of cancentrine and its dehydro derivatives is unique among the isosquionoline alkaloids in that it combines a cularine unit with a rearranged morphine system in a novel and unexpected manner. It remains, in the late 1990s, as a formidable synthetic challenge. 4. Synthesis and Alkaloid Transformations Although I had served . . . as a professor at the founding of the Carleton University. 1 had not really been at home as a professor until I came to Waterloo. [ I ]

After his retirement from Uniroyal in 1966, Manske was named Adjunct Professor at the University of Waterloo where his structural elucidation

7

Me

Y Me

1. Hofmann

2. H 2 I R

MeO

3. CH2N2

Me0

Me0 22 (Cancentrine)

SCHEME

23 (Dihydromethine-Omethylether)

15. Degradation of the “golden-yellow” alkaloid, cancentrine.

1. R.

H. F.

MANSKE: F I I T Y

YEARS OF ALKALOID CHEMISTRY

35

research continued but the emphasis shifted somewhat to the total synthesis of benzylisoquinoline alkaloids and their interconversions. Much of this work was carried out in collaboration with R. Rodrigo, who joined Manske first as a post doctoral fellow and later established himself as a highly innovative synthetic chemist. In the area of synthesis, the construction of the ochrobirine ring system (144) was followed, two years later, by the total synthesis of the alkaloid itself (254).Also, general methods for the synthesis of the spirobenzylisoquinoline (259,262,164) and phthalideisoquinoline (264) skeleta were established and elegant total syntheses of the benzazepine alkaloid, rhoeadine (160), and the benzilic alkaloid, cryptopleurospermine (165), were achieved. Furthermore, methods were developed for the conversion of the protoberberine ring system into spirobenzylisoquinolines (157), into protopines (158), and into rhoeadines (161).

D. THELYCOPODIUM ALKALOIDS Leo Marion had earlier found nicotine in Asclepias syrica, the common milkweed, and we found it in most of the lycopodiums which yielded some thirty new alkaloids. The structures of these have been largely laid bare by Wiesner, by MacLean, and by Ayer. . . . [ I ]

An odyssey which began in 1942 in collaboration with L. Marion, led to the publication of twelve papers on the isolation of alkaloids of the Lycopodium species (club mosses) (Scheme 16) (68,75,80,82,83,87,88,93, 94,225). They also initiated the structural investigation of lycopodine (70,203) and annotinine (93); however, the structure of annotinine, the first alkaloid to have its structure established was elucidated by chemical degradation by Wiesner in 1957 and confirmed in the same year by Przbylska and Marion by X-ray crystallographic analysis of annotinine bromohydrin. The structural investigation of other alkaloids isolated by Manske and Marion was carried out largely by Canadian chemists, of whom the late K. Wiesner (New Brunswick), W. A. Ayer (Alberta), R. H. Burnell (Laval), and D. B. MacLean (McMaster) were major participants. Their overall efforts brought to Canada, in the late 1960s, a worldwide reputation in this area of natural product research. The Lycopodium alkaloids, with their surprisingly large number of new and unusual ring systems (Scheme 16), have provided synthetic chemists worldwide with a challenging playground. The resulting ingenious achievements in total synthesis have enriched the field of organic chemistry and are an appropriate measure of the impact of the initial work by Manske and Marion; equally appropriately, they have

36

MACLEAN AND SNIECKUS

Lycopodine

Annotinine

Me

Fawcettimine

p-Obscurine

Cemuine

Annotine

Luciduline

Lucidine B

SCHEME 16. Representative Lycopodiurn alkaloids.

E. MISCELLANY A few gems were found in the mountain of ore. N-Acetylornithine crystallized copiously during the extraction of the dried tubers of Corydalis ochofensiswith methanol in a Soxhlet apparatus. 3-Methoxylpyridine was obtained from the more volatile fraction of the alkaloids from Therrnopsis rhombifoliu, a legume collected from the ancestral farm in Alberta. [l] “Lobinaline from Lobelia cardinalis was of some special interest for two reasons. . . . Its isolation in a state of high purity could be easily achieved because it formed a monohydrochloride that was virtually insoluble in cold water. It was the only Lobelia alkaloid that had two nitrogens. . . . (11

Alkaloids derived from plants that do not elaborate isoquinoline alkaloids, included lobinaline (Scheme 17), from Lobelia cardinalis (46,134), several alkaloids from Therrnopsis rhombifoliu (79) including rhombifoline,

1. R. H. F.

MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

Lobinaline

37

Rhom bifoline

SCHEME 17. Structures of Lobinaline and Rhombifoline isolated by Manske.

in addition to 3-methoxypyridine (72), and lycoctonine from Delphinium brownii (40,86).The work on Delphinium was expanded by Marion at NRC and added another impressive chapter to the Canadian school of natural product research [6]. Natural products, other than alkaloids, also attracted his attention. For example, he isolated acetylornithine from C. ochorensis ( 3 3 ,an inositol isomer from two species of Calycanrhus (63),and a number of triterpenes from Lycopodium lucidulum (155). F. HETEROCYCLIC CHEMISTRY . . . he is also credited with a few hors-d’oeuvres such as a novel method of synthesis of indoleacetic acid, an improved synthesis of isoquinolines, a new synthesis of tryptamine. . . . (71

The scientific contributions of Manske were not confined to alkaloids but extended into several areas of general heterocyclic chemistry. H e wrote review articles on the synthesis and reactions of quinolines (RZ)and isoquinolines (R2). Together with M. Kulka, his collaborator at Uniroyal, he contributed a chapter, “The Skraup Synthesis of Quinolines” to Organic Reactions (R4). These were comprehensive and definitive contributions to the literature at that time and still provide valuable background for contemporary workers in these areas of heterocyclic chemistry. Aside from these reviews, Manske reported on modifications of the Skraup quinoline synthesis (64,99) and, in a definitive paper, described the preparation and characterization of seven monomethyl and twenty-one dimethylquinolines (69) which proved to be useful reference materials in the commonly used vigorous S and Se degradation studies of alkaloids. In a series of investigations, Kulka and Manske synthesized the twelve isomeric pyridocarbazoles (92,97,102,206,222,223)using quinolines (112), N-substituted tetrahydroquinolines and hydrazino isoquinolines (96) as key intermediates (e.g., Scheme 18). Still others were obtained from

38

MACLEAN AND SNIECKUS

3-carbethoxy-4-hydroxy-l l Kpyrido[2,3-a]carbazole

SCHEME18. An example of the synthesis of a pyridocarbazole by Manske and Kulka.

P-aminoethyl carbazoles by the application of the Bischler-Napieralski reaction (202).A considerable number of original intermediates were produced in these syntheses. The pyridocarbazoles proved to be valuable reference compounds in the later structural elucidation of the antitumor alkaloids, ellipticine and olivacine [23]. His contributions to indole chemistry, aside from his work with Robinson at Manchester, stemmed from his interest in plant growth hormones and his structural work on calycanthine. For example, he used a combination of the Japp-Klingemann and the Fischer indole reactions to prepare a series of 3-indolyl-w-substituted carboxylic acids which were examined as plant growth regulators (18,32,32).As his laboratory notebooks reveal (Fig. 8), for a number of years he supplied nurseries and agricultural research centers with 3-indolyl acetic acid and 3-indolyl butyric acid [24] (Fig. 6). The discovery of the dihydrodibenz[b,f]oxepin ring system within the structure of cularine prompted Manske to explore methods for its synthesis. A number of substituted dibenzoxepins and -0xepinones were prepared (205,124) whose availability assisted in the structural elucidation of the alkaloid. Thus, the oxidation of dibenz[b,f]oxepin itself afforded diphenyl ether-2,2’-dicarboxylic acid and xanthone derivatives, behavior which was exactly analogous to the oxidation of the Hofmann degradation of cularine (vide supra, Scheme 12). As a measure of the respect in which he was held by the scientific community, a special issue of the Canadian Journal of Chemistry was dedicated to his memory [25] (Figs. 9-12).

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

39

FIG.8. A page from laboratory book of R. H. F. Manske showing an account of 3-indolyl w-substituted carboxylic acids prepared and sold to nurseries and agricultural research centers.

40

MACLEAN AND SNIECKUS

FIG.9. R. H. F. Manske in Dominion Rubber Co. laboratories, Guelph, Ontario. ca 19441945. He maintained that a pipe was not a fire hazard on the grounds that the ash acted like the gauze in a Davy Lamp. He never had any fires. (Courtesy D. Brewer.)

V. Editorship With age and weakening resistance there came upon me the urge to edit some books. . . . [2]

Because of his broad experience in all aspects of alkaloid chemistry, it was appropriate that Manske was chosen to be editor of the series, The Alkaloids, Chemistry and Physiology (Fig. 13). This became the definitive treatise on the subject and is still referred to as ‘Manske’ among committed alkaloid chemists. With the same meticulous care exhibited in his research, Manske solicited contributions from chemists directly involved in research on particular classes of alkaloids to ensure accurate, expert, and current coverage. H e himself wrote on alkaloids with which he was intimately familiar and, in the later years, instituted a continuing chapter on miscellaneous alkaloids (see Publications Lists, Section XI). Of the Volumes 1-20 with Manske on the spine, Volumes 1-4 were coedited with H. L. Holmes

1. R.

H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

41

FIG.10. Gathering for a seminar at the Dominion Rubber Co. laboratories, 1965. Front row, left to right: A. Harrison, R. H. F. Manske: second row, head above Manske: M. Kulka. (Courtesy D. Brewer.)

and Volumes 17-20 bear his name, posthumously, with R. G. A. Rodrigo. Rereading the Prefaces of Volumes 1-16 offers, in addition to the Manske mastery of crisp and correct English, an instructive portrait of alkaloid chemistry over a 25-year period. As an editor, Manske was known to be precise, demanding, critical but ultimately objective and impartial. From experience (V. S.), we know that there had to be a very good reason for a tardy manuscript and that the English grammar and syntax was carefully evaluated (at times, with the help of his son-in-law, H. MacCallum, a Professor of English at the University of Toronto). There were not a few manuscripts which were entirely rewritten by Manske. On the other hand, we also know that he listened carefully to counterargument and admitted openly his errors when proven wrong. These characteristics, clearly evident in Volumes 1-16 of the series, contributed not insignificantly to making The Alkaloids unsurpassable as a comprehensive account of alkaloid research. H e also served as Associate Editor

42

MACLEAN AND SNIECKUS

FIG. 11. R. H. F. Manske (third from right) with his research group, Dominion Rubber Co.. December 1963. From left to right: M. Kulka, A. E. Ledingham, W. Boos. R. H. F. Manske, K. McPhee, and G. Rozentals. (Courtesy D. Brewer.)

(1939-1948) of the Journal of the American Chemical Society and of Organic Reactions (Vol. 7 , 1953) with equal diligence.

VI. The Scientist and Society

In summary the burden of my message has been that he who professes science is truly a scientist only if he strives to achieve an awareness of his place in society as a whole. If the buries himself in the confines of his discipline and neither knows nor cares about the broad vista of the world about him he has failed as a man; and if he does not apply the objective, that is scientific, method to matters other than to those of his narrow discipline he has failed as a man. Indeed the scientist has failed as a man if he has not made it a sine qua non of his life to question authority, be it of Mohamet or of Darwin. [26b]

1.

R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY

43

FIG.12. Doris and Richard Manske at his retirement party from the Dominion Rubber Co. (Uniroyal Ltd.), September 1966. (Courtesy D. Brewer.)

In 1963, Manske was invited to assume the office of Vice-president of the Chemical Institute of Canada (CIC) (now Canadian Society of Chemistry, CSC) and, by custom, that of President, in the following year. Even though he “. . . felt at that time . . . that I was the last one of a series of chemists who had been asked to fill the post,” he considered the offer as a great compliment, accepted it, and, characteristically, used it to address a topic of his interest. As Vice-president and President (1964), Manske provided, in lectures, a forum, and a presidential letter [26], thoughtprovoking messages regarding the role of science and scientists in society. His thesis, that “objectivity and objectivity alone should guide them in making decisions and that such a modus vivendi should be applicable to matters other than scientific,” provoked a great deal of discussion at a time when the social responsibility of the scientist was a widely debated topic. More than 10 years later and shortly before the end, in a lecture entitled “Science, Society, and Survival or Time is Running Out” he attempted again “. . . to have scientific society expend some of its expertise on the

44

MACLEAN AND SNIECKUS

FIG.13. Note by R. H. F. Manske concerning the autobiographical handwritten manuscript [ I ] . “Dear Victor-This is hurried. crude, and perhaps not legible. Please suffer it and treat it firmly and not necessarily kindly. Unless I get a stop order I will continue to scribble. Sincerely Dick M” shows the highly individual style and flair of the Editor of The Alkaloids.

socio-political issues of our times.” [9]. Although disappointed (“To my regret nothing came of it.”), Manske maintained strong and outspoken principles in this matter, as the following quotes clearly indicate. I . . . maintain that a society that functions only on subjectivity and emotions is an anachronism at a time when so much factual knowledge is available. [ I ] If we leave the decisions to politicians and theologians we will inherit a society which scientists will not like and we will have only ourselves to blame. [ l )

Not only politicians and theologians received Manske’s wisdom and wit. Whenever he believed that his scientific training would allow a knowledgeable contribution, he attempted to establish a reasoned dialogue. Thus he participated in a lengthy debate on the question of the sale of Canadian wheat to Red China and, in the editorial pages of his home town newspaper, entered into a lively discussion on science and religion [15]. On the last topic, he delivered a series of lectures at the University of Waterloo. Throughout his life, he maintained an alert interest in the world around him and a concern for human relations.

1. R.

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VII. Naturalist, Orchidist, Musician, and Cuisinier The beautiful yellow buffalo bean proved thirty years later to be Thermopsis rhombifoliu and an excellent source of alkaloids. [l]

As a growing youth, Manske saw first-hand “one of the last large areas of the world where Nature had maintained an ecological balance for many millenia unspoiled by man.” From observing the colorful succession of blooms of the prairie crocus in the Spring to the birds of game, of song, and of predation which “appeared not in flocks but in clouds as they sped south,” in the Fall, his strong sense of, and sensitivity to, nature was deeply established. H e collected shrubs, trees, and herbaceous plants and planted them near the farm but it was not until he arrived at Queen’s that he “. . . learned that botany and biology were serious subjects of study and that plants produced many chemical compounds which also merited serious study.” At NRC in Ottawa, he carried out numerous plant collecting expeditions for the alkaloid isolation work and became acquainted with leading botanists. Soon after assuming the position of Director of Research, Dominion Rubber Co., Manske purchased a house with an adjacent greenhouse on five acres of bare land. Here his interest in orchids flourished. He purchased orchids from other parts of the world (“My second acquisition . . . few of which flowered and all proved to be junk.”), cultivated others, developed a commercial venture in Cuttleya and Cymbidium orchids [23], and experimented with raising new hybrids. In the latter venture, he achieved “a modest triumph,” the registration of an orchid, named Ne Touche Pus, with the World Orchid Association, London, England (Fig. 14). Manske expressed deep concern for human ecology in his lectures and writings [1,9,26]. He also practiced it. The bare land of his property was reforested and became a bird sanctuary. On fine winter mornings, he was seen feeding and watching birds and, to maintain ecological balance, sniping at a few black crows with his .22 rifle. H e similarly developed and maintained a wildlife sanctuary near the Dominion Rubber Co. laboratories. Vividly remembered by his co-workers was the fine morning when Manske strode into the lab carrying the .22, opened a window, and made short work of the turtle which had been rapidly depleting the fish population in the nearby pond [15]. My first contact with organized noise was at the country dances with fiddlers. [l]

Manske tested his skills on a fiddle at an early age. However, following his exposure to the gramophone recording of Misha Elman playing the Minuet in G of Beethoven, he found “Those nasty C-sharps . . . beyond

FIG.14. (a) R. H. F. Manske with a prize orchid (Courtesy Kitchener-Waterloo Record, April 16, 1973); (b) in his greenhouse adjacent to his home in Guelph, Ontario, Canada. He grew 1,500 orchids.

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my competence.” Nonetheless, his love for the violin was enhanced when he experienced live performances by the great masters, Kreisler, Heifetz, and Elman, during his studies at Manchester. Although chemistry became his commitment, music was a constant life thread and this was impressively displayed in his home; modern stereo equipment and a large library of records greeted friends who (a) could accept the absence of compositions of Chopin, Puccini, and Wagner; and (b) would recognize that the beginning of music is a signal for the end of conversation [23]. Many organic chemists claim excellence in cooking based on the similarity of some techniques to those in the laboratory; Manske practiced the culinary art as seriously as his science. Although this became quickly evident in discourse, he made a definitive statement on his considerable knowledge with a book, published posthumously under the anagrammatic pseudonym of Marcand H. Kreish [27]. This unique and lively little volume, describing 29 preps, tried and tested, admittedly without vigilance of the editorial board of Organic Synthesis, shows the combination of Kreish’s masterful scientific writing (the Experimental Section) and provides samples of his wit and humor. Three excerpts are adequately illustrative: On dill pickles: There is ample evidence that our Western civilization is falling to pieces, not the least of which is the appearance of cucumbers that masquerade as dill pickles. Palates that no longer rebel against TV dinners, ready-mix cakes, or instant hash, have been conditioned to consume a concoction that is made by soaking cucumbers in a mixture of dill, vinegar, salt, and God knows what. [27]

On modern bread: It is made from some of the finest wheat in the world, from which at least some of the essentials of nutrition are abraded, and it is then passed through an assembly line emerging white, sliced, and wrapped, with neither flavor nor texture to invite ingestion. [27]

On pork chops: I know of no cook who can take a third-rate material and make a delicacy out of it. On the other hand, I have experienced many third-rate dishes resulting from slovenly manipulation of first-rate material. [27]

VIII. Concluding Remarks Richard Manske was a man with a tremendous appetite for life. He was always busy with his chemistry, his hobbies, or his family. He lived his life

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to the fullest, enjoyed it all, and transmitted his zest for living and learning to all who knew him well [28]. It [the manuscript] is a chronicle to record my thanks that history conspired to give me a profound experience, appreciated only adequately in retrospect. denied to most, and never to be experienced again. [ l ]

Acknowledgments

In retrospect. we are grateful that Dr. Manske undertook the challenge of writing his autobiography [ l ] for. without it, some of the richness of his life would have remained unstated. We express our heartfelt thanks to the chemists at Uniroyal, Walter Boos, David Brewer, Ashley Harrison, Marshall Kulka. and Archie Ledingham, who generously gave their time for interviews [15].and especially David Brewer for some of the photographs. Russell Rodrigo read an early draft and gave valuable advice. Cory Burgener, interviewed by Anne Snieckus, filled in some important, previously unrecorded, gaps on the life of her father. Anna Roglans and Guobin Miao provided invaluable help in the preparation of the manuscript. V. S. thanks the Alexander von Humboldt-Stiftung for a Fellowship and Professor Dieter Hoppe at the University of Munster for his hospitality and Freundschaft. During this tenure, by the grace of electronic communication, this manuscript was completed.

Curriculum Vitae of R. H. F. Manske

Personal Data: Born in Berlin, Germany, September 14, 1901. Emigrated with parents to Canada, 1906. Citizenship: Canadian. Married Jean Gray, 1924: deceased 1959; Married Doris Williams, 1960. Children: Barbara, Cory. Education: 1923, B.Sc., Queen’s University, Kingston, Ontario, Canada. 1924, M.Sc., Queen’s University. 1926, Ph.D., Manchester University, Manchester, England. Professional Experience: 1926-1927, Research Chemist, General Motors Corp., Detroit, Michigan, USA. 1927-1929, Eli Lilly Research Fellow, Yale University, New Haven, CT, USA. 1929-1930, Sterling Fellow, Yale University. 1930-1943, Associate Research Chemist and then Head, Organic Chem-

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istry Section, National Research Council of Canada, Ottawa, Ontario, Canada. 1943-1966, Director of Research, Dominion Rubber Co. (Uniroyal Ltd.). 1963-1964, President, Chemical Institute of Canada. 1966-1977, Adjunct Professor, University of Waterloo, Waterloo, Ontario, Canada. Awards and Honors: 1923-1924, NRC Bursary, Queen’s University. 1925-1927, Exhibition Scholar, Manchester University. 1935, Fellow, Royal Society of Canada. 1937, D.Sc., Manchester University. 1954, Centenary Lecturer, The Chemical Society, London 1959, Medal, Chemical Institute of Canada. 1960, DSc. (Honorary), McMaster University. 1967, Honorary Fellow, Chemical Institute of Canada. 1972, Morley Medal, Cleveland Section of the American Chemical Society. 1975, A. C. Neish Lecturer, NRC of Canada, Halifax, Nova Scotia, Canada. Publications: 167 papers on the structural elucidation and synthesis of alkaloids. Miscellaneous: Sometime Member of the Editors, J. Am. Chem. SOC. Author, review articles, Chem. Rev., Biol. Revs., Chem. Ind. (London). Author, chapter on alkaloids, Encyclopaedia Brittanica. Editor and part author, The Alkaloids, Vols. 1-16. Associate Editor, Organic Reactions, Vol. 7.

Footnotes

Footnote callouts appear in text in brackets. 1. Aside from quotes which are referenced, a number of quotations, so marked in the text, are from two manuscripts intended to be part of a biography (“. . . this is not an autobiography but rather an attempt to write the history of events to which I was largely a spectator and in which I was only occasionally a participant.”). This course of action was determined by the comparative poverty of our expression. R. H. F. Manske manuscripts (V.S.): typed, 35 pp., dated June 7,1973, and handwritten, 43 pp.. dated July 26,1976. 2. R. H. F. Manske, Chemistry in Canada, June 1959, p. 74. 3. Chromatography, although introduced early in the twentieth century, was mainly used, as the name implies, for the separation of colored substances.

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4. In teaching undergraduates at Waterloo, Manske was quick to point out, with a twinkle in his eye that “in all respects” is a redundant ending to this sentence. 5. “I had heard some suggestions that,. . . computers will be called upon to devise synthetic routes, if not ultimately to do the actual synthesis. During a particular vivid nightmare I was programming a computer to synthesize. . . calycanthine. . . . I seemed to have been successful. When I awoke in a cold sweat I did not have calycanthine but P-methylindole, one of the pyrolytic decomposition products of the alkaloid. I could actually smell it and when I turned on the radio I heard the somber strains of the taurine Adeste Fecales. R. H. F. Manske, Chemistry in Canada, January 1977, p. 5. 6. R. U. Lemieux, and 0. E. Edwards, LCo Edmond Marion 1899-1979. In Biographical Memoirs of Fellows of the Royal Society, 1980,26,357. See also W. Eggleston, National Research in Canada. The NRC, 1916-1966. Clarke, Irwin, Toronto, 1978,p. 363. 7. L. Marion, Chemistry in Canada, June 1959, p. 74. 8. The choice was between Corydalis (Heligrammite) and Dicentra (Bleeding Heart), according to family legend. Dicentra species were also under intensive study at that time. Burgener, Cory, personal communication. 9. R. H. F. Manske, Chemistry in Canada, June 1977, p. 17. 10. J. Hine, Physical Organic Chemistry, McGraw-Hill, New York, 1%2, p. 258. All undergraduates are exposed to the hydrocyanation chemistry, see, e.g. T. W. G. Solomons, “Organic Chemistry,” 5th ed. Wiley, New York, 1992,p. 705. For an insightful account of Lapworth’s contributions, see M. D. Saltzman, Chemistry in Britain, June 1986,p. 543. 11. See, however, R. Robinson, Memoirs of a Minor Prophet, Elsevier, Amsterdam, 1976, p. 155. 12. See R. H. F. Manske, The Alkaloids, 1%5,8, 581. 13. N. J. Leonard, The Alkaloids, 1950, I , 107. See also D. S. Tarbell, and A. T. Tarbell, Roger Adams, Scientist and Statesman. American Chemical Society, Washington, DC, 1981. 14. This series also included the investigation of several papaveraceous plants. 15. V. Snieckus, Morley Medal nomination for R. H. F. Manske, 1972. 16. T. Kametani, M. Ihara, T. Honda, H. Shimanouchi, and Y. Sasada, J. Chem. SOC. ( C ) , 1971,2541. 17. T. R. Govindachari, B. R. Pai, H. Suguna, and M. S. Premila, Heterocycles, 1977,IZ. 1811. 18. R. Suau, M. Valpuesta, M. V. Silva, and A. Pedrosa, Phytochemistry, 1988,27, 1920. 19. H. Shimanouchi, Y. Sasada, T. Honda, and T. Kametani, J. Chem. SOC.Perkin Trans. II, 1973, 1226. 20. S. McLean, and M.-S. Lin, Tetrahedron Lett., 1964, 3819; S. McLean, M.4. Lin, A. C. MacDonald, and J. Trotter, ibid, 1966, 185. 21. G. Blask6, N. Murugesan, S. F. Hussain, R. D. Minard, M. Shamma. B. Sener, and M. Tanker, Tetrahedron Lett., 1981,22,3135. 22. D. B. MacLean, The Alkaloids, 1985,26,241; W. A. Ayer, and L. S. Trifinov, ibid., 1994, 45, 233. 23. M. Kulka, and A. Gillies, Chemistry in Canada, June 1963, p. 17. 24. V.S. procured a little brown bottle of 3-indolyl-propionic acid (Fig. 6) during his post doctoral tenure (1965-1966) at the NRC laboratories, Ottawa, which he brought to Waterloo a month before his first meeting with Manske. 25. Can. J. Chem. 1979, No. 12, pp. 1545-1749. 26. (a) R. H. F. Manske, Chemistry in Canada, June 1%3, p. 25; (b) R. H. F. Manske, ibid., March 1964, p. 12. 27. M. H. Kreish, “I Cook as I Please.” Exposition Press, Hicksville, NY, 1978, 47 pp. Reviewed V. Snieckus, J . Chem. Educ., 1979,56,A182. 28. D. B. MacLean, The Alkaloids, 1979, 17, xi.

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Publications of R. H. F. Manske 1. A Modification of the Gabriel Synthesis of Amines. H. R. Ing and R. H. F. Manske, J. Chem. Soc. 2348-2351 (1926). 2. Harmine and Harmaline. Part IX. A Synthesis of Harmaline. R. H. F. Manske, W. H. Perkin, Jr., and R. Robinson, J. Chem. Soc. 1-14 (1927). 3. The Decomposition of P-3-Indolylpropionic Azide. R. H. F. Manske and R. Robinson, J. Chem. Soc. 240-242 (1927). 4. A Synthesis of Rutaecarpine. Y. Asahina, R. H. F. Manske, and R. Robinson, J. Chem. SOC. 1708-1710 (1927). 5. Formation and Decomposition of Ketone Cyanohydrins, with Special Reference to Some Compounds Recently Classified as Such. A. Lapworth, R. H. F. Manske, and A. Robinson, J. Chem. Soc. 2052-2056 (1927). 6. The Alkylation of a Cyano-P-Alkylacrylic Esters and a Phenyl-0-Alkylacrylonitriles. J. A. McRae and R. H. F. Manske, J. Chem. Soc. 484-491 (1928). 7. The Conditions Determining the Thermodynamic Stability of Cyanohydrins of Carbonyl Compounds. Part 1. Some Effects of (a) Substitution in Aromatic Aldehydes and (b) Ring Formation. A Lapworth and R. H. F. Manske, J. Chem. Soc. 2533-2549 (1928). 8. Synthesis of Ephedrine and Structurally Similar Compounds. 1. A New Synthesis of Ephedrine. R. H. F. Manske and T. B. Johnson, J. Am. Chem. Soc. 51,580-582 (1929). 9. Synthesis of Ephedrine and Structurally Similar Compounds. 11. The Synthesis of Some Ephedrine Homologs and the Resolution of Ephedrine. R. H. F. Manske and T. B. Johnson, J. Am. Chem. SOC.51,1906-1909 (1929). 10. Synthesis of Ephedrine and Structurally Similar Compounds. 111. A New Synthesis of Ortho-diketones. R. H. F. Manske and T. B. Johnson, J. Am. Chem. SOC. 51, 22692272 (1929). 11. A Modification of the Curtius Synthesis of Primary Amines. R. H. F. Manske, J. Am. Chem. SOC. 51,1202-1204 (1929). 12. Calycanthine. 1. The Isolation of Calycanthine from Meratia praecox. R. H. F. Manske, J. Am. Chem. Soc. 56, 1836-1839 (1929). 13. The Conditions Determining the Thermodynamic Stability of Cyanohydrins of Carbonyl Compounds. Part 11. Dissociation Constants of Some Cyanohydrins Derived from Methyl Alkyl and Phenyl Alkyl Ketones. A. Lapworth and R. H. F. Manske, J. Chem. Soc. 1976-1981 (1930). 14. The Occurrence of D-Mannose in Seaweed and the Separation of L-Fucose and DMannose. R. H. F. Manske, J. Biol. Chem. 86,571-573 (1930). 15. The Synthesis of Indolylbutyric Acid and Some of its Derivatives. R. W. Jackson and R. H. F. Manske, J. Am. Chem. SOC. 52,5029-5035 (1930). 16. An Attempted Synthesis of a Tricyclic System Present in Morphine. R. H. F. Manske, J . Am. Chem. Soc. 53, 1104-1111 (1931). 17. Calycanthine. 11. The Degradation of Calycanthine to N-Methyltryptamine. R. H. F. Manske, Can. J. Res. 4,275-282 (1931). 18. The Synthesis of Some Indole Derivatives. R. H. F. Manske, Can.J. Res. 4,591-595 (1931). 19. A Synthesis of the Methyltryptamines and Some Derivatives. R. H. F. Manske, Can. J. Res. 5,592-600 (1931). 20. The Alkaloids of Senecio Species. I. The Necines and Neck Acids of S. refroisus and S. jacobaea. R. H. F. Manske, Can. J. Res. 5,651-659 (1931). 21. Alkaloids of Fumariaceous Plants. I. Dicentra canadensis Walp. R. H. F. Manske, Can. J. Res. 7,258-264 (1932).

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22. Alkaloids of Fumariaceous Plants. 11. Dicentra cucullaria (L.) Bernh. R. H. F. Manske, Can. J. Res. 7,265-269 (1932). 23. Alkaloids of Fumariaceous Plants. 111. A New Alkaloid, Bicuculline, and its Constitution. R. H. F. Manske, Can. J. Res. 8, 142-146 (1933). 24. Alkaloids of Fumariaceous Plants. IV. Adlumina fungosa Greene. R. H. F. Manske, Can. J. Res. 8,210-216 (1933). 25. Alkaloids of Fumariaceous Plants. V. The Constitution of Adlumine. R. H. F. Manske, Can. J. Res. 8,404-406 (1933). 26. Alkaloids of Fumariaceous Plants. VI. Corydalissempervirens (L.) Pers. R. H. F. Manske, Can. J. Res. 8,407-411 (1933). 27. Alkaloids of Fumariaceous Plants. VII. Dicentra eximia (Ker) Torr. R. H. F. Manske, Can. J. Res. 8,592-599 (1933). 28. Alkaloids of Fumariaceous Plants. VIII. Corydalis aurea, Willd. and the Constitution of Bicucine. R. H. F. Manske, Can. J. Res. 9,436-442 (1933). 29. Alkaloids of Fumariaceous Plants. IX. Dicentra formosa Walp. R. H. F. Manske, Can. J. Res. 10, 521-526 (1934). 30. Alkaloids of Fumariaceous Plants. X. Dicentra oregana Eastwood. R. H. F. Manske, Can. J . Res. 10,165-770 (1934). 31. Reaction Products of Indoles with Diazoesters. R. W. Jackson and R. H. F. Manske, Can. J. Res. 13B, 170-174 (1935). 32. A Synthesis of Indolyl-Valeric Acid and the Effects of Some Indole Acids on Plants. R. H. F. Manske and L. C. Leitch, Can. J. Res. 14B, 1-5 (1936). 33. The Alkaloids of Senecio Species. 11. Some Miscellaneous Observations. R. H. F. Manske, Can. J . Res. 14B, 6-11 (1936). 34. Alkaloids of Fumariaceous Plants. XI. Two New Alkaloids, Corlumine, and Corlumidine and Their Constitutions. R. H. F. Manske, Can. J. Res. 14B, 325-327 (1936). 35. Alkaloids of Fumariaceous Plants. XII. Corydalis scouleri Hk. R. H. F. Manske, Can. J. Res. 14B, 347-353 (1936). 36. Alkaloids of Fumariaceous Plants. XIII. Corydalis sibirica Pers. R. H. F. Manske, Can. J. Res. 148,354-359 (1936). 37. The Natural Occurrence of Acetylornithine. R. H. F. Manske, Can. J. Res. 15B, 84-87 (1937). 38. Alkaloids of Fumariaceous Plants. XIV. Corypalline, Corlumidine, and Their Constitutions. R. H. F. Manske, Can. J. Res. 15B, 159-167 (1937). 39. Alkaloids of Fumariaceous Plants. XV. Dicentra chrysantha Walp. and D. ochroleuca Engelm. R. H. F. Manske, Can. J. Res. 15B, 274-277 (1937). 40. An Alkaloid from Delphinium brownii Rydb. R. H. F. Manske, Can. J. Res. 16B, 57-60 (1938). 41. Anolobine, an Alkaloid from Asimina triloba Dunal. R. H. F. Manske, Can. J. Res. 16B, 76-80 (1938). 42. The Alkaloids of Fumariaceous Plants. XVI. Some Miscellaneous Observations. R. H. F. Manske, Can. J. Res. 16B, 81-90 (1938). 43. The Alkaloids of Fumariaceous Plants. XVII. Corydalis caseana A. Gray. R. H. F. Manske and M. R. Miller, Can. J. Res. 16B, 153-157 (1938). 44. The Alkaloids of Fumariaceous Plants. XVIII. Fumaria officinalis L. R. H. F. Manske, Can. J . Res. 16B, 438-444 (1938). 45. Calycanthine. 111. Some Degradation Experiments. L. Marion and R. H. F. Manske, Can. J. Res. 16B, 432-437 (1938). 46. Lobinaline, an Alkaloid from Lobelia cardinalis L. R. H. F. Manske, Can. J. Res. 168, 445-448 (1938).

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47. The Alkaloids of Senecio Species. 111. Senecio integerrimus, S. longilobus, S. spartioides, and S. ridellii. R. H. F. Manske, Can. J. Res. 17B, 1-7 (1939). 48. The Alkaloids of Senecio Species. IV. Erechtites hieracifolia (L.) Raf. R. H. F. Manske, Can. J. Res. 178, 8-9 (1939). 49. A Synthesis of a-Naphthyl-Acetic Acid and Some Homologues. R. H. F. Manske and A. E. Ledingham, Can. J. Res. 178, 14-20 (1939). 50. The Alkaloids of Fumariaceous Plants. XIX. Corydalis ophiocarpa Hook. F. et Thorns. R. H. F. Manske, Can. J. Res. 17B, 51-56 (1939). 51. The Alkaloids of Fumariaceous Plants. XX. Corydalis micrantha (Engelm.) Gray and Corydalis crystalha Engelm. R. H. F. Manske, Can. J. Res. 178, 57-60 (1939). 52. The Alkaloids of Fumariaceous Plants. XXI. Corydalis lutea (L.) DC. R. H. F. Manske, Can. J. Res. 178, 89-94 (1939). 53. The Alkaloids of Fumariaceous Plants. XXII. Corydalis ochroleuca Koch. R. H. F. Manske, Can. J. Res. 178, 95-98 (1939). 54. Calycanthine. IV. A Structural Formula. R. H. F. Manske and L. Marion, Can. J. Res. 178, 293-301 (1939). 55. The Alkaloids of Papaveraceous Plants. XXIII. Clauciumflavum Crantz. R. H. F. Manske, Can. J. Res. 18B, 75-79 (1940). 56. The Alkaloids of Fumariaceous Plants. XXIV. Corydalis ochotensis Turcz. R. H. F. Manske, Can. J. Res. 18B, 75-79 (1940). 57. The Alkaloids of Fumariaceous Plants. XXV. Corydalis pallida Pers. R. H. F. Manske, Can. J. Res. 18B, 80-83 (1940). 58. The Alkaloids of Fumariaceous Plants. XXVI. Corydalis claviculata (L). DC. R. H. F. Manske, Can. J. Res. 18B, 97-99 (1940). 59. The Alkaloids of Fumariaceous Plants. XXVII. A New Alkaloid, Cheilanthifoline, and Its Constitution. R. H. F. Manske, Can. J. Res. 18B, 100-102 (1940). 60. The Alkaloids of Fumariaceous Plants. XXVIII. Corydalis nobilis Pers. R. H. F. Manske, Can. J. Res. 188,288-292 (1940). 61. The Alkaloids of Fumariaceous Plants. XXIX. The Constitution of Cryptocavine. R. H. F. Manske and L. Marion, J. Am. Chem. SOC.62,2042-2044 (1940). 62. The Alkaloids of Fumariaceous Plants. XXX. Aurotensine. R. H. F. Manske, Can. J. Res. 18B, 414-417 (1940). 63. A New Source of Cocositol. R. H. F. Manske, Can. J . Res. 19B, 34-37 (1941). 64. A Further Modification of the Skraup Synthesis of Quinoline. R. H. F. Manske, F. Leger, and G. Gallagher, Can. J. Res. 19B, 318-319 (1941). 65. The Alkaloids of Fumariaceous Plants. XXXI. Corydalis montana (Engelm.) Britton. R. H. F. Manske, Can. J. Res. 20B, 49-52 (1942). 66. The Alkaloids of Papaveraceous Plants. XXXII. Styfophorum diphyllum (Michx.) Nutt.. Dicranostigma franchetianum (Prain) Fedde, and Glaucium serpieri Heldr. R. H. F. Manske, Can. J. Res. 20B, 53-56 (1942). 67. The Alkaloids of Fumariaceous Plants. XXXIII. Corydalis cheilantheifolia Hemsl. R. H. F. Manske, Can. J. Res. 20B, 57-60 (1942). 68. The Alkaloids of Lycopodium Species. I. Lycopodium complanatum L. R. H. F. Manske and L. Marion, Can. J. Res. U)B, 87-92 (1942). 69. The Synthesis and the Characterization of the Monomethyl and the Dimethyl-Quinolines. R. H. F. Manske, L. Marion, and F. Leger, Can. J. Res. 20B, 133-152 (1942). 70. The Alkaloids of Lycopodium Species.11. Some Degradation Experiments with Lycopodine. L. Marion and R. H. F. Manske, Can. J. Res. 20B, 153-156 (1942). 71. The Alkaloids of Papaveraceous Plants. XXXIV. Hunnemanniafumariaefolia Sweet and the Constitution of a New Alkaloid Hunnemanine. R. H. F. Manske, L. Marion, and A. E. Ledingham, J. Am. Chem. SOC.64,1659-1661 (1942).

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72. The Natural Occurrence of 3-Methoxy-pyridine. R. H. F. Manske, Can. 1. Res. 2oB, 265-267 (1942). 73. The Alkaloids of Fumariaceous Plants. XXXV. Corydalis platycarpa Makino. R. H. F. Manske, Can. J. Res. 21B, 13-16 (1943). 74. An Alkaloid from Menispermum canadense L. R. H. F. Manske, Can. J. Res. 21B, 17-20 (1943). 75. The Alkaloids of Lycopodium Species. 111. Lycopodium annotinum L. R. H. F. Manske and L. Marion, Can. J. Res. 21B, 92-96 (1943). 76. The Alkaloids of Fumariaceous Plants. XXXVI. Corydalis thalictrifolia Franch and the Constitution of a New Alkaloid, Thalictrifoline. R. H. F. Manske, Can. J . Res. 21B, 111-116 (1943). 77. The Alkaloids of Fumariaceous Plants. XXXVII. Dactylicupnos macrocapnos Hutchinson. R. H. F. Manske, Can. J. Res. 21B, 117-118 (1943). 78. The Alkaloids of Papaveraceous Plants. XXXVIII. Bocconia arborea Wats. R. H. F. Manske, Can. J. Res. 21B, 140-143 (1943). 79. The Alkaloids of Thermopsis rhombifolia (Nutt.) Richards. R. H. F. Manske and L. Marion, Can. J. Res. 21B, 144-148 (1943). 80. The Alkaloids of Lycopodium Species. IV. Lycopodium tristachyum Pursh. L. Marion and R. H. F. Manske, Can. J. Res. 22B, 1-4 (1944). 81. The Alkaloids of Lycopodium Species. V. Lycopodium obscurum L. R. H. F. Manske and L. Marion, Can. J. Res. 22B, 53-55 (1944). 82. Some Derivatives of Dialkoxy-phthalides. R. H. F. Manske and A. E. Ledingham, Can. J. Res. 22B, 115-124 (1944). 83. The Alkaloids of Lycopodium Species VI. Lycopodium clavatum L. L. Marion and R. H. F. Manske, Can. J. Res. 22B, 137-139 (1944). 84. The Alkaloids of Fumariaceous Plants. XXXIX. The Constitution of Capaurine. R. H. F. Manske and H. L. Holmes, J. Am. Chem. SOC.67,95-103 (1945). 85. Some Derivatives of Vicinal Trialkoxy-benzene. R. H. F. Manske, A. E. Ledingham, and H. L. Holmes, Can. J. Res. 23B, 100-105 (1945). 86. Identity of the Hydrolytic Base Obtained from Delphinium brownii Rydb. with Lycoctonine. L. Marion and R. H. F. Manske, Can. J. Res. 24B, 1-4 (1945). 87. The Alkaloids of the Lycopodium Species. VII. Lycopodium lucidulum Michx. (Urostachys lucidulus Herter). R. H. F. Manske and L. Marion, Can. J. Res. 24B, 57-62 (1946). 88. The Alkaloids of Lycopodium Species. V111. Lycopodium sabinaefolium Willd. L. Marion and R. H. F. Manske, Can. J. Res. 24B, 63-65 (1946). 89. The Alkaloids of Fumariaceous Plants. XL. Corydalis cornuta Royle. R. H. F. Manske, Can. J. Res. 24B, 66-67 (1946). 90. Calycanthine. V. On Calycanine. L. Marion, R. H. F. Manske, and M. Kulka, Can. J. Res. 24B, 224-231 (1946). 91. Alkaloids of Fumariaceous Plants. XLI. The Constitution of Capaurimine. R. H. F. Manske, J. Am. Chem. SOC.69, 1800-1801 (1947). 92. The Synthesis of Some Carbazole Derivatives. R. €3. F. Manske and M. Kulka, Can. J. Res. 25B, 376-380 (1947). 93. The Alkaloids of Lycopodium Species. IX. Lycopodium annotinum var. acrifolium. Fern. and the Structure of Annotinine. R. H. F. Manske and L. Marion, J . Am. Chem. SOC. 69,2126-2129 (1947). 94. The Alkaloids of Lycopodium Species. X. Lycopodium cernuum. L. L. Marion and R. H. F. Manske, Can. J. Res. 26B, 1-2 (1948). 95. Some Anomalous Reactions of Phenylmagnesium Chloride. R. H. F. Manske and A. E. Ledingham, Can. J. Res. 27B, 158-160 (1949).

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96. The Synthesis of Some Isoquinolines. R. H. F. Manske and M. Kulka, Can. J. Res. 278, 161-167 (1949). 97. The Synthesis of Some Pyridocarbazoles. R. H. F. Manske and M. Kulka, Can. J. Res. 278, 291-296 (1949). 98. The Alkaloids of Papaveraceous Plants. XLII. Dendromecon rigida Benth. R. H. F. Manske, Can. J. Res. 278, 653-654 (1949). 99. The Preparation of Quinolines by a Modified Skraup Reaction. R. H. F. Manske, A. E. Ledingham, and W. Ashford, Can. J. Res. 278,359-367 (1949). 100. The Alkaloids of Fumariaceous Plants. XLIII. The Structures of Cularine and of Cularimine. R. H. F. Manske, J. Am. Chem. SOC. 72,55-59 (1950). 101. The Alkaloids of Fumariaceous Plants. XLIV. Corydalis incisa (Thunb). Pers. and the Constitutions of Adlumidine and Capnoidine. R. H. F. Manske, J. Am. Chem. SOC.72, 3207-3208 (1950). 102. P-Aminoethylcarbazoles. R. H. F. Manske and M. Kulka, Can. J . Res. 28B, 443-452 (1950). 103. The Alkaloids of Lycopodium Species. XI. Nature of the Oxygen Atom in Lycopodine; Some Reactions of the Base. D. B. MacLean, R. H. F. Manske, and L. Marion, Can. J. Res. 28B,460-467 (1950). 104. The Alkaloids of Fumariaceous Plants. XLV. Coreximine, a Naturally Occurring Coralydine. R. H. F. Manske, J. Am. Chem. SOC. 72,4796-4797 (1950). 105. Synthesis and Reactions of Some Dibenzoxepines. R. H. F. Manske and A. E. Ledingham, J. Am. Chem. SOC.72,4797-4799 (1950). 106. Synthesis of Some Pyridocarbazoles. R. H. F. Manske and M. Kulka, J. Am. Chem. SOC. 72,4997-4999 (1950). 107. 3-Bromometameconine. R. H. F. Manske, J. A. McRae, and R. Y. Moir, Can. J. Chem. 29,526-535 (1951). 108. The Alkaloids of Fumariaceous Plants. XLVI. The Structure of Glaucentrine. R. H. F. Manske, E. H. Charlesworth, and W. R. Ashford,J. Am. Chem. SOC.73,3751-3753 (1951). 109. The Alkaloids of Fumariaceous Plants. XLVII. The Structure of Coreximine. R. H. F. Manske and W. R. Ashford, J. Am. Chem. SOC.73,5144-5145 (1951). 110. The Alkaloids of Fumariaceous Plants. XLVIII. The Structure of Corpaverine. R. H. F. Manske, J. Am. Chem. SOC. 74,2864-2866 (1952). 111. The Synthesis of Pyridocarbazoles. M. Kulka and R. H. F. Manske, Can. J. Chem. 30, 711-719 (1952). 112. The Nitration of Some Quinoline Derivatives. M. Kulka and R. H. F. Manske, Cun. J. Chem. 30,720-724 (1952). 113. Hydroxypyridocarbazoles. M. Kulka and R. H. F. Manske, J. Org. Chem. 17, 15011504 (1952). 114. Cyclodehydration of o-Phenoxyphenylacetic Acids to Dihydrodibenz[b,f]-oxepinones. M. Kulka and R. H. F. Manske, J. Am. Chem. 75,1322-1324 (1953). 115. The Alkaloids of the Lycopodium Species. XII. Lycopodium densum Labill. R. H. F. Manske, Can. J. Chem. 31,894-895 (1953). 116. The Alkaloids of Fumariaceous Plants. XLIX. Thalictricavine, a New Alkaloid from Corydulis tuberosa DC. R. H. F. Manske, J. Am. Chem. SOC. 75,4928 (1953). 117. The Alkaloids of Papaveraceous Plants. L. Dicranostigmu lactucoides Hook F. et Thorns. and Bocconiu pearcei Hutchinson. R. H. F. Manske, Can. J. Chem. 32, 83-85 (1954). 118. The Identity of Cryptocavine and Cryptopine. A. F. Thomas, L. Marion, and R. H. F. Manske, Can. J. Chem. 33,570-571 (1955). 119. The Alkaloids of Fumariaceous Plants. LI. Corydalis solida (L) Swartz. R. H. F. Manske. Can. J. Chem. 34, 1-3 (1956).

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120. Lycopodium Alkaloids. VII. The Reaction of Annotinine with Phenyllithium. G. S. Perry, D. B. MacLean, and R. H. F. Manske, Can. J. Chem. 36,1146-1150 (1958). A By-product in the Preparation 121. 1,6-Bis(y-Carbethoxypropyl)-2,3,7,8-Dibenzopyrocoll. of y-(3-Indolyl)butyric Acid R. H. F. Manske and W. R. Boos, Can. J. Chem. 38,620621 (1960). 122. The Genus Oceanopapaver, R. H. F. Manske, Nature 200,1123 (1963). 123. Diphenylmethane-3,3’-DicarboxylicAcid. R. W. Beattie and R. H. F. Manske, Can. J. Chem. 42,223-224 (1964). 124. Studies on the Alkaloids of Menispermaceous Plants. CCXIX. Dauricine from Menispermum canadense L. R. H. F. Manske, M. Tomita, K. Fujitani, and Y. Okamoto, Chem. Pharm. Bull. 13, 1476-1477 (1965). 125. The Alkaloids of Fumariaceous Plants. LII. A New Alkaloid, Cularicine and Its Structure. R. H. F. Manske, Can. J. Chern. 43,989-991 (1965). 126. The Alkaloids of Papaveraceous Plants. LIII. Eschscholtzia californica Cham. R. H. F. Manske and K. H. Shin, Can. J. Chem. 43,2180-2182 (1965). 127. The Alkaloids of Papaveraceous Plants. LIV. The Structure of Eschscholtzine. R. H. F. Manske, K. H. Shin, A. R. Battersby, and D. F. Shaw, Can.J. Chem. 43,2183-2189 (1965). 128. The Structure of Corpaverine. T. Kametani, K. Ohkubo, I. Noguchi, and R. H. F. Manske, Tetrahedron Lett. 3345-3349 (1965). 129. The Nature of Corpaverine. T. Kametani, K. Ohkubo, and R. H. F. Manske, Tetrahedron Left. 985-988 (1966). 130. The Alkaloids of Fumariaceous Plants. LV. The Structure of Cularidine. R. H. F. Manske, Can. J. Chem. 44,1259-1260 (1966). 131. The Alkaloids of Papaveraceous Plants. LVI. A New Alkaloid, Eschscholtzidine, and Its Structure. R. H. F. Manske and K. H. Shin, Can. J. Chem. 44, 1259-1260 (1966). 132. The Elucidation of the Structures of Ochotensine and Ochotensimine. S. McLean, M.-S. Lin, and R. H. F. Manske, Can. J. Chem. 44,2449-2454 (1966). 133. The Configuration and Conformation of Cularine. N. S. Bhacca, J. Cymerman Craig, R. H. F. Manske, S. K. Roy, M. Shamma, and W. A. Slusarchyk, Tetrahedron 22, 1467-1475 (1966). 134. The Examination of Lobinaline and Some Degradation Products by Mass Spectrometry. D. M. Clugston, D. B. MacLean, and R. H. F. Manske, Can. J. Chem. 45,39-47 (1967). 135. Optical Rotatory Dispersion and Absolute Configuration. XII. The Argemonine Alkaloids. R. P. K. Chan, J. Cymerman Craig, R. H. F. Manske, and T. 0. Soine, Tetrahedron 23,4209-4214 (1967). 136. The Structure and Configuration of Caseamine and Caseadine. Two Novel Tetrahydro Protoberberines from Corydalis caseana. A Gray. C.-Y. Chen. D. B. MacLean, and R. H. F. Manske, Tetrahedron Lett. 349-353 (1968). 137. Nuclear Overhauser Effect Studies on Fumaria Alkaloids. J. K. Saunders, R. A. Bell, C.-Y. Chen, D. B. MacLean, and R. H. F. Manske, Can.J. Chem. 46,2876-2878 (1968). 138. The Structures of Three Alkaloids from Fumaria oflcinalis L. J. K. Saunders, R. A. Bell, C.-Y. Chen, D. B. MacLean, and R. H. F. Manske, Can.J. Chem. 46,2873-2875 (1968). 139. The Alkaloids of Fumariaceous Plants. LVII. Miscellaneous Observations. R. H. F. Manske, Can. J. Chem. 47, 1103-1105 (1969). 140. Some Benzophenanthridine Alkaloids from Bocconia arborea. D. B. MacLean, D. E. F. Gracey, J. K. Saunders, R. Rodrigo, and R. H. F. Manske, Can. J. Chem. 47, 19511956 (1969). 141. Structure of Sibiricine, an Alkaloid of Corydalis sibirica. R. H. F. Manske, R. Rodrigo, D. B. MacLean, D. E. F. Gracey, and J. K. Saunders, Can.J. Chem. 47,3585-3588 (1969). 142. The Structure of Ochrohirine. R. H. F. Manske, R. Rodrigo, D. B. MacLean, D. E. F. Gracey, and J. K. Saunders, Can. J. Chem. 47, 3589-3592 (1969).

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143. Structures of Three Minor Alkaloids of Fumaria officinalis. D. B. MacLean, R. A. Bell, J. K. Saunders, C. Y. Chen, and R. H. F. Manske, Can. J. Chem. 47,3593-3599 (1969). 144. Synthesis of an Analog of Ochrobirine. R. H. F. Manske and Q. A. Ahmed, Can. J. Chem. 48, 1280-1282 (1970). 145. The Structure of Cancentrine: A Novel Dimeric Benzylisoquinoline. G. R. Clark, R. H. F. Manske, G. J. Palenik, R. Rodrigo, D. B. MacLean, L. Baczynskyj, D. E. F. Gracey, and J. K. Saunders, J. Am. Chem. SOC.92,4998-4999 (1970). 146. Structural and Conformational Studies on Tetrahydroprotoberberines. C. K. Yu, D. B. MacLean, R. G. A. Rodrigo, and R. H. F. Manske, Can. J. Chem. 48,3673-3678 (1970). 147. A New Tetrahydroprotoberberine Alkaloid from Corydalis caseana. A. Gray, C. K. Yu, D. B. MacLean, R. G. A. Rodrigo,and R. H. F. Manske, Can. J. Chem. 49,124-128(1971). 148. The Structures of Fumarofine. C. K. Yu, J. K. Saunders, D. B. MacLean, and R. H. F. Manske, Can. J. Chem. 49,3020-3024 (1971). 149. Cancentrine 11. The Structure of Cancentrine. R. G. A. Rodrigo, R. H. F. Manske, D. B. MacLean, L. Baczynskyj, and J. K. Saunders, Can. J. Chem. 50,853-861 (1972). 150. Cancentrine 111. Dehydro Derivatives. D. B. MacLean, L. Baczynskyj, R. Rodrigo, and R. H. F. Manske, Can. J. Chem. 50,862-865 (1972). 151. The Absolute Configuration of Some Spirobenzylisoquinoline Alkaloids. M. Shamma, J. L. Moniot, R. H. F. Manske, N. K. Chan, and K. Nakanishi, J. Chem. SOC.Chem. Commun. 310-311 (1972). 152. An Unusual Oppenhauer Oxidation of (+)-Ophiocarpine. V. Smula, R. H. F. Manske, and R. Rodrigo, Can. 1. Chem. 50, 1544-1547 (1972). 153. The Structure of Epiapocavidine, a New Tetrahydroprotoberberinefrom Corydalis tuberosa. R. H. F. Manske, R. Rodrigo, D. B. MacLean, and L. Baczynskyj, Anales de la Real Sociedad Espanola de Quimica 68, 689-695 (1972). 154. The Total Synthesis of (5)-Ochrobirine. B. Nalliah, Q. A. Ahmed, R. H. F. Manske, and R. Rodrigo, Can. J. Chem. U,1819-1824 (1972). 155. The Triterpenes of Lycopodium lucidulum Michx. K. Orito, R. H. F. Manske, and R. Rodrigo, Can. J. Chem. 50,3280-3282 (1972). 156. Cancentrine. IV. Acetolysis Products of Cancentrine Methiodide. R. Rodrigo, R. H. F. Manske, V. Smula, D. B. MacLean, and L. Baczynskyj, Can. J. Chem. 50, 3900-3910 (1972). 157. A Photolytic Protoberberine-Spirobenzylisoquinoline Rearrangement. B. Nalliah, R. H. F. Manske, R. Rodrigo, and D. B. MacLean, Tetrahedron Lett. 2795-2798 (1973). 158. Transformations of 13-Oxoprotoberberinium Metho Salts. 11. Conversion to protopine analogs. B. Nalliah, R. H. F. Manske, and R. Rodrigo, Tetrahedron Lett. 1765-1768 (1974). 159. New Synthesis of Spirobenzylisoquinoline Alkaloids. S. 0. de Silva, K. Orito, R. H. F. Manske, and R. Rodrigo, Tetrahedron Lett. 3243-3244 (1974). 160. Photosensitized Oxidation of an Enaminoketone. The Total Synthesis of a Rhoeadine Alkaloid. K. Orito, R. H. F. Manske, and R. Rodrigo, J. Am. Chem. SOC.%, 19441945 (1974). 161. Transformations of 13-Oxoprotoberberinium Metho Salts, 111. Biogenetically Patterned Conversions of Rhoeadines. B. Nalliah, R. H. F. Manske, and R. Rodrigo, Tetrahedron Lett. 2853-2856 (1974). 162. A New Synthesis of Spirobenzylisoquinolines.Analogs of Sibiricine and Corydaine. H. L. Holland, D. B. MacLean, R. G. A. Rodrigo, and R. H. F. Manske, Tetrahedron Lett. 4323-4326 (1975). 163. Fumaramine and Bicucullinine: Two Minor Alkaloids of Corydalis ochroleuca Koch. R. G . A. Rodrigo, R. H. F. Manske, H. L. Holland, and D. B. MacLean, Can. J. Chern. 54,471-472 (1976). 164. 3,4-Methylenedioxyphthalide-a-carboxylicAcid; Its Use in the Total Synthesis of Isoqui-

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noline Alkaloids. B. C. Nalliah, D. B. MacLean, R. G. A. Rodrigo, and R. H. F. Manske, Can. J. Chem. 55,922-924 (1977). 165. The Synthesis of Cryptopleurospermine, a Benzilic Alkaloid of Crypfocarya pleurosperrna. G. C. Dunmore, R. H. F. Manske, and R. Rodrigo, Heterocycles 8,391-395 (1977). 166. Solidaline. A Modified Protoberberine Alkaloid from Corydalis solida. R. H. F. Manske, R. Rodrigo, H. L. Holland, D. W. Hughes, D. B. MacLean, and J. K. Saunders, Can. J. Chem. 56,383-386 (1978). 167. Benzolactams. 11. Synthesis of Tetrahydrobenz[d]Indeno[l,2-b]Azepines and Their 120x0-Derivatives. K. Orito, H. Kaga, M. Itoh, S. 0. de Silva, R. H. F. Manske, and R. Rodrigo, J. Heterocycl. Chem. 17,417-423 (1980).

REVIEWS R1. The Chemistry of Quinolines. R. H. F. Manske, Chem.Rev. 30, 113-144 (1942). W . The Chemistry of Isoquinolines. R. H. F. Manske, Chem.Rev. 30, 145-158 (1942). R3. Sources of Alkaloids and Their Isolation. R. H. F. Manske, The Alkaloids 1,l-14 (1950). R4. The Skraup Synthesis of Quinolines. R. H. F. Manske and M. Kulka, Org. React. 7, 59-98 (1953). R5. The Biosynthesis of Isoquinolines. R. H. F. Manske, The Alkaloids 4, 1-6 (1954). R6. The Protoberberine Alkaloids. R. H. F. Manske, The Alkaloids 4,78-118 (1954). R7. The Aporphine Alkaloids. R. H. F. Manske, The Alkaloids 4, 119-146 (1954). R8. The Protopine Alkaloids. R. H. F. Manske, The Alkaloids 4, 147-167 (1954). R9. The Cularine Alkaloids. R. H. F. Manske, The Alkaloids 4, 249-252 (1954). R10. a-Naphthaphenanthridine Alkaloids. R. H. F. Manske, The Alkaloids 4,253-264 (1954). R11. The Lycopodium Alkaloids. R. H. F. Manske, The Alkaloids 5,295-300 (1955). R12. Minor Alkaloids of Unknown Structure. R. H. F. Manske, The Alkaloids 5, 301-332 (1955). R13. The Ipecac Alkaloids. R. H. F. Manske, The Alkaloids 7, 419-422 (1960). R14. The Isoquinoline Alkaloids. R. H. F. Manske, The Alkaloids 7,423-432 (1960). R15. The Lycopodium Alkaloids. R. H. F. Manske, The Alkaloids 7, 505-508 (1960). R16. Minor Alkaloids of Unknown Structure. R. H. F. Manske, The Alkaloids 7, 509-521 (1960). R17. The Carboline Alkaloids. R. H. F. Manske, The Alkaloids 8, 47-53 (1965). R18. The Quinazolinocarbolines. R. H. F. Manske, The Alkaloids 8, 55-58 (1965). R19. The Alkaloids of Calycanthaceae. R. H. F. Manske, The Alkaloids 8, 581-589 (1965). R20. The Alkaloids of Geissospermum. R. H. F. Manske and W. A. Harrison, The Alkaloids 8, 679-691 (1965). R21. The Alkaloids of Pseudocinchona and Yohimbe. R. H. F. Manske, The Alkaloids 8, 694-723 (1965). R22. The Cularine Alkaloids. R. H. F. Manske, The Alkaloids 10,463-465 (1965). R23. Papaveraceae Alkaloids. R. H. F. Manske, The Alkaloids 10,467-483 (1965). R24. a-NaphthaphenanthridineAlkaloids. R. H. F. Manske, TheAlkaloids 10,485-489 (1965). R25. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 10, 545-595 (1965). R26. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids U, 455-512 (1970). R27. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 13, 397-430 (1971).

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R28. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 14, 507-573 (1973). R29. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 15, 263-306 (1975). R30. Alkaloids of Dendrobium. Symp. Sci. Aspects of Orchids (H. H. Szmant and J. Wemple, eds.), pp. 122-125, Chemistry Department, University of Detroit, 1974. R31. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 16, 511-556 (1977).

MISCELLANEOUS M1. The Isoquinoline Alkaloids, Centenary Lecture. R. H. F. Manske, J. Chem. SOC.,29872990 (1954). M2. Fifty Years with Alkaloids. R. H. F. Manske, Chemistry in Canada, 74-78 (June 1959). M3. Message from the President. R. H. F. Manske, Chemistry in Canada, 10 (September 1963). M4. Society and the Scientist. R. H. F. Manske, Chemistry in Canada, 25-30 (June 1963). M5. Society and the Scientist. R. H. F. Manske, Chemisfry in Canada, 1246 (March 1964). M6. Alkaloids. R. H. F. Manske, Encyclopaedia Britannica (macropedia),Vol. 15, pp. 871884, (1985).

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CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS ATTA-UR-RAHMAN AND M.

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H. E. J. Research Institute of Chemistry University of Karachi Karachi- 75270, Pakistan I. Introduction

111.

IV.

V. VI.

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B. Steroidal Alkaloids of the Buxaceae .......................................... 63 C. Steroidal Alkaloids of the Liliaceae ................................. D. Steroidal Alkaloids of the Solanaceae ................................................. 69 ................... 72 E. Steroidal Alkaloids from Terrestrial Animals F. Steroidal Alkaloids from Marine Organisms ...................... Physical Properties ........................................................... ................75 A. NMR Spectra ...................................... ................................................................. 81 .......................................... 89 Biogenesis ................................................... 90 A. Steroidal Alkaloids of the Apocynaceae and Buxaceae ........................... 92 B. Steroidal Alkaloids of the Liliaceae and Solanaceae ............................... Some Synthetic Studies and Chemical Transformations ............ Pharmacology ... .................. ................... 98 A. Steroidal Alk ................... 98

D. Steroidal Alkaloids of the Solanaceae ........ ...................... E. Steroidal Alkaloids from Terrestrial Animals ..................................... References

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I. Introduction

Steroidal alkaloids are an important class of secondary metabolites that occur in plants and also in certain higher animals and marine invertebrates. THE ALKALOIDS, VOL. 50 0099-9598/98 $25.00

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They possess the basic steroidal (cyclopentanophenanthrene) skeleton with a nitrogen atom incorporated as an integral part of the molecule, either in a ring or in the side chain. Unlike most other classes of alkaloids, steroidal bases are not derived from amino acids. Biogenetically, they are considered to be derived from steroids or triterpenoids, and they are therefore often referred to as “steroidal amines” rather than as proper alkaloids. Because of their structural similarities with anabolic steroids, steroidal hormones, and corticosteroids, steroidal alkaloids have been targets of pharmacological investigations. Recent interest in the field has also been due to the increasing worldwide demand for steroidal raw materials, as well as due to the shortage of diosgenin, the most important starting material for the steroid industry. Many steroidal alkaloids can be converted into valuable bioactive steroidal hormones by simple chemical and microbial conversions. Several corticosteroids used against skin diseases can be obtained by the chemical conversion of structurally related steroidal alkaloids. With the advent of new and more sensitive spectroscopic, bioassay, and isolation techniques, the field of steroidal alkaloids has witnessed a renaissance in the last decade. Plants of the families Apocynaceae, Buxaceae, Liliaceae, and Solanaceae continue to be the richest sources of steroidal alkaloids and the objects of active chemical research. The isolation of a large number of steroidal alkaloids from marine invertebrates and amphibians has added a new dimension to this area. While much work has been done on the chemistry and pharmacology of steroidal bases, surprisingly little effort has been directed to the total synthesis and biosynthesis of this important class of natural products. Steroidal alkaloids are generally divided into six groups based on their occurrence. The four major groups of the steroidal alkaloids that are of plant origin are (A) steroidal alkaloids of the Apocynaceae, (B) steroidal alkaloids of the Buxaceae, (C) steroidal alkaloids of the Liliaceae, and (D) steroidal alkaloids of the Solanaceae. In addition, there is an important group of steroidal alkaloids (class E) derived from amphibians, such as Salarnandru and Phylfobares. In recent years, a number of alkaloids have also been isolated from marine animals such as Zoanthid, Cephalodiscus, and Rifterelfa species (class F). A number of important reviews and monographs on the chemistry and pharmacology of steroidal alkaloids have been published (2-20). The present chapter presents a brief overview of the subject, highlighting some major contributions during the past 10 years, and is not intended to be a comprehensive review.

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63

11. Isolation and Structure Elucidation

A. STEROIDAL ALKALOIDS OF THE APOCYNACEAE Phytochemical investigations on various plants of the family Apocynaceae, such as Holarrhena, Paravallaris, Funtumia, Kibatalia, and Malouetia, have resulted in the isolation of over 150 new steroidal alkaloids. Most of the earlier work in this area was performed by Goutarel and co-workers in France in the 1960s and early 1970s ( 6 ) . The majority of the steroidal bases isolated from plants of the genera Holarrhena, Paravallaris, Funturnia, and Malouetia are of two structural types: the conanine type and the pregnane type (with generally one N) having the basic skeleta 1and 2, respectively. Conessine (3) was the first, and the most common, member of conaninetype alkaloids isolated from plants of the genera Holarrhena, Malouetia, and Funtumia. Interest in this compound is due to its C-18 substituted steroidal nature, which can lead to important hormones through fairly simple chemical conversions (21). During the past 10 years a number of new conanine-type alkaloids have been isolated, including holonamine (4) (22),regholarrhenine A (9, B (6), and C (7) (23). Siddiqui etal. in Pakistan working on H. pubescens (syn. H. antidysentrica) have also isolated several conanine-type steroidal bases in recent years (24). Some conanine derivatives such as 12a-hydroxynorcona-N(18)J ,4trienin-3-one (8) and lla,l2a-dihydroxynorcona-N(18),1,4-trienin-3-one (9) have also been isolated from the stem bark of Funtumia africana (Benth.) Stapf. (22). Paravallaris macrophylla Pierre has yielded a new steroidal alkaloid, 20-epi-kibataline (lo), the structure and stereochemistry of which were determined by X-ray diffraction analysis (25). B. STEROIDAL ALKALOIDS OF THE BUXACEAE A number of genera of the family Buxaceae, such as Buxus, Sarcococca, and Pachysandra, have been found to be rich in alkaloidal content. Some of these alkaloids were also found to be biologically active. A number of reviews have been published on this class of steroidal alkaloids (6,9,12,13,28). The genus Buxus comprises evergreen shrubs, which grow throughout the areas from Eurasia to South Africa, Malaysia, Indonesia, and North and Central America. The genus Buxus has proved to be one of the richest

64

A'ITA-UR-RAHMAN A N D CHOUDHARY

,

( 8 ) R1 = H. Rz = OH (9) R' = Rz =OH

30

(7)Regholarrhenine C

(5) Regholarrhenlne A: R = Me (6) Regholarrhenlne B; R = H

(41 Holonamlne

31

(10) PO-epi-Klbatallne

1121

.... NHMe

HlC\H

(11) Cyclobuxine-D

R = NH2 or =

sources of steroidal alkaloids, having so far yielded more than 200 new isolates. Of the 12 Buxus species investigated so far (B. balearica, B. harlandi, B. hildebrandtii, B. hyrcana, B. koreana, B. rnadagascarica, B. rnalay-

2. CHEMISTRY A N D

BIOLOGY OF STEROIDAL ALKALOIDS

65

ana, B. microphylla, B. papillosa, B. rolfei, B. wallichiana, and B. sempervirens), B. sempervirens and B. papillosa have yielded the largest number of new alkaloids. Although these plants are generally considered to be more of ornamental value than of medicinal value, a number of patents have been issued for their curarine-like action, usefulness in tuberculosis (18), and .activity against the HIV virus (19). Their structural resemblance to steroidal hormones provides further incentive for continuing research in this field. Much of the earlier work on various Buxus species was conducted by the research groups of Kupchan (B. sempervirens), Nakano (B. microphylla and B. koreana), Goutarel ( B . balearica, B. rolfei, and B. malayana), Doepke, and more recently, by our research group in Pakistan (B. papillosa and B. hildebrandtii). The first alkaloid, cyclobuxine-D (ll),isolated from B. microphylla in 1964, was recognized as a prototype of a new class of steroidal bases that contain a cyclopropane ring and a substitution pattern at C-4 and C-14 that is biogenetically intermediate between the lanosterol- and cholesterol-type steroids. Buxus alkaloids generally have either of two basic skeleta, 12 (derivatives of 9p,19-cycl0-4,4,14a-trimethyl-5a-pregnane) and 13 [derivaIn each skeleton, tives of abeo-9 (10 + 19)-4,4,14a-trimethyl-5a-pregnane]. certain modifications are observed due to the absence of one or both methyl groups (the C-4 and C-14 methyls), the presence of different oxygen functions, and the location of double bonds. An interesting structural variation is the presence of the tetrahydrooxazine ring in a number of steroidal alkaloids isolated from B. papillosa (C. K. Schnieder) and B. sempervirens L. Representative examples are harappamine (14) and moenjodaramine (15) from the leaves of B. papillosa (26). Several new alkaloids with the 9 (10 +-19) abeo-pregnane skeleton have been isolated from a number of different Buxus species, such as papilamine (16) (27). Occasionally, either one or both double bonds were also found to be reduced. A few alkaloids with a triene system (with an additional double bond between C-1-C-2) were also isolated from Buxus plants. A new series of steroidal alkaloids containing a tetrahydrofuran ring incorporated in their structures has been isolated from B. hildebrandtii and B. papillosa. For example, @-buxafuranamine (17)and Olo-buxafuranamine (18) have been isolated by us from B. hildebrandtii of Ethiopian origin (28). A number of reviews containing spectral generalizations of Buxus alkaloids have been published during the past 10 years (12,13,18,28).These generalizations include diagnostic features that can be deduced from mass spectrometry, 'H NMR and 13CNMR spectroscopy, UV and IR spectrophotometry, and specific optical rotations, and they are very useful in the

66

A l T A - U R - R A H M A N p N D CHOUDHARY

\.....rNRMe

MeHN (14) Harappamine. R = H (151 Moenjodaramine. R = CH,

(171 06-Buxafuranamlne

30

(16) Papllamine

(181 O'o-Buxafuranamlne

31

(191Buxane

9 y

MelN

NMe2

H

/

H _.

R (20) Pachysamine-A

(21) Saracoclne: A5.6

(221 Saracodine

2.

CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS

67

structure elucidation of new steroidal alkaloids of this class. We have also proposed a new system of nomenclature for Bums alkaloids based on the skeleton called “buxane” (19) (29). Plants of the genus Pachysandra, another genus of family Buxaceae, are also known to contain steroidal alkaloids of the simple pregnane type with two nitrogen atoms, such as pachysamine-A (20) isolated from P. terminalis Sieb. et Zucc. The earlier work on this plant was essentially all contributed by the Japanese group led by Kikuchi at Kyoto (30). Sarcococca species were also found to contain pregnane-type steroidal alkaloids. Representative examples include saracocine (21)and saracodine (22) isolated from S. pruniformis Lindl. (syn. S. saligna) (32). The groups of Kohli et al. and Chattejee er al. in India were the initial contributors in this area. The alkaloids found in the genera Sarcococca and Pachysandra are simple pregnane derivatives lacking methyl substitution at C-4 and C-14. Structurally, they are very close to the steroidal alkaloids of the family Apocynaceae. c . STEROIDAL ALKALOIDS OF THE LILIACEAE The family Liliaceae includes the genera Veratrum, Fritillaria, Petilium, Korolkowia, Rhinopetalum, Lilium, Zygadenus, and Notholiron. Over 300 new steroidal alkaloids have been isolated from the various genera of this family ( I , 7,9,22,24-20). A number of them have attracted considerable attention because of their interesting pharmacological properties, and a few have also been used clinically. For example, some alkaloids of Veratrum and Zygadenus are used for the treatment of hypertension. Most of the phytochemical investigations were focused on plants of genus Veratrum and Fritillaria. Structurally, steroidal alkaloids isolated from the family Liliaceae can be divided into three broad classes: the jerveratrum type (23), the cerveratrum type (24) and the solanidine type (25). The jerveratrum-type alkaloids usually occur in different Veratrum and Fritillaria species. They have a tetracyclic steroidal moiety bound to a piperidine ring (ring E). These alkaloids generally contain one to four oxygen atoms and occur as free alkamines or as monoglycosides. Jervine (26) is the most abundant jerveratrum base, having been isolated from several Veratrum species (16,18,32). The cerveratrum alkaloids form the second largest subclass of steroidal bases. They bear a CZ7skeleton with six rings that are often highly oxygenated. The common sites for oxygenation are indicated by arrows on the basic cerveratrum skeleton 27. Over 100 members of this class have been reported in the literature. The highly oxygenated members of the series

68

A'lTA-UR-RAHMAN AND CHOUDHARY 27

26

21

I241 Cerveratmm-type

15 2

3

1261 Jervine 27

HO

(271

(30)Spirasolane-type

usually occur in Veratrum and Zygadenus species, while compounds with fewer oxygen atoms are found in Fritillaria, Petilium, and Korolkowia species. All or some of the hydroxyl groups may be esterified with naturally

2.

CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS

69

occurring acids such as benzoic, 2-methylbutanoic, acetic, 2‘methylbutenoic, and 2’,3’-dihydroxy-2’-methylbutanoic acids. Imperialine (Kashmirine) (28) is a simple cerveratrum-type base isolated from several Fritillaria and Petilium species (26,33,34). A number of X-ray diffraction studies carried out on cerveratrum alkaloids have helped in understanding the structure of this seemingly complex class of secondary metabolites with up to 17 asymmetric centers (27). A number of solanidine-type (25) steroidal bases have also been isolated from plants of the families Liliaceae and Solanaceae, including the genera Veratrum, Rhinopetalum, and Notholiron. Solanidine (29) is the most common example of this structural class, isolated from various species of Fritillaria (26,35), Veratrum (26), and Rhinopetallum (36,37),as well as from Solanum chacoense Bitter (26).

D. STEROIDAL ALKALOIDS OF THE SOLANACEAE The plant family Solanaceae has yielded several types of steroidal bases, and over 200 alkaloids have been isolated from various species of Solanum (2,4,5,8,20,22,14-26,19,20) and Lycopersicon (Lycopersicum) (26). All these alkaloids possess the CZ7cholestane skeleton and can be divided into five structural types: solanidine (25), the spirosolanes (30), solacongestidine (31), solanocapsine (32), and jurbidine (33) ( 4 ) . There is a great deal of overlap between the structural types of steroidal alkaloids isolated from the plant families Solanaceae and Liliaceae. Nearly 350 plant species of both families have been found thus far to contain steroid alkamines (aglycones) or their glycosides. However, the jerveratrum- and cerveratrum-type C27nor-steroidal alkaloids have not been found in plants of the family Solanaceae (26). About 50 members of the spirosolane-type alkaloids are known. These alkaloids have a methylpiperidine ring (ring F) with the a-position joined to C-22 of the steroid moiety to form an oxazaspirane unit. Both saponins (alkamine glycosides) and sapogenins (aglycone alkamines) are known in this class. Spirosolanes are important intermediates in the industrial production of hormonal steroids because of their closely related pregnane structures. This was demonstrated 30 years ago when Sat0 et al. announced the chemical transformation of the spirosolane alkaloids solasodine (34) and tomatidine (35) into 3/3-acetoxypregna-5,16-dien-20-one and its 5,6-dihydro derivative (4,38). Solasodine (34) is an important member of this class isolated from many Solanum species (26),and has been receiving increased interest as a starting material for the commerical production of steroidal drugs. It has also been regarded as the “diosgenin of the next decade” ( 4 ) .

70

A’ITA-UR-RAHMAN AND CHOUDHARY

(311 Solacongeslldlne-type

18

2

3

1321 Solanocapsine-type

(331 Jurbidlne-type

21 18

i k !

Solasodine (34)has remained an important target of synthetic studies over the past 10 years. A number of derivatives of 34,such as N-cyano and A-nor-3-aza derivatives and degradative products, were prepared in order to obtain “new physiologically active steroids.” The related steroidal alka-

2.

CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS

71

*..*.* ",*.

.....

(381 Etiolinine, R = Glc(4 + 1)Clc

OH

Y

(401 Solanocapslne

= OH: Rz = Me: R3 = Et. AZ2lN) (42)R' = Me: R2 = R3 = H (41) R'

HO

139) 3-O~-Lycotrlaoslde

OH

&YR ..A

HN

B

H (44)

R =H

(451 Samandrine. R =

OH

(43)Jurubidlne

loid solasodenone (36) was degraded to progesterone in 65% overall yield (39). A number of glyco-derivatives of solasodine, such as solaradixine, solashbanine, solaradine, robustine, and ravifoline, have been isolated from various species of the genus Solanurn (16,20).

72

ATTA-UR-RAHMAN A N D CHOUDHARY

Over 50 members of the solacongestidine type of steroidal alkaloids have been isolated, mostly from Solanum and Veratrum species. A representative example is etioline (37), which was isolated from Solanum capsicastrum Link., S. spirale, S. havanense, Veratrum lobelianum, and V. grandiporum (I690). Leaves, roots, and stems of Solanum havanense Jacq. have furnished Psolamarine and the new glycoside etiolinine (38) (41). Solanidine-type alkaloids (25) have been isolated mostly from plants of the genus Solanurn, but a few have also been isolated from plants of the Liliaceae genera Veratrum, Rhinopetalum, Fritillaria, and Notholiron (20,16). About 40 members of this class, including both alkamines (aglycone) and glycoalkaioids, are known. Solanidine (29) is the most important member of this class, being isolated from many plants of the genera Rhinopetalum, Veratrum, Solanum, and Fritillaria. Stems of S. lyratum have yielded a mixture of new steroidal glycoalkaloids, including 3-0-p-lycotriaoside (39) (42). Only a few members of solanocapsine-type (32) alkaloids are known, almost all of which were isolated from Solanum plants. Solanocapsine (40) is an important member of this class, isolated from S. capsicastrum Link. S. hendersonii hort., and S. pseudocupsicon L. (43). Recently, phytochemical investigations of the roots of Taibyo Shinko No. 1 (a hybrid between Lycopersicon esculentum Mill. and L. hirsutum Humb. et Bonpl.), which is a tomato stock highly resistant to soil-borne pathogens, has resulted in the isolation of two new solanocapsine-type alkaloids, 22,26-epi-imino-16~,23-epoxy-23a-ethoxy-5a,25a~-cholest-22(N)-ene-3/3,20a-diol (41) and 22,26-epi-imino-16a,23-epoxy-5a,22pHcholestane-3/3,23a-diol (42) (44). Jurubidine-type (33) bases also form a relatively small group of Solanum alkaloids, of which jurubidine (43) is an example (45). E. STEROIDAL ALKALOIDS FROM TERRESTRIAL ANIMALS

Over 30 new steroidal alkaloids have been isolated from various species of Salamandra, Phyllobates, and Bufo (5,16).These alkaloids are generally found in secretions from the skin glands of these amphibians and appear to protect the skin against fungal and bacterial infections. The crude mixture obtained by the evacuation of the skin glands of Salamandra species contained several novel alkaloids which have the basic skeleton 44. Two structural features of these alkaloids are of interest: a cis junction between rings A and B, and the presence of an expanded ring A with the formation of an isoxazolidine system. Samandrine (45) is the major alkaloid isolated from the skin extracts of S. maculosa taeniata (31) and other Salamandra species (16,46).

2.

73

CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS

(48) Batrachotoxin (46) Bufotallin a: R = H (47) Bufotallin b : R = Me

0

0

(491 Zoanthamine

( 50)

Zoanthenamine

%o

\N

0

( 52)

28-Deoxy-zoanthenamine

Biogenetically, these alkaloids are derived from mevalonate via cholesterol. The expansion of ring A results from the cleavage of the C-2, C-3 bond and the insertion of a nitrogen atom, which is itself derived from glutamine. Two new, basic bufotallin derivatives (46 and 47) have been isolated from the skin of the Formosan toad (Bufo melanosfictus) (47). The skin

74

AlTA-UR-RAHMAN AND

CHOUDHARY

secretions of Phyllobates species of highly colored frogs (poison-dart frogs) contain over a dozen steroidal alkaloids of a novel skeleton. Batrachotoxin (a), a bioactive steroidal base, was isolated from five species of Phyllobates (48). Batrachotoxin (48) is a powerful Na+-channel potentiator and some synthetic and biosynthetic studies have also been focussed on this alkaloid (49). Homobatrachotoxin, a naturally occurring derivative of 48 has also been isolated from the skin and feathers of a bird, the hooded pitohui (Pitohui dichrous) (46). F. STEROIDAL ALKALOIDS FROM MARINE ORGANISMS Considerable research effort is currently being focussed on the discovery of new bioactive natural products from marine animals. A number of new and novel steroidal alkaloids have been isolated in the process, mostly from marine invertebrates. Many of them are believed to be of dietary or microbial origin. A series of new alkaloids has been isolated from a new species of a colonial zoanthid of the genus Zoanthus collected from various coasts of the Indian ocean. This series includes zoanthamine (49) (50), zoanthenamine (50), zoanthamide (51)(51), 28-deoxy-zoanthenamine (52),22-epi28-deoxy-zoanthenamine (53) (52), and zoanthaminone (54) (53). These zoanthamine-type alkaloids are of unknown biosynthetic origin, although some elements may suggest a triterpenoidal origin. Cephalostatins, a new series of bioactive, dimeric steroidal alkaloids, have been isolated from the marine hemicordate worm Cephalodiscus gilchristi (order Cephalodiscida). The structure of cephalostatin 1 (55) was determined by X-ray diffraction analysis (54). Fifteen members of this series, cephalostatins 1-15, have been isolated by Pettit et al. (55,56).Cephalostatins apparently arise in nature by the condensation of two 2-amino-3oxosteroid units to yield dimeric steroidal molecules connected by a pyrazine ring. Recently, another class of highly cytotoxic, dimeric and steroidal alkaloids structurally related to the cephalostatins has been isolated from the lipophilic extract of the tunicate Ritterella tokioka Kott (Polyclinidae). Thirteen members of the series, designated ritterazines (ix., ritterazines A-M) (56a),have been isolated so far by Fusetani et al. in Japan (57-59). A marine sponge of the genus Plakina has yielded two new antimicrobial steroidal alkaloids, namely plakinamine A (69) and plakinamine B (70). The structures of these two novel steroidal bases were deduced mainly by spectroscsopic techniques (60). Scheuer et al. have recently isolated two new steroidal alkaloids, lokysterolamine A (71)and lokysterolamine B (72),from an unidentified species

2.

CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS

(53)22-epi-28-Deoxy-zoanihenamine

75

(54) Zoanthaminone

of the genus Corticium collected in Sulawesi, Indonesia. These alkaloids bear a skeletal relationship to plakinamine A (61).

111. Physical Properties

A. NMR SPECTRA

Since the majority of steroidal alkaloids generally have a hydrocarbon skeleton with few functional groups, their 'H NMR spectra are usually not very informative and one has to rely on a combination of spectroscopic

76

A'ITA-UR-RAHMAN AND CHOUDHARY

(56) Ritterazine A, R IQI

Ritternrine

n

(60)Ritterazine E, R

27

1y OH

=

P = =

H; 22R u. m e Me: 2 2 s

(57) Ritterazine B, R 1 = OH. R2

=

H, R3 = H; 22R

(61) Ritterazine F, R 1 = OH, RZ = H, R3 = H; 2 2 s (62) Ritterazine G.R' = OH, R2 = H, Ai4; 22R

(63) Ritterazine H, R1,R2 = 0, RZ = H; 22R (64) Ritterazine I. R 1 , RZ = 0, R3 = OH: 2 2 s

techniques to deduce the structural type. Most of the methyl and methylene protons of the cyclopentanophenanthrene skeleton resonate in the range of S 1.0-2.5 in their 'H NMR spectra. This serious overlap of proton signals makes it difficult in the majority of cases to clearly assign the chemical shifts to individual protons. However, with the advent of two-dimensional NMR spectroscopic techniques such as COSY, NOESY, TOCSY, HMQC, and HMBC, it is now possible to obtain more structural information from these NMR experiments (12,13,16,62-64). 1. Buxus Alkaloids

The presence of a cyclopropyl moiety in cycloartenol-type Buxus alkaloids confers certain characteristic spectral properties. In the case of an unsubstituted triterpenoid skeleton, the C-19 cyclopropyl methylenic protons appear strikingly upfield in the region of 6 0.1-0.5 as AB doublets

2.

CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS

/Plw+"0X

77

I

H

.-ft\ft'/"'W I

H

I

,* n

(66) Ritterazine K, R1 = H. R2 = OH: 22R

(67) Ritterazine L, R' = H, R2 = H: 22R (68) Ritterazine M, R1 = H, R2 = H; 2 2 s

(69)Plakinamine A; R, = H.R, =

6H (70)Plakinamlne 8 ; R, = Me, R, =

(71)Lokysterolamine A: R = NMe, (72)Lokysterolamine B; R = NHAc

(J = 4.0 Hz) (65). Alkaloids such as 73, which contain a C-11 or C-1 keto function in ring A or D, often have no cyclopropyl signals in this upfield region due to the electron-withdrawing effect, which consequently shifts

78

A'ITA-UR-RAHMAN A N D CHOUDHARY

H 21

\

H 21

,...%me,

(73)N-Benzoyl-0-acetylcyclobuxoline-F

\

.,.,*me2

(74)Cyclobuxapaline-C

Med (75) Verabenzoamine

H

(76) Noriatifoline

(77)Malouetafrine

them to the region between 6 0.9 and 1.5. Compounds bearing C-1, C-2 and C-11, C-12 double bonds or C-1 and C-11 hydroxy groups exhibit only one-half of the AB doublets at about 6 0.6, the other proton being shifted

2.

CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS

79

downfield (13).Compounds with C-6, C-7 double bonds such as 74 exhibit a pronounced upfield shift of the cyclopropyl protons to -6 0.4-0.1. This is due to the P-oriented cyclopropyl methylene protons lying in the shielding region of the 6,7-double bond, as in cyclobuxapaline-C (66).The 'H NMR spectra of Bums bases generally show five methyl signals, one of which (C-21 methyl) appears as a doublet at 6 1.0. The C-31 methyl is often oxygenated as C H 2 0 H , CH20Ac, or C H = O (67). 2. Cerveratrum- Type Alkaloids The cerveratrum-type alkaloids contain several oxygen functionalities. They generally possess three methyl groups, one of which (C-19) is always tertiary and appears at 6 0.9 while the C-21 and C-27 secondary methyl groups resonate as doublets at -6 1.0 and 0.8, respectively. Verabenzoamine (79,an alkaloid isolated from V. album, contains a hydroxyl function at C-16, causing a slight downfield shift of the C-21 methyl to 6 1.14 (68).The presence of other downfield methyl signals is often due to the presence of an acyl group. Several, one-proton multiplets resonate downfield in the region of 6 3.5-5.5, and are characteristic of oxygen-bearing methine protons geminal to the oxygen function. In general, the oxygenation sites are C-3, C-4, C-6, C-14, C-15, C-16, and C-20. Several of the cerveratrum alkaloids have some of the hydroxyl groups esterified. A downfield shift of about 6 1.0 is generally observed for the methine protons geminal to the acyl functions, in comparison to the corresponding OH-bearing alkaloids, as expected (69). 3. Conanine-Type Alkaloids

Only two methyl signals (C-19 and C-21 methyls) are visible in the 'H NMR spectra of conanine-type bases. The doublet for the C-21 secondary methyl protons appears at -6 1.3. This doublet resonates slightly upfield (-6 1.1) if the five-membered nitrogen-containing ring is completely saturated, as in norlatifoline (76) isolated from Funtumia latifoliu Stapf. (70). The C-18 imine proton in malouetafrine (77)and related alkaloids resonates downfield as a close doublet at 6 7.6 (J = 3.0 Hz), exhibiting allylic coupling with the C-20 methine proton (71). 4. Jerveratrum- Type Alkaloids

The 'H NMR spectra of jerveratrum-type bases of the general skeleton of type 23 show several characteristic signals. There are two tertiary methyl groups (C-18 and C-19) and two secondary methyl groups (C-21 and C-27) in these alkaloids. The doublets for the secondary methyl protons generally resonate between 6 0.70-1.20 (J = 7.0 Hz),and the C-21 methyl generally resonates downfield of the C-27 methyl. The C-18 allylic methyl proton resonates as a close doublet at 6 2.0 displaying a small allylic

80

ATI'A-UR-RAHMAN A N D CHOUDHARY

(78)Stenanzine

k

(79)Hupehenisine

(801Solanogantamine

coupling. Reduction of the C-11 conjugated carbonyl group in ring C, if present, results in the shielding of the C-18 methyl protons by about 0.25 ppm. The C-22 methine proton geminal to the nitrogen atom often appears as a double doublet at about 6 2.7, while the C-23 methine proton resonates as a multiplet at 6 3.3 as in jervine (25) (72). Both these protons appear slightly upfield if the ether bridge between C-17 and C-23 is cleaved, as in stenanzine (78) (73). The C-3 proton geminal to the hydroxyl group resonates at 6 3.7 if a C-5, C-6 double bond is present. An upfield shift of about -6 0.5 is observed if ring B is completely saturated, as in the case of hupehenisine (79)(74). 5. Solanidine- Type Alkaloids

The solanidine-type steroidal alkaloids generally show four methyl signals in their 'H NMR spectra, two of which are secondary (C-27 and C-21) and appear as doublets; the doublet for the C-21 methyl generally resonates upfield of the C-27 methyl doublet. The multiplet for the C-3 methine proton geminal to the hydroxyl or amino group appears at -6 3.7 or 2.8, respectively. The presence of another downfield signal at 6 3.8 is generally due to the C-23 methine proton when a hydroxyl group is present at C-23, as in solanogantamine (80) (75).

2.

CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS

81

6. Spirosolane- Type Alkaloids

The 'H NMR spectra of spirosolane-type steroidal alkaloids contain doublets for two secondary methyls (C-21 and C-27) resonating between S 0.8-0.96 and singlets for two tertiary methyls (C-19 and C-18) which appear at -6 1.1 and -0.8, respectively. Spirosolane-type alkaloids possess spiro-linked piperidine and tetrahydrofuran moieties which give them a characteristic spectral pattern. A downfield multiplet at -8 4.2 is due to the C-16 methine proton geminal to the oxygen of the tetrahydrofuran ring as in solasodine (34) (76,77).The C-26 methylene protons geminal to nitrogen resonate in the range of S 2.6-2.8. The C-22 quaternary carbon, resonating at 6 -99.0 in the 13CNMR spectra, is characteristic of all spirosolane alkaloids (62).

B. MASS SPECTRA The mass fragmentation pattern of steroidal alkamines is characteristically different from that of other classes of natural products. The majority of steroidal alkaloids are saturated cyclic hydrocarbons with nitrogen either in the side chain or incorporated in the ring systems. The molecular compositions of these compounds provide useful information about the individual structural types. For example, compounds with more than one nitrogen usually belong to the conanine (see alkaloids of the Apocyanaceae), Bums (see alkaloids of the Buxaceae), and pregnane (see alkaloids of the Buxaceae) classes, while compounds with only one nitrogen atom may be members of cerveratrum, jerveratrum, solanidine (alkaloids of the Liliaceae and Solanaceae), secosolanidine (alkaloids of the Solanaceae), and other structural types. Similarly, alkaloids containing several oxygen atoms may either be sugar derivatives or belong to a highly oxygenated class, such as the cerveratrum-type alkaloids (alkaloids of the Liliaceae). The molecular fragmentations are generally triggered by the presence of a heteroatom or double bonds. The diagnostic and most abundant ions are formed by the cleavage of bonds (Y to the nitrogen atoms in steroidal alkaloids. These ions help in the identification of different structural classes, the positions of functional groups, and the position and types of unsaturation in different compounds. Mass spectrometric studies of a large number of steroidal alkaloids have provided some important generalizations that are extremely useful in structure elucidation. Some of these are summarized below. A number of review articles on the mass spectrometry of steroidal alkaloids have been published (71,78-8I).

82

AITA-UR-RAHMAN AND CHOUDHARY

1. BUMSAlkaloids

The mass spectra of Buxus (Buxaceae) bases differ significantly from the spectra of other steroidal alkaloids due to the presence of a cyclopropyl ring in their skeleta. The lower mass region of the mass spectra is particularly informative due to the fragments resulting from the cleavage of the nitrogen-containing side chains. These fragments predominate in the mass spectra of all Buxus bases. The majority of Buxus alkaloids have a basic nitrogen in the side chian at C-17 or C-3, and the fragmentation occurs between the carbon atoms a and fl to the nitrogen atom. Compounds bearing monomethylamino substituents at C-20, e.g., cyclobuxoviricine, yield a base peak at m/z 58 due to the ion, CH3-CH=N+(H)CH3, while compounds containing the dimethylamino group substituted at C-20, such as 81, invariably show the base peak at m / z 72 due to the trimethyliminium ion (Scheme 1) (Z2,13,18,82).The fragment ions resulting from the cleavage of the nitrogen-containing side chain on ring D are more abundant than fragments arising from the cleavage of ring A, when the latter contains a nitrogen substituent at C-3. Alkaloids such as 82 which contain nitrogen only at C-3 display the base peak at m / z 57 or 71 in the mass spectrum, depending on whether they contain a monomethylamino or dimethylamino substituent at C-3 (Scheme 2) (Z2,Z3). Alkaloids bearing oxygenated functionalities on ring D exhibit characteristic peaks in their mass spectra. For example, the mass spectrum of buxanolidine (83), which contains a hydroxyl group on ring D, showed a prominent ion at m/z 129 (C7HI5NO)resulting from the cleavage of ring D along with the C-17 side chain. This hydroxy group may be attached to C-15, C-16, or C-17 of ring D. Another fragment at m/z 115 (ChHI3NO)established that the OH was present on a six-carbon fragment, so that it could be

/

m/z 72

fQl1

SCHEME 1.

2. CHEMISTRY A N D

BIOLOGY OF STEROIDAL ALKALOIDS

83

R = Me,m / z 71 (lOoO/o) R = H. m / z 57 (100%) SCHEME 2.

attached to C-15 or C-16. The ion at d z 85 (CSHIIN)with no oxygen arose by the cleavage of the N-containing group of ring D along with C-17, and, since it was devoid of oxygen, indicated the presence of the O H group at C-16. Alkaloids bearing OAc instead of -OH on ring D at C-16 such as 16a-acetoxybuxabenzamidenine(83) isolated from B. papillosa show peaks at m/z 157 and 171 (Scheme 3) (22,23,83). 2. Cerveratrum- or Cevine-Type Alkaloids

The presence of a C-27 carbon skeleton with six fused rings, one nitrogen, and at least one oxygen is a characteristic common to all cevine-type steroidal bases. Many other cevine-type bases also contain an ether bridge between C-9 and C-4. The mass spectra of cerveratrum alkaloids contain a significant peak at d z 112 or 111 resulting from the cleavage of ring E at the N-C-18 and C-20-C-22 bonds. Rings E and F usually have little structural

.

.N/Me

m / z 72

0

II

-C33) 16a-Acetoxy-buxabenzamidenine

SCHEME 3.

84

A’ITA-UR-RAHMAN A N D CHOUDHARY

variation in this class, and the substitution pattern in the remaining skeleton does not significantly alter the overall fragmentation patterns so that the principal fragments generally occur at m/z 112 and 98. Fragment ions at m / z 125 and 124 are also quite common in this class, resulting from the cleavage of the N-C-18 and C-20-C-17 bonds. The mass spectra of C-3 glycocevine and highly oxygenated (4 to 15 oxygen atoms) members of this class often lack a M+ ion and contain an M+-acyl ion. Characteristic losses of acyl moieties, such as acetyl, benzoyl, and methylbutyryl, are also apparent in their mass spectra. The mass fragment at d z 112 of zygacine (& aI cerveratrum-type ), of steroidal base isolated from Zygadenus gramineus (Liliaceae), is shown in Scheme 4 (84). 3. Conanine-Type Alkaloids

Conanine-type steroidal alkaloids display two prominent ions in their mass spectra: the ion at m/z M+-15 and the one at m / z 56 + R, where R is the substituent at the nitrogen atom in ring E. For example, the mass spectrum of 5a-conanine (85), an important member of this class, exhibited the base peak at m / z 300 (M+-15) and a large peak at m/z 71 (56 + CH3) (Scheme 5) (85).Alkaloids of this series which contain a 18,20-imino group generally exhibit the base peak at m/z 121 resulting from the cleavage of ring C at C-13-C-12 and C-14-C-8 bonds. Holanamine (86) is an example of this class (86) (Scheme 6).

m / z 112 OH

184) Zygacine SCHEME 4.

2. CHEMISTRY

85

A N D BIOLOGY OF STEROIDAL ALKALOIDS

Me

I

4

m/z 71

(85) 5a-Conanine

m / z 300

SCHEME 5.

4. Jerveratrum- Type Alkaloids

The jeveratrum-type steroidal alkaloids generally contain only one nitrogen and up to three oxygen atoms. Several characteristic fragments in their mass spectra are very useful in the structure elucidation of new compounds of the series. Jervine (26), isolated from various Veratrum species (Liliaceae), and related bases with piperidine and tetrahydrofuran rings exhibit a prominent peak (sometimes the base peak) at d z 110 in their mass spectra. The cleavage of the C-20-C-22 bond and the opening of the tetrahydrofuran ring result in the formation of fairly large ions at d z 125 and 124, which then lose the C-21 secondary methyl group to yield the ion at d z 110 (Scheme 7) (87). The mass spectra of veratramine-type steroidal alkaloids, such as stenanzine (78) isolated from Rhinopetalurn stenantherurn, show the most abundant ion at d z 114, again resulting from the a-cleavage of the piperidine (C-22-C-20) side chain (Scheme 8) (73). +.

(86)Holanamine SCHEME 6.

86

ATTA-UR-RAHMAN A N D CHOUDHARY

I

(26)Jervine

H

SCHEME 7.

5. Pregnane-Type Alkaloids It is well known that the fragmentation in nitrogen-bearing substances is preferentially initiated by the cleavage of the bond between the carbons a and P to the nitrogen atom. For example, the mass spectrum of 3dimethylaminopregnane shows characteristic ions at mfz 84 and 110, resulting from the cleavage of ring A at the site shown in structure 87 (such as saracodine) (Scheme 9) (82,88). Like Buxus bases, many pregnane-type alkaloids also contain monomethylamino or dimethylamino substituents at C-20. Their mass spectra also show the base peak at m f z 58 or 72, respectively, resulting from the cleavage of the C-17-C-20 bonds. 6. Salamandra Alkaloids

Salamandra alkaloids have a 3-aza-A-homo-5P-androstane skeleton with only one nitrogen and up to three oxygen atoms. The mass spectra of Salamandra (samanine-type) bases generally contain a prominent fragment ion at m/z 85 resulting from the cleavage of ring A. This ion comprises a five-membered ring with oxygen and nitrogen atoms incorporated in the ring system (Scheme 10) (89).

+' _ m / z 429

HO

(781 Stenanzine

0

SCHEME 8

H

I

HoQ

m / z 114

2.

CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS

87

m/z 84

R = N(Me)COMe SCHEME9.

7. Secosolanidine-Type Alkaloids

The mass spectra of verarine (SS), hosukinidine, veralinine, and veramiline contain several characteristic fragment ions that facilitate the identification and classification of these compounds. The abundant fragment ion at m/z 98 arises from the cleavage of the C-20-C-22 bond, while the considerably more intense fragment ion at m/z 125 is formed by the cleavage of the C-20-C-17 bond. This ion often appears as the base peak in the mass spectra of the alkaloids of this type such as hosukinidine (89) (Scheme 11) (90). 8. Solanidine-Type Alkaloids

The mass spectrum of solanidine (29), an important member of this class isolated from different species of Veratrurn, Fritillaria (Liliaceae), and Solanum (Solanaceae), etc., provides only a few characterizable ions. The principal fragment ions in this class of alkaloids are at m/z 204 and 150, R

+.

CH2 m / z 85

SCHEME10.

88

A'ITA-UR-RAHMAN AND CHOUDHARY

m / z 98 SCHEME11.

the latter being the base peak. The ion at m / z 204 is a conjugated immonium ion resulting from the cleavage of rings B and D with a hydrogen rearrangement from C-12 to C-14. The base peak at m / z 150 is formed by the cleavage of the C-15-C-16 and C-17-C-13 bonds, followed by hydrogen transfer from C-20 to C-15 (Scheme 12) (91). Alkaloids containing OH groups on the piperidine ring F, such as solanopubamine and solanogantamine, display an ion at m/z 166 (150 + 0) as the base peak.

Ho

& 4

(29)Solanidine

m/z 150 SCHEME 12.

rn/z 204

2. CHEMISTRY

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A N D BIOLOGY OF STEROIDAL ALKALOIDS

9. Spirosolane-Type Alkaloids

The mass spectra of the spirosolane alkaloids exhibit many characteristic fragment ions that facilitate their identification. The base peak at d z 114 in the lower mass region represents the ion resulting from the cleavage of the tetrahydrofuran ring at the C-20-C-22 and C-20-0 bonds. Another important fragment in the mass spectrum, at d z 138, results from the cleavage of ring D. Scheme 13 shows the key fragments of solasodine (34), a common alkaloid isolated from various species of Solanum (Solanaceae) (92). The mass fragmentation patterns of the glycosidic spirosolanes are generally not reported in the literature. C.

X-RAYCRYSTALLOGRAPHY

Steroidal alkaloids, which usually possess at least four rings and up to 17 asymmetric centers, present a considerable challenge to the structure chemist. While a number of new spectroscopic techniques are now available

m / z 125

m / z 415 (34) Solasodine

H

’\

H

m / z 114 SCHEME 13.

90

ATTA-UR-RAHMAN AND CHOUDHARY

to assign structures to unknown compounds, X-ray crystallography remains the most powerful1 tool for three-dimensional structure determination. In the last decade, the structures of a large number of steroidal alkaloids were determined or confirmed by X-ray crystallographic analyses, including chuanbeinone (Fritifluriu dufuvuyi Franch) (92),delavinone hydrochloride (F. defuvayi) (93), ebeiedine 3,6-diacetate (F. ebeiensis G. D. Yu and P. Li) ( 9 4 , ebeienine (94),imperialine (F. imperialis L.) (95),isobaimonidine (96,97),protoveratrine C (Verutrum album L.) (98),N-3-isobutyryl cylobuxidine F (99),shinonomenine (ZOO), tortifoline (F. tortifofiu) (ZOZ), ussurienine (F. ussuriensis Maxim) (Z02), veratrenone (Z03),veratridine perchlorate (ZO4), verticine N-oxide (ZO5), verticinone hydrochloride (ZO6), verticinone methobromide (F. verticiffutuWild.) (109, zoanthamine (5O), zoanthaminone (53),veramarine (V. album) (ZO8), holonamine (ZO9),and 20-epi-kibataline ( 2 10). IV. Biogenesis Triterpenes and plant steroids are the biosynthetic precursors of all of the classes of steroidal alkaloids and alkamines (9,20,23,ZZZ).Incorporation of the nitrogen atom in steroidal skeleta generally takes place in the later stages of their biosynthesis. This was the hypothesis presented immediately after the isolation of the first steroidal base and was founded on the structural similarities of the two groups of secondary metabolites. This assumption was further supported by the isolation of the structurally related, non-nitrogen-containing steroidal analogues from some plant species. The biosynthesis of the steroidal alkaloids follows the general pathway of steroid or triterpene biosynthesis in plants, starting from acetyl coenzyme A via the principal intermediates mevalonic acid, isopentenyl pyrophosphate, farnesyl pyrophosphate, squalene, cycloartenol (or lanosterol in animals), and cholesterol. Cholesterol, or biogenetic equivalents of it, is the precursor of both the C2,-steroidal sapogenins and alkaloids such as the cerveratrum, jerveratrum, Sufumundru, pregnane, and many other types of steroidal alkaloids, whereas the triterpene cycloartenol (90) is the biosynthetic precursor of the Buxus alkamines (Scheme 14). A. STEROIDAL ALKALOIDS OF THE APOCYNACEAE AND BUXACEAE Alkaloids of the plant families Apocynaceae and Buxaceae form very large groups of steroidal bases. Although some biosynthetic studies have been conducted on other classes of steroidal bases, little experimental work has been done on these classes of bases.

2. CHEMISTRY A N D

BIOLOGY OF STEROIDAL ALKALOIDS

91

H

HO

HO (90)Cycloartenol

Monoamino and diamino steroids SCHEME 14.

Khuong-Huu et al. in 1972 suggested that the reactive ll-keto-9fl,19cyclo system encountered in some Buxus alkaloids might be the biogenetic precursor of bases containing the conjugated transoid 9(10 += 19) abeodiene sysem, as, for example, in buxaquamarine (91) (122).

MeN

I

HZC,

,CHz

0

(91)B uxaquamarine

MeN H (92)

R

= H

(93)R = OH

(94) Paravallarine, R = H (95) Paravallaridine, R = OH

92

ATTA-UR-RAHMAN A N D CHOUDHARY

It was also proposed earlier that the biosynthesis of these alkaloids involves the intermediacy of C-3 or C-20 ketones bearing the steroidal skeleton. The occurrence of C-3 steroidal ketones such as 92 and 93 in Pnravalfaris microphyffahaving the characteristic 18 20 lactone function of paravallarine (94) and paravallaridine (95) further supported this hypothesis (113). B. STEROIDAL ALKALOIDS OF THE LILIACEAE A N D SOLANACEAE

Mitsuhashi and co-workers proposed Schemes 15 and 16 for the biosynthesis of various classes of steroidal alkaloids based on their biosynthetic work on Veratrum grandiflorum which is known to contain five different classes of steroidal alkaloids (114). Cholesterol, or a biogenetic equivalent of cholesterol, is the precursor of both the C27-steroidal sapogenins and the alkaloids (115). According to present knowledge, the biosyntheses of these sapogenins and alkaloids, which often occur together in plants, are closely related. A number of nitrogen-free steroidal sapogenins, such as dormantinol(96) and solasapogenin (97), have been isolated from Solanum and Veratrum species containing steroidal bases. Earlier investigations indicated that acetate, mevalonate, and cholesterol, as well as cycloartenol and lanosterol, are significantly incorporated into tomatidine, solasodine, solanidine, and solanocapsine and/or the spirostanols (115-127). In other biosynthetic investigations, radioactive labeled (25R)-26-aminocholesterol administered to Solanum laciniatum was found to be incorporated to a high extent into solasodine (113, whereas the corresponding 160-hydroxy derivative showed only a low level of incorporation (118). These results suggested that in the biosynthesis of the Cz7-steroidal alkaloids, the introduction of nitrogen occurs immediately after hydroxylation at C-26. This has also been confirmed in the biosynthesis of solanidine (29) in V . grandiflorum, where amino acid arginine act is the nitrogen source (114).

V. Some Synthetic Studies and Chemical Transformations

Despite the substantial pharmaceutical importance of steroidal alkaloids, little synthetic work has been done in this area. Efforts have been largely focused on chemical and microbial transformations to pharmacologically more potent compounds.

2. CHEMISTRY A N D

93

BIOLOGY OF STEROIDAL ALKALOIDS OH

HO

(96) Dormantinol

Cholesterol

I

t

0

HO

HO

,.."

Dormantinone

H

'...

HO Rubijervlne

Hakurirodine

""U

(37) Etioline (Solacongestidine-type)

(28) Solanidine

SCHEME 15.

94

ATTA-UR-RAHMAN AND CHOUDHARY

H

HO

HO Epirubijervine (Solanidine-type)

HO

HO

‘

‘* HO

\

’ 16

.’. 20

f

22 25

/ HO

’ (26)Jervine [Jerveratrum-type]

SCHEME16.

\

HO

Veratramine

H

H

2. CHEMISTRY A N D

95

BIOLOGY OF STEROIDAL ALKALOIDS

H

AcO

AcO

'

(98)

(991

H

I

AcO ( 100)

Me,N

R =H (103)R= NO, (102)

AcO

&

96

ATFA-UR-RAHMAN A N D CHOUDHARY

.....,,,H

H2N d'

A (107)Funturnine

.....

1,111

k

{fip k

I1091 R = H

(110) R = I

(108) Ac

(111)R =CO,H (112)R = CON, (113) R = lsocyanato

In order to prepare biologically active steroidal alkaloids some model substances structurally related to Bums alkaloids have been synthesized, e.g., 3~-acetoxy-16a(l-nitro-l-methoxycarbonylmethyl)-20-0~0pregn-5-ene (98), (24S)-3~-acetoxy-22-aza-23-oxo-24-nitro-l6,24-~yclocho-

2.

CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS

97

la-5,17-diene (~),3~-acetoxy-22-aza-23-hydroxy-24-nitro-l6,24-cyclochola5,17,22,24-tetra-ene (loo), and 3~-acetoxy-24-amino-22-aza-23-oxo-16,14cyclocholaJ,17,24-triene (101)(119). Dev has reviewed the partial synthesis of a number of Buxus alkaloids starting from cycloartenol (90) (120). Conessine (102)furnished the 6-nitro derivative 103 and the 5a-nitrosooxyd-oxime 104 when it was warmed with fuming nitric acid and sodium nitrite in glacial acetic acid. Alternatively, when conessine was oxidized by fuming nitric acid in chloroform and ether at O'C, the hydroxyketone 105 and the 5P-nitro-6-ketone lo6 were formed (121). The partial synthesis of funtumine (107)from 3P-hydroxypregn-5-en-20one has been achieved (122). The steroidal amide 108 yielded a mixture of products, such as the spirosolane (109)and its iodo derivative 110,on photolysis with mercuric acetate and iodine, or with iodosobenzene diacetate and iodine. The structures of these products were established by X-ray crystallography. This work represents the first intramolecular functionalization of an amide to yield a lactam, and has potential application in the synthesis of steroidal alkaloids structurally related to solasodine (123).The partial synthesis of the steroidal glycoside kryptogenin 3-0-P-chacotrioside from methyl protodioscin has been reported. The former glycoside was seen as a potential intermediate in the synthesis of solanidine-3-0-P-chacotrioside(i.e., a-chaconine) (124). A,B-Perhydroindole analogues of the 28-acetylspirosolane series of steroidal alkaloids have been synthesized. The norsecospirosolane 111 (R = C02H) was treated with C1C02Me in acetone-Et3N, and then with NaN3, to give 112 (R = CON3), which rearranged in toluene at 100°C to give 113 (R = isocyanato). Cyclization of the latter in refluxing acetonitrile containing aqueous NaHC03 gave the indolosteroidal compound 114, which was reduced by NaBH4to give the pyrrolidinospirosolane (115)(125). 3-Hydroxy-4-keto-steroidal alkaloids isolated from various species of Solanum exhibit interesting pharmacological properties. To obtain these the following two routes 4-keto-steroidal alkaloids from solasodine (a), were attempted: (a) allylic acetoxylation of (22S,25R)-22,26-N-Cbz-epi-iminocholest-5en-3PJ6P-diol-acetate (116);and (b) hydroboration of (22S,25R)-16P-acetyl-22,26-N-Cbz-epi-iminocholest-4-en-3-one (117). The first route yielded (22S,22R)-3P-hydroxy-16P-acetoxy-22,26-N,NCbz-epi-iminocholestan-5,6-oxido-4-one (118), while the second one afforded two products, i.e., (22S,25R)-3/3-hydroxy-l6P-ethoxy-22,26-N-Cbzepi-imino-5a-cholestan-4-one (119) and its 16P-acetoxy homologue (120) (126).

98

A'lTA-UR-RAHMAN A N D CHOUDHARY

Cbz

Cbz

I

HO' (1171

0

H2N (121) Irehdiamine-A

/

U

CH,HN

*oA,H

OCH,

H 1122) Mitiphylline

MezN (123) ConessIne

VI. Pharmacology

A . STEROIDAL ALKALOIDS OF THE APOCYNACEAE Irehdiamine-A (El),a pregnane-type alkaloid isolated from Funrumia elusticu, has exhibited potentializing activity on hepatocarcinogenesis in

2.

CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS

99

rats (127).Binding of irehdiamine-A also resulted in the uncoiling of closed circular DNA (128). Mitiphylline (l22),an amino-glycocardenolide isolated from Holarrhena mitis by Goutarel et af.,has been shown to possess cardiotonic activity (129). Steroidal alkaloids isolated from the Apocynaceae may be converted to pharmacologically active steroidal hormones by simple chemical or microbial transformations. For example, funtumine (107), isolated from H. febrifuga and H. fiatifofia, can be converted to androstanedione, while holamine isolated from H. jloribunda can be converted to androst-4-ene3,17-dione (230).Conessine (l23),the major alkaloid of H. antidysenterica, is reported to be active against both intestinal and extra-intestinal amoebiasis (131). B. STEROIDAL ALKALOIDS OF THE BUXACEAE Extracts of Buxus sempervirens (Boxwood) are reputed to have activity against syphilis, rheumatism, dermatitis, rabies, malaria, cancer, and tuberculosis. A British patent for curative action in tuberculosis was issued to Merck and Co. The crude alkaloidal extract of this plant also inhibited the activity of the enzyme cholinesterase (18,232).The ethanolic extract of the plant was also found to inhibit reverse transcriptase activity of HIV in vitro. The investigators claim that Buxus (stem, root, bark) extracts can be used as a drug for the treatment of AIDS and other diseases that involve the tumor necrosis factor (TNF). The active principles were found to be the cycloartenol and the steroidal alkaloids cyclobuxine D and buxamine (233). Cyclobuxine-D (11)has been used as a complexing agent to investigate the reversible helix-coil transitions of DNA (18). Cyclobuxine (11)was found to have a protective effect against 60 min ischemia and subsequent 30 min reperfusion in the isolated rat heart model. Ischemia induced a marked decline in the contractile force and a gradual rise in resting tension. Reperfusion of the heart for 30 min resulted in a poor recovery of the contractile force. When the heart was perfused in the presence of cyclobuxine, a significant suppression of mechanical failure was seen. Ischemia also induced an immediate release of ATP metabolites and a release of creatine phosphokinase during reperfusion. Cyclobuxine inhibited the release of ATP metabolites, and slightly prevented the release of creatine phosphokinase during reperfusion. The ultrastructural damage induced by ischemia and subsequent reperfusion were significantly suppressed by cyclobuxine (134). Cyclosufforbuxinine-M (W),another Bwrus alkaloid, showed a marked inhibitory activity on the cholinesterase in horse and human serum (235). The antiulcer (gastroprotective) activity of steroidal alkaloids from Pachysandra terminalis Sieb. has also been investigated (136).

100

A"A-UR-RAHMAN

A N D CHOUDHARY

CH* (124) Cyclosufforbuxinine-M

0

HO

0

k

1128) Solanine

OH

GlcO

6H (127)Tomatine

(129)Capsirnine-3-OP-D-glucoside

2. CHEMISTRY

A N D BIOLOGY OF STEROIDAL ALKALOIDS

101

C. STEROIDAL ALKALOIDS OF THE LILIACEAE Plants of the genus Veratrum have been noted for their pharmacological activity for centuries. The powder of the root of V. viride is used for the treatment of toothache. The boiled, thin root slices in vinegar are considered to be useful against Herpes milliaris. This plant was used by several Native American Indian tribes to kill lice, as a catarrah remedy, against rheumatism, and as an insecticide. Some of the alkaloids are now widely used in the treatment of hypertension (137-139). Protoveratrine is useful in the treatment of various stages of hypertension. Intravenous injection of protoveratrine causes pronounced bradycardia and blood pressure lowering effects by stimulation of vagel afferents, but some side effects are also known (98). Imperialone (125), an alkaloid isolated from Petilium species, has shown high M-cholinolytic activity on the heart, which was accompanied by sensitization of M-cholinoreceptors in the secretory cells of lacrimal, salivary, and gastric glands and M-receptors in the smooth muscles of the intestine and urinary bladder. Based on current data, imperialone can be regarded as an M2-cholinolytic having M3- and M4-cholinopotentiating properties. Imperialone is a potent agent used for selective cardiac M2-receptor blockade and for enhancement of the functional activity of the smooth muscles and secretory organs that have M3- and M4-cholinoreceptors (140). Ebeinone (126), another ceveratrum-type steroidal alkaloid isolated from the bulbs of F. imperialis, showed anticholinergic activity (1 &ml) as manifested by the blockade of acetylcholine responses on the isolated guinea pig ileum and atria (242). D. STEROIDAL ALKALOIDS OF THE SOLANACEAE

C2,-Steroidal alkaloids of the family Solanaceae are of great potential interest as starting materials in the manufacture of steroidal hormone analogues. Many solanidine- and secosolanidine-type alkaloids isolated from various plants of this family have exhibited pronounced antibacterial and antifungal activities. Tomatine (l27),a glycospirosolane-type alkaloid isolated from many species of Solanum and Lycopersicum, inhibits the growth of several types of Gram-positive and Gram-negative bacteria and the pathogenic fungus Candida albicans, but it has no effect on human pathogenic Actinomyces (81). Solanine (128), an alkaloid isolated from several different Solanum species, is found to be a mitotic poison and inhibits human plasma cholinesterase. Solasodine (34) and its glycosides exhibit bradycardiac activity similar to that of veratramine (81). Capsimine-3-O-P-~-glucoside(129), a new steroidal alkaloid isolated from the root bark of S. capsicasfrurn, exhibited strong activity against

102

AlTA-UR-RAHMAN A N D CHOUDHARY

(130)Solamargine

CCI4-induced liver damage in ICR male mice (0.1 mg/kg). The steroidal alkaloids etioline, capsimine, capsicastrine, and naringgenin showed pronounced in v i m cytotoxicity against the human cancer cell lines PLC/PRF/ 5 and KB (142). A review has been published on the role of glycoalkaloids in the resistance of potato plants toward insect pests (243). Glycoalkaloids isolated from S. chacoense have the ability to impart resistance to the Colorado beetle (144). Solamargine (130), a glycoalkaloid of the spirosolane type isolated from the ripe berries of S. khasianum and other Solanum species, has exhibited antifilarial activity. The alkaloid can kill 100% adults and microfilariae (mf) of Sectaria cervi at a dose of 4 mg/ml in 60 and 80 min, respectively. It has been observed that when solamargine (130) was administered orally (10 mg/kg) to rats, in which S. cervi adults were administrated intraperitoneally, the blood mf count was reduced by more than 30% after the first phase of the treatment for 10 days. At a dose of 100 mg/kg solamargine can kill 100% of adult worms without any toxicity (145).

E. STEROIDAL ALKALOIDS FROM TERRESTRIAL ANIMALS an alkaloid isolated from several species of frogs Batrachotoxin (a), (Phyflobatesgenus), is the most potent cardiotoxin known and produces

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ventricular fibrillation and tachycardia in the cat heart at the 2 nM level. Batrachotoxin and its derivatives have also been investigated for controlling membrane permeability, since they are extremely potent and irreversible activators of so-called “sodium channel” in excitable membranes (246). Samandrine (45) isolated from European Fire Salamander (Sulumandru salamundru) and alpine Salamander (S. utru) is a potent, centrally active neurotoxin with a lethal dose of about 70 pg. It is also a potent local anesthetic and cardiac depressant (48). F. STEROIDAL ALKALOIDS FROM MARINE ORGANISMS

Cephalostatins are dimeric steroidal alkaloids isolated from the marine worm Cephulodiscus gilchristi. They were evaluated against a diverse group of 60 human cancer cell lines. These alkaloids were found to be powerful inhibitors of human cancer cell lines and in the murine P388 lymphocytic leukemia (PS system) (Ed50 10-7-10-9 pglml) (54-56). Another series of structurally related dimeric steroidal alkaloids called ritterazines (56-68) isolated from the tunicate Ritterellu tokioku also showed potent cytotoxicity against P-388 murine leukemia cells with IC50values between 0.01 and 10 pg/ml (57-59). Colonial zoanthids, which occur as dense mats on intertidal rocks, can eject sprays of water when they are disturbed. If the spray comes in contact with a victim’s eyes, it causes lachrymation followed by prolonged redness and pain. Zoanthamine (49), zoanthenamine (50), zoanthamide (51), and 28-deoxy-zoanthenamine (52), alkaloids isolated from the Zoanthid species, possess inhibitory activity in the phorbol myristate acetate (PMA)-induced mouse ear inflammation assay, as well as analgesic activity (52). Lokysterolamine A (71), an alkaloid isolated from the marine sponge Corticium species, was found to have in vitro activity in the mouse lymphoid neoplasm (P-388), human lung carcinoma (A-549), human colon adenocarcinoma (HT-29), and human melanoma (MEL-28) assays. In addition, it showed medium immunomodulatory activity (LcVIMLR > 187) and antimicrobial and antifungal activity against B. subtilis and Cundidu ulbicuns (62).

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-CHAFTER 1

BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS" ARNOLD BROW School of Pharmacy University of North Carolina Chapel Hill, North Carolina 27599

XUE-FENG PEI Laboratory of Bioorganic Chemistry National Institirtes of Health Bethesda, Maryland 20892 1. Introduction

...................................................................................

109

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11. Analytical Criteria

111. Unnatural Alkaloid A. Simple Tetrahydroisoquinolines ..................................................... B. 1-Benzyltetrahydroisoquinolines .............. ............................. C. I-Phenethyltetrahydroisoquinolines .......... D. (+)-Emetine and (+)-2.3-Dehydr E. (+)-Dihydroquinine ................. ........................ F. Unnatural Alkaloid Enantiomers s ...................... IV. (+)-Morphine .. ................................................... V. (+)-Physostigminc .................................................. VI. (+)-Colchicine ............................................................................... VII. (+)-Nicotine .... ................................................... VIII. Conclusions ..........................................................

112 112 I13 I I4 I I6 117

118 118

123 I28 133 I35 136

I. Introduction The plant alkaloids morphine, scopolamine, reserpine, physostigmine, to mention a few which are widely used in medicine, are single enantiomers of high optical purity. Studies of these alkaloids for more than a century have

* This paper is dedicated to Professor Dr. Vladimir Prelog from the Laboratorium for Org. Chemie. ETH-Zentrum, Zurich. Switzerland. on the occasion of his 90th birthday. THE ALKALOIDS. VOL.. SO 00YY-YSYX/98 $25.00

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Copyright 0 IYYX by Academic Press All rights of reproduction in any form reserved.

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revealed that proper stereochemistry and proper absolute configuration (enantioselectivity) are most often required to provide the desired pharmacological effect which is essential for their various medical uses. Enantioselectivity also plays an important role in the biosynthetic reactions controlled by enzymes. This is shown in Fig. 1 with the biosynthesis of (-)-morphine from (S)-norcoclaurine, elaborated in detail at the isoquinoline stage by Zenk and his group in Munich, Germany. As a consequence of this, it becomes a postulate, that chiral drugs, rather than racemic mixtures, should be targets in drug research ( I ) . This knowledge also relates to antipodal isomers of biologically active alkaloids (enantiomers), and to the question of how these “unnatural” isomers would perform in biochemical reactions and in pharmacological assays. For some time we have studied unnatural alkaloid enantiomers, and the results reviewed here, are generally in line with the view that the pharmacological effect of natural isomers is enantioselective. However, unnatural enantiomers may also have a pharmacological effect of their own, and these are often worth exploring.

11. Analytical Criteria

The chemical purity of drugs available to treat clinical disorders is carefully controlled by the United States Food and Drug Administration. Chiral drugs must be optically pure. Any undesired enantiomer present may be toxic, or have a pharmacological action of its own, and it is important to quantitate its presence. This is possible by several available techniques. Measuring specific rotations in solvents of different polarity has been replaced by C D and O R D data, is amplified by NMR methods, and, most effectively, by chromatographic analysis on chiral columns ( I ) . A few examples may illustrate this principle: Colchicide (10-demethoxycolchicine) showed significant activity in P388 leukemia, suggesting that it might be a potentially useful anticancer agent (2). Samples of colchicide prepared by the original procedure were found to be contaminated with 1.3%of thiocolchicine. This impurity could have accounted for the observed activity, since material prepared by a novel route, which was free of thiocolchicine, was not active ( 3 ) . Morphinans of the unnatural (+)-series, in contrast t o their enantiomers of the (-)-series which are chemically connected with natural morphine, were found to be inactive as analgesics in vivo ( 4 ) . The compounds of the (+)-series, however, possess useful antitussive properties and, when optically pure, are free of the side effects of their

3.

BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS

(-) -0ripavine

(-)-Thebaine

111

(-)-Neopinone

1 (-)-Morphinone

(-)Codeine. R=CHB (-)-Morphine, R=H

(-)-Codeinone

FIG. 1. Biosynthesis of morphine in plants. * These metabolic conversions are highly stereoselective. ** R. Lenz and M. H. Zenk, Terrahedron Lett 35, 3897 (1994).

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(-)-enantiomen. The synthesis of alkaloid enantiomers is now well advanced and allows these compounds to be made, if needed, by classical synthesis, or by processes using biotechnology, on an industrial scale (1).

111. Unnatural Alkaloid Enantiomers

A. SIMPLE TETRAHYDROISOQUINOLINES The tetrahydroisoquinolines shown in Table I are substituted by a methyl group at C-1, occur in optically active form in the Cactaceae (5), and are formed in small amounts in mammals (6). Data on the acute toxicities of both enantiomers of salsoline and isosalsoline, and their respective N-methyl analogs were obtained by different routes of administration. All these compounds were considerably less toxic when given orally (7). These optically active compounds did not show antiParkinson activity in the reserpine-reversal assay in mice and were devoid of significant antihypertensive activity. Stereoselective competitive inhibition of MAO-A was observed with the (R)-enantiomers of salsolinol, salsoline, N-methylsalsoline, salsolidine, and isosalsolidine (Ba), which are now available by the separation of racemic mixtures on cyclodextrin columns

TABLE I SIMPLE T E T R A H Y D R O l S O Q U l N O l ~ l NALKALOIDS ~~

Alkaloids

R'

R?

R3

Refs

Salsolinol Salsoline Isosalsoline Carnegine N-Methylsalsoline N-Methylisosalsoline Salsolidine

H H H CH3 CHI CH3 H

H H CH1 CH3 H CH3 CH3

H CH3 H CH3 CH? H CH3

(6,8b)

(7,8a) (7.8a.Rb) (7,8a) (7.8a) (7) (8a.9)

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113

(8b). The (R)-enantiomers of these alkaloids were generally more potent than the (S)-enantiomers. The finding that both enantiomers of salsolinol are excreted in the urine of alcoholics, but in different amounts, is noteworthy ( 6 ) . B. 1-B ENZY LTETRAHYDROISOQUINOLINES The alkaloids listed in Table I1 occur in nature in optically active form, and ( S ) - and (R)-reticuline are crucial intermediates in the biosynthesis of morphine in the poppy plant, Pupuver somniferum (see Fig. 1). Tetrahydropapaaeroline (THP), the condensation product of dopamine with the aldehyde of its own oxidative degradation is formed in vivo on incubation with M A 0 preparations ( 6 ) .Norreticulines, now readily available in enantiomeric form by synthesis ( ] I ) , and the reticulines obtained on N-methylation of norreticulines were the most important compounds to be evaluated in relevant assays (13). Both enantiomers of THP, and of norreticuline, when evaluated in v i m for their binding to adrenergic and dopaminergic receptors, and for antinociceptive activity in the hot-plate assay in mice, showed significant differences (14). Enantioselectivity appeared to be less apparent for the

TABLE I1 ALKALOIDS OF NATURAL A N D UNNATURAL CONFIGURATION"

R2q

~

3

0

$H2

OR4

Alkaloids

R'

Tetrahydropapaveroline Tetrahydropapaverine Reticuline Norreticuline Norarmepavine Norcoclaurine

H H CH3 H H H

R2

R7

R"

"The natural alkaloids of plant origin usually have the (S)-configuration.

RS

Refs.

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a-adrenergic or dopamine receptors. (S)-Tetrahydropapaveroline inhibited the binding of a radiolabeled ligand to beta-adrenergic receptors one hundred times more than the (R)-isomer. All of the analgesic activity resided in (S)-norreticuline which had about one-third of the potency of morphine, while the corresponding (R)-enantiomer was inactive. Single and repeated doses of the enantiomers of norreticuline and reticuline, when injected into rats s.c., did not show any appreciable analgesic effect in the writhing test at doses up to 30 mg/kg ( 6 ) . The enzymatic 0-methylation of the enantiomers of tetrahydro-isoquinoline-1-carboxylic acids, and of several 1-benzyltetrahydro-isoquinolinesof importance in the biosynthesis of morphine (see Frg. l ) , with (S)-adenosylL-methionine catalyzed by mammalian catechol 0-methyltransferase showed interesting differences between the natural and unnatural isomers. The 0-methylation of optically active 4-deoxy-norcoclaurine-1-carboxylic acids (15), and that of racemic norcoclaurine-1-carboxylicacid (16),yielded exclusively 7-0-methylated products suggesting that these acids are unlikely intermediates in the biosynthesis of morphine. A different result was found with the 1-benzyltetrahydro-isoquinolinesshown in Table 111. The 0-methylation of (S)-norcoclaurine in the presence of the mammalian enzyme gave predominantly (S)-coclaurine (80% at C-6 versus 20% at C-7), and this result agrees well with data reported for the 0-methylation occurring in plant species (17), but it differs substantially from the results obtained for the (R)-enantiomer (24% C-6 versus 76% C-7) (26). Bioconversion of (S)-3’-hydroxy-N-methylcoclaurineinto (S)-reticuline in the opium poppy occurs with high enantioselectively (18), but gave a different result when repeated in vitro with the mammalian enzyme (19). Only the (R)-3’-hydroxy-N-methylcoclaurineyielded appreciable amounts of ( R ) norreticuline (44% of C-4’ 0-methylated product). (R)-Norreticuline, which affords (R)-reticuline on methylation, may be a key intermediate in the biosynthesis of mammalian morphine ( 6 ) .

C. 1-PHENETHYLTETRAHYDROISOQUINOLINES Simple alkaloids of this class are relatively rare (20). Alkaloidal analogs were investigated in great detail in connection with methopholine (racemic 1pchlorophent hyl-2-methyl-6,7-dimet hoxy- 1,2,3,4-tetrahydroisoquinoline), which was developed at Roche as an analgesic and was found to be similarly potent as codeine and having a spasmolytic activity resembling that of papaverine (21). Methopholine and its analogs are readily available by synthesis and were resolved into their enantiomers (22). The analgesic activity resides entirely with the (R)-enantiomers (23).The specific rotation of aromatic halogenated compounds in this series vary greatly. Negative

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TABLE 111 ENZYMATIC OMETHYLATION OF OFTICALLY ACTIVE COCLAURINES WITH RADIOLABELED SADENOSYLMETHIONINE I N THE PRESENCE OF MAMMALIAN COMT

Q OH

OH

(9

(R)

Norcoclaurine 6-OMe 7-OMe

80% 20%

24% 76%

6-OMe 7-OMe

HO

Q4' H

(9 R = H

R

=

CH3

31% 45% 14% 86%

OH

OH

(R)

3'-Hydroxy-N-Nor- and N-methylcoclaurines 4'-OMe 32% 4'-OMe 3'-OMe 27% 3'-OMe 4'-OMe 44% 4'-OMe 3'-OMe 56% 3'-OMe

values were observed for all of the compounds when measured below 360 nm in methanol, and they yielded on catalytic dehalogenation the same compound (24). Most interesting are the pharmacological data of the optically active phenpropylamines obtained from the optically active methopholines on Hofmann degradation. The compounds obtained after catalytic hydrogenation of the vinyl group, and shown in Fig. 2, have a reverse pharmacological profile (25). Only the amine derived from the inactive (+)-(S)-methopholine showed significant analgesic activity. The isomer obtained from the analgetically active (-)-(R)-enantiomer was inactive. These phenpropylamines can freely rotate around the C-C bonds giving rise to a multitude of conformers. It is interesting to note that the

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CH30

6

61 (S)-Methopholine, not active

(Rplethopholine, active

1) CH#Acetone 2)AgZOlHfl, 150-170 OC

I

CI (R)-Phenpropylamine, not active

Cl (9-Phenpropylamine, active

FIG.2. Hofrnann degradation of enantiorners of rnethopholine.

oxidative degradation of the two analgetically active species, the ( - ) - ( R ) methopholine and the (S)-phenpropylamine, would lead, if executed, to amino acid enantiomers.

D. ( +)-EMETINE AND

(+)-2,3-DEHYDROEMETlNE

The alkaloid emetine is an active ingredient of Ipecac (Cephaelis ipecacuanha) used by South American Indians for the treatment of amoebic dysentery. The chemistry of natural emetine, now available by total synthesis, has been reviewed (26). In both the emetine series and the synthetic 2,3dehydroemetine series (27), it was shown that the amebicidal effect was associated with the alkaloids of natural configuration only, and not by the isomers shown in Fig. 3. Unnatural (+)-emetine, prepared by total synthesis, was found to be markedly less toxic than the natural alkaloid in rats (s.c. 700 mg/kg versus 25 mg/kg, respectively), and inactive as an amebicide in v i m and in vivo (28,29). The testing of (+)-2,3dehydroemetine prepared in optically pure

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CH3O

117

CH3O

NH H # L O C H 3

OCH3 :H3 (+)-Ernetine

(+)-2,3-Dehydroernetine

FIG.3. Structures of (+)-emetine and (+)-2.3-dehydroemetine.

form showed the antiamebic effect to be highly enantioselective, and restricted to the (-)-enantiomer (30).

E. ( +)-DIHYDROQUININE Quinine, medically used as an antimalarial, and quinidine, medically used as an antiarrhythmic drug, have been used in medicine for many years, and a concise review of the Cinchona alkaloids is available (31). Racemic dihydroquinine and its two enantiomers prepared by total synthesis (32), when assayed in mice infected with Plasmodium berghei, had the same antimalarial activity equal to that of quinine (33). This result parallels similar findings reported for the enantiomers of the widely used antimalarial mefloquine, which is also a racemic mixture (34). The structures of these aminoalcohols are shown in Fig. 4. It is conceivable that these aminoalcohols operate via an identical mechanism, but detailed experimental data are unfortunately lacking.

8

&FH5

CH3O 0

\

CF3

/

CF3 (+)-Dihydroquinine

W-Mefloquine

FIG.4. Structures of antimalarial aminoalcohols.

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TABLE IV SELECTION OF UNNATURAL ALKALOID ENANTIOMERS Alkaloids

Refs.

(S )-Tetrahydroharmine (1 R)-a-Hydroxybenzyltetrahydroisoquinoline ( + )-Coralydine (+ )-0-Methylcorytenchirine (I?)-( + )-Cherylline (R)-l,2-Methylenedioxyapomorphine (R)-l,2-Dihydroxyapomorphine ( + )-Perhydrohistrionicotoxin

F.

UNNATURAL

ALKALOID ENANTIOMERS

OF

DIVERSE STRUCTURES

Many naturally occurring alkaloids of medicinal importance have been the target of chemical synthesis and are reviewed in the literature (35). Table IV lists several unnatural alkaloids which were obtained from racemic precursors by chemical resolution, or by separation and alcoholysis of diastereomers obtained with optically active l-phenylethylisocyanates ( 9 ) . They all are fully characterized. Optically active tetrahydroharmines racemize under acidic conditions (36). Both apomorphine analogs were less active than apomorphine in a variety of assays (40). Contraction of frog sciatic nerve muscle preparations were similarly stimulated by both enantiomers of perhydrohistrionicotoxin (41). In reviewing the biological data reported so far on unnatural alkaloid enantiomers we can see that the biochemical and pharmacological activities, with the exception of the antimalarial effect of the Cinchona alkaloids (33) and some of the electrophysiological properties of histrionicotoxins (42), were in most cases enantioselective. To further support this view we decided to investigate the unnatural enantiomers of several medically important alkaloidal drugs in more detail.

IV. (+)-Morphine Sinomenine, a major alkaloid from Sinomenium acurum, belongs to the (+)-series of opioids, enantiomeric to that of natural (-)-morphine (Fig. 5); its conversion into (+)-morphine was reported by Goto’s group (42a-c), and into (+)-morphinans by Sawa et al. (43).A markedly improved synthesis of (+)-morphine from (-)-sinomenine was later reported by an NIH

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(-)-Sinornenine

119

(+)-Morphine

FIG. 5. Natural (-)-sinomenine and unnatural (+)-morphine.

group (44), but is best accomplished today by the Rice total synthesis of opioids using the readily available (S)-configured tetrahydroisoquinolines as the crucial intermediates (45). The Rice total synthesis of natural and unnatural opioids was first probed with racemic materials (46). After the successful chemical resolution of an intermediate tetrahydroisoquinoline, this project was followed with a practical synthesis of natural and unnatural opioids (45). Some of the chemical reactions in the Rice total synthesis of the unnatural opioids (+)morphine, (+)-codeine, and (+)-heroin are shown in Fig. 6. The desired

(9-Tetrahydroisoquinoline

(9-Hexahydroisoquinolineformamide

(S)-Octahydroisoquinoline-6-one

I (+)-Morphine, R'=R2=H (+)-Codeine. R'=H. R2=CH3 (+)-Heroine. R'=R2=Ac

(+)-Dihydrocodeinone

(+)-Brornonordihydrothebainone

FIG.6. Key compounds in the Rice total synthesis of (+)-morphine and (+)-codeine.

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(S)-tetrahydroisoquinoline was obtained by Bischler-Napieralski cyclization of an appropriate amide, reduction of the 3,4-dihydroisoquinoline,and chemical resolution of the tetrahydroisoquinoline with tartaric acid. Birchreduction of the (S)-enantiomer and formylation of the product gave the desired (S)-hexahydroisoquinolineformamide. Transketalization and bromination followed by deketalization gave the (S)-octahydro-isoquinolin-6one which was brominated at the orfho-position of the phenethyl substituent. Bromo-directed Grewe cyclization was accomplished with triflic acid to give the desired, optically active, (+)-bromo-N-formyl-nordihydrothebainone in 60% yield, and the corresponding amine on acid hydrolysis. Closure of the oxygen bridge was effected with a slight excess of bromine followed by treatment with aqueous base. Hydrogenation of the (+)-bromonordihydrocodeinone over Pd catalyst in the presence of formaldehyde directly gave (+)-dihydro-codeinone. Chemical reactions already described by Rapoport (47) then led to (+)-codeine (44), and, on treatment with boron tribromide, to (+)-morphine (44,48). Treatment of (+)-morphine with acetic anhydride gave (+)-heroin (44). An alternate conversion of (+)-dihydro-codeinone into (+)-codeine has been described (49). In addition to the Rice total synthesis of unnatural opioids on a larger scale, there are alternatives. The biomimetic total synthesis of (-)-codeine from (R)-norreticuline (50), if repeated with the (S)-enantiomer (ZZ) also would lead into the unnatural (+)-opioids discussed above. Another possible entry into unnatural (+)-opioids is available with the Overman total synthesis of enantiomeric opium alkaloids which, however, was not carried out on a larger scale (51). Preliminary pharmacological investigations of (+)-morphine prepared by Goto’s group showed that it was devoid of analgesic activity in the hot plate and tail flick assays, whereas the natural alkaloid was highly potent in both assays (42a-c). Several of the unnatural (+)-opioids, including (+)morphine, (+)-dihydromorphinone, and (+)-dihydrocodeine, however, showed significant antitussive activity. The early work of Goto on unnatural (+)-opioids signaled a distinct recognition of the enantiomers by receptor molecules, an avenue which was explored by Rice and his colleagues at the NIH. The Rice report on the stereospecific and nonstereospecific effects of (+)- and (-)-morphine, giving evidence for a new class of receptors, opened a new chapter in the understanding of opioid enantiomers (52). The unnatural (+)-morphine had minimal activity in three opiate assays in v i m : it was 10,000-fold weaker than its natural (-)-enantiomer in its ability to displace [3H]-dihydromorphine from binding sites in rat brain homogenates. In electrically stimulated guinea pig ileum, (+)-morphine did not inhibit contractions at a dose one hundred times greater than that of (-)-morphine, or of (-)-normorphine that is normally effective in

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inhibiting contractions, and (+)-morphine did not antagonize the action of (-)-morphine or (- )-normorphine in this assay. Furthermore, in the assay for adenylate cyclase activity in neuroblastoma x glioma hybrid cell homogenates, (+)-morphine had less than 1/1000th of the inhibitory potency of (-)-morphine. Finally, (+)-morphine did not antagonize the inhibitory action of (-)-morphine in the adenylate cyclase assay. The p-opiate receptors involved possess a high degree of stereoselectivity. They are blocked by naloxone, mediate analgesia and the endogenous ligands for these receptors are the endorphins and the encephalins (52). In the in vivo assays, (+)-morphine was microinjected into the periaqueductal gray region (a site known to mediate morphine analgesia) of drugnaive rats producing minimal analgesia but the hyper-responsitivity usually observed after microinjection of (-)-morphine. Also, when (+)-morphine was microinjected into the midbrain reticular formation of drug-naive rats, rotation movements similar to those following microinjection of (-)morphine occurred. These behaviors are not blocked by naloxone, and suggest that there are at least two classes of receptors, one stereoselective and blocked by naloxone and the other only weakly stereoselective and not blocked by naloxone. It was speculated that precipitated abstinence may be due, in part, to a selective blockade of the receptors of the former class, but not of the latter (52). Opioid and nonopioid enantiomers selectively attenuate N-methyl-Daspartate (NMDA) neurotoxicity, with (+)-morphine being considerably more potent than the (-)-enantiomer (53). Another study reported that some unnatural opiates, including (+)-morphine which does not interact with the classical opiate receptors, interact with the phencyclidine receptor, which is known to antagonize the actions of glutamic acid mediated by the NMDA excitatory amino acid receptor (54). It will be interesting to see how these nonopiate selective receptor reactions converge, and whether nonopioid enantiomers might provide a useful therapeutic approach to clinical syndromes involving NMDA receptor mediated neurotoxicity. Metabolism of (-)-morphine and its (+)-enantiomer in v i m , comparing glucuronidation and N-demethylation, was investigated (55). It was found that natural (-)-morphine, with hepatic microsomal enzyme preparations from control rats, and rats pretreated with phenobarbital were metabolized as follows: natural (-)-morphine was primarily glucuronidated at the phenolic OH-group, whereas (+)-morphine was primarily conjugated at the alcoholic OH-group. The rate of N-demethylation of (-)-morphine was about twice as high as that of the (+)-enantiomer. Phenobarbital treatment led to a three- to four-fold increase of the glucuronides, but not to a change in the N-demethylation. In contrast, pretreatment with morphine decreased the N-demethylation process of both enantiomers by 80%. This study

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demonstrates that there is an inherent substrate stereoselectivity for morphine metabolism in rat hepatic uridine-5’-diphosphate-glucuronyltransferase, but it is not considered critical for the receptor-mediated analgesia. Systemic administration of (-)- and (+)-morphine given to adult rats was used to study the motivational properties for taste and place conditioning (56). Whereas place conditioning was preferred by opioids binding to the preceptor, conditional taste aversion was seen by both of the enantiomers. Codeine is the methyl ether of natural (-)-morphine, but since it is present in raw opium only to the extent of 0.8-2.5%, it is largely produced from (-)-morphine by O-methylation (45). Codeine alone, and in combination with other drugs is widely used as an antitussive. Similar properties were found to be associated with synthetic morphinans of the (+)-series represented by dextromethorphan which until today seems to dominate the market ( 4 ) . It is not surprising that (+)-opioids, including (+)-codeine now available by the Rice total synthesis, were investigated for antitussive activity. Much of this work was done by Harris and his associates at the Medical College of Virginia in Richmond, who benefited from the material prepared by Rice and his colleagues. The Harris study nicely supported the theory that the effects of opiates on the cough reflex, are based on a different receptor mechanism (57). The investigators reported that natural (-)-codeine was active in the mouse tail-flick test as well as in the hot plate test whether given p.0. or S.C.(EDsO 4.1 mg/kg S.C. and 13.4 mg/kg p.0. in the first test versus 20.7 mg/kg S.C.and 20.5 mg/kg P.o., respectively, in the second test). The (+)-enantiomer of codeine was inactive in both tests up to 100 mg/kg, but did cause hyperexcitability, convulsions, and ultimately death. Although (-)-codeine was more potent than (+)-codeine in inhibiting the cough reflex in anesthetized cats, the (+)-enantiomer did have activity (EDs0 0.27 mg/kg i.v. for (-)enantiomer and 1.61 mg/kg i.v. for the (+)-enantiomer). In these animals, (-)-codeine did not significantly affect the cardiovascular parameters at the doses tested, whereas (+)-codeine caused a significant and transient decrease in blood pressure and heart rate. In another study by Harris et al., it was reported that from a series of p-opiate agonists/antagonists, except morphine, the opiates with the natural configuration were more potent antitussive agents than their unnatural antipodal isomers, but the differences were much smaller than those found for other opiate-receptor-mediated actions (58). It was suggested that the antitussive effect of opiates may be regulated by another type of receptor exhibiting a lesser degree of stereoselectivity than that required by the p receptor. (-)-Naloxone, prepared from natural thebaine, is in many assays a pure narcotic antagonist with no agonist activity. The (+)-enantiomer. prepared by a multistep synthesis from natural (-)-sinomenine, when exam-

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123

ined in three assay systems was found to have no more than 1/10001/10,00Oth of the activity of (-)-naloxone and can thus serve to test the stereoselectivity of the biochemical and pharmacological actions of (-)naloxone (59). There are no biological data available on (+)-heroin, the enantiomer of (-)-heroin prepared from (+)-morphine by acetylation (44). The work done on the enantiomers of opiates in search for better analgesics and antitussive agents clearly demonstrated, as early as 1960, that developing chiral drugs has merits and that useful information is obtained in studying the enantiomers of biologically active molecules. In the case of analgesics it became obvious that there is no need to deal with racemic mixtures obtained by synthesis, since the (+)-enantiomen of the target molecules were inactive. In the case of antitussives related to opioids the opposite became true. The (-)-enantiomer responsible for the side effects of natural morphine can be replaced with the (+)-enantiomer. The latter, if taken as recommended, does not show the undesired side effects of the opiates, and lacks the potential of addiction and physical dependence.

V. (+)-Physostigmine In the introduction of his colorful story on “The Ordeal Bean of Old Calabar: The Pageant of Physostigma venenosum in Medicine,” Bo Holmstedt writes: Many drugs have played a role not only in the cure and alleviation of disease, but also as tools in elucidating physiological and pharmacological mechanisms (60).Physostigmine, also called eserine, is an alkaloid of the Calabar bean of Physostigma venenosum Balf. of West Africa, and it is certain that we could not have advanced in our understanding of basic cholinergic mechanisms without studying physostigmine, although its role in medicine is perhaps less known than that of atropine, muscarine, and nicotine. The transmission of impulses throughout the cholinergic nervous system is dependent on acetylcholine as the chemical mediator (61). Compounds that produce a similar effect in preventing the normal hydrolysis of acetylcholine by cholinesterases are called anticholinesterases, or acetylcholineblocking agents. One of the first compounds to be recognized as an acetylcholine-blocking agent was physostigmine. It is used clinically as its soluble salicylate in the treatment of glaucoma by reducing intraocular tension. Because of its miotic properties it is employed after atropine to return the pupil to its normal size. These pharmacological effects are produced by the competitive inhibition of cholinesterases by blocking the active site on

124

BROSSI AND PEI

the enzyme by carbamoylation. Physostigmine similarly inhibits in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the former is present in red blood cells. in the brain, and in nerve tissues, and the latter is present in blood serum, pancreas, and liver. To help in the further elucidation of the structural requirements of the acetylcholine active centers, Robinson et al. have prepared the enantiomer of the natural alkaloids, namely (+)-physostigmine and (+)-physovenine, the latter being the antipodal isomer of the ether alkaloid physovenine which is also occurring in Physosfigma venenosiim (62). The Robinson synthesis of unnatural (+)-physostigmine, shown in Fig. 7, is grosso mod0

I

I

CH3 CH3 (+)-Eserethole, R = Et (+)-Esermethole, R = CH3

x CH3 (+)-Eseroline: R = H (+)-Physostigrnine: R = CH3NHCO

X=l

FIG.7. Syntheses of (+)-physostigrnine.

CH3

3.

BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS

125

identical with that of the natural alkaloid reported by Julian and Pikl (63a), and greatly improved later (63b-d). A chemical resolution was executed at the stage of eserethole, with (+)-tartaric acid to give the (+)-enantiomer following work reported by Kobayashi (64). Reaction of (+)-eserethole with aluminum chloride gave (+)-eseroline which was reacted with methylisocyanate as reported earlier to give (+)-physostigmine (65). With an easy chemical resolution of Julian's 3-methylaminoethyloxindole this route is now further simplified (66). The key compound (+)-eseroline has recently been obtained by an alternate synthesis, also shown in Fig. 7. In this synthesis, the chemical resolution was accomplished with a carbinolamine obtained by total synthesis on reacting it with ditolyltartaric acid. The resulting optically active quaternary salts, on treatment with aqueous sodium hydroxide, readily converted into the desired optically active carbinolamine, and its methiodide on reaction with methylamine gave the important optically active esermethole which is 0-demethylated to give eseroline (67). Unnatural (+)-physostigmine and its analogs are best prepared today by a modification of the Julian total synthesis developed at Georgetown University in Washington, DC, during 1992-1994 (63c,d; 68). Details, explained in Fig. 8, showed that the nitrile of the 0-methyl ether series on chromatography using microcrystalline cellulose triacetate (MCTA) as stationary phase, on elution with 96% ethanol as mobile phase, yielded first the desired faster running (-)-(3aR)-enantiomer needed to prepare the tricyclic (+)-3aR)-esermethole in 45% yield (63c). Similar good enantiomeric separation also was achieved with the corresponding amides, originally prepared from an oxindole-3-acetic acid of proper configuration (69).

-* I CH3

M

e

O

'

N

e

+ I

O

I

CH3

CH3

R = CN, CONHCH3. CONHBn

CH3 R

R = H, CH3, En

FIG.8. Practical synthesis of unnatural

(-t)-physostigmine.

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Conversion of the nitrile and amides into the desired (+)-esermethole was accomplished by classical reactions (65a,b). The unnatural alkaloids and their analogs made to ascertain their biological activities were (+)-physostigmine (62,70,73), (+)-phenserine (67,68), (+)-N'-norphenserine (68), and (+)-physovenine (72). The phenserines were included since their corresponding (-)-(3aS)-enantiomers belong to a series of compounds which selectively inhibit AChE, are long acting, and less toxic than the corresponding physostigmines (70). The first studies assessing the anticholinesterase activity of (+)-physostigmine and (+)-physovenine measured the in vitro activity in inhibiting erythrocytic AChE (62). It showed that both of these enantiomers were practically devoid of inhibitory activity. Robinson, in this important study, concluded that the asymmetry of the molecules, caused by optical inversion at C3a, may adversely be affected by binding of the inhibitor to the enzyme. He suggested that the opening of ring C, to give the 3H-indoleninium cation, may occur at the enzyme surface, and that this reaction may be responsible for the anti-acetylcholinesterase activity. Since the opening of ring C on acid catalysis requires nonphysiological conditions, this is, in the opinion of the reporters, unlikely to happen. Enantioselectivity in the inhibition of AChE and BChE by physostigmine enantiomers was later confirmed with enzyme preparations from the electric eel (72), and from various other tissues, including human erythrocytes (73). Both studies confirmed that only natural (-)-physostigmine interacts with the enzymes, and that the unnatural (+)-enantiomer is largely inactive. A similar result was obtained with (+)-phenserine, the enantiomer of the highly selective AChE-inhibitor phenserine, a phenylcarbamate analog of physostigmine (70), but the data are less convincing in the NI-nor series where a hydrogen atom substitutes for the N-methyl group (67,68). Several nor-compounds of both enantiomeric series were compared and the results are shown in Table V. The inhibitory data of the physostigmines showed the (-)-enantiomer to be almost equipotent in inhibiting AChE and BChE (28 nM versus 16 nM). Significant greater selectivity was noted for the phenserines, with the (-)-enantiomer being much more potent in inhibiting AChE than BChE (22 nM versus 1552 nM, 70-fold selectivity). This contrasts with the results measured for the enantiomers of the N'-norphenserines, which showed relatively little difference between the enantiomen in the inhibition of AChE; similar selectivity to AChE against BChE for the (-)-enantiomer (25 nM for AChE versus 623 nM for BChE, 25-fold selectivity), but considerably greater selectivity to AChE for the (+)-enantiomer (67 nM for AChE versus 5923 nM for BChE, 88-fold selectivity). The optical purity of the compounds of the (+)-series was measured at the stages of the intermediates N'-benzylnoresermethole and Nl-benzylnor-

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TABLE V OF NATURAL AND UNNATURAL PHYSOSTTGMINE, PHENSERINE, N'VALUES NORPHYSOSTIGMINE, NI-NORPHENSERINE, AND PHYSOVENINE VERSUS HUMANERYTHROCYTE ACHE AND HUMANASM MA BCHE 1% (nM)

Compound

AChE

(-)-Physostigmine (?)-Physostigmine (+)-Physostigmine (-)-Phenserine (t)-Phenserine ( + )-Phenserine ( - )-N'-Norphenserine ( ?)-N'-Norphenserine ( + )-A"-Norphenserine ( - )-N'-Norphysostigmine ( + )-N'-Norphysostigmine (-)-Physovenine ( )-Physovenine (+)-Physovenine

28 70 >10,000 22 '

75

3500 25 47

67 21 193 27 30 56

BChE

Refs.

16 35 4000

1552 5610 >10,000 623 1659 5923 2.0 203 4 4

56

eseroline by HPLC on a chiral stationary phase, and the compounds were found to be at least 98% (ee). These differences in assays measuring the inhibition of binding to the enzyme with enantiomers of N-CH3 and N-H substituted analogs are most puzzling and require further study. It is at the moment not clear whether these differences result from a steric effect ( N CH3 versus N-H), differences in basicities (tertiary amine versus secondary amine), differences in the formation of a hydrogen bond of the substrate with the enzyme (E-H.a.N-CH3 versus N-H-a-E),or other factors. Ring-opening of physostigmines as speculated by Robinson (62), and discarded because it occurs under nonphysiological conditions (74), also would have to explain a similar behavior of ring-C 0-ether analogs represented by physovenine (71), and the S-ether isosteres (75). Several indoline carbamates were prepared as illustrated in Fig. 9, and tested. Although these carbamates had anticholinesterase activity, it was less than that observed with the tricyclic compounds. The NIH-modification of the Julian total synthesis of natural (-)-physostigmine gave access to substantial amounts of materials needed to develop the unnatural (+)-series (69). Albuquerque and his colleagues evaluated (+)-physostigmine as an antidote to poisoning with organophosphates (77), and in order to study the damage at the neuromuscular synapse by mechanisms not related to cholinesterase carbamoylation (78). It was found that unnatural (+)-physostigmine, which had a much lower AChE inhibitory

128

BROSSI AND PEI

1 6

1

NaBHmeOH

H2/R02/CF3COOH

PhNHCOO 0

6

PhNHCOO 0

NHCH3

N(CH3)2

I CH3

I CH3

FIG.9. Ring-opening of phenserine to indolines.

activity than the (-)-enantiomer, was able to protect the animals exposed to lethal doses of the organophosphate sarin (77). Although higher doses of (+)-physostigmine were necessary, the degree of protection by the unnatural antipode was similar to that of the natural alkaloid. Treatment of rats with atropine and (+)-physostigmine protected the animals against a lethal dose of the organophosphate, although at a higher dose. The protective effect of (+)-physostigmine, in conclusion, does not seem to depend on the inhibition of AChE, but on a direct blockade at the nicotinic acetylcholinesreceptor and its ion channel (77). Enantiomers of physostigmine and its analogs are now available by total synthesis (63a-d), making it possible to evaluate them in a variety of biological assays.

VI. (+)-Colchicine

Natural (- )-colchicine from the plant Cofchicum autumnale, the autumn crocus, or meadow saffron, and the glory lily Gloriosa superba, is an ancient and well-known drug used in the treatment of gout (79). Colchicine exerts its biological effect by its binding to tubulin forming a colchicine-tubulin complex which disrupts microtubule assembly and therefore affects mitosis and other microtubule-dependent functions. The chemistry and pharmacology of colchicine has repeatedly been reviewed (80). The colchicine binding to tubulin is highly selective €or the conformational states of colchicine,

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BIOLOGICAL ACTIVITY OF U N N A T U R A L ALKALOID ENANTIOMERS

129

and requires the phenyl-tropolone system to be (as)-configured (a]), as evidenced by the presence of strong negative Cotton effects at 260 nm and at 340-350 nm in the CD spectra of the natural colchicinoids (82-84). The arrangement of the two aromatic moieties in a counterclockwise helicity in natural colchicine (Fig. 10) and derived allo-congeners has been confirmed by X-ray analysis of several representative compounds (80). The importance of the (as)-configuration of the phenyl-tropolonic unit for interaction with tubulin and bovine serum albumin (85) has been confirmed by a Swedish group (86).It was shown that only (-)-(as)-deacetamidocolchicine, lacking the chiral acetamido group at C-7, and obtained by chromatographic optical resolution on a chiral column, did inhibit tubulin polymerization, whereas the (+)-antipodal isomer was completely inactive. Unnatural (+)-colchicine, the enantiomer of natural (-)-colchicine, shows positive Cotton effects at 260 nm and 340 nm in the CD spectrum, which remain unchanged on addition to a solution of tubulin (2: 1) (82). Unnatural (+)-colchicine played an important role in assessing the stereoselectivity in the interaction of (-)-colchicine with tubulin and other proteins. The compound was first prepared by Corrodi and Hardegger in 1957 (87), and the details are summarized in Fig. 11.

FIG.10. X-ray structure of natural (-)-colchicine.

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: : : CH3O : q = H * - p h

-

bH Optically Active Schiff Base of Deacetylcolchiceine

OH Ketimine

bH 10-Demethylcolchicone

OH Racemic Deacetylcolchiceine

CH30 Colchicone

FIG.11. Racemization of (-)-deacetylcolchicine.

Deacetylcolchiceine, readily available from colchicine on hydrolysis with aqueous mineral acids gave, on reaction with benzaldehyde, a Schiff base which on equilibration with methanolic potassium hydroxide gave, among other products, racemic deacetylcolchiceine (80). This compound was resolved with (+)-10-camphorsulfonic acid, and the antipodal isomers converted after 0-methylation, separation of the ether isomers, and N-acetylation into (-)- and (+)-colchicine, and (-)- and (+)-isocolchicine. It was later found that 0x0-deacetamido-colchiceine(the enol of colchicone), resulting from the hydrolysis of the ketimine formed during the equilibration, was another major product (88). An improved method to prepare unnatural (+)-colchicine from natural (-)-colchicine followed initial experiments reported by Blade-Font (89), and is detailed in Fig. 12 (90). Colchicine in refluxing acetic anhydride gives

3.

cH3 -

BIOLOGICAL ACTIVITY OF U N N A T U R A L ALKALOID ENANTIOMERS

N<

CH3O

*

131

CH30

OAc

CH30

CH30

(- )-Colchicine

1). 0.1 N HCVAcOH 2). 20 % H2SO4

4

2). CH2N2

CH3O

OH

W-Trifluoroacetyldeacetylcolchicine

I

u-Deacetylcolchiceine

::::qNH Aq. K2C03/(CH&CO

CH30

1 ) dCarnphorsulfonicAcid CH30 c H

-

0

CH30

0-Deacetylcilchicine

2). Ac20

3

q

N

H

A

c

CH30

-

0

CH3O (+)-Cobhicine

FIG.12. Improved procedure for the preparation of unnatural (+)-colchicine.

a triacetate which has lost the chirality at C-7, and on hydrolysis with 0.1 N HCl gave (+)-colchiceine. Heating the racemate with 20% sulfuric acid in acetic acid yielded racemic deacetylcolchiceine which, on treatment with trifluoroacetic anhydride in the presence of sodium carbonate, afforded the racemic trifluoroacetamide. 0-Methylation with diazomethane in methanol gave, after workup and chromatography, the desired ether isomer as the faster running compound, followed by the iso-isomer (90). Hydrolysis of the trifluoroacetamide with aqueous potassium carbonate gave racemic deacetylcolchicine which was resolved with (+)-10-camphorsulfonic acid in methanol. The less soluble salt, on treatment with ammonia, gave (+)deacetylcolchicine, and unnatural (+)-colchicine on treatment with acetic anhydride (50).

132

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In agreement with earlier findings (87), (+)-colchicine crystallized from chloroform, whereas the enantiomeric (- )-colchicine could be obtained crystalline only from ethyl acetate. The C D spectra of these solvated enantiomers do not fully conform in solution (91),but they did after the samples were dried at 70°C in high vacuum (80). Optically pure (+ )-colchicine prepared by Corrodi and Hardegger, when assayed in vitro as an inhibitor of mitosis, was found to be only 1/100th as potent as the natural alkaloid (92).It was recognized at that time that these alkaloids not only express chirality at C-7, but at the same time through their molecular asymmetry (93), an important detail investigated and fully confirmed later (82).Unnatural (+)-colchicine prepared from (+)-deacetylcolchicine, when assayed for inhibition of tubulin, showed a low potency (32% versus 90% for the (-)-enantiomer), and it was much less toxic in mice when given i.m. (123 mg/kg versus 3.6 mg/kg for the (-)-enantiomer) (94). The lower potency of (+)-colchicine on comparison with the (-)enantiomer was also noted in its affinity for three antisera prepared by coupling deacetylcolchicine to bovine serum albumin (85). There is great promise that the elegant total synthesis of natural (-)-colchicine by Banwell which introduced chirality by an optically active reducing agent followed by SN2 replacement of the (7R)-configured alcohol by an azide ion will offer a new route to colchicinoids of unnatural configuration (95). Secothiocolchicinoids, shown in Fig. 13, with a six-membered ring B were obtained from deacetylthiocolchicine by a Demjanov rearrangement and they have the phenyltropolone moiety in an (aR)-arrangement (96). The alcohol, and the derived methylene compound which is optically inactive, are believed to equilibrate in solution and to interact with tubulin as the (as)-atropisomers (96). Continuation of such efforts with the inclusion of both enantiomers will improve our understanding of the effects which conformational isomers of colchicinoids and their chemical analogs exert in their binding to tubulin. Such information is highly desirable to develop antitumor agents belonging to this class of spindle-toxins.

FIG.13. Secothiocolchicine and 6-methylene analogue.

3.

BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS

133

VII. (+)-Nicotine (-)-Nicotine is the natural enantiomer of nicotine, and (+)-nicotine the synthetic, nonnatural enantiomer. Nornicotine is a minor tobacco alkaloid in most species of Nicotiana (97), and may also be a metabolite of nicotine (Fig. 14) (98). Nicotine has long played an important role in furthering our understanding of the cholinergic system. There are several early reports in which the effects of unnatural (+)-nicotine on the nicotinic receptors and toxicities were compared to those of natural (-)-nicotine (99-105). Although the relative potency of the (+)-enantiomer varied with investigators and with the purity of the agents used, (+)-nicotine is qualitatively less potent than the natural (-)-nicotine on the stimulation andlor blockade of the nicotinic receptors in the peripheral nervous system. It should be noted that nicotine free base is hygroscopic and subject to autoxidation and absorption of COz. (+)-Nicotine di-d-tartrate and (-)-nicotine di-l-tartrate, however, are anhydrous and stable salts suitable for biological studies (103).The optically pure (+)-nicotine can be prepared by resolution of (+)-nicotine (103), which can be obtained by racemizing natural nicotine (106), or by synthetic methods. Numerous total syntheses of racemic nicotine have been reported, but the early synthesis, described in Fig. 15 ( 1 0 3 , still seems most practical. 3-Cyanopyridine, prepared by sulfonation of pyridine followed by treatment with potassium cyanide, reacted with a Grignard reagent to give ppyridyl-y-ethoxypropyl ketone. The ketone ws converted to the oxime, and the oxime reduced to an amine. After the cleavage of the ethyl ether with 48% HBr, the aminoalcohol was cyclized by treatment with NaOH to give racemic nornicotine. The racemic nicotine was obtained from the nornicotine on N-methylation with MeI. Resolution of racemic nicotine has proved tedious because of the unstability of nicotine free base. Complete resolution was achieved not long ago with d-tartaric acid combined with di-p-toluoyl-l-tartaric acid, leading to optically pure (+)-nicotine (103).

R = CH3, (-)-Nicotine R = H, (-)-Nornicotine

R = CH3, (+)-Nicotine R = H. (+)-Nornicotine

FIG.14. Nicotines and nornicotines.

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BROSSI AND PEI

R = H, (&Nornicotine R =CH3, (+)-Nicotine

FIG.15. Synthesis of racemic nicotine and racemic nornicotine.

The nornicotine enantiomers have been resolved recently via HPLC on a chiral column (108). Optically pure (+)-nicotine could also be prepared from (+)-nornicotine by N-methylation. It was reported that (+)-nicotine is less toxic than its natural antipode. The intravenous acute LDS0value in mice is 2.75 mg/kg for the (+)-nicotine compared with 0.38 mg/kg for the (-)-nicotine (203).(+)-Nicotine is much less potent than (-)-nicotine in raising blood pressure in anesthetized rats, and in the isolated guinea-pig ileum, with a potency ratio of 0.06, and 0.019, respectively (203). For the ganglionic nicotine receptor on cat superior cervical ganglion (stimulation and blockade), the relative potency of (+)nicotine is 0.2 of that of (-)-nicotine. Both (+)- and (-)-nicotine, however, had the same blocking effect for the muscle-type nicotinic receptor on the neuromuscular junction of rat diaphragm (105). In the adrenergic nerve terminals of the isolated rabbit pulmonary artery, (-)-nicotine produced sympathomimetic effects by releasing norepinephrine from those terminals. (+)-Nicotine, on the other hand, did not produce such effects, but instead inhibited the effects due to (-)-nicotine (105). Because (+)-nicotine had no effect on the response to exogenously applied norepinephrine and blocked the 3H-efflux induced by (-)-nicotine, the inhibitory effect of (+)nicotine was attributed to its presynaptic action. Such an inhibitory effect of (+)-nicotine was noncompetitive, on the basis of the shift of the concentration-contraction curve obtained with (-)-nicotine. Simultaneous application of both enantiomers produced no inhibition of the response to (-)nicotine, irrespective of the concentration of (+)-nicotine. Occurrence of the inhibitory effect of (+)-nicotine was not prevented by hexamethonium, a competitive nicotinic antagonist. (+)-Nicotine did not inhibit the responses of the artery to electrical transmural stimulation. These results

3. BIOLOGICAL EFFECTS OF

ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS

135

TABLE VI DISPLACEMENT OF ( -)-[3H]NICOTINE NlCOTlNES A N D NORNlCOTlNES IN RATBRAIN

SOLUBlLlZATION ON

BINDING BY

(+)-Nicotine (-)-Nicotine (+ )-Nornicotine (-) -Nornicotine

Cortex

K , (nM) Hippocampus

Cerebellum

31.6 1.44 41.4 56.4

47.1 1.87 37.6 46.3

97.5 3.83 30.6 33.0

~

K, = affinity constant.

indicate that (+)-nicotine inhibits the response to (-)-nicotine by acting neither on the nicotinic receptor nor on excitation-secretion coupling mechanisms (105). (+)-Nicotine may possibly act at a site other than the nicotinic receptors, and induce desensitization of nicotinic receptors. The binding properties of the enantiomers of nicotine and nornicotine were studied in solubilized preparations of rat cortex, hippocampus, and cerebellum (109). The (-)-nicotine was more potent than (+)-nicotine in the assays, but (-)-nornicotine is less potent than (-)-nicotine. It is interesting to note that the nornicotines show no enantioselectivity in the binding assay (Table VI).

VIII. Conclusions

Investigating the antipodal isomers of biologically active alkaloids is challenging and useful for several reasons: it will require a practical resolution of racemic intermediates, or an efficient asymmetric synthesis; it will show whether the natural alkaloid is enantioselective in its pharmacological action; it will signal whether the unnatural enantiomer has a pharmacological quality of its own which may be potentially useful; and it will give qualitative information whether the toxicities of the two enantiomers are significantly different. It is obvious that such information has to be based on proper analytical data, and data allowing for the quantitation of the optical purity of the two enantiomers. Should the antipodal isomer have potentially valuable pharmacological properties of its own, not covered by its natural enantiomer, it is suggested that this be further evaluated with an appropriate and acceptable formulation, such as a pharmacologically acceptable salt (HCl, H2S04, H3P04, fumarate, tartrate, maleate, etc.).

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Further study of (+)-physostigmine, or analogues of the (+)-series may ultimately lead to valuable information regarding the nicotinic acetylcholine-receptor-channel, or even to a new drug for treating cholinergic disorders, or organophosphate poisoning. Developing an analog of (+)morphine as an antitussive agent has to show improvements over dextromethorphan which is widely accepted. Antipodal isomers of alkaloids having the same pharmacological effect and practically identical toxicities, as observed with antimalarial aminoalcohols, may well set the stage for developing a racemic drug. It is hoped that this review of unnatural alkaloid enantiomers will stimulate further research in this area, and will support the conviction that chiral is better based on experimental data from unnatural antibiotics, sugars, peptides, steroids, and amino acids, to mention a few.

Acknowledgments

The authors wish to thank Ms. Sheng Bi for her considerable help in the preparation of the manuscript. We would also like to thank Dr. B. Witkop, Institute Scholar of the National Institutes of Health, for his most valuable comments and suggestions.

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80. 0. Boy6 and A. Brossi. The Alkaloids, 41, 125 (1992); and refs. therein. 81. V. Prelog and G. Helmchen, Angew, Chem. Int. Ed. Engl. 21,567 (1982);The aS absolute configuration of the phenyl-tropolone system in natural (-)-colchicine follows the chirality rules which consider the order of the ortho-ortho’ substitutents of the central bond. M. F. Mackay, Department of Chemistry, La Trobe University, Bundoora, Victoria 3083, Australia, informs us that natural (-)-colchicine according to rules established in 1966 by Cahn, Ingold, and Prelog would have aR absolute configuration of the phenyl-tropolone system. In assigning the absolute axial configuration to colchicinoids it is important therefore, to explain on what ruling it is based. 82. H. J. C. Yeh, M. Chrzanowska, and A Brossi, FEBS Lett. 229,82 (1988). 83. A. Brossi, 0.BoyC, A. Muzaffar, H. J. C. Yeh, V. Toome, B. Wegrzynski, and A. Brossi, FEBS Lett. 262, 5 (1990). 84. E. A. Pyles and S. B. Hastie, J. Org. Chem. 58,2751 (1993). 85. J. Wolff, H. G. Capraro, A. Brossi, and G. H. Cook, J. Biol. Chem. 255, 7144 (1980). 86. U. Berg, J. Deinum, P. Lincoln, and J. Kvassman, Bioorg. Chem. 19, 53 (1991). 87. H. Corrodi and E. Hardegger, Helv. Chim. Acta 40, 193 (1957). 88. M. A. Iorio, A. Brossi, and J. V. Silverton, Helv. Chim. Acta 61, 1213 (1978). 89. A BladC-Font, Tetrahedron Lett. 2977 (1977). 90. R. Dumont, A. Brossi, and J. V. Silverton, J . Org. Chem. 51,2515 (1986). 91. A Brossi, J. Nut. Prod. 48, 878 (1985). 92. H. Lettr6 and R. Lettri, Narunvissenschafien 80, 180 (1966). 93. D. Werner and K. H. Donges, Planta Medica 22,306 (1972). 94. M. ROsner, H. Capraro, M. Iorio, A. E. Jacobson, L. Atwell, A. Brossi, T. H. Williams, R. H. Sik, and C. F. Chignell, J. Med. Chem. 14, 257 (1981). 95. M. G. Banwell, “Pure and Applied Chemistry,” 1995, in press. 96. L. Sun, A. T. McPhail, E. Hamel, C. M. Lin, S. B. Hastie, J. J. Chang, and K. S. Lee, J . Med. Chem. 36,544 (1993). 97. F. Saitoh, M. Noma, and N. Kawashima, Phytochemistry 24,477 (1985). 98. J. W. Gorrod and P. Jenner, Int. J. Clin. Pharmacol. Biopharm, 12, 180 (1975). 99. C. S. Hicks and D. A. Sinclair, Aust. J. Exp. Bio. Med. Sci. 25, 83 (1947). 100. C. S. Hicks, M. E. Mackay, and D. A. Sinclair, Aust. J. Exp. Bio. Med. Sci. 25,353 (1947). 101. R. B. Barlow and T. J. Hamilton, Br. J. Pharmacol. 25, 206 (1965). 102. L. G. Abood, K. Lowy, A. Tometsko, and H. Booth, J. Neurosci. Res. 3,327 (1978). 103. M. D. Aceto, B. R. Martine, 1. M. Uwaydah, E. L. May, L. S. Harris, C. Izazola-Conde, W. L. Dewey, T. J. Bradshaw, and W. C. Vincek, J. Med. Chem. 22, 174 (1979). 104. L. T. Meltzer, J. A. Rosecrans, M. D. Aceto, and L. S. Harris, Psychopharmacology 68, 283 (1980). 105. S. Ikushima, I. Muramatsu, Y. Sakakibara, K. Yokotani, and M. Fujiwara, J . Pharmacol. Exper. Therap. 222, 463 (1982). 106. A. Pictet and A. Rotschy, Ber. Dtsch. Chem. Ges. 33,2353 (1900). 107. L. C. Craig, J. Am. Chem. SOC. 55,2854 (1933). 108. D. S. Gamey, J. T. Wesicak, M. W. Decker, J. D. Brioni, M. J. Buckley, J. P. Sullivan, G. M. Carrera, M. W. Holladay, S. P. Arneric, and M. Williams, J. Med. Chem. 37, 1055 (1994). 109. X. Zhang and A. Norberg, Naunyn-Schmiedeberg’s Arch. Pharmacol. 348,28 (1993).

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-CHAPTER A

THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS JOHN W. DALY Laboratory of Bioorganic Chemistry National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892 I. Introduction ................................................................................... 11. Samandarines 111. Batrachotoxin IV. The Pumilioto A. Pumiliotoxins and Allopumiliotoxins

141

C. Other Pumiliotoxin-Class Alkaloids ................................................ Histrionicotoxins Gephyrotoxins ... Decahydroquinolines ....................................................................... Cyclopenta[b]quinolizidines Epibatidine ..................... Pseudophrynamines .. Pyrrolizidine Oximes Coccinellines ........... Bicyclic “Izidine” Alkaloids .............................................................. A. Pyrrolizidines B. 3,5-Disubstitute ines ...................................................... C. 5.8-Disubstituted Indolizidines ...................................................... D. 5,6,8-Trisubstituted Indolizidines E. Quinolizidines ........................................................................... XIV. Monocyclic Alkaloids ... XV. Summary and Prospects References ....................................................................................

149

V. VI. VII. VIII. IX. X. XI. XII. XIII.

152

159 159 160 161 163 163 164 165 167

I. Introduction

Alkaloids are normally thought of as nitrogenous secondary metabolites produced by and stored in plants. However, amphibian skins have in the past few decades proven to be a source of a diverse array of alkaloids THE ALKALOIDS. VOL. SO Ix)w-959x/Yx 525.00

141

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unprecedented in the plant kingdom. Such skin alkaloids apparently serve the amphibians in chemical defense against predators. Structures of amphibian alkaloids have been reviewed in detail, most recently in 1993 (I).The distribution of various alkaloids among some 40 species of frogs of the neotropical family Dendrobatidae was presented in 1987 (2). Synthetic efforts leading to amphibian alkaloids have been reviewed, most recently in 1986 (3).Since the 1993 review, many more alkaloids have been detected in amphibian skin extracts using gas chromatographic (GC) mass spectral and GC-Fourier-transform infrared (FTIR) spectral analyses; further structural classes have been defined, and most importantly evidence has been obtained that indicates that alkaloid-bearing amphibians, with the exception of the European fire salamander, probably do not synthesize their skin alkaloids, but instead rely on dietary sources and merely efficiently sequester and store for extended periods alkaloids that they obtain from ants, beetles, millipedes, and probably other small arthropods, whose identities remain shrouded in mystery. The amphibian skin alkaloids, thus, would represent a remarkable instance of a chemical ecology, wherein the amphibian is wholly dependent on dietary arthropods as a source of the alkaloids that comprise the active principles in its defensive skin secretions. The identities of the ultimate source of the unique so-called “dendrobatid alkaloids,” a term coined from the family name Dendrobatidae of the frogs from which they were discovered, remain a challenge for future research. Such “dendrobatid alkaloids” include the batrachotoxins, the pumiliotoxins and related congeners, the histrionicotoxins, the gephyrotoxins, the decahydroquinolines, the cyclopentaquinolizidines, and epibatidine.

11. Samandarines

The poisonous nature of the striking black and yellow European fire salamander (Salamandra salamandra) has been known since ancient times. The active principles were discovered to be highly toxic alkaloids in the 1860s, but it was not until the pioneering studies of Clemens Schopf, begun in the early 1930s, that the steroidal structures were realized and elucidated. Such structure elucidation was completed prior to the emergence of mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy as powerful analytical techniques, and, thus, was dependent on classical methods, involving chemical degradation and UV and IR spectral analysis. X-Ray crystallographic analysis played a significant role in the later stages of this research. Fortunately, relatively large quantities of alkaloids could be

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obtained from the parotid glands of the salamanders. The major alkaloid from the salamander was samandarine (1).By 1961 the structures of samandarine and several congeners had been determined and were reviewed in detail ( 4 ) . A total of nine samandarine-class alkaloids have been isolated; all but three have the oxazolidine ring system of samandarine and all have a seven-membered nitrogen-containing steroidal A-ring (ZJ).

Samandarines are known in Nature only from the European fire salamander and are apparently synthesized by the salamander (personal communication, G. Habermehl, 1988). The samandarines represent the first example of an “animal alkaloid.” Limited pharmacological studies on these extremely toxic substances indicate that they are powerful local anesthetics (see Ref. I ) .

Ill. Batrachotoxins Two brightly colored dendrobatid frogs (Phyllobates aurotaenia and Phyllobates bicolor) of the rain forests of the Pacific coast in Colombia were known to have extremely toxic skin secretions based on the use of such secretions to poison blow-darts by native peoples of that region (see Ref. 6). The nature of the active principles was unknown until studies were initiated at NIH in 1962, involving field collection of such frogs, and leading ultimately to the isolation and structure elucidation of the steroidal alkaloids batrachotoxin (2), homobatrachotoxin (3), batrachotoxinin A (4), and some minor congeners (7-9). Efforts at structure elucidation (see Ref. 3 ) relied heavily on mass spectrometry and NMR spectroscopy, but ultimately it was the X-ray analysis of ap-bromobenzoate of batrachotoxinin A that revealed the steroidal moiety of these alkaloids (7). The nature and site of the Ehrlich-positive pyrrole moiety of 2 and 3 were deduced from spectral properties and confirmed through the synthesis of 2 by esterification of (4) with 2,4-dimethylpyrrole carboxylic acid (8). Extracts of 5000 skins had

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yielded only 46 mg of batrachotoxinin A, 11 mg of batrachotoxin, and 16 mg of homobatrachotoxin. 0 R=

H3C

N I

R=

H

I

H

4

R = H

Batrachotoxin proved to be a specific and potent activator of voltagedependent sodium channels in nerve and muscle and, as such, both it and a radioactive batrachotoxinin-A benzoate have become widely used as pharmacological research tools (see Ref. 1). Fortunately, a new species of dendrobatid frog containing much higher levels of batrachotoxins was discovered in a remote river drainage in western Colombia in the early 1970s. The frog was named Phyllobates terribilis in view of its extraordinary toxicity. Batrachotoxins isolated from this frog ( 9 )represent the sole source of these valuable research tools to the present time, since the syntheses of such alkaloids are multistep and impractical for large quantities. Batrachotoxins are known to occur at high levels only in the skins of the three true poison-dart frogs ( P . aurotueniu, P. bicolor, P. terribilis), which are to this day still used to poison blow-darts by the indigenous peoples of western Colombia. Much lower levels of batrachotoxins are found in the skin of the other two species (P. lugubris, P. vittatus) of the genus. Batrachotoxins have not been detected in other dendrobatid frogs and, indeed, their presence in the skin was one taxonomic character leading to the definition of Phyllobates as a monophyletic genus. Such frogs are insensitive to batrachotoxin, due to an altered sodium channel that does not respond to batrachotoxin by opening (10). Remarkably, Phyllobates frogs raised in terraria on a diet of fruit flies and crickets had no trace of batrachotoxin in their skin (10). The lack of alkaloids in these captiveraised frogs was the first indication that skin alkaloids in dendrobatid frogs might have a dietary origin, but this was not fully appreciated until years later, in part because the wild-caught frogs maintained significant skin levels

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of batrachotoxins for up to 6 years in captivity (20). It is now realized that dendrobatid frogs have the ability to sequester dietary alkaloids into the skin and to retain such alkaloids for extended periods ( I I ) , probably because frogs eat their skin during shedding, thus “recycling” any skin alkaloids. The insensitivity of frogs of the genus Phyllobates to the action of batrachotoxins would permit them to ingest putative batrachotoxincontaining arthropods with impunity. Remarkably, one of the batrachotoxins, namely homobatrachotoxin (3) has now been discovered to be present in the skin and feathers of New Guinean birds of the genus Pitohui (22). Such birds are recognized as being toxic by the natives of Papua New Guinea. Whether there is a requisite dietary source, or whether the bird has its own biosynthetic pathway of homobatrachotoxin is unknown.

IV. The Pumiliotoxin Class The initial studies on batrachotoxin from the Colombian poison-dart frogs attracted the attention of a herpetologist, Charles W. Myers, who was interested in a brightly colored and extremely variable dendrobatid frog, Dendrobates pumilio, in Panama. Thirty years of collaborative field work by Myers and Daly on dendrobatid frogs in the rain forests of Central and South America ensued (23). The collaboration has resulted in the detection of over 400 alkaloids in amphibian skin extracts and the discovery of over a dozen new species of dendrobatid frogs. It began with the investigation of the levels and nature of toxic alkaloids in skin extracts from the Panamanian dendrobatid species Dendrobates pumilio, and the possible correlation of toxicity with brightness of coloration for populations of this extremely variable frog. There was no correlation and the skin alkaloids did not include batrachotoxins, but were instead much simpler substances (14). In this initial study three major skin alkaloids were isolated in quantities of less than 2 mg each. Mass spectra of the more toxic pumiliotoxins A and B indicated formulas of C19H33N02and C19H33N03rrespectively. NMR spectral analyses were not definitive and the compounds were incorrectly suspected to be steroidal in nature, like the samandarines and batrachotoxins. Further quantities were later isolated from some 250 skins and analysis indicated that pumiliotoxins A and B were closely related bicyclic alkaloids with two double bonds, differing only in the presence of one or two hydroxyl groups, respectively, in a side chain (25). Instability and limited supplies thwarted efforts to prepare a crystalline salt for X-ray analysis and modern

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2D-NMR techniques had not yet been developed, so that a decade after the initial studies, the structures of pumiliotoxins A and B were still only partially defined. During this 10-year period, many further “dendrobatid alkaloids” were discovered and alkaloid profiles were delineated for many species and populations of dendrobatid frogs, using gas chromatography and mass spectral analysis. A code system, employing in boldface the nominal molecular weight and a letter, when necessary, for each alkaloid was introduced in an attempt to cope with the hundreds of alkaloids being detected in dendrobatid frog skin extracts. A. PUMILIOTOXINS AND ALLOPUMILIOTOXINS A number of the “dendrobatid alkaloids” appeared to be related in structure to pumiliotoxins A and B in exhibiting diagnostic prominent mass spectral fragment ions at d z 166 (C&16NO+) and d z 70 (C4H8N+). These alkaloids, now totalling almost thirty, were grouped into a subclass called pumiliotoxins. A further set of alkaloids exhibited diagnostic prominent mass spectral fragment ions at d z 182 ( C I O H ~ ~ Nand O ~ )d z 70, indicating, in consort with other data, the presence of an additional hydroxyl group in the bicyclic ring system of the pumiliotoxins. These alkaloids, now totalling almost twenty, were grouped into a subclass called allopumiliotoxins. Finally in 1978, extracts from 750 skins of an Ecuadoran dendrobatid frog, Epipedobates tricolor, yielded some 21 mg of pumiliotoxin 251D.This was unexpected, the extracts having been obtained in hopes of isolating and analyzing a trace alkaloid with analgetic activity, detected in early extracts from seven of these frogs (see Epibatidine below). Most of the pumiliotoxin 251D had apparently been lost during evaporations in the earlier fractionation. The hydrochloride of pumiliotoxin 251D was obtained crystalline and X-ray analysis revealed the structure 5 (16). A reinterpretation of the mass and NMR spectra in light of the structure and spectra of 251D allowed structures to be advanced for many of the pumiliotoxins, which have been confirmed and refined by derivatization, degradation, and synthesis (see Ref 2). NMR analyses on allopumiliotoxins isolated from extracts of 1080 skins of Dendrobates pumilio defined structures of several alkaloids of this subclass (I 7). Structures for pumiliotoxin 251D, pumiliotoxin A (307A), pumiliotoxin B (323A), allopumiliotoxin 267A, and the allopumiliotoxin 339A are shown in 5-9 respectively. All are relatively common in dendrobatid frogs. Structures of many of the other 50 pumiliotoxins and allopumiliotoxins are based only on GC-mass spectral and GCl T I R spectral data. The latter technique has proven an invaluable complement to GC-mass spectrometry in defining the structures of alkaloids that

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are often found in skin extracts in small amounts in complex mixtures consisting of dozens of alkaloids. Pumiliotoxins/allopumiliotoxinshave characteristic Bohlmann bands and “fingerprint” regions in F l J R spectra. Pumiliotoxins and allopumiliotoxins are the most widely distributed of all alkaloids found in amphibian skin. Most are CI6-or C19-compoundsand have isoprene units in their structures. They have cardiotonic and myotonic activity apparently due to enhancing sodium channel function (see Ref. 2 ) .

OH

cH3

251D

307A

323A

267A

339A

CH, 267C

“‘OH

10

In frogs of the neotropical family Dendrobatidae, the pumiliotoxins and/ or allopumiliotoxins are major alkaloids in most species from the genera Dendrobates, Epipedobates, and Minyobates, while being absent or trace alkaloids in species of the genus Phyllobares. They are major alkaloids in a dendrobatid frog. Dendrobates auratus, introduced into Hawaii in 1932 (18). In 1984, the first report of pumiliotoxins/allopumiliotoxinsin skin extracts from nondendrobatid frogs and toads appeared (29). Indeed, this report was the first to demonstrate that “dendrobatid alkaloids” occurred in nondendrobatid amphibians. Pumiliotoxins/allopumiliotoxinsoccur in all species of frogs as yet examined of the genus Pseudophryne of the endemic Australian family Myobatrachidae (20,21) and in all species of frogs as yet examined from the genus Manteffaof the endemic Madagascan subfamily Mantellinae (22,23). In toads of the South American genus Melanophryniscus of the family Bufonidae, a pumiliotoxin 267C (lo), which was at that time unknown in dendrobatid species, was discovered and its structure defined by NMR spectroscopy (19). It also occurred in the Australian

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Pseudophryne and the Madagascan Mantella. Pumiliotoxins/allopumiliotoxins also occur in the two species of toads of the genus Melanophryniscus that have been examined (24). Pumiliotoxins/allopumiliotoxinshave not been detected in skin extracts of some 70 other amphibian genera, nor for that matter have any alkaloids been detected in any of these other genera. The wide distribution of pumiliotoxins/allopumiliotoxins,coupled with their absence in captive-raised dendrobatid frogs (11,18,25), suggests that any putative dietary source for such alkaloids must be widely distributed over the world in both tropical and subtropical regions. The nature of such a small dietary arthropod is unknown. Pumiliotoxins/allopumiliotoxinsare as yet unknown in Nature, save in amphibian skin from four of the six genera of neotropical dendrobatid frogs, in skins from one genus of Madagascan mantelline frogs, in skins from one genus of Australian myobatrachid frogs, and in skins from one genus of bufonid toads. As yet only dendrobatid frogs have been demonstrated to have the ability to accumulate pumiliotoxins (and other alkaloids), as provided in their diet, into their skin (11).

B. HOMOPUMILIOTOXINS Homopumiliotoxins are closely related in structure to the pumiliotoxins, but have a quinolizidine ring system rather than an indolizidine system. Only one, namely homopumiliotoxin 2236 (ll),has been isolated in sufficient quantities of NMR spectral analysis (26), since, unlike the pumiliotoxins/ allopumiliotoxins, the homopumiliotoxins have been minor or trace alkaloids when detected in skin extracts. Thus, for the homopumiliotoxins, as for many amphibian alkaloids present as minor or trace components in complex mixtures of alkaloids, diagnostic features of GC-mass spectra and GC-FTIR spectra have been critical to the classification and postulation of structures. The homopumiliotoxins exhibit diagnostic prominent mass spectral ions at d z 180 (CI1Hl8NO+)and d z 84 (C5HloN+)and have a characteristic FTIR pattern, particularly in the Bohlmann band region (24). There are now about 15 alkaloids that can be assigned to the homopumiliotoxin subclass.

223G

U 11

Homopumiliotoxins, like the pumiliotoxins/allopumiliotoxins,are known in nature only from amphibian skin, but, unlike the pumiliotoxins/allopumi-

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liotoxins, they occur only sporadically and at low levels. Homopumiliotoxins have been detected in certain dendrobatid species and in some species of Mantella and Melanophryniscus, but not in Pseudophryne. C. OTHER PUMILIOTOXIN-CLASS ALKALOIDS The existence of three other subclasses of pumiliotoxin alkaloids have been proposed, namely 6,10-dihydropumiliotoxins (22), dehydrohomopumiliotoxins (22), and 8-deoxypumiliotoxins (27). Alkaloids of the first two putative subclasses have been detected only in Madagascan frogs of the genus Mantella, and the proposed structures must be considered very tentative until sufficient material is obtained for NMR spectral analysis. The 8deoxypumiliotoxin 251H (12) was recently isolated from extracts obtained in 1976 in quantities (ca. 1 mg) sufficient for NMR spectral analysis (27). Such 8-deoxypumiliotoxins exhibit diagnostic prominent mass spectral ions at d z 150 (CloH16N+) and d z 70 (C4HsN+).As yet, 8-deoxypumiliotoxins have been detected only in neotropical dendrobatid frogs and in Madagascan frogs of the genus Mantella.

12

Another apparent pumiliotoxin subclass has recently been detected as trace alkaloids in extracts of Mantella (23). The prominent mass spectral ions are at d z 166 (CloHI6NO+)and d z 84 (C5H10N+).Tentative structures have not been proposed and for convenience this group of alkaloids was referred to as “isopumiliotoxins” (23).

V. Histrionicotoxins

During the initial fieldwork in the 1960s on the dendrobatid poison-dart frog Phyllobates aurotaenia, analysis of extracts from a few skins of a microsympatric dendrobatid frog, Dendrobates histrionicus, had revealed the presence of C19-alkaloids with prominent fragment ions at d z 218 (Cl4HZ0NO+) and d z 96 (C6HloN+).In search of a source for such alkaloids,

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Myers and Daly in 1970 targeted a population of Dendrobates histrionicus in southwestern Colombia known to be extremely abundant. Extracts from 400 skins afforded two major alkaloids, histrionicotoxin (13)and isodihydrohistrionicotoxin (14),which were crystallized as hydrochloride salts and the novel structures revealed by X-ray analyses (28). Subsequently, from additional extracts, further histrionicotoxins were isolated and their structures determined by NMR spectral analysis. Most were C19-compounds, differing only in the degree and nature of unsaturation in the side chains, but some C17-compounds,such as 259A (15),and some CI5-compounds, such as 235A (16), were also found. All show a major fragment ion at d z 96 (C6HION+) and most show a significant fragment ion corresponding to a-cleavage of the side chain next to the nitrogen. GC-FTIR spectra of histrionicotoxins and their phenylboronate derivatives provided valuable data, particularly with respect to the nature of the unsaturation in side chains (29). A total of 16 histrionicotoxins have been detected from dendrobatid frogs. Unlike the pumiliotoxins/allopumiliotoxins,the histrionicotoxins could arise from a precursor with a linear carbon skeleton, The toxin designation proved inappropriate, since histrionicotoxins have relatively low toxicity. They are potent noncompetitive blockers of nicotinic receptor-channels and as such have proved to be useful tools, both in natural and radiolabeled form (see Ref. I). Many of the other amphibian alkaloids, including the gephyrotoxins, the decahydroquinolines, indolizidines, pyrrolidines, and piperidines also are noncompetitive blockers of nicotinic receptor-channels (see Ref. I).

Histrionicotoxins have been detected in Nature only in dendrobatid frogs. They have not been detected in the tiny dendrobatid frogs of the genus Minyobates, nor in alkaloid-containing frogs of the nondendrobatid genera

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Pseudophryne, Mantella, and Melanophryniscus. Interestingly, histrionicotoxins do not occur in the dendrobatid frog Dendrbbates auratus introduced into Hawaii in 1932, even though the founding population of that frog in Panama contains significant levels of histrionicotoxins (18). In some dendrobatid frogs, a set of highly unsaturated C19-histrionicotoxinsoccur together, while others contain nearly exclusively the C19-alkaloidoctahydrohistrionicotoxin, and in a few the C19-histrionicotoxinsare replaced by CIS-and CI7-histrionicotoxins. The lack of any histrionicotoxins was one consideration in defining the Colombian species Dendrobates lehmanni as a new species, since all populations of Dendrobates histrionicus, among which Dendrobates lehmanni was at that time included, had histrionicotoxins as prominent alkaloids (30). Dendrobatid frogs have the ability to accumulate into their skin histrionicotoxins provided to them in the diet (11).Feeding leaf litter insects from a Panamanian site at which the dendrobatid frog Dendrobates auratus occurs did result in low levels of histrionicotoxins in some of the frogs raised in terraria on such insects (25). It seems likely that an arthropod source of the histrionicotoxins occurs only in the New World tropics, based on the absence of histrionicotoxins in Old World alkaloid-containing frogs, in New World semitropical toads, and in the dendrobatid frog introduced into Hawaii.

VI. Gephyrotoxins

Another alkaloid isolated along with the histrionicotoxins from the Colombian dendrobatid frog Dendrobates histrionicus proved on X-ray analysis of a crystal of the hydrobromide salt to be a tricyclic alkaloid, which was named gephyrotoxin (17) (31). There are questions concerning the absolute configuration of gephyrotoxin (see Refs. 1,3). Only two gephyrotoxins have been detected.

/

HO

17

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DALY

Gephyrotoxins are known in Nature only from a very few dendrobatid species of the genus Dendrobates, where they always occur along with a set of CI9-histrionicotoxins. It seems likely that whatever small arthropods are the source of the histrionicotoxins, they will also prove to be the source of gephyrotoxins.

VII. Decahydroquinolines The initial studies on Dendrobates purnilio in the late 1960s had resulted in the isolation of three alkaloids, two of which, pumilotoxins A and B, were the first members of the pumiliotoxin class to be characterized. The third was a decahydroquinoline cis-195A (18),whose structure was determined by X-ray crystallography (32).This alkaloid was at that time referred to as pumiliotoxin C, but that name proved unsatisfactory on two accounts; first, the alkaloid is relatively nontoxic, and second, the name made for confusion with pumiliotoxins A and B, which are toxic and which are structurally unrelated to the decahydroquinoline class of amphibian alkaloids.

cis-195A

H

18

The mass spectra of such 2,5-disubstituted decahydroquinolines are dominated by a-cleavage resulting in loss of the 2-substituent. In some cases, a loss of 43 amu, corresponding to loss of carbons 6,7, and 8 of the alicyclic ring is significant (unpublished results): Mass spectral data, often in conjunction with FTIR spectral analysis, suggests the presence of 30-40 decahydroquinolines to be present in amphibian skin extracts. All could be derived from a precursor with a linear carbon skeleton. The FTIR spectra provide information as to the relative configurations at carbons 2, 4a, and 8a, but not at carbon 5 (33,34).The structures of four representative decahydroquinolines isolated from extracts of dendrobatid frog skin and analyzed by NMR are shown in 19-22. Both cis- and trans-fused decahydroquinolines

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occur. The absolute configurations of cis-195A (18) and trans-219A (20) are as shown, as determined by X-ray analyses (32,35).

19

&& I

21

,r&JJ H y

N

I H

H

cis-243A

trans-269AB

20

22

The major decahydroquinolines isolated from dendrobatid frogs include CI3-(cis-l95A), C15- (cis- and trans-219A), c 1 7 - (cis- and trans-243A), and C19- (trans-269AB) compounds. The CI5-,c17-, and C19-decahydroquinolines, like the C15-,C17-, and C19-histrionicotoxins,have highly unsaturated side chains. 2,5-Disubstituted decahydroquinolines, such as cis-l95A, are as yet unreported in Nature except from amphibian skin. Decahydroquinolines occur in a wide range of dendrobatid frogs, often together with histrionicotoxins. Like the histrionicotoxins, decahydroquinolines appear to be absent or virtually absent in the tiny dendrobatid frogs of the genus Minyobates. Decahydroquinolines do occur in frogs of the Madagascan genus Mantella and in bufonid toads of the genus Melanophryniscus. Neither of these genera have histrionicotoxins. Decahydroquinolines are not present in myobatrachid frogs of the genus Pseudophryne. Decahydroquinolines are readily taken up into the skin of the dendrobatid frog Dendrobates auratus when provided in the diet (IZ). Low levels of the C19-decahydroquinoline 269AB were found in dendrobatid frogs raised on leaf-litter insects in Panama (25).Interestingly, the decahydroquinoline cis-195A was a major alkaloid in skin extracts from frogs at the leaf-litter site, but was not detected in frogs raised on insects collected using Berlese funnels from leaf-litter at the same site.

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VIII. Cyclopenta[b]quinoliidines Structure elucidation of the batrachotoxins, pumiliotoxins, histrionicotoxins, gephyrotoxins, and decahydroquinolines were facilitated by the ease with which large numbers of frog skins could be obtained in the 1960s, 1970s, and early 1980s. Since that time, international conservation efforts have made it impossible in most cases for scientists to obtain permits to collect more than a limited number of frogs, in spite of the fact that many dendrobatid frogs are incredibly abundant. Thus, the isolation of quantities of the remaining minor and trace alkaloids for NMR spectral analysis became difficult in the 1980s and characterization of such alkaloids had to rely almost wholly on mass, FTIR spectral, and microchemical (perhydrogenation, methylation, acylation, and boronate formation) analyses. Fortunately, the sensitivity and analytical potential of NMR increased remarkably during the 1980s,and submilligram quantities of alkaloids have now become amenable to structure elucidation. A tricyclic alkaloid, detected in a new species of a Colombian dendrobatid frog, Minyobates bornbetes, was one such alkaloid, whose structure elucidation awaited the advent of more sensitive and more powerful NMR instrumentation and techniques. The alkaloid and its congeners were unusual in exhibiting base peaks of an odd mass, for example, at d z 111 (Cl7Hl3N+)for the major alkaloid 251F. From 100 skins of frogs collected in 1983, sufficient alkaloid 251F (ca 300 pg) was isolated in 1990 for NMR spectral analyses (36). The major tricyclic alkaloid 251F (23)proved to be a cyclopenta[b]quinolizidine. Tentative structures for eight other such alkaloids were deduced from mass and FTIR spectral data. The proposed structure of 251F has now been confirmed by synthesis (37).

251F

23

Such cyclopenta[b]quinolizidines are known in Nature from only two populations of the tiny dendrobatid frog Minyobates bornbetes. If cyclopenta[b]quinolizidines come from a dietary source, then the lack of such alkaloids in other dendrobatid frogs is remarkable.

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IX. Epibatidine An interesting alkaloid, present in trace amounts in skin extracts of the Ecuadoran dendrobatid frog Epipedobates tricolor, was discovered in the late 1970s because of its striking biological effects. Thus, injection of small amounts of extract obtained in 1974 from seven frogs elicited a Straub-tail reaction in mice, a reaction normally diagnostic for an opioid-class alkaloid, such as morphine. A further 750 skins were obtained in 1976 and using a Straub-tail assay with chromatographic fractionation, the alkaloid responsible proved to be a trace constituent containing a chlorine atom and having a probable empirical formula of CllCI3N2C1.The quantity of this alkaloid, 20W210,isolated (ca 700 pg) was insufficient in the early 1980s to obtain definitive NMR data. The purified alkaloid, later to be named epibatidine, was shown to have analgetic activity in mice some 200-fold greater than that of morphine, but, unlike morphine, the activity was not blocked by naloxone. By serendipity these extracts provided sufficient pumiliotoxin 251D and 8-deoxypumiliotoxin 25lH to define structures for two other classes of “dendrobatid alkaloids” (16,27).Further extracts of the Ecuadoran frog obtained in 1979 and in 1987 had much lower amounts of the analgetic alkaloid; a major disappointment. International restrictions placed late in 1987 prevented further large-scale collecting of any dendrobatid frogs in spite of the incredible abundance of many of the species. Another disappointment was the discovery that the frog Epipedobates tricolor raised in captivity had no skin alkaloids. Thus the remaining sample of purified epibatidine represented the only means to ever discover the structure of the analgetic alkaloid. In 1990, it was determined to undertake the NMR analysis. In order to avoid further chromatographic losses, the combined impure sample of epibatidine was converted to an N-acetyl derivative and small amounts of contaminating tertiary amines (pumiliotoxins) were removed by acid extraction. NMR analysis of the N-acetyl derivative revealed the structure of epibatidine to be that of the chloropyridyl azabicycloheptane 24 (38).Synthetic material was later used to elucidate the basis for the analgetic activity. Epibatidine proved to be an extremely potent nicotinic agonist (39) and is now the focus of active investigation in many laboratories.

24

156

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Epibatidine is unknown in Nature except from Ecuadoran dendrobatid frogs of the genus Epipedobates. The levels vary greatly among the four species in which it has been detected and even in different populations of the source species Epipedobates tricolor. A dietary source is suspected, but there is no clue as to what small arthropod might be involved. It is possible that epibatidine, like its structural relative, nicotine, has a plant origin.

X. Pseudophrynamines The GC-mass spectral analysis of a single skin of an Australian myobatrachid frog, Pseudophryne semimarmoruta, in the early 1980s revealed the presence of two alkaloids, one of which was a pumiliotoxin and the other an allopumiliotoxin (29).This result prompted the collection of additional specimens and species of the genus Pseudophryne in 1987. Such extracts yielded pumiliotoxins/allopumiliotoxins,and also a new class of alkaloids not seen in dendrobatid frogs. NMR analysis revealed the structures of two major alkaloids as pseudophrynamine A (25) and pseudophrynaminol (26) (20,21). The former could be converted by methanolysis into pseudophrynaminol and an ester, which was identical to the alkaloid 286A (27), also isolated from the Pseudophryne frogs. 0

I H

t CH3

I

CH3

I

H

H37cH20H 25

I

I

H

CH3

H

CH3

286A

26

27

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Pseudophrynamines are as yet unknown in Nature except in the skin of nocturnal frogs of the Australian myobatrachid genus Pseudophryne, and have not been detected in any of the alkaloid-containing frogs that are diurnal, namely, the dendrobatid frogs, the mantelline frogs, and the bufonid toads of the genus Melunophryniscus. Whether the presence of pseudophrynamines is associated with a nocturnal prey item or whether the source is endemic to Australia remain unsolved questions.

XI. Pyrrolizidine Oximes The Panamanian dendrobatid frog Dendrobutes pumilio provided not only pumiliotoxins A and B and decahydroquinoline cis-l95A, but from extracts obtained in 1983,a set of three new tricyclic alkaloids. The originally proposed tentative amidine structures of these new alkaloids (26) were later concluded to be incorrect based upon GC-FTIR spectral analysis. At that point further NMR spectral analyses delineated the structures as being those of pyrrolizidine oximes (40), of which the predominate member in dendrobatid skin extracts is the 0-methyloxime 236 (28).The corresponding oxime 222 and two hydroxy analogs of 236 also occur in dendrobatid frogs. The structure of 236 has been confirmed by synthesis (41).

236

28

Pyrrolizidine oximes are known in Nature only from amphibian skin. They are major alkaloids in recent extracts from several dendrobatid species. Minor or trace amounts of pyrrolizidine oximes have been detected in a myobatrachid frog, in mantelline frogs, and in a bufonid toad. Although oximes are unknown in arthropods, a close relative of the pyrrolizidine oximes, nitropolyzonamine (29), is a constituent in defensive secretions of a millipede (42). Nitropolyzonamine and another millipede alkaloid polyzonimine (30) have been detected as trace alkaloids in some dendrobatid extracts (unpublished results).

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30

29

It seems highly likely that the pyrrolizidine oximes in dendrobatid frog skin originate from small neotropical millipedes. Indeed, after being raised on Panamanian leaf-litter arthropods, the major alkaloid in the dendrobatid frog Dendrobates auratus was the pyrrolizidine oxime 236 (25).The emergence of pyrrolizidine oximes as significant skin alkaloids in certain populations of Panamanian Dendrobates pumilio and Dendrobates auratus may reflect an increased availability of alkaloid-containing millipedes as the habitat has changed from 1970 to the present time.

XII. Coccinellines

The tricyclic alkaloid precoccinelline (31) has now been identified as a minor component by GC-mass and GC-FTIR spectral analyses in extracts from a number of species of dendrobatid frogs, where it has been given the code number 193C (I). Coccinellines, including precoccinelline, are well known as alkaloids in ladybug and other beetles (43), where they presumably serve as defensive substances.

31

Some of the other tricyclic alkaloids, detected in extracts of amphibian skin, are probably related in structure to the coccinellines. One of these, alkaloid 205B, was isolated from extracts of the Panamanian dendrobatid frog Dendrobates pumilio and a tentative structure was proposed (26). Further NMR and FTIR spectral analyses of 205B are in progress.

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Precoccinelline occurs in several dendrobatid frogs and in a bufonid toad of the genus Mefanophryniscus (24). In a population of the dendrobatid frog Dendrobares auratus introduced into Hawaii, it is a major alkaloid (18).It seems almost certain that precoccinelline and perhaps other similar tricyclic alkaloids have their origin in dendrobatid frog skin from small dietary beetles. Indeed, precoccinelline was a significant alkaloid in dendrobatid frogs raised on arthropods from leaf-litter in Panama (25). XIII. Bicyclic “Izidine” Alkaloids

A wide range of simple bicyclic alkaloids, for which we might use the term “izidine” alkaloids, have been found in skin extracts from dendrobatid frogs, mantelline frogs, and bufonid toads of the genus Melanophryniscus ( I ) . They are absent in myobatrachid frogs of the genus Pseudophryne. The “izidine” alkaloids detected in amphibian skin include 3,5-disubstituted pyrrolizidines, 3,5-disubstituted indolizidines, 5,8-disubstituted indolizidines, 5,6,8-trisubstituted indolizidines, and 1,4-disubstituted quinolizidines. Many probably originate from dietary ants. A. PYRROLIZIDINES A variety of 3,5-disubstituted pyrrolizidines have been identified in extracts from’dendrobatid and mantellid frogs and in bufonid toads of the genus Mefanophryniscus ( I ) . Identification has been based on GC-mass and GC-ITIR spectral analyses and in some instances comparison to synthetic material. Major mass spectral fragment ions are due to a-cleavage and the FTIR spectra show virtually no Bohlmann bands. Structures of pyrrolizidine cis-223H (32) and cis- and rrans-251K (33) and (34)are shown. The absolute configurations are not known. About 15 alkaloids detected in extracts from amphibian skin, appear to be 3,5-disubstituted pyrrolizidines; some are present as two diastereomers. 3,5-Disubstituted pyrrolizidines are present as venom constituents in myrmicine ants, and indeed cis-223H proved identical on GC analysis with a (5Z,8E)-3-heptyl-5-methylpyrrolizidinefrom the thief ant (Solenopsis sp.) (44), whereas trans-25lK was identical on GC analysis with a (5E,8E)3-butyl-5-hexylpyrrolizidinefrom a Venezuelan ant (Megalomyrmex modestus) (45). It seems highly likely that all of the 3,5-disubstituted pyrrolizidine alkaloids in frog skin owe their presence to a diet of ants containing such alkaloids. Dendrobatid frogs, in particular, are known to consume large numbers of ants and have been referred to as ant specialists (46).

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DALY

cis-223H

R/ 32

cis-251K

trans-251K

33

34

B. 3,5-DISUBSTITUTED INDOLIZIDINES In 1978,3,5-disubstituted indolizidine structures were proposed for three alkaloids found in the dendrobatid frog Dendrobates histrionicus (15).The structures were based on mass spectral data and biosynthetic speculation. The structure of one, namely 223AB ( 3 9 , was subsequently confirmed by demonstrating its identity with synthetic (5E,9E)-3-butyl-5-propylindolizidine (47). Since that time, three of the four diastereomers of indolizidine 223AB have been detected in amphibian skin (see Ref. I). Remarkably, while the sole diastereomer of 223AB in a Colombian Dendrobates histrionicus was the 5E,9E isomer 35 (48), the sole diastereomer in Panamanian Dendrobates speciosus proved to be the 52,92 isomer 36 (I).

qJ

v=

5E,9E-223AB

qJ

v: 52,92-223AB

35

[oH5E.9E;!9AB

36

woH @J WJ CH3

v=

6H3

6

5E,9E-239CD

5E,9E-l95B

5Z,9Z-195 B

38

39

40

About 15 alkaloids detected in extracts of amphibian skin appear to be 3,5-disubstituted indolizidines, some of which are represented by as many

4. THE

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as four diastereomers. Structures of many are based only on diagnostic features of the mass and FTIR spectra. The major fragment ions are due to loss of either one or the other side chain. Loss of methyl is a relatively minor event. A fragment ion at d z 124 is often present due to a McLafferty rearrangement during cleavage of the second side chain. Bohlmann bands in the FTIR spectra are relatively weak and broad for the 5 2 , 9 2 diastereomer, and are even weaker for the 5E,9E and other diastereomers (24). Certain members of this class of amphibian alkaloids, such as the 223AB isomers (see previous discussion), 5E,9E-239AB (37) 5 E,9E-239CD (38) and 5E,9E-195B (39) have been isolated and their relative configuration defined by NMR spectral analysis (35,48). The absolute configurations of 35, 37, 38, and 39 are also known based on comparison with synthetic enantiomers (see Ref. I). The presence of simple 3- or 5-substituted indolizidines in alkaloid fractions from amphibian skin has been proposed, based solely on mass spectral analysis (1). However, the compounds could instead be 3,5-disubstituted pyrrolizidines with one substituent being a methyl group. Unfortunately, the original alkaloid samples have proven to be insufficient in amount for GC-FTIR spectral analysis. 3,5-Disubstituted indolizidines represent another alkaloid class present as venom constituents in myrmicine ants. Indeed, monomorine I from ants of the genera Monornoriurn and Solenopsis (49,50) is diastereomeric to the indolizidine 5E,9E-l95B (39) found widely in dendrobatid frogs. All four diastereomers of 195B, including the diastereomer 52,92-195B (40) identical by GC analysis with monomorine I, were present in extracts from a bufonid toad of the genus Melanophryniscus (24). Recently, both monomorine I and the amphibian diastereomer 5E,9E-l95B were found in a Puerto Rican myrmicine ant (51). Dendrobatid frogs (Dendrobates auratus) fed Pharoah’s ant (Monornorium pharaonis) efficiently accumulated the ant alkaloid monomorine I and two minor 3,5-disubstituted indolizidines into their skin (12). Indolizidine 5E,9E-l95B was a major alkaloid in a dendrobatid frog (Dendrobates aurutus) raised in outside cages in Hawaii (18). It therefore seems likely that the 3,5-disubstituted indolizidines found in amphibian skin are the result of sequestration from myrmicine ants. 3 3 Disubstituted indolizidines occur in dendrobatid frogs of the genera Dendrobates and Phyllobates, but apparently not in the genera Epipedobates and Minyobates ( I ) . They occur in mantelline frogs and in bufonid toads of the genus Melanophryniscus. c . 5,8-DISUBSTITUTED INDOLIZIDINES Another new class of amphibian alkaloids was established on the basis of NMR analysis of a minor alkaloid 207A isolated in the mid-1980s from

162

DALY

258 skins of the Panamanian montane dendrobatid frog Dendrobates speciothe 5-substituted-8-methyl indolizidine 207A (41) is shown, as are the structures of other members of this class, namely 203A,205A,235B', and 235B"(42-45)that have been isolated in quantities sufficient for NMR analysis (33,35,52, see also Ref. I ) . Absolute configurations of 41-44 are known, based on comparison to synthetic enantiomers (see Ref. I ) . About 40 alkaloids detected in extracts of amphibian skin appear to be 5,8-disubstituted indolizidines, based on diagnostic features of the mass and FTIR spectra. The 5-substituted-8-methylindolizidines have a base peak at m l z 138 ( G H I 6 N + )and a diagnostic fragment at m l z 96 (C6HloNt),arising from the m/z 138 ion by a retro-Diels-Alder process. Another group of 5,8-disubstituted indolizidines appears to have 8-substituents other than methyl and, therefore, yield base peaks of d z 152 or higher, dependent on the 8-substituent. All yield the diagnostic retro-Diels-Alder ion at m l z 96. One such alkaloid is the relatively widely occurring indolizidine 217B (46).A sharp and intense band at about 2785 cm-' in the F U R is diagnostic for the 5,8-disubstituted indolizidine class, virtually all of which have H-5 and H-9 in a cis-relationship (22,2433). The 5,8-disubstituted indolizidines and the 5,6,8-trisubstituted indolizidines (see following section) appear to be unique in Nature to amphibian skin and have not been reported from an arthropod. They are very widespread in dendrobatid frogs and have also been found in mantelline frogs and bufonid toads of the genus Melanophryniscus (I). sus (52).The structure of

207A

203A

205A

41

42

43

2358'

2358"

2178

44

45

46

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NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS

163

D. 5,6,8-TRISUBSTITUTED INDOLIZIDINES There now appear to be a class of 5,6,8-trisubstituted indolizidines exemplified by the relatively widespread dendrobatid alkaloid 223A (47).NMR spectral analysis indicates the structure shown (53).The mass spectral base peak, due to a-cleavage, is at d z 180 (C12H22N+) and there is a further retro-Diels-Alder fragment ion at m/z 124 (C8HI4N+).The FTIR spectrum exhibits, as in the case of the 5,8-disubstituted indolizidines, a sharp, intense Bohlmann band at about 2785 cm-'. There are some half-dozen other bicyclic alkaloids from amphibian skin that appear likely to be 5,6,8-trisubstituted indolizidines, since they exhibit a base peak plus the diagnostic retro-Diels-Alder fragment ion at d z 124.

223A

47

E. QUINOLIZIDINES The existence of a quinolizidine class of alkaloids in extracts of amphibian skin has been.proposed based on mass and FTIR spectral data (22,24). The simplest members were proposed to be 4-substituted l-methylquinolizidines, as exemplified by 217A (48) and 231A (49),which show a mass spectral base peak corresponding to a-cleavage at d z 152 (CloH18N+) for 217A,and at d z 166 (ClIH20N+) for 231A,along with a retro-Diels-Alder fragment at d z 110 (C7HI2N+) that was proposed to be diagnostic for this class of amphibian alkaloids. Alkaloids of this proposed 1,4-disubstituted quinolizidine class show a somewhat broader and less intense Bohlmann band (22,24) than do the 5,8-disubstituted and 5,6,8-trisubstituted indolizidines. Over 20 alkaloids detected in extracts from amphibian skin were tentatively assigned to a 1,4-disubstituted quinolizidine class, based on mass spectra and in a few cases on mass and FTIR spectra (I).However, it now seems likely that some may prove to be 4,6-disubstituted quinolizidines, some may prove to be 5,6,8-trisubstituted indolizidines, and some may even prove to contain another bicyclic ring system. Isolation and NMR analysis of alkaloids that have been tentatively assigned to the quinolizidine class have a high priority in this research area. The alkaloids that are tentatively proposed as 1,4-disubstituted quinolizidines are relatively common in dendrobatid frogs, in mantelline frogs, where

164

DALY

217A

231A

48

49

217A and 231A are major alkaloids in one species (22,23),and in bufonid toads of the genus Melanophryniscus (24). Such alkaloids have not been reported in any arthropod.

XIV. Monocyclic Alkaloids 2J-Disubstituted pyrrolidines and 2,6-disubstituted piperidines occur in amphibian skin, but usually only as trace constituents (I). However, a pyrrolidine 197B (50) was a major alkaloid component in skin extracts of a Colombian dendrobatid frog, Dendrobates histrionicus and was identified (48).Identification and a later deteras a trans-2-butyl-5-pentylpyrrolidine mination of absolute configuration were by comparison to synthetic samples (see Ref. I). The piperidine 241D (51) was a major alkaloid component in skins extracts of the Panamanian dendrobatid frog, Dendrobates speciosus (52). The structure was defined by NMR spectral analysis and later confirmed by synthesis (54). The other 2,5-disubstituted pyrrolidines, about five in total, and the other 2,6-disubstituted piperidines, about 18 in total, in skin extracts have been defined based only on diagnostic features of mass and FTIR spectra. In the case of the piperidines, both cis- and transisomers are often present together in skin extracts from dendrobatid frogs, and can be distinguished by Bohlmann bands in the FTIR spectra (34). Cis-and trans-pyrrolidines are easily distinguished by FIYR spectra after N-methylation (34).

trans-1978

241D

50

51

4.

THE NATURE A N D ORIGIN OF AMPHIBIAN ALKALOIDS

165

2,5-Disubstituted pyrrolidines and 2,6-disubstituted piperidines are well known as venom constituents in myrmicine ants (55,56). The ant pyrrolidines are all trans, as is the case for the pyrrolidine 197B from amphibian skin, while the ant piperidines are usually cis/trans mixtures, as often is the case of piperdines detected in amphibian skin extracts. Many of the ant piperidines have a 2-methyl substituent and many of the piperidines detected in amphibian skin appear to have a 2-methyl substituent. The pyrrolidinedpiperidines seem somewhat restricted in their amphibian distribution, being most common in dendrobatid species of the genus Dendrobates, and are rare or absent in other dendrobatid genera and in other alkaloid-bearing amphibians (Mantella, Melanophryniscus, Pseudophryne). While it seems highly likely that such pyrrolidines and piperidines, when found in amphibian skin, originate from dietary ants, it should be noted that a Costa Rican dendrobatid frog (Dendrobates auratus) did not accumulate trans-2-heptyl5-hexenylpyrrolidine when fed Pharoah’s ants containing that pyrrolidine, nor did Dendrobates auratus accumulate a piperidine in a mixture of alkaloids provided on dusted fruit flies ( 2 2 ) .

XV. Summary and Prospects During the past 30 years, a coordinated program of field work, isolation, structure elucidation, synthesis, and pharmacological evaluation has led to detection of a total of over 400 alkaloids from amphibian skin, several of which have become valuable pharmacological tools. The research has led to the discovery of nearly a dozen new species of frogs, to the introduction of unique new alkaloid structures, and most recently to evidence that probably none of these alkaloids are produced by the frogs themselves, but instead are taken up from dietary arthropods, including ants, beetles, and millipedes. Such dietary alkaloids appear to be sequestered unchanged into secretory skin glands of the frog, where they then serve as a chemical defense for their new host. Ironically, such alkaloids apparently failed to protect the arthropods, and, indeed, may cause the frogs to target as prey such alkaloid-containing arthropods. The research has changed over the years; initially large-scale collections of amphibians were permitted, and chromatographic isolation of major alkaloid constituents on the 10-50 mg scale was followed by NMR analysis, and often by crystallization and X-ray crystallography. With such paradigms, structures for the major classes of amphibian skin alkaloids, namely the batrachotoxins, the pumiliotoxins, allopumiliotoxins, homopumiliotoxins, histrionicotoxins, gephyrotoxins, and decahydroquinolines were

166

DALY

lished during the 1970s. The challenge remained for the 1980s of the minor and trace alkaloids, where the amounts that could be isolated even from hundreds of frog skins often was in the submilligram scale. But during the 1980s, the sensitivity and power of 2D-NMR spectroscopy greatly increased and structures of 5,8-disubstituted indolizidines, epibatidine, a cyclopentyl [blquinolizidine, the pyrrolizidine oximes, and pseudophrynamines were established with quantities insufficient to attempt crystallization. During the late 1980s and early 1990s, sensitive GC-FTIR analysis was used to complement the information gleaned from mass spectral analysis, the latter also providing deuterium exchange data. Microchemical perhydrogenation, methylation, acylation, and phenylboronation provided further information on the nature and position of various structural entities. With such composite data almost three-quarters of the over 400 alkaloids detected in amphibian skin extracts can be assigned or tentatively assigned to one of some nearly 20 structural classes. HPLC separations and NMR analyses of such alkaloids on the submilligram scale continue in order to verify structures. Syntheses have in the past confirmed some structures and undoubtedly will continue to confirm or dismiss further structures. A current major problem is that permission to collect more than a token few frogs is now nearly impossible. Most of the blame for this lies with the International Commission for Trade in Endangered Species, which in 1987, in violation of its own guidelines, placed all of the dendrobatid frogs on a threatened list in spite of evidence to the contrary for most of the dendrobatid species. Thus, many of the trace alkaloids, which might be isolated in sufficient quantity for NMR analysis from 100 or more skins, will never be available at that level because of limitations imposed on scientific collecting. The demonstration that dendrobatid frogs raised in captivity on fruit flies and crickets have no alkaloids in the skin, and that such frogs readily sequester alkaloids provided to them in their diet unchanged into skin, strongly suggests that, with the exception of the samandarines, the more than 400 alkaloids that have been demonstrated in amphibian skin come from dietary sources. A challenge for further research is the identification of what tiny arthropods are the source of the batrachotoxins, the pumiliotoxins, homopumiliotoxins and related congeners, the histrionicotoxins, the gephyrotoxins, the decahydroquinolines, the cyclopenta[b]quinoliidines, epibatidine, and the literally hundreds of trace alkaloids whose structures are unknown or tentative. Such tiny arthropods might be expected to be the source of a treasure-trove of alkaloids that presumably could be obtained in quantities sufficient for structure elucidation and pharmacological evaluation. The amphibians in question eat only small mobile creatures, being cued to feed by movement, but there is the possibility that the dietary trail will lead through small arthropods to alkaloid-containing plants eaten by

4. THE NATURE AND

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167

such arthropods. Plant alkaloids have been identified as minor alkaloid components in extracts from 1000 skins of the dendrobatid frog Phyllobates terribilis (9). These were the indole alkaloids chimonanthine and calycanthine and a dipyridylpiperidine, noranabasamine. Morphine has been detected as a trace alkaloid in the skin of a bufonid toad, Bufo marinus (57). Of the over 400 alkaloids detected in amphibian skin, about 60 are likely to be ant-derived alkaloids, namely the 3,5-disubstituted pyrrolizidines, the 3,5-disubstituted indolizidines, the 2,5-disubstituted pyrrolidines, and the 2,6-disubstituted piperidines. Precoccinelline undoubtedly originates from small beetles, as probably do some dozen other tricyclic alkaloids. The pyrrolizidine oximes, nitropolyzonamine, and polyzonimine undoubtedly come from small millipedes. This leaves over 300 alkaloids of amphibian skin with an unknown biological source, certainly a major challenge for further research.

Acknowledgments

The author acknowledges his great debt to all who have contributed so much to the past three decades of research on “amphibian alkaloids.” In particular, I wish to express gratitude to my biologist colleague and mentor in field work, Dr. Charles W. Myers, to the chemists, Drs. Takashi Tokuyama, Thomas F. Spande, and H. Martin Garraffo, who have contributed so much over, the years, to the X-ray crystallographer, Dr. Isabella Karle, who revealed structures of some of these alkaloids, to the pharmacologists, Drs. Edson X. Albuquerque and Fabian Gusovsky, who were instrumental in defining sites of action of many of these alkaloids, and to Dr. Bernard Witkop who started me on this long journey.

References

1. J. W. Daly, H. M. Garraffo, and T. F. Spande, in “The Alkaloids” (G. A. Cordell, ed.), Vol. 43, pp. 185-288. Academic Press, New York, 1993. 2. J. W. Daly, C. W. Myers, and N. Whittaker, Toxicon 25, 1023 (1987). 3. J. W. Daly and T. F. Spande, in “Alkaloids: Chemical and Biological Perspectives” (S. W. Pelletier, ed.), Vol. 4, pp. 1-274, Wiley, New York, 1986. 4. CI. Schopf, Experientia 17,285 (1961). 5. G. Habermehl in “The Alkaloids” (R. H. F. Manske, ed.), Vol. 9, pp. 427-439. Academic Press, New York, 1967. 6. F. Marki and B. Witkop, Experientia 19, 329 (1963). 7. T. Tokuyama, J. Daly, B. Witkop, I. L. Karle, and J. Karle, J. Am. Chem. Soc. 90, 1917 (1968).

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T. Tokuyama, J. Daly, and B. Witkop, J. Am. Chem. Soc. 91,3931 (1969). T. Tokuyama and J. W. Daly, Tetrahedron 39,41 (1983). J. W. Daly, C. W. Myers, J. E. Warnick, and E. X. Albuquerque, Science 208,1383 (1980). J. W. Daly, S. I. Secunda, H. J. Garraffo, T. F. Spande, A. Wisnieski, and J. F. Cover, Jr., Toxicon 32, 657 (1994). 12. J. A. Dumbacher, B. Beehler, H. M. Garraffo, T. F. Spande, and J. W. Daly, Science 258, 799 (1992). 13. C. W. Myers and J. W. Daly, Scient. Am. 248, 120 (1983). 14. J. W. Daly and C. W. Myers, Science 156,970 (1967). 15. J. W. Daly, G. B. Brown, M. Mensah-Dwumah, and C. W. Myers, Toxicon 16,163 (1978). 16. J. W. Daly, T. Tokuyama, T. Fujiwara, R. J. Highet, and I. L. Karle, J. Am. Chem. Soc. 102,830 (1980). 17. T. Tokuyama, J. W. Daly, and R. J. Highet, Tetrahedron 40,1183 (1984). 18. J. W. Daly, S. I. Secunda, H. M. Garraffo, T. F. Spande, A. Wisnieski, C. Nishihira, and J. Cover, Jr., Toxicon 30, 887 (1992). 19. J. W. Daly, R. J. Highet, and C. W. Myers, Toxicon 22, 905 (1984). 20. T. F. Spande, M. W. Edwards, L. K. Pannell, J. W. Daly, V. Erspamer, and P. Melchiorri, J. Org. Chem. 53, 1222 (1988). 21. J. W. Daly, H. M. Garraffo, L. K. Pannell, T. F. Spande, C. Severini, and V. Erspamer, J. Nut. Prod. 53, 407 (1990). 22. H. M. Garraffo, J. Caceres, J. W. Daly, T. F. Spande, N. R. Andriamaharavo, and M. Andriantsiferana, J. Nut. Prod. 56, 1016 (1993). 23. J. W. Daly, N. R. Andriamaharavo, M. Andriantsiferana, and C. W. Myers, Am. Mus. Novitates No. 3177, 1 (1996). 24. H. M. Garraffo, T. F. Spande, J. W. Daly, A. Baldessari, and E. G. Gros, J. Nut. Prod. 56,357 (1993). 25. J. W. Daly, H. M. Garraffo, T. F. Spande, C. Jaramillo, and A. S. Rand, J. Chem. Ecol. 20, 943 (1994). 26. T. Tokuyama, N. Nishimori, A. Shimada, M. W. Edwards, and J. W. Daly, Tetrahedron 43, 643 (1987). 27. P. Jain, H. M. Garraffo, T. F. Spande, H. J. C. Yeh, and J. W. Daly, J. Nut. Prod. 58, 100 (1995). 28. J. W. Daly, L. Karle, C. W. Myers, T. Tokuyama, J. A. Waters, and B. Witkop, Proc. Nut. Acad. Sci. USA 68, 1870 (1971). 29. T. F. Spande, H. M. Garraffo, J. W. Daly, T. Tokuyama, and A. Shimada, Tetrahedron 48,1823 (1992). 30. C. W. Myers and J. W. Daly, Bull. Am. Mus. Nut. Hist. 157, 173 (1976). 31. J. W. Daly, B. Witkop, T. Tokuyama, T. Nishikawa, and I. L. Karle, Helv. Chim. Actu 60, 1128 (1977). 32. J. W. Daly, T. Tokuyama, G. Habermehl, I. L. Karle, and B. Witkop, Justus Liebigs Ann. Chem. 729,198 (1969). 33. T. Tokuyama, T. Tsujita, A. Shimada, H. M. Garraffo, T. F. Spande, and J. W. Daly, Tetrahedron 47,5401 (1991). 34. H. M. Garraffo, L. D. Simon, J. W. Daly, T. F. Spande, and T. H. Jones, Tetruhedrom 50, 11329 (1994). 35. T. Tokuyama, N. Nishimori, I. L. Karle, M. W. Edwards, and J. W. Daly, Tetruhedron 42,3453 (1986). 36. T. F. Spande, H. M. Garraffo, H. J. C. Yeh, Q-L. Pu, L. K. Pannell, and J. W. Daly, J. Nut. Prod. 55, 707 (1992). 37. D. F. Taber and K. K. You, J. Am. Chem. SOC. 117,5757 (1995). 8. 9. 10. 11.

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38. T. F. Spande, H. M. Garraffo, M. W. Edwards, H. J. C. Yeh, L. Pannell, and J. W. Daly, J. Am. Chem. SOC.114, 3475 (1992). 39. B. Badio and J. W. Daly, Mol. Pharmacol. 45,563 (1994). 40. T. Tokuyama, J. W. Daly, H. M. Garraffo, and T. F. Spande, Tetrahedron 48,4247 (1992). 41. K. D. Hutchinson, J. V. Silverton, and J. W. Daly, Tetrahedron 50, 6129 (1994). 42. J. Meinwald, J. Smolanoff, A. T. McPhail, R. W. Miller, T. Eisner, and K. Hicks, Tetrahedron Lett. 2367 (1975). 43. W. A. Ayer, M.J. Bennet, L. M. Browne, and J. T. Purdham, Can. J. Chem. 54,1807 (1976). 44. T. H. Jones, M. S. Blum, H. M. Fales, and C. R. Thompson, J. Org. Chem. 45,4778 (1980). 45. T. H. Jones, M. S. Blum, H. M. Fales, C. R. F. Brandao, and J. Lattke, J. Chem. Ecol. 17, 1897 (1991). 46. C. A. Toft, J. Herpetol. 15, 139 (1981). 47. T. F. Spande, J. W. Daly, D. J. Hart, Y.-M. Tsai, and T. L. MacDonald, Experientia 37, 1242 (1981). 48. J. W. Daly, T. F. Spande, N. Whittaker, R. J. Highet, D. Feigl, N. Nishimori, T. Tokuyama, and C. W. Myers, J. Nut. Prod. 49,265 (1986). 49. F. J. Ritter, I. E. M. Rotgans, E. Talman, P. E. J. Verwiel, and F. Stein, Experientia 29, 530 (1973). 50. T. H. Jones, R. J. Highet, M. S. Blum, and H. M. Fales, J. Chem. Ecol. 10,1233 (1984). 51. T. H. Jones, J. A. Torres, T. F. Spande, H. M. Garraffo, M. S. Blum, and R. R. Snelling, J. Chem. Ecol. 22, 1221 (1997). 52. M. W. Edwards, J. W. Daly, and C. W. Myers, J. Nut. Prod. 51, 1188 (1988). 53. H. M. Garraffo, P. Jain, T. F. Spande, and J. W. Daly, J. Nut. Prod. 65, 2 (1997). 54. M. W. Edwards, H. M. Garraffo, and J. W. Daly, Synthesis 11, 1167 (1994). 55. T. H. Jones, M. S. Blum, and H. M. Fales, Tetrahedron 38, 1949 (1982). 56. T. H. Jones, P. J. DeVries, and P. Escoubas, J. Chem. Ecol. 17,2507 (1991). 57. K. Oka, J. D. Kantrowitz, and S. Spector, Proc. Nut. Acad. Sci. USA 82, 1852 (1985).

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-CHAPTER L

BIOCHEMISTRY OF ERGOT ALKALOIDS-ACHIEVEMENTS AND CHALLENGES* DETLEF GROCER Institute for Plant Biochemistry Halle (Saale), Germany

HEINZG. FLOSS Department of Chemistry University of Washington Seattle, Washington 981 95

I. Introduction 111. The Natural Ergot Alkaloids ............................................................. 173 A. Structural Types ........................... .......................................... 174 B. Lysergic Acid Derivatives ............................................................ 174 C. Clavine Alkaloids and Secoergolines .............................................. 176 D. New Alkaloids ...,..................................... IV. Producing Organisms ...................................... A. Biology of Ergot Fungi ................................................................ 182 B. Other Fungi ........................ ....,...............,..............,. C. Higher Plants ...................... .................................................. 183 V. Biosynthesis ................................................................................... 183 A. Biosynthesis of the Ergoline Ring System ........... 184 193 B. Biosynthesis of Lysergic Acid Derivatives ....................................... C. Enzymology of Ergoline Alkaloid Formation ................................... 198 VI. Biotechnological Production ................................. ....................... 201 A. Directed Fermentation ................................... ....................... 201 B. Bioconversion of Ergot Alkaloids .................................................. 202 VII. Pharmacological Properties of Ergolines .............................................. 204 A. Biological Activities Mediated by Neurotransmitter Receptors ............. 206 B. Ergolines with Antitumor and Antimicrobial Properties ..................... 207 ..................... 208 VIII. Future Challenges .................... A. Enzymology and Molecular Genetics .............................................. 208 B. Regulation ................................................................................ 210 C. Evolutionary Aspects .................................................................. 210 References .................................................................................... 212

* Dedicated to Dr. Dr. h.c.mult. Albert Hofmann, the great pioneer of ergot research, on the occasion of his 90th birthday. THE ALKALOIDS, VOL. 50 0099-9598/98 $25.00

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Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved.

172

GROGER AND FLOSS

I. Introduction Ergot alkaloids comprise a group of indole alkaloids which are predominantly found in various species of the ascomycete Claviceps. In pharmacopeias, the sclerotia of Claviceps purpurea (Fr.) Tulasne parasitizing on rye, Secale cereale L., are designated as ergot or Secale cornutum. Now, the term ergot is used in a broader sense to describe the sclerotia of various Claviceps species growing on different host plants or their saprophytic mycelia. Ergot fungi are the oldest known producers of mycotoxins. In contrast to other mycotoxicoses, ergotism is today practically eliminated. Due to their many fascinating features, there is a continuing and extensive interest in these secondary metabolites. Thus the chemistry of ergot alkaloids has presented many challenges to organic chemists. A number of natural alkaloids and semisynthetic ergolines are important drugs which are widely used in clinical medicine. Moreover, ergot alkaloids have been an important stimulus in the development of new drugs by providing structural prototypes of molecules with pronounced pharmacological activities. The chemistry of ergot alkaloids, including newly detected alkaloids, has been described in Volumes VIII ( 1 ) and XV (2) of this treatise. A recent review in Volume 38 ( 3 ) covered the major synthetic work in the ergoline field. In the present review, a picture of our current knowledge of the formation of ergot alkaloids in Nature will be given. Extensive work has been done on this subject, which proved to be unexpectedly complex and full of surprises. Biotechnological aspects and some current trends in ergot alkaloid pharmacology will also be covered. Another purpose is to draw attention to unsolved questions which merit further investigation.

11. Historical Background

In classic antiquity, ergot was apparently not known, although there are some hints in the old literature. In the Middle Ages, however, severe epidemics occurred in Central and Western Europe in both man and animals. The animal poisonings resulted from ingestion of ergot-infected grasses, while in man the toxic effects were caused by bread made from rye contaminated with ergot. The symptoms in man were known as “ignis sacer” or “holy fire.” Ergot was first mentioned as a remedy in 1582 in the Kreuterbuch of Adam Lonicer ( 4 ) , and in 1808 the American physician J.

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Stearns introduced ergot into official medicine (5). The life cycle of Claviceps purpurea was described in 1853 in a classical paper by Tulasne ( 6 ) , and the first chemically pure, homogeneous ergot alkaloid, ergotamine, was isolated in 1918 by Stoll (7). In 1934 lysergic and isolysergic acid were obtained as degradation products of ergot alkaloids by Jacobs and Craig (8). Lysergic acid diethylamide (LSD) was first prepared in 1938 and its extremely potent hallucinogenic activity was reported in 1943 (9 ).The basic principles of the parasitic cultivation of rye ergot were established by von BCkCsy in 1935 (10) and by Hecht in 1941 (12).After World War 11, in Japan the first representative of a new class of natural ergoline derivatives, agroclavine, was isolated from, most remarkably, saprophytic cultures of grass ergot (22,23).The first total synthesis of lysergic acid was accomplished by Kornfield et al. in 1954 (24) and the total synthesis of the cyclol-type alkaloid ergotamine was described in 1961 by Hofmann and his colleagues (25). A phytochemical sensation was the structure elucidation of the active hallucinogenic principles of “ololiuqui,” the Aztec name for the seeds of Morning Glory (Zpornoea sp.), an old magic Mexican drug, which proved to be amides of lysergic acid and other ergolines (26). The large-scale production of simple lysergic acid derivatives in submerged culture was described by Arcamone et al. in 1961 (27). A hypothesis on the biosynthetic origin of the ergoline ring system by condensation of tryptophan with an isoprenoid C-5 unit was proposed by the groups of Mothes and Weygand in 1958 (28). Subsequently, the first radioactive feeding experiments with saprophytic cultures of grass ergot were performed, demonstrating the incorporation of [D-l4C]tryptophaninto elymoclavine (29).For further historical information the reader is referred to several comprehensive reviews (20-25).

111. The Natural Ergot Alkaloids

The first pharmaceutical-chemical investigation of ergot was published in 1816 by the French pharmacist Vauquelin (26). A crystalline alkaloid preparation, “ergotinine cristallisee,” was obtained by Tanret in 1875 (27). A special landmark in ergot alkaloid chemistry, as mentioned, was the isolation of ergotamine by A. Stoll in 1918, a pioneer in this field (7). Now quite a number of natural ergolines are known which have been isolated from different sources. Some general information on ergot alkaloids and “new alkaloids” which have been isolated since 1989 are summarized below. For a detailed description of individual alkaloids some earlier compilations should be consulted (2,3,28).

174

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A. STRUCTURAL TYPES Ergot alkaloids are 3,4-substituted indole derivatives. An essential structural element of ergot alkaloids is the tetracyclic ergoline ring system (1) (29) or slight modifications thereof. Most of the naturally occurring ergot alkaloids are derivatives of 8-substituted 6-meth~l-A~.~or A9."-ergolene. On the basis of their structures they can be divided in two major classes: (a) Amide derivatives of lysergic acid (2) and the stereoisomeric isolysergic acid (3). The amide portion can be a small peptide or a simple alkylamide. A structural isomer of lysergic acid is paspalic acid (4). In this Asergolene the hydrogen atom at C-10 has the a-configuration, trans to 5-H. (b) The clavine alkaloids, or clavines, are hydroxy- and dehydro-derivatives of 6,8-dimethyl-ergolenes and the corresponding ergolines. In the stereoisomeric chanoclavines the D-ring is open between N-6 and C-7.

'H

1

2

3

4

B. LYSERGIC ACIDDERIVATIVES 1. Peptide Alkaloids

Peptide ergot alkaloids are composed of lysergic acid and a peptide moiety. They are divided into two major groups. The "classic" ergot alkaloids possessing a cyclol structure are called ergopeptines (30).The ergopep-

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tams (31) contain a noncyclol lactam and, in contrast to the ergopeptines, a D-proline. a. Ergopeptine Alkaloids. These alkaloids are characterized by a modified tripeptide containing proline and an a-hydroxy-a-amino acid which has undergone cyclol formation with the carboxyl carbon of L-proline. The amino acids present in the cyclol peptide portion characterize the different ergopeptines. In Table I the cyclol-type alkaloids are grouped in some form of a “periodic table” (32).

b. Ergopeptam Alkaloids. The first member of a new group of peptide ergot alkaloids, N-[N-(d-lysergyl-L-valy1)-L-phenylalanyl-D-proline lactam, later designated as ergocristam, was isolated in the 1970s (33,34).Ergot alkaloids of this noncyclol type are called ergopeptams (Table 11). They occur only in traces in sclerotia and saprophytic cultures. Their biochemical relevance has still to be elucidated. Members of the ergotamam and ergoxam group, corresponding to the ergotamine and ergoxine group, respectively, have not yet been found in Nature.

TABLE I ERCOPEP~INE ALKALOIDS“

R’

Ergotamine group R = CH3

Ergoxine group R = CzHS

Ergotoxine group R = CH(CH3)2

CHZ-C~HS CHz-CH(CH3)z CHCHS-C~HS CH(CH3)2 CH2CH3

Ergotamine (5) a-Ergosine (6) P-Ergosine (7) Ergovaline (8) Ergobine (9)

Ergostine (10) a-Ergoptine (11) P-Ergoptineb (12) Ergonine (13) Ergobutine (14)

Ergocristine (15) a-Ergokryptine (16) P-Ergokryptine (17) Ergocornine (18) Ergobutyrine (19)

The derivatives of isolysergic acid characterized by the ending -kine are not listed here, e.g., ergotaminine. Not yet found in nature.

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GROGER AND FLOSS

TABLE I1 ERGOPEPTAM ALKALOIDS“

HRI CHz- GHs CHz-CH(CH3)z CH(CH3)CzHs CWCHdz CHzCH?

Ergotoxam group R = CH(CH& Ergocristam (20) a-Ergokryptam (21) P-Ergokryptam (22) Ergocornam (23) Ergobutyram”

Ergoannam group R = CH(CH3)CzHs a,P-Ergoannam”(25) P,P-Ergoannam (26)

“ T h e derivatives of isolysergic acid characterized by the ending -inam are not listed here. Not yet found in nature.

2. Simple Lysergic Acid Derivatives From the “water soluble fraction” of rye ergot the propanolamide of lysergic acid (27) was isolated in 1935 (35-38) which exhibited a pronounced oxytoxic activity. It was variously designated as ergometrine, ergobasine or ergonovine. Abe et al. obtained a compound from ergot which gave upon hydrolysis lysergic acid, pyruvic acid, and valine. The name ergosecaline and, tentatively, the structure 28 were assigned to this compound (39). Saprophytic cultures of Cluviceps paspali were the source for the isolation of lysergic acid a-hydroxyethylamide (29),lysergic acid amide (ergine) (30), and isolysergic acid amide by Arcamone el al. in 1961 (17) (Table 111). C. CLAVINE ALKALOIDS A N D SECOERGOLINES Agroclavine (31) was found in sclerotia and saprophytic cultures of ergot parasitizing on Agropyrurn sernicostaturn Nees in Japan (12,Z3). It was the first member of a new class of ergot alkaloids called clavine alkaloids or clavines. In the clavines C-17 has a lower oxidation state than in the lysergic acid derivatives, and the double bond in the D-ring may be in the 8,9- or

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TABLE 111 SIMPLE LYSERGIC ACIDDERIVATIVES

R CH3

I

-NH

Compound Ergonovine (27) (Ergobasine, Ergometrine)

- C -H

I

CH,OH

CH3

I

-NH

- CH -OH -NH,

Lysergic acid a-hydroxyethylamide (29) Lysergic acid amide (30) (Ergine)

9,lO-position or may be lacking altogether. In the tricyclic 6,7-secoergolines ring D is not closed. Prominent members of this group are the chanoclavines. Chanoclavine-I, originally designated as chanoclavine, was discovered by Hofmann et al. in 1957 (41). Later it was found to be one of several stereoisomers (42). In chanoclavines-I and -11 the hydrogens at positions 5 and 10 are in trans and cis arrangements, respectively. Chanoclavine-I (37)is an essential intermediate in the biosynthesis of tetracyclic ergolines. Clavines are found also in fungi outside the genus Claviceps and in higher plants. Some representatives of the major types of the more than 30 known clavine alkaloids are depicted in Fig. 1.

178

GROCER AND FLOSS

dCC gH & 8, 8-Ergolenes

Ergolines

CH20H

&H.cH3 /

&2H3 /

H0

H'

31 Agroclavhe

COzH

/

H'

32 Elymoclavine

H'

33 Festuclavine

9-Ergolenes

HO,

CHzOH

HO,

CH3

&$CH3

CHzOH

&$H3

CHzOH

CH3 /

/

/

N

N H0

H'

35 Penniclavine

34 Fumigaclavine

6,7-Secoergolenes

"CH3

N H0

cH3

/

36 Setoclavine

37 Chanoclavine-I

0

N H'

38 ChanoclavineLl

FIG.1 . Various types of clavine alkaloids.

D. NEWALKALOIDS Ergot fungi, endophytes of grasses, and some species of higher plants are the sources of new ergot alkaloids. The structures of ergolines which were elucidated mostly during the period of 1989-1994 are summarized as follows. 1. Dehydroelymocluvine (39)

From the roots of an African plant Securiducu longipedunculutu (Polygalaceae) an alkaloid fraction was isolated. The natives of Guinea Bissau use extracts of this plant in religious rites, due to their psychotropic effects. The structures of two alkaloids were determined by electron ionization (EI) and fast atom bombardment (FAB) mass spectrometric measurements

5.

BIOCHEMlSTRY OF ERGOT ALKALOIDS

179

as elymoclavine (32), and dehydroelymoclavine (39). Other evidence supporting these identifications is lacking (43). 2. Elymoclavine-O-fi-~-fructofuranosyl-(2 4 I)-O-fi-o-

fructofuranoside (40)

Elymoclavine fructosides have been isolated (44) from saprophytic cultures of Claviceps purpurea and grass ergot cultivated in sucrose media. Besides the known (45) elymoclavine-0-0-D-fructofuranoside, another glycoside was also isolated. Acidic hydrolysis gave elymoclavine, and the UV spectra showed the presence of a ASv9 double bond. From these results and interpretation of MS and ‘H and I3C NMR spectra the structure 40 was deduced. 3. 8-Hydroxyergine (41) and 8-Hydroxyerginine (42a)

8-Hydroxyergine (41) and 8-hydroxyerginine (41s) were the main alkaloids found in the culture broth of a Clavicepspaspali strain after a fermentation period of 28 days (46).The structures of 4 1 and 41a have been proposed based mainly on physical data (UV, MS, ‘H and I3C NMR). CqOH

I

H‘

39 Dehydmelymclavine

40 Elymoclavine-O-~-D-fructofuranosyl-(2+1)0-

Q$

p-D-fructofuranoside

“CH,

H‘ 41

@=OH, Ri=CONH2) %-HY&OX-

41. (R=CONHz, Rl=OH)

S-HY~IQXY-

180

GROCER AND FLOSS

4. 10-Hydroxy-cis- and 10-hydroxy-trans-paspalicacid amides (42)and (43)

During the post-production phase of a Claviceps paspali strain cis- and trans-10-hydroxypaspalicacid amides (42 and 43) accumulated. Based on their mass spectra, 42 and 43 were recognized as isomers of 8-hydroxyergines. Facile elimination of H 2 0 from the molecular ion and abundant peaks at d z 170 and 171 support the presence of an OH group at C-10. Comparison of the I3C NMR spectra of the A8y9-ergines bearing a 10hydroxy substituent with those of the corresponding cis/truns pairs of dihydrolysergic acid derivatives and agroclavine/agroclavine-Iallowed the assignment of the C/D ring stereochemistry of 42 and 43 (47). 5. 0-12’-Methylergocornine (44)and 0-12’-methyL-u-ergokryptine(45)

Some minor alkaloids isolated from the saprophytic culture of a Claviceps purpurea strain (48) have been characterized as the first naturally occurring ergopeptines possessing an OCH3 group at C-12‘, namely O-l2’-methylergocornine (44) and 0-12’-methyI-a-ergokryptine (45). The structure elucidation was based mainly on physical data (MS, M’, d z 575 for 44,589 for 45; I3C-NMR: 12’-OCH3 at S 49.2 ppm in 44,48.8 ppm in 45). CONH2

I

42 (R=-OH)

IO-Hydroxy4s-paspaIic acid arnide

43 (R= ...w OH)

10-Hydroxy-trans-paspspalicacid arnide

H‘ 44 (R=CH(CH3h) 0-12’ Methylergocomine 45 (R=CH&H(CH3)2) 0- 12’ Methylergokqptine

H’ 46 Ergobalansine.

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6. Ergobafansine (46)

The grass species, Cenchrus echinatus L., is native to the tropics and subtropics. Very often it is infected by fungal endophytes, e.g., Bafansia species. Powell et af. (49) isolated from Bafansia-infected C. echinatus an ergot alkaloid, ergobalansine (46),and its C-8 epimer, ergobalansinine. The same alkaloids were also produced by saprophytic cultures of Bafansia obtecta and B. cyperi. Ergobalansine proved to be a peptide derivative of lysergic acid, but differs from other known ergopeptines in that the characteristic proline residue has been replaced by an alanine residue. The structure has been elucidated by analysis of the mass spectra (M+,d z 521, prominent ions at d z 267,221,207,192,180,167, and 128) and by 'H and I3C NMR spectroscopy. Most surprisingly, the same alkaloids have also been found, in addition to other ergolines, in the seeds and epigeal parts of Ipornoea piurensis, a South American morning glory species (50). 7. Ergobine (9)

Ergobine (9) was isolated in trace amounts from submerged cultures of a Cfaviceps purpurea strain (51). The structure was established through chemical degradation, UV, 'H NMR, and mass spectrometry, and amino acid analysis. Its isolation completes the series of natural ergopeptines having a-aminobutyric acid as the second amino acid of the peptide moiety. 8. Ergogaline (47)

A minor alkaloid was recently isolated from sclerotia of a particular Cfaviceps purpurea strain (52). The structure was primarily established by X-ray crystallography and other physical methods (IR, MS, 'H, and I3C NMR). Ergogaline (47; C33H43N505; m.p. 182") is a new naturally occurring member of the "ergotoxine" family containing L-homoisoleucine in the peptide moiety. Apparently 47 is the first natural product containing this unique amino acid.

47 Ergogaline

48 Cycloclavine

182

GROCER AND FLOSS

IV. Producing Organisms A. BIOLOGY OF ERGOT FUNGI Ergot alkaloids have been known for a long time as constituents of fungi of the genus Claviceps, which belongs to the order Clavicipitales (53) and to the class of the Ascomycetes. About 40 species of the genus Claviceps, which are plant parasites, have been described (5435). In the compilation of Brady (56),host plants belonging to the families Juncaceae, Cyperaceae, and Gramineae are listed. Within the various Claviceps species different biochemical “races” have been distinguished, based on their alkaloid content. The genus Claviceps may be divided biochemically into three groups (57): 1. Claviceps species parasitizing on Agropyron and Pennisetum host plants. They form only clavine alkaloids. 2. Ergot fungi of the Claviceps paspali type, which produce clavines and simple lysergic acid derivatives. 3. Claviceps purpurea and related fungi, which are able to synthesize clavines, simple lysergic acid derivatives, and peptide ergot alkaloids. The life cycle of the most prominent Claviceps species, C. purpurea was described more than 100 years ago (6,58).Several reviews on the biology of Claviceps have been published (32,59,60,62).

B. OTHER FUNGI Spilsbury and Wilkinson (62) discovered the first ergolines in fungi outside the genus Claviceps. They isolated fumigaclavine A and B and festuclavine from Aspergillus &migatus Fres. Fumigaclavine was also found in Rhizopus nigricans. Later, various clavines were isolated from different Aspergillus and Penicillium species. Of special interest are the rugulovasines from Pencillium species which feature a benz[c,d]indole skeleton with a spirobutanolide side chain (63-65). Relevant literature on this topic has been compiled by Narayan and Ra6 (66). Toxicoses caused by endophyte-infected grasses are a serious problem for animal breeding in many parts of the world. The loline- and ergottype alkaloids produced by these fungal endophytes are responsible for substantial losses to cattle and sheep producers. Balansia species and Epichloe typhina from toxic pasture grasses produce clavine alkaloids, inter alia 6,7-secoagroclavine. Moreover, E. typhina was found to synthesize ergovaline (8) and its stereoisomer ergovalinine (67-70). Tall fescue (Fes-

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ERGOT ALKALOIDS

183

tuca arundinacea Schreb) infected with Sphacelia typhina (Acremonium coenophialum) contained ergopeptine alkaloids, predominantly ergovaline (8) (71,72). Ergobalansine (46) is synthesized by Balansia obtecta, an endophyte infecting the annual grass species, Cenchrus echinatus L. (49). The perennial grass, Stipa robusta, which is indigenous to the Southwestern United States is often contaminated with Acremonium. Extraction of infected grass yielded chanoclavine-I, ergonovine, 8-hydroxylysergicacid amide and, in a remarkably high concentration, lysergic acid amide and isolysergic acid amide (73).

C. HIGHER PLANTS From the seeds of various Convolvulaceae, including Zpomoea violacea L. and Rivea corymbosa (L.) Hall, Hofmann and Tscherter isolated ergine (30),isoergine and chanoclavine (37)(16), followed later by elymoclavine (32),lysergol and ergometrine (27)(7475). Since then, a number of authors have demonstrated the occurrence of various ergolines in different species of the family of twining plants (Convolvulaceae), mostly in Zpomoea, Argyreia, and Strictocardia. Interestingly, cycloclavine (48), representing a novel type of clavine, was isolated from Zpomoea hildebrandtii Vatke (76).Even peptide ergot alkaloids were obtained from Zpomoea. Ergosine (7)and its epimer ergosinine are constituents of Zpomoea argyrophylla Vatke (73, and ergobalansine (46) has recently been isolated from Zpomoea piurense (50). The latter and cycloclavine (48) have so far not been detected in Claviceps species. Some compilations of ergolines found in Argyreia, Zpomoea, and Rivea have been published (78-80). The first and only report on the occurrence of clavines in a plant not belonging to the Convolvulaceae family appeared in 1992 (43). From a chemotaxonomical point of view an independent confirmation of this finding would be extremely desirable.

V. Biosynthesis The biosynthesis of ergot alkaloids has been studied for nearly 40 years. A number of hypotheses on the origin of lysergic acid were published in the 1950s, (85). Finally, in 1958 Mothes et al. (18), as well as Birch (81), proposed that the ergoline ring is built up from tryptophan, a C5 isoprene unit and a methyl group (Scheme l ) , and this proposal was quickly confirmed experimentally. Most remarkable, at the time, was the finding that mevalonic acid (50) is not only a precursor of typical isoprenoids, but also

184

GROGER AND FLOSS

49

SCHEME1.

participates in the formation of other secondary metabolites, like alkaloids. The very first experiments in ergot alkaloid biosynthesis were done with sclerotia growing parasitically on rye plants (28J9). Since the early 1960s, when saprophytic alkaloid-producing strains became available, practically all laboratories have used fermentation procedures for such biosynthetic studies. In this chapter, an outline of the general picture of ergot alkaloid biosynthesis will be presented. Emphasis will be placed on some complex and unexpected reactions uncovered by the experimental work and on results obtained during the last decade. A. BIOSYNTHESIS OF THE ERCOLINE RINGSYSTEM

By the use of isotope techniques it was established in the late 1950s and in the 1960s that ergot fungi synthesize the ergoline ring system from three major precursors, tryptophan (49), an isoprenoid C5-unit (52), ultimately derived from mevalonic acid (SO), and a methyl group provided by methionine. Formation of the rings C and D of the ergoline system was studied with multiple labeled mevalonic acid samples and specificpotential intermediates. The biogenetic interrelationships of clavine and lysergic acid alkaloids were also studied intensely. Since 1970 crude and purified enzyme preparations catalyzing individual steps of ergot alkaloid formation have become available. Summarizing all the results, the ergoline biosynthetic pathway can now be formulated as depicted in Scheme 2. It starts with the isoprenylation of tryptophan (49) to 4-(y,y-dimethylallyl)tryptophan (DMAT) (53)which is subsequently methylated to 54. In a reaction which is not yet completely understood, the tricyclic chanoclavine-I (37)is formed, which in turn is oxidized to the corresponding aldehyde (55). In the next step, the first tetracyclic clavine, agroclavine (31), is synthesized which can be oxidized at C-17 to elymoclavine (32) and further to lysergic acid (2).

5. BIOCHEMISTRY OF

54

185

ERGOT ALKALOIDS

62

37

* 7H20H

-@ /

CH3

H'

-

H'

31

32

*

LysergicAcid(2)

H3

Isotopic Labels

SCHEME2.

The experimental evidence for various steps of the ergoline alkaloid pathway and mechanistic aspects will be discussed in detail in the following. 1. Isoprenylation of Tryptophan

The formation of the ergoline ring system requires decarboxylation, Nmethylation and isoprenylation of 49. Tryptamine and Nw-methyltrypt-

186

GROCER A N D FLOSS

amine were not incorporated into ergot alkaloids (88,89).This rules out decarboxylation as the first step. Feeding experiments (90)indicated that L-tryptophan is a more immediate precursor than the D-isomer, and that it is incorporated with retention of the a-hydrogen and the amino nitrogen. In the course of the reaction at the a-carbon of the tryptophan side chain an inversion of configuration takes place. Since it was ruled out (92) that tryptophan is activated for the condensation with dimethylallyl pyrophosphate (52) by hydroxylation at the 4-position, a direct isoprenylation of 49 was indicated. The initial connection of the isoprene unit could be either the a-position of 49 or at the 4-position of the indole ring (Scheme 3). The two potential precursors were synthesized in labeled form, 57 by Weygand et af. (92) and DMAT (53) by Plieninger and Liede (93). DMAT (53) was always incorporated much better than 57, suggesting the intermediacy of 53 in ergoline ring biosynthesis (94,95). Moreover DMAT was isolated from ergot cultures incubated in the absence of oxygen (96) or in the presence of ethionine (97).Finally, an enzyme preparation was isolated catalyzing the condensation of tryptophan and dimethylallyl pyrophosphate to give 53 (98).

52

49

A

d 2 ($iH H'

N

N H'

57 SCHEME 3.

53

5.

187

BIOCHEMISTRY OF ERGOT ALKALOIDS

2. Interrelationships of Clavines The oxidative reaction sequence chanoclavine + agroclavine + elymoclavine + lysergic acid (Scheme 4) was proposed in 1958 (99). Experimental support for the irreversible conversion of agroclavine into elymoclavine (32)was provided by Agurell and Ramstad (200-102) and by Baxter et al. (203). The latter alkaloid in turn was shown by Mothes et al. (104)to be a progenitor of lysergic acid derivatives. The conversion of chanoclavine-I (37)into these tetracyclic alkaloids was first demonstrated

HO

CH3

HO

CH#H

$\CH3 /

/ $\CH3

N

N

H'

H' 35

36

t

d:cH3 @ 99 CHzOH

FH3

CH2OH

I

"CH3

"CH3

\

H'

H'

HMN

31

31

32

H'

SCHEME 4.

188

GROCER AND FLOSS

in 1966 (105,106). Hydroxylation of 31 and 32 leads to setoclavine (36) and penniclavine (39, respectively, and their C-8 epimers. These reactions involve hydroxylation at C-8 with a shift of the double bond into the 9,lOposition and are catalyzed by peroxidases. Besides Claviceps, a number of other fungi, as well as plant homogenates, can catalyze these hydroxylations (107). An alternative route of clavine transformations was proposed by Abe’s group (108,109), but their results have never been confirmed in other laboratories. 3. Cis-Trans Zsomerizations in Clavine Alkaloid Biosynthesis

It has been clearly established (106) that [2-’4C]mevalonate (SO) specifically labels the CH3 group at the 8,9-double bond of chanoclavine-I (37) which occupies the trans position relative to the vinyl hydrogen, whereas in the tetracyclic agroclavine (31) and elymoclavine (32) it labels C-17, which is located cis to this hydrogen (103,110) (Scheme 2). These results suggested the occurrence of cis-trans isomerizations during the formation of tetracyclic ergolines from mevalonic acid. The labeling pattern of agroclavine and elymoclavine obtained after feeding [ 17-14C]-or [7-14C]chanoclavine-I showed that the hydroxymethyl group of 37 becomes C-7 of 31 and the C-methyl group of 37 becomes C-17 of 31 (106,111). These findings demonstrate the occurrence of one cis-trans isomerization between chanoclavine-I and agroclavine. It was suggested that a second cis-trans isomerization occurs during the formation of chanoclavine-I from mevalonate (111). This was deduced from the fate of the two diastereotopic hydrogens at C-4 of mevalonate, the pro4R hydrogen is retained during elymoclavine formation (Scheme 2), suggesting that the isopentenyl pyrophosphate isomerase reaction takes the “normal” steric course in ergot. This means that 52 and 53 should carry the label from C-2 of mevalonate in the methyl group that is located cis to the vinyl hydrogen at the allylic double bond. This was subsequently proven by feeding [Z-I4CH3]-53and showing by degradation of 37,31, and 32 that the label was located in the hydroxymethyl group of chanoclavineI, but at C-7 of agroclavine and elymoclavine (112,113).Therefore, two cistrans isomerizations must occur in ergoline biosynthesis, the first between DMAT and chanoclavine-I, and the second between chanoclavine-I and agroclavine. 4. Formation of Ring C: Modification of the Isoprene Unit

The first alkaloid of the ergoline pathway starting from DMAT is the tricyclic chanoclavine-I (37). Despite many experimental efforts, the exact mechanism by which .ring C is closed is still not completely understood. It is known (114) that H-5 is retained, that the configuration at C-5 is inverted, that one hydrogen is lost from C-10, and that 37 is the product of the ring

5.

189

BIOCHEMISTRY OF ERGOT ALKALOIDS

closure. There is no correlation between the stereochemistry of hydrogen abstraction from C-10 (ergoline numbering) and the stereochemistry of the chanoclavine isomer resulting from the cyclization; all of the different clavines and chanoclavine isomers are formed with retention of the pro5s and loss of pro-% hydrogen of mevalonate (225-227). Desoxychanoclavine-I, its N-nor-derivative, and its N-methyl derivative are not precursors of tetracyclic clavines and paspalic acid (228),suggesting that oxygenation of one of the allylic methyl group precedes ring C closure. Therefore, Plieninger’s group (229) synthesized (E)-4-(4’-hydroxy-3’methyl-2’-butenyl)-tryptophan (56) labeled in the hydroxymethyl group and fed this promising precursor to a Claviceps culture. They reported incorporation into both agroclavine and elymoclavine. However, later (222) it was found that only elymoclavine is labeled and, surprisingly, that the label is located at C-17, not at C-7 as expected. Subsequently, Arigoni’s group (122,223) synthesized both the E and 2 isomers of 4’-hydroxyDMAT, [14CH3]-56and [‘4CH3]-58.Both compounds labeled elymoclavine at C-7, but did not label agroclavine. This labeling pattern would result if 56 were processed as if the hydroxy group were not present, undergoing the two cis-trans isomerizations and eventually generating 32 directly in the cyclization which normally produces 31 (Scheme 5 ) . The mode of

I

@ /

H :

N H‘

H’ 56 E-OH-DMAT

58 Z-OH-DMAT

CH20H

I

56

59

32 SCHEME 5.

190

GROGER AND FLOSS

incorporation of the 2 isomer, [14CH3]-58,has never been adequately explained; it may involve initial isomerization to [14CH3]-56.In any case, the results demonstrated that the incorporations of 56 and 58 are artefacts of feeding compounds that are not intermediates on the normal biosynthetic pathway. Searching for a potential intermediate between DMAT and chanoclavine-I, Kozikowski et af. (120) synthesized both diastereomers of the diol 60,deuterium-labeled in the N-methyl group, but found no incorporation into elymoclavine by Claviceps strain SD58 (Scheme 6). Subsequently, the same group (121) synthesized the monohydroxylated DMAT derivative [N-CD3]-61which clearly labeled elymoclavine (32) when fed to Claviceps strain SD58. Trapping experiments, however, failed to identify 61 as a genuine constituent in the ergot fungus but, surprisingly, instead revealed the presence of the diene 62 and its ready formation from 61. Later, Kozikowski’s group (122) succeeded in the synthesis of 62. Deuterium-labeled 62 was efficiently incorporated into elymoclavine, and its natural occurrence in Claviceps was confirmed in a trapping experiment and by its detection in the culture medium of strain SD58. All of the data indicate that 61 is not on the normal biosynthetic pathway to the ergot alkaloids. Rather, it is channeled into the pathway by, probably nonenzymatic, dehydration to the true intermediate 62. A plausible pathway for ring C formation can now be formulated as shown in Scheme 6: 62 is probably formed from 54 by hydroxylation at the benzylic position of the isoprenoid moiety to give 64, followed by a 1,Cdehydration to 62. The diene 62 could then be epoxidized by cytochrome P-450 to the vinyl oxirane 63 which has been proposed (113) to undergo decarboxylative ring closure via an SN2’ process to give chanoclavine-I. The benzylic alcohol 64 is apparently a rather labile compound. It has not yet been found in nature, and attempts to synthesize it chemically have so far been unsuccessful. Circumstantial evidence for its likely formation may, however, be seen in the isolation of the clavicipitic acids (64a), a pair of diastereomeric shunt products of ergoline biosynthesis (122a-d). Another feature of ergoline formation was investigated by Kobayashi and Floss (123). They demonstrated unequivocally that the oxygen atoms of both chanoclavine-I and elymoclavine are derived from molecular oxygen. These findings and other results support the view that the formation of ring C of the ergot alkaloids proceeds by a mechanism involving a potential carbocation at the benzylic carbon (C-10, ergoline numbering) and a potential carbanion at C-a of the amino acid side chain, e.g., in a reactive species generated from 63.

5. The N-Methylation Step The N-methylation step must occur between DMAT (53) and chanoclavine-I. Originally, methylation after ring C formation seemed attractive,

5. BIOCHEMISTRY OF

191

ERGOT ALKALOIDS

& CHzOH

H '

63

H'

H ' 37

SCHEME6.

because this would have allowed the involvement of pyridoxal5'-phosphate catalysis in the decarboxylation/C ring closure reaction. NorchanoclavineI and -11 were detected in Cluviceps cultures (1249, but feeding experiments

192

GROGER AND FLOSS

with both labeled compounds gave no incorporation into tetracyclic clavines. Therefore the methylation step must occur before or simultaneously with closure of ring C. This idea was supported by the detection of N methyl-DMAT (54) in ergot cultures and from some preliminary feeding experiments with 54 (225).Clear-cut evidence came from experiments with double labeled ['5N-CD3]-54, which was efficiently incorporated into elymoclavine without cleavage of the bond between the nitrogen and methyl group (226).No incorporation was observed with the corresponding tryptamine derivative (87). Summarizing these results we may conclude that methylation of the amino group of 53 is the second step in ergoline biosynthesis and, by implication, that the decarboxylation and C ring closure do not involve pyridoxal phosphate catalysis (87). 6. Formation of Ring D

A number of mechanisms have been proposed for the closure of ring D in ergoline biosynthesis. A potential candidate as an intermediate in the conversion of 37 into 31 seemed to be paliclavine (65), an alkaloid isolated in 1974 (227). Subsequent feeding experiments with [N-rnethyl-'4C]chanoclavine-I and [N-rnethyl-'4C]paliclavine showed no incorporation of 65 into paspalic acid or the tetracyclic clavines (228). These results rule out an SN2' reaction of paliclavine as the mechanism for closure of ring D. Instead, there is substantial evidence that chanoclavine-I is converted into 31 via chanoclavine-I aldehyde (55). Feeding experiments revealed that 55 is a more efficient precursor of tetracyclic ergolines than is chanoclavine-I. [17-3H,4-'4C]Chanoclavine-I was converted by Claviceps into elymoclavine with complete, stereospecific loss of one of the labeled hydrogens, HR, from C-17 (229,230).Finally, a blocked mutant of Claviceps was isolated which accumulates 55 (232). All this evidence leaves little doubt that chanoclavine-I aldehyde is a true intermediate in ergoline formation (Scheme 2). Mechanistic aspects of the closure of ring D were investigated extensively (222,229).Incorporation experiments with [7-'4C,9-3H]chanoclavine-I and (4R)-[2-'4C,4-3H]mevalonate yielded labeled elymoclavine and lysergic acid hydroxyethylamide with only 70% tritium retention. A mechanism was proposed to account for the partial loss of tritium, which envisioned an intermolecular recycling of the vinyl hydrogen in the tricyclic substrate. Substantial evidence supports such a process. The most conclusive evidence comes from double labeling experiments in which a mixture of [2-13C]-and [4-D2]mevalonic acid was fed to ergot cultures. The appearance in the tetracyclic alkaloids, but not in chanoclavine-I, of molecules containing both I3Cand deuterium, according to the mass spectra, demonstrates clearly the intermolecular transfer of the hydrogen from the 9-position. The results

5. BIOCHEMISTRY

193

OF ERGOT ALKALOIDS

on the cyclization/isomerization of chanoclavine-I to agroclavine can be summarized as follows (87): The hydrogen at C-10 is completely retained; the hydrogen at C-9 is partly eliminated; low rates of alkaloid production correlate with low tritium retention; (c) the hydrogen at C-9 seems to undergo an intermolecular transfer during the reaction; and (d) the original pro-R hydrogen from C-17 of chanoclavine-I is eliminated. Further experimental data and comprehensive discussions and mechanistic interpretations have been presented (87,129). B. BIOSYNTHESIS OF LYSERGIC ACIDDERIVATIVES A number of lysergic acid derivatives are pharmacologically very active compounds which are widely used in medicine. It is therefore somewhat surprising that our knowledge of the exact mechanism of their biosynthesis is still fragmentary. The earlier work on this topic has been reviewed (85-87); some of it and more recent results are discussed in the following. 1. Lysergic Acid and Its Derivatives

Agroclavine (31)and elymoclavine (32)are precursors of lysergic acid (2).However, the exact sequence of the steps from 32 to 2 is not yet known. Lysergene, lysergol and penniclavine are not intermediates on this pathway (131,132). Labeled paspalic acid (4) was incorporated into lysergic acid amides (133,134), but it is not clear if 4 is a natural intermediate because it can isomerize spontaneously in aqueous solution very easily. Another to A9*l0at the possibility is the isomerization of the double bond from aldehyde stage. Lysergaldehyde has not yet been synthesized, but its enol acetate, 6-methyl-8-acetoxymethylene-9-ergoline, could be prepared (135). This compound was incorporated into lysergic acid amide alkaloids. While this observation does not constitute proof, it is at least consistent with the assumption that lysergaldehyde is a true intermediate. In this context it was proposed that lysergaldehyde, rather than lysergic acid, is converted to the CoA ester en route to the lysergic acid amide alkaloids (Scheme 7). This speculation explains the observation that no A8q9-amidealkaloids are found in nature and that 4, but not lysergic acid, accumulates in appropriate Cluviceps purpurea strains (87,132).The idea is supported by experiments (136) in which an ergotamine (5)-producing strain was grown in an atmoThe lysergyl fragment of 5 showed the same "0enrichment sphere of as the cyclol oxygen, favoring a pathway involving direct formation of an A8q9

194

GROGER AND FLOSS

H'

H'

65

64a

SCHEME7.

activated derivative of lysergic acid from an aldehyde intermediate without further dilution of the l80of elymoclavine. On the other hand, results obtained by Keller's group (137,138) support the alternative idea that Dlysergic acid is a free intermediate in the biosynthesis of ergot peptide alkaloids. Lysergic acid a-hydroxyethylamide (29) is a typical constituent of C h i ceps paspali strains. Ergonovine (27) is accumulated in sclerotia and sapro-

5.

195

BIOCHEMISTRY OF ERGOT ALKALOIDS

phytic cultures of various Cfuviceps species, although the biosynthesis of these simple lysergic acid amides is not yet well understood. A number of potential precursors were not incorporated into 29 (102,139),but [UI4C]alanine labeled the carbinolamide moiety of 29 (102). Radioactivity from [2-14C]alaninewas incorporated primarily into the carbinolamide carbon and I5N from ~-[U-'~C,'~N]alanine into the amide nitrogen (140,242). Alanine was also incorporated into the L-alaninol part of ergonovine, but feeding experiments with alaninol gave ambiguous results (242,143).Lysergylalanine (66) was suggested by Agurell (102) to play a key role in the biosynthesis of lysergic acid amides. However, lysergyl-~-[2-'~C]alanine did not label 29 significantly and showed a small, albeit specific, incorporation into ergonovine (27) (97.4% of radioactivity at C-2 and C-3 of the alaninol side chain) (244,145).The low incorporation rate and the lack of proof of formation of lysergylalanine in Cfuviceps puspuli makes it questionable whether 66 is a normal intermediate in ergonovine biosynthesis (245) (Scheme 8 ) .

H'

cs;'-

/CH3 OH

66

#+\ Ergotarmne (5)

. 0

CH3 ,NH-CH\/

&

\CH3

/

N

H'

H'

27

29

SCHEME 8.

CH20H

196

GROGER AND FLOSS

2. Biosynthesis of the Peptide Moiety of Ergot Alkaloids Knowledge about the biosynthesis of the cyclol part of the classical ergot alkaloids, e.g., ergotamine and ergotoxines, is rather fragmentary and it is a particular challenge to biochemists to solve this intriguing problem. Several reviews on this topic have been published (85,87,246, 247). Biogenetically, the cyclol alkaloids may be viewed as modifications of linear peptides, e.g., ergotamine: d-lysergyl-alanyl-L-phenylalany1-Lproline, ergocornine: d-lysergyl-valyl-L-valyl-L-proline. Numerous feeding experiments (142,148-152) revealed that lysergic acid and the cyclol-specific amino acids, valine, leucine, phenylalanine, and proline are specifically incorporated into the appropriate parts of the corresponding peptide alkaloids. Also, the a-hydroxy-a-amino acid moiety is derived from the corresponding a-amino acid, valine in the case of ergotoxines and alanine in the case of ergotamine. The mechanism of this formal hydroxylation reaction was clarified in Floss' laboratory. A 2,3-dehydroamino acid intermediate was ruled out by deuterium labeling experiments (253). The two other alternatives are ( a ) dehydrogenation to the imine followed by the addition of water; or (b) direct hydroxylation at the a-position.

It was shown that the oxygen in the cyclol ring is derived from molecular oxygen, favoring alternative (b) (236). Many mechanistic possibilities may be considered for the assembly of the peptide portion of the cyclol alkaloids. Chain elongation may start from the lysergic acid or from the proline end. Are individual amino acids added successively, or are intermediates (di- and tripeptides) assembled and then attached to a starter molecule? How is a linear peptide intermediate modified to give the diketopiperazine and finally the cyclol structure? What types of enzymes are involved in this reaction sequence, and do some steps occur spontaneously? Results answering some of these questions are summarized below. As proposed by Agurell(202), lysergylalanine (66) should be the precursor for ergotamine and ergosine and, by analogy, lysergylvaline for the ergotoxine alkaloids (254). However, labeled lysergylalanine and lysergylvaline were not incorporated intact into ergopeptines, but only after hydrolytic breakdown (245,252). Similarly, labeled dipeptides, diketopiperazines, and tripeptides fed to various Claviceps strains were not incorporated intact. For example, labeled L valyl-L-proline, L-leucyl-L-proline lactam, L-valylL-proline lactam (255), ~-valyl-~-valyl-~-[U-~~C]proline (156),and L-valyl-L(257)labeled the alkaloids regardless of whether they leucyl-~-[U'~C]proline had the right sequence or not. The isotope distribution in the alkaloids clearly indicated cleavage of the precursors prior to incorporation. Furthermore, ra-

5.

197

BIOCHEMISTRY OF ERGOT ALKALOIDS

dioactivity was also found in the protein fraction and in the free amino acid pool. It was also demonstrated (257)that washed mycelia of Cluwiceps are capable of hydrolyzing the added peptides, e.g., leucyl-proline lactam. Although all these negative results do not prove the absence of discrete peptide intermediates, they did lead to the suggestion (256)that peptide chain assembly and elaboration of the cyclol structure takes place in a concerted fashion on a multienzyme complex in analogy to peptide antibiotic formation (258).Chain growth could start at the C-terminal end, as suggested by the higher specific activity in the a-hydroxyvaline moiety, compared to the valine moiety of ergocornine (250,252).Taking ergocristine synthesis as an example (Scheme 9) successive activation and transfer reactions would form a lysergyltripeptide (67) covalently linked to an SH-

---L-proline, L-phenylalanine,

EIU-SH

EIU-SH

2

L-valine, d-lysergic acid, ATF’

I;’ I

9

0,

Lys-N-CH

H3c1 . C0 H *’ c H 6 p

C

I I C-N,COC==o 0” H‘ \CHI

1 . Hydroxylation Lys-N-C’ 2. CYCIOI fomaEon I

‘c I

C-N,COCao

0”

I

I

68

Ergocristine (15) I

I

Epimerization

Lys = d-Lysergx Acid

c-N, 0”

0

H‘ \CH2

/LO

C H‘ ‘FH2

SCHEME 9.

198

GROGER AND FLOSS

group through the carboxyl end of proline. Release from the multienzyme complex by internal displacement of the sulfur would lead simultaneously to the formation of the lactam ring as in 68. Hydroxylation of the a-carbon of the valine adjacent to lysergic acid, followed by cyclol formation are the final steps in the synthesis of 15. Favoring this proposal is the isolation of the D-proline analogue of 68, which might arise by the facile nonenzymatic epimerization of the L-prolyl-L-phenylalanyl lactam 68 -+ 69 (33). The multienzyme hypothesis is also supported by inhibitor experiments which indicate that ergopeptine formation is a nonribosomal process (159). The mechanism of peptide bond formation is still unknown, and no “cyclolsynthetase complex” has been characterized from ergot fungi. C. ENZYMOLOGY OF ERGOLINE ALKALOID FORMATION Although tracer studies with radioactive and stable isotopes are a valuable tool, they are only a prelude for the further elucidation of a biosynthetic pathway by characterization of the enzymes catalyzing individual steps in the reaction sequence. Studies at the enzyme level are also necessary for detailed studies of the reaction mechanisms involved. Our knowledge of the enzymes involved in ergoline alkaloid formation is unfortunately still very fragmentary. 1. Dimethylallyltryptophan Synthase

The first ergoline pathway-specific enzyme was detected in Claviceps strain SD58 in 1971 by Heinstein et af. (98). The enzyme was also isolated and partially characterized from two other alkaloid-producing Claviceps strains (260).Later, dimethylallyl pyrophosphate: L-tryptophan dimethylallyltransferase (DMAT synthase) was purified to apparent homogeneity (161) and was described as a monomeric protein with a molecular mass of 70-73 kDa. Cress er af. (162) used the same Claviceps strain and obtained DMAT synthase in homogenous crystalline form, showing that the enzyme contains two similar or identical subunits of 34,000 molecular weight. DMAT synthase was active in the absence of divalent metal ions. Ca2+seems to be an allosteric effector which deregulated the enzyme at concentrations above 20 mM. Recently, Gebler and Poulter (163)purified DMAT synthase to apparent homogeneity and came to the conclusion that this enzyme is an a2 dimer with an Mr of 105 kDa. The chemical mechanism of the reaction catalyzed by DMAT synthase was clarified by Shibuya et af. (264)in extensive studies. The isoprenylation of tryptophan catalyzed by DMAT synthase involves displacement of the allylic pyrophosphate moiety by C-4 of the indole ring with inversion of configuration at C-1 of dimethylallyl pyrophosphate (DMAPP). The geometry of the allylic double bond is retained and no scrambling of labeled

5.

BIOCHEMISTRY OF ERGOT ALKALOIDS

199

hydrogens between the two methyl groups was observed. The results are fully consistent with a mechanism for DMAT synthase involving direct attack of DMAPP on C-4 of the indole apparently through a stabilized allylic carbocation or ion pair as intermediate. Furthermore, the results support earlier conclusions of Arigoni's group (212,113)that the conversion of mevalonate into DMAT in Claviceps is not completely stereospecific, apparently due to some stereochemical infidelity in the isopentenyl pyrophosphate isomerase reaction. The mechanism of the prenyl transfer reaction catalyzed by DMAT synthase was also studied with analogs of both substrates (265).The authors came to the conclusion that the prenyl transfer reaction catalyzed by DMAT synthase is an electrophilic aromatic substitution and is mechanistically similar to the electrophilic alkylation catalyzed by farnesyl diphosphate synthase. 2. N-Methyltransferase

The second pathway-specific step in ergoline biosynthesis is catalyzed by S-adenosylmethionine:dimethylallyltryptophan N-methyltransferase, which was detected in crude cell-free extracts of a clavine producing strain (266). DMAT N-methyltransferase has a sharp pH maximum at 8.0-8.5, and its activity is related to the age and alkaloid production of Claviceps cultures. Using the chiral methyl group methodology (267,268) it was found that the methylation step in ergoline formation proceeds with net inversion of methyl group configuration. The process most likely involves a direct migration of the methyl group of SAM to the amino nitrogen of DMAT in a ternary enzyme complex via an SN2transition state (269). 3. Chanoclavine-I cyclase

Three groups ( 2 70-2 74) have obtained crude enzyme preparations from different Claviceps strains which catalyze the conversion of chanoclavineI (37) into agroclavine (31) and/or elymoclavine (32). The reaction required ATP, NADH or NADPH, and Mg2'. One group (273) found that the reaction is not strictly dependent on O2 and added FAD. Ogunlana et al. (270) detected elymoclavine, Erge et al. (273) solely agroclavine, and Heinstein et al. (274) a mixture of both as reaction products. ChanoclavineI cyclase also converts chanoclavine-I aldehyde, but not isochanoclavineI or dihydrochanoclavine into agroclavine (273). The time-course of the appearance of this enzyme in Claviceps cultures closely resembles that determined for DMAT synthase (273,274).No purification of this cyclase has so far been reported. 4. Agroclavine/Elyrnoclavine Hydroxylase

Tracer experiments have established the oxidative sequence agroclavine (31) + elymoclavine (32) + lysergic acid (2). Enzymes catalyzing individual

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steps of this sequence have been reported. Hsu and Anderson (175) obtained conversion of 31 into 32 with the 60-80% ammonium sulfate fraction of a Claviceps cell-free extract. Surprisingly, a mammalian microsomal system was also found to hydroxylate C-17 of 31 (176).Later, the conversion of agroclavine to elymoclavine by a microsomal fraction from two Claviceps strains in the presence of NADPH was reported (177). The same group (178) demonstrated the conversion of elymoclavine into paspalic acid (4) by the particulate fraction of an ergotamine producing strain. Maier et al. (179) subsequently characterized microsomal oxygenases from various Claviceps strains, which were designated as agroclavine 17-mono-oxygenase and elymoclavine 17-mono-oxygenase. Carbon monoxide (177,178) and cytochrome c (179) were found to inhibit the hydroxylations. These and other properties suggest that both clavine-specific enzymes are cytochrome P-450 dependent. Lysergol was not converted to 2 by elymoclavine 17-mono-oxygenase, indicating this enzyme is highly specific (1 79). Based on in vivo experiments, Sieben et al. (180) concluded that the substrate specificity of agroclavine hydroxylase is high with respect to the 8,9-double bond and to the tertiary nitrogen status of N-6, whereas the specificity is low for variations in the pyrrole partial structure.

5. Enzymes Related to Peptide Ergot Alkaloid Formation The enzymatic formation of peptide ergot alkaloids has been studied by two groups. The first crucial step is the activation of ergopeptine-specific amino acids and d-lysergic acid. Maier et al. (181) observed d-lysergyl-CoA formation in a crude Claviceps cell-free extract. It is not clear whether this reaction is involved in alkaloid formation since there is little, if any, correlation between the activity of the lysergyl-CoA forming enzyme and alkaloid production. Later, it was shown that protoplasts of Claviceps purpurea synthesize peptide-type alkaloids de novo (137,282). The incorporation of labeled amino acids into ergopeptines was stimulated by addition of d-lysergic acid. Maier et al. (183-185) obtained partially purified enzyme preparations from various ergopeptine-producing Claviceps strains which catalyzed the cell-free incorporation of the appropriate amino acids into peptide alkaloids. From a Sepharose 6B column an ergopeptine synthetase was eluted with an Mr value of about 195,000 ? 5,000. In contrast to the protoplast system, addition of agroclavine or elymoclavine to this enzyme reaction mixture stimulated alkaloid formation more strongly than addition of lysergic acid. Keller et al. (186) purified a d-lysergic acid-activating enzyme about 145fold from a Claviceps strain. The enzyme catalyzed both d-lysergic aciddependent ATP-pyrophosphate exchange and the formation of ATP from d-lysergic acid adenylate and pyrophosphate. The same enzyme also acti-

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vates dihydrolysergic acid, although to a lesser extent. The Mr was estimated at between 135 and 140 kDa. This activating enzyme was later purified to near homogeneity and an Mr of about 245,000 was found (238). The discrepancy in the molecular weight estimations has not been clarified. The lysergic acid activating enzyme was unable to activate the other ergotamine specific amino acids and did not catalyze the synthesis of d-lysergic acid containing peptides. An ammonium sulfate fractionated enzyme preparation was also obtained which catalyzed the incorporation of d-lysergic acid into two lysergylpeptides. These were tentatively identified as N[N-(dlysergy1)-L-alanyll-~-phenylalanyl-~-proline lactam and the corresponding D-proline isomer. This rather unstable enzyme fraction did not catalyze the formation of cyclol-type alkaloids. It is evident that there are many open questions about the enzymology of peptide ergot alkaloid formation, which call for further investigations in order to solve these interesting problems.

VI. Biotechnological Production

For the commercial production of medicinally important ergot alkaloids various methods are available: (a) Isolation from sclerotia of C. purpurea. The “classical” parasitic cultivation of ergot is still in use in some European countries; (b) Extraction from saprophytic cultures of different Claviceps species, mainly C. purpurea, C. paspali, and C. fusiformis. Ergot fungi are easily cultivated in surface culture or under submerged conditions. However, strain improvement by mutation/selection to obtain high-yielding ergot strains and the optimization of fermentation conditions for the production of suitable alkaloids in bioreactors is a tedious process; (c) Partial synthesis. Lysergic acid and clavine alkaloids serve as starting material for partial synthesis. For more details the reader is referred to selected reviews (32,59,85). A. DIRECTED FERMENTATION

The biosynthesis of the peptide moiety of peptide ergot alkaloids is at least partially controlled by the relative amino acid concentration in the internal pool of the cells. The addition of appropriate amino acids (287) or their analogs (288) to the culture broth determines significantly the

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composition of the alkaloid mixtures in a given strain. By directed fermentation, unnatural alkaloids can be obtained which may possess interesting pharmacological properties. Apparently, the enzyme system catalyzing ergopeptine formation, a hypothetical cyclol synthetase complex, is not as strictly specific as multienzyme complexes in peptide antibiotic synthesis. Cultivation of a strain producing ergotoxine alkaloids showed that the relative concentrations of the precursors, leucine and valine, influenced the distribution of the individual alkaloids. In a leucine-rich and in a valinerich medium the amounts of a-ergokryptine and ergocornine, respectively, were increased (287). An ergocristine-producing strain utilized added 4chlorophenylalanine and 4-fluorophenylalanine for the formation of the corresponding ergocristine analogs. Other amino acids and various analogs were also incorporated into ergopeptines of various strains (288).An ergosine analog with a modified proline moiety was obtained by feeding Lthiazolidine-4-carboxylic acid to an ergosine producing Cfuviceps strain. The new alkaloid, “thiaergosine,” was identified as 2’P-methyl-5’cr-isobutyl-9’-thiaergopeptine (69) (289).Flieger ef uf. (290) increased the production of 6-ergokryptine (17)by adding D,L-isoleucine at a concentration of 0.3% to the culture medium of a saprophytic ergotoxine-producing ergot strain. Apart from a four-fold increased level of 17 the mycelium was found to contain three new alkaloids that amounted to 40% of the total alkaloids. These were identified as a 5’-epimer of P-ergokryptine, P-ergokryptam (22), and the lactam alkaloid, P,P-ergoannam (26),which contains isoleucine as the first amino acid of the peptide moiety. Unnatural peptide ergot alkaloids, ergorine (70),ergonorine (71),and ergonornorine (72),were also obtained by Crespi-Perellino et uf. (292) when they fed norvaline to an ergocornine-producing Cfuvicepsstrain. These alkaloids form a new unnatural series carrying n-propyl substituents at C-5’ of the cyclol moiety. Moreover, 72 has the n-propyl group both at C-2’ and C-5’ and represents the first example of an ergopeptine with an unnatural amino acid adjacent to lysergic acid (Fig. 2). B. BIOCONVERSION OF ERGOT ALKALOIDS Bioconversion of ergot alkaloids has been explored both in the search for alkaloids with new or more useful pharmacological properties and to facilitate the production of alkaloids already used in therapy. Clavines, lysergic acid derivatives, and semisynthetic ergolines were used as substrates. Besides Cfuviceps strains, other microorganisms, rat liver homogenates and isolated enzymes were applied as biocatalysts. A few examples will be given below. Agroclavine (31)and elymoclavine (32)are converted to the stereoisomeric setoclavines (36)and penniclavines (35),respectively

5.

H'

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H' 69 Thiaergosine

70 (RI= Me;R2 = n-Pr) Ergorine 71 (RI= i-Pr; R2 = n-Pr) Ergonorine 72 (R,= R2 = n-Pr) Ergonomorine

Pr = Propyl FIG.2. Peptide alkaloids obtained by directed fermentation.

(207,192).Many filamentous fungi and other microorganisms, e.g., Aspergillus carbonarius, Cladosporium fulvum, Streptomyces griseus, Nocardia rubra, and also plant homogenates (193), catalyze these oxidations, which are mainly mediated by peroxidases. The C-8 oxidation of 8,9-ergolenes is accompanied by a shift of the double bond into the 9,10-position; the intermediates are the 10-hydroxy- or 8,9-epoxy derivatives (194). Submerged cultures of Cfaviceps strain SD58 transformed l-alkyl-, l-benzyl-, l-hydroxymethyl-, 2-halo-, 2,3-dihydro-, and 6-ethyl-6-noragroclavine to the corresponding elymoclavine derivatives (180). Some Fusarium species and strains of Streptomyces griseus catalyze the 8,9-double bond isomerization of elymoclavine to lysergol (107). Kren et al. (295) isolated a new C. purpurea strain with high invertase activity. Free and immoblized cells were used to catalyze fructosylation of elymoclavine, chanoclavine, and lysergol. Enzymatic synthesis of P-N-acetylhexosaminisides of clavines and of ergonovine was described, using P-N-acetylhexosaminidasefrom Aspergillus oryzae (296). Microbial transformations of the powerful hallucinogen, lysergic acid diethylamide (LSD) (73),have been studied by Ishii et al. (297-299). Especially the N-6 and the amide-N alkyl substituents were attacked. Streptomyces lavendulae demethylated only the N-6 position, yielding nor-LSD (74). S. roseochromogenes transformed only the amide-N alkyl group to yield lysergic acid ethylamide (75), lysergic acid vinylamide (76),and lysergic acid ethyl 2-hydroxyethylamide (77).A semisynthetic ergoline with pronounced dopaminergic activity, lergotrile (78), was used as substrate for

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about 40 biotransformation organisms. With Streptornyces species only N demethylation leading to norlergotrile (79) was observed (200) (Fig. 3). Hydroxylation at C-12 (80) or C-13 (81) takes place in mammalian systems, but was not achieved with micro-organisms (201).A comprehensive review on this topic was published by Kren (202).

VII. Pharmacological Properties of Ergolines

The first therapeutic application of ergot was mentioned in the famous “Kreuterbuch” of A. Lonicer in 1582 (4). He described the use of ergot sclerotia for inducing child-birth and for stopping post-partum bleeding. The scientific evaluation of the pharmacological properties of ergot alkaloids started with the isolation of ergotamine in 1918 (17).Genuine and semisynthetic ergot alkaloids exhibit an astonishingly broad spectrum of biological activities: uterotonic activity, increase and decrease of bloodpressure, induction of hyperthermia and emesis, dopaminergic and neuroleptic activity, control of the secretion of pituitary hormones. Comprehensive reviews of this fascinating topic are available (22,23,203-206).

H‘

H’

FIG.

3. Bioconversions of some ergolines.

5.

82 Nicergoline

NB N , C * H ,

BIOCHEMISTRY OF ERGOT ALKALOIDS

&$

83 Brornocriptine

/

H'

CH3-I-H'

N

\

CH3 84 Lisuride

86 1-Cyclopropylmethyl13-brornofestuclavine

85

LY 53857

87 1 -Allyld-norfcstuclavine

FIG.4. Pharmacologically active ergolines.

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A. BIOLOGICAL ACTIVITIES MEDIATED BY NEUROTRANSMIITER RECEPTORS The highly diversified activities of ergolines may be partially explained by the following assumptions (207): 1. Ergot alkaloids interfere at more than one type of specific receptor site. 2. The population of receptors to which ergot alkaloids have access varies from organ to organ. 3. Affinity and intrinsic activity vary from alkaloid to alkaloid as a function of their chemical structure. The structure analogy between the tetracyclic ergolines and the neurotransmitters serotonin, noradrenaline, and dopamine may contribute to the ability of different ergot alkaloids to interfere with various specific receptors. Furthermore, the structural differences between the ergolines and these biogenic amines may explain why some of the ergot alkaloids act as partial agonists and/or antagonists on receptor sites of neurotransmitters (207). Clavines and simple lysergic acid amides possess a high affinity for serotoninergic binding sites (5-HT receptors) and the peptide-type ergot alkaloids show, in general, a high affinity for a-adrenergic receptors. In some cases, pronounced effects of slight structural changes in a given ergoline alkaloid on the pharmacological activity have been observed. For example, when the double bond at position 9,lO of the lysergic acid moiety of ergotamine is hydrogenated the sympathicolytic-adrenolytic effects are specifically enhanced, whereas vasoconstriction is diminished. Dihydroergotamine (DHE) is in use as a prophylactic agent for the treatment of migraine and some forms of vascular headache (208). Besides DHE, other dihydro derivatives of ergopeptine alkaloids show interesting pharmacological activities. Dihydroergotoxine is an antihypertensive agent and improves cerebral metabolism. It stimulates dopamine and serotonin receptors. Later, another ergoline derivative, nicergoline (82), was developed which is a potent blocking agent for a,-adrenoreceptors. Nicergoline is also used as an antihypertensive agent and acts like dihydroergotoxine on cerebral metabolism (209). Bromination of a-ergokryptine in position 2 increases the dopamine agonist activity and lowers the oxytocic and vasoconstrictor side effects. Bromocriptine (83) was the first clinically useful prolactin inhibitor which is used in the treatment of galactorrhea, puerperal mastitis, prolactindependent mammary carcinoma, and Parkinsonism (220). Lisuride (84), an 8-aminoergoline with a simpler structure than 83, has pronounced prolactininhibitory activity and is used clinically in the same way as bromocryptine. Moreover, it shows remarkably high serotonin antagonist activity and is also used in the treatment of migraine.

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The interactions of genuine and semisynthetic ergolines with serotonin (5-hydroxytryptamine) receptors have been studied intensively (206). The 5-HT receptor family is rather heterogenous with regard to their biochemical, molecular biological, and pharmacological properties. The 5-HT receptors are divided into seven main groups which comprise several subtypes. One group should be mentioned. Therapeutic indications for ~ - H T antag~A onists in the treatment of CNS diseases include depression, anxiety, schizo~A antagophrenia, and sexual disorders (212).Nonergoline ~ - H T receptor nists show a lack of specificity and selectivity. A prototype of ergoline 5HTZAreceptor antagonists proved to be compound LY 53857 (85), which showed high antagonistic activity for vascular 5-HT2Areceptors and negligible q-adrenergic, histaminergic, and dopaminergic activity (213,214). This compound carries an isopropyl group at N-1 and an ester group at C-8.

B. ERGOLINES WITH ANTITUMOR A N D ANTIMICROBIAL PROPERTIES

It is well documented that various ergolines with high antiprolactin activity due to their dopaminergic action, e.g., 83 and 84, are useful agents in the treatment of mammary tumors. On the other hand, some ergolines reveal in vitro cytostatic effects comparable to clinically used antitumor antibiotics. These antitumor properties are apparently not mediated by the interaction with neurotransmitter receptors, but the mechanism of action seems to be a fundamentally new one for ergolines. Cytostatically active ergolines are agroclavine (31) and festuclavine (33) and derivatives thereof. Other clavines and simple lysergic acid derivatives are inactive, suggesting a requirement for a methyl group at C-8 of the ergoline system (215, 216). Some festuclavine derivatives, e.g., 1-cyclopropylmethyl-13-bromofestuclavine (86), have pronounced antineoplastic activity in vivo (217,218) and are now undergoing clinical trials. Some of the antitumor active ergolines show mutagenic effects in the Ames test, but certain clavine derivatives exhibiting low mutagenicity are among the most active cytostatic agents (219,220). Inhibitory effects on nucleoside uptake by human lymphoid leukemia cells and on incorporation into DNA and RNA, respectively, were observed (221). However, the exact mechanism of action of tumor inhibition by ergolines remains to be clarified. Eich’s group (222,223) also investigated the antibiotic activities of simple ergolines. Some derivatives of 31 and 33, especially 1-allyl-6-norfestuclavine (87) showed remarkable in vitro activity against some pathogenic and nonpathogenic bacteria and the yeast Candida afbicans.

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VIII. Future Challenges Ergot has been called a treasure house for drugs (224). This pool of pharmacologically active agents is by no means exhausted. The tremendous increase of our knowledge of neurotransmitter receptors will stimulate further research on ergoline alkaloid pharmacology. The search for more effective compounds will continue, especially for agents with high specificity and high selectivity. Specificity means affinity for a given receptor type and selectivity refers to the differentiation between various subtypes of a receptor. Promising candidates are inter alia some tetracyclic clavines, 6,7secoergolines, and compounds possessing the ergoline pharmacophore combined with a highly selective dopamine receptor agonist of the D1-subtype, e.g., the benzoergolines (206).

A. ENZYMOLOGY A N D MOLECULAR GENETICS

Our knowledge about the enzymes involved in ergoline alkaloid biosynthesis is woefully incomplete. The best characterized enzyme is dimethylallyltryptophan synthase, catalyzing the first pathway-specific step in ergoline formation. The gene, dmaW, encoding this prenyltransferase and a nearfull-length cDNA were cloned and sequenced. The sequence of dmaW and its cDNA indicates that the gene encodes a 455 amino acid polypeptide with a predicted molecular mass of 51,824 Da and a possible prenyl diphosphate binding motif, Asp-Asp-Ser-TyrAsn. The cDNA was expressed in yeast; extracts of the transformants containing the sense construct catalyzed the formation of DMAT (225). The expression of enzymatically active recombinant DMAT synthase from Claviceps in yeast is a landmark in ergot alkaloid biochemistry and sets an example for further studies on ergot alkaloid-related enzymes. The heterologous expression of dmaW will further aid in clarifying the mechanism of this intriguing reaction and in unravelling the complex regulation of ergoline biosynthesis; it may also eventually improve the biotechnological production of ergot alkaloids. However, this accomplishment must only be a beginning. Purification to homogeneity, characterization, cloning, and expression of other clavine-specific enzymes is urgently needed. Some enzymes of this pathway have not even been detected yet, e.g., enzymes converting N-methyl-DMAT (54) into chanoclavine-I (37).Further studies at the enzymatic and molecular genetic level will help in the

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elucidation of some fascinating reaction mechanisms and will provide the basis for a deeper understanding of the physiology and regulation of ergot alkaloid formation. Enzymes catalyzing individual steps of the ergoline pathway are either cytoplasmic or compartmentalized in microsomes. The intracellular trafficking of these alkaloidal intermediates is another point of interest. Similarly, the mechanism of formation of the simple lysergic acid derivatives is poorly understood; none of the enzymes catalyzing the biosynthesis of ergometrine or lysergic acid a-hydroxyethylamide have been detected so far. The most intriguing unsolved problem is the enzymology of cyclol-type alkaloid formation. Purification of a hypothetical “cyclol synthetase” has not yet been achieved. The d-lysergic acid activating enzyme isolated by Keller el al. (238) did not activate the other amino acids involved in ergotamine biosynthesis. Thus, the question arises whether the hypothetical cyclol synthetase is composed of two multienzymes? One enzyme could activate d-lysergic acid, and the other the three amino acids typical for a given peptide alkaloid. The second multienzyme would presumably also catalyze transfer reactions leading to an enzyme-bound lysergyltripeptide, in analogy to the enzymology of peptide antibiotics, e.g., gramicidin S formation (226). The intermediate peptides are most likely attached to the enzyme via a 4’-phosphopantheteine, as has been demonstrated in peptide antibiotic biosynthesis (258,226). Release from the enzyme is probably coupled with lactam formation. It remains to be established whether hydroxylation of the a-carbon of the amino acid adjacent to lysergic acid occurs on this multienzyme, conceivably resulting in direct release of the final cyclol. Alternatively, this hydroxylation, followed by spontaneous or enzymatic cyclol formation, may be catalyzed by a separate enzyme operating on the free ergopeptam released from the multienzyme. As an alternative to this involvement of two multienzyme complexes it is possible that all reactions necessary for ergot peptide alkaloid formation are catalyzed on a single polypeptide chain. In contrast to the prokaryotic enzymes, eukaryotic peptide synthetases exist mostly as single, multifunctional enzymes (227). The first example of this is L-a-aminoadipoyl-Lcysteinyl-D-valine (ACV) synthetase which catalyzes the formation of the precursor tripeptide of the penicillin and cephalosporin antibiotics (228). A second, more dramatic example by cyclosporin synthetase, a 1.4 MDa single protein (229) catalyzing the 40 steps necessary to assemble and modify a peptide of 11 amino acids (230). A definitive answer to these questions in the case of peptide ergot alkaloids probably requires cloning, sequencing, and analysis of the cyclol synthetase gene(s).

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B. REGULATION A fair amount of work has been carried out on the physiology and regulation of ergoline alkaloid formation in Cluviceps cultures. A number of regulatory mechanisms have been identified and studied at the cellular and enzymatic level (78,231,232).Most important among these is the recognition of tryptophan not only as a precursor of ergot alkaloids, but also as an inducer of their synthesis. In this regulatory role, tryptophan can be replaced by a wide range of analogs, most of which do not serve as precursors of the alkaloids. The induction has a limited time window and leads to elevated levels of alkaloid synthesis and alkaloid-synthesizing enzymes. The ergot alkaloids were one of the first systems in which the phenomenon of substrate induction of secondary metabolite formation was demonstrated. Another regulatory phenomenon encountered in this biosynthesis is feedback inhibition of alkaloid synthesis by the endproduct, elymoclavine. Again this was demonstrated both in vivo and at the enzymatic level. Many other physiological studies on ergot alkaloid formation were carried out in the 1960s and 1970s, which, due to space limitations, cannot be reviewed here. Virtually all these investigations on the physiology and regulation of ergoline biosynthesis were carried out several decades ago. The interesting phenomena they uncovered must be re-examined in the light of our vastly increased knowledge of molecular and cellular biology and with the improved tools of molecular genetics available today. Only then can we expect to acquire an understanding of the intricate control mechanisms governing ergoline alkaloid biosynthesis at the molecular level. C . EVOLUTIONARY ASPECTS

Ergoline alkaloids have been isolated predominantly from Cluviceps species, from several other fungal genera, and from members of one higher plant, family, the Convolvulaceue. Apparently these alkaloids are not formed in prokaryotic organisms, in contrast to the maytansines, which occur in several families of higher plants and in bacteria (233).The rather isolated occurrence of ergolines in fungi and in one higher plant family raises the question whether nature has twice independently “invented” the ability to synthesize these alkaloids, or whether the biosynthetic machinery has evolved only once and then passed on from a fungus to the plant or vice versa by lateral gene transfer. A priori, independent evolution of the same complicated biosynthetic pathway more than once would seem to require extraordinary selective pressure which is difficult to envisage in the case of secondary metabolites like the ergot alkaloids. Thus, this scenario

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does not appear very likely, suggesting lateral gene transfer as the more plausible alternative. It seems more likely that the genes encoding ergoline biosynthesis have evolved in a fungus and were then passed on to the plant, rather than the reverse. Clustering of pathway-specific genes would be advantageous for a horizontal gene transfer, and such clustering is quite common in fungi (234,235),but not in higher plants. Little factual information is available to answer these important questions. Virtually all of our knowledge about ergoline biosynthesis stems from investigations with Claviceps strains. Only very preliminary studies have been performed using Zpomoea plants, which showed that the same precursors, tryptophan and mevalonic acid, are used to assemble the ergoline system (236). We need more information on the biosynthetic pathway to ergolines, on the enzymes involved, and on the genes encoding them in Claviceps, but particularly in higher plants. Comparison of these features should reveal whether the biosynthetic pathways in the fungus and in the plant show signs of a common genetic ancestry or whether they appear to be unrelated. If the deduced amino acid sequences of key ergoline-specific biosynthetic enzymes show significant similarity, this would argue in favor of a common evolutionary origin, and hence a lateral gene transfer. Conversely, if there is no such homology we may conclude that convergent evolution has led to genes with different nucleotide sequences, but with similar functions (237).In this case, or if even the biosyntheticpathways are radically different, parallel evolution of the genetic information to synthesize ergolines may have evolved independently in taxonomically distant organisms. If, as seems more likely, the ability to synthesize ergoline alkaloids has been dispersed by horizontal gene transfer, the question then arises how this process has occurred and why it has been so selective. More detailed comparisons of the genes encoding the pathway in fungi and plants may help answer this and related questions, which have general far-reaching importance beyond the specific case of the ergot alkaloids. The unanswered questions outlined above, the importance of ergolines as therapeutic agents, and the concern about ergot poisoning in cattle and potentially in man, should ensure that the ergot alkaloids will continue to command great interest and provide a fertile ground for scientific investigation for many years to come.

Acknowledgments

We are greatly indebted to Mrs. Heide Pietsch, Halle, for typing the manuscript, and to Mr. Paul R. Shipley, Seattle, for drawing the figures for this chapter.

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References

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-CHAPTER L

NATURAL POLYAMINE DERIVATIVES-NEW ASPECTS OF THEIR ISOLATION, STRUCTURE ELUCIDATION, AND SYNTHESIS ARMINGUGGISBERG AND MANFRED HESSE Organisch-chemisches Instifur der Universith’t Zurich 8057 Zurich, Switzerland

I. Introduction ..................................................................................... 11. Alkaloids with the Spermidine Skeleton

.................................................

219 221

A. Alkaloids of Oncinotis Species

A. Biogenetic Cons B. Synthesis of the

References ........................

I. Introduction

Since very early times, polyamines have aroused the interest of scientists. As early as 1678, during microscopic examinations of animal sperm, Lewenhoeck (today written as Antoni van Leeuwenhoek, 1632-1723) obtained crystals (I),that were later (2) interpreted to be spermine phosphate. About 100 years later, in 1791, Vauquelin (3) verified this crystallization without knowing about the work of L,eeuwenhoek. After nearly another 100 years, in 1865, Boettcher ( 4 ) repeated the confirmation. However, only after the comprehensive analyses of Rosenheim et al. ( 5 ) was the structure of spermine brought to light. The structure was established by a synthesis and the comparison of crystals of its phosphate with those of the natural phosphate (2,5). In retrospect, we can only be amazed that the structure of such an uncomplicated, saturated, small molecule was elucidated so late, even though the means of carrying out such an elucidation had been available THE ALKALOIDS, VOL. 50 OOW-Y5Y8/YX $25.00

219

Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved.

220

GUGGISBERG AND HESSE

some 50 years earlier. However, as was so often the case, the chemical and physical behavior of spermine was incompatible with the usual methods used for a structure elucidation. This was especially true of spermine's polarity, solubility in water, and its instability in oxygen. Spermine (7) is a typical representative of the polyamines. These compounds are a class of organic compounds, in which the chain of a saturated hydrocarbon contains varying numbers of secondary amino groups (cf. Scheme 1).In polyamines, these amino groups are usually separated from H2N

/\/\/ NH2

1, Putrescine (PA 4, 1,4-diaminobutane)

2, Cadaverine (PA 5, 1,5-diarninopentane) H2N-Nw

H

NH2

3, Spermidine (PA 34)

H2NMN-NH2

H

4, syrn-Norspermidine(PA 33) H2N

N -NH2 H

5, N-(3-Aminopropyl)-l,5-diaminopentane (PA 35)

-

6, Thermine (PA 333)

H2N

- N H

H N\/\/NH2

7, Spermine (PA 343)

8, Thermospermine (PA 334)

SCHEME 1. Some natural polyamines (PA). The number represents the methylene groups between the nitrogen atoms (7).

6. NATURAL

POLYAMINE DERIVATIVES

221

one another by 3, 4, or 5 methylene groups. Besides 7, spermidine (3) and putrescine (1) are found particularly frequently in natural substances. Polyamines with another composition, or even another structure, are also known, e.g., cadaverine (2), sym-norspermidine (4), N-(3-aminopropyl)1,5-diaminopentane (S), thermine (6), or thermospermine (8) ( 6 ) .The occurrence of polyamines is not restricted to animals, but seems to encompass all living things. Apart from the unsubstituted bases, many N-alkylated, N-acylated and, particularly, (4-hydroxypheny1)propenoic acid derivatives have been isolated from natural sources, or their presence therein has been demonstrated (6-22). N-Acylated and N-methylated derivatives of polyamines have been found in animals (particularly in spider venom, see later), while both types, i.e., N-alkylated, as well as N-acylated, derivatives, are known in plants. The appearance of polyamines in specific plants will be described in detail in the following sections.

11. Alkaloids with the Spermidine Skeleton

Spermidine (3) is found in many plant families as the basic component of a few natural products. Among these genera are Cannabis, Codonocarpus, Equisetum, Lunaria, Maytenus, Oncinotis, Peripterygia, and Pleurostylia ( 6 ) .Recently, the diversity of the structures of natural spermidine derivatives in Oncinotis tenuiloba Stapf. was demonstrated in a particularly impressive way (see Scheme 2). OF ONCINOTIS SPECIES A. ALKALOIDS

Aside from the known bases, inandenin-12-one (10) and inandenin13-one, the corresponding alcohols, inandenin-12-01, and inandenin-13-01, were isolated and identified (22). Neooncinotine (11) and oncinotine (13) were previously isolated from Oncinotis species (Apocynaceae) ( 6 ) and detected in 0. tenuifoba (23). New, however, is the occurrence of N ( 4 ) benzoylspermidine (9) (14), whose structure was derived by spectroscopic means and confirmed by synthesis. Also new is the isolation of oncinotin12-one (14) and oncinotin-ll-one (15), (25) and, for the first time ever, a natural product containing both a spermidine and a spermine, namely, tenuilobine (12) (26). This compound represents something completely new. It contains two different bases, whose amides are connected via a hexadecanoic diacid. This is the fatty acid which, together with spermidine, forms all of the natural macrocyclic lactam alkaloids in Oncinotis species.

222

GUGGISBERG AND HESSE

H2N

NH2

9 N(4)-Benzoylspermidine

12 Tenuilobine

i"

11 Neooncinotine

10

Inandenin-12-one

NH2

H2N

13 R1=R2=H2,Oncinotine 14 R'=H2, R2=0, Oncinotin-12-one

15 R'=O, R2=H2, Oncinotin-11-one

SCHEME2. Types of alkaloids from Oncinotis tenuiloba Stapf.

6.

NATURAL POLYAMINE DERIVATIVES

223

Tenuilobine (12) behaves, as expected, like other 1,3-diaminopropane derivatives that have a free primary amino and a tertiary amido group (17): when the free base is allowed to stand for a long time at room temperature, two base-catalyzed rearrangements occur stepwise (verified by thin-layer chromatography). Through these rearrangements first one and then both base residues form secondary amides from which 1,38-diamino-4,9,13,30,34penta-aza-octatriacontane-14,29-dione(22) is finally obtained (18). The structure of 12was clarified by spectroscopic methods and proven by synthesis (Scheme 3) (18). Starting with monomethyl hexadecanedioate (16)and the end-positioned, doubly protected spermidine 17,the monoamide 18 was synthesized, which was saponified to the acid 19. Repetition of the amide formation step, but now with the appropriate spermine derivative, yielded the penta-protected tenuilobine, which gave 12 after deprotection. By using KAPA (KH/1,3diaminopropane) (17), 12 can be rearranged to the straight-chain pentaaza derivative 22. The rearrangement product 22 was not found in Oncinotis tenuiloba. Since tenuilobine (12),as well as the macrocyclic inandeninones (e.g., lo), consist of a CI6-chainlinked with the corresponding polyamine portion, a biogenetic relationship can be taken into consideration (Scheme 4). A common precursor, such as the mono-amide 19,may be reduced enzymatically to give the intermediate 23. Now, cyclization (pathway a) and appropriate oxidation of the carbon chain (introduction of the keto group) would result directly in the formation of 10,which bears an exocyclic 4-aminobutyl group. In the case of oncinotine (13),the assumed oxidation process would lead to inandenin-10-one, which was shown (19) to be transformed in vitro into 13 via a second cyclization process involving N(5) and the C(10) keto group. The generation of neooncinotine (11) could be explained analogously. Here, cyclization would occur at the 4-aminobutyl group of the precursor 23 (pathway b). This is presumed to be a minor process, since oncinotine (13) is accompanied by only small amounts of neooncinotine (11)(13,20). It is also possible that the dicarboxylic acid in 19 is unsaturated, allowing the formation of the keto group via an intermediate epoxide. Oncinotin-11-one (14)is another biogenetic, and also chemically, interesting alkaloid. On acetylation (Ac20/NaOAc), the compound forms the monacetylation product 24, as well as the diacetyl derivative 25 (Scheme 5). Compound 25 is the partial “retro Michael” reaction product in which the bicyclo[l5.4.0]henicosane system in 14 has been opened to give a cyclohenicosane derivative. Catalytic hydrogenation produced N,N’diacetylinandenin-12-one (26),which was in accord with the corresponding compound synthesized from inandenin-12-one (10)(22).For the unequivocal assignment of the structure of 25, i.e., the determination of the position

224

GUGGISBERG AND HESSE

CmCH3

I (CHz)i4 I COOH 16

+

0 (CHz)?i-NHZ 1-Methyl-2-chloropyridiniumI iodide/Et3N, CH2C12,A II * ROOC-(CH2)14-C-N NH 96% (CH2)d-NHZ

I

I I

(CH2)d -NHZ

z l

18 R=CH3

17

(CHZIB-NHZ

19 R=H

MeOH/2N aq. NaOH

quant. (CHZ)~-NHZ

I

1-Methyl9-chloropyridinium iodide, EtSN, CH2Cl2

(CH2)4

I I

NZ

91Yo

1'

R\

(CH2)s-NHZ 20 I

?

,H

H\ N

(CH 2 ) A

N H' 'R

F

21 R=Z

93%

12 R=H

a7% SCHEME 3. Synthesis of tenuilobine (12) and isotenuilobine (22).

6.

NATURAL POLYAMINE DERIVATIVES

225

Oncinotine (13) lnandeninones (e.g., 10)

Tenuilobine (12)

Neooncinotine (11)

SCHEME 4. A possible biogenetic pathway for the major alkaloids of 0. tenuiloba.

of the C,C-double bond, catalytic hydrogenation with D2 was carried out and the D2 derivative, D2-26, was subjected to the well-known Schmidt degradation (21) (Scheme 6). Similarly, the acetyl derivative of the alkaloid 14 itself was also degraded with NaN3/H2S04.A comparison of the results from the MS and G U M S analysis of the degradation products with those from the corresponding degradation of the undeuterated N,N'-diacetylinandenin-12-one (26) yielded the indicated structure (25). Apart from 14, there is another compound in Oncinotis tenuiloba which is an isomer of 14, but which was obtained in such small quanities that its structure could not be determined with any certainty. Therefore, the structure was confirmed by carrying out the synthesis of the postulated compound, oncinotin-11-one (15) (Scheme 7) (22). The starting material was 3,7-diazabicyclo[5.4.0]undecan-2-one (27), which was alkylated to 29 with N,N'-dibenzylaminoalkyne(28). Catalytic hydrogenation of the triple bond gave 30, a protected spermidine derivative, in which the piperidine ring of the desired molecule was already present, but from which the Clo alkyl chain bonded to C(16) was still missing. This latter group was introduced by an organo-lithium alkylation with a THP-protected o-bromoalkanol. The resulting monocyclic compound 31 was transformed via the alcohol 32 into the amino acid 33. The cyclization was achieved via its acid chloride according to the dilution principle and the final yield was 31%. The

226

GUGGISBERG A N D HESSE

NaOAdAgO 2d, 23"

LNHAC SCHEME 5. Conversion of an oncinotine skeleton to an inandenine nucleus.

deprotected compound 34 proved to be identical with the natural product 15 (22). Recently, an asymmetric synthesis of oncinotine (13) has been described (23,24). Aside from the macrocyclic spermidine derivatives of the inandenines and the oncinotines, spermidine alkaloids with 13-membered rings also occur in nature. So far, about 30 13-membered spermidine alkaloids have been isolated from plants and had their structures elucidated (6,25-28).

D

LNHAC

LNHAC

NH,

COOCH~

NH2

a) NaN$HZSO4 b) 2 N HCI, 150" c) Org. layer: CH2N2/Et20

SCHEME6. Schmidt degradation of oncinotine-12-one (14) and N,N-diacetyl-inandenin-12-one(26).

COOCH~

mcozEt

COzEt

fiCN

L

N

H

EtOH

N C, ' . c

75%

U

MeONa (2 eq.) EtOH, A

67% 27

1

1) t-BUOK (1.3 eq.) 2) CI-CHz-CC-CHz-NBnz28 DMF

67% HdRh-C(5%) THF

cl"j3

t

znB N7@ J

53% 30 29

71%

31

32 Jones Ox. Acetone 89%

-

HOOC 33

Nan2

+ Li-(CHZ),,-O-THP

H

N

w

N

B

n

z

34

15

SCHEME7. Synthesis of oncinotine-11-one (15).

6.

NATURAL POLYAMINE DERIVATIVES

229

The classification of these alkaloids can be made according to different points of view. With respect to their biogenetic formation, they can be divided into two groups: those whose formation involves an interaction between spermidine and an aliphatic carboxylic acid (the aliphatic group) and the aromatic group, which is represented by the 3-phenylpropenoic acid derivatives of spermidine. These macrocyclic spermidine alkaloids can be distinguished by the structure of their ring skeleta. In the celacinnines, the primary amine of the C3N moiety of spermidine forms the amide group; to this category belong the compounds (+)-loesenerine and (-)-celacinnine, as well as their derivatives. In contrast to the celacinnines (Scheme 8), the amide group of dihydroperiphyllines contain the C4N moiety of spermidine. Among others, (+)-palustrine and (- )-dihydroperiphylline, as well as their derivatives, belong to this latter type. The following discussion will be based upon the classification principle just described. Both the aliphatic and aromatic groups contain representatives of the other, the ring-isomeric group of compounds. CLASSOF SPERMIDINE ALKALOIDS B. THECELACINNINE 1. The Loesenerines

In 1987, (+)-loesenerine (35) was isolated from the plant Maytenus loeseneri Urb. (Celastraceae) and its structure was elucidated by spectroscopic methods (25). One year later, (+)-17,18-didehydroloesenerine (36) and (+)-16,17-didehydroloesenerin-18-ol(37) were extracted from the same plant material and it was established that both belonged to the same group of alkaloids (26). At the same time, (+)-myricoidine (38) and (+)dihydromyricoidine (39) were isolated from the plant Clerodendron rnyricoides Vatke (Verbenaceae) (Scheme 8) (27). All five alkaloids possess the same ring and side-chain skeleton. Compounds 35,36,38, and 39 have only one chiral center at C(8), while 37 has a second chiral center at C(18). The relative configuration of 35 was determined by comparing its optical rotation with that of the model compound, (+)-(R)-3-methoxybut-l-ene(40) (29). Since both compounds had the same positive rotation direction, 35 was assigned the (R)-configuration (25). On the same basis it was concluded that 38 and 39 also had the (R)configuration. The N-(9)-nitroso derivatives of 35,36, and 37 each produced the same Cotton effect, whereby it was deduced that both chiral centers of 36 and 37 were R. The correlation between 35 and 40 appeared to us to be completely inadequate, so we wanted to clarify the absolute configuration of 35 by employing an asymmetric synthesis. We were able to show that 35 has the (S)-configuration, and, that therefore, the configuration at C(8) of the derivatives 36,37,38, and 39 must also be changed.

230

GUGGISBERG AND HESSE

6H 37 (+)-16,17-Didehydroloesenerin-l8-ol 35 36 38 39

R=Ac, 17,l 8=H2 R=Ac, R=H R=H, 17,18=H2

(+)-Loesenerine (+)-17,18-Didehydroloesenerine (+)-Myricoidine (+)-Dihydromyricoidine

0

41 R’=H, R2=(€)-PhCH=CHC0 42 R’=H, R2=(Z)-PhCH=CHC0 43 R’=H, R2=PhC0 44 R’=H, R2=(Furan-3-yl)carbonyl 45 RLH, R~=OH 46 RLH, R~=AC 47 RLOH, R2=(~-phcH=cHco

48 R=H 50 R=OH

(-)-Pleurostyline (-)-7-Hydroxypleurostyline

(-)-Celacinnine (-)-Celallocinnine Celabenzine (-)-Celafurine (+)-( S)-Mayfoline (-)-( S)-N’-Acetyl-N’-deoxy mayfoline 7-Hydroxycelacinnine

49 7’-Hydroxy-7, 8-dihydropleurostyline

SCHEME8. The loesenerine type spermidine alkaloids. (The absolute configuration of compounds 35-38 and 51 is corrected and that of 45 and 46 confirmed by synthesis).

6.

NATURAL POLYAMINE DERIVATIVES

231

51 (+)-Cyclocelabenzine 52 R=H (+)-lsocyciocelabenzine 53 R=OH (+)-Hydroxyisocyclocelabenzine

SCHEME8. Continued

In 1993, the synthesis of (5)-tetrahydromyricoidine [( +)-(54)], an alkaloid of the celacinnine type, was reported (30)(cf. Scheme 9). Starting with the lactam 57, the synthesis of the alkaloid (5)-tetrahydromyricoidine (?)54 was carried out by using two ring enlargement reactions. For the construction of the bicyclic lactam, the method of Wasserman and co-workers (32) was used. This method includes the conjugated addition of the perhydropyridazine 56 to the a,P-unsaturated ester 55 to give the bicyclic product. Thus, treatment of methyl (E)-Zdecenoate (55) (32) with 56 (33, 34) by heating in toluene for 30 h gave 9-heptyl-l,6-diazabicyclo[4.3.0]nonan-7one (57) in a yield of 91%. The cleavage of the bridging N,N-bond of the bicyclic compound with sodium in liquid ammonia was carried out according to the method of Kemp and co-workers (35)to give compound 58. The amino group in lactam 58 could be protected successfully by the Boc group (36,37). Thus, treatment of lactam 58 with B o q O in the presence of a catalytic amount of 4-(dimethylamino)-pyridine afforded compound 59 in a yield of 89%. Treatment of the sodium salt of lactam 59 with acrylonitrile, followed by catalytic reduction of the nitrile function in compound 60, gave the amine 61. Because of the labile Boc function, acidic conditions were avoided in the hydrogenation steps and NH40H in EtOH was used as the solvent. Although the yield of the reduction step was low, only 42%, the alkylation of the amide proceeded almost quantitatively. Deprotection of the secondary amine to give 62 was readily accomplished by exposing compound 61 to trifluoroacetic acid (TFA). Treatment of 61 with TFA at 20" for 10 min, evaporation of the TFA, dissolving the residue in 2,4-lutidine, and refluxing the solution for 1.5 h gave 54 in a yield of 62%. Another synthetic aim was the production of optically active loesenerine (34). This synthesis was planned under the assumption that (+ )-loesenerine

232

GUGGISBERG AND HESSE

60

59

58

H$Kat.

62

61

(4-54

R= -(CH2),Me

SCHEME9. Synthesis of (?)-tetrahydromyricoidine [( +)-54] (30).

has the (@-configuration. Therefore the synthesis of (R)-loesenerine was carried out as shown in Scheme 10. This compound proved not to be the natural enantiomer (38-40). By coupling the chiral a,S-unsaturated ester (E,S)-63 with the perhydropyridazine 56, followed by cyclization in toluene, the bicyclic diastereoisomeric pair (R,S)-65 and (S,S)-65 were obtained in a ratio of 12: 1 (Scheme 10). The side-product 64 can be explained in terms of oxidation by air. The desired diastereoisomer (R,S)-65, in a yield of 81.3%, was separated from (S,S)-65 by extensive chromatography. The relative configurations of (R,S)-65 and (S,S)-65 were determined by X-ray crystallographic analyses.

6.

233

NATURAL POLYAMINE DERIVATIVES

The transformation of (R,S)-65 via the 9-membered 66 into the 13membered intermediate product 69 shows an analogy to the synthesis of the racemic tetrahydro-myricoidine [( +)-(54)] (Scheme 9). After the introduction of N-protecting groups, the aldehyde 73 could be generated. The alkyl group was then introduced in the (Z)-configuration by a Wittig reaction. Unfortunately, a partial racemization was observed during this reaction step. The cleavage of the Z-protecting group from compound 74 was achieved with Me3Sil in CH3CN (41). (-)-Dihydromyricoidine (38) and its N-acetylation product, and therefore also the (-)-loesenerine (34)that had been obtained, proved to be the enantiomers of the natural alkaloids (40). Thus the assignments of the absolute configurations that were given in Scheme 8 were reversed. 2. Mayfoline

(+)-(S)-Mayfoline (45) (42) and ( - ) - ( S ) - N ( 1)-acetyl-N(1)-deoxymayfoline (46)(43) were isolated from Maytenus buxifolia (A. Rich.) Griseb. (Celastraceae) and structurally characterized. The structure of 46 was confirmed by a synthesis of the racemate (44). Both of the alkaloids 45 and

56

4.2% 64

6.8%(S,S)-65

81.3% (R,Sj-65

SCHEME 10. Asymmetric synthesis of (+)-(R)-loesenerine (35) (40).

234

GUGGISBERG AND HESSE

(R,S)-65

THF 86%

f

I

1)EtONdEtOH RT, 30 rnin; 15h HV quant. 2)Acrylonitrile Toluene, 0”

NH2

-

HdRa-Ni, 50 psi, NH3aq in EtOH 76%

91Yo

0

0

0

69

70

I

Z-CI, THF NEt(i-Pr)2 96%

-

Ho$%N-Bo~H%,. ’%H

HO

I

~

CSA, MoLsieve 90% MeOH,

->(” 0

72

~ ‘“H

N

I 71

SCHEME 10. Continued

-

B

O

6. NATURAL POLYAMINE DERIVATIVES

235

74

38 (-)-Dihydromyricoidine

34 (-)-Loesenerine

SCHEME 10. Continued

46 have similar ORD-spectra, and were assigned as S absolute configuration by comparison of their ORD-spectra with those of (R)-a-phenylethylamine and (R)-a-phenylpropylamine, whose absolute configurations are known. A recently performed asymmetric synthesis has confirmed the (S)configuration of natural (+)-mayfoline (45) (Scheme 11) (45).

0

0

76

75

f'CN

NaOEt, Acrylonitrile

H

R

H

81 HdRa-Ni EtOH

79 R=H 80 R=Et

78

I (-..SNH2 - (--SN 73%

0

;"&y, 86%

ph&*

H

ph&*'

H

82 orAc20 Davis Reagent, 75% CHpCI2 85%

SCHEME 11. Synthesis of (+)-(S)-mayfoline (45).

6 . NATURAL POLYAMINE DERIVATIVES

237

(+)-(S)-Mayfoline (45) contains an hydroxyamino group, and it represents the only occurrence of this group in this family of 13-membered spermidine alkaloids. The optically active key intermediate, the 9membered azalactam 78, was prepared from optically active (-)-(3S)methyl-3-amino-3-phenylpropanoate(75) (46,47) and 2-ethoxypyrroline (76) in high yield. One of two methods developed for the formation of the nine-membered lactam 78 made use of the bicyclic 4-0x0-tetrahydropyrimidine 77. Reductive cleavage of (2S)-(+)-2-phenyl-2,6,7,8-tetrahydro-3H-pyrrolo[1,2alpyrimidin-4-one (77) was carried out by reacting it with three to four equivalents of NaBH3CN in the presence of HOAc (r.t., 2 h), followed by the usual work-up procedures. The product, (4S)-( - )-4-phenyl-13diazanonan-2-one (78) was obtained in yields of slightly greater than 30%. Results have shown that this route allows for the retention of chirality during the reductive cleavage without substantial racemization (32).Other products of this reductive ring expansion were the azalactam 79 and its N ethylated derivative 80. The unprecedented reduction-alkylation of the latter compound in liquid carboxylic acid solution has not appeared in the literature, although the use of NaBH4 has been reported (48-50). The Nalkylation of the amido group and its use for ring enlargment was discussed earlier in this chapter, so the conversion of 78 to 83 was performed without racemization. Lactam 83 was then converted to mayfoline (45) and the dihydroxy compound 84, by hydroxylation with Davis’ reagent (= ( 2 ) truns-2-(butylsulfonyl)-3-phenyloxaziridine),as well as to N ( 1)-acetyl-N( 1)deoxymayfoline (85) by acetylation using Ac20/pyridine (52,52). (-)-Celacinnine (41) and (-)-celallocinnine (42) were initially isolated from Muytenus arbutifoliu Wilczek (Celastraceae), and subsequently, (-)celacinnine, as well as celabenzine (43) and (-)-celafurine (44), were found in Tripterygiurn wilfordii Hook. f. (Celastraceae) (5339.(-)-Celacinnine (41) was the first spermidine alkaloid of this type for which the structure was already known (Scheme 8 ) . In addition to (-)-celacinnine (41) and (-)-celallocinnine (42), (-)-pleurostyline (48) has been isolated from Pleurosrylia ufricanu Loes (Celastraceae) (55). In 1992, 7-hydroxycelacinnine (47), 7‘-hydroxy-7’,8’-dihydropleurostyline (49) and ( -)-7-hydroxy-pleurostyline (50) were isolated from the leaves of Pleurostyliu oppositu Wall. (28), in addition to the previously known compounds, (-)-pleurostyline (48),(-)-celacinnine (41), and (-)celallocinnine (42). The difference between 50 and (-)-pleurostyline (48) is that the former carries an alcohol function in the a-position with respect to the lactam carbonyl group. The relative configuration of this hydroxy group was deduced from NOE experiments. Compound 47 probably exists as the racemate, because it does not display any Cotton effect and the optical rotation is zero. The [&IDvalue of celabenzine (43) is also zero (54).

238

GUGGISBERG AND HESSE

In addition to celabenzine (43), Wagner and Burghart isolated (+)cyclocelabenzine (51), (+)-isocyclocelabenzine (52), and (+)-hydroxyisocyclocelabenzine (53)from Maytenus rnossambicensis (Klotzsch) Blakelock var. rnossarnbicensis. Their structures were elucidated by spectroscopic methods, particularly through 'H- and I3C-NMR spectroscopy. However, no assignment of the configuration at each of the two chiral centers was made (56). An asymmetric synthesis of (+)-cyclocelabenzine (51) (57,58) enabled the absolute configuration of each of its chiral centers to be assigned as (S)-C(8) and (R)-C(13). During the planning of the asymmetric synthesis of cyclocelabenzine (51), it was assumed that the center at C(8) probably had the (S)configuration, like all other structurally known spermidine alkaloids. Therefore, (S)-P-phenylalanine was employed as the chiral building block. The configuration at the second center, C(13), was not predetermined, so that both options, R or S, were left open. 2-Phenyl-propan-l,3-diamine(86) served as the starting material and was transformed via 87 to its N-phthaloylN'-ethoxycarbonyl derivative 88. The insertion of the C4 moiety of the spermidine residue into the 1,3-propanediamine part was achieved by alkylation with 1-bromo-4-chlorobutane to give 89. In the presence of POC13at relatively high temperatures, 89 could be transformed into the isoquinolone 90, into which, after cleavage of the phthaloyl protecting group, (-)-(S)N-Boc-P-phenylalanine was inserted. The ring closure between the amine N-atom of phenylalanine and the C4 end could not be achieved by the alkylation of either the chloro- (91) or the iodo-compound 92. Therefore, a roundabout route via the alcohol 93, the aldehyde 94, and the Schiff base was chosen. The latter was finally reduced with NaBH3CN to yield the target molecule. The product proved to be a mixture of diastereoisomers, which could be separated chromatographically into the natural, albeit oily, (+)-isomer and the crystalline, synthetic (-)-isomer. The relative configuration of the (-)-isomer was determined by an X-ray crystallographic analysis, and on the basis of the known absolute configuration of the P-phenylalanine that had been employed, the absolute configuration of both diastereoisomers could then be assigned as (-)-(8S,13S)- and (+)(8S,13R) - 4,5,6,7,8,9,12,13- octahydro - 8 - phenyl- 2H-2,7,11- benzotriaza cyclopenta-decine-2,13-methano-l,lO-(3H, 1lH)-dione(51)(57,58).

C. THE DIHYDROPERIPHYLLINE CLASSOF SPERMIDINE ALKALOIDS This group of 13-membered ring alkaloids is isomeric with the loesenerines mentioned earlier in this section. The lactam is built up from the primary amine of the C4 unit of the spermidine moiety instead of from the C3 unit.

6iw 0

COOH

/

ToluenenHF

\ 86

NH2

87

1. CICOOEt, MeOH 2. Toluene 61YO

t N=Phth

Br-(CH2)&I NaH, DMF,

@N=phth

70% COOEt

89

1

COOEt

88

POC13, 155-170", 16h 55%

@?h

1. H2N-NH2 H20, EtOH, 60" 2. (-)-(S)-N-Bx-Rphenylalanine, Mukaiyama Reagent, NEt3, CHpCI2

*

57%

0 90

91

CI

Nal, Acetone, 12h

95%

0 93

HO

HMPT, H20, loo", 2.5 h

0 92

I

79%

SCHEME12. Synthesis of (+)-(8S,13R)-cyclocelabenzine (51).

240

GUGGISBERG AND HESSE

41%

1. CF,COOH, 0.5 h, r.t. 2. NEt,, MeOH, pH8 3. NaBH3CN,2 days

51

51 (+)-(ES, 13R)-Cyclocelabenzine

(-)-(8S, 139-Cyclocelabenzine

20 la], =+29.2"

= -165.1"

amorph

M.P. 242-243"

SCHEME12.

Continued

(+)-Palustrine (95) was originally isolated from the plant Equisetum palustre L. (Equisetaceae) in 1948 (59). (+)-Palustridhe (96) and 18deoxypalustrine (97) were later found as accompanying materials to 95

6. NATURAL

241

POLYAMINE DERIVATIVES

(60). Compound 95 was the first alkaloid of this type that had been isolated and shown to have a 13-membered ring skeleton. The analogy of the alkaloids of this group with the previously mentioned substances, particularly with myricoidine (38) and 17,18-didehydroloesenerine (36),suggests the hypothesis that the same 2,4,7-decatrienoic acid is used by nature for the synthesis of both types of compounds. The postulated reaction steps are shown in Scheme 13 and lead to a palustrine with a double bond in the 14,15-position. This reaction route is currently being investigated (62). A complete synthesis of (+)-palustrine (95) was achieved by Wasserman et af. (62). Interestingly, the spectroscopic data of the synthetic substance

-C02H

-

-

c 'H2N

N H

NH2

Spermidine (3)

(+)-Palustrine (98)

SCHEME 13. A postulated biogenetic pathway of (+)-palustrine (98).

95 R’=H, &OH

former structure of (+)-Palustrine

98 established structure of (+)-Palustrine

96 R’=CHO, R2=OH (+)-Palustridine 97 R1=R2=H 18-Deoxopalustrine

1H 5\

H - 17.-LOH

eCH3

H

100 (+)-Anhydrocannabisativine

99 (+)-Cannabisativine

101 102 103 104 105

eCH3

R=(€)-PhCH=CHCO (-)-Periphylline R=(Z)-PhCH=CHCO (-)-lsoperiphylline 2,3-H2, R=(€)-PhCH=CHCO (-)-Dihydroperiphylline 2,3-H2,R=149 mass units Perimargine 2,3-H2, R=151 mass units Dihydroperimargine

0

H

Ph 106 (-)-Neoperiphylline

SCHEME13. Continued

Ph

6.

NATURAL POLYAMINE DERIVATIVES

243

were not identical with those of the natural product (63). Wasserman speculated that the double bond in the natural substance had been incorrectly determined. Therefore, he suggested structure 98 as an alternative for (+)palustrine, (62). The results of chemical degradation and mass spectrometric fragmentation reactions indicate that the C,C-double bond can only be situated in the piperidine ring. In consideration of the finding that two vinyl protons are present in the molecule, there is, apart from structure 95 with the double bond between C(15)-C( 16), only one other possible alternative, and that is that the double bond lies between C(14)-C(15), as is the case in structure 98. In fact, in a parallel work, Natsume and Ogawa (64) synthesized in a stereoselective manner Al4*I5-(+)-palustrine (98), which was shown to be identical with the natural (+)-palustrine, terminating a longtime structural uncertainty. (+ )-Cannabisativine (99) and (+)-anhydrocannabisative (100)were isolated from Cannabis sativa L. (Moraceae). The relative configuration of (+)-99 was determined by an X-ray crystal-structure analysis (65). Later, the absolute configuration could also be deduced from an enantioselective total synthesis of (-)-cannabisativine (66,67). An additional group of compounds were isolated by Hocquemiller et al. from Peripterygia marginata Loes. (Celastraceae) (68). The first alkaloid, (-)-periphylline (101), became known in 1974, and the others, (-)isoperiphylline (102),(-)-dihydroperiphylline (103),perimargine (104), dihydroperimargine (105),and (-)-neoperiphylline (106),were discovered 3 years later. Perimargine and dihydroperimargine (105)were obtained as an inseparable mixture. The masses of the unknown parts of these compounds, obtained mass spectrometrically, were 149 and 151 amu, respectively. The absolute configuration of (-)-periphylline (101)was determined by a comparison of its ORD-spectrum with those of the spermine alkaloids (+)-chaenorhine (110,Scheme 14) and (-)-homaline (69),whose absolute configurations were already known at that time; see next section.

111. Spermine Alkaloids

A. BIOCENETIC CONSIDERATIONS Natural derivatives of spermine (7) and phenylpropenoic acid, including their hydroxylated and methoxylated descendants, are known (6). In many cases, the phenylpropenoyl group is located on the neighboring nitrogen atoms, N(1) and N(5), of one of the two 1,3-diaminopropane units. The biogenetic formation of the alkaloid aphelandrine (lll), depicted in Scheme

H Hh-N -i-NH2

@

n

@@

R R

108 R=R’=H, Verbascenine

NH

111 112 113 114 115 116

R=OH, Aphelandrine R=OH, Orantine, 17, 18 di(epi) R=OCH3, 0-Methylorantine R=OH, 30-OCH3, Ephedradine B R=OCH3, 30-OCH3, Ephedradine C R=OH, 26-OCH3, Ephedradine D

H3C0 110 Chaenorhine

109 R=OH, Chaenorpine

SCHEME 14. Alkaloid skeleta derived from N( l), N(S)-di(3-phenylpropenoyl)-spermine (lCn), a biogenetic approach.

6.

NATURAL POLYAMINE DERIVATIVES

245

14, has been studied extensively. It is formed from spermine [and also from the smaller polyamines, spermidine (3) and putrescine (l)] and phenylpropenoic acid (or phenylalanine and tyrosine, respectively) in the plant Aphelandra tetragonu (Vahl.) Nees, from whose roots the alkaloid was isolated (70). It is remarkable that all four of the alkaloid types depicted in Scheme 14 have the same absolute configuration ( S ) at the benzylic C-atom, even though the compounds were isolated from plants that belong to completely different families: Aphelandra (111,109,113 were isolated) and Encephalosphaeru ( l l l ) , are both members of the Acanthaceae, Premna (111) is from the Verbenaceae, Chaenorhinum (109,110),and Verbascum (108), the Scrophulariaceae, as well as Ephedru (ll2,114,115,116) from the Ephedraceae. For the cyclization of 1,5-di(3-phenylpropenoyl)spermine (107), the large ring is initially formed (linkage of groups A and B, Scheme 14) by a &addition (Michael reaction). At first glance, this coupling appears to be particularly unfavorable, but the formation of 17-membered ring does indeed ensue from the addition of the primary amino group in the @position with respect to a secondary amide, which, in addition, is conjugatively bonded to a phenol. The ring closure becomes plausible when we assume that the spermine derivative forms a complex in which the four oxygen atoms are arranged about a central atom, that atom being a metal ion (e.g., Mg2', Ca2+).In this case, the centers A and B lie within bonding distance of one another (!) and this makes the ring closure easily understandable. If no HO- or CH30-residues are present on either of the benzene rings, then no further ring formation will occur: the spermine 3-phenylpropenoyl derivative, remains monocyclic. Indeed, that is the reason for the occurrence of verbascenine (108). Otherwise, a phenolic oxidation occurs at the orthoposition with respect to the existing oxygen function and this can lead to additional ring closures, as can easily be seen from the structures of the alkaloids depicted in Scheme 14. A phenolic oxidation is also responsible for the fact that after the roots of Aphelundra tetragonu are ground, the alkaloid content decreases rapidly and after standing in the air for about 8 h in the presence of air, aphelandrine (111) can no longer be detected. It has been shown that a hydroxylation reaction occurs at the ortho-position with respect to the existing hydroxy group in 111 (72). Additional oxidation results in the formation of quinones and subsequent products that are no longer extractable. Presumably, when the whole plants of Verbuscum nigrum L. and Chaenorhinum minus (L.) Willk. et Lge. are dried, phenolic oxidation is responsible for the complete disappearance of the spermine alkaloids which had been detected earlier and isolated from the fresh plants (cf. (71,72)). The spermine derivative 107 depicted in Scheme 14 does not possess the unique substitution pattern of naturally occurring di(3-phenylpropenoy1)-

246

GUGGISBERG A N D HESSE

spermine. Presumably, homaline is formed from N(5),N(10)-di(3-phenylpropenoy1)spermine. This is an alkaloid which occurs in Homafium pronyeme (Flacourtiaceae). We presume that the biogenetic route put forward in Scheme 14 also makes the importance of these alkaloids for the plant plausible. The spermine derivative 107 is excellent for complexing cations (73,74). Concerning the possibility that the complexation of metal ions is the prerequisite for the ring closure, we can postulate for the biosynthesis of these types of alkaloids in plants the following model. The fact that the alkaloids are rapidly degraded when the integrity of the plant cells is ruptured (71) shows that they are compartmentalized inside the cells. The intracellular localization of a broad number of different types of alkaloids in plants was shown to be in the central vacuole (75). In vacuoles, different ions, including Ca2+,are also accumulated (76). If we assume that acylated spermine derivatives can be transported into the vacuole, the cyclization (ring closure) could occur there with the help of metal ions. It is highly possible that the macrocyclic alkaloid cannot repass the tonoplast which would explain its accumulation in the roots. At the site at which the metal ion is to be used, the complexing ability of the spermine derivative must be decreased. This occurs via ring closure to give compounds of the type lOB, and then through the subsequent phenolic oxidation to the alkaloids 109-116,whose complexing abilities are presumably less than that of 108 and, more importantly, less than that of 107 (74). The thus-formed alkaloid, e.g., aphelandrine (lll), is deposited in the roots of Aphefandra tetragona. However, it does appear that it still takes part in the metabolism of the plant, because the amount of the alkaloid (determined from the ratio of the amount of the alkaloid to the dried weight of the root) changes: it increases before flowering and decreases again afterward (77). The mechanism of this remobilization is completely unclear. Nevertheless, the finding that a spermine coumaroyl-transferase is located in the shoots, but not in the roots, of Aphefandra tetragona indicates that the acylated polyamines may be transported in the plant (78).Which enzymes or whether endophytic fungi are involved in the degradation is currently the subject of experimental investigations (79). B. SYNTHESIS OF THE MONOCYCLIC SPERMINE ALKALOIDS VERBACINE, VERBALLOCINE, VERBASCENINE, PMETHOXYCINNAMOYL-BUCHNERINE, AND BUCHNERINE

The idea was to make all macrocyclic spermine alkaloids that are derived from N( l),N(S)-di(3-phenylpropenoyl)spermine (107)accessible through a

6.

NATURAL POLYAMINE DERIVATIVES

247

generally applicable synthesis. In order to attain this objective, two key steps are necessary. First, a 17-membered compound of the type 108 must be synthesized according to Scheme 14. The formation of this macrocycle must be such that derivatives with phenolic hydroxy and methoxy groups can also be constructed. The second step in a general synthesis is the formation of the second ring, whereby, according to the substitution pattern, various phenolic oxidations are conceivable; cf., for example, the formation of 109,110, and 111from 108. Scheme 15 depicts a general synthesis corresponding to the first step in the formation of monocyclic spermine derivatives of the type 108. The problem has already been approached from another direction to produce 3-phenylpropenoic acid derivatives by two reactions with primary amines: the amine is added at the P-position and then a 3-phenylpropenoic acid amide is formed by aminolysis. However, it was found that this possibility for derivatization could not be carried out experimentally (80). If, however, a 3-phenylpropenoate of the type 117is heated with propane1,3-diamine, the desired compound of type 118 is produced (Scheme 15). In these compounds, all four N-atoms of the spermine skeleton are indeed present, but the central C4 moiety is still missing. This can be achieved in good yields by a cyclization with 1,4-dibromo- or 1,4-dimesyloxybutane. By suitable alteration of the protecting group in 120, compounds of the type 121 can be produced, into which the N-(3-phenyl)propenoyl substituents can be introduced. If the aminal protecting groups are now removed, we arrive at the end product of the type 122. In this way, the natural alkaloids verbacine (81),verballocine (82), verbascenine (72),p-methoxycinnamoylbuchnerine (82),and buchnerine (82) could be synthesized (83). As can be seen from Scheme 14, the compounds of type 122 correspond with those of 107 and are therefore possible biogenetic precursors of the aphelandrine (111)-chaenorhine (110) alkaloid family, which would result from a phenolic oxidation. The experimental investigation of this relationship is in progress (84).

IV. 3-Phenylpropenoyl Derivatives of Spermine and Spermidine

During the search for the biological precursors of aphelandrine (111) in Aphelandra plants, N ( 1),N(5)-di[4-hydroxy-(3-phenylpropenoyl)]spermine was isolated from the anthers (pollen sacs) of A . chamissioniuna Nees, which can be separated very cleanly from the other parts of the plant. This compound was identified using synthetic materials (85). Furthermore,

R

118 a R=H b R=OCH3

SCHEME 15. General synthesis of monocyclic spermine alkaloids (83).

NH2

6.

NATURAL POLYAMINE DERIVATIVES

249

somewhat surprisingly, the related spermidine derivatives, N(l),N(5)-

di[4-hydroxy(3-phenylpropenoyl)]spermidine, N(5),N(lO)-di[4-hydroxy(3-phenylpropenoyl)]spermidine, and N ( l),N(5),N( lO)-tri[4-hydroxy-( 3phenyl-propenoyl)]spermidine were also isolated and identified using synthetic products (8). No evidence for the presence of polyamines or their derivatives could be found in the female parts of the flowers. The significance of this observation cannot yet be assessed. The compounds in Scheme 14 that have one or two C,C-double bonds in the 3-phenylpropenoyl residue, isomerize on radiation with UV light (253.7 nm) to the ( E ) - [chaenorpine (109) and chaenorhine ( l l O ) ] or to the (2)-isomers [di(4-hydroxy-3-phenylpropenoyl)spermine].On irradiation with daylight or after chromatography under laboratory light, the starting compounds are obtained once more (86). In the case of the noncyclized di(3-phenylpropenoyl)spermine, mixtures of the ( E , E ) - , (E,Z)-, ( 2 , E ) - , and (2,Z)-isomers are observed. The same also applies to the corresponding spermidine derivative (87), which has recently been synthesized and spectroscopically studied (88,89). In addition, a few of these compounds can act as starting substances for phenolic oxidations, as described earlier for the spermidine derivatives. The known alkaloids, the lunarines (90) and the codonocarpines (92) are formed in this way (84).

V. Polyamines from Spiders, Wasps, and Marine Sponges Apart from the occurrence of polyamines and their derivatives in plants, in recent years, polyamine toxins have been isolated in increasing numbers from the venom of tropical spiders. Of particular interest were spiders of the genera Agelenopsis (92), Araneus, Argiope (93), Hololena, and Nephila (94). The molecular weights of these substances lie between 300 and 1000 Da, while those of the peptide toxins from these venoms, which, until recently had been the sole focus of attention, have masses of about 3000 Da. A few examples of these polyamine toxins are shown in Scheme 16 (7,95,96). Two of them have been taken from a recent review article. The number of methylene groups between two neighboring N-atoms is between 3 and 5, while, so far, the maximum number of amine N-atoms to be found in one molecule is 11. In addition, a-amino acids are sometimes included. The head region of the spider toxins frequently contains hydroxylated carboxylic acids that are bonded as amides to the actual polyamine. During the isolation of these compounds from the venom secretion, it must be born in mind that it is necessary to separate numerous (somewhere

Structure

Molecular weight

Trivialname and synonyms

Species

NPTX-1

Nephilla clavata (spider)

0

SCHEME16. Polyamine toxins from spiders (7), wasps (7), and marine sponge (95).

lanthoxin 433, PhTX-433, PTX-433)

6.

NATURAL POLYAMINE DERIVATIVES

251

between 10 and 50) individual components, which have very similar structures and which appear in quite different quantities in the mg range. The structure elucidation of such homologous and isomeric substances turns out to be extremely difficult. Aside from the analytical aspects and the identification of these compounds, their access by synthetic means plays an increasing role. With respect to the pharmacology of this class of alkaloids, see (97). In looking for a versatile synthetic approach to these compounds and their analogs, we aimed at synthesizing the penta-N-protected polyamine thermo-pentamine (128a,PA 3343) derivative, containing five independently removable N-protecting groups, which should allow the regioselective introduction of an acyl moiety (98,99). The selective protection of polyamines is a rather laborious and uneconomic task. Therefore, we decided to investigate a nonlinear approach to the construction of the target compound. Treatment of commercial 3-bromopropylamine hydrobromide 123 with pyridine-2-sulfonyl chloride gave the pyridine-2-sulfonamide derivative 124 (Scheme 17). Compound 124 was treated with diallylamine in the presence of diisopropylethylamine at 80°C in toluene to give the diallylamino derivative 125. Successive treatment of the latter with sodium hydride and l-bromo-3-chloropropane in DMF afforded the building block 126. Following literature methods (100,101), tert-butyl N-(8-arnino-4-benzyl-4-azaoct-l-yl)carbamate(127) can be prepared in five steps from commercially available starting materials, such as acrylonitrile and benzylamine, or in three steps from N-Boc-1,3diaminopropane (102) and N-(bromobuty1)phthalimide. Coupling of the two building blocks 126 and 127 using KF/Celite, or diisopropylethylamine (DIPEA) in the presence of catalytic amounts of sodium iodide gave the desired polyamine 128b. Finally, treatment of the polyamine l28b with trifluoroacetic anhydride and triethylamine at -20°C afforded the target polyamine 128a. Independent deprotection of each N-protecting group was then investigated (Scheme 18). These compounds are possible synthons for further natural polyamines. During the last decade a large number of natural occurring polyamine derivatives was discovered in plants, animals and in micro-organisms. General interest has increased in this the oldest group of natural products which have only one part in common, the basic polyamine back-bone, and show a large variation in their structures. Even so, according to the number of amino groups, the poly amine back-bone is not homogeneous, it varies between two and eleven amino groups. Because of their different origin, which includes different biogenetic pathways and because of their different, and in many cases remarkable, pharmacological behavior, a large number

252

GUGGISBERG AND HESSE

2pyrsO2CI K2C03 98%

HBr' H2NMBr

HN-Br

I

I

So2-2pyr 124

123

CI MNM

-

N(all~l)~

I

NaH, DMF, Br(CH2)3CI 97%

(allyl),NH, DIPEA, Toluene 90%

HN-

N(allyl),

I

SO2-2pyr 125

Nal, DIPEA, Toluene, A 66%

+

B ~ ~P' "M-~H N

-

I

Bn

127

I

B

8"

o

c

H

N

~

N

~

N

M

I

R

N

/

~

(

a

l

l

y

~

)

2

I

SO2-Ppyr

128a R=COCF3

DIPEA=Diisopropylethylamine

SCHEME 17. Synthesis of the penta-A'-protected pentamine 128.

of structural variations have been synthesized. Other polyamine derivatives are known to have no pharmacological activity at all [e.g., aphelandrine (lll)]. On the other hand, the function of the polyamines and their derivatives in nature is still not rationalized and not well understood. We hope that brilliant ideas and experiments of scientists will help to clarify their role in nature and so contribute, in general, to our understanding of the role of alkaloids in nature as well.

z

B

m

z I,

2

254

GUGGISBERG A N D HESSE

Acknowledgments

This work was generously supported by the Schweizerischen Nationalfonds and the Dr. Helmut Legerlotz-Stiftung. We thank Dr. Anthony Linden for linguistic assistance, Dr. Stefan Bienz for scientific discussions. and Jorg Heerklotz for drawing the formulas. We are also grateful to our co-workers for their scientific contributions, and especially for making unpublished results available: Dr. Laurent Bigler, Dr. Martin K. H. Doll, Dr. Catherine GoulaouicDubois, FrCdBric Gabriel, Ursula Hauserrnann, Albert Horni, Wenqing Hu, Dr. Jae Kyoung Pak, Dr. Elke Reder, Dr. Katja Schultz, Dr. Jiangao Song, Dr. Christa Werner.

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6. NATURAL

POLYAMINE DERIVATIVES

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256 71. 72. 73. 74. 75. 76. 77.

GUGGISBERG AND HESSE

M. Todorova, C. Werner, and M. Hesse, Phytochemistry 37, 1251 (1994). K. Seifert, S. Johne, and M. Hesse, Helv. Chim.Acta 65, 2540 (1982). E. Kimura, Tetrahedron 48, 6175 (1992).

L. Bigler and M. Hesse, unpublished results. P. Matile, Ann. Rev. Plant Physiol. 29, 193 (1978). K. S. Schumaker and H. Sze,J. Biol. Chem. 261, 12172 (1986). C. Werner, C. Hedberg, A. Lorenzi-Riatsch, and M. Hesse, fhytochernistry 33, 1033 (1993). 78. C. Hedberg, M. Hesse, and C. Werner, Plant Science 113, 149 (1996). 79. F. Gabriel, C. Werner, and M. Hesse, unpublished results. 80. B. Ganem, Acc. Chem. Res. 15,290 (1982). 81. K. Drandarov, Tetrahedron Lett. 36, 617 (1995). 82. S. Lumbu and C. HootelC, J. Nut. Prod. 56, 1418 (1993). 83. A. Guggisberg and M. Hesse, Helv. Chim.Acta in preparation. 84. A. Guggisberg, W. Hu, and M. Hesse, in preparation. 85. F. Veznik, A. Guggisberg, and M. Hesse, Helv. Chim.Acta 74,654 (1991). 86. B. F. Tawil, A. Guggisberg, and M. Hesse, J. fhotochem. Photobiology 54, 105 (1990). 87. W. Hu and M. Hesse, Helv. Chim.Acta 79,548 (1996). 88. W. Hu, Ph. D. Thesis, University of Zurich, in preparation. 89. W. Hu and M. Hesse, Helv. Chim.Acta, in preparation. 90. C. Poupat, H.-P. Husson, B. C. Das, P. Bladon, and P. Potier, Tetrahedron 28,3103 (1972). 91. R. W. Doskotch, A. B. Ray, and J. L. Beal, J. Chem. Soc., Chem. Commun. 300 (1971). 92. V. J. Jasys, P. R. Kelbaugh, D. M. Nason, D. Phillips, K. J. Rosnack, N. A. Saccomano, J. G. Stroh, and R. A. Volkmann, J. Am. Chem. Soc. 112,6696 (1990). 93. T. Budd, P. Clinton, A. Dell, I. R. Duce, S. J. Johnson, D. L. J. Quicke, G. W. Taylor, P. N. R. Ushenvood, and G. Usoh, Brain Research 448,30 (1988). 94. T. Chiba, T. Akizawa, M. Matsukawa, H. Pan-Hou, and M. Yoshioka, Chem. fharm. Bull. Jpn. 42, 1864 (1994). 95. N. Ushio-Sata, S. Matsunaga, N. Fusetani, K. Honda, and K. Yasumuro, Tetrahedron Lett. 37, 225 (1996). 96. K. D. McCormick and J. Meinwald, J. Chem. Ecology 19,2411 (1993). 97. H. L. Mueller, R. Roeloffs, and H. Jackson, in “The Alkaloids” (G. A. Cordell, eds.), Vol. 46, p. 63. Academic Press, San Diego, 1995. 98. C. Goulaouic-Dubois, A. Guggisberg, and M. Hesse, Tetrahedron 51, 12573 (1995). 99. J. K. Pak and M. Hesse, unpublished results. 100. R. J. Bergeron and J. S. McManis, J. Org. Chem. 53,3108 (1988). 101. V. J. Jasys, P. R. Kelbaugh, D. M. Nason, D. Phillips, N. A. Saccomano, and R. A. Volkmann, Tetrahedron Lett. 29,6223 (1988). 102. W. J. Fiedler and M. Hesse, Helv. Chim. Acfa 76, 1511 (1993).

-CHAPTER 7-

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS TONIM. KUTCHAN Laboratorium fur Molekulare Biologie Universitat Munchen 80333 Miinchen, Germany

...... .................................... A. Uses of Monoterpenoid Indole Alkaloids .........................................

I. Introduction

11. Monoterpenoi

B. Enzymatic Synthesis of Ajmaline C. Enzymatic Synthesis of Vindoline ................................................... D. Molecular Genetics of Tryptophan Decarboxylase .............................

258 259 259 259 263 265

................ 281 IV. Bisbenzylisoquinoline Alkaloids A. Uses of Bisbenzylisoquinolin B. Enzymatic Synthesis of Berb

B. Enzymatic Synthesis of Scopolamine ...........

E. Molecular Genetics of Hyoscyamine 6P-Hydroxylase

....................................

A. Uses of Acridone Alkaloids B. Enzymatic Synthesis of Furo

304

................................ THE ALKALOIDS. VOL. SO 00YY-Y598/98 52s.00

251

Copyright Q 1998 hy Academic Press All rights of reproduction in any form reserved.

258

KUTCHAN

I. Introduction

The current status of the alkaloid branch of the field of natural products reflects the many new advances in analytical chemistry and pharmacology. Only minimal quantities of pure alkaloid are now necessary for a complete structure elucidation by mass spectral and nuclear magnetic resonance spectroscopic analyses. Absolute stereochemistry can be unambiguously assigned by crystal structure determination. The pharmacological activities of crude plant extracts or pure substances are determined by fully automated systems enabling millions of data points to be collected per year in industrial screening programs. We excel1 at identifying biological activity and at elucidating the structure of the compound possessing this activity. What now remains to be done? It is clear the vast majority of the estimated 350,000 plant species remain uninvestigated for these purposes. It is also clear that the factor that limits the number of biological activities for which we can test is the number of cloned human target enzymes and receptors that are available. As the underlying biochemical bases for diseases and symptoms continue to be discovered and the various components cloned, our number of test systems will increase. What happens, though, when a small quantity of an alkaloid of complex chemical structure from a rare plant is discovered to be physiologically active, but the alkaloid still has to pass through clinical trials? Or clinical trials have been successfullycompleted, but now enough material is needed to satisfy market demand? What happens when the source plant does not show good plantation characteristics and a chemical synthesis is not commercially feasible? In order to develop an answer to these questions, we must understand how a plant synthesizes alkaloids and how this biosynthesis is regulated. Given this knowledge, we can develop biomimetic syntheses that are a combination of chemical and enzymatic steps. We can alter the metabolism of the plant to change the alkaloid profile. We can also influence the regulation of alkaloid biosynthesis in cell culture to produce a desired alkaloid. The manipulations necessary to develop an alternate system of production for a plant-derived pharmaceutical each requires the techniques of molecular genetics. There is a very applied aspect of this type of experimentation, pharmaceutical biotechnology, which is the development of new sources of pharmaceuticals that are too complex in structure to be chemically synthesized for industrial use. There will also be a major contribution to our basic knowledge concerning the regulation of alkaloid biosynthesis in plants and concerning the mechanisms by which alkaloid biosynthetic enzymes efficiently catalyze transformations that are not readily chemically reproduced. Since 1988,molecular genetics has been successfully

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

259

applied to the alkaloid field. Summarized herein are the contributions that molecular biology has made to our understanding of the biosynthesis of selected alkaloids in plants and toward the development of alternate production systems for these alkaloids over the past 7 years.

11. Monoterpenoid Indole Alkaloids INDOLE ALKALOIDS A. USESOF MONOTERPENOID

Containing over 1800 members of rich structural diversity, the monoterpenoid indole alkaloids are also a particularly fruitful source of physiologically active molecules. Historically, the use of Rauvol~?aserpentina as a medicinal plant for the treatment of a wide variety of ailments in India can be traced back 3000 years (1). In more recent times, the chemical structures of the biologically active components from plants have been arduously elucidated. In fact, in the latter part of this century, the monoterpenoid indole alkaloid-producing plants have proven to be a rich source of drugs that later became registered pharmaceuticals (Fig. 1). Select examples are the antineoplastic agents vinblastine (1)and vincristine (2), the adrenergic blocker and aphrodisiac yohimbine (3), the antihypertensives ajmalicine (4) and ajmaline (5), the rat poison and homeopathic drug strychnine (6), the antimalarial quinine (7), and the vasodilator vincamine (8). Of this collection of drugs, only vincamine (8) is prepared chemically for industrial use. The remainder are still isolated from the plants that produce them. The study of the biosynthesis of the monoterpenoid indole alkaloids catapulted when medicinal plant cell suspension cultures were introduced as the experimental system. The first enzyme specific to monoterpenoid indole alkaloid biosynthesis to be identified was strictosidine synthase [EC 4.3.3.21, which condenses tryptamine (9) and secologanin (10) to form the first alkaloidal intermediate 3a(S)-strictosidine (ll),from C. roseus cell suspension cultures (2,3).Rapid success was made thereafter in elucidating the enzyme catalyzed biosynthesis of several important indole alkaloids.

B. ENZYMATIC SYNTHESIS OF AJMALINE The biosynthesis of the monoterpenoid indole alkaloids in plants begins with the decarboxylation of the amino acid L-tryptophan (12) by tryptophan decarboxylase [aromatic L-amino acid decarboxylase; E C 4.1.1.281 to form tryptamine (9) ( 4 ) (Fig. 2). Tryptamine (9) is then condensed with the secoiridoid secologanin (lo), derived in multiple enzymatic steps from

260

KUTCHAN

a+...I& H

“‘yt

WJ

I HO ’“‘Cqm, R

R= CH, CH,

(Ell Ully) (1)

Vlnblaatlne

Velbe

Vlncrlrtlne

Oncovln (Ell Ully) (2)

H

I R= C=O

C a t h u u t h w roaeua

b Vincdmlne (8)

Yohlmblne (9) Yohlmbln Splegel (Kall-Chemle)

Cetal (Parke Davia)

Vissa minor

Coryrantbe johimbe

\

Ba(S)-Strlctoaidlne (11)

Quinine (7) Llmptar (Marion Menell Dow)

CinChOM OffiCfMf&

/ \

Ajmallclns (4)

Lamuran (Boehrlnger Mannhelm)

Rauwolfia rerpcntina

Ajmallne (6) Qlluryimal (Glullnl Pharma)

Rauwolfia rerpcntisa

FIG.1. Pharmaceutically useful monoterpenoid indole alkaloids derived from 3a(S)-strictosidine (11). The structural types are as follows: l,2--bis ibogamine/aspidosperma; 3yohimbine; 4-heteroyohimbine; 5-ajmaline; 6-strychnos; 7-cinchona; 8-eburnamine.

m H y

Tryptophy':

\

decarboxyiase

li L-Tryptophan (12)

;I Strictosidine

Tryptamine (9)

synthase

C

O

--

H

Geranioi (13)

Ba(S)-Strictosidine (11)

1

Secologanln (10) Strictosidine gluwsidase

OHC

Qlc

C-OCH,

-

Dehydrogeissoschizine oxidoreductase \

CH, \

CHsW

Qeissoschizine (16)

--

a-

4,21-Dehydrogeissoschizine (15)

l

Slrictosidipe-agiywne (14)

oI1 Poiyneuridine aldehyde esterase

Vinorine synthese H

I

co2

Poiyneuridine aldehyde (1 7)

AcCoA

CoA

T \

Vinorine (19)

16-epi-Vellosimine (18)

Vinorine hydroxyiase 170Acetyiajmaian acetyiesterase H

_reduc'ase(s) NADPH

H

17QAcetyl-norajmaline (21)

Norajmaiine (22)

0,

Vomiienlne

7 Ac

1

NADPH

m\ Vomilenine (20)

Norajmailne N-methyl-

SAHJ

\

OH

Ajmailne (5)

FIG.2. Biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline (5) in Rauvolfia serpentina.

18

262

KUTCHAN

geraniol(l3) ( 5 - 3 , to form 3a(S)-strictosidine (11) (2,3).Strictosidine can then be enzymatically permutated in a species-specific manner to form a multitude of diverse structures as depicted in Fig. 1. The biosynthetic pathway that leads to ajmaline in R. serpentina cell suspension cultures is now almost completely elucidated at the enzyme level (1).This pathway, as it currently stands, is summarized in Fig. 2. The glucose moiety of strictosidine (11) is hydrolyzed by specific glucosidases to form a highly unstable aglycone (14) (8). Strictosidine /3-glucosidases [EC 3.2.1.1051 have been found to occur only in those plant cell cultures that produce monoterpenoid indole alkaloids, suggesting that they are specific for this gluco-alkaloid (8,9). Strictosidine aglycone (14) opens to a highly reactive dialdehyde that over several, as yet undetermined, steps yields 4,21-dehydrogeissoschizine(15) (9). The transformation to the next alkaloid, geissoschizine (16) (ZO), proceeds through the reduction of the iminium ion by an oxidoreductase [EC 1.3.1.361 (1). The next conversion step transforms geissoschizine (16) into the sarpagan ring system of polyneuridine aldehyde (17). The formation of the sarpagan bridge is thought to involve a microsomal cytochrome P-450 enzyme due to the dependence of the formation of this skeleton on the presence of NADPH in the cell free extract (9). The reaction is also inhibited by carbon monoxide, ketoconazole, and cytochrome c. The ester moiety of polyneuridine aldehyde (17) is cleaved by a specific esterase, polyneuridine aldehyde esterase, and undergoes subsequent decarboxylation to 16-epi-vellosimine (18) (11). It is not yet clear whether the loss of C 0 2 is spontaneous or enzyme mediated, but since the enzyme is not inhibited by EDTA, a typical inhibitor of many metal ion-dependent decarboxylases, the decarboxylation is thought to proceed without enzyme mediation (12). Polyneuridine aldehyde esterase has been purified, well characterized, and partially sequenced (1). The sarpagan- is converted to the ajmalan-skeleton in the next reaction step by vinorine synthase (13). The formation of vinorine (19) from 16epi-vellosimine (18) requires acetyl CoA as co-substrate. The supposed intermediate of this reaction, deacetylvinorine, could not be isolated and may, therefore, remain bound to the enzyme prior to acetylation (14). Vomilenine (20) results from the introduction of a hydroxyl group at C21 through the action of a cytochrome P-450 mono-oxygenase on vinorine (19) (15). This enzyme activity is dependent on NADPH and O2 and is inhibited by carbon monoxide, ketoconazole, metyrapone, and cytochrome c. The CO inhibition is reversible by light. Taken together, these pieces of evidence strongly support the identity of this enzyme as a cytochrome P450 (15). The next two steps in the pathway to ajmaline are NADPH-dependent reductions. It is not yet known whether one or two oxidoreductases are

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

263

responsible for these conversions, but the sequence of events is thought to be reduction of the imine followed by reduction of the 19,20-double bond to 17-0-acetyl-norajmaline (21) (16).The next conversion is the hydrolysis of the 17-0-acetate moiety to norajmaline (22) followed by methylation at N-1 to ajmaline (5) (1). There is very little difference in the affinity of the acetylesterase for 17-0-acetyl-norajmaline (21) and for 17-0-acetyl-ajmaline. Likewise, the methyltransferase shows similar K Mvalues for 17-0-acetyl-norajmaline (21) and norajmaline (22) such that it is difficult to discern the exact order of the last two transformation steps in vivo (1). Beginning with tryptamine (9) and secologanin (lo), ajmaline ( 5 ) is biosynthesized in Rauvolfa cell cultures by at least ten enzymes requiring five moles of NADPH, one mole of acetyl CoA, and one mole of S-adenosyl-L-methionine per mole of product formed.

c . ENZYMATIC SYNTHESIS

OF VINDOLINE

The Iboga-type indole alkaloid catharanthine (23) and the Aspidospermatype indole alkaloid vindoline (24) are the immediate precursors that are oxidatively coupled to form the chemotherapeutic agent vinblastine (1) in Catharanthus roseus plants (Fig. 3). It has been suggested that tabersonine (25) is the precursor to both the Iboga and Aspidosperrna classes of alkaloid (10). The enzymatic synthesis of vindoline (24) from tabersonine (25) is now almost completely elucidated using a combination of C. roseus plants and cell suspension cultures. The proposed pathway is depicted in Fig. 3. The first conversion of tabersonine (25) is a hydroxylation step at C-16 catalyzed by a cytochrome P-450 monooxygenase to form 16hydroxytabersonine (26) (17). The reaction is dependent on NADPH and O2and is inhibited by clotrimazole, miconazole, cytochrome c, and carbon monooxide. This first enzyme of vindoline (24) biosynthesis is found both in cell culture and in young leaves. The newly introduced hydroxyl moiety is methylated in the next step along the pathway. 16-Hydroxytabersonine 16-0-methyltransferase [EC 2.1.1.941 requires S-adenosyl-L-methionine as cosubstrate and, as for tabersonine 16-hydroxylase, is found both in cell cultures (17) and young leaves of C. roseus plants (18). The second hydroxylation in the conversion of tabersonine (25) to vindoline (24) is thought to be a hydration of the 2,3-double bond to form 28. The enzyme that catalyzes this conversion has not yet been characterized. The N-methyltransferase [EC 2.1.1.991 that forms desacetoxyvindoline (29) is the first enzyme of the pathway to be found only in differentiated plant material and not in cell culture (19). It could be localized to the chloroplasts of C. roseus leaves and is specifically associated vith the thylakoids (20).

264

KUTCHAN

NADPH. O*

Tobrrronlno (25)

lE-Hydmxyl~borronlno (28)

18-Yothoxytabmonlne (27)

Hydrmlon

j i

D.amylvlndollno (30)

D s r a c e t o ~ n d o l l n e(29)

1E-YolhoXy-2.3-dlhydro3-hydroxyIaberaonlne (28)

a/ ok

' "2 m, cop(,

FIG.3. Biosynthetic pathway leading to the monoterpenoid indole alkaloid vindoline (24) in Cutharunthus roseus.

In view of these results, it is clear why Curharunthus cell cultures do not produce vindoline (24). The third hydroxylation in the pathway is the next step. Desacetoxyvindoline (29)is acted upon by a cytosolic 2-oxoglutarate-dependentdioxygenase [EC 1.14.11.111 resulting in hydroxylation at C-4 (21) to form deacetylvindoline (30). The enzyme requires, in addition to the co-substrate 2-oxoglutarate, ascorbate, ferrous ions, and molecular oxygen for activity. The mechanism of desacetoxyvindoline 4-hydroxylase has been investigated and the order of substrate binding found to be 2-oxoglutarate first, followed by O2 and desacetoxyvindoline (29) (22). The order of products released is deacetylvindoline (30) followed by C 0 2 and finally succinate. As with the N-methyltransferase, desacetoxyvindoline 4-hydroxylase is also absent

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

265

from Catharanthus cell cultures. The 4-hydroxylase has been purified to near homogeneity from C. roseus leaves and tryptic peptides have been prepared and sequenced (22). The final transformation is the acetylation of deacetylvindoline (30) to vindoline (24). The enzyme deacetylvindoline acetyltransferase [EC 2.3.1.1071 that catalyzes this reaction is cytosolic and requires acetyl CoA as cosubstrate (23,24). It is the third enzyme along the pathway that is absent from cell cultures. Beginning from the branch point intermediate tabersonine (25), vindoline is biosynthesized in C. roseus leaves through the action of six enzymes. The first two enzymes of the pathway are found in both cell cultures and leaves, while the last three enzymes are restricted to leaf material. Three of the six transformations are hydroxylations. These three introductions of oxygen into the Aspidosperma skeleton appear to proceed by three different mechanisms. The first mono-oxygenation is carried out by a microsomal cytochrome P-450, the second hydroxylation is supposed to be hydration of a double bond by an as yet unidentified enzyme and the third is catalyzed by a 2-oxoglutarate-dependentdioxygenase. The overall pathway requires and one mole one mole of NADPH, two moles of S-adenosyl-L-methionine, of acetyl CoA per mole of vindoline (24) formed. It is now clear that cell cultures of C. roseus cannot produce the bisindole alkaloid vinblastine (l),because at least the last three enzymes required for vindoline (24) biosynthesis are absent from cell culture (27). The enzymatic formation of vinblastine (1) from vindoline (24) and catharanthine (23) in C. roseus plants has itself been a point of contention. Although horseradish peroxidase (25), hemin and microperoxidase (heme undecapeptide) (26), and nonspecific peroxidases in C. roseus (27) are capable of catalyzing the oxidative coupling of the two monomeric units to 3’,4’-anhydro-1, it has not yet been unequivocally demonstrated that this reaction is mediated by a substrate- and species-specific enzyme in vivo in Catharanthus. The enzymatic transformations of alkaloid biosynthesis that have to date been thoroughly characterized have all been highly substrate specific enzymes. These enzymes have, in addition, been found to occur only in those species that accumulate the corresponding alkaloids. If nonspecific peroxidases are catalyzing the formation of 3’,4’-anhydro-1 in vivo in C. roseus, this would be a unique exception to this trend. D. MOLECULAR GENETICS OF TRYITOPHAN DECARBOXYLASE Tryptophan decarboxylase catalyzes the decarboxylation of the aromatic amino acid L-tryptophan (12) to tryptamine (9) (Fig. 4). In monoterpenoid indole alkaloid-producing plant species, tryptamine can then serve as a

266

KUTCHAN

Tryptophan decarboxylase H

H

Tryptamine (9)

L-Tryptophan (12)

FIG.4. Reaction catalyzed by tryptophan decarboxylase.

substrate for the enzyme strictosidine synthase, which catalyzes the first committed step in the biosynthesis of this class of alkaloids (2,3) (Fig. 2). The cDNA encoding tryptophan decarboxylase was isolated from a cDNA expression library of C. roseus prepared from poly(A)' RNA of developing seedlings (28). Antibodies raised against the purified enzyme were used to screen the library. The tryptophan decarboxylase cDNA encodes a protein of 500 amino acids (Table I) with a calculated molecular mass of 56,142 Da. The tryptophan decarboxylase amino acid sequence from C. roseus shows similarities to aromatic L-amino acid decarboxylase from Drosophila melanogaster (39% identity). Most decarboxylases require pyridoxal phosphate linked to the &-aminogroup of a lysine residue for enzymatic activity. By sequence comparison with the pyridoxal phosphate binding sites of pig kidney L-dopa decarboxylase, D. melanogaster L-dopa decarboxylase, and feline L-glutamate decarboxylase, lysine-319 correlates well as the cofactor binding site in the C. roseus enzyme (Table I) (28). The cDNA was expressed in Escherichia coli to produce enzymatically active tryptophan decarboxylase as a final proof of identity of the clone.

TABLE I AMINO ACIDSEQUENCE OF TRYPTOPHAN DECARBOXYLASE FROM C. roseus MGSIDSTNVA MSNSPVGEFK PLEAEEFRKQ AHRMVDFIAD YYKNVETYPV LSEVEPGYLR KRIPETAPYL PEPLDDIMKD IQKDIIPGMT NWMSPNFYAF FPATVSSAAF LGEMLSTALN SVGFTWVSSP RALEKLGPDS MVEDDVAAGY RHYLDGIERV DFKNWQIATG NFSLVCFRLK HVRRWJDLIQ

AATELEMIVM IGKLVCYGSD VPLFLCATLG DSLSLSPHKW RKFRSLKLWL PDVSSLHVEE KLTDDLLKEA

DWLAQILKLP QTHTMFPKTC TTSTTATDPV LLAYLDCTCL ILRSYGVVNL VNKKLLDMLN

KSFMFSGTGG KLAGIYPNNI DSLSEIANEF WVKQPHLLLR QSHIRSDVAM STGRVYMTHT

GVIQNTTSES RLIPTTVETD GIWIHVDAAY ALTTNPEYLK GKMFEEWVRS IVGGIYMLRL

Swiss-hot accession number P17770. EMBWGenBankDDBJ Databases accession number 304521. Lysine in boldface is the proposed binding site for the pyridoxal phosphate cofactor.

ILCTIIAARE FGISPQVLRK AGSACICPEF NKQSDLDKW DSRFEIWPR AVGSSLTEEH

7. MOLECULAR GENETICS OF

PLANT ALKALOID BIOSYNTHESIS

267

The tryptophan decarboxylase transcript was shown to accumulate in C. roseus cell suspension cultures exposed to a variety of elicitors (29,30). Auxin reduces transcription of the gene, as demonstrated by run-off transcription experiments with C. roseus nuclei (31). The cDNA has been heterologously expressed in tobacco plants, and presence of the transgene increased the level of tryptamine (9) (32) and, surprisingly, the level of tyramine (45) (33). It has also been utilized in the metabolic engineering of Brassica nupus (canola) (34). In oil seed crops such as canola, indole glucosinolates (31) in the seed protein meal limit use of the meal as animal feed due to decreased palatability. The tryptophan decarboxylase transgene in canola redirects tryptophan (12)pools away from indole glucosinolate biosynthesis and into tryptamine formation (Fig. 5a). The mature seed of the transgenic canola plants contains reduced indole glucosinolates (31), but no tryptamine (9), making it more suitable for use as animal feed. An additional example of the redirection of metabolic pathways in plants was achieved by introducing the C. roseus tryptophan decarboxylase cDNA into Solanum tuberosum cv DCs i d e (potato) (35). In this case, redirection of tryptophan (12) into tryptamine (9) resulted in a decrease in both tryptophan (12)and phenylalanine (32) pools in the transgenic potato tubers (Fig. 5b). This in turn led to a decrease in soluble and cell wall associated phenolics. Tubers from the transgenic plants were also more susceptible to infection by the pathogenic fungus Phytophthora infestans. These results suggest that regulation of the shikimate pathway can be modulated to down-regulate phenylpropanoid biosynthesis by introduction of a single gene outside of either pathway. These two examples of metabolic engineering of foodstuff plants by an alkaloid biosynthetic gene from a medicinal plant provide fine examples of how wide the scope of application for alkaloid genes can be.

E. MOLECULAR GENETICS OF STRICTOSIDINE SYNTHASE Strictosidine synthase catalyzes the stereospecific condensation of the primary amino group of tryptamine (9) and the aldehyde moiety of the iridoid glucoside secologanin (10) to form the first monoterpenoid indole alkaloid 3a(S)-strictosidine (11) (Fig. 6) (2,3). There is biotechnological interest in strictosidine synthase because, although tryptamine (9) and secologanin (10) can be chemically condensed, both diastereomers vincoside (3fl(R)-ll) and strictosidine (11) are formed. Only strictosidine ( l l ) , the exclusive product of the enzymatic reaction, serves as a precursor for the monterpenoid indole alkaloids (2,3), and could be used in biomimetic syntheses of known and of new alkaloids. Strictosidine synthase has been

268

KUTCHAN

a H

.7

lndole glucoslnolate (31) **'*

Tryptophan decarboxylase

H L-Tryptophan (12)

H Tryptamine (9)

b

6H Shikimic acid

1 I

-- -

---I-L'--

-&

H

L-Phenylalanlne (32)

L-Tryptophan (12)

I Q

Q

J

I

Tryptophan decarboxylase

I I I

9 Phenylpropanolds

~

N

H

.

H

Tryptamlne (9)

FIG.5. Introduction of the tryptophan decarboxylase cDNA into: (a) canola to divert metabolic pools of L-tryptophan (12) away from indole glucosinolate biosynthesis and into tryptamine (9): and (b) potato to redirect L-tryptophan (12) into tryptamine (9)in order to determine the overall effects on aromatic amino acid biosynthesis.

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

269

H Tryptamine (9)

3a(~)-Strictosidine (1 1)

Secologanin (1 0)

FIG.6. Reaction catalyzed by strictosidine synthase.

purified to apparent homogeneity from both C. roseus and R. serpentina cell suspension cultures (36,37).The C. roseus enzyme occurs in at least four isoforms (36). The enzyme from both sources is stable, requires no addition of cofactor for activity, and is readily immobilized (37-39) for the production of strictosidine (11). The cDNA encoding strictosidine synthase has been isolated from R. serpentina cell suspension cultures by screening of a A-phage library with an oligonucleotide based on the amino acid sequence of a tryptic peptide prepared from the homogeneous enzyme (40). A partial cDNA ( 4 2 ) and finally a full length clone (29) were subsequently isolated from C. roseus. The R. serpentina cDNA encodes a protein of 344 amino acids, 27 of which constitute a signal peptide (Table 11). The C. roseus cDNA encodes a protein that is eight amino acids longer (352), 31 of which are the signal peptide. The position of signal peptide cleavage was determined by computer analysis (42) to yield proteins with identical amino termini (Table 11) from both species. These two enzymes show 80% amino acid homology, which may reflect that both species are members of the Apocynaceae. DNA gel blot analysis using the R. serpentina cDNA as a hybridization probe showed that not all species that are known to contain the enzyme strictosidine synthase contain a highly similar gene, presumably due to differences in codon usage (43). Strictosidine synthase from R. serpentina has been functionally expressed in E. coli and the baker’s yeast Saccharomyces cerevisiae and in an insect cell culture (Spodoptera frugiperda Sf9 cells) (44,45). The predicted site of signal peptide cleavage could be verified with baculovirus expression of the Rauvolfia cDNA in insect cell culture (45). Expression of strictosidine

270

KUTCHAN

TABLE I1 AMINO ACIDSEQUENCE COMPARISON OF STRICTOSIDINE SYNTHASE FROM R. serpentina" AND FROM C. roseusb

J. MAKLSDSQTM -ALFTV-FLL FLSSSLAL-- SSPILKEILI EAPSYAPNSF TFDSTNKGFY SSSSS K F S A D NF E KS M V FMF L TSVQDGRVIK YEGPNSGFVD FAYASPYWNK AFCENSTDAE KRPLCGRTYD ISYNLQNNQL T F P DYK S M YIVDCYYHLS WGSEGGHAT QLATSVDGVP FKWLYAVTVD QRTGIVYFTD VSTLYDDRGV SIH SPE Q G H C K Y --QQIMDTSD KTGRLIKYDP STKETTLLLK ELHVPGGAEV SADSSFVLVA EFLSHQIVKY GVEE N R M I G V NR WLEGPKKGTA EVLVKIPNPG NIKRNADGHF WVSSSEELDG NMHGRVDPKG IKFDEFGNIL S F T S GQ VSR G EVIPLPPPFA GEHFEQIQEH DGLLYIGTLF HGSVGILVYD ---KKGNSFV SSH s s DHDN Y YE

Q

Top sequence Swiss-Prot accession number P15324. EMBWGenBanklDDBJ Databases accession number Y00756. Bottom sequence Swiss-Prot accession number P18417. EMBLEenBanWDDBJ Databases accession number X61932. Only the C. roseus residues that differ from the R. serpenrina sequences are shown. The arrow indicates the position of signal peptide cleavage. The asparagine residues in boldface mark the putative N-linked carbohydrate binding sites.

synthase in insect cell culture as a preprotein resulted in secretion of the processed enzyme into the medium. A facile purification of the enzyme could be developed from the insect cell culture medium that yielded 4 mg/ liter enzymatically active enzyme in near homogeneous form. This is the equivalent amount that could be purified from more than 300 liters of R. serpentina cell suspension culture grown to stationary phase. This enriched source of enzyme in immobilized form has been used for the production of strictosidine (Z.-W. Shen and T. M. Kutchan, unpublished). The C. roseus enzyme has been heterologously expressed in tobacco and in E. coli (46,47).In the tobacco plant, the heterologous enzyme was clearly localized to the vacuole (46). The gene for strictosidine synthase, strl, has been isolated from R. serpentina (48).A single gene codes for the enzyme in both R. serpentina (48)

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

271

and in C. roseus (29).This suggests that the isoforms of the enzyme isolated from C. roseus are all derived from one gene through post-translational modification. The gene translation sequences indicate that the enzyme from Rauvoljia contains one putative N-linked carbohydrate attachment site and that from Catharanthus contains two such sites (Table 11). The occurrence of isoforms in C. roseus may be due to heterogeneity in the carbohydrate structure at the second site. The strictosidine synthase gene transcript accumulates predominantly in the roots and leaves of both plants, but it can also be detected in flowers and stems indicating that the alkaloids may be synthesized throughout the plant. The abundance of the C. roseus transcript can be increased in cell cultures exposed to a variety of elicitors (29,30). Auxin reduces transcription of the gene in cell culture as shown by radiolabeled run-off transcripts synthesized by C. roseus nuclei (29). The molecular genetics of monoterpenoid indole alkaloid biosynthesis continues to progress. Although not yet in the public domain, cDNAs encoding desacetoxyvindoline 4-hydroxylase (Fig. 7) have been isolated from C. roseus and sequenced (F. Vazquez-Flota, E. De Carolis, A. M. Alarco, and V. De Luca, unpublished). The clones show extensive amino acid sequence homology to other 2-oxoglutarate dependent dioxygenases such as hyoscyamine 6fl-hydroxylase from Hyoscyarnus niger (49). As for several other enzymes of alkaloid biosynthesis, desacetoxyvindoline 4-hydroxylase is encoded for by a single gene. This hydroxylase is one of the three enzymes of vindoline (24) biosynthesis that are known to be absent from cell culture and is therefore one of the reasons that this alkaloid cannot be produced in culture. The isolation of this gene will ultimately yield knowledge about the genetic factors that are controlling vindoline (24) biosynthesis in Catharanthus.

a, H,CO

'

Desacetoxyvindoline

4

N i H3c HO *CO&H3

2-Oxoglutarate

Few, 02

Desacetoxyvindoline (29)

Succinate

co,

Deacetylvindoline (30)

FIG.7. Reaction catalyzed by desacetoxyvindoline 4-hydroxylase.

272

KUTCHAN

111. Tetrahydrobenzylisoquinoline Alkaloids

A. USESOF TETRAHYDROBENZYLISOQUINOLINE ALKALOIDS The tetrahydrobenzylisoquinolinealkaloids hold a special position in the alkaloid field because the search for useful drugs of defined structure from plants began with the isolation of morphine (33)from dried latex of the opium poppy Papaver somniferum in 1806 by Serturner (50). The tetrahydrobenzylisoquinoline class of alkaloids contains such varied physiologically active members (Fig. 8) as the narcotic analgesic morphine (33),the antitussive and narcotic analgesic codeine (34),the antitussive noscapine (39,the vasodilator papaverine (36),the antibacterial berberine (27)used in the treatment of eye and intestinal infections, the sedative corydaline (38) used in the form of an extract of Corydalis cava, the antibacterial sanguinarine (39)used as a toothpaste and oral rinse additive in the form of an extract from Sanguinaria canadensis, and, finally, protopine (40)used as an extract of Fumaria oficinalis in the treatment of liver and gall bladder disease. Of this collection of pharmaceuticals, only papaverine (36) is synthetically produced for industrial use. The remainder of the alkaloids presented in Fig. 8 are either purified from plants or are used in the form of a crude extract. All of these industrially useful natural products are biosynthesized from (S)-reticuline (41). Major strides have been made in the elucidation of the enzymatic synthesis of tetrahydrobenzylisoquinoline alkaloids due in large part to the use of plant cell suspension cultures that have been optimized for the production of select alkaloids. Not only has the tetrahydrobenzylisoquinoline class of alkaloids yielded the first alkaloid to be isolated (morphine (33)),but also the first two alkaloid biosynthetic pathways, those of berberine (37)and macarpine (42),to be completely elucidated at the enzymic level.

SYNTHESIS OF BERBERINE B. ENZYMATIC The biosynthesis of the tetrahydrobenzylisoquinoline alkaloids in plants begins with a metabolic grid (Fig. 9) that eventually leads to formation of the first tetrahydrobenzylisoquinoline alkaloid (S)-norcoclaurine (43) (52,52).The pathway proceeds from two molecules of L-tyrosine (44)that are acted upon by either tyrosine decarboxylase [EC 4.1.1.251 to form tyramine (45)or by a phenol oxidase [either E C 1.10.3.1 or 1.14.18.11 to form L-dopa (46)(53), both units which can serve as precursor to the isoquinoline portion of benzylisoquinolines (5455). L-Dopa (46)can then be decarboxylated by dopa decarboxylase [EC 4.1.1.251to form dopamine

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

273

Protopine (40)

Codeine (34)

UX,,

. d

‘ 0 Sanguinarine (39) Viadent (Horner)

Noscapine (35) Capval (Dreluso)

Papaver aomniferum

\

/

Sanguinaria canadensis

Papaverine (36) BP-Papaverine (Burlington)

Phytonoxon N (Stelgemald)

Papaver somniferum

Corylalir cava

Corydcline (38)

Berberlne (37) Berber11 (Mann)

Berbcris vulgaris

FIG.8. Pharmaceutically useful alkaloids derived from the tetrahydrobenzylisoquinoline alkaloid (S)-reticuline (41). The structural types are as follows: 33,34-morphinan: 35phthalidisoquinoline: 36-benzylisoquinoline: 37-berberine: 38-protoberberine: 39benzo[c]phenanthridine; @-protopine.

274

KUTCHAN

Ho

Tyrosine deurboxylaae

Tyramine (45)

CU+* HO

Dopamlne (47)

coz

)

HO

I ,

(S)-Normclaurlne aynthaae

Ho

L-Tyrorlne (44)

HO

L-Dopa (46)

\

4

(S)-Norcoclaurlne (43)

coz

Tranraminare

HO

p-Hydroxyphenyiacetaldehyde (49)

SAM

Normciaurine 6OMethyl-

HO p-Hydroxyphenylpyruvatlc acid (48)

.

SAH

trenderase

Coclaurine N-Methyltranrlermae

(S)-Kmethylcoclaurlne 3-hydroxylare

--i-i-

NADPH. 0,

SAH

SAM

HO

(S)-3'-Hydroxy-IY-

(S)-IY-Methylcoclaurlne (51)

(S)-Coclaurine (50)

methylcoclaurlne (52) 3'-Hydroxy-Nmethylwclaurine lofare

Smulerlne

HIW

no Oz

\

H A

SAM

SAH

OCHa (S)-Reticullne (41)

(S)-Scoulerine (53)

(S)-Tetrahydro-

H A Berbarlne (37)

0,

(S)-Tetrahydmcolumbamlne (54)

<% , '

0%

0%

(S)-Canadina (55)

FIG.9. Biosynthetic pathway leading to berberine (37) in Berberis species.

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

275

(47). Dopamine (47) can also be formed by action of a phenol oxidase on tyramine (45). It is difficult to determine which of these pathways is predominant in a given plant because all of the enzyme activities are present in protein extracts (53). The benzyl moiety of (S)-norcoclaurine (43) is formed by transamination of L-tyrosine (44)by tyrosine aminotransferase [EC 2.6.1.51 to p-hydroxyphenylpyruvate (48) which is next decarboxylated to p-hydroxyphenylacetaldehyde (49) (53).Dopamine (47) and p-hydroxyphenylacetaldehyde (49) are then condensed to form the first tetrahydrobenzylisoquinolinealkaloid by (S)-norcoclaurine synthase [EC 4.2.1.781 (56,57).(S)-Norcoclaurine (43) is 6-0-methylated by norcoclaurine 6-0-methyltransferase [EC 2.1.1.1281 to (S)-coclaurine (50) (58,59) that is in turn N-methylated to (S)-N-methylcoclaurine (51) by (S)-coclaurine N-methyltransferase [EC 2.1.1.115; ( R , S ) tetrahydrobenzylisoquinoline N-methyltransferase] (60). (S)-N-Methylcoclaurine (51) is hydroxylated at C-3’ by the cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3‘-hydroxylase to form 52 (61) that is finally methylated by 3’-hydroxy-N-(S)-methylcoclaurine 4‘-0methyltransferase [EC 2.1.1.1161 to the branchpoint intermediate of benzylisoquinoline alkaloid biosynthesis, (S)-reticuline (41) (62). This biosynthetic pathway leading to (S)-reticuline (41) is a revision of an earlier accepted pathway that postulated the condensation of dopamine (47) with 3-hydroxy-49 to form 3‘-hydroxy-43 ((S)-norlaudanosoline) as the first alkaloidal intermediate in tetrahydrobenzylisoquinoline biosynthesis (63-65). The enzyme (S)-norcoclaurine synthase catalyzes the formation of both 43 and 3’-hydroxy-43 and was originally called (S)-norlaudanosoline synthase (56,57).The pathway was revised, however, based on the failure to incorporate L-dopa (46)and dopamine (47) into the benzyl portion of this class of alkaloids ( 5 4 , the absence of (S)-norlaudanosoline (3’-hydroxy43) in plant extracts as determined by even radioisotope dilution analysis (65), by the extremely efficient incorporation of the trihydroxylated (S)norcoclaur:-e (43) into the tetrahydroxylated (S)-reticuline (41), and by the relative substrate specificities of the enzymes involved in the formation of (S)-reticuline (41). The 6-0- and the N-methyltransferases are relatively unspecific enzymes. They will methylate the tetra-oxygenated homologs of (S)-norcoclaurine (43) as well, which could explain the incorporation of (3’-hydroxy-43) into (S)-reticuline (41) (55). (S)-Norlaudanosoline (3’-hydroxy-43) is now viewed as an unnatural alkaloid in the plant kingdom. In Berberis, sp. the N-methyl group of (S)-reticuline (41) is oxidized by the berberine bridge enzyme [EC 1.5.3.91 (66) to the berberine bridge carbon C-8 of (S)-scoulerine (53) (67). During the course of catalysis, the berberine bridge enzyme consumes one mole of O2 and produces one

276

KUTCHAN

mole of H 2 0 2per mole of product formed. The enzyme is compartmentalized in a smooth membranous vesicle with a specific gravity of p = 1.14 g/cm3 (68). The specific pathway from (S)-scoulerine (53)that leads to berberine (37) then proceeds with O-methylation by (S)-scoulerine 9-0methyltransferase [EC 2.1.1.1171 to (S)-tetrahydrocolumbamine (54) (69). The 3-O-methyl moiety of tetrahydrocolumbamine (54) is converted to the methylenedioxy bridge of canadine (55) by the microsomal cytochrome P450-dependent oxidase canadine synthase [EC 1.1.3.361 (70). The final step in the biosynthesis of berberine (37)is catalyzed by (S)tetrahydroprotoberberine oxidase [EC 1.3.3.81 (72,72). The enzyme consumes one mole of O2and produces one mole of H 2 0 2per mole of berberine (37)formed and is compartmentalized in smooth vesicles, together with the berberine bridge enzyme (68). This oxidase is specific for the (S)enantiomer, but will accept a series of tetrahydroprotoberberines and 1benzylisoquinoline alkaloids as substrates (72). The purified enzyme was shown to contain a covalently bound flavin and is inhibited by dicoumarol and the flavonoid morine, by the reaction endproducts, and by Hg2+,Ag”, and Cd2+ ions. This course of enzymic reactions from two moles of Ltyrosine (44)to one mole of berberine (37) consumes four moles of Sadenosylmethionine and two moles of NADPH. The stereochemistry of one-carbon transformations in berberine biosynthesis has been studied by chiral methyl group methodology and 3H NMR spectroscopy (73). The transfers of the methyl group of Sadenosylmethionine to either oxygen or nitrogen during the methyltransferase reaction occurs with inversion of configuration. The formation of the berberine bridge catalyzed by the berberine bridge enzyme involves the removal of a hydrogen from the N-methyl group and its replacement by the phenyl group in an inversion mode. And finally, the aromatization of ring C of the protoberberine nucleus catalyzed by (S)-tetrahydroprotoberberine oxidase involves the nonstereospecific removal of a hydrogen from C-8, the berberine bridge. The regulation of berberine biosynthesis in cell suspension cultures of Thalictrum glaucum has been analyzed by comparing the levels of enzymes present in cultures that do produce berberine (37) and in near isogenic cultures that do not accumulate this alkaloid (74). The methyltransferase activities are greatly reduced in the alkaloid-free strain and are the basis for the inability of this culture to biosynthesize alkaloids that occur after (S)-norcoclaurine (43) along the pathway: The missing enzymes are norcoclaurine 6-O-methyltransferase, (S)-coclaurine N-methyltransferase, 3’hydroxy-N-methylcoclaurine4’-O-methyltransferase, and (S)-scoulerine 9O-methyltransferase. The fact that all four members of one class of enzymes in one biosynthetic pathway are greatly reduced or absent from a cell

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

277

culture strain that has lost the ability to synthesize an alkaloid suggests that these methyltransferase genes are under coordinate control in this particular species. In a Thalictrum minus cell suspension culture strain, two enzymes of berberine biosynthesis, norcoclaurine 6-0-methyltransferase and (S)-tetrahydroprotoberberine oxidase, were found to be induced by the cytokinin 6-benzylaminopurine (75-77) and thereby to stimulate alkaloid biosynthesis. C.

ENZYMATIC SYNTHESIS OF (R)-CANADINE A N D CORYDALINE

The origin of the tetrahydroprotoberberines with an R-configuration at C-14, such as (R)-canadine (56) and corydaline (57) of C. c a w , has also been elucidated at the enzymic level. As is the case for the (S)-tetrahydroprotoberberines, the R-epimers are derived from ( S ) scoulerine (53) via (S)-reticuline (41). A reversible NADPH-dependent oxidoreductase system catalyzes the reduction of the 7,8-bond in berberine (37) to form 7,8-dihydroberberine (58) that is subsequently stereospecifically reduced to (R)-canadine (56) (78) (Fig. 10). Likewise, (S)-tetrahydrocolumbamine (54) can be oxidized by (S)-tetrahydroprotoberberine oxidase (Fig. 11) to columbamine (59) that is then 0-methylated by columbamine 2-0-methyltransferase [EC 2.1.1.1 181 to palmatine (60) (79). Palmatine (60) can be reduced by the reversible NADPH-dependent reductase to 7,8-dihydropalmatine (61) (80).The final steps in corydaline biosynthesis are a NADPH-dependent reduction and an S-adenosylmethionine-dependent methylation. D. ENZYMATIC SYNTHESIS OF MACARPINE The benzo[c]phenanthridine alkaloids sanguinarine (39), chelirubine (62), and macarpine (42) are accumulated in cell cultures of Eschscholzia californica (47) in response to treatment with elicitor substances (81).This class of highly oxidized alkaloids possesses antimicrobial (82,83),sedative

FIG.10. Biosynthesis of (R)-canadine (56) in Corydalis cuva.

278

Hzs KUTCHAN

(S)-Terrahydro. protoberberlne

Columbamlns 20rnemyllranrlarare

/ 02

(S)-Tetrahydroc4lumbamlne (54)

Corydallne (57)

HZ02

\

OCHs

7-s SAM

SAH

EH,

Columbamlne (59)

Palmatine (60)

7.8-Dlhydropalmatine (61)

FIG.11. Biosynthesis of corydaline (57) in Corydalis cava.

(84-86), and antiviral (87) activities, they function as inhibitors of Na'K+ dependent ATPase (88) and interact with vasopressin (V,) receptors (89). With such a plethora of biological activities, these alkaloids can function in plants as deterents to herbivores and pathogens. Of these three alkaloids that accumulate in the Eschscholziu cell cultures, macarpine (42) predominates (90,91). Dihydrochelirubine (63) had been previously identified as the main, constitutively accumulated, alkaloid of another cell culture strain of E. culifornica (92).It was principally with elicited cell suspension cultures of E. cafifornica that the biosynthesis of macarpine (42) was completely elucidated at the enzymic level. The pathway proceeds as follows. The biosynthesis of (S)-reticuline (41) is as given in Fig. 9. In E. culifornica, as in Berberis, (S)-reticuline (41) is oxidatively converted to ( S ) scoulerine (53) by action of the berberine bridge enzyme (67,81). The pathway (Fig. 12) diverges at this point from that of berberine (37) biosynthesis. Instead of methylation, methylenedioxy bridge formation takes place. (S)-Scoulerine (53) is oxidized by the first of a series of six cytochrome P-450 enzymes that are specificly involved in the biosynthesis of benzophenanthridines. The 10-methoxy moiety of (S)-scoulerine (53) is converted to the methylenedioxy bridge of (S)-cheilanthifoline (64) by the cytochrome P-450-dependent oxidase cheilanthifoline synthase [EC 1.1.3.331 (93,94). The second methylenedioxy bridge is closed by the action of a second cytochrome P-450-dependent oxidase, stylopine synthase [EC 1.1.3.321, on (S)-cheilanthifoline (64) (93,94).Both of these microsomal enzymes require O2 and NADPH for activity and are inhibited by typical cytochrome P-

(S)-Scoularlne (53)

(S)-Reticuline (41)

(S)-Chsllanlhifollne (64) NADPH 0,

N-Methylrtyloplns

I

Stylopine rynthare

N-Methyl transferare

NADPH SAH

(S)-(cls)-N-Methylttylopine (66)

Protopine (40)

NADPH 0,

1

SAM

(S)-Stylopine (65)

Prolopine 6-hydroxylase

Spontaneous

Dlhydrobenrophsnanlhridins OxIdaso

-

7-i0,

HzOt

Dlhydrosangulnarine (88)

6-Hydroxyprotaplne (67)

Sanguinarine (39)

\

NADPH

/--0

/--O

Dlhydrobenzophananthridine

-7-T

0,

SAH

SAM

Dlhydrochellrublne (63)

Chellrubine (62)

r o

100Methyltranstorare

-7.Y H*Oz

Dlhydrorangulnarlne 10-hydroxylare

10-Hydroxydlhydrosangulnarlne (89)

NADPH 12-hydroxylase f-0

/--0 Dlhydrobenrophananthrldlne oxidare

7-r '4

12-Hydroxydihydrochelirubine (70)

Dihydromacarplne (71)

H A

Macarplne (42)

FIG.12. Biosynthetic pathway leading t o macarpine (42) in various members of the Papaveraceae.

280

KUTCHAN

450 inhibitors such as ketoconazole, prochloraz, cytochrome c, and carbon monoxide. Stylopine (65) is then N-methylated by (S)-tetrahydroprotoberberine cis-N-methyltransferase [EC 2.1.1221 to (S)-cis-N-methylstylopine (66) (95). The next step is catalyzed by the cytochrome P-450-dependent monooxygenase (S)-cis-N-methylstylopine hydroxylase [EC 1.14.13.371 (96).This microsomal enzyme requires O2 and NADPH for the insertion of a hydroxyl group onto C-14 of (S)-cis-N-methylstylopine (66) that results in formation of protopine (40). Protopine (40)is hydroxylated at C-6 by the fourth cytochrome P-450 of the series, protopine 6-hydroxylase [EC 1.14.13.551, to 6-hydroxyprotopine (67), which spontaneously rearranges to the first molecule with the benzo[c]phenanthridine skeleton, dihydrosanguinarine (68) (97,98). Oxidation to the orange fluorescing alkaloid sanguinarine (39) is mediated by dihydrobenzophenanthridine oxidase [EC 1.5.3.121 (99), a copper containing enzyme that requires O2for activity and concomitantly produces H202during catalysis (200). This oxidase is inhibited by the copper chelator sodium diethyldithiocarbamate, as well as by KCN, sodium azide, dithiothreitol, and mercaptoethanol. Sanguinarine (39) is an endproduct alkaloid, but dihydrosanguinarine (68) can be further transformed to 10-hydroxydihydrosanguinarine (69) through oxidation by the fifth cytochrome P-450dependent enzyme, dihydrosanguinarine 10-hydroxylase [EC 1.14.13.561 (202). 10-Hydroxy-dihydrosanguinarine(69) is methylated by 10-hydroxydihydrosanguinarine 10-0-methyltransferase [EC 2.1.1.1191to dihydrochelirubine (63), which can then be oxidized by dihydrobenzophenanthridine oxidase to the yellow fluorescent alkaloid chelirubine (62) (99,200). Chelirubine (62) is also an endproduct of this pathway, but dihydrochelirubine (63) can be hydroxylated by the final cytochrome P-450 monooxygenase, dihydrochelirubine 12-hydroxylase [EC 1.14.13.571, to 12-hydroxydihydrochelirubine(70), which is in turn methylated by 12hydroxydihydrochelirubine 12-0-methyltransferase [EC 2.1.1.1201 to dihydromacarpine (71) (202). The oxidation of dihydromacarpine (71) to the red fluorescent macarpine (42) is the final transformation step catalyzed also by dihydrobenzophenanthridine oxidase (99,200).From two molecules of L-tyrosine (44) to the most highly oxidized benzophenanthridine macarpine (42), six molecules of S-adenosylmethionine,seven molecules of NADPH, and ten molecules of molecular oxygen are consumed. During the process of elicitation of benzophenanthridine alkaloid biosynthesis in E. cdifornica cell suspension cultures, seven of the enzymes that lead from (S)-reticuline (41) to macarpine (42) are induced (202-203). These seven are the vesicular berberine bridge enzyme and the six aforementioned microsomal cytochrome P-450 enzymes. The remaining enzymes, which are cytosolic, increase only two- to three-fold in response to

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

281

treatment of the cultures with elicitor. In contrast to the control of berberine (37) biosynthesis in T. glaucum, where the presence of methyltransferases in the cultures appears to be rate limiting (74), in E. californica, the level of the membrane-associated enzymes is apparently the regulating factor. Induction of the biosynthesis of benzophenanthridine alkaloids in cell culture, in response to elicitor treatment, was first reported for fungal elicitation of P. somniferum (104) and for S. canadensis (100). In E. californica, alkaloid biosynthesis is stimulated by a series of biotic and abiotic elicitor substances (81), including the general plant signal tranducer methyl jasmonate (105,106).Treatment of the cell cultures with the inducer methyl jasmonate typically resulted in a 10-15-fold increase in enzyme activities of the berberine bridge enzyme, and of the cytochromes P-450 involved in benzophenanthridine biosynthesis (103). Dihydrobenzophenanthridine oxidase activity was either two-fold stimulated or remained constant after elicitation.

E. ENZYMATIC SYNTHESIS OF MORPHINE Along with RauvolJia sp. and Catharanthus roseus, P. somniferum serves as one of the most important sources of pharmaceutical alkaloids. Morphine (33) is one of the major alkaloids isolated from plants for industrial use. Annually, approximately 160 tons of morphine (33) are purified and 90-95% of that amount is methylated to codeine (34), which then is used either directly, or is chemically converted to a variety of derivatives that find use as antitussives and analgesics (Fig. 13). The elicit production of morphine (33) for acetylation to heroin (72) (Fig. 14) reaches almost ten times that amount, totally over 1200 tons per year. There would potentially be tremendous commerical, as well as social, value in establishing an alternative source for codeine (34) for legitimate use. The enzymatic synthesis of morphine (33) is almost completely elucidated. The biosynthesis of (S)-reticuline (41) in P. somniferum proceeds through (S)-norcoclaurine (43), as in Berheris and Eschschofzia (Fig. 9) (107). (S)-Reticuline (41) must first be converted to (R)-reticuline (73) before the phenanthrene ring with the correct stereochemistry at C-13 can be formed. The inversion of stereochemistry at C-1 of (S)-reticuline (41) occurs by oxidation of this benzylisoquinoline to the 1,2-dehydroreticulinium ion (74) by action of an (S)-reticuline oxidase (K. Hirata and M. H. Zenk, unpublished) (Fig. 15). The 1,2-dehydroreticulinium ion (74) is then stereospecifically reduced to the R-epimer by 1,2-dehydroreticulinium ion reductase [EC 1.5.1.271(108). Unlike many oxidoreductases, this NADPHrequiring enzyme does not catalyze the physiologically reverse reaction in vitro.

282

KUTCHAN

w 0

@

*..

ti WH,

Ho.."

:*BNq ti

0

PNcH, Dlhydrocodeine

a

Dlhydrocodelnone

Mukatuuln Monaunt Paracodln

o ,,

Ho".'

Blocodone Dlcodld Hycodan Robidone

Remedawn Tlamon

/

HFO*

\ /

Codlupr Codelne (34)Cod1 OPT C o d i P ~ ~ ~ s r Codlpront l~ Pactlnfant ~rya~ol Tu-ameo Tuulpact Tussorelerd

o * P N C H ,

H&-E-0

'

Acetyldlhydrowdeinone Acedlcon

Antitussives Analgesics

\

Morphine (33)

Codeine (34)

'$BNma OH

Dolvlran

Buprenorphlne

Mipralave Mlgraeflux Mlgrine-Kranlt Tilretta

% ' NCH,

*.

ti

>*

0

14-Hydroxydihydrocodeinone Dlnarkon Endone Oxanest Roxicodone Supeudol

w

.

b

HF'&W,

"Orfin Buprenet

Finibron Prefln

.-....--.-

Tmmnidr

Etorphine

FIG.13. Commercially useful antitussives and analgesics synthetically derived from morphine (33).

(R)-Reticuline (73) is next acted upon by the microsomal NADPHdependent cytochrome P-450 salutaridine synthase [EC 1.1.3.351, which catalyzes the stereospecific carbon-carbon phenol coupling between C-12 and C-13 (109,110). The discovery of this enzyme defined a new role for

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

283

Heroin (72)

FIG.14. Structure of the narcotic heroin (72).

cytochromes P-450 in alkaloid biosynthesis, because unlike most of the described enzymes of this class, salutaridine synthase functions as an oxidase rather than as a mono-oxygenase. There is no concomitant incorporation of oxygen into the substrate during the course of the catalytic reaction. The characterization of salutaridine synthase as a cytochrome P-450 supported the hypothesis of Barton and Cohen on the mechanism of oxidation of plant phenols by single electron transfer that would afford phenolic radicals that, by radical pairing, could form new C-Cand C - 0 bonds ( 2 2 2 ) . The substrate- and stereospecificity of the reaction refuted the possibility that this reaction could be catalyzed in vivo by nonspecific phenol oxidases, laccases, or peroxidases (122). Salutaridine (75) is then reduced by the NADPH-dependent salutaridine reductase [EC 1.1.1.2481 to form salutaridinol (76) (113,224). The next step is the closure of the oxide bridge between C-4 and C-5 to form the first morphinan in the pathway, thebaine (77). The enzyme that catalyzes this reaction, salutaridinol 7-0-acetyltransferase [EC 2.3.1.1501, requires acetyl CoA as a co-substrate and produces salutaridinol-7-0acetate, which undergoes spontaneous allylic elimination at pH 8-9 to produce the pentacylic morphinan ring system (ZZ5,Z 26). Thebaine (77) is demethylated by an as yet uncharacterized enzyme to neopinone (79). Neopinone (79) is not further enzymatically transformed, but exists in an equilibrium with its positional isomer codeinone (80) (127). Codeinone (BO), on the other hand, is further modified by codeinone reductase [EC 1.1.1.2471 to codeine (34)(118,119).In vitro, as codeinone is reduced, the equilibrium is continually driven from neopinone (79) toward codeinone (80) until the substrates are depleted. The demethylation of codeine (34)to morphine (33)is a reaction that has been characterized in

284

KUTCHAN

Dehydrooxidase

HO

reductase

HO

0

NADPH (S)-Reticullne (41)

NADP' (R)-Reticuline (73)

1,2-Dehydroreticulinium ion (74)

Salutaridlne '1

NADP+

I I

NADPH

b

NCH,

Salutarldine synthase NADPH.02 H3C0

Ha*

OH

0 Salutaridlne (75)

Saiutaridinoi (76)

(R)-Retlcullne (73)

Salutarldlnol

Hs* Demethylation HO

IJ I

H,CO

fi

NCH,

w

HG-E-0 H

-

NCH,

0

n

Selutaridinol-7Qacetate (78)

Thebaine (77)

Neopinone (79) Equilibrium

Codeinone reductase

Demethylation

NADP+

Morphine (33)

Codeine (34)

NADPH

Codeinone (80)

FIG.15. Biosynthetic pathway leading to morphine (33) in Pupaver somniferum.

mammalian liver (I20),but has not yet been directly detected in the poppy plant. Each of the known enzymes of morphine (33) biosynthesis has been detected in both P. somniferurn plants and cell suspension cultures.

7.

285

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

OF TYROSINE/DOPA DECARBOXYLASES F. MOLECULAR GENETICS

Tyrosine/dopa decarboxylase catalyzes the formation of the protoalkaloids tyramine (45) and dopamine (47) from the corresponding L-amino acids (Fig. 16). Four plant cDNAs encoding members of the tyrosine decarboxylase family were first isolated from fungal elicited Petroselinum crispum cell suspension cultures (222).These cDNAs were actively expressed in E. coli and were shown to preferentially decarboxylate L-tyrosine (44),and to a lesser extent L-dopa (46).L-Tryptophan (12) and L-phenylalanine (32) were not accepted as substrates by these enzymes (222).cDNA and genomic clones representing two gene families of tyrosine/dopa decarboxylases from the opium poppy, P. somniferum, have also been isolated (222). Heterologous expression in E. coli of two cDNAs that are representative of the two gene families indicated that both families of enzymes from P. sornniferum are able to decarboxylate L-dopa (46)as well as L-tyrosine (44),but not L-tryptophan (12) or L-phenylalanine (32) (222,223).The best conversion rates were obtained using L-dopa (46)as substrate, although both enzymes have an identical K M of 1 mM for both L-dopa (46)and Ltyrosine (44).These substrate specificities support the findings of a metabolic grid at the early stages of tetrahydrobenzylisoquinoline alkaloid biosynthesis (Fig.9). Genomic DNA blot hybridization analysis suggests that there are six to eight genes in the TyrDCl and four to six genes in the TyrDC2 gene families (122).The two gene families share 73% amino acid identity (Table 111) within P. somniferurn. TyrDCl and TyrDC2 are 64%and 62% identical to tyrosine decarboxylase from Petroselinum crispum (221),52% and 51% identical to tryptophan decarboxylase from C. roseus (28), and 38% and

Tyrosine HO

,":

decarboxylase

L-Tyrosine (44)

HO

p

N

H

2

Tyramine (45)

HorC COZ

Dopa

\

f *

NH2

HO

decarboxylase

L-Dopa (46)

HO

Dopamine (47)

FIG.16. Reactions catalyzed by tyrosine/dopa decarboxylase.

286

KUTCHAN

TABLE 111 AMINO ACIDSEQUENCE COMPARISON OF TYROSINEDOPA DECARBOXYLASES TyrDClaAND TyrDC2’ FROM P. somniferum

MGSLPANNF- E-SMSLCSQN PLDPDEFRRQ GHMIIDFLAD YYKNVEKYPV RTQVDPGYLK NTEDVL N SAFGVT E RD S E R KRLPESAPYN PESIETILED VTNDIIPGLT HWQSPNYFAY FPSSGSIAGF LGEMLSTGFN T Q TE Y V WGFNWMSSP AATELESIVM NWLGQMLTLP KSFLFSSDGS SGGGGVLQGT TCEAILCTLT V D F K N E --S AARDKMLNKI GRENINKLW YASDQTLSAL QKAAQIAGIN PKNFLAIATS KATNFGLSPN V R K F ENS AA H GR G HC RK SLQSTILADI ESGLVPLFLC ATVGTTSSTA VDPIGPLCAV AKLHGIWVHI DAAYAGSACI S E EYEM V A I V P TREV E CPEFRHFIDG VEDADSFSLN AHKWFFTTLD CCCLWKDSD SLVKALSTSP EYLKNKATDS E PS A N R E KQVIDYKDWQ IALSRRFRSM KLWLVLRSYG IANLRTFLRS HVKMAKHFQG LIGMDNRFEI C G V T N T E L M R V WPRTFAMVC FRLKPAA-I- FRKKIV-EDD HIEA---QTN E--VNA-KL- ---LESVNAS T L PKT K W D N G HQNG NGWPLRDE NL L N N QVY T T GKIYMTHAW GGVYMIRFAV GATLTEERHV TGAWKVVQEH TDAILGALGE -DVCS IY IL A L KFS A FSS sv EMBL/GenBank/DDBJ Databases accession number U08597. EMBUGenBanklDDBJ Databases accession number U08598. Only the TyrDC2 residues that differ from those of TyrDCl are shown.

a

37% identical to dopa decarboxylase from D. melanogasfer (124,125) at the amino acid level, respectively. Both TyrDCl and TyrDC2 transcripts could be detected by RNA gel blot analysis in roots. Only the TyrDC2 transcript, though, is found in the stem, sepal, and carpel, and in callus culture (122). This differential expression of TyrDC genes in the poppy plant has been further investigated by in sifu hybridization (126). This method is more sensitive than RNA gel blot analysis and allows, in addition, RNA transcripts to be localized to individual cells within an organ. TyrDCl transcripts were detected only in root tissue, while TyrDC2 transcripts were predominantly in stem, and also in root tissue. Both TyrDCl and TyrDC2 transcripts were associated with vascular tissue in mature roots and stems, where they are restricted to the metaphloem and protoxylem. This places tyrosine/ dopa decarboxylases in close proximity to the laticifer cells that are known to accumulate large quantities of dopamine (47) (over 2 mg/ml latex) (127), and is consistent with the observation that tyramine (45) can serve as a precursor to the morphinan alkaloids (128).

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

287

The benzophenanthridine alkaloid sanguinarine (39) has been reported to accumulate exclusively in the roots of P. somniferum (126),but has been previously isolated from root, stem, leaf, and flower of various varieties of P. somniferum, from root, stem, and leaf of Papaver setigerum (129) and from the capsule of the closely related, thebaine (77)-producing species, Papaver bracteaturn (130). Arguably, sanguinarine already was isolated from opium as early as 1870 (131).This apparent co-distribution of benzophenanthridine and morphinan alkaloids in the Papaver plant may make a correlation between TyrDC gene family and the biosynthesis of a specific alkaloid class difficult. Further molecular genetic analyses on the opium poppy have dealt with the isolation (132) and cloning of the genes (133-135) encoding latexspecific proteins. The members of this multigene family encode polypeptides that are approximately 20 kDa in size and that accumulate in the vesicles of laticifer cells. These major latex proteins accumulate only in laticifers, however, their function remains unelucidated.

G. MOLECULAR GENETICS OF THE BERBERINE BRIDGE ENZYME The berberine bride enzyme (Fig. 17) catalyzes the stereospecific oxidative conversion of the N-methyl group of (S)-reticuline (41)into the berberine bridge carbon, C-8, of (S)-scoulerine (53) (66,67)along the biosynthetic pathway that leads to the protoberberine (Fig. 9) and benzophenanthridine (Fig. 12) alkaloids. This reaction catalyzed by the berberine bridge enzyme has not been achieved chemically and finds no equivalent in nature. It is therefore of substantial biochemical interest to know how this enzyme catalyzes C-C bond formation. In addition, the enzyme is elicitor-inducible in E. californica cell cultures. This implies that regulation of berberine bridge enzyme transcript accumulation may also control benzophenanthridine alkaloid accumulation. An analysis of the promoter of the 66e1 gene

H3c0T Berberine

HO

,,+/

H311%

H""

/

/

' (S)-Reticuline (41)

OCHI

0 2

H A

\

(S)-Scoulerine (53)

FIG.17. Reaction catalyzed by the berberine bridge enzyme.

OH

(3.34,

288

KUTCHAN

may reveal, in part, how alkaloid biosynthesis is regulated in response to pathogen attack. The cDNA encoding the berberine bridge enzyme has been screened from a cDNA bank prepared from polyA+ RNA isolated from elicited cell suspension cultures of E. californica using oligonucleotides based on peptide amino acid sequences of the purified native enzyme (136). Translation of the nucleotide sequence (Table IV) confirms the presence of a signal peptide (by comparison with the N-terminal amino acid sequence of the native enzyme) that directs the enzyme into the endoplasmic reticulum. This is in agreement with the finding that the berberine bridge enzyme accumulates in smooth vesicles that are believed to arise from the endoplasmic reticulum (68). Inspection of the amino acid sequence also revealed that there are three consensus sequences for the covalent attachment of N-linked carbohydrate on the enzyme (Table IV). The first of these sites, Asn-His-Thr, contains a modified asparagine residue as confirmed by amino acid sequences analysis (236). There is a 25% homology of the berberine bridge enzyme with the 6-hydroxy-~-nicotineoxidase [EC 1.5.3.61 gene of Arthrobacter oxidans (137). The bacterial enzyme contains a covalently bound FAD and the berberine bridge enzyme contains the correct consensus sequence for flavin attachment. This was the first hint as to the identity of a cofactor for this redox reaction. In order to better analyze the enzyme, it was heterologously expressed in yeast and in insect cell culture (45). Using a baculovirus expression vector, 4 mg pure berberine bridge enzyme could be isolated from 1 liter TABLE IV AMINO ACIDSEQUENCE OF THE BERBERINE BRiocE ENZYME FROM E. californica

.1 MENKTPIFFS LSIFLSLLNC ALGGNDLLSC LTFNGVRNHT VFSADSDSDF NRFLHLSIQN PLFQNSLISK PSAIILPGSK QaT-EE -GGS m V S I U S E T A W E SGSTLGELYY AITESSSKLG FTAGWCPTVG TGGHISGGGF GMMSRKYGLA VPEKVTVFRV LKTVAKSTFD DLTKEPLPSK AWNQSEQKKK ISRSWGESYF

ADNWDAILI TKNVAIDEAT LLFPELGLVE AFYGLLERLS TEFLDWLEKV LSNYERLIRA

DANGAILDRQ SLLHKWQFVA EDYLEMSWGE KEPNGFIALN YEFMKPFVSK KTLIDPNNVF

AMGEDVFWAI EELEEDFTLS SFAYLAGLET GFGGQMSKIS NPRLGYVNHI NHPQSIPPMA

RGGGGGVWGA VLGGADEKQV VSQLNNRFLK SDFTPFPHRS DLDLGGIDWG NFDYLEKTLG

IYAWKIKLLP WLTMLGFHFG FDERAFKTKV GTRLMVEYIV NKTVVNNAIE SDGGEWI

Swiss-Prot accession number P30986. EMBLIGenBanklDDBJ Databases accession number M771.50. The three asparagine residues in boldprint are the putative N-linked carbohydrate binding sites. The histidine in boldface is the site of covalent attachment of F A D to the enzyme. The peptide containing the F A D that was isolated for laser-desorption time-of-flight mass spectrometric analysis is underlined. The arrow indicates the position of signal peptide cleavage.

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

289

of insect cell culture. To obtain this quantity of pure protein, it would be necessary to extract and process more than 300 liters of elicited cell suspension cultures of E. californica. With milligram quantities of enzyme available, one molecule of FAD was found to be covalently bound to His-104 of the berberine bridge enzyme (138). The location of the modified amino acid residue was determined by site-directed mutagenesis and by laser-desorption time-of-flight mass spectrometry of the peptide Leu-83 to Glu-133 (Table IV). The flavin moiety was identified by spectral measurements (fluorescence emission and absorbance spectroscopy) and by mass spectrometry. A series of 23 1benzylisoquinoline and protoberberine alkaloids were tested as substrates, and from this study information about the reaction mechanism could be gained. During the conversion of the N-methyl group of (S)-reticuline (41) into the C-8 of (S)-scoulerine (53), the methylene iminium ion 81 must exist as an intermediate (Fig. 18). It cannot readily be determined whether

p. \ r ,N-CH,

MeO

H

O

TOH

' (S)-Reticuline (41)

OMe

\

(S)-Scoulerine (53)

FIG. 18. Proposed mechanism of the conversion of (S)-reticuline (41) to (S)-scoulerine (53) catalyzed by the berberine bridge enzyme.

290

KUTCHAN

this intermediate is formed by two, single electron abstractions or by hydride abstraction, but the second half of the reaction, closure of ring C, proceeds with nucleophillic attack by C-2’ of the phenyl ring, consistent with the inversion of configuration during the course of (S)-scoulerine (53) formation as determined by 3H NMR (73). An analysis of the time course of the elicitation process using the cDNA clone as a hybridization probe in RNA gel blot experiments revealed that the berberine bridge enzyme transcript reaches maximum levels within 6 h after the addition of a yeast cell wall preparation to E. californica cell suspension cultures. Enzyme activity increases up to 22 hours after elicitation, and total benzophenanthridine alkaloids continue to accumulate for several days (236). This scenario is indicative of de n o w transcription, as observed for stress-induced phenylpropanoid genes (239,140). The gene bbe2 encoding the berberine bridge enzyme has been isolated from E. californica and sequenced (K. Hauschild and T. M. Kutchan, unpublished). As was observed for strl (encoding strictosidine synthase) from R. serpentina (48), it occurs as a single copy gene containing no introns. The bbe2 gene is induced by methyl jasmonate, which was shown to stimulate accumulation of low molecular weight compounds in a large number of plant species in culture, including benzophenanthridines in Eschscholzia (106,242). Identifying the cis elements and trans factors necessary for elicitor-induced transcription of bbe1 should help to elucidate the complex defense response in this plant.

IV. Bisbenzylisoquinoline Alkaloids

A. USESOF BISBENZYLISOQUINOLINE ALKALOIDS The bisbenzylisoquinoline class of alkaloids is comprised of two tetrahydrobenzylisoquinoline alkaloids connected by one to three ether linkages formed by the phenolic coupling of the monomers. The structure of a prototype of this class of natural products, (+)-tubocurarine (82) (Fig. 19), which is the peripheral muscle relaxant isolated from tube-curare, contains two ether linkages with the tetrahydro-benzylisoquinoline monomers combined in a head-to-tail orientation. Tube-curare is used by certain South American Indians as a traditional arrow poison. In modern medicine, (+)-tubocurarine chloride finds use as a neuromuscular blocking agent that secures muscular relaxation during surgical operations. This family of alkaloids is rich in pharmacologically active constituents that range in activity from antimalarial (dehatrine (83) (142)) to cytotoxic (( +)-thalicarpine

7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

291

Dehatrine (83) Beilschmiedia madang

(+)-Tubocurarine (82) Chondodendron tomentosum Tubarine (Burroughs Wellcome)

(+)-Thalicarpine (84) Thalictrum minus

Aromoline (85) Berberis stolonifera

lsotetrandrine (86) Lirnaciopis loangensis

FIG.19. Physiologically active bisbenzylisoquinoline alkaloids.

(84)), vasodilatory (aromoline (85) (143)) and antiparasitic (isotetrandrine (86) (144)).The structural variations in these alkaloids include substitutions

on the phenyl rings, the regiospecificity of the ether linkages and the stereochemistry of the isoquinoline moieties at C-1. The best understood enzymatic synthesis is that of berbamunine (87) and guattegaumerine (88), both of which are produced in cell suspension cultures of Berberis srofonifera (145).

B. ENZYMATIC SYNTHESIS OF BERBAMUNINE The single most important step in the biosynthesis of bisbenzylisoquinoline alkaloids is ether linkage formation through phenol coupling. Biotechnological production of this pharmaceutically important class of alkaloids

292

KUTCHAN

requires correct stereo- and regio-selectivity of ether bond formation. As for the formation of salutaridine (75) from (R)-reticuline (73)in morphine biosynthesis (Fig. 15), the type of enzyme that catalyzes formation of the carbon-oxygen phenol coupling in bisbenzylisoquinoline alkaloid biosynthesis is a cytochrome P-450 (209). This enzyme, berbamunine synthase [.EC 1.1.3.341, oxidatively couples one molecule of (S)-N-methylcoclaurine (51)and one molecule of (R)-N-methylcoclaurine (89) to form berbamunine (87) (Fig. 20) without concomitant incorporation of oxygen into the substrate. The enzyme is microsomal and requires O2and NADPH for activity. Berbamunine synthase has been purified to homogeneity from microsomes of B. stolonifera cell suspension cultures and the sequence of the amino terminus was determined (146). The enzyme was found to catalyze the formation of both the R,S-dimer berbamunine (87) and the R,R-dimer guattegaumerine (88) in a ratio of 9: 1 and was inhibited by prochloraz, tetcyclacis, ketoconazole, cytochrome c, and carbon monoxide (209).The carbon monoxide difference spectrum of the dithionite-reduced enzyme unambiguously demonstrated that the enzyme is a cytochrome P-450. In addition, the presence of catalase or superoxide dismutase in the reaction mixture had no effect on enzyme activity supporting the finding that this catalyst is not a peroxidase. The biosynthesis of the berbamunine synthase substrate (S)-N-methylcoclaurine (51) proceeds as given in Fig. 9. However, the enzymatic synthesis of its diastereomer, (R)-N-methylcoclaurine (89), remains unelucidated.

c. MOLECULAR GENETICS OF BERBAMUNINE SYNTHASE A cDNA encoding berbamunine synthase was isolated from a cDNA library prepared from B. stolonifera cell suspension cultures (247). An oligonucleotide primer based on the N-terminal amino acid sequence of the purified native oxidase was used as a screening probe. The cDNA was assigned to a new family, CYP80, of cytochromes P-450 based on the translation of the amino acid sequence as compared to the known sequences. Inspection of the amino acid sequence revealed at least one notable difference in berbamunine synthase as compared with other cloned cytochrome P-450 mono-oxygenases. This amino acid difference relates to structurefunction studies on the bacterial P-450 cam that suggest that three amino acid residues of Helix I, Gly-248, Gly-249, and Thr-252, are essential for the formation of the molecular oxygen binding pocket (148). Mutation of Thr-252 to Ala or Val abolished the insertion of oxygen into the substrate (249). The amino acid equivalent of Thr-252 is present in berbamunine synthase, although the enzyme is not a mono-oxygenase (Table V). How-

H3m yen, 7. MOLECULAR

no

/

\

/

293

GENETICS OF PLANT A L K A L O I D BIOSYNTHESIS

OH

H 3 1 0/3 J l c n 3

''VH

o \

\

no

\ ;iDPH

Berbamunine (87)

(R)-N-Methylcoclaurine (89)

Berbamunine synthase (C-0 Phenol coupling)

no H

no

6,

O

y

\

(S)-Norcoclaurine (43)

(S)H-Methylcoclaurine (51)

(S)-Coclaurine ( 5 0 )

I

H 3 1 1 3 n3113 -HO Ho

H,CO

\

I

NCn3

H,CO

1

""nCH,

\

.

"'H CH,

no

3/

no '.4

on

(R)-Reticuline (73)

(S)-Reticuline (41)

(S)-3-Hydroxy-Nmethylcoclaurine (52)

synthase

0 Salutaridine (75)

FIG.20. Comparison of the C - 0 phenol coupling reaction catalyzed by the cytochrome P450 berbamunine synthase along the biosynthetic pathway leading to the bisbenzylisoquinoline alkaloids and the C-Cphenol coupling reaction catalyzed by the cytochrome P-450salutaridine synthase along the biosynthetic pathway leading to the morphinan alkaloids.

294

KUTCHAN

TABLE V AMINO ACIDSEQUENCE OF THE BERBAMUNINE SYNTHASE FROM B. stolonifera

MDYIVGFVSI QKYGPLIHLK NSYWKKGRKI LGHVVFSKDV FKLLIKIWEG WALAQLIKNP HRCMETCQVM PFGSGRRICP VIPKVRI

SLVALLYFLL FGLHSSIFAS LHTEIFSQKM FEYSDQSDEV EVLARRANRN DKLAKLREEL GYTIPKGMDV GRPLAVRIIP

FKPKHTNLPP TKEAAMEVLQ LQAQEKNRER GMDKLIHGML PEPKDMLDVL DRWGRSSTV HVNAHAIGRD LVLASLVHAF

SPPAWPIVGH TNDKVLSGRQ VAGNLVNFIM MTGGDFDVAS IANDFNEHQI KESHFSELPY PKDWKDPLKF GWELPDGVPN

LPDLISKNSP PLPCFRIKPH TKVGDWELR YFPVLARFDL NAMFMETFGE LQACVKETMR QPERFLDSDI EKLDMEELFT

PFLDYMSNIA IDYSILWSDS SWLFGCALNV HGLKRKMDEQ GSDTNSNIIE LYPPISIMIP EYNGKQFQFI LSLCMAKPLR

Swiss-Prot accession number P47195. EMBLIGenBanklDDBJ Databases accession number U09610. P. G. and T in boldface are the residues of Helix I that are presumably involved in forming the oxygen binding pocket of the cytochrome P-450.The nonconserved residue in CYP80 is underlined. The C in boldface is the cysteine of the heme-thiolate ligand.

ever, the equivalent position of Gly-248 is occupied by a Pro in berbamunine synthase. Because this amino acid residue is one of three highly conserved residues of the O2 binding pocket that are thought to be essential for the insertion of oxygen into the substrate, this may indicate how CYP80 functions as an oxidase instead of as a mono-oxygenase. CYP80 has been actively expressed in insect cell culture using a baculovirus expression vector (147). Either the insect cell cytochrome P-450 reductase, or that from porcine liver, or B. stolonifera cell suspension cultures, can transfer electrons to this cytochrome P-450, resulting in enzyme activity. The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine (89) to form the R,R-dimer guattegaumerine (88) or one molecule each of (R)-N-methylcoclaurine (89) and ( S ) - N methylcoclaurine (51) to form the R,S-dimer berbamunine (87) (Fig. 21). The ratio of products formed, however, was reversed with respect to the native enzyme. The heterologously expressed enzyme typically produced guattegaumerine (88) and berbamunine (87) in a ratio of 9: 1. During the course of catalysis, H202was not formed as a reaction product, suggesting that the electrons abstracted from the substrates are ultimately released from the enzyme as two molecules of H 2 0 (T. Amann and T. M. Kutchan, unpublished). Berbamunine synthase could be obtained in near-homogeneous form (5 mg/l) from insect cell microsomes. In order to obtain this amount of enzyme from B. stolonifera cell cultures, over 18,000 liters of suspension cells would have to be extracted. A single gene probably codes for berbamunine synthase in the B. stoloniferu genome, although two more weakly hybridizing DNA fragments result-

HmT -H 3 m 7.

MOLECULAR GENETICS OF PLANT ALKALOID BIOSYNTHESIS

CYP80

HO

HO

\

/

295

CH,

/

'

OH

(R)-N-Methylcoclaurine (89) (S)-N-Methylcoclaurine (51)

' O H

R-pH R-aH

Guattegaumerine (88) Berbamunine (87)

0

\

R-pH R-aH

FIG.21. Reaction catalyzed by berbamunine synthase.

ing from the genomic DNA gel blot experiments may represent either other members of a small gene family or simply other cytochrome P-450enzymes from this species.

V. Tropane and Nicotine Alkaloids A. USESOF TROPANE A N D NICOTINE ALKALOIDS The tropane class of alkaloids are found mainly in the plant family Solanaceae, and include the anticholinergic drugs, hyoscyamine (90) and scopolamine (91). Solanaceous plants have been traditionally used for their medicinal, hallucinogenic, and poisonous properties, due in large part to the tropane alkaloids. Both alkaloids find use today in modern medicine (Fig. 22) in anticholinergic preparations. In addition, atropine, the racemate of hyoscyamine (90),is used to dilate the pupil during eye examinations. The narcotic, topical anesthetic and psychostimulant cocaine (92) is a tropane alkaloid found outside of the Solanaceae in Erythroxylum coca (Erythroxylaceae). Although cocaine (92), and the derivative tropacocaine (93), are effective topical anesthetics, they are no longer marketed due to illicit use as psychostimulants. Biosynthetically related to tropane alkaloids, and also found in the Solanaceae, nicotine (94) finds use as a powerful insecticide, as well as in chewing gum and in epidermal patches that help to ease the difficulties associated with cigarette addiction. The nicotine (94) used industrially today is made available as a by-product of the tobacco industry. The hyoscyamine (90) and scopolamine (91) used in the pharmaceutical industry are isolated from plantation-grown Duboisia plants. The biosynthesis of both nicotine and

KUTCHAN

296

0 Hyoscyamine (90)

Scopolamine (91)

Hyoscyamus n i g e r

A t r o p a belladonna

Bellergal@ (Sandoz)

Scopoderm TTS@(Ciba)

Nicotine (94)

Nicotiana tabacum Nicorette@(Much Pharma)

"CH3

N

/

~

~

~

. 0-c

Fl

H

Cocaine (92) Erythroxylon c o c a

Tropacocaine (93)

FIG.22. Physiologically active alkaloids derived from the N-methyl-A'-pyrrolinium ion (98).

tropane alkaloids has not yet been completely elucidated at the enzymic level, but good progress has been made, especially along the pathway leading to scopolamine (91).

B. ENZYMATIC SYNTHESIS OF SCOPOLAMINE The biosynthesis of tropane and nicotine alkaloids begins with the conversion of the primary amino acid L-arginine (95)to L-ornithine (96)by arginase [EC3.5.3.11 followed by decarboxylation of L-ornithine (96)to putrescine (97) by ornithine decarboxylase [EC 4.1.1.171 (Fig. 23) (1.50). In higher plants, putrescine (97)can be metabolized to higher polyamines, or can be conjugated with cinnamate derivatives or fatty acids. In tobacco, putrescine (97)can be metabolized to nicotine (94)and in Atropa, Hyoscyamus, and

Ornithine decarboxylase O&lTHdQ Hd n CYN

coz

L-Omlthine (96)

Putrescine N-methyltransfefare

Diamine oxidare

r * W T7. H

Putrescine (97)

SAM

% '

SAH

N -Methylputrescine (99)

0,

H,O,

t?4

C+'a

NH,

4-Methylaminobutanal (100) /Spontaneous

4 I

1$2:

Nicotine (94)

Acetoacetic add (102)

1

n

Tropinone (101)

Hygrine (104)

(98)

103

NADPH Tropinone reductase I NADP*

Esterification

7 Littorine (108) HO Tropine (105) Phanyliactic acid (106)

I fic0,i-i \

Aromatlc lactate dehydrogenase

Hyoscyamine (90)

Transaminasec H

NHZ

L-Phenylalanine (32)

I

intramolecuiai mipration

Phenylpyruvic acid (107)

I1

Hyoscyamine

8phydroxylese

Scopolamine (91)

FIG.23. Biosynthetic pathway leading to scopolamine (91) in select members of the Solanaceae.

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Duboisia species it serves as a precursor to the tropane alkaloids. On the pathway to the N-methyl-A'-pyrrolinium ion (98) that is a precursor to nicotine (94)in tobacco and scopolamine (91)in Hyoscyamus, putrescine (97)is methylated to N-methylputrescine (99) by putrescine N-methyltransferase [EC 2.1.1.531 (151-153). N-Methylputrescine (99) is next oxidized by diamine oxidase [EC 1.4.3.61 and the 4-methylaminobutanal (100)formed cyclizes spontaneously to the N-methyl-A'-pyrrolinium ion (98)(154). This early stage of the pathway proceeds differently in Datura, and the corresponding enzymology remains to be carried through with this plant family (155).

The enzymes that convert the N-methyl-A'-pyrrolinium ion (98)to tropinone (101)remain unelucidated, but the sequence is thought to be condensation with a derivative of acetoacetic acid (102) to form 103 that then undergoes decarboxylation to hygrine (104) (156). Hygrine is then converted to tropinone (101).The NADPH-dependent reduction of tropinone (101)to tropine (105)is catalyzed by tropinone reductase I [EC 1.1.1.206; tropine dehydrogenase] (157). Cultured roots of Hyoscyamus niger contain two tropinone reductase activities, tropinone reductase I that participates in scopolamine (91)biosynthesis and tropinone reductase I1 [EC 1.1.1.2361, which reduces tropinone (101)with the opposite stereochemistry compared to tropinone reductase I, producing +tropine (158,159). Cocaine (92)is a well-known tropane alkaloid that is a $-tropine ester (Fig. 22), but is produced in Erythroxylum species, rather than in the Solanaceae. The next step in the pathway to scopolamine (91) is the condensation of tropine (105)with phenyllactate (106)(160,161),which is derived from L-phenylalanine (32)through transamination by phenylalanine aminotransferase [EC 2.6.1.581 to phenylpyruvate (107)followed by reduction by an (R)-aromatic lactate dehydrogenase [EC 1.1.1.2221 (162).The first tropane alkaloid formed along the pathway is thus littorine (108),an alkaloid found in tropane-producing solanaceous plants (160,163). Littorine (108) then undergoes intramolecular rearrangement, thereby serving as direct precursor to hyoscyamine (90) (160,164,165).Although the enzymes that catalyze the formation of hyoscyamine (90) from tropine (105) and phenyllactate (106) have not yet been investigated, the stereochemical course of the intramolecular rearrangement has been studied in detail (166,167). During migration of the tropinyl carboxylate, an inversion of configuration occurs at both migration termini. The biosynthesis leading to the first tropane alkaloid in the pathway is a revision of a long-accepted pathway in which group migration on L-phenylalanine (32)was proposed (168, and references therein). Hyoscyamine (90) is in turn converted to scopolamine (91) through the action of hyoscyamine 60-hydroxylase [EC 1.14.11.111 (169). The enzyme requires 2-oxoglutarate, ferrous ions, ascorbate, and

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299

molecular oxygen for activity. The enzyme is bifunctional, catalyzing both the mono-oxygenation of hyoscyamine (90) to 60-hydroxy-90 and epoxide formation to form scopolamine (91). Protein blot analysis of extracts from various tissues of H . niger using antibodies raised against the native enzyme showed that hyoscyamine 6P-hydroxylase is abundant in cultured roots and plant roots, but is absent in leaf, stem, calyx, cell cultures, and cultured shoots (270).Immunohistochemical studies using monoclonal antibody and immunogold-silver enhancement detected the enzyme only in the pericycle cells of young roots. C. MOLECULAR GENETICS OF PUTRESCINE N-METHYLTRANSFERASE Cuban cigar tobacco varieties bred in the 1930s to have a low nicotine (94) content were used in backcrosses to establish a genetically stable breeding line with a low-alkaloid content for the production of low-nicotine cigarettes. This low-nicotine variety, LA Burley 21, and the parental strain, Burley 21, which is isogenic at all but two low-nicotine loci, were used to isolate a cDNA encoding putrescine N-methyltransferase (252-253). This enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to an amino group of putrescine (97), the first committed step in the biosynthesis of nicotine and tropane alkaloids (Fig. 24). The availability of the near-isogenic Burley strains made it possible to isolate a cDNA encoding this enzyme by subtractive hybridization (172).The deduced amino acid sequence of one of the clones (Table VI), obtained by this method of screening for differences in the composition of expressed genes in two plant varieties, is 73% identical to spermidine synthase [EC 2.5.1.16; putrescine aminopropyltransferase] from humans, 70% identical to that from mouse, and 58% to spermidine synthase from E. coli. In contrast, this clone that encodes putrescine N-methyltransferase is less homologous to N- and 0-methyltransferases from various organisms, plants included. Due to these differential homologies, it is suggested that putrescine N-methyltransferase has evolved from tobacco spermidine synthase during the diversification of solanaceous plants. The cDNA codes for Putrescine N-Methyltransferase H,N

H2N

SAM Putrescine (97)

SAH

CH,

N -Methylputrescine (99)

FIG.24. Reaction catalyzed by putrescine N-methyltransferase.

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TABLE VI AMINO ACIDSEQUENCE OF PUTRESCINE N-METHYLTRANSFERASE FROM N. tabacum

MEVISTNTNG STIFKNGAIP -SE S L H KHFEHREKQE TSEQQNGTIS HDNGNELLGS SDSIKPGWFS EFSALWPGEA FSLKVEKLLF QGKSDYQDVM LFESATYGKV RYPSIEKIDI VDSSDPIGPA VNYAWTTAPT AAFILPSFAR

LTLDGAIQHT VEIDDVVVDV KDLFERPFFE YPTGVIGYML SMIES

ENGGFPYTEM SRKFFPYLAA AVAKALRPGG CSTEGPEVDF

IVHLPLGSIP NFNDPRVTLV WCTQAESIW KNPVNPIDKE

NPKKVLIIGG LGDGAAFVKA LHMHIIKQII TTQVKSKLGP

GIGFTLFEML AQAGYYDAII ANCRQVFKGS LKFYNSDIHK

EMBLlGenBanklDDBJ Databases accession number D28506. Six putative N-linked carbohydrate binding sites are printed in boldface. Four heptapeptide repeats are underlined.

a protein of 375 amino acids that contains four repeats of a heptapeptide sequence. There is a putative N-glycosylation site in each of these repeats, as well as two additional sites outside of these regions, and all concentrated within 66 amino acids of the amino terminus (Table VI). Heterologous expression of this tobacco cDNA in an E. coli mutant that lacked the spermidine synthase gene resulted in the accumulation of Nmethyl putrescine (99), thereby confirming the identity of the cDNA as that for putrescine N-methyltransferase. The transcript for the enzyme accumulates predominantly in root tissue of the wild-type tobacco plant, consistent with this organ being the major site of nicotine (94) biosynthesis. This corresponds also to the site of tropane alkaloid biosynthesis found for other members of the Solanaceae (270). Removal of the flower heads and young leaves (the decapitation procedure used among tobacco growers to increase root growth and nicotine levels in plants) induced transcription of the gene in the root (272). Genomic DNA gel blot analysis detected two to five strongly hybridizing DNA fragments in the Nicotiana tabacum and Nicotiana sylvestris genomes, suggesting that putrescine N-methyltransferase is represented by a small gene family in tobacco. D. MOLECULAR GENETICS OF TROPINONE REDUCTASE-I Along the biosynthetic pathway that leads specifically to the tropane alkaloid scopolamine (91), tropinone reductase I converts the 3-keto group of tropinone (101) to the 3a-hydroxyl of tropine (105) (Fig. 25). The cDNA encoding this enzyme was isolated from Datura stramonium by screening a cDNA library prepared from hairy roots with oligonucleotides based on peptide amino acid sequences from purified native tropinone reductase I

7.

301

MOLECULAR GENETICS OF PLANT A L K A L O I D BIOSYNTHESIS

+

HO

H&-N

Tropine (105)

0

TroPhJne

Tropinone (101)

OH H

1/, -Tropine (109) FIG.25. Reactions catalyzed by tropinone reductase I and tropinone reductase 11.

(172). The cDNA was expressed as a P-galactosidase fusion protein in E. coli and found to have tropinone reductase I activity, thereby confirming its identity. DNA gel blot analysis identified hybridizing DNA fragments in the nuclear DNA of the tropane alkaloid-producing species H. niger and Atropa belladonna, but not in tobacco, a species in which tropane alkaloids are not biosynthesized and in which the enzyme is not expected to occur. A cDNA for a second reductase, tropinone reductase 11, was also isolated in these experiments (172).Tropinone reductase I1 also reduces tropinone (101), but with a stereospecificity opposite to that of tropinone reductase I. Hence, the 3-keto group of tropinone is converted to the 3P-hydroxyl of +-tropine (109). +-Tropine (109) is not a precursor of the tropane alkaloids in the Solanaceae, but rather has the same stereochemistry as the Pconfigured tigloidine, 3P-acetoxytropane, and the calystegins. The deduced amino acid sequences of the two cDNAs are 64%identical over 260 amino acid residues and share sequence similarities with other enzymes in the short-chain, nonmetal dehydrogenase family (Table VII). In a series of mutagenesis experiments, specific domains of these two reductases were interchanged and the resultant chimeric proteins were

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TABLE VII AMINO ACIDSEQUENCE COMPARISON OF TROPINONE REDUCTASE I" A N D TROPINONE REUUCTASE II" FROM D.srramonium MEESKVSMMN CNNEGRWSLK GTTALVTGGS KGIGYAIVEE LAGLGARVYT CSRNEKELDE MA N E C R G s s Q ND

I__________________________

1

....................................

CLEIWREKGL NVEGSVCDLL SRTERDKLMQ TVAHVFDGKL NILVNNAGW IHKEAKDFTE TQ S F K A S S Q E N NHH I Y Y V ______________ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ) I _ _ _ _ _ _ _ _ _ _ - 111 _ _ KDYNIIMGTN FEAAYHLSQI AYPLLKASQN GNVIFLSSIA GFSALPSVSL YSASKGAINQ MD V I VS AL V YEAV G VL H F ER E SL SI

_ _ _ _ _ _ _ _ _ _ _ I I _ _ _ _ _ _ _ _ _ - - _ _ _ _IV_ _____-___________ _ _ _ _ _ - - - - - - - - - - -

MTKSLACEWA KDNIRVNSVA PGVILTPLVE TAIKKNPHQK EEIDNFIVKT PMGRAGKPQE L RC F G G A S MT -QD E NLNKL DRC ALR M E K

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I I _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ v_ ____ _ __ _____ - _ _ _ _ _ _ _ _ _

VSALIAFLCF PAASYITGQI IWADGGFTAN GGF W LM C LA M V V _ _ _ _ _ _ _ _ _ _ _ _ I I _ _ _ _ _ _ _VI ___-_-___--~ " EMBL/GenBank/DDBJ Databases accession number L20473. EMBL/GenBank/DDBJ Databases accession number L20474. Only the tropinone reductase 11 residues that differ from those of tropinone reductase I are shown. Six amino acid residues that are highly conserved in short-chain nonmetal dehydrogenases are in boldface, The tropinone reductase cDNAs were divided into six regions (I-VI) for structure function studies with chimeric proteins. Peptides IV and V confer the stereospecificity of the reduction.

expressed in E. coli in order to identify the domain that confers the stereospecificity of the catalytic reaction (273). The wild-type enzymes exhibit absolute stereospecificity in reducing tropinone (101). A series of chimeric enzymes could be produced, however, that have relaxed stereospecificity. The carboxy-terminal peptides of about 120-amino acids, in which 53 residues are different between tropinone reductase I and tropinone reductase 11, appear to determine the stereospecificity of the reaction. E. MOLECULAR GENETICS OF HYOSCYAMINE 6P-HYDROXYLASE

Hyoscyamine 6P-hydroxylase catalyzes the final two steps in the biosynthetic pathway leading from hyoscyamine (90) to the medicinally important alkaloid scopolamine (91) (Fig. 26) (270).The cDNA encoding this bifunctional enzyme was isolated from a cDNA library prepared from polyA+ RNA of cultured roots of H. niger (49). The library was screened with oligonucleotides that were based on internal peptide fragments of the native enzyme. The cDNA encodes a protein of 344 amino acids (Table VIII)

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Hyoscyamine Bp-hydroxylase

"CH,

2-Oxoglutarate

0

02

Hyoscyamine (90)

Succinate

co2 6p-Hydroxyhyoscyamine (1 10)

Scopolamine (91)

FIG.26. Reaction catalyzed by hyoscyarnine 60-hydroxylase.

and shows homology to other members of the 2-oxoglutarate-dependent dioxygenase family. Heterologous expression of the hyoscyamine 6P-hydroxylase cDNA in E. cofi demonstrated that the dioxygenase first hydroxylates hyoscyamine in the 6P-position and subsequently catalyzes epoxide formation, although the epoxidation reaction proceeds markedly slower than hydroxylation in the heterologous host (174). RNA gel blot analysis of the tissue-specific expression of the hyoscyamine 6P-hydroxylase gene corroborates the results obtained by immunohistochemical localization of the enzyme (170) to the root pericycle in scopolamine-producing species of Hyoscyamus, Atropa, and Duboisia. Similarly, the hyoscyamine 60-hydroxylase transcript is localized to roots and cultured roots of H. niger, but is not found in stem, leaves, or cell cultures of this same species. These results explain why it has not been possible to produce substantial quantities of scopolamine in cell culture. A promotef analysis of the genes encoding putrescine N-methyltransferase and hyoscyamine 60-hydroxylase should help with the elucidation of the TABLE VIII AMINO A C I DSEQUENCE OF HYOSCYAMINE 6p-HYDROXYLASE FROM H . n i p 3 MATFVSNWST KSVSESFIAP LQKRAEKDVP VGNDVPIIDL QQHHHLLVQQ ITKACQDFGL FQVINHGFPE ELMLETMEVC KEFFALPAEE KEKFKPKGEA AKFELPLEQK A K L W E G E Q L SNEEFLYWKD TLAHGCHPLD QDLVNSWPEK P A K Y R E W A K YSVEVRKLTM RMLDYICEGL GLKLGYFDNE LSQIQMMLTN YYPPCPDPSS TLGSGGHYDG NLITLLQQDL PGLQQLIVKD ATWIAVQPIP TAFVVNLGLT LKVITNEKFE G S I H R W T D P TRDRVSIATL IGPDYSCTIE PAKELLNQDN PPLYKPYSYS EFADIYLSDK SDYDSGVKPY KINV Swiss-Prot accession number P24397. EMBLiGenBanklDDBJ Databases accession number Mh27 19

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underlying mechanisms of tissue-specific expression of tropane alkaloids in the Solanaceae. The contemporary commercial source of scopolamine (91) is Duboisia crosses, cultivated originally in Australia. Other tropane alkaloid-producing species such as Atropa, however, accumulate hyoscyamine (90) instead of the pharmaceutically used scopolamine (91) as the major alkaloid. It was of interest to ask whether expression of a transgene in this medicinal plant would alter the alkaloid pattern such that more of the pharmaceutically useful alkaloid, scopolamine (91),would be produced. The cDNA encoding hyoscyamine 6P-hydroxylase from H. niger was, therefore, introduced into A. belladonna using either Agrobacterium tumefaciens- or Agrobacterium rhizogenes-mediated transformation. Elevated levels of scopolamine (91) were detected in the resultant transgenic plants (1.2% dry weight in transgenic leaves compared to trace levels in control leaves) (175) and hairy roots (0.3% dry weight compared to approximately 0.03% dry weight in the controls) (176), thereby demonstrating for the first time the viability of metabolic engineering of medicinal plants and plant organ cultures.

VI. Acridone Alkaloids A. USESOF ACRIDONE ALKALOIDS The acridone class of alkaloids now contains over 100 members, found to occur only in species of the Rutaceae. Although the acridone alkaloid acronycine (111) (Fig. 27), from the bark of Acronychia baueri, shows an extremely broad spectrum of in vivo antitumor activity, this class of alkaloids, in general, does not currently find use in the pharmaceutical industry. Extracts of the acridone alkaloid-producing plant Ruta graveolens, however,

Acronycine (111) Acronychia baueri

FIG.27. Structure of the physiologically active acridone alkaloid acronycine (111).

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are marketed in Germany in preparations such as DienaplexB (Phytopharma), Cefalymphat (Cefak), and Melilotus-Echtroplex" (Weber & Weber), which are recommended for relief of cramps and the discomforts of rheumatism. Biosynthetically, the acridone alkaloids are derived from anthranilic acid (1l2), which in plants also serves as a precursor to the amino acid Ltryptophan (12)and the furoquinoline alkaloids, as well as auxin. Progress has been made elucidating the enzymes of acridone alkaloid biosynthesis, chiefly using cell suspension cultures of R. graveolens. B. ENZYMATIC SYNTHESIS OF FUROFOLINE-I Anthranilate synthase [EC 4.1.3.271 catalyzes the formation of anthranilic acid (112)from chorismic acid (113)by elimination of the enolpyruvyl side chain accompanied by the transfer of an amino group from glutamine (177, and references therein) (Fig. 28). This enzyme is the first enzyme specific for the tryptophan portion of the shikimate pathway in higher plants. Anthranilate synthase is composed of two, nonidentical subunits. The asubunit binds chorismate and catalyzes aromatization and the &subunit transfers the amino group from glutamine. In the absence of p-subunit, NH3 can serve as the amino moiety donor, albeit less efficiently. Anthranilate synthase is localized to plastids and its activity is feedback inhibited by tryptophan. In select members of the Rutaceae, the biosynthesis of acridone alkaloids branches from the tryptophan pathway at the level of anthranilic acid (112). In cell cultures of R. graveolens, the synthesis of acridone alkaloids is elicitor-induced and the resultant alkaloid accumulation correlates with the induction of anthranilate synthase activity (278). The next step in the pathway to the acridones is the first committed step of alkaloid biosynthesis in this plant. Anthranilic acid (112)is methylated to N-methylanthranilic acid (114) by anthranilate N-methyltransferase [EC 2.1.1.1111 (179). The activity of this enzyme is also elicitor-inducible in cell suspension and organ cultures of R. graveolens. N-Methylanthranilic acid (114) is then activated to Nmethylanthraniloyl-CoA (115)(180). Although a specific N-methylanthranilate: CoA ligase activity has not yet been detected in R. graveolens cell cultures, anthranilate: CoA ligase [EC 6.2.1.321 has been cloned from an aromatic acid metabolizing Pseudomonas strain (182), so it is likely that the enzyme that transfers CoA at the expense of ATP to N-methylanthranilic acid (114)in R. graveolens will eventually be identified and the corresponding gene will be isolated. N-Methylanthraniloyl-CoA (115) specifically serves as a substrate for acridone synthase, which catalyzes the condensation of 115 with three

306

KUTCHAN Anthranllate N -methyitransferare

HO

aqH NHCH,

/ Gin1

Glu.

pyruvate

Anthranlllc acld (112)

SAM

Chorismic acid (113)

SAH

N-Methylanthranilic acid (114)

i COA

: llpation

i

+ d: Acrldone

+

4CoASH+3CQ2

OH

AH,

3

1,3-Dihydroxy-N-methyIacridone (116)

HO&-COSCoA

N -Methyl-

anthraniloyl CoA (115)

synthase Hg2: Mn2'

Rutacridone synthase

Glycocitrine-11 (1 18)

0

OH

Rutacridone (1 17)

Furofoline- I

Furofoline-I (1 19)

FIG.28. Proposed biosynthetic pathway leading to the acridone alkaloid furofoline-I in Ruta graveolens.

molecules of malonyl-CoA in the presence of ATP and Mg2+ions leading to 1,3-dihydroxy-N-methylacridone (116)(282). The enzyme is a homodimer with a subunit M, of 40 kDa. Enzyme activity is inhibited by the antibiotic cerulenin, which inhibits 2-ketoacyl-ACP synthase, the formation of acetate-derived secondary metabolites and the chalcone synthase reaction (283),as well as by 1,3-dihydroxy-N-methylacridone (116),the reaction endproduct (284). The formation of the acridone nucleus as catalyzed by acridone synthase apparently proceeds with the mechanism observed for flavonoid and stilbene biosynthesis (285). Microsomal preparations of R. gruveolens cell cultures convert 1,3-dihydroxy-N-methylacridone (116)to rutacridone (117)(184). This reaction is dependent on the presence of isopentenyl pyrophosphate, Mg2+or Mn2+ ions, NADPH and O2 and is inhibited by the typical cytochrome P-450

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inhibitors tetcyclacis and ketoconazole. Glycocitrine-II(ll8) is an interme(116) to rutacridiate in the conversion of 1,3-dihydroxy-N-methylacridone done (117), although an unequivocal conversion of glycocitrine-I1 (118) to rutacridone (117) by the microsomal preparations was not observed. It is clear, though, that the formation of rutacridone (117) from 1,3-dihydroxyN-methylacridone (116) involves a prenyltransferase activity, as well as a cyclase activity. The microsomal preparation also converted rutacridone (117) to furofoline-I (119), but the enzymes catalyzing the multistep conversion of 1,3-dihydroxy-N-methylacridone(116) to furofoline-I (119) await purification and characterization. C. MOLECULAR GENETICS OF ANTHRANILATE SYNTHASE

Two cDNAs encoding the a-subunits of anthranilate synthase, a1 and a2, have been isolated from a cDNA library prepared from polyA' RNA isolated from young shoots of R. gruveolens by screening with a highly conserved fragment of the yeast a-subunit gene (286). The 520 amino acids at the carboxy terminus of both R. gruveolens a-subunits (Table IX) are 34% identical to the a-subunit from bacteria and yeast and more than 70% identical to the a1 and a 2 sequences from Arubidopsis (287). The anthranilate synthase a-subunit in R. gruveolens is synthesized as a cytosolic pre-protein and is transported into the stroma of plastids. The presence of a functional transit peptide could be experimentally demonstrated with pea chloroplasts. Both the a l - and a2-subunit mRNAs are constitutively expressed at low levels in Ruru cell cultures, but the al-subunit mRNA was 100-foldinduced within 6 h after addition of a Rhodotorulu rubru extract, an elicitor to the cell cultures. The aZsubunit mRNA did not respond to this elicitor treatment. A differential function for the two a-subunit genes was proposed based on the elicitation experiments. It is suggested that the a2-subunit is involved in L-tryptophan (12) biosynthesis and that the al-subunit is dedicated to the defense-response of acridone alkaloid biosynthesis in R. gruveolens. Three genes encoding the anthranilate synthase &subunit have been isolated from Arubidopsis (288). If the &subunits of Arubidopsis and R. gruveolens are as homologous as the a-subunit genes, it should be possible to readily complete the characterization of the genes encoding both subunits of anthranilate synthase in Rum.

D. MOLECULAR GENETICS OF ACRIDONE SYNTHASE The cDNA encoding acridone synthase has been screened from a cDNA library prepared from polyA' RNA isolated from elicited cell suspension cultures of R. gruveolens using oligonucleotides based on peptide sequences

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TABLE IX AMINO ACIDSEQUENCE COMPARISON OF ANTHRANILATE SYNTHASE al“ A N D d bSUBUNITS FROM R. graveolens

MSAAATSMQS LKFSNRLVPP SRRLSPVPNN VTCNNLPKSA APVRTVKCCA SSWNSTINGA YRLRTLKC --MITLNVET PPLTRSQL S TF V SAASV NFNDRVAT R WRPNSLSLTT AATTNGASAA SNGASTTTTT WSDATRFID SSKRANLVPL YRCIFADHLT PVLAYRCLVQ S K SASTSAST ASP PSPSL VDQS NFHEA - KG I EDDKETPSFL FESVEPGRI- STVGRYSWG AHPVMEVIAK DNMVTVMDHE KGSLVEEWD RDA SQA SI Q A1 IV E IL G QRT QF E DPMEIPRRIS EDWKPQIIDD LPEAFCGGWV GFFSYDTVRY VEKKKLPFSK APQDDRNLAD DV M G L E Y FS T P MHLGLYNDVI VFDHVEKKVY VIHWVRLNQQ SSEEKAYAEG LEHLERLVSR VQDENTPRLA D Y V A E N D M N R N H IVP K R AF V D PGSIDLHTGH FGPPLKKSNM TCEEYKMAVL AAKEHIQAGD IFQIVLSQRF ERRTFADPFE S A E E L S K R KERS VYRALRVVNP SPYMTYMQAR GCVLVASSPE ILTRVKKNKI VNRPLAGTAR RGRTTEEDEM I S 1 L I R T I K RK LV LETQLLKDAK QCAEHVMLVD LGRNDVGKVS KSGSVKVEKL MNVERYSHVM HISSTVTGEL I EP I H FKE N E QDNLSCWDAL RAALPVGTVS GAPKVKAMEL IDELEVNRRG PYSGGFGGIS FTGDMDIALA L H TS I K T L LRTIVFQTGT RYDTMYSYKN ATKRRQWVAY LQAGAGIVAD SDPDDEHREC QNKAAGLARA M A DVD E I H A Q E A IDLAESAFVN KSSS S IE - - * EMBLlGenBanklDDBJ Databases accession number L34344. EMBLlGenBanklDDBJ Databases accession number L34343. Only the a2 subunit residues that differ from those of the a1 subunit are shown.

of the native enzyme (182, 289). The derived amino acid sequence (Table X) encodes a protein of 391 amino acids and is 72% homologous to chalcone synthase [EC 2.3.1.741 and 60% homologous to resveratrol synthase [2.3.1.95]. The Ruta cDNA was heterologously expressed in E. coli where it catalyzed exclusively the condensation of N-methylanthraniloyl-CoA (115) with three molecules of malonyl-CoA to form 1,3-dihydroxy-N-methylacridone (116). Despite strong structural similarities to chalcone synthase, the heterologously expressed enzyme does not accept 4-coumaroyl-CoA as a substrate. Acridone synthase contains the peptide sequence WGVLFGFGPGLT known as the “chalcone synthase/resveratrol synthase signature motif”

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TABLE X AMINO ACIDSEQUENCE OF ACRIDONE SYNTHASE FROM R. graveolens MESLKEMRKA QMSEGPAAIL AIGTATPDNV FMQADYPDYY FRMTKSEHMT ELKDKFRTLC EKSMIRKRHM CFSEDFLKAN PEVCKHMGKS LNARQDIAW ETPRLGNEAA LKAIKEWGQP K S S I T H L I F C SSAGVDMPGA DYQLTRILGL NPSVKRMMIY QQGCYAGGTV VRLAKDLAEN NKGSRVLWC SELTAPTFRG PSPDAVDSLV GQALFADGAA ALWGADPDS SIEFlALYYLV SASQMLLPDS DGAIEGHIRE EGLTVHLKKD VPALFSANID TPLVEAFKPL GISDWNSIFW IAHPGGPAIL DQIEEKLGLK EDKLRASKHV MSEYGNMSSS CVLFVLDEMR SRSLQDGKST TGQGLDWGVL FG FGP GT , DE T I V L R S V P I E A

EMBLlGenBanklDDBJ Databases accession number U4088. The putative binding site for N-rnethylanthraniloyl-CoA(115) is printed in boldface. The underlined sequence is the "chalcone synthasehesveratrol synthase signature motif."

(190), as well as a sequence similar to the putative 4-coumaroyl-CoA binding sequence embedding a cysteine residue in chalcone synthase and resveratrol synthase (192) (Table X). This is proposed to be the N-methylanthraniloyl-CoA (115)binding site of acridone synthase based on these similarities to chalcone synthase and resveratrol synthase. RNA gel blot analysis of R. graveofens cell suspension cultures demonstrated that the level of the acridone synthase RNA transcript is increased in response to fungal-elicitor treatment (289). Transcript levels reach a maximum within 3 h after the addition of elicitor to the cell cultures, consistent with the accumulation of acridone alkaloids under similar treatment (178). Heterologously expressed acridone synthase has now been crystallized (U. Matern, personal communication) making it the first enzyme of alkaloid biosynthesis to undergo X-ray crystallographic analysis.

VII. Conclusions and Future Prospects The enzymatic synthesis of approximately ten alkaloids to date is either completely or nearly completely elucidated. The majority of these alkaloids, ajmaline, vindoline, berberine, corydaline, macarpine, morphine, berbamunine, scopolamine, and nicotine, are of pharmaceutical interest and those that are currently in the pharmacopea are still isolated for industrial use from the plants that produce them. The future for research on these alkaloids lies in the development of alternate systems of production, such as plant cell culture or microbial systems, that are independent of weather,

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blight, and politics, as well as in the development of plants with an improved alkaloid spectrum for a more efficient production of pharmaceutical alkaloids isolated from field-grown plants. The design of these alternate systems and optimized plants requires the techniques of molecular manipulation provided by molecular genetics. Meaningful molecular genetic manipulations require, in turn, knowledge of alkaloid biosynthetic pathways at the enzymic level. To this end, substantial progress has been made with the select alkaloids listed herein, but there remains much to be discovered about the enzymatic synthesis of pharmaceutically important alkaloids such as camptothecin, quinine, and emetine, to list only a few examples. cDNAs have now been isolated for more than ten enzymes of alkaloid biosynthesis and the rate of cloning is sure to increase in the coming years. Simultaneous with the isolation of genes is the development of heterologous expression in bacterial, yeast, and insect cell culture systems of single enzymes, and soon short pathways, to be used for the partial biomimetic syntheses of alkaloids. Our understanding of the regulation of alkaloid gene expression by elicitation or tissue-specific expression will also improve as the promoters of alkaloid biosynthetic genes are analyzed. With regard to the metabolic engineering of plants, transgenic A m p a plants (transformed with the hyoscyamine 6P-hydroxylase cDNA) provide the first example of how medicinal plants can be successfully altered using molecular genetic techniques to produce increased quantities of a medicinally important alkaloid, in this case, scopolamine. The transformation of canola with the tryptophan decarboxylase cDNA demonstrates how alkaloid biosynthetic genes can be used to improve crop species, in this case, through the reduction of indole glucosinolates in seed. This field is currently limited by our ability to transform and regenerate medicinal plants and certain crop species. To date, expertise in this important area lags well behind that for tobacco, petunia, and even cereal crops, etc. The production of alkaloids in tissue and cell culture was an area of research that received much attention in the past because it promised an alternate source of pharmaceuticals. However, many important compounds such as vincristine, vinblastine, pilocarpine, morphine, and codeine among many others are not accumulated to any appreciable extent in culture. The reason for this is thought to be the tissue-specific expression of alkaloid biosynthetic genes, because in select cases root and embryoid cultures, as well as plants regenerated from nonproducing callus cells, contain the same alkaloid profile as the parent plant. This indicates that de-differentiated cells, such as those found in suspension culture, do not contain the correct cell type for the activation of all alkaloid biosynthetic genes. It may be possible in the future to dissect the complex regulation of alkaloid biosyn-

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thesis into single factors that control this tissue-specific expression in order to deregulate production of alkaloids in cell culture. The future of the 200-year-old alkaloid field is bright. We can look forward to genetically engineered micro-organisms and eukaryotic cell cultures that produce alkaloids, to metabolically engineered medicinal plants with tailored alkaloid spectra, to the production of pharmaceutically important alkaloids in plant cell culture, and even to the enzymatic synthesis of new alkaloids.

Acknowledgments

Our molecular genetic work described herein was supported by a grant from the Bundesminister fur Forschung und Technologie and by Sonderforschungsbereich 369 of the Deutsche Forschungsgemeinschaft, Bonn.

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-CHAPTER P

PSEUDODISTOMINS: STRUCTURE, SYNTHESIS, AND PHARMACOLOGY ICHIYA NINOMIYA, TOSHIKO KIGUCHI, A N D TAKEAKI NAITO Kobe Pharmaceutical University Higashinada, Kobe 659, Japan I. Introduction A. Isolation of Pseudod B. Structure Elucidation of Pseudodistomins .......................................... 111. Synthesis ........... ........................ ............. A. Synthesis of Tetrahydro-pseudodistomin ................... B. Total Synthesis of Pseudodistomins and Their Analogs ......................... IV. Biogenesis ........................ V. Pharmacolo ...... References .......................................................................................

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I. Introduction

Since the end of last decade, the level of interest regarding biologically active components from marine organisms has noticeably increased leading to the initiation of research programs in certain countries that have specific links toward natural resources in the rim of the Pacific basin, particularly in Australia, USA, and Japan. Among a number of various types of marine products isolated, we have been particularly interested in the marine alkaloids with a piperidine ring, the pseudodistomins. This review deals with their occurrence, structure, synthesis, and pharmacology. The particular reason why we selected this group of alkaloids is based not only on their biological activity but also their characteristic structures were suited to the synthetic strategy with which we have been involved for several years. In the course of our synthetic work, we also happened to be involved in the revision of some of the structures originally proposed, and then to structureactivity studies of the biologically active alkaloids. THE ALKALOIDS. VOL. SO WW-YSYRiYR $2S.(NI

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11. Isolation and Structure

A. ISOLATION OF PSEUDODISTOMINS 1. Pseudodistomins A and B (1987)

Kobayashi and co-workers ( 1 ) have been engaged in the search for biologically active substances from marine tunicates which are abundant on the coasts of the southern islands of Japan. In their search, they succeeded in the isolation of a new class of nitrogen-containing compounds (2), which possess very potent antineoplastic and calmodulin antagonistic activities, from the orange-colored tunicate Pseudodistorna kanoko Tokioka et Nishikawa (family Polyclinidae), collected at Ie Islands, Okinawa by scuba divers at depths of 5-10 m. The methanol-toluene (3 :1) extract of Pseudodistoma kanoko was partitioned between toluene and water. The chloroform extract of the aqueous layer, which showed potent cytotoxicity against L1210 murine leukemia cells, was subjected to flash column chromatography on silica gel (CHCl3/n-BuOH/AcOH/H20, 1.5 :6: 1: 1) followed by reversed phase HPLC [ODS, (CH3CN/H20/TFA, 500: 500: l)] to give a ca. 1:1 mixture of pseudodistomins A (1)and B (2) as a colorless oil. The constituents had the same molecular weight [FABMS, d z 295 (M + H)+] and very close HPLC retention times under various solvent systems, and both were quite susceptible to oxidation on exposure to air and to E/Z-isomerization of the double bonds. Acetylation afforded a mixture of two acetates which were separated by reversed-phase HPLC (ODS, 88%MeOH) to give two acetates 3 and 4, in the yields of 0.012% and 0.018% of wet weight, respectively. They are designated as pseudodistomin A (1) and B (2). 2. Pseudodistomin C (1995) In 1995, Kobayashi’s group (3) again carried out the extraction of bioactive components from the same tunicate Pseudodistoma kanoko, looking for new components, and obtained a third alkaloid designated as pseudodistomin C (8). Extraction with methanol-toluene (3 :1) was carried out and the extract was partitioned between toluene and water. The toluene-soluble material was subjected to silica gel flash column chromatography followed by preparative TLC to afford pseudodistomin C (8) (0.03% yield, wet weight). The fraction containing pseudodistomins from the silica gel column was acetylated (AqO/pyridine) and the acetylated mixture was purified by reversed-phase HPLC to give pseudodistomin C acetate (9) together with pseudodistomin A and B acetates (3) and (4). This acetate 9 was used for characterization and spectral studies.

8. PSEUDODISTOMINS:

STRUCTURE, SYNTHESIS, AND PHARMACOLOGY

319

B. STRUCTURE ELUCIDATION OF PSEUDODISTOMINS

The first paper regarding this series of natural products appeared in 1987 (2) on the isolation and structure proposal of pseudodistomins A (1) and B (2). The structures of pseudodistomins A (1) and B (2) were proposed based mainly on the assignment of their 'H and 13C NMR spectra, in addition to the conversions to tetrahydro-pseudodistomin, which was achieved from both alkaloids 1 and 2 by catalytic hydrogenation. Tetrahydro-pseudodistomin played a crucial role in determining both the skeletal structure of these alkaloids and also the structural correlation of the two alkaloids. I . Structure of Tetrahydro-pseudodistomin

Hydrogenation of the dienyl group in the side chain of both pseudodistomins A and B acetates (3 and 4) afforded tetrahydro-pseudodistomin acetate (9, thereby establishing that alkaloids 1 and 2 have the same skeleton and also the same absolute configuration at the asymmetric centers C-2, C-4, and C-5. The relative stereochemistry of the piperidine ring was determined by analyzing the ' H NMR spectrum of tetrahydropseudodistomin acetate (5). By using various NMR techniques, decoupling experiments, comparison with the J values of 4,5-dihydroxy-pipecolic acids, and difference nOe experiments, the structure of the acetate 5 was suggested. The intramolecular hydrogen bonding between the axially oriented NH on C-5 and the nitrogen at position-1 is reported to stabilize the conformation of the piperidine ring. The absolute stereochemistry was determined by applying the nonempirical dibenzoate chirality method of the bis( p-bromobenzoyl) derivative 6. As a result, the absolute configuration of both pseudodistomins A (1) and B (2) was concluded to be 2R, 4R, and 5s. 2. Structure of Pseudodistomin B The structure determination of pseudodistomins A (1) and B (2)was carried out primarily with pseudodistomin B acetate (4) due to its abundance. It was obtained as a colorless oil and showed a molecular formula of C24H40N204 (HREIMS, r d z 420.2974, A -1.2 mmu) and [Q]D +35" (c 1, MeOH). The increase in the molecular weight by 126 amu and the IR bands at 1740 and 1630 em-' suggested that the acetate 4 possesses one 0acetyl and two N-acetyl groups compared with 2. In the 'H NMR spectrum, several protons were observed as broad signals or signals split in a 1:4 ratio, indicating the presence of two slowly interconverting conformations due to the amide rotation of the N-acetyl group. Interpreation of the 'H and 13CNMR spectra obtained using various techniques, including COSY

320

NINOMIYA, KIGUCHI, A N D NAITO OR

ANHR 5

3

1 R=H: proposed structure for pseudodistomin A 3 R=Ac

** . -

6NHR'

&NHR2 R'

5 R'=R~=AC

E

~

E

\. \.

5

3

bsc

-'.

16

R'=Ac. R2=pBrC6H4C0

R

2 R=H: proposed structure for pseudodistornin B 4 R=Ac

SCHEME 1. Proposed structures of pseudodistomins A (1) and B (2). Reagents: (a) H2, PdC; (b) KOH, MeOH; (c) p-brornobenzoyl chloride, DMAP, pyridine.

and two-dimensional 'H-13C shift correlation experiments, suggested the structure. The side chain of a trideca-3'3 '-diene was deduced also from the 'H and I3C NMR spectra. From the above assignment of the spectral evidence, the structures of the two pseudodistomins A (1)and B (2) were proposed as 2(R)-(trideca-3'(E),s'(Z)-dienyl)-4(R)-hydroxy-5(S)-aminopiperidine (1) and 2(R)-(trideca-3'( E),5'(E)-dieny1)-4(R)-hydroxy-S(S)amino-piperidine (2). However, these proposed structures were later revised when the natural and synthetic samples were compared (4).The position of the dienyl group in the side chain was later firmly deduced by ozonolysis of newly supplied samples of pseudodistomin B (1)and then A (2) (9,which were collected later, and which yielded the piperidine fragment 7 with a six-carbon side chain. Thus the structures were firmly established by shifting the position of the dienyl group from the proposed 3'- and 5'- to the 6'- and 8'-positions.

3. Structure of Pseudodistomin A

DI.([

Pseudodistomin A acetate (3), colorless oil, +36" ( c 1,MeOH), was shown to have the same molecular formula, C24H40N204,as that of 4. The 'H and 13CNMR spectra showed that the structure of 3 was different from that of 4 only in the olefin geometry within the side chain. The coupling constants of 53f,4f = 15 and 55t,6, = 11 Hz implied that the double bonds of 3 possessed the 3'E and 5'Z configurations. From NMR measurements,

8.

PSEUDODISTOMINS: STRUCTURE, SYNTHESIS, AND PHARMACOLOGY

321

8 ' 6

0 3'

R

a, b. c

GNHA

\

2 R=H: revised structure for pseudodistomin B 4 R=Ac

1 R=H: revised structure for pseudodistomin A 3 R=Ac

OR

8 R=H: pseudodistomin C 9 R=Ac

OAc

10

SCHEME2. Structures of pseudodistomins A (l),B (2), and C (8). Reagents: (a) 0,; (b) NaBH4; (c) AczO, pyridine.

along with the structural correlation with tetrahydro-pseudodistomin acetate (5) and pseudodistomin B (2), the structure of pseudodistomin A (1) was proposed to be an isomer of 2 with respect to the geometry of the dienyl group at the 3 ' ( E ) ,S'(Z)-positions, which was subsequently revised to the 6'E- and 8'2-configurations ( 5 ) . The 6'E- and 8'2-configurations were deduced from the assignment applying a combination of the HOHAHA spectrum, 'H-'H COSY spectrum, and the coupling constant data of the acetate 3. 4. Structure of Pseudodistomin C

Pseudodistomin C acetate (9) (3) was obtained as a colorless oil having [a],, +85" ( c 0.98, CHC13) and was shown to have the molecular formula

C26H40N204 by the HREIMS data ( d z444.2925, M+A -4.3 mmu), implying that 9 contains two carbons more than pseudodistomin A and B acetates (3 and 4). The UV spectrum showed an absorption maximum at 235 nm with a molar absorption ( E 37,000) being almost twice as large as those of

322

NINOMIYA, KIGUCHI, A N D NAITO

3 and 4, thereby suggesting the presence of two conjugated diene chromophores in the structure of 9. The 'H NMR spectra of pseudodistomins A and B acetates (3 and 4) in CDC13 showed the signals of two conformers in solution, but all the protons were assignable. In contrast, the 'H NMR spectrum of 9 in CDC13showed such broad resonances that no signals could be assigned. 2D-NMR experiments ('H-'H COSY, HSQC, and HMBC) of 9 were therefore carried out in a CD30D solution, which showed relatively well-resolved signals. Since the 'H NMR spectrum of the natural alkaloid 8 in C5D5N solution had better resolution, 'H-'H COSY and HSQC spectra of 8 were recorded in this solution. From these spectral data, pseudodistomin C (8) was suggested to consist of a piperidine moiety and a doubly unsaturated side chain. The piperidine unit has the same substituents (4-hydroxyl and 5-amino groups) as those of pseudodistomins A (1) and B (2) and the side chain attached to C-2 contains two dienes. The 'H-'H COSY spectrum of 8 showed a clear crosspeak between a piperidine-ring proton on C-2 (&, 3.40) and an olefinic proton at C-1' (&, 5.83). Thus, one of the two dienes was shown to be located at the C-l'-C-4' positions. To clarify the position of the second diene, ozonolysis was carried out on 8, to afford the product 10 on NaBH4 reduction and acetylation. The diacetate of lS-pentanediol was also detected on the basis of reversedphase TLC and HPLC analyses. The second dienyl group was therefore deduced to be located at the C-8'-C-llf positions. The geometries of these two double bonds were inferred to be all E from the coupling constants, 5,'.2' = 53,,4, = 15.4 Hz, and the l3C chemical shifts of the allylic methylenes (C-5', C-7', and C-12'; Sc 31.7-32.4). In order to obtain unambiguous evidence of the stereochemistry of the piperidine ring moiety of pseudodistomin C (8), the acetate 10, which was obtained by HPLC purification of the ozonolysis product from 8, was synthesized in an optically pure form (Section III.A.5).

111. Synthesis

A. SYNTHESIS OF TETRAHYDRO-PSEUDODISTOMIN As described in the previous section, pseudodistomins A (1)and B (2) were readily hydrogenated at the dienyl group in their side chains to afford an identical tetrahydro-pseudodistomin, which played a key role in their initial structure determination. Four syntheses of this structure were reported, almost at the same time, in 1992-1994.

8. PSEUDODISTOMINS:

STRUCTURE, SYNTHESIS, AND PHARMACOLOGY

323

1. Natsume's Synthesis (1992)

Natsume et al. had developed a new type of carbon-carbon bond forming reaction using singlet oxygen and succeeded in applying this reaction to hydropyridines (6) and then to tetrahydro-pseudodistomin (7). After paving the route by preliminary studies, the synthesis of tetrahydropseudodistomin was initiated from 4-trimethylstannylpyridine (11) (8), which underwent smooth alkylation with Grignard reagent only at the C2 position, due to the presence of a stannyl group at the C-4 position, to form the 1,2-dihydropyridine derivative 12 in 84% yield. The product was then subjected to sensitized photooxygenation in dichloromethane and the

0 Piperidine ring @ 2-Alkyl p u p @ 5-Amino Group @ 4-Hydroxyl group

-

Pyridine Alkylation of pyridine ring Photooxygenation using singlet oxygen and 1,3-heteroatom transposition Hydroboration oxidation

a

d R

84% 11

66%

R"

R

98%

Z

2

2

12

13

14

R=C13H27

0

-

@ NHCOC13

e, f

76%

75%

R\'

2

2 15

OH

g, h

16

R"

2 17

n

18

19

5 tetrahydropseudodistomin acetate

20

SCHEME 3. Natsume's synthesis of (2)-tetrahydropseudodistomin acetate (5). Reagents: (a) C17HZ7MgBr.ZC1: (b) '0,: ( c ) NaBH3CN, SnCl,; (d) (COOH),; (e) C13CCN. NaH; ( f ) xylene, A; (g) NaOH: (h) (TMS),NK, ZCI: (i) BH3.THF Hz02, NaOH; (k) Collins reagent: (I) KB(CHMeEt)3H (m) Hz. Pd-C (n) Ac20, pyridine.

u)

324

NINOMIYA, KIGUCHI, AND NAITO

resulting mixture was treated successively with sodium cyanoborohydride and tin( 11) chloride. The piperdine derivative 13 was obtained in 66% yield. The stannyl group was removed by heating 13 with oxalic acid in THF-H20 to produce trans1-substituted 2-tridecy1-1,2,3,6-tetrahydropyridin-3-01 14 in 98% yield. The 1,3-transposition ( 9 ) of the functional groups of 14 to the 5-amino function in 15 with retention of the stereochemistry was carried out by treating 14 with trichloroacetonitrile in the presence of sodium hydride in THF, followed by heating in xylene, to form the trichloroacetamide 15 in 76% yield. This compound was then converted to the carbamate 16 by alkaline hydrolysis and treatment with potassium bis(trimethylsily1)amide in THF, followed by the addition of benzyloxycarbonyl chloride. Introduction of a hydroxyl group was effected by hydroboration of 16 to give the 4a-hydroxy derivative 17 in 75% yield, which was then converted to the required 4phydroxy derivative 19 by oxidation and reduction. Finally, removal of the protective group from 19 under catalytic conditions yielded tetrahydropseudodistomin (20)in 84% yield. The acetate 5 of 20 was identical with the derivative obtained from the natural alkaloid. This synthesis therefore provided concrete evidence for the skeleton of the proposed structures of pseudodistomins A (1) and B (2),except for the position and geometry of the dienyl group in the side chain. 2. Ninomiya’s Synthesis by Enamide Photocyclization (2 992) Evaluating the structures of tetrahydro-pseudodistomin and the pseudodistomins, Ninomiya et al. considered a strategy of synthesizing pseudodistomins by applying the enamide photocyclization technique (20) aimed at an extension of their synthesis to a wide variety of pseudodistomin analogs in order to develop a general synthetic route to these biologically active antineoplastic derivatives. Their synthetic strategy was to form first a piperidine ring with a cis 5-amino-4-hydroxy moiety by photocyclization of the enamide having a five-membered oxazole ring and the long carbon chain at the C-2 position which could be formed by substituting a methylthio group ( 2 2 J 2 ) . Thus the enamide 22 was prepared from the thioimidate 21 by acylation with 2-phenyloxazole-4-carbonylchloride and subjected to reductive photocyclization involving irradiation in the presence of sodium borohydride in acetonitrile-methanol (9 :1).A mixture of the photocyclized lactams 23a and 23b was obtained in 59% and 7% yields respectively. Irradiation of the (methy1thio)lactam 23 with a high-pressure mercury lamp (Pyrex filter) in the presence of allyltributyltin in toluene-acetonitrile (7 :3) for 3 days (23) gave the desired a-allyllactam 25 in 40% yield, along with a small amount of the p-allyllactam 24 (21%). Treatment of the allyllactam 25 with borane-THF complex, followed by hydrogen peroxide, resulted in the conversion of three functional groups in the molecule in 64% yield.

8. PSEUDODISTOMINS: STRUCTURE, SYNTHESIS,

AND PHARMACOLOGY

325

The conversion included oxidation of the ally1 group to the propanol, reduction of the lactam carbonyl group to the amine, and a ring-opening reaction of the oxazoline moiety to the cis-amino-alcohol structure. Selective protection of the primary hydroxyl group of the diol26 by a silyl group, debenzylation by hydrogenolysis in the presence of Pearlman's catalyst, acetylation, and desilylation gave the triacetylalcohol 28 in four steps in overall 50% yield. Oxidation of the alcohol 28 with pyridinium chlorochromate in the presence of sodium acetate gave the unstable aldehyde which was then condensed with E-2-decenylphosphorane under Wittig conditions to give a mixture of dienes in 46% yield from 28, which, without separation, @ Piperidine ring @ 5-Amino group @ 4-Hydroxyl group @ 2-Alkyl group

I-

h Reductive photocyclization of enamide Photoallylationwith allyltributyltin

0

Me

a

-

b

C

MeSWNBn 21

Bn

Bn 22

23a 7p-SMe 59% (from 21) 23b 7a-SMe 7% (from 21)

COCl Ph d, e 64%

*

HOa,..

Bn

Bn

Bn

24 21%

25 40%

26

27

28

5 tetrahydropseudodistornin acetate

SCHEME4. Ninomiya's synthesis of (2))-tetrahydropseudodistominacetate (5). Reagents: (a) Et3N; (b) hv, NaBH4, MeCN-MeOH; (c) allyltributyltin, hu, MeCN-toluene (3: 7); (d) BH,.THF (e) H202.NaOH; ( f ) TBSC1, imidazole, DMF; ( 9 ) H2. Pd(0H)Z-C; (h) Ac20, pyridine; (i) AcOH, THF, H 2 0 ; (j)PCC, AcONa, CH2C12; (k) (E)-2-decenyltriphenylphosphonium bromide, NaH, T H F (1) H2, Pd-C.

326

NINOMIYA, KIGUCHI, AND

NAITO

was directly subjected to catalytic hydrogenation in the presence of 10% palladium on carbon to afford, as a single product, 5 in 42% yield from 28, which was identical with the compound derived from the natural alkaloid on comparison of IR, 'H and I3C NMR spectra. 3. Knapp's First Asymmetric Synthesis (1993)

The first asymmetric synthesis of tetrahydro-pseudodistomin was reported by Knapp's group ( I d ) , after a lengthy route of over 20 steps which featured the use of D-serine as the chiral source, and dibenzyltriazone as a protective group for the amino group. This route established both the skeletal structure and the absolute stereochemistry of the piperidine ring moiety of the alkaloids 1and 2. D-Serine possesses a three-carbon chain with functionality and absolute stereochemistry that are appropriate for elaboration to C-4,5,6 of the pseudodistomins. Furthermore, Knapp et al. (15) had already converted the amino group of several a-amino acid derivatives to the dibenzyltriazone, a nonpolar protective group that withstands a variety of functional group transformations and C-C bond forming reactions and also slows the racemization of the derived a-aminoaldehydes. Thus, ethyl D-serinate hydrochloride was 0-silylated and converted to its dibenzyltriazone derivative 29. Reduction of the ester 29 followed by the Mitsunobu reaction (16) with Zn(N3)*gave the azide 30. The azide group was reduced and protected as its N-trifluoroacetate 31.0-Desilylation and Swern oxidation of the alcohol 32 led to the protected 2,3-diaminopropanal 33. That no racemization of intermediates had occurred was demonstrated by reduction of 33 back to 32; the (S)-Mosher ester of 32, whether from 31 or 33, had de >98%. Lewis acid-promoted addition (17) of allyltrimethyltin to the aldehyde 33 as in Scheme 5 , occurred with stereoselectively (8 : 1) according to the Cram rule to afford the homoallylic alcohols 34a and 34b; pure 34a was obtained after crystallization. After hydroxyl protection, the carbon chain was extended by oxidative cleavage of the alkene and then Wittig reaction to afford a 7: 1 mixture of the cis and trans unsaturated esters 35a and 3 9 , which were readily separated. Methanolic sodium borohydride removed the Ntrifluoroacetate from either pure 35a or mixture 35ab and triggered an intramolecular Michael reaction in which the piperidine 36 was formed as the only cyclized product in both cases. The stereochemistry of 36 was determined unambiguously from 'H-'H coupling constants. Treatment of 35a with methanolic sodium methoxide afforded 36 stereoselectively, but in only 25% yield. The indication that the piperidine 36 exists in a chair form, with the bulky dibenzyltriazone group and the twocarbon chain occupying equatorial positions and the protected hydroxyl group in an axial position, helps to rationalize the high kinetic stereoselectivity in the cyclization (Fig. 1).

8. PSEUDODISTOMINS: STRUCTURE, SYNTHESIS, AND @ 5-Amino group @ 4-Hydroxyl group

I

@ Piperidine ring @2-AWlgroup

TBSO

OH

II-

Michael addition

29

,-N R

= -N

En

NHCOCF3

30

31

32

>O

lVVVl

34a threo : 34b etythro (8: 1)

37

f

L N

11.

'NHCOCF~

HO

TBSO

N3

D-serine

33

D-Serine (Lewis acid-promoted addition of allylnimethyitin)

a. b

COOH

327

PHARMACOLOGY

..

3

H

35a trans : 35b cis (7:l)

38

36

20 R'=H u c 5 R'=Ac; tetrahydropseudodistomin acetate

SCHEME5. Knapp's synthesis of (+)-tetrahydropseudodistomin acetate (5). Reagents: (a) LiBH,; (b) Zn(N3)2(pyridine)z,PPhl, DEAD; (c) H2, Pd-C: (d) (CFSCO)~O:(e) TBAF ( f ) (COC1)2, DMSO, DIPEA; (g) allyltrirnethyltin,BF3.Etz0, CH~CIP: (h) TBSOTE (i) Os04, NMO; (j) NaI0,; (k) Ph3P = CHC02Me; (1) NaBH4: (m) (Boc),O; (n) LiBH4; ( 0 ) Swern CH = PPh3; (4)H2, Pd-C (r) TBAF (s) TFA; (t) aq. diethanolamine: oxidation; (p) CIOHZI (u) Ac20, pyridine.

Completion of the synthesis of the target compound 5 is shown in Scheme 5. On protecting the 3-amino group of 36 with a tert-butoxycarbonyl group, the ester in the side chain was reduced to give the primary alcohol 37. The remainder of the side chain was attached by means of Swern oxidation and a Wittig reaction with undecylidenetriphenylphosphorane, followed by hydrogenation to give the protected tetrahydropseudodistomin 38. Sequential deprotection was carried out by using tetra-n-butylammonium fluoride to remove the O-(tert-butyldimethylsilyl) group, trifluoroacetic acid to

328

NINOMIYA, KIGUCHI, A N D NAITO

Bn . . =-N

/-N

>O L N Bn

FIG.1. Stereoselectivity in the cyclization.

remove N-tert-butoxycarbonyl group and aqueous diethanolamine at pH 3 to cleave the dibenzyltriazone group, and as a result to afford crude tetrahydropseudodistomin (20). Acylation of 20 gave the acetate (+)-5 [[aID +36.9" (c 0.8, MeOH)], [lit [2]; +33" (c 1, MeOH)], whose structure was confirmed by spectral analysis. Further, Knapp et al. have carried out conformational analysis by using NMR spectra and the Macro Model program on the acetate 5 having a chair form with the Sacetamido, 4-acetoxy, and 2-undecyl groups in axial/ equatorial and axial orientations, respectively, due to severe steric interaction between the 2-undecyl and 1-acetyl groups (Fig. 2). 4. Ninomiya's Second Synthesis by 1,3-Cycloaddition of a Nitrone (1994)

During the course of synthetic studies directed to pseudodistomins and tetrahydropseudodistomin, serious problems arose from the fact that the proposed structures of the alkaloids needed to be revised. It became apparent that spectral evidence would not be enough to establish the structures unambiguously. Therefore, it seemed necessary to synthesize a series of analogs which could be the structure of alkaloids, and thus Ninomoya et al. planned to develop another synthetic route with wide applicability in order to investigate the structure-activity relationships. As the key reaction, Ninomiya et al. selected the 1,3-cycloaddition of a nitrone with an olefin

FIG.2. Conformation of tetrahydropseudodistomin acetate (5).

8. PSEUDODISTOMINS: STRUCTURE, SYNTHESIS,

AND PHARMACOLOGY

329

for the construction of 2-substituted 5-aminopiperidin-4-01s (18). Nitrone cycloaddition has been known as a useful reaction for the construction of 1,3-amino alcohol systems widely found in natural products (29). Initially, the cycloaddition of the nitrone derived from tetradecanal to racemic 2aminobut-3-en-1-01 was investigated. Tetradecanal (39) was condensed with N-benzylhydroxylamine to give the nitrone 40 which on cycloaddition to racemic 2-(N-tert-butoxycarbonylamino)but-3-en-l-o1 (41) (20) afforded a 2 : 3 : 3 :5 mixture of four adducts 42-45 in 81% combined yield which was readily separated by chromatography. The stereostructures of these four adducts 42-45 were unambiguously confirmed by the following ring transformation of the isoxazolidines to the readily assignable piperidine derivatives. On treatment with methanesulfonyl chloride-pyridine, the adducts 43 and 45 gave the corresponding mesylates 47 and 49, while the most nonpolar adduct 42 and the third adduct 44 afforded the bicyclic compounds 46 and 48, respectively, as a result of concomitant ring formation. On treatment of two mesylates 47 and 49 with hydrogen in the presence of Pearlman's catalyst, the following, smooth, three-step sequence of reactions, cleavage of the N - 0 bond, debenzylation, and N-alkylation, occurred to give the piperidinols 51 and 53 in 6280% yields. Similarly, the two quaternary salts 46 and 48 were converted into the piperidinols 50 and 52 in 80-83% yields. Deprotection of the N-tert-butoxycarbonyl group in the piperidinols 50-53 gave the 5-amino-4piperidinols 20,54-56, respectively, in quantitative yields, which were characterized as their corresponding acetates for identification. From these conversions, tetrahydropseudodistomin (20) was also synthesized; initially by simple deprotection of 50 by trifluoroacetic acid, and second from 53 by a combination of oxidation (chromium trioxide-pyridine) and reduction with K-selectride. The above experiments were carried out with the racemates, and then also successfully applied to the asymmetric synthesis of (2R,4R,5S)tetrahydropseudodistomin (20) as follows. Cycloaddition of the nitrone 40 with (+)-2-aminobut-3-en-l-o1 41, prepared from D-methionine, gave four optically active adducts 42-45 in 79% combined yield, which were readily separated. The most nonpolar adduct, (-)-42, was subjected to mesylation, catalytic hydrogenolysis, and then deprotection to afford the desired (2R,4R,5S)-20 in 67% overall yield. Acetylation of the piperidinol 20 with acetic anhydride-pyridine gave the acetate (+)-5, m.p. 82-84°C (EtzO), [uID+70.3" (c. 0.64, MeOH), [lit [2]; +33" (c 1, MeOH)] [lit [14]; +36.9" (c 0.8, MeOH)] which was identical with an authentic sample of (+)tetrahydropseudodistomin acetate (5) (IR, 'H and 13CNMR). However, a large difference was observed between the values of the optical rotation of tetrahydropseudodistomin acetate (5) obtained from natural (as an oil)

330

NINOMIYA, KIGIJCHI, A N D NAITO

@ 5-Amino group @ 2-Alkyl group @ 4-Hydroxyl group @ Piperidine ring

-

D-Methionine

I-

1,3-Dipolarcycloaddition and ring transformation

I

R

H

a

NHBoc

‘L0OBn

RAO

-b

&OH

c-

MeS3LOOH D-methionine

I b

I t

J NHBoc

NHBoc

Bn 42 cis-erythro 13%

Bn 43 cis-fhreo 17%

&?

R“

I

I

t

1

NHBoc

N

NHBoc

R“ S N O

44 trans-erythro 19%

mNHBoc mNHBoc NHBoc

NHBoc

R“

H

Bn 45 trans-fhreo

N Bn 46

d

J

Ms’

??OMSBn 47

dj K i l 4 3 )

F242)

R

R“ ?+OMS N Bn

N Ms‘ Bn

48

1-:2

49

44)

GNHR2 IjrNHR2 OR’

R“

R1

R

R’

50 RLH, R~=BOC

.( 20 R’=R*=H

5 R1=R2=Ac; (+)-tetrahydro pseudodistornin acetate

1

9. h. i. f 66% 2o

SCHEME6. Ninomiya’s synthesis of (+)-tetrahydropseudodistomin acetate (5). Reagents: (a) PhCHZNHOH, toluene, molecular sieves: (b) toluene, A; (c) MsCl, pyridine; (d) Hz, Pd(OH),-C; (e) TFA: ( f ) A c 2 0 ,pyridine;(g) (BOC)~O; (h) CrOypyridine; (i) KB(CHMeEt)3H.

8. PSEUDODISTOMINS: STRUCTURE, SYNTHESIS,

AND PHARMACOLOGY

331

and synthetic (crystals) sources. This asymmetric synthesis of tetrahydropseudodistomin (20) is an attractive alternative to the 24-step synthesis of Knapp (Section III.A.3). 5. Kobayashi’s Synthesis of a Key Intermediate for Pseudodistomin C (1995)

At the end of 1995, Kobayashi el al. (3) isolated the third component of this class, designated as pseudodistomin C from the same Okinawan tunicate Pseudodistoma kanoko and proposed a structure based on chemical degradation and synthesis of the degradation product 10. Pseudodistomin C has two dienyl moieties in the C-2 side chain at the 1’,3’- and 8’,10’-positions. In order to obtain unambiguous evidence of the stereochemistry of the piperidine moiety and the position of two dienyl groups, ozonolysis was carried out to afford the acetate 10, which plays a crucial role in both structure elucidation and synthesis. Kobayashi et al. (3) reported their synthesis of this key degradation product 10 as follows. They selected the oxazolidine homoallyl alcohol 57 (21) as the starting compound, prepared from L-serine. A 1: 1 mixture of the diastereomers at C-4 was transformed, in four steps, into the oxazolidine alcohol 58. The Mitsunobu reaction on 58, followed by the exchange of the protective group, afforded the carbamate 59, which was then subjected to amide-mercuration (22) to give the (2R)- and (2s)-piperidine derivatives 60 and 61 in the ratio 54 :46. The stereochemistries of C-2 of 60 and 61 were clearly assigned on the basis of the NOESY spectra of 62 and 63,which were obtained by deprotection of the tert-butoxycarbonyl group, respectively. The piperidine derivatives 60 and 61 were oxidatively demercurated to give the primary alcohols 64 and 66, which were then transformed, in four steps, into the tetraacetates 65 and 67, respectively. The ‘H NMR spectrum of the acetate 10, obtained from a natural specimen of pseudodistomin C (S), and that of 65, were identical, but the sign of the optical rotation was opposite, thereby proving that the product 65 was an enantiomer of the acetate 10. Kobayshi recommenced their synthesis from D-serine and succeeded in the synthesis of the derivative identical with 10.

B. TOTAL SYNTHESIS OF PSEUDODISTOMINS AND THEIRANALOGS The structures of pseudodistomins A (1)and B (2)isolated in 1987 were proposed, based mainly on spectral evidence. However, the synthesis of the compound having the proposed structure for pseudodistomin B, followed by another synthesis of 2, both by Ninomiya’s group, confronted the situation that the proposed structures for both alkaloids required revision. The problems remaining to be solved related to the location of the conjugated dienyl moiety in the side chain.

332

NINOMIYA, KIGUCHI, AND NAITO

@ 5-Amino group @ 4-Hydroxyl group

@ Piperidine ring @ 2-Akyl group

I-I

L-Serine or o-serine (Nucleophilic addition of Grignard reagent ) Amide mercuration

66

OAc

-

.. Z

Ac

AC

64

65

67

SCHEME7. Kobayashi’s synthetic study of pseudodistomins. Reagents: (a) p-TsOH, MeOH; (b) pivaloyl chloride, pyridine; (c) DMP, BF3.0Et2; (d) KOH, MeOH; (e) phthalimide, DIAD, PPh3; ( f ) H2NNH2;(8) ZCI; (h) Hg(OTFA),; (i) NaHC03; (j) NaBr; (k) TFA; (1) NaBH.,, 0,; (m) TFA; (n) Ac20; (0)Hz. Pd-C (p) AcZO,pyridine.

This happened due to an insufficient amount of natural product available at the time for thorough investigation. During the course of this synthesis, Kobayashi’s group ( 4 ) reported additional chemical evidence, including the most important reaction, ozonolysis, which afforded the piperidine derivative 7 with a six-carbon side chain at the C-2 position, and thereby concluded the position of the dienyl group to be at the C-6’-and 8’-positions.

8. PSEUDODISTOMINS:

STRUCTURE, SYNTHESIS, AND PHARMACOLOGY

333

Total syntheses of both the originally proposed and revised structures for the two alkaloids 1 and 2 have been achieved, thus firmly establishing the structures of the natural products. 1. Total Synthesis of Pseudodistomin B

a. Synthesis of the Proposed Structure. Total synthesis of pseudodistomin B (2), according to the structure proposed, was based essentially on the strategy described in the synthesis of tetrahydropseudodistomin, as shown in Scheme 4 (Section III.A.2), where the intermediate 3’E,5’Ederivative 68 was prepared (11). The aldehyde, synthesized by oxidation of 28 with pyridinium chlorochromate in the presence of sodium acetate, was reacted with the Wittig reagent prepared from 2-decenyl bromide to give a mixture of products. Only compound 68 was isolated homogeneously and had the same structure as that of natural pseudodistomin B acetate (4). Comparison of their 13C NMR spectra clearly showed chemical shift differences for various carbons as shown in Fig. 3. In the I3C NMR spectrum, the synthetic sample 68 with the 3’E,5’Estructure has a resonance at 29.3 ppm for the carbon at the 2’-position due to the influence of the N-acetyl group on the piperidine ring, being shifted to higher field even though it is an allylic position. This gives an important hint regarding the assignment of the 13C-NMRpeaks. In addition, the peaks of the three terminal carbons in the alkyl side chain appear as a normal n-propyl group. At the time, three possibilities were considered for the structure of pseudodistomin B, as shown in Fig. 4. However, when Kobayashi et af.( 5 )carried out the ozonolysis on pseudodistomin B and obtained the piperidine 7 with a six-carbon side chain, the structure was revised as having a 6’,8’-dienyl group, which fits with one of the structure expected above.

GNHAc a,b

~

E

HOA,..

46’0 Ac

28

C

,

a mixture of 3€,52diene and 3% 5’E-diene

E

H 5’3’

W

Ac

1

:

l

68 proposed structure for pseudodistomin B acetate

SCHEME 8. Ninomiya’s synthesis of the proposed structure for (+)-pseudodistomin B ace(b) (E)-2-decenyltriphenylphosphoniumbromide, tate. Reagents: (a) PCC, AcONa, CH2CI~; NaH. THF.

334

NINOMIYA, KIGUCHI, AND NAITO

Natural pseudodistomin B acetate (4) (100 MHz in CDCI,)

14.1

31.8

Synthetic 3 E . 5’E-diene 68 (125 MHz in CDC13)

FIG.3. Comparison of the I3C NMR data of the synthetic 3’E,S’E-diene 68 with natural pseudodistomin B acetate (4).

b. Synthesis of the Revised Structure (Total Synthesis of Pseudodistomin B). With the revised structure which carries a dienyl group at the 6’- and 8’-positions, total synthesis of pseudodistomin B (2) was planned starting from the key intermediate 28, which could be coupled with a dienyl Grignard reagent (23) to elongate the chain to the alkaloid (3). Tosylation of the alcohol 28 afforded the tosylate 69 which was then coupled with the Grignard reagent prepared from (E,E)-3,5-decadienylbromide in the pres-

OH

A

8 6 / /

J

N H

FIG.4. Possible structures for pseudodistomin B.

8. PSEUDODISTOMINS: STRUCTURE, SYNTHESIS,

AND PHARMACOLOGY

335

ence of dilithium tetrachlorocuprate at -20°C. The reaction proceeded smoothly to give a 2 :1 mixture of acetates 4 and the corresponding alcohol 70 in 61% combined yield. Acylation of the alcohol 70 with acetic anhydride and pyridine gave the acetate 4 which was identical with the natural product by comparison of their IR, 'H and 13CNMR spectra. Thus, total synthesis of pseudodistomin B was finally achieved following a revision of the proposed structure. 2. Total Synthesis of Pseudodistomin A Taking the revision of the structure of pseudodistomin B into consideration, and also aiming at the synthesis of a series of compounds for further study regarding their structure-activity relationships, Ninomiya et al. have applied the new methodology of 1,3-dipolar cycloaddition of a nitrone, as described in Section III.A.4, to the synthesis of pseudodistomin A.

a. Synthesis of the Proposed Structure. The proposed structure for pseudodistomin A (1)had a 3'ES'Z-dienyl group in the side chain. The only difference with pseudodistomin B (2) was the configuration of the 3'E,S'E-moiety in pseudodistomin B (2). Therefore, total synthesis was carried out using the same intermediate 74, but with a different combination of protective groups. This intermediate 74 was prepared in three ways: (1) via the route including enamide photocyclization and introduction of a three-carbon chain and oxidation to 26 and its deprotection (24); (2) and (3) 1,3-cycloaddition of a nitrone to an olefin, prepared from D-methionine, and leading to the isoxazolidines 72 and 73, which were, respectively, ring transformed to 74 (25). The primary hydroxyl group in 74 was oxidized with chromium trioxide-pyridine to the corresponding aldehyde which was then subjected to the Emmons-Horner reaction with triethyl phosphonoacetate to afford the unsaturated ester 75 with a five-carbon side chain with one double bond and a terminal ester group. The ester group was reduced with

C 4 H S W M g B r NHAc

Li2CuC14.THF, -2OOC L

Ac TSCI, EtN, 28 R=H DMAP (-69R=TS 70%

SCHEME9. Synthesis of pseudodistomin B acetate (4).

OR

ANHAC

336

NINOMIYA, KIGUCHI, AND NAITO

1,4-butanediol

TBSO NHBoc

-

P

a

NHBoc TBSOA,..

o

H

b. C, d. e

*OH

+

othertwo

+

T B S O ~ , ,

N'

Bn 72 cis-erythro 1 1%

71

NHBoc

isomers

Bn 73 trans-three0 21%

\

62Oh

&NHBn HoA*.*

N

-

NHBoc

h. c. d. e 40% HOL/\,..

Bn

i

75

74

26

&NHBOC

37% HOW,..

15%

BOC 76

6.';

k, I. m.n C7H15

3€,SE-diene

E \-A,-*

5'3'

Ac

5

:

l

68

77 proposed structure for pseudodistominA acetate

SCHEME 10. Ninomiya's synthesis of the proposed structure for (2)-pseudodistomin A acetate. Reagents: (a) toluene. A; (b) MsCI. pyridine: (c) Hz. Pd(OH),-C; (d) (Boc),O; (e) TBAF, THF; ( f ) Cr03.pyridine; (g) KB (CHMeEt)3H; (h) TBSCI; (i) (EtO),P(O)CH,COOEt, NaH, THF; (j) DIBAH; (k) MnOz; (I) octyltriphenylphosphonium bromide, n-BuLi; (m) TFA. CH2C12; (n) AczO, DMAP, pyridine.

diisopropylaluminum hydride to give the compound 76 with an unsaturated alcohol side chain. Again, the primary alcohol was oxidized with manganese dioxide, followed by the Wittig reaction with triphenyl n-octylphosphorane, deprotection, and reacetylation to afford a mixture of two compounds, 77 and 68 (5 : l),which were separated and purified. This completed the synthesis of the proposed structure for pseudodistomin A acetate having a 3'E and 5'Z-dienyl group in the side chain (23). Comparison of the I3CNMR spectra of natural pseudodistomin A acetate (3)and the synthetic compound 77 as summarized in Table I, clearly showed that they are not identical, which necessitated a revision of the proposed structure for this alkaloid, particularly regarding the position of the dienyl group. Reassignment of their NMR spectra suggested the revision by shifting the position of the dienyl group from the 3',5'- to the 6',8'-positions, as in the case of pseudodistomin B (2).

8. PSEUDODISTOMINS: STRUCTURE, SYNTHESIS, AND

PHARMACOLOGY

337

TABLE I COMPARISON

OF THE

"c NMR DATAOF THE DIENE MOIETY IN PSEUDODISTOMIN A OF THE

ACETATE (4) AND THE SYNTHETIC 3'E,S'Z-DIENE (n) Alkaloids

Natural pseudodistomin B acetate (4) (100 MHz) Synthetic 3'E,5'Z-diene (77) (125 MHz) A

Chemical Shifts of the Diene Moiety (ppm, in CDC13) 125.8 126.5 -0.7

128.4 128.1 0.3

130.1 131.0 -0.9

134.0 132.3 1.7

b. Synthesis of the Revised Structure (Total Synthesis of Pseudodistomin A). According to the reassignment of the spectral data of pseudodistomin A, Ninomiya et al. (24) carried out a total synthesis of pseudodistomin A acetate (3), having a revised structure with a dienyl group at the 6',8'position. The condensation of the tosylate 69 with the Grignard reagent prepared from deca-3E,5Z-dienyl bromide, in the presence of dilithiocuprate at -2o"C, gave the compound 3, with the revised structure for pseudodistomin A acetate (3), in 66% yield. Similarly, a Grignard reaction was camed out with the isomeric reagent prepared from deca-32,5'E-dienyl bromide to yield the product 78 isomeric with pseudodistomin A acetate (3). Again, comparison of the "C NMR spectra of the synthetic compound 3 with the 6'E,8'Z-dienyl group with that of the natural alkaloid indicated complete superimposition, as shown in Table 11. At the same time, Kobayashi et al. (5) reported the results of ozonolysis of the newly supplied pseudodistomin A (1)and obtained the acetate 7 with a six-carbon side chain. This experiment unambiguously established

1, LipCuC14.THF, -20°C

C 4 H v M g B r 2, AqO, pyridine

8 6 '

3 TSO\/\,.. 69 Ac

2.AcpO, pyridine 55%

78

SCHEME 11. Synthesis of pseudodistomin A acetate (2).

338

NINOMIYA, KIGUCHI, AND NAITO

TABLE I1 COMPARISON OF THE I3C NMR DATAOF THE DIENE MOIETY IN PSEUDODISTOMIN A AND B AND THE CORRESPONDING SYNTHETIC 6‘,8’-DIENES ACETATE Chemical Shift of Diene Moiety (pprn, in CDCl3)

Alkaloids Natural pseudodistomin B acetate (4) (to0 MHz) Synthetic 6’E,8’E-diene (4) (125 MHz) A Natural pseudodistomin A acetate (2) (100 MHz) Synthetic b’E,d’Z-diene (2) (125 MHz) A Synthetic 6’ZJ’E-diene (78) (125 MHz) A

130.2 130.2 0.0 125.8 125.9 -0.1 125.6 0.2

130.6 130.6 0.0 128.4 128.6 -0.2 128.9 -0.5

131.9 132.0 -0.1 130.1 130.3 -0.2 129.7 0.4

132.6 132.6 0.0 134.0 134.3 -0.3 134.9 -0.9

the position of the dienyl group at the 6’E,8’Z-positions and therefore the structure of pseudodistomin A (1).

IV. Biogenesis

Of the three pseudodistomins A (l),B (2), and C (8) thus far isolated from the tunicate Pseudodistoma kanoko, the structures of the two alkaloids A (1)and B (2) were established by synthesis. However, surprisingly, the stereochemistry of the third alkaloid, pseudodistomin C (8), was found not to be identical with the others. The absolute configuration of pseudodistomin C (8) was determined by comparison with the sign of the optical rotation of the key synthetic compound 64 as 2S, 4S, and 5R, the latter prepared from L-serine. This result was quite unexpected, since the piperidine alkaloids isolated from the same tunicate possess different stereochemistries at the C-4 and C-5 positions. Therefore, the difference in the structures of the pseudodistomins suggests that they are biogenetically classified as sphingosine derivatives, and that a precursor of pseudodistomins A (1)and B (2) may be reasonably assumed to be D-(+)-erythro-sphingosine; the absolute configurations of the (4R)-hydroxyl and (5s)-amino groups are coincident with those of the corresponding positions of D-( +)-erythro-sphingosine containing (3R)-hydroxyl and (2s)-amino groups. It was, however, quite surprising to find that pseudodistomin C (8) possesses the 4S,SR-~onfigurations.Pseudodistomin C (8) therefore has to be derived from the unusual L-(-)erythro-sphingosine (Czo-homolog).The biogenetic cyclization process for

8. PSEUDODISTOMINS:STRUCTURE, SYNTHESIS, AND

PHARMACOLOGY

339

the alkaloids 1,2,and 8 is assumed to give the same stereochemistry at C2 of the piperidine ring, since all of the alkaloids 1, 2, and 8 possess the same 2s-configuration. Precedence for the existence of enantiomeric isomers within the same organism have recently been reported. Leucetamol A (79) was isolated from sponge Leucetta microraphis, and this amino alcohol was described as a racemate; the 2S,3R- and 2R,3S-isomers were concurrently present in the sponge (26). From a sponge Xestopongia species, an amino alcohol 80 was isolated and was shown to have the 2R,3S-configuration by synthesis (27). Crucigasterins (80) were also isolated from a tunicate Pseudodistoma crucigaster and were reported to possess the 2R,3S-configurations. Thus, the amino alcohols 80 and 81 were proposed to be biosynthetically derived from the unusual D-alanine. These facts may provide some rationale for the coexistence of pseudodistomins A (l),B (2), and C (8), which have opposite absolute stereochemistries at the C-4 and C-5 positions, in the extract of the tunicate Pseudodistoma kanoko. From the above discussion, Kobayashi et al. (3,28) have tentatively mapped a biogenetic pathway of the pseudodistomins as shown in Scheme 12. Therefore, the biosynthetic study on the tunicates remains as an interesting future problem. H02CL/\OH iH2

HQCyoH NH2

H 1 pseudcdistomin A 3 pseudodistomin B

H 8 pseudodistomin C

SCHEME 12. A biogenetic pathway for the pseudodistomins.

340

NINOMIYA, KIGUCHI, A N D NAITO

OH

OH 79 leucetarnol A (racemate)

OH

OH

-

-

-

-S

1

NH2

NHZ 81 crucigasterins

80

FIG.5. Natural amino-alcohols.

V. Pharmacology

The orange-colored tunicate (400 g, wet weight), collected at the Ie Islands, Okinawa, was kept frozen until needed. The methanol-toluene (3 : 1) extract of Pseudodistoma kanoko was partitioned between toluene and water. The chloroform extract of the aqueous layer, which showed potent cytotoxicity against the L1210 murine leukemia cell system, was purified. Two of the new marine alkaloids, pseudodistomins A (1) and B (2) were tested for cytotoxic activity against L1210 and L5178Y murine leukemia cells in vitro. Pseudodistomins A (1) and B (2) were cytotoxic and exhibited IC50values of 2.5 and 0.4 pg/ml against L1210 cells and of 2.4 and 0.7 pg/ml against L5178Y cells, respectively (2). Pseudodistomin C (8) also exhibited cytotoxicity against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro (ICso values of 2.3 and 2.6 pg/ml, respectively) (3). Studies on the antitumor activity of 1 and 2 in vivo are currently in progress. Both pseudodistomins A (1) and B (2) also exhibited inhibitory activity of calmodulin-activated brain phosphodiesterase. The ICso values of 1 and 2 were 3 X lo-’ M. Pseudodistomins A (1) and B (2) were about three times more potent than W-7, a well-known calmodulin antagonist. Pharmacological results were also reported on the components from the other tunicates, such as Pseudodistoma cruciparta (29),Pseudodistoma novaezelandiae (30),and Pseudodistoma crucigaster (27).

8. PSEUDODISTOMINS:

STRUCTURE, SYNTHESIS, AND PHARMACOLOGY

341

Acknowledgment

The authors are grateful to Mss. Yoko Yuumoto and Miho Ikai for their contributions to the synthetic work on the pseudodistomins.

References

1. J. Kobayashi and M. Ishibashi, in “The Alkaloids” (A. Brossi and G. A. Cordell. eds.), Vol. 41, p. 41. Academic Press, New York, 1992. 2. M. Ishibashi, Y. Ohizumi, T. Sasaki, H. Nakamura, Y. Hirata, and J. Kobayashi, J. Org. Chem. 52,450 (1987). 3. J. Kobayashi, K. Naitoh, Y. Doi, K. Deki. and M. Ishibashi, J . Org. Chem. 60,6941 (1995). 4. T. Kiguchi, Y. Yuumoto, I. Ninomiya, T. Naito, K. Deki. M. Ishibashi, and J. Kobayashi, Tetrahedron Lett. 33, 7389 (1992). 5. M. Ishibashi, K. Deki, and J. Kobayashi, J . Nat. Prod. 58, 804 (1995). 6. M. Natsume, J. Synth. Org. Chem. Japan 44,326 (1986). 7. I. Utsunomiya, M. Ogawa, and M. Natsume, Heterocycles 33,349 (1992). 8. Y. Yamamoto and A. Yanagi. Chem. Pharm. Bull. 30, 1731 (1982). 9. L. E. Overman, J . Am. Chem. SOC.98,2901 (1976). 10. I. Ninomiya and T. Naito, in “The Alkaloids” (A. Brossi, ed.), Vol. 22, p. 189. Academic Press, New York, 1983. 11. T. Naito, Y. Yuumoto, I. Ninomiya, and T. Kiguchi, Tetrahedron Lett. 33, 4033 (1992). 12. T. Naito, Y. Yuumoto, T. Kiguchi, and 1. Ninomiya, J. Chem. SOC., Perkin Trans. I , 281 (1996). 13. H. Fliri and C.-P. Mak, J. Org. Chem. 50, 3438 (1985); G. E. Keck, E. J. Enholm, J. B. Yates, and M. R. Wiley, Tetrahedron 41,4079 (1985). 14. S. Knapp and J. J. Hale, J. Org. Chem. 58,2650 (1993). 15. S. Knapp, J. J. Hale, M. Bastos, A. Molina, and K. Y . Chen, J. Org. Chem. 57,6239 (1992). 16. M. C. Viaud and P. Rollin, Synthesis 130 (1990). 17. Y. Yamamoto, Acc. Chem. Res. 20,243 (1987). 18. T. Naito, M. Ikai, M. Shirakawa, K. Fujimoto, 1. Ninomiya, and T. Kiguchi, J. Chem. SOC., Perkin Trans. 1, 773 (1994). 19. P. N. Confalone and E. M. Huie, in “Organic Reactions” (A. S. Kende, ed.). Vol. 36. p. 3. Wiley, New York, 1988; E. Breuer, in “Nitrones, Nitronates and Nitroxides in The Chemistry of Functional Groups” (S. Patai and Z. Rappoport, eds.), p. 139. Wiley, New York, 1989. 20. Y. Ohfune and N. Kurikawa, Tetrahedron Lett. 25, 1071 (1984). 21. P. Gamer and J. M. Park, Org. Synth. 70, 18 (1991). 22. H. Takahata, H. Bandoh, and T. Momose, J. Org. Chem. 57,4401 (1992). 23. G. Fourquet and M. Scholosser, Angew. Chem. Int. Ed. Engl. 13, 82 (1974). 24. T. Kiguchi, Y. Yuumoto, I. Ninomiya, and T. Naito, Hererocycles 42, 509 (1996). 25. T. Kiguchi, M. Ikai, M. Shirakawa, I. Ninomiya, and T. Naito, The 115th Annual Meeting of the Pharmaceutical Society of Japan, p. 69. Sendai, 1995.

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26. F. Kong and D. J. Faulkner, J. Org. Chem. 58,970 (1993). 27. E. A. Jares-Erijman, C. P. Bapat, A. Lithgow-Bertelloni, K. L. Rinehart, and R. Sakai, J. Org. Chem. 58,5732 (1993). 28. J. Kobayashi and M. Ishibashi, Heterocycles 42, 943 (1996). 29. N. Dematte, A. Guerriero, R. D e Clauser, G. De Stanchina, F. Lafargue, V. Cuomo, and F. Pietra, Comp. Biochem. Physiol., B: Comp. Biochem. 81, 479 (1985). 30. N. B. Perry, J. W. Blunt, and M. H. C. Munro, Ausr. J. Chem. 44, 627 (1991); D . Enders and M. Finkam, Liebigs Ann. Chem. 551 (1993).

-CHAPTER b

SYNTHESIS OF THE ASPZDOSPERMA ALKALOIDS J. EDWINSAXTON School of Chemistry The University of Leeds Leeds LS2 9JT, U K I. Introduction ................................................................................... 11. The Aspidospermine Group ...................... 111. Vindorosine and Vindoline

V. The Vindolinine Group .............................................. VI. The Meloscine Group .. ........................

343

366

VIII. The Kopsine Gr References ......

I. Introduction

For the purpose of this chapter the Aspidosperma alkaloids will include all those based on the aspidospermidine (1)and vincadifformine (2) ring systems, together with those such as meloscine (3), which are derived by obvious rearrangement of one or other of these systems. The last summary of synthesis in this area was published in 1979 (I). During the last 16 years these alkaloids have continued to exert a fascination on, and offer a challenge to, organic chemists engaged in the art of synthesis, and several notable and ingenious synthetic routes have been developed, either by the application of modern sophisticated synthetic methods, or by brilliant attempts to mimic the presumed biosynthesis of these alkaloids in the laboratory. Several of the most talented contemporary exponents of the art of organic synthesis have made outstanding contributions in this area, and such is the volume of work published in the period under discussion that it will be possible only to discuss in brief outline what has been achieved. All of the major alkaloids have now yielded to synthesis, including those of the meloscine and kopsine groups, which, in terms of their ring systems, would appear to offer the greatest challenge. THE ALKALOIDS. VOL SO 00YY-YSYX/YX $2500

343

Copyright 0 I Y Y 8 by Academic Press All rights of reproduction in any form reserved.

344

SAXTON

(+)-Aspidosperrnidine (1)

(-)-Vincadifformine (2)

Meloscine (3)

11. The Aspidospermine Group

In the years 1963-1980 synthesis in this group was dominated essentially by Stork’s classical, elegant synthesis of aspidospermine itself (2), and by its numerous variants, and by the work of Ban and his co-workers. Since then, other new, radical approaches have been developed by Magnus, Overman, and their collaborators, among others. The synthesis by Magnus and his co-workers (3) proceeds via a transient indole-2,3-quinodimethane(4) which spontaneously undergoes a thermal electrocyclic ring closure to give the tetracycle ( 5 ) containing the desired cis ring junction. A Pummerer reaction on (5) gave the trifluoroacetate (6),which was converted into ( 5 ) aspidospermidine [( +.)-1]by cyclization, reduction, and deprotection stages (Scheme 1). Conceptually, the most original approach to the Aspidosperma alkaloids is the tandem aza-Cope rearrangement-Mannich cyclization route developed by Overman et al. ( 4 ) . Originally introduced in a synthesis of 11-methoxytabersonine (q.v.) its versatility allows it to be modified to afford syntheses of several other alkaloids in this group, including N,acetylaspidoalbidine (7) (Scheme 2). In this synthesis the tricyclic urethane (8) was first constructed by coupling of the hydropyrindinone derivative (9) with the dianion derived from the trimethylsilylcyanhydrin (10) (5). A

9. SYNTHESIS

OF THE ASPIDOSPERMA ALKALOIDS

i Ts

345

a-N

Me

Ts

phsiANA Ts

I

Ts

iii,iv

t

I

vi. vii

(?)-Aspidospermidine((?)-l]

SCHEME 1. Reagents: (i) PhSCH2CH2NH2;(ii) PhCI, 140°C; (iii) m-CPBA, NaHC03, CH2C12,0°C; (iv) TFAA, CH2C12.0°C; (v) PhCI, 130°C; (vi) Raney Ni, EtOH; (vii) LiAIH4.

Wittig reaction on (S), followed by hydrolysis of the urethane and amide functions and reaction with formaldehyde, gave an oxazoline (ll),which on acid treatment underwent an aza-Cope rearrangement, followed by an internal Mannich reaction and further cyclization, to give the indolenine (12). Obvious stages then gave (+)-deoxylimapodine (13), which had

346

SAXTON

already been converted into (t)-Na-acetylaspidoalbidine(7) by oxidation with mercury(I1) acetate (6). (N.B. The structures 7 and 13 represent the enantiomers of the natural alkaloids.) The earlier synthesis of Na-acetylaspidoalbidine,by Ban and coworkers, relied on the pivotal tetracyclic lactam (14), prepared by a novel photoisomerization of the lactam (15), followed by the conventional addition of ring D. Alkylation of (14) gave the intermediate (16), which on reduction and deprotection gave (2)-quebrachamine [(+)-17]; alternatively, partial reduction of 16, deprotection, further reduction, and acetylation afforded a new synthesis of (5)-N-acetylaspidospermidine(18)(7). When the lactam (14) was subjected to a Claisen condensation with dimethyl oxalate, a twocarbon unit was attached to C-20; conventional manipulation of the product then afforded ( +)-deoxylimapodine (13) and ( 5)-Na-acetylaspidoalbidine (7). In yet another application, oxidation of (14) with oxygen and LDA at low temperature resulted in the introduction of a hydroxyl group to C-20, which thereby allowed the synthesis of (+)-deoxyaspidodispermine (19) to be completed (Scheme 3) (6). Pearson and Rees have contributed an ingenious synthesis of limaspermine (20) by taking advantage of the fact that iron carbonyl complexes of alkoxycyclohexadienes behave as stable equivalents of cyclohexenone ycations. The initial synthesis (8)required some 30 stages, but in a subsequent modification (9) limaspermine was prepared in seven stages fewer from phydroxyphenylacetic acid. This abbreviated route consists essentially of a new approach to the Stork-type tricyclic ketone 21, and the synthesis was completed by Stork’s method, followed by demethylation of the ether functions. Meyers and Berney (20) have reported an asymmetric synthesis of the tricyclic ketone 22, which thus constitutes a formal synthesis of (+)aspidospermine, the enantiomer formerly regarded as unnatural, but now known to occur in Aspidosperma pyrifolium Mart. (22). Other work reported in recent years includes syntheses of (-)-aspidospermidine (ent-1) (22),(5)-aspidospermidine [(+)-l] (23),(+)-aspidospermidine (1) (14), Na-benzylaspidospermidine( 2 9 , (t)-eburcine (23) and (+)-16-epieburcine (26).

III. Vindorosine and Vindoline Owing to its obvious importance as one of the components of the oncolytic alkaloid vinblastine, vindoline (and its 11-demethoxy analogue, vindorosine), have naturally attracted considerable attention. Buchi’s celebrated

i-iii

c-1-BUCOHN

I

IV

- vi

AC

ti

SCHEME2. Reagents: (i) BuLi, THF, -70°C. then add 9; (ii) 3~ HCI, MeOH, pH 6.5; (iii) LiOH, MeOH, H 2 0 ; (iv) Ph3P=CH2,23"C; (v) KOH, EtOH, H20,210°C (vi) (CH20),, Na2S04,23°C; (vii) CSA, Na2S04, PhH, 80°C; (viii) LiAIH4; (ix) Na, NH3; (x) HC02NH4, Pd-C; (xi) Ac20.

348

SAXTON

Quebracharnine (17)

I

ii-vi

(14)

t

xiii, xiv

1

xviii, ix, x

xvi, x vii, ix, x, ix, xii (f)-Deoxylimapcdine (13) Et

I

(f)-N-Aoetylaspidoalbidine(7)

AC

ix, xii (1 8 )

Ac Deoxyaspidodisperrnine(19)

SCHEME3. Reagents: (i) MeOH, hv, (ii) PhCOC1, NEt3; (iii) dihydropyran, CSA; (iv) LDA, CICH2CH2CH21;(v) MeCH2CH2NH2,CH2C12,r.t.; (vi) NaH, KI, 18-crown-6; (vii) LDA, THF, HMPA; (viii) EtI, -60°C; (ix) LiAlH4, THF; (x) 10% HCI, THF; (xi) LiAIH4; (xii) Ac20, py; (xiii) LiAIH4 (excess), dioxane, heat: (xiv) Ht, H2O; (xv) LDA, (C02Me)2;(xvi) NH2NH2,KOH, HO(CH2)20(CH2)20H.100-200°C; (xvii) CH2N2;(xviii) 0 2 , LDA, -78°C.

synthesis (27) has been reexamined by several groups of workers, and five new syntheses of the tetracyclic intermediate 24 (28J9) or its 11-methoxy derivative (20,21) have been reported, together with a notable synthesis of Buchi's pentacyclic ketone 25a (22).

9. SYNTHESIS

OF THE ASPIDOSPERMA ALKALOIDS

349

COU Limaspermine (20)

(21)

g-)&$@ .

Et

H'

H

Me02

(22)

Me

16 C02Me

Eburcine (23)

0

The new synthesis of vindorosine and vindoline by Langlois and his collaborators (23) also consists of an independent synthesis of the ketones 25a, b, but some variations in the later stages of a Buchi-type synthesis were also introduced. Following an exploratory investigation in which the vindorosine intermediate 25b was prepared, the synthesis of 25a was achieved from 7-methoxy-N,-methyl-3,4-dihydro-/3-carboline in an overall yield of 26% (Scheme 4). The rearrangement reactions of 26a to 27a and of 26b to 27b proceeded in the remarkably high yields of 70% and 73%, respectively. In the final stages of the vindorosine-vindoline synthesis, Langlois and co-workers used a different reagent for the introduction of the oxygen functionality to C-16. Whereas Biichi had oxidized the ketoesters 28a, b by means of hydrogen peroxide in the presence of strong base,

iii

I

I xiii,xiv

xv, iii

( f l $H

R

\+

M N e 3

0-

0

4

tt

Et

(37)

(26a) R = OW, u-H at C-3 (26b) R = H. U-H at C-3 (32) R = OMe, 0-H at C-3

(34a)R = O M e (34b) R = H

Me

H

Me0 c

0

2

Et

(35)

1

(27a) R = OMe (27b) R = H

I

xix

v, vi

‘Et

-

‘Et Me0

(2%) R = O W (25b) R = H

(28a) R = O M e (28b) R = H

2

0

(36)

?-

Vindorosine (30)R = H Vindoline (31) R = OMe

SCHEME4. Reagents: (i) MeSOCH2Li,THF, DMSO; (ii) TsOH, THF, H20; (iii) NaBH3CN, MeOH; (iv) Raney nickel, MeCOMe; (v) (Me0)2C0,NaH; (vi) methylketene ethyl trimethylsilyl acetal, Bu,NF (vii) m-CPBA; (viii) Zn, AcOH, H20; (ix) LiAlH4, THF, -78°C; (x) Ac20, py; (xi) PhPOCI2, heat; (xii) NaHC03, H20; (xiii) KH, MeI, D M F (xiv) KH, Ac20, DMF; (xv) LDA, THF, -78” to 0°C; (xvi) NaOMe, MeOH; (xvii) r-BuOCI, CH2C12,O”C,then DBU; (xviii) NaBH3CN. TFA, MeOH; (xix) CH20, NaBH3CN, AcOH, MeCN.

9.

SYNTHESIS OF THE ASPIDOSPERMA ALKALOIDS

351

Langlois and co-workers oxidized the trimethylsilyl ethers 29 with rn -chloroperbenzoic acid, but they then had to interpose a reduction stage to remove the &-oxide function simultaneously introduced. Reduction and acetylation stages then gave vindorosine (30) and vindoline (31). The stereoselective synthesis of (-)-vindoline by Feldman and Rapoport (24) involved, in the early stages, the enantioselective preparation of the tetracyclic P-ketosulfoxide 32 from L-aspartic acid, via the tetrahydropyridine derivative 33. However, rearrangement of 32 to the pentacyclic intermediate 27a was accompanied by racemization, presumably via the reverse Mannich intermediate 34a. The product 27a was therefore the same as Langlois’ ketone, prepared from the racemic 3a-H epimer 26a. In order to avoid racemization, an alternative route was devised, in which the ketoester 35 was used as the substrate for rearrangement, by treatment of its 7chloroindolenine derivative with base. In this way, the r6les of C-2 and the future C-16 were reversed, i.e., C-2 became electrophilic and C-16 nucleophilic in the 7-chloroindolenine derivative, and the tendency of 35 and its relatives to racemize via a reverse Mannich reaction was suppressed. The product 36 of the rearrangement was then converted into the ketoester 28a, the late intermediate in the synthesis of vindoline by Langlois and coworkers (Scheme 4). It was subsequently shown (25) that the P-ketosulfoxide 26b itself, prepared from optically pure precursors, is racemic. The racemization must therefore occur during its preparation, and not during the rearrangement reaction to the ketone 27. In contrast, esters of type 37 are configurationally stable. Various workers have investigated the possibility of preparing vindorosine and vindoline by partial synthesis from more accessible alkaloids in this group. The first of these was Kutney’s conversion of ( 2 ) vincaminoridine (38), via 11-methoxy-N-methylvincadifformine, into vindoline, by a rather lengthy sequence of functional group transpositions (26).

Vincaminoridine (38)

Owing to the relative abundance of tabersonine (39), methods have also been explored for its conversion into both vindorosine (30)and vindoline

352

SAXTON

(31). The method of Danieli er af. (27a) involved oxidation of 39 by means of benzeneseleninic anhydride, which gave the C-17 hydroxy derivative (ma), presumably via hydration, during workup, of the N,, C-17 didehydro derivative initially obtained, the stereochemistry of attack being determined by the adjacent ethyl group. Oxidation of the product 40a at C-16, also at the &face, by rn-chloroperbenzoic acid was accompanied by oxidation of Nb, with the formation of 41; reductive methylation, removal of the N oxide function, and acetylation then gave vindorosine (30) (Scheme 5). The conversion of tabersonine into vindoline, prompted by the relative inaccessibility of 11-methoxytabersonine (42), required the initial introduction of a methoxyl group into position 11. Since this could not be achieved on tabersonine itself, it was first converted by reduction with sodium cyanoborohydride into 2,16-dihydrotabersonine, and thence into its N,-acetyl derivative (27b). Preparation of the 11-methoxy derivative then became an exercise in aromatic chemistry. Fortuitously, the dehydrogenation of the 2J6-dihydro-ll-methoxytabersonineso obtained simultaneously introduced the desired C-17 hydroxyl group, to give the intermediate (40b), and the synthesis of vindoline was then completed as before. The brilliant enantioselective synthesis of vindorosine and vindoline by Kuehne and his collaborators (28), the first to be recorded, is an extension of their synthesis of tabersonine (39) and 11-methoxytabersonine (42) (q.v.). The methods employed for the conversion of these alkaloids into vindorosine and vindoline resembled in principle those used by Danieli er al. However, since the starting materials for these syntheses are available in R, S, and racemic forms, both enantiomers and the racemic forms of these alkaloids are accessible by total synthesis. The most recent synthesis (29) of the vindoline ring system involves a radical new approach in which rings C and E are formed by an ingenious tandem cyclization-cycloaddition of a transient carbenoid intermediate 43, generated from the diazo-imide 44 by treatment with rhodium acetate (Scheme 6). This carbenoid presumably cyclizes to a dipole 45 which subsequently cycloadds across the indole 8-bond to give the hexacyclic product 46. Removal of the amide carbonyl group via the thioamide, followed by hydrogenolysis of the C-21 to oxygen bond in acid solution, then gave desacetoxy-17-oxo-14,15-dihydrovindorosine (47), whose structure and stereochemistry were unequivocally established by X-ray crystal structure analysis. The potential of this new route for the total synthesis of vindorosine and vindoline is obvious; indeed, the 11-methoxy derivative of 47 has already (1978) been converted into vindoline by Kutney and co-workers. Although it does not strictly belong in this section, mention may be made at this point of the first total synthesis (30) of (5)-obscurinervidine (48),

9. SYNTHESIS

353

OF THE ASPIDOSPERMA ALKALOIDS

(-)-Tabersonine (39) R = H 1I -Methow-tabersonine(42) R = OMe

(40a) R = H (40b) R=OMe

0-

(41) R = H

Catharosine R = H

Iv Vindorosine (30)R = H Vindoline (31) R=OMe

SCHEME 5 . Reagents: (i) PhSeOOSeOPh; (ii) m-CPBA; (iii)CH20, NaBH3CN, pH 4.2; (iv) Raney nickel; (v) AczO, NaOAc, r.t.

354

SAXTON

C02Me I

vi-viii

SCHEME 6. Reage?ts: (i) (Im),CO; (ii) H02CCH2C02Me,i-PrMgCI; (iii) N-methylindole 3-acetyl chloride, 4 A mol. sieves: (iv) MsN3, NEt3; (v) R ~ , ( O A C (cat.), )~ PhH, 50°C; (vi) Lawesson’s reagent, heat; (vii) Raney nickel; (viii) H2, Pt02, MeOH, HCI.

since it shares some features with the vindorosine syntheses, notably the application of the Takano cyclization for the generation of rings A-D, and the attachment of ring E by Biichi’s method.

9. SYNTHESIS OF THE

355

ASPIDOSPERMA ALKALOIDS

1

Obscurinervidine (48) 57a

R

R

H

H

-

2

57b

Me

H

57c

H

OW

Vincadifforrnine (2)

-

DBUWt

Minovine

Ervinceine

IV. The Vincadifformine Group

.

Synthetic work in this area has in recent years been dominated by the versatile biomimetic synthesis developed by Kuehne and his collaborators, together with outstanding original contributions from Overman and Magnus. Kuehne’s original concept involved the construction of a spirocyclic quaternary ammonium salt 49 which, it was anticipated, would fragment to yield a secodine derivative 50 on treatment with base; this would, in turn, spontaneously cyclize to give ( 2)-vincadifformine (2). This approach was brilliantly and elegantly exploited and high yields of the anilinoacrylate alkaloids were obtained. In the first synthesis (31) of (2)-vincadifformine the quaternary ammonium salt 49 was obtained from the indoloazepine ester 51, which was itself prepared by two independent methods, via the p-carboline derivative 52 or the y-carboline derivative 53; evidently, both must proceed via the common intermediate 54 (Scheme 7). Extension to the synthesis of 11-methoxy-vincadifformine(ervinceine) required the intermediate 55, which was readily obtained by synthesis from N-benzyl4-piperidone via the y-carboline derivative 56 (32). Subsequently, a much improved synthesis was developed (32),in which the secodine precursors were spirocyclic tetrahydro-P-carbolinium salts, e.g., 57a-c, rather than indoloazepine esters; these can readily be prepared from tetrahydro-p-carboline derivatives, themselves obtained by reaction of the appropriate tryptamine with pyruvic ester. By this means

(53)R = H

li

(56) R = O

M

1

CI ii NCH2Ph

R

(55)R = OMe

1

iii, iv

R

@ -4Q ’

N

H

\

R

’ N

H

co2m

Vincadifformine (2) R = H Ervinceine

Et

co2w

(50)

R = OMe

SCHEME7. Reagents: (i) r-BuOCI, PhH, NEt3: (ii) TICH(C02Me),, PhH; (iii) H2, Pd-C, AcOH; (iv) Br(CH2),CHEtCH0, TsOH, MeOH, Nz. 40°C; (v) NEt3, MeOH, 60°C.

9. SYNTHESIS OF THE

ASPIDOSPERMA ALKALOIDS

357

(t)-vincadifformine (4)-2,(?)-minovine (N,-methylvincadifformine),and (+-)-ervinceine (11-methoxy-vincadifformine) were synthesized in relatively high yield, in essentially two stages from tryptamine. Indoloazepine esters, e.g., 58, can also condense with aldehydes at NI, and the &position of the indole ring to give bridged tertiary bases, and in a further demonstration of the versatility of this synthesis, Kuehne and his collaborators have used this reaction in a synthesis of 3-0x0-vincadifformine (59)(33), which was obtained in 85% yield from the bridged epimeric azepines 60,presumably via the oxosecodine derivative 61 (Scheme 8). These syntheses emphasize the fugitive nature of the secodines, and underline the difficulties inherent in their isolation; however, since the primary purpose in preparing them is as precursors of the anilinoacrylate alkaloids, their spontaneous cyclization is, if anything, an advantage. Nevertheless, Raucher and co-workers have synthesized secodine (62)itself (34) and, during a synthesis of minovincine (63),Kuehne and Earley prepared 20,21-didehydro-19-oxosecodine(64),which proved to be the first example of a stable, isolable secodine derivative (35). Kuehne and his collaborators have adapted this approach to the synthesis of tabersonine (39),and three syntheses have been recorded (28,36a-c). Chronologically, the first of these (36a) involved the condensation of the

3-0x0-vincadifforrnine(59)

(61)

SCHEME 8. Reagent: (i) OHCCHEtCH2CH2CO2Me.PhMe, Nz. 4

A mol. sieves, heat.

358

SAXTON

Secodine (62)

co Me 2

Minovincine (63)

indoloazepine ester 58 with the lactol chloride 65 as an initial stage, and the second (36b) the condensation of 58 with the epoxyaldehyde 66. The latter was dramatically improved when the aldehyde 66 became available in its enantiomeric forms, but the synthesis was not regioselective, owing to ambiguity in the stage which involved the opening of the epoxide function, and a further improvement was achieved (28) when the lactol chloride 65 was prepared in optically active (at the asterisked carbon atom), as well as racemic, forms. A notable feature of these syntheses was the diastereoselectivity observed, the stereochemistry being controlled by that at the future C-14. The initial pentacyclic product isolated was 14-hydroxyvincadifformine, which on dehydration gave tabersonine (39)(Scheme 9). Since the lactol chloride (65)was available in R, S, and racemic forms, the synthesis of both enantiomeric forms, and the racemate, of tabersonine was achieved (28). Similarly, by use of the methoxylated indoloazepine ester 67the synthesis of both enantiomers, and the racemate, of ll-methoxytabersonine (42) was completed. Kuehne's third synthesis of tabersonine (39)proceeded via the critical secodine intermediate 68,which was constructed from the indoloazepine ester 58 and the sensitive 0-chlorodivinyl ketone 69. Thermal cyclization of 15-oxosecodine (68)then gave 15-0x0-vincadifformine(70), which could not be reduced and dehydrated to tabersonine (39)since the related alcohol is a neopentyl alcohol with an equatorial hydroxyl group, which is prone to rearrangement. Instead, 15-0x0-vincadifformine was brominated and reduced to a 14,15-bromohydrin, which gave tabersonine (39)on elimina-

9. SYNTHESIS

14-Hydroxyvincadinormine R = H

14-Hydroxyewinceine R = OMe

OF THE ASPIDOSPERMA ALKALOIDS

359

Tabemnine (39) R = H 11-Methoxy-tabemnine (42) R = OMe

SCHEME 9. Reagents: (i) boric acid, MeOH, heat, 24 h; (ii) PPh3, MeCN, CC4, 70°C.

tion of hypobromous acid by means of McMurry’s reagent (Scheme 10) (36c). More recently, Kuehne et al. have completed two syntheses of minovincine (35,37) by a similar biomimetic approach; and two other syntheses of vincadifformine consist essentially of alternative routes to the secodine intermediate 50 (38,39). Simple modifications of these routes have afforded syntheses of 3-0x0-vincadifformine (40),its ethyl ester analog (42) and its N,-methyl derivative, 3-oxominovine (40). Independent syntheses of vincadifformine, tabersonine, and their 3-OX0 derivatives (42,43) have also been recorded, together with a synthesis of 18,19-didehydrotabersonine (44).

360

SAXTON

..

- Et

cw

v-viii

Et H Tabersonine (39)

-

SCHEME 10. Reagents: (i) CH2=CHMgBr, THF, Ar; (ii) (COC1)2,CH2CI2,DMSO, -60°C. Ar; (iii) MeOH; (iv) PhMe, Ar, heat. 10 h; (v) i-PrzNH, n-BuLi, THF, hexane; (vi) (CH2. C H Z N H ~THF, ) ~ , -78°C. then Br2. C H A X (vii) NaBH4, MeOH;, (viii) LiAIH4, TiC13, THF. Ar, heat, 2 hr.

Yet another adaptation of Kuehne's biomimetic synthesis has resulted in the preparation of (2)-cylindrocarine (71) ( 4 3 , a member of the aspidospermine group.

Cylindrocarine (71)

9. SYNTHESIS

OF THE ASPIDOSPERMA ALKALOIDS

361

11-Methoxy-tabersonine (42), important as an intermediate in vindoline synthesis, has been synthesized by three groups of workers; of these syntheses, that owing to Kuehne et al. (28) has been summarized above. In the first synthesis, Overman et al. (46) introduced their ingenious tandem aza-Cope-Mannich cyclization route for the preparation of the tabersonine ring system. This involved the construction of the intermediate 72, as shown in Scheme 11; this was then reacted with paraformaldehyde to give an oxazolidine 73, which rearranged thermally without added acid, and subsequently cyclized, to give 1l-methoxy-l,2,14,15-tetradehydroaspidospermidine (74) stereospecifically and in high yield. Evidently, traces of formic acid in the reaction mixture were sufficient to catalyze the formation of 75 from the oxazolidine 73; this then suffered an aza-Cope rearrangement to give an intermediate 76 which was ideally set up for an internal Mannich reaction. Cyclization of the product afforded the imine 74, which, on methoxycarbonylation, gave 11-methoxy-tabersonine (42). Magnus and co-workers’ first contribution in this area was a synthesis (the first) of 3-0x0-tabersonine (77) (47).This was followed by an investigation (48) in which it was established that their synthetic approach was consistent with the presence of a methoxyl group at position 11. An additional refinement in the eventual synthesis (49) was the incorporation, at the outset, of an asymmetric unit which thereby ensured that the synthesis was stereoselective. The intermediate 78 was prepared, as outlined in Scheme 12, and the asymmetric unit was then discarded by a reverse DielsAlder fragmentation, to give the pentacyclic enone 79. Since the phenylthio group could not be removed without affecting the 14,15-double bond, the latter was deliberately saturated during the desulfurization stage; it was then reintroduced, and the ester group attached to C-16 was incorporated by Vilsmeier formylation, oxidation, and esterification, and the synthesis of (-)-11-methoxy-tabersonine (42) was completed by removal of the urethane ester group (49). Finally, in this group, Overman et al. (50)have used the N-acetylaspidoalbidine intermediate (12) in a synthesis of deoxoapodine (80).

V. The Vindolinine Group

Synthetic work in this small group is entirely due to Levy and his collaborators, who have reported two partial syntheses of tuboxenine (81) (SZ), and one of vindolinine (82) and 16-epi-vindolinine (83) (52). The vindolinine synthesis involved the cyclization, by sonochemical means, of

q

cok

COMe

i-iv

V

*

Q

Et

1

SPh

SOPh

vi

x, xi

xii. xiii

*

(72)

OM

(76)

(74)

(75)

1l-Methoxy-labersonine(42)

SCHEME 11. Reagents: (i) PhSCHCICH2CH2CI,ZnBr2, CH2CI2,25°C; (ii) NaI, MeCOEt, Ar, heat; (iii) NH3, CHCI3, r.t., 2 days: (iv) CIC02Me, PhNEt2, PhMe; (v) m-CPBA, CHCI,; (vi) o-C6H4CIZr CaC03, 165°C;(vii) BuLi, THF, -78°C; (viii) HCI, HzO, Et20,0°C; (ix) LiOH, MeOH, r.t.; (x) Ph3PMeBr, BuLi, T H F (xi) 40% KOH, MeOH, heat, 8 h; (xii) (CH20)n, PhMe, Na2S04; (xiii) heat, 6 h; (xiv) LDA, CIC02Me, THF, -78°C.

* a

Me0

/ N

bMe 2

&Me 2

0

h2Me

(78)

S

* Et

&Me

I

2

...

Xlll -xvI

(-)-11-Methoxy-tabemnine (42)

SCHEME12. Reagents: (i) EtAIC12; (ii) resolution; (iii) BuMe2SiC1, imidazole, DMF; (iv) Zn, CH2Br2,Tic&, THF; (v) KF, H20, THF (vi) (COC1)2, PhMe, lO"C, then DBMP, PhMe, heat; (vii) m-CPBA, NaHC03, CH2C12, H20; (viii) TFAA, DBMP (ix) 210°C; (x) Raney nickel (W-2), EtOAc; (xi) ( p-PhOC&PS2)2, THF, 0°C; (xii)p-MeC&SOCl, i-Pr2NEt,PhMe, 110°C; (xiii) MeI; (xiv) NaBH.,, MeOH (xv) POCl3, DMF (xvi) 2~ NaOH, (xvii) NaC102, H20, H2NS03H,MeCOMe, CH2=CMeOAc; (xviii) CH2N2;(xix) 1M NaOMe, MeOH, r.t.

364

SAXTON

19-iodo-tabersonine (84), prepared from vindolinine, which gave a mixture of vindolinine (82) and 16-epi-vindolinine (83), together with their 19epimers (Scheme 13). The yields and proportions of the four epimers de-

* Et H

COMe 2

3-0x0-tabersonine (77)

H

CoMe 2

Tuboxenine (81)

Deoxoapodine (80)

Me H

CoMe 2

19-lodotabersonine (84)

\ e M & \

H

\

H

Vindolinine (82)

/

N H

tJ9

16-Epivindolinine (83)

SCHEME13. Reagents: (i) ultrasound (500 W, 20 KHz), THF, Na, Ar, 0°C; (ii) ultrasound (60 W, 45 KHz), THF. Na, Ar, 0°C.

9. SYNTHESIS OF

365

THE ASPIDOSPERMA ALKALOIDS

pended on the conditions used; at lower ultrasonic intensities vindolinine (82) and 16-epi-vindolinine (83) were obtained in a 1 : 2 ratio, but at higher intensities all four 16J9-epimers were formed.

c Q ~ ; NHCOBu o c H 2 p h -

2

-

2

vi-x

H

o

H

Meloscine (3) p-H at C-16 Epimeloscine (85) a-H at C-16

SCHEME 14. Reagents: (i) KOH, EtOH, HzO, 130°C; (ii) (CHzO),, CSA, PhH, heat; (iii) (Me2CH),C6H2SO2N,, n-Bu4NBr. 18-crown-6, PhH, H 2 0 , KOH; (iv) MeOH, Et20, hv. (v) KOH, EtOH, H20, 150°C; (vi) Na, NH,; (vii) TsCI. py. CHCI,; (viii) o-02NC6H4SeCN, NaBH,, EtOH; (ix) rn-CPBA, CH2C12, -70°C; (x) Me2S. NEt,, r.t.

366

SAXTON

VI. The Meloscine Group Overman et d ’ s synthesis (50,53) of meloscine (3)and epimeloscine (85) shared common starting materials with their synthesis of N-acetylaspidoalbidine (7).The oxazolidone 86,on hydrolysis with alkali, gave the precursor of the methylene-iminium ion 87 which, when heated with acid, underwent tandem aza-Cope rearrangement and internal Mannich cyclization to give the tricyclic ketone 88 (Scheme 14). Photochemical rearrangement of the derived a-diazoketone resulted in ring contraction, to give the amide-ester 89, and the synthesis was completed by hydrolysis and lactam formation, separation of epimers, and introduction of the terminal double bond.

VII. The Aspidofractinine Group Ban’s second synthesis of aspidofractinine (90) (54) is a variant of his original synthesis ( 5 9 , which was described in Vol. 17 (I), and essentially consists of improvements in the later stages. Levy and co-workers’ remarkably brief synthesis (56a) makes use of the previously prepared oxindole ketoester 91, which was cyclized directly to 3,19-dioxo-aspidofractinine(92)by polyphosphoric acid. Sequential reduction of the carbonyl groups then gave aspidofractinine (90) (Scheme 15), and reduction by means of lithium aluminum hydride gave 19-hydroxyaspidofractinine (56b). Gramain and his collaborators have more recently completed two syntheses of 19-oxo-aspidofractinine, and therefore, in a formal sense, (+)aspidofractinine; in both syntheses the final stage involved oxidative cyclization of 19-0x0-aspidospermidine (57). Inevitably, the incorporation of functional groups into the aspidofractinine ring system, as in (-)-kopsinine (93),pleiocarpine, and kopsijasmine requires a more elaborate synthetic approach. The synthesis of the first of these, 93,together with (-)-kopsinilam (94),was achieved by Magnus and Brown in an investigation ultimately aimed at the synthesis of the dimeric alkaloid, pleiomutine (58).The tetracyclic base (99,prepared by the indole quinodimethane route from N,-protected 2-methylindole-3-aldehyde, was acylated by means of (+)-R-p-tolylsulfinylacetic acid; the desired diastereoisomer was then separated and elaborated to the diene 96, the absolute configuration of which was deduced by application of the Weiss homoannular diene rule, a conclusion that was subsequently confirmed by X-ray crystal structure analysis of the sulfoxide 97. A Pummerer rearrangement

9. SYNTHESIS OF

THE ASPIDOSPERMA ALKALOIDS

367

Aspidofractinine (90) SCHEME 15. Reagents: (i) PPA, heat; (ii) TsOH, PhMe, heat, 15 hr; (iii) Me30BF4;(iv) NaH, DMF; (v) TsOH, PhMe, heat; (vi) HSCH2CH2SH, BF3, AcOH; (vii) Raney nickel, EtOH; (viii) LiAIH4.

on 97 gave N,-protected 5,22,-dioxo-kopsane (98), the 0-dicarbonyl system in which was cleaved, the carboxyl group thus released was esterified, and the N,-protecting group was removed, to give (-)-kopsinilam (94). Reduction of the lactam carbonyl group then gave (-)-kopsinine (93) (Scheme 16). In another notable communication from Kuehne and co-workers, the synthesis of (5)-kopsinine (93) and several related alkaloids was reported (59). In this synthesis, the pentacyclic intermediate 99 was built up by the familiar biomimetic route; alkylation, followed by oxidative elimination, then gave the diene N-oxides 100a-c, which reacted with

368

SAXTON

,”

-0,

’N

’CI

..

R

0

- CI

i, ii R

R =p-M”OSH4S9

1

iii, iv

Ar =p-MeC H

(95)

6 4

n

-

ArS---

ArS

---

v, vi

R R

vii - ix

X

R

I

xi -xiii

(98)

n

xiv, xv

H

H (-)-Kopsinilam (94) CqMe

(-)-Kopsinine (93)

SCHEME 16. Reagents: (i) (+)-R-p-tolylsulfinylacetic acid, C6HIIN=C=N-CH2CH2NMe (CH2CH2)206 T s ; (ii) separation of diastereoisomers; (iii) TFAA, CH2CI2;(iv) PhCI, 130°C; (v) CH2=CHCH2Br, KN(S~MC,)~; (vi) 100°C; (vii) HN=NH; (viii) m-CPBA: (ix) 240°C (x) TFAA: (xi) KOH, MeOH, (xii) Li, NH3, -78°C; (xiii) MeOH, HCI: (xiv) Lawesson’s reagent: (xv) Raney nickel.

9. SYNTHESIS

OF THE ASPIDOSPERMA ALKALOIDS

369

phenyl vinyl sulfone to give the hexacyclic sulfones 10la-c, with concomitant reduction of the N-oxide function. Hydrogenation-hydrogenolysis of lOla with Raney nickel then gave (+)-pleiocarpinine (102), and similar treatment of lOlb or lOlc gave (2)-kopsinine (93). Oxidation of (?)pleiocarpinine completed the synthesis of (2)-aspidofractine (103), and N,-methoxycarbonylation of ( 2)-kopsinine gave (+)-pleiocarpine (104). Finally, thermal cyclization of ( 5)-pleiocarpinine and (5)-kopsinine afforded ( 2)-N,-methylkopsanone (105) and (+)-kopsanone (106), respectively (Scheme 17). Independent syntheses of the diene ester 107 and its N-methyl derivative (60,61) constitute additional syntheses of these alkaloids, while Wenkert’s syntheses of N,-methoxycarbonyl-17-oxo-aspidofractinine(62) and ( 2 ) aspidofractinine (23c) also proceed via a ring C diene. The synthetic contributions of Magnus and his collaborators were taken a stage further with a characteristically ingenious synthesis of ( 5 ) kopsijasmine (108) (63). The diene intermediate 109, prepared earlier (64) during the synthesis of kopsan-22-one, was converted by alkylation, internal Diels-Alder cycloaddition, reduction, and oxidation stages, into the sulfoxide 110 which, in spite of the fact that elimination of phenylsulfinic acid would have given an anti-Bredt lactam, gave the acetate 111 when heated in the presence of silver acetate and acetic acid. Standard stages then led to (5)-kopsijasmine (log), the major point of interest in these last few stages being the use of sodium and anthracene to remove the arylsulfonyl group (the use of sodium and naphthalene also resulted in the reduction of the 16,17-double bond) (Scheme 18).

VIII. The Kopsine Group

Synthetic work in this subgroup is, so far, entirely due to Magnus and coworkers. The first investigation (64,65),which resulted in the synthesis of kopsan22-one (106), involved the preparation of the intermediate 109, on to which the additional rings were attached as in the preparation of 110, except that in this case the alkylation stage was performed with ally1 bromide. Diels-Alder cycloaddition then gave the heptacyclic intermediate 112, which has the kopsane ring system rather than the alternative fruticosane skeleton. Elimination-readdition of phenylsulfininc acid from the related sulfoxide gave the isomeric sulfoxide 113, and a second Pummerer rearrangement then gave the dioxo compound 114. Standard stages then led to kopsan-22-one (106) and kopsan-5,22-dione (115) (Scheme 19).

SCHEME 17. Reagents: (i) PhSeNEt,, N2, 20°C (ii) boric acid, CHzCIz, Nz, heat; (iii) 2 X m-CPBA, (iv) NaH, MeI, DMF; (v) NaH,PhCH2Br, DMF (vi) PPh3; (vii) PhS02CH=CH2, 100"C,12 h; (viii) Raney nickel, H20, EtOH, heat; (ix)MeOH, sealed tube, 200°C; (x) PhNEt3 Mn04; (xi) C1CO2Me,NazC03, CH2C12,N2, r.t.

vi

M"sc

I

XN.

xv

$5

K o p s i j j i n e (lOe)

SCHEME 18. Reagents: (i) KH; (ii) CH2=CC1CH21; (iii) PhMe, heat; (iv) TsNHNH2, NaOAc, THF,EtOH, H20; (v) rn-CPBA, NaHC03; (vi) AgOAc, AcOH, 205°C; (vii) LiOH, H20,THF (viii) Jones' reagent; (ix) MeOH, NaOH, (x) DBN, DME, heat; (xi) Na, anthracene, DME, -30°C; (xii) CIC02Me, K2C03, Et3NBuCI; (xiii) CH2N2, THF, Et2O; ( x ~ v )BH3.THF (xv) 6 M HCI, heat.

0

-- a Ar

Ar

0

c--

\

/ Ar

Ar

'a

(109)

x-xii

Ar

Ar

(113)

& H

I

v. xiii

XN

H

Kopsan-522dione (115)

Kopsan-Zane (106) Ar = p-MeOCgtO

thrwshout

SCHEME 19. Reagents: (i) H2N(CH&CH=CHCI, 4 A mol. sieves; (ii) C13CCH20COCl, i-Pr2NEt,PhC1,120"C; (iii) Zn, AcOH, THF, H2O; (iv) PhSCH2COCl. m-CPBA; (v) TFAA, CH2CI2; (vi) 130"C, PhCI; (vii) KN(SiMe&, THF (viii) CH2=CHCH2Br; (ix) 100°C; (x) TsNHNH2, NaOAc, EtOH; (xi) m-CPBA; (xii) 230°C; (xiii) TFAA, PhCl, 130°C; (xiv) Li, NH3, THF (XV)Moffatt oxidation; (xvi) LiAIH4.

9. SYNTHESIS OF THE

O

a

373

ASPIDOSPERMA ALKALOIDS

I-Ill

O

@

3 NC02Me

&NAr (114) Ar = 0 SC H OMe-p 2 6 4

iv, v

ONco2&

@Nco2k

ix, vii

t y

N c o p

Kopsine (116)

SCHEME 20. Reagents: (i) NaOH, MeOH. THF, then HCI. H 2 0 ;(ii) Na, CloHx,DME, then CIC02Me. H20, K2C03, PhCHZNEt3CI; (iii) Me2CHCH2O2CCI.NEt3, NaBH4, T H F (iv) oNCSeC6H4N02,PBu3, T H F (v) H202; (vi) Os04. NMO, f-BuOH, THF, H 2 0 ; (vii) (COC1)2. DMSO, NEt3, CH2C12; (viii) LDA, THF, -78°C; (ix) BH3. THF, then 5 M HCI.

374

SAXTON

Finally, Magnus et al. (66) have used the intermediate 114 in a synthesis of kopsine (116)itself. Base-catalyzed fission of the /3-dicarbonyl system in 114,followed by a sequence of standard stages, led to the 16,22-methylene compound 117,which was hydroxylated, then oxidized (Swern) to the ahydroxyaldehyde 118. The kopsine skeleton was reformed by basecatalyzed formation of the 6,22-bond, and kopsine (116)was obtained by removal of the lactam carbonyl group, and a final Swern oxidation (Scheme 20). This synthesis also constitutes a formal synthesis of isokopsine (119),

6H

lsokopsine (119)

Frutiiosine (120) 16&OH F r u t i m i n e (121) 16a-OH

fruticosine (UO), and fruticosamine (Ul), all of which have been previously prepared from kopsine (67).

References

1. G. A. Cordell, in “The Alkaloids” (R. H. F. Manske and R. Rodrigo, eds.), Vol. 17, pp. 199-384. Academic Press, New York, 1979. 2. G. Stork and J. E. Dolfini, J. Am. Chem. SOC.85,2872 (1963). 3. T. Gallagher, P. Magnus, and J. C. Huffman, J. Am. Chem. SOC.104, 1140 (1982); T. Gallagher and P. Magnus, Tetrahedron 37, 3889 (1981); C. Exon, T. Gallagher, and P. Magnus, J. Am. Chem. Soc. 105,4739 (1983); T. Gallagher, P. Magnus, and J. C. Huffman, J. Am. Chem. SOC.105,4750 (1983). 4. L. E. Overman, M. Sworin, L. S. Bass, and J. Clardy, Tetrahedron 37,4041 (1981). 5. L. E. Overman, G. M. Robertson, and A. J. Robichaud, J. Org. Chem. 54,1236 (1989); J. Am. Chem. SOC.1l3,2598 (1991). 6. K. Yoshida, Y. Sakuma, and Y . Ban, Heterocycles 25,47 (1987). 7 . Y. Ban, K. Yoshida, J. Goto, and T. Oishi, J. Am. Chem. SOC.103,6990 (1981); Y. Ban, K. Yoshida, J. Goto, T. Oishi, and E. Takeda, Tetrahedron 39,3657 (1983). 8. A. J. Pearson, Tetrahedron Lett 22, 4033 (1981); J. Chem. SOC., Perkin Trans. I , 1255 (1979); A. J. Pearson and D. C. Rees,J. Am. Chem. SOC. 104,1118 (1982); A. J. Pearson and D. C. Rees, J. Chem. Soc., Perkin Trans. I , 2467 (1982). 9. A. J. Pearson, D. C. Rees, and C. W. Thornber, J. Chem. SOC.,Perkin Trans. I , 619 (1983).

9. SYNTHESIS 10. 11. 12. 13.

14. 15.

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

27.

28. 29. 30.

31.

32. 33. 34. 35. 36.

OF THE ASPIDOSPERMA ALKALOIDS

375

A. I. Meyers and D. Berney. J. Org. Chem. 54,4673 (1989). A. A. Craveiro, F. J. A. Matos, and L. M. Serur, Phytochemistry 22, 1526 (1983). M. Node, H. Nagasawa, and K. Fuji, J. Am. Chem. Soc. 109, 7901 (1987). (a) E. Wenkert, K. Orito, D. P. Simmons, N. Kunesch, J. Ardisson, and J. Poisson, Tetrahedron 39,3719 (1983); (b) P. Le Menez, N. Kunesch, S. Liu, and E. Wenkert, J. Org. Chem. 56,2915 (1991); (c) E. Wenkert and S. Liu, J. Org. Chem. 59, 7677 (1994). D. Desmaele and J. d’Angelo, J. Org. Chem. 59, 2292 (1994). N. Benchekroun-Mounir, D. Dugat, and J.-C. Gramain, Tetrahedron Lett. 33,4001 (1992); N. Benchekroun-Mounir, D. Dugat, J.-C. Gramain, and H.-P. Husson, J. Org. Chem. 58, 6457 (1993). K. Yoshida, S. Nomura, and Y. Ban, Tetrahedron 41,5495 (1985). G. Buchi, K. E. Matsumoto, and H. Nishimura, J. Am. Chem. Soc. 93,3299 (1971); M. Ando, G. Buchi, and T. Ohnuma, J. Am. Chem. SOC.97,6880 (1975). J. D. Winkler, R. D. Scott, and P. G. Williard, J. Am. Chem. SOC.112, 8971 (1990). S. J. Veenstra and W. N. Speckamp, J. Am. Chem. SOC.103,4645 (1981). S. Takano, K. Shishido, M. Sato, and K. Ogasawara, Heterocycles 6, 1699 (1977); 13, 307 (1979). Y.Ban, Y.Sekine, and T. Oishi, Tetrahedron Len. 151 (1978). M. Natsume and I. Utsunomiya. Chem. Pharm. Bull. 32,2477 (1984); I. Utsunomiya and M. Natsume, Heterocycles 23, 223 (1985). R. Z. Andriamialisoa, N. Langlois, and Y.Langlois, J. Chem. Soc., Chem. Commun. 1118 (1982); J. Org. Chem. 50, 961 (1985). P. L. Feldman and H. Rapoport, J. Org. Chem. 51,3882 (1986); J. Am. Chem. Soc. 109, 1603 (1987). M. Dardaine and N. Langlois, Tetrahedron Lett. 33, 3641 (1992). J. P. K u t n q , U. Bunzli-Trepp, K. K. Chan, J. P. de Souza, Y. Fujise, T. Honda, J. Katsube, F. K. Klein, A. Leutwiler, S. Morehead, M. Rohr, and B. R. Worth, J. Am. Chem. Soc. 100,4220 (1978). (a) B. Danieli, G. Lesma, G. Palmisano, and R. Riva, J. Chem. Soc., Chem. Commun. 909 (1984); (b) B. Danieli, G. Lesma, G. Palmisano, and R. Riva, J. Chem. Soc., Perkin Trans. I , 155 (1987). M. E. Kuehne, D. E. Podhorez, T. Mulamba, and W. G. Bornmann, J. Org. Chem. 52, 347 (1987). A. Padwa and A. T. Price, J. Org. Chem. 60,6258 (1995). J. P. Brennan and J. E. Saxton, Tetrahedron Lett. 26, 1769 (1985); Tetrahedron, 43, 191 (1987); J. W. Blowers, J. P. Brennan, and J. E. Saxton, J. Chem. SOC., Perkin Trans. I , 2079 (1987). M. E. Kuehne, D. M. Roland. and R. Hafter, J. Org. Chem. 43,3705 (1978); M. E. Kuehne, T. H. Matsko, J. C. Bohnert, and C. L. Kirkemo, J. Org. Chem. 44, 1063 (1979). M. E. Kuehne, J. A. Huebner, and T. H. Matsko, J. Org. Chem. 44,2477 (1979). M. E. Kuehne, T. H. Matsko, J. C. Bohnert, L. Motyka, and D. Oliver-Smith, J. Org. Chem. 46,2002 (1981). S. Raucher, J. E. Macdonald, and R. F. Lawrence, J. Am. Chem. SOC. 103,2419 (1981). M. E. Kuehne and W. G. Earley, Tetrahedron 39,3715 (1983). (a) M. E. Kuehne, F. J. Okuniewicz, C. L. Kirkemo, and J. C. Bohnert. J. Org. Chem. 47, 1335 (1982); (b) M. E. Kuehne and D. E. Podhorez, J. Org. Chem. 50,924 (1985);

376

SAXTON

M. E. Kuehne, J. C. Bohnert, W. G. Bornmann, C. L. Kirkemo, S. E. Kuehne, P. J. Seaton, and T. C. Zebovitz, J. Org. Chem. 50,919 (1985); (c) M. E. Kuehne, W. G. Bornmann, W. G. Earley, and I. Marko, J. Org. Chem. 51, 2913 (1986). 37. M. E. Kuehne and W.G. Earley, Tetrahedron 39,3707 (1983). 38. G. Kalaus, M. Kiss, M. Kajtir-Peredy, J. Brlik, L. Szab6, and Cs. Szhtay, Heterocycles 23,2783 (1985). 39. M.-C. Barsi, B. C. Cas, J.-L. Fourrey, and R. Sundaramoorthi, J. Chem. SOC., Chem. Comrnun 88 (1985). 40. G. Kalaus, C. P. Dinh, M. KajtBr-Peredy, J. Brlik, L. Szab6, and Cs. Szintay, Heterocycles 31,1183 (1990). 41. B. Danieli, G. Lesma, G. Palmisano, D. Passarella, and A. Silvani, Tetrahedron 50, 6941 (1994). 42. G. Kalaus, I. Greiner, M. KajtBr-Peredy,J. Brlik, L. Szab6, and Cs. Szintay,J. Org. Chem. 58,1434 (1993). 43. J. Uvy, Y.J. Laronze, J. Laronze, and J. Le Men, Tetrahedron Led 1579 (1978). 44. J. W.Blowers, J. E. Saxton, and A. G. Swanson, Tetrahedron 42,6071 (1986). 45. J. P. Brennan and J. E. Saxton, Tetrahedron 42, 6719 (1986). 46. L. E. Overman, M. Sworin, and R. M. Burk, J. Org. Chem. 48,2685 (1983). 47. M. Ladlow, P. M. Cairns, and P. Magnus, J. Chem. Soc., Chem. Commun. 1756 (1986). 48. K. Cardwell, B. Hewitt, and P. Magnus, Tetrahedron Len. 28,3303 (1987). 49. K. Cardwell, B. Hewitt, M. Ladlow, and P. Magnus,J. Am. Chem. SOC.110,2242 (1988). 50. L. E. Overman, G. M. Robertson, and A. J. R0bichaud.J. Am. Chem. SOC.,113,2598 (1991). 51. G. Hugel, J. Cossy, and J. Lbvy, Tetrahedron Lett. 28, 1773 (1987); D. Cartier, M. Ouahrani, G. Hugel, and J. LBvy, Heterocycles 27,657 (1988). 52. G. Hugel, D. Cartier, and J. Uvy, Tetrahedron Len. 30,4513 (1989). 53. L. E. Overman, G. M. Robertson, and A. J. Robichaud, J. Org. Chem. 54,1236 (1989). 54. H. Kinoshita, T. Ohnuma, T. Oishi, and Y.Ban, Chem. Lett. 927 (1986). 55. Y.Ban, Y.Honma, and T. Oishi, Tetrahedron Len. 1111 (1976). 56. (a) D. Cartier, M. Ouahrani, and J. LBvy, Tetrahedron Lett. 30, 1951 (1989); (b) D. Cartier, D. Patigny, and J. LBvy, Tetrahedron Lett. 23, 1897 (1982). 57. M. Dufour, J.-C. Gramain, H.-P. Husson, M.-E. Sinibaldi, and Y. Troin, Tetrahedron Len. 30,3429 (1989);J. Org. Chem. 55,5483 (1990). 58. P. Magnus and P. Brown, J. Chem. SOC., Chem. Commun. 184 (1985). 59. M. E. Kuehne and P. J. Seaton, J. Org. Chem. SO, 4790 (1985). 60.M. Ogawa, Y.Kitagawa, and M. Natsume, Tetrahedron Lett. 28,3985 (1987). 61. E. Wenkert and M. J. Pestchanker, J. Org. Chem. 53,4875 (1988). 62. P. Le MBnez, J. Sipi, N. Kunesch, E. C. Angell, and E. Wenkert, J. Org. Chem. 54, 3216 (1989). 63. P. Magnus, I. R. Matthews, J. Schultz, R. Waditschatka, and J. C. Huffman, J. Org. Chem. 53,5772 (1988). 64.T. Gallagher and P. Magnus, J. Am. Chem. SOC.105,2086 (1983). 65. P. Magnus, T. Gallagher, P. Brown, and J. C. HuffmanJ. Am. Chem. Soc. 106,2105 (1984). 66. P. Magnus, T. Katoh, I. R. Matthews, and J. C. Huffman, J. Am. Chem. SOC.111,6707 (1989). 67. T. R. Govindachari, K. Nagarajan, and H. Schmid, Helv. Chim. Acta 46,433 (1963); A. Guggisberg, M. Hesse, W.von Philipsborn, K. Nagarajan, and H. Schmid, Helv. Chim. Acta 49,2321 (1966); A. Guggisberg, T. R. Govindachari, K. Nagarajan, and H. Schmid, Helv. Chim. Acta 46, 679 (1963).

-CHAPTER 1-

SYNTHETIC STUDIES IN ALKALOID CHEMISTRY CSABA SZ~NTAY Institute of Organic Chemistry Technical University, and Central Research Institute for Chemistry H-1525 Budapest, Hungary 1. 11. 111. IV. V. VI. VII.

VIII. IX. X. XI.

Introduction ..... ............. ......................... Synthesis of Ipecacuan oids ........................... Synthesis of Yohimbine Alkaloids ........ ........................... Synthesis of Corynantheidine Alkaloids ........ Synthesis of Rauwolfia Alkaloids ............................... Synthesis of Berbanes ...................................................................... Synthesis of Vincamine and Structurally Related Alkaloi A. Synthesis of (+)-Vincarnine and (-)-Vincarnone .... B. Interconversions ........ ................................................... C. Synthesis of Tacamine D. Synthesis of Cuanzine .............. Synthesis of Aspidosperma Alkaloids .................................................. Synthesis of Alkaloids from Catharanthus roseus ................................... Synthesis of Morphine .................. ...... Synthesis of ................................. References ...................................................

371

383 385

396 399

400 405 401 411

I. Introduction

The Institute of Organic Chemistry was founded in 1913 by the late Professor GCza ZemplCn, previous co-worker of Nobel Laureate Emil Fischer. Thus I was breast-fed with a devotion for the chemistry of natural products since 1950, in which year I was fortunate to join the group of Professor ZemplCn. In the following, I would like to write about the results that our school has achieved in the field of alkaloid chemistry, and by doing so I’d like to show how, by pursuing the internal logic of a given research project, we can open up project topics in this new wonderful area. In the late 1950s, the theoretical problem of tautomeric equilibrium existing in the case of pseudobasic aminocarbinols was one of the main THE ALKALOIDS, VOL. 50 0099-9598/98 $25.00

371

Copyright 8 1998 by Academic Press All rights of reproduction in any form reserved.

szANTAY

378

fields we were interested in ( I ) . The basic model for study was cotarnine (1, R = CH3), and the question to be answered was the following: can the compound exist in all three (a, b and c) tautomeric forms in equilibrium, and how does that equilibrium depend on the nature of the substituents attached to the nitrogen, in other words on the basicity of the compound (Scheme l)? As it turned out, the equilibrium can exist between a and b or between b and c, but no system was found in which all three forms are present at the same time. For example, if R represents a 2,4-dinitrophenyl group, we were able to separate the tautomers b and c by simple crystallization, and thereafter reequilibrate them (I). Among many of the R groups investigated we envisaged a P-keto side chain (-CH2-CH2-CO-CH3) on the nitrogen. In order to prepare the desired compound the hydrochloric acid salt of 3,4-dihydro-6,7-dimethoxyisoquinoline (2) was heated with methyl vinyl ketones. In that case, not only the C-N, but also a C-C bond was formed through intermediate 3, and to our surprise we could isolate the salt of the corresponding benzo[a]quinolizidine derivative 4 in excellent yield (2) (Scheme 2). A thorough study of the mechanism proved the equilibrating nature of this reaction, and the fact that it is acid-catalyzed (3). We immediately realized that the easy access to these important precursors set the stage for pursuing the total syntheses of biologically highly active alkaloids having a benzo-, or an indoloquinolizidine structural unit. Even the exploration of the mechanism of the reaction proved to be extremely useful while working on the enanrioselecrive synthesis of emetine. Thus, purely theoretical studies paved the route to our much more practical investigations and to our cooperation with the pharmaceutical industry.

b

a

1 SCHEME1.

C

10. SYNTHETIC

379

STUDIES IN ALKALOID CHEMISTRY

3

4 SCHEME2.

11. Synthesis of Ipecacuanha Alkaloids

The ipecacuanha alkaloid emetine (8) has been used for the treatment of amoebic disentery, amoebic hepatic ulcers, and other ailments since the beginning of this century. Its commercially feasible total synthesis is therefore an attractive goal ( 4 ) . In this respect, especially the work of Brossi et al. (Hoffmann-La Roche Inc.), and of Openshaw and Whittaker (The Wellcome Research Laboratories) should be emphasized. Compound 4 (R = C2H5) suggested itself as an excellent precursor for the said synthesis. The conversion of its racemic form into the required enantiomer was first performed by Openshaw and Whittaker by an asymmetric transformation of the second kind applying camphenesulfonic acid derived from unnatural camphene. For the same purpose we used simple, natural tartaric acid in acetone, where the salt with the required absolute configuration crystallized with one equivalent of acetone in 90% yield (5). The rationalization of this highly enantioselective asymmetric transformation of a compound with two stereocenters is based on the theoretical studies (3) performed on the reaction shown in Scheme 3. Through the use of a Horner-Wittig reaction and subsequent catalytic hydrogenation the ester 5 was obtained in good yield. Reduction of ester 5 with diisobutylaluminum hydride resulted in the first synthesis of the natural alkaloid (-) -protoemetine (6). The latter

380

SZANTAY

5 / 1

7

R=H

cephaeline

8

R=CH,

emetine

6 protoemetine

9 tubulosine

SCHEME3.

compound was transformed by a Pictet-Spengler-type reaction under quasiphysiological conditions into (-)-cephaeline (7) and subsequently into (-)-emetine (8) thus achieving a new and highly stereoselective total synthesis of this important alkaloid (6,7), the production of which was scaled up for industrial use in the Chinoin Pharmaceutical Works. When protoemetine was reacted with 5-hydroxytryptamine instead of 3hydroxy-4-methoxy-phenethylamine under similar conditions, the alkaloid tubulosine (9) was obtained, again in a largely stereoselective way (8). This latter example leads us to the huge area of indole alkaloids and their synthesis.

III. Synthesis of Yohimbine Alkaloids When a similar reaction to that depicted in Scheme 2 was performed using 3,4-dihydro-P-carboline (10) instead of dihydroiso quinolines 2,indo-

10. SYNTHETIC STUDIES

38 1

IN ALKALOID CHEMISTRY

12 SCHEME 4.

loquinolizidines (11) or derivatives of a new type of ring system 12 were obtained as the main products according to the conditions used (9) . It is worth noting at this point, that on reacting 2 as a base with methyl vinyl ketone, a similar lP-dipolar cycloaddition takes place, and the isoquinoline analogues of 12 can be isolated (10). In order to build up the yohimbine skeleton, another functionality at the side chain R in ketone 11 was needed. Thus, we used the ketoester derivative 13 as the appropriate precursor. As in the case of the emetine synthesis, the next step was a Horner-Wittig reaction followed by reduction. A subsequent Dieckmann condensation, reduction, and resolution gave (+)yohimbine (14) and (-)-@-yohimbine (15) in a simple way (ZZJ2). To perform a regioselective Dieckmann cyclization, instead of the diester intermediate, the corresponding nitrile ester 16 was also used for the synthesis of yohimbine. Its hydrogenation afforded, in addition to the trans-product, the corresponding cis-derivative 17. It stood to reason

13

14 yohimbine SCHEME 5.

15 P-yohimbine

SZANTAY

382

17

16

18 alloyohimbine SCHEME6.

therefore to utilize the cis-isomer 17 for the preparation of yohimbines belonging to the do-series, especially since such bases had not been heretofore synthesized. The internal Dieckmann condensation of 17 and subsequent transformation of the nitrile group into an ester, followed by reduction of the keto group afforded the first total synthesis (13-15) of alloyohimbine (18). This synthesis also made it possible for us to revise the stereostructure of alloyohimbine, and epi-alloyohimbine, heretofore incorrectly represented in the literature. Further epimers were also prepared (16). The synthesis of yohimbine alkaloids was further tuned for an enantioselective approach (17-21).Using a similar technique as in the case of the emetine synthesis, the racemic ketoester 13 was transformed in a procedure applying asymmetric transformation of the second kind into (+)-13 with the help of (+)-tartaric acid. The obtained ketoester (+)-13 has the correct absolute configuration for the synthesis of all of the above-mentioned yohimbines in their natural, optically active form.

10. SYNTHETIC

STUDIES IN ALKALOID CHEMISTRY

383

IV. Synthesis of Corynantheidine Alkaloids Again, the easily accessible ketone 11 (R = C2H5)was used to prepare the alkaloid (-)-corynantheidine (20).The ketone was allowed to react with cyanoacetic acid methyl ester followed by reduction with sodium borohydride and methanolysis giving 19 in excellent yield. During the condensation, the configuration of C-3 was inverted, thus we arrived at the d o - t y p e compound. A selective reduction of 19 at low temperature with LiAlH4 afforded the formyl ester, the sodium salt of which was methylated with dimethyl sulfate in almost quantitative yield providing the required racemic corynantheidine, which was resolved to give the natural product 20. The above reaction sequence has some interesting features: (a) it was the first case in which a malonic ester unit was selectively reduced to a formyl ester unit; (b) the methylation with dimethyl sulfate, carried out in heterogeneous phase, was not accompanied by any quaternization at the nitrogen and gave exclusively the required E-isomer; and (c) every step of the synthesis proceeded with very high stereoselectivity (22,23). It is noteworthy that through an unusual epimerization of the appropriate intermediate we could also switch from the allo-, to the normal-series of this alkaloid family (24,25), and synthesize dihydrocorynantheine (22),an alkaloid isolated from Corynanthe yohimbe, through the intermediate 21. I should like to mention at this point that a similar condensation reaction of cyanoacetic acid esters with the benzo[a]quinolizidine-type ketones and further elaboration resulted in our structure elucidation of the alkaloids ankorine, alangicine, and alangimarckine (14).

SCHEME 7.

SZANTAY

384

21

22

dihydro-corynantheine

SCHEME 8.

V. Synthesis of RauwolJia Alkaloids

The Rauwolfia alkaloids reserpine (23a), deserpidine (23b), and a few semisynthetic derivatives, are still widely used drugs. In order to achieve the total synthesis of these important natural products, all that we needed was a slight modification of our yohimbine syntheses.

n

0

I

i

y

OAC

23a b

26 SCHEME 9.

R=OCH, R=H

10. SYNTHETIC STUDIES

IN ALKALOID CHEMISTRY

385

We have to introduce a second functionality into the side chain of ketone 13 at the a-position with respect to the ester function. Thus ketone 24 was used as a suitable precursor. It was allowed to react with malononitrile, and the product reduced by NaBH4 furnishing 25 in good yield. The next six reaction steps leading to 26 were performed without isolation of the intermediates in an overall yield of 60% (!). Compound 26 was then transformed in several steps (28-34) to deserpidine (23b).

VI. Synthesis of Berbanes Berbanes are isoquinoline derivatives, but at the same time they can be regarded as depyrrolo-yohimbine derivatives. Several such compounds were synthesized (35,36) following the above discussed routes in order to investigate their biological effects. As a result, a new class of selective a2-adrenoceptor antagonists has been found (37). Compounds 27a, b for example proved to be outstanding new chemical entities in this respect. It has to be emphasized that the biological effects depend very much on the stereochemistry of the berbanes. Derivatives with normal-, or pseudoarrangements were ineffective, while in the allo-series many potent compounds were found. Even changing the configuration of the hydroxyl group in 27 causes a significant loss of biological activity.

27a b

R=COOCH3 R=H

SCHEME10

386

SZ~~NTAY

VII. Synthesis of Vincamine and Structurally Related Alkaloids A. SYNTHESIS OF (+)-VINCAMINE AND (-)-VINCAMONE (+)-Vincamine (30),an alkaloid isolated from Vinca minor, proved to be a useful pharmaceutical, and is on the market as a specific brain vasodilator agent. Its stereoselective total synthesis was achieved by reacting the so-called Wenkert enamine 28 with a-acetoxyacrylic acid ester followed by catalytic reduction and partial hydrolysis giving 29a, which was subsequently oxidized to racemic vincamine. The latter compound was resolved with dibenzoyl tartaric acid yielding the natural product 30 (38). An enantioselective modification of the synthesis was achieved by using a-acetoxyacrylic acid (-)-menthy1 ester as a reaction partner instead of the methyl ester (39). After transesterification of the 29b thus obtained into 29a (menthol can be recovered), and subsequent oxidation, the natural (+)-vincamine was gained in 41% e.e. Thus the inexpensive natural (-)menthol gave the required absolute configuration of the endproduct. To improve the economy, i.e., the “optical yield,” of the vincamine synthesis, several modifications were introduced. In one approach, the intermediate 29c was resolved with (-)-D-dibenzoyl-tartaric acid and the (-)29c thus prepared was transformed into (+)-vincamine (M), (-)-vincamone (33) or (+)-apovincaminic acid ester (32a), respectively, through oxime 31 (R = CH3) (4441). The unwanted enantiomer (+)-29c was racemized in a rather unusual way and reintroduced into the synthetic sequence. When (+)-29c was oxidized with sodium dichromate in acetic acid, not only the carbon-nitrogen

28 29a

I I

R’

SCHEME 11.

R2

30 vincamine

10. SYNTHETIC STUDIES

(-) 29c

387

IN ALKALOID CHEMISTRY

-

ROOC

....I$.& b

a 31

(+)30

- 31

R=CH, b R=CZHS cavinton

(+)32a

(-) 33

(-)

-vincamone

SCHEME12.

double bond was formed, but the CI-Cl2,, bond was also broken in the course of the oxidation affording racemization, and so yielding racemic 34b. Since no racemization was caused by any other oxidizing agent tried (e.g., Hg”, Pb’” gave only the optically active imminium salt), we coined the name “chromic effect” (42) for the unique behavior of the dichromate

sz ANTAY

388

anion. Thus, through the sequence (+)-29c + (+)-34b + (2)-29c + (-)29c + 31 + (+)-30all the material was used. A very useful, practical method was found by using the concept of the refro-Michaelreaction. After resolution of the salt 34c,obtained by interaction of enamine 28 and acrylic acid ethyl ester, the unwanted enantiomer was heated in solution in the presence of base effecting the refro-Michael reaction. In this way, the achiral enamine 28 was recovered and reintroduced into the reaction sequence (49). The retro-Michael reaction can be carried out even more easily if the leaving group has more than one electron-withdrawing functionality. Thus 28 was allowed to react with methylene malonic ester giving rise to the racemic tetraester, which was resolved with (+)-dibenzoyltartaric acid. The desired enantiomer was catalytically reduced and treated with base giving the appropriate optically active intermediate for vincamine synthesis (4350). The unwanted enantiomer was transformed back to 28 in the presence of base in over 80% yield at ambient temperature. A new pentacyclic intermediate, the tetrahydropyrano-indoloquinolizine 36 was obtained from racernic 35 and formaldehyde (48), and resolution of 36 was also achieved. The unwanted enantiomer could again be readily transformed back to the precursor 28. Reduction of (1S)-36followed by basic treatment yielded (-)-35.

34a

b C

R=H R=CH~ R=C~HS

SCHEME 13.

10. SYNTHETIC STUDIES

389

IN ALKALOID CHEMISTRY

A chiral synthesis of the vinca alkaloids was carried out starting from Ltryptophan. In this case, several obstacles had to be overcome (43-45). From L-tryptophan methyl ester 40b the corresponding optically active immonium salt 37 was easily prepared. However, when the enamine 38, needed for further elaboration was made through basification of 37, a complete racemization occurred no matter how cautiously it was carried out. Racemization is triggered by the generation of a stable C-6 carbanion adjacent to the ester group, while enamine formation requires a C-1 carbanion, which is conceivable in a kinetically controlled reaction. Since the latter species should be captured without much delay, it is necessary to select more electrophilic olefins than those used previously. A kinetically controlled reaction may be favored by a strong and bulky base (e.g., KOBu') used in catalytic quantities. Under such conditions, methyl acrylate does not react with 37,while the more reactive diethyl methylenemalonate or acrolein do. The reaction of 37 with diethyl methylenemalonate afforded, with exclusive

c 3

38

37

34a b

39 SCHEME14.

R=H R=CH,

SZANTAY

390

diastereoselectivity, 39 in almost quantitative chemical yield and without any loss of optical activity. The crucial point in the enantioselective syntheses starting from tryptophan esters is the elimination of the derived chiral center after its temporary utilization. In our case, a two-step hydrolysis, and decarboxylation yielded compound 34a, which, on further elaboration through 31 (R = CH3), gave rise to natural (+)-vincamine (30).It was a very fortunate situation that the inexpensive, natural L-tryptophan gave the correct absolute configuration of (+)-30,which could not have been foreseen in advance. In another chiral approach starting from L-tryptophan we have observed an unexpected phenomenon. When L-tryptophan methyl ester (40b) was allowed to react with aldehyde diester 41 a complete racemization occurred and only racemic 43 was obtained. However, if the free acid was made to react with 41 no racemization was detected and after esterification optically pure (+)-43was obtained. Searching for the reason for this dramatic difference, we were able to isolate from the latter reaction the optically active intermediate 44. Thus the formation of a lactone ring is responsible for the preservation of the original configuration. The (+)-lactam 44 was transformed into the previously discussed 34a (47). In addition to (+)-vincamine (M), the alkaloid (-)-vincamone (33)and apovincaminic acid ethyl ester (32b)(CavintonB) are also on the market

41

40a R = H b R=CH3

40b+41 40a+41

43

- 4

(*I 43 (+) 44

(+) 43

44 SCHEME 15.

10. SYNTHETIC STUDIES

IN ALKALOID CHEMISTRY

391

for similar clinical indications. That is the reason why in the above discussion intermediates having either methyl, or ethyl ester group were used. The concept we employed in the synthesis of emetine and yohimbine, i.e., cycloaddition to a C = N double bond, also proved to be efficient in the synthesis of vincamone (46). A direct transformation of the key intermediate 31 into (-)-vincamone (33) was achieved, after hydrolysis, through straightforward aerial oxidation in the presence of Cu2+and Mn2+catalysts, with the intermediacy of the oxime of 33 (52). As we can see, we spent many years playing around with different synthetic approaches, like playing “etudes” on a musical instrument. But as a reward, our syntheses of all three products (3033, and 32b) were scaled up for industrial use in the Chemical Works of Gedeon Richter, Ltd. B. INTERCONVERSIONS The interconversion of the alkaloids discussed above presents an attractive synthetic challenge. While the transformation of vincamine (30)into vincamone (33) is relatively easy to achieve (53),the inverse approach is far more demanding. This latter goal was attained using the following

45

SCHEME 16.

SZANTAY

392

reaction sequence (54). Vincamone was transformed through the corresponding dioxo compound into 45. On treatment of the latter compound with excess diazomethane, (+)-vincamine was obtained in 32% yield. Again, the transformation of vincamine into apovincamine (32a)requires only the elimination of water, which can easily be achieved by several methods. The reverse process, however, requires more delicacy. Since apovincaminic acid esters are easily available through total synthesis (43),their efficient conversion into (+)-vincamine is a very attractive target. When (+)-apovincamine (32a)was dissolved in concentrated hydrochloric acid and subsequently treated with an aqueous solution of sodium nitrite, the crystalline hydrochloride of the 15a-chloro derivative 46 was formed in 76% yield, with no other isomers being detectable (55). The very high regio- and stereoselectivity of the reaction is remarkable. When the N oxide 47a was reacted with thionyl chloride in benzene, 46 was obtained. A similar reaction was carried out with ethyl ester 4% as well. The presence of the N-oxide function proved to be imperative for this process. Taking into account the above experimental results, it is clear that further studies of the reaction sequence are required in order to put forward a plausible mechanism for these highly selective transformations. The chloro compound 46 was catalytically reduced to (+)-vincamine (55). Speaking of N-oxides, it is worth mentioning that the N-oxide derivatives of the three alkaloids (30,33,and 32b)gave, rather unexpectedly, dimers when treated with acetic acid anhydride. This surprise stems from the fact that the Polonovsky reaction, and its modification by Potier, are widely used in alkaloid chemistry, and usually lead to the corresponding iminium salts. For example, on converting eburnamonine (optical antipode of 33) into its N-oxide and on treatment of the latter with trifluoroacetic acid anhydride, the corresponding iminium salt was obtained, which, on reduction, gave rise to trans-eburnamonine. (Note that eburnamonine and vincamone are enantiomers.) In our case, when vincamone N-oxide (48)was treated with acetic acid anhydride, crystals of the dimeric indole derivative 51 precipitated in 52%

4% b

46 SCHEME 17.

R=CH~ R=C,H,

10. SYNTHETIC

STUDIES IN ALKALOID CHEMISTRY

393

0

49

48

49+50

50

SCHEME 18.

yield (56,57). In the first step in an E,-type trans diaxial elimination reaction, i.e., under kinetic control the iminium salt 49 is formed, which is in equilibrium with its enamine form 50, and their interaction gives rise to 51. Turning back to the interconversion of alkaloids, we were also able to transform (58) (+)-vincamine (Ma) into (-)-criocerine (52a), one of only four known tetrahydrofuranyl ring-bearing eburnane alkaloids, isolated from Criocerus dipludeniiflorus. Upon treatment of M a with iodine, iodocriocerine was obtained in 77% yield, which by acidic treatment was transformed, as described earlier in the literature, into 52a in essentially quantitative yield. From (+)-vincine, (30b) which is the 11-methoxy derivative of vincamine, following a similar route, (- )-craspidospermine (52b), an alkaloid from Craspidospermum verticillatum, was prepared in excellent yield. In the last step, instead of acidic treatment, a Pd-C-formic acid reduction was applied to remove the iodine (59). Vincine itself was also synthesized through the regioselective bromination of the appropriate intermediate of vincamine synthesis (60), and substitution of bromine at position 11 by a methoxy group (61). In a similar way, the alkaloid (- )-vincinone (11-methoxyvincamone) was also prepared (62). With criocerine (52a) a similar dimerization was carried out as with vincamine, described above (63). Alkaloid 52a was dissolved in acetic acid. The work-up after 1 day yielded the dimer 53 in which the configuration

394

SZANTAY

30a

b

R=H R = O C H ~ vincine

52a

b

R=H

criocerine

R = OCH3

craspidospermine

SCHEME19.

of C-19' is such that C-18 occupies the thermodynamically favored position, i.e., H-19' is &oriented in ring D'. When using trifluoroacetic acid instead of acetic acid, the novel dimer 54 was obtained in 32% yield. The formation of the new tetrahydropyranyl ring in 54 can only be accommodated by a highly strained structure in which C-18 assumes a quasi-axial position with respect to ring D', i.e., H19' is in the a steric orientation. A possible reaction path 52a + 53 + 54 was excluded by consideration of the fact that 53, on treatment with trifluoroacetic acid, gave only 55 in 63% yield and no amount of 54 could be detected, i.e., the configuration at C-19' was retained. To rationalize the formation of the dimer 54, two basic questions should be answered:

(1) Why do the products differ when trifluoroacetic acid is used instead of acetic acid? and (2) Why does the configuration of C-19' differ in 53 and 54? A possible reaction sequence may be envisaged by assuming that the monoprotonated form 56 attacks the enamine 52a at C-18 forming 57 and, to a small extent, the sterically congested 58. Both 57 and 58 can be diprotonated in the strong trifluoroacteic acid, but only to a negligible extent in acetic acid, and this triggers further

10. SYNTHETIC STUDIES

395

IN ALKALOID CHEMISTRY

10

11

19

18

-

2 52a

1-m

60

54

I

I-

i

/ 53

55

SCHEME 20.

transformations. Thus the further protonation of 58 gives 59 and similarly 57 can also be protonated. However, only 59 exhibits the steric arrangement necessary for the formation of a tetrahydropyranyl ring system, which, when generated, freezes the less stable compound in its form 54. At the same time, 57 equilibrates back to its components, thus shifting the whole system toward 54. Dimer 54 proved to be a rather interesting compound. By virtue of its highly strained structure, it was stable enough only to facilitate structure elucidation, but it transforms spontaneously into dimer 60 in solution at ambient temperature in 1-2 days, and much faster by mild heating, in over 90% yield. The transformation 54 4 60 is clearly a symmetry-allowed

SZANTAY

396

56

H+

-H+

59

58 SCHEME 21.

cycloreversion, i.e., a retru Diels-Alder reaction, the driving force being the relief of the congested system (64). While the Diels-Alder-type cycloaddition is widely used in the synthesis of indole alkaloids, the spontaneous retru-Diels- Alder reaction under such mild conditions is a rather rare phenomenon.

C. SYNTHESIS OF TACAMINE The alkaloids tacamine (61a) and apotacamine (61b) isolated from Tabernaemontana eglandulos, are isomers of vincamine and apovincamine. It

10. SYNTHETIC

STUDIES I N ALKALOID CHEMISTRY

397

stood to reason therefore that our experiences gained during the syntheses of the latter compounds should be used for preparing the new alkaloids. In order to minimize the possibility of dialkylation, iminium salt 62a was reacted after basification with the bulky tert-butyl acrylate (62b). After reduction of 62b with NaBH4, and subsequent treatment of the appropriate stereoisomer with P0Cl3, lactam 63 was obtained, which was allowed to react with tert-butyl nitrite, followed by methanolysis. When oxime ester 64 thus obtained was boiled in dilute acetic acid in the presence of sulfuric acid and sodium pyrosulfate ruc-tacamine (61a) was obtained while boiling it in methanol-sulfuric acid gave rac-apotacamine (61b), in 28% and 47% yield, respectively. The key intermediate 64 was also prepared more easily by reacting the enamine derived from 62a with bromopyruvate oxime ester followed by reduction with NaBH4 (65). D. SYNTHESIS OF CUANZINE

The alkaloid cuanzine (65b) was isolated from Voacanga chulotiana. Owing to its significant pharmacological effects, combined with a substantial

R%

61a

R' OH

R2 H

tacamine

64

63 SCHEME 22.

398

SZANTAY

structural challenge, cuanzine has emerged as a highly attractive target for synthetic investigations. In order to determine the importance of the methoxy group with respect to the biological effects, as well as to gain experience with a model compound, initially demethoxycuanzine (65a) was synthesized. The key intermediate 66a was allowed to react with the oxime of bromopyruvic acid ethyl ester, giving the oxazine 67a. On catalytic reduction the latter compound yielded both the cis- (40%) and the trans-epimers. Transesterification of the cis-isomer 68a and subsequent treatment with sodium metabisulfate and sulfuric acid in aqueous acetic acid gave 65a (66,68). For the synthesis of cuanzine (65b) itself, first a new method for the preparation of 7-methoxytryptamine was developed (69),which was transformed into ruc-cuanzine and its side alkaloid, decarbomethoxy apocuanzine using the reaction sequence discussed above (70).

65a b

6611 b

R=H R=OCH3

R=H R=OCH3

65

67a b

R=H R=OCH3

68a b SCHEME23.

R=H

R=OCH3

10. SYNTHETIC STUDIES

399

IN ALKALOID CHEMISTRY

VIII. Synthesis of Aspidospema Alkaloids Because we had an easy access (71) to 2-(ethoxycarbonyl)tryptamine (69), we wanted to use it for syntheses of the Aspidosperma and Pseudoaspidosperma alkaloids vincadifformine (70a), '4'-vincadifformine (70b), tabersonine (70d), and other epimers; particularly since 70a can serve as a precursor of the commercially important vincamine. At the outset, 69 was transformed into ester 71. The strategy we planned to use was as follows. The derivative 71 on reaction with suitably substituted aldehydes would give, via the unisolated secodine intermediate 72, which plays an important role in Kuehne's biomimetic alkaloid syntheses, the tetracyclic compounds of formula 73. Formation of the fifth ring would afford the target compounds. Carrying out the plan as shown in Scheme 25, we obtained 3-oxovincadifwhich, on reduction of the 0x0-group, can be transformed formine (~OC), into vincadifformine (70a). We developed a pathway involving a suitable reaction partner to 71 that would allow the preparation of vincadifformine more directly than before. The first task was to choose an appropriate aldehyde, which had to have a leaving group that would not to be so reactive as to take part in the cycloaddition, but would at the required moment, and under suitable conditions, be prone to form the fifth ring. These conditions were met by the

69

R'

70a

SCHEME24.

C2H5

RZ

X

H

H2

400

69

SZANTAY

- G:"" +

COOCH3-

COOCHj

[

%kCOWH.' 00%

71

72

COOCH,

-@H

7 0 ~3-oxovincadifformine

COOCH3

73

71

+

~

~

~

&OCOC6H5 c H

,

N

~

-

-

70a vincadifformine

COOCHj

74 SCHEME 25.

benzoyloxy group. Thus using aldehyde 74 we achieved our aim as depicted in the following scheme: In order to obtain tabersonine (70d) and 3-oxo-tabersonine (70e) a double bond (A14*'5) had to be introduced somewhere in the reaction sequence. To this effect, 70c was readily transformed into 3-thioxovincadifformine, which was made to react with p-toluenesulfinyl chloride followed by reduction or oxidation of the thiolactam group. In addition to the alkaloids vincadifformine, pseudovincadifformine, 20epi-pseudovincadifformine, tabersonine, and 3-oxotabersonine, the related alkaloid-like molecules, e.g., 3-oxovincadifformine, 14-epi-pseudovincadifformine, 21-oxo-pseudotabersonine,etc., were also synthesized (72-75). Extension of our strategy was also used for the formal synthesis of the alkaloid 12-demethoxy-N(1)-acetylcylindrocarine (76), isolated from Aspidosperma cylindrocarpon.

IX. Synthesis of Alkaloids from Catharanthus roseus The alkaloids vinblastine (75a) and vincristine (75b) are widely used in the chemotherapy of cancer. They are presumably synthesized in the plant through a coupling of the alkaloids catharanthine (76) and vindoline (87).

10. SYNTHETIC

401

STUDIES IN ALKALOID CHEMISTRY

cx.J&JL H$XXC

Catharanthine part

........................................................ Vindoline part

75a b

R=CH,

vinblastine

R=CHO

vincristine

SCHEME 26.

The total synthesis of these bisindole alkaloids has been achieved by the outstanding achievements of Potier et al., Kutney et al., Kuehne et al., Attaur-Rahman et al., and other excellent scientists, mainly through the coupling of the monomeric alkaloids. Since the vindoline content of the plant is far higher than that of catharanthine, the total synthesis of 76 was an important challenge to solve. At the outset, 3-ethylpyridine was reduced and acylated followed by a Diels-Alder reaction with a-chloro-acryloyl chloride. A subsequent treatment with methanol gave 77a. After deprotection to 77b and acylation with the mixed anhydride of indole-3-acetic acid and pivalic acid, the amide 78 was obtained. By irradiation the latter compound was transformed into 79, which was reduced by NaBH4/BF3 system in one step and in almost quantitative yield to catharanthine. Resolution was performed at an early stage, i.e., before the indole part was introduced, in good yield, with isoquinuclidine base 77b using (+)dibenzoyl-D-tartaric acid. By this short sequence the first total synthesis of the natural, optically active (+)-catharanthine was achieved (77-80). I feel that it is appropriate at this stage to make a short detour to mention some unexpected reactions in this field. The compounds containing the isoquinuclidine skeleton were very prone to rearrangements. For example, compound 80 under solvolytic conditions afforded the new heterocyclic ring system 81, which was easily further transformed to 82 (82,82). However, if the stereoisomeric isoquinuclidine, containing the chlorine in the endo-position was acylated with the above-mentioned mixed anhydride, an entirely different rearrangement took place, depicted in the

SZANTAY

402

7% b

78

R = C02CH C 6H5 R=H 0

76

79

(+)-catharanthine

SCHEME 27.

scheme below, yielding 83, which on irradiation in methanol gave 84 (83-84). On irradiation of 78, in addition to 0x0-catharanthine (79) the side products 85 and 86 were also isolated (82). A new and interesting oxidative rearrangement was observed (85) also with vindoline (87), when treated with Mn02, as depicted in Scheme 31. Especially the formation of product 88, isolated together with several other products, was remarkable. The aspidosperma + eburnan skeletal

80

81 SCHEME 28.

82

10. SYNTHETIC

403

STUDIES IN ALKALOID CHEMISTRY

83

84 SCHEME 29.

rearrangement was already well known starting from vincadifformine or tabersonine, but not in the case of vindoline. Turning back to the synthesis of the bisindole alkaloids, it has been found that the product formed after the coupling of catharanthine and vindoline, with the help of ferric chloride, can be stabilized as its borane complex (86), making the synthesis more manageable. The nitration of vincristine (75b) also gave intriguing results (87),since one of the products obtained (31%)has the structure 89, i.e., the substitution took place at position 7'. The attempted reduction of the nitro-group of compound 89 provided an interesting outcome, because as a result it was substituted by hydrogen and the original vincristine was recovered. Reduction of nitro compounds results generally in the cleavage of the nitrogen-oxygen bond rather than that of the carbon-nitrogen bond. In our case, the reduction was performed either by sodium dithionite (69%) or NaBH4-Pd in methanol (64%). It was found that compound 89 has very low toxicity, while vincristine possesses rather high toxicity. If we could apply 89 as a per 0s remedy and reduce it to vincristine at the site of the tumor to be attacked, a new approach to avoid serious side effects may be found.

86

85 SCHEME 30.

SZANTAY

404

h.lo4_H

Y

3

88

87 vindoline SCHEME31

One of our findings has shown that oxidation of vinblastine (75a) or vincristine (7%) give a 9-aspidosperma-aspidosperma-typeskeleton (90) via transannular cyclization. Acid catalysis in turn triggers an aspidosperma + eburnan skeletal rearrangement of 90 into 91 (88-89). Such a rearrangement is well known when starting from the “monomeric” vincadifformine, tabersonine, or vindoline, but not thus far in the case of the more complex bisindoles of type 75. For the “monomeric” structures the rearrangement in all cases proceeds with retention of configuration at each point of ring annelation. However, in exploring the synthetic scope and stereochemical details of these transformations involving various constitutional and stereoisomeric analogues, we have found that C-14’ is particularly vulnerable to epimerization during the 90 -+ 91 rearrangement. Moreover, the stereostructural identification of compounds of type 91 has proved to be a notoriously challenging task, which was solved by using model compounds and through detection of a “hidden” exchange partner in NOE analysis (89).

75b

nitr.-

‘red.

89 SCHEME32.

10. SYNTHETIC

STUDIES IN ALKALOID CHEMISTRY

405

H+ __c

Me

I5

90

91

s : R = M e ; b:R=CHO

SCHEME 33.

X. Synthesis of Morphine

Our interest in developing commercially feasible syntheses of natural products turned our attention toward morphine (92), which is still the most frequently used analgesic in surgery and in the final stages of cancer. Furthermore, new semisynthetic products from thebaine (97) are the modern stars among analgesics or narcotic antagonists. As soon as these products can be industrially produced by total synthesis at a reasonable price, narcotic control will also be easier. An enantioselective production of R-( +)-norreticuline (93a), the proper enantiomer for the synthesis of natural morphine, was achieved through reduction of the prochiral iminium compound by using a chiral reducing agent (92). (Note that the previously executed studies were done with the racemic derivative.) The regioselective para-ortho' oxidative coupling of norreticuline is regarded as the key step of any biomimetic morphine synthesis. To achieve this goal reticuline was converted to the protected derivative 94. The most pronounced protecting effect of the 6'-halogen was observed when tetraethylammonium [bis(trihalogen-acyloxy)]-iodate-type reagent was used in the oxidative cyclization. In this way, we amved at the 1-bromo-Nacylnorsalutaridine ( 9 9 , which was the only isolable product, in 58% yield without forming any isomers (90-93). Compound 95 was easily transformed into thebaine (97) via salutaridine (96). the conversion of which into morphine has already been described by Rice. In a theoretically interesting achievement, reticuline (93b) was directly converted into salutaridine (96) using lead tetraacetate in the presence of

SZANTAY

406

92

bn

93a b C

96

R=H R=CH, R=CO,C,H,

salutaridine

95

94

97 thebaine SCHEME34.

trichloroacetic acid (92). Robinson’s seminal suggestions on the biogenesis of morphine have been further refined by Barton, and have been supported by in vivo experiments involving the oxidation of reticuline to salutaridine. The literature is replete with unsuccessful attempts to realize this reaction in the laboratory.

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The first and hitherto sole successful transformation of this type was carried out by Barton, who oxidized tritium-labeled 93b to 96 using potassium ferricyanide. The product was detected by an isotope dilution technique and was present in 0.03% yield. We described the first in vitro replication of the in vivo process in preparative quantities. By the above-mentioned procedure, salutaridine was isolated in white, crystalline form in 2.7% yield, which was two orders of magnitude higher than reported earlier. It is worth mentioning that on treatment of 93c with manganese tris(acet0nylacetate) apara-para' coupling was achieved in 32% yield. After deprotection and methylation of the nitrogen the alkaloid pallidine (98) was obtained (94), the dextrorotatory antipode of which was isolated from Corydalis pallida. Experience thus shows that varying the oxidizing agents, protection techniques, and further reaction conditions, we can fairly well steer the selectivity of the phenolic coupling.

XI. Synthesis of Epibatidine Epibatidine (99) was isolated from the skin extract of an Ecuadorian poison frog Epipedobates tricolor in 1992 by Daly et al. and mentioned as representing a new class of amphibian alkaloids. According to the biological investigations, epibatidine proved to be several hundred times more potent as analgesic than morphine and operates via a nonopioid mechan-

OH

93c

98 pallidine SCHEME35.

408

SZANTAY

ism. Further biological studies required synthetic epibatidine. The need triggered a competition among laboratories, and in a relatively short time several synthetic approaches were reported (95). The main goal of our synthetic strategy was to create a practical route to the natural epibatidine (99) on a large scale. Hence we wanted to

101

100

Br0

103

102

Jy3m3

cl

105

104 H N

99

cl

99' SCHEME 36.

10. SYNTHETlC

STUDIES IN ALKALOID CHEMISTRY

409

use commonly available starting materials, and well-known and wellcontrollable chemical transformations. Nitromethane was allowed to react with methyl vinyl ketone to give compound 100. After bromination and subsequent quaternization with triphenylphosphine the salt 101 was obtained. Wittig reaction of the related phosphorane with chloropyridine aldehyde gave rise to 102,treatment of which with KF-alumina furnished 103. Reduction of the keto group followed by mesylation afforded 104 and subsequent reduction of the nitro group gave amine 105, which on heating resulted in the endo-isomer of epibatidine (99’). On boiling the latter compound in tert-butanol in the presence of potassium tert-butoxide epimerization occurred and racemic epibatidine was obtained (96). To avoid the need for epimerization in the last step we simply changed the two functionalities on the cyclohexene ring, i.e., a kind of “Umpolung” was applied. In that case, the nitro group was first reduced in compound 103, and subsequent treatment with ethylene glycol furnished the protected amino ketone 106. After diacylation with tosyl chloride to 107,the

n 0

-& 0

103

CI

106

c1

c1

107

108

N

H

109

99

SCHEME37.

SZANTAY

410

THF, rt. 3 days

THF. rt. 3 days

-

(-) 103

SCHEME 38.

protective group was removed yielding the ditosylated amino ketone 108. The oxo-function was replaced by an amine via reductive amination to afford 109. Base-catalyzed ring closure provided 99 (97). Our enantioselective synthetic approach was based upon the enantioselective intramolecular Michael addition of prochiral compound 102 leading to the chiral 103, induced by chiral bases. Up till now a-phenylethylamine enantiomers have proved to be best bases to catalyze the enantioselective reaction. Compound 102 was obtained in over 80% e.e., then transformed into the natural product (-)-99. As far as the mechanism is concerned, we could isolate at low temperature an intermediate as a result of the addition of the amine on the double bond of the unsaturated ketone, which on further heating was transformed to the optically active (-)-lo3 (97).

Acknowledgments

I can’t help finishing this review without expressing my deep appreciation to all of my co-workers, many of whom became in the meantime middle-

10. SYNTHETIC

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411

aged professors. Their names can be found in the references, but I should like to thank especially Professor Toke, Professor Szabo, Professor NovAk, Professor Kalaus, the late Dr. Barczai-Beke, Dr. Honty, Dr. Blask6, Dr. Tbth, Dr. Dornyei, Dr. Szentirmay, Dr. Kolonits, Dr. Soti, Dr. Sapi, Dr. Bolcskei, Dr. Vedres, Dr. Moldvai, Dr. Kardos-Balogh, Dr. Incze, Dr. Temesvdri-Major, and my son, Dr. SzBntay, Jr., for their contributions. Without their enthusiastic and devoted work during the last decades the above-discussed results would not be available now. Last, but not least, the name of the late Professor Beke (t1963), from whom I have learned a lot, should also be mentioned here with my great gratitude.

References*

1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

D. Beke and Cs. Szantay, Liebigs Ann. Chem. 640,127 (1961). D. Beke and Cs. Szantay, Chem. Ber. 95, 2132 (1962). Cs. Szantay and J. Rohaly, Chem. Ber. 98,557 (1965);Magy. Kkm.Folydirat. 70,478 (1964). Cs. Szantay, in “Recent Developments in the Chemistry of Natural Carbon Compounds” (R. Bognhr, V. Bruckner, G. Fodor, and Cs. Szintay, eds.), Vol. 2, p. 63. Akadkmiai Kiado, Budapest, 1967. Cs. Szantay. M. Barczai-Beke, and I. Jelinek, Hung. Pat. HU 17.745, Chem. Abstr. 93, 168148 (1980). Cs. Szantay, L. Toke, and P. Kolonits, J. Org. Chem. 31, 1447 (1966). Cs. Szantay, L. Toke, and G. Blaskb, Acta Chim. Hung. 95, 81 (1977). Cs. Szantay and Gy. Kalaus, Chem. Ber. 102,2270 (1969). Cs. Szantay, L. Toke, K. Honty, and Gy. Kalaus, J. Org. Chem. 32,423 (1967). Cs. Szantay and L. Novak, Chem. Ber. 100,2018 (1967). Cs. Szantay, L. Toke, and K. Honty, Tetrahedron Lett. 22, 1665 (1965). L. Toke, K. Honty, and Cs. Szantay, Chem. Ber. 102,3248 (1969). L. Toke, K. Honty, L. Szabo, G. Blasko, and Cs. Szantay,J. Org. Chem. 38,2496 (1973). L. Toke, Zs. Combos, G. Blasko, K. Honty, L. Szabb, J. Tamas, and Cs. Szantay, J. Org. Chem. 38, 2501 (1973). L. Toke, and Cs. Szantay, Heterocycles 4, 251 (1976). K. Honty, E. Baitz, Gacs, G. Blasko, and Cs. Szintay, J. Org. Chem. 47, 5111 (1982). Cs. Szantay, K. Honty. L. Toke, and L. Szabo, Chem. Ber. 109, 1737 (1976). G. Blasko, H. Knight, K. Honty, and Cs. Szantay, Liebigs Ann. Chem. 1986, 655. Cs. Szantay, G. Blaskb, K. Honty, and G. Dornyei, in “The Alkaloids” (A. Brossi, ed.), Vol. 27, p. 131. Academic Press, New York, 1986. Cs. Szantay and K. Honty, in “The Monoterpenoid Indole Alkaloids” (J. E. Saxton, ed.), Chapter 4, p. 161. Wiley, New York, 1994. E. W. Baxter and P. S. Mariano, in “Alkaloids: Chemical and Biological Perspectives” (S. W. Pelletier, ed.), Vol. 8, p. 197. Springer-Verlag, New York, 1992.

*All the relevant citations of the background literature can be found in the original papers mentioned above. I apologize to the authors for not repeating them in the present text, but by doing so would lead to an unmanageable number of references.

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22. Cs. Szhntay and M. Birczai-Beke, Chem. Ber. 102,3963 (1969). 23. L. Pkkanyi, M. Czugler, A. Kalman, G. Domyei, Cs. Szantay, and M. BBrczai-Beke, Crystal Srruct. Comm. 8, 523 (1979). 24. M. Bbrczai-Beke, G. Dornyei, M. KajtQ, and Cs. Szintay, Tetrahedron 32, 1019 (1976). 25. M. Barczai-Beke, G. Dornyei, G. TBth, J. Tamas, and Cs. Szintay, Tetrahedron 32, 1153 (1976). 26. Cs. Szantay, 8. Szentirmay, and L. Szab6, Tetrahedron Lett. 42,3725 (1974). 27. Cs. Szintay, 8. Szentirmay, L. Szabb, and J. Tamas, Chem. Ber. 109,1737 (1976). 28. Cs. Szintay, G. Blask6, K. Honty, L. Szab6, and L. Toke, Heterocycles 7, 155 (1977). 29. Cs. SzBntay. L. Toke, G. Blask6, K. Honty, and L. Szab6, Lectures in Heterocyclic Chemistry, Vol. IV, S-25 (1978). 30. Stereoselective Synthesis of Natural Products 1979, p. 28. Excerpta Medica, Amsterdam, 1979. 31. Cs. Szhtay, G. Blasko, K. Honty, E. Baitz Gacs, and L. Toke, Liebigs Ann. Chem. 1983, 1269. 32. Cs. Szantay, K. Honty, G. Blask6, E. Baitz Gacs, and P. Kolonits, Liebigs Ann. Chem. 1983, 1278. 33. Cs. Szantay, G. Blask6, K. Honty, E. Baitz Gics, J. Tamb, and L. Toke, Liebigs Ann. Chem. 1983, 1292. 34. Cs. Szantay, G. Blask6, K. Honty, L. Szabo, and L. Toke, in “Indole and Biogenetically Related Alkaloids,” p. 202. Academic Press, New York, 1980. 35. I. Tbth, G. BozsBr, L. Szabo, J. Tamis, E. Baitz Bbcs, and Cs. Szdntay, Liebigs Ann. Chem. 1987, 1021; and citations therein. 36. G. Domyei, Cs. Szantay, and L. Szabb, Heterocycles 39,449 (1994). 37. E. S. Vizy, I. Toth, G. T. Somogyi, L. Szabb, L. G. Hdrsing, and Cs. Szintay, J. Med. Chem. 30,1355 (1987). 38. Cs. SzAntay, L. Szab6, and Gy. Kalaus, Tetrahedron Lett. 191 (1973). 39. Cs. Sziintay, L. Szab6, and Gy. Kalaus, Tetrahedron 33,1803 (1977). 40. L. Szab6, Gy. Kalaus, and Cs. SzBntay, Archiev der Pharmazie 316,629 (1983). 41. Cs. Szintay, L. Szab6, Gy. Kalaus, P. Gybry, J. Sipi, K. N6grBdi, in “Organic Synthesis. Today and Tomorrow” (IUPAC) (B. M. Trost and C. R. Hutchinson, eds.), p. 285. Pergamon Press, New York, 1981. 42. Gy. Kalaus, 8. Szentirmay, L. Szabo, and Cs. Szhntay, Tetrahedron Lett. 25,2373 (1979). 43. L. Szab6, J. Stipi, Gy. Kalaus, Gy. Argay, A. KalmBn, E. Baitz-Gacs, J. Tamas, and Cs. Szintay, Tetrahedron 39,3737 (1983). 44. L. Szabb, J. Sapi, K. Nbgrldi, Gy. Kalaus, and Cs. Szantay, Tetrahedron 39,3749 (1983). 45. Cs. Szantay and A. Nemes, in “The Monoterpenoid Indole Alkaloids” (J. E. Saxton, ed.), p. 437. Wiley, New York, 1994. 46. L. Novik, J. Rohaly, Cs. Szantay, and L. Czibula, Heterocycles 6, 1149 (1977). 47. Gy. Kalaus, I. Greiner, L. Szab6, and Cs. Szantay. Unpublished results. 48. J. Kreidl, Cs. Szintay, L. Szab6, M. Farkas, Gy. Kalaus, K. Nbgrhdi, A. Nemes, J. MCsziros, and Zs. Aracs, French Patent, 2648 841; Chem. Absrr. 115,71988 (1991). 49. Cs. Szhntay, L. Szab6, Gy. Kalaus, and Cs. Szintay, Jr., Hungarian Patent 204 526 (1992). 50. Cs. Szantay, L. Szab6, Gy. Kalaus, J. Sapi, J. Kreidl, A. Nemes, B. Stefk6, J. MCszBros, and I. Juhasz, Hungarian Patent 205 109 (1992); and Hungarian Patent 205 110 (1992). 51. Cs. Szantay, Gy. Kalaus, M. Farkas, L. Czibula, Gy. Visky, L. Szab6, J. Kreidl, A. Nemes, and I. Juhlsz, Hungarian Patent 191 401 (1984). 52. Cs. Szintay, L. Szabb, Gy. Kalaus, J. Kreidl, M. Farkas, L. Czibula, B. Stefkb, Gy. Visky, and J. MBszaros, European Patent 159 160 (1985); Chem. Absrr. 104,225088 (1986). 53. Cs. Szhtay, L. Szab6, J. Kreidl, Gy. Kalaus, T. Keve, P. Turcsinyi, I. Polgdr, M. Farkas, and K. Lakszner, Hungarian Patent HU 166475; Chem. Abstr. 80,710002 (1974).

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54. J. Sapi, L. Szabo, E. Baitz-Gacs, Gy. Kalaus, and Cs. Szantay, Tetrahedron 44,4619 (1988). 55. I. Moldvai. Cs. Szantay. Jr., K. Rissanen, and Cs. Szantay, Tetrahedron 48,4999 (1992). 56. 1. Moldvai, A. Vedres, G . Toth, Cs. Szantay, Jr., and Cs. Szantay, Tetrahedron Lett. 27, 2775 (1986). 57. I. Moldvai, Cs. Szantay, Jr., G . Toth. A. Vedres. A. Kalman, and Cs. Szantay, Rec. Trav. Chim. Pay-Bas 107, 335 (1988). 58. 1. Moldvai, Cs. Szantay, Jr., and Cs. Szantay, Synth. Comm. 21, 965 (1991). 59. I. Moldvai, Cs. Szantay, Jr.. and Cs. Szantay, Synth. Comm. 22, 509 (1992). 60. L. Szabo. L. Dobay. Gy. Kalaus. E. Gacs-Baitz, J. Tamas, and Cs. Szantay, Arch. Pharm. 370, 781 (1987). 61. Cs. Szantay, L. Szabo, T. Keve, Gy. Kalaus, L. Dancsi. F. Vezekenyi, T. Acs, and J. Galambos, Hungarian Patent HU 185305 (1983). 62. L. Szab6, Gy. Kalaus, and Cs. Szantay, Acta Chim. Hung. 131, 541 (1994). 63. 1. Moldvai, Cs. Szantay, Jr., G . Tarkanyi. and Cs. Szintay. Tetrahedron 51, 9103 (1995). 64. Cs. Szantay, Jr., I. Moldvai, G . Tirkanyi, and Cs. Szantay. In preparation. 65. L. Szab6, E. Marvanyos, G . Toth, Cs. Szantay Jr.. Gy. Kalaus. and Cs. Szintay, Heterocycles 24, 1517 (1986). 66. F. Soti, M. Kajtar-Peredy, G . Keresztury, M. Incze. Zs. Kardos-Balogh, and Cs. Szhntay, Tetrahedron 47, 271 (1991). 67. F. Soti, M. Incze, Zs. Kardos-Balogh, and Cs. Szantay. in “Studies in Natural Product Chemistry” (Atta-Ur-Rahman, ed.), Vol. 8, p. 283. Elsevier, Amsterdam, 1991. 68. F. Soti. Zs. Kardos-Balogh. M. Incze, G . Keresztury, G . Czira, and Cs. Szantay, Tetrahedron 48, 5015 (1992). 69. F. Soti, M. Incze. 2s.Kardos-Balogh, M. Kajtar-Peredy, and Cs. Szantay. Synth. Comm. 23, 1689 (1993). 70. F. Soti, M. Kajta:a;r-Peredy, Zs. Kardos-Balogh, M. Incze, G . Keresztury, G . Czira, and Cs. Szantay, Tetrahedron 50,8209 (1994). 71. Cs. Szantay, L. Szabo, and Gy. Kalaus. Synthesis 354 (1974). 72. Gy. Kalaus, M. Kiss, M. Kajtar-Peredy, J . Brlik. L. Szabo. and Cs. Szantay, Heterocycles, 23, 2783 (1985). 73. Gy. Kalaus, Chau Phan Dinh, M. Kajtar-Peredy, J. Brlik, L. Szabo, and Cs. Szantay, Heterocycles 31, 1183 (1990). 74. Gy. Kalaus. I. Greiner, M. Kajtar-Peredy, J . Brlik. L. Szabo, and Cs. Szintay, J . Org. Chem. 58, 1434 and 6076 (1993). 75. Gy. Kalaus, I. Juhasz, I . Greiner, M. Kajtar-Peredy, J. Brlik, L. Szabo, and Cs. Szantay, Liebigs Ann. Chem. 1245 (1995). 76. Gy. Kalaus, I. Vago. I. Greiner. M. Kajtar-Peredy, J. Brlik, L. Szabb, and Cs. Szantay, Nut. Prod. Lett. In press. 77. Cs. Szantay, T. Keve, H. Bolcskei, and T. Acs, Tetrahedron Lett. 24, 5539 (1983). 78. Cs. Szantay. T. Keve. H. Bolcskei, T. Acs. and G . Megyeri, in “Natural Products Chemistry” (R. I. Zalewski and J. J. Skolik, eds.), p. 125. Elsevier, Amsterdam, 1985. 79. Cs. Szantay and H. Bolcskei, in “Trends in Medicinal Chemistry” (H. van der Coot, Gy. Domany L. Pallos, and H. Timmermann, eds.), p. 185. Elsevier, Amsterdam, 1989. 80. Cs. Szintay, H. Bolcskei, and E. Gats-Baitz, Tetrahedron 46, 1711 (1990). 81. Cs. Szantay, E. Gacs-Baitz, T. Keve, H. Bolcskei, and G . Megyeri, Heterocycles 23, 1885 (1985). 82. Cs. Szantay, H. Bolcskei, E. Gacs-Baitz, and T. Keve, Tetrahedron 46, 1687 (1990). 83. E. Gacs-Baitz, H. Bolcskei, and Cs. Szantay, J . Chem. Sac., ferkin Trans 2, 213 (1994). 84. H. Bolcskei. E. Gics-Baitz, and Cs. Szantay, Pure & Appl. Chem. 66, 2179 (1994). 85. H. Bolcskei, E. Gics-Baitz, and Cs. Szantay. Tetrahedron Left. 30,7245 (1989). 86. Cs. Szantay, Jr., M. Balazs, H. Bolcskei, and Cs. Szantay, Tetrahedron 47, 1265 (1991).

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87. L. Szabo, Cs. Szantay, E. Ghcs-Baitz, and M. Mik, Tetrahedron Len 36, 5265 (1995). 88. K. Honty, Cs. Szantay, Jr., P. Kolonits, A. Demeter, and Cs. Szantay, Tetrahedron 49, 10421 (1993). 89. Cs. Szanta, Jr., A. Demeter, K. Honty, P. Kolonits, and Cs. Szantay, Mugn. Rex Chem. 31,773 (1993); Cs. Szantay, Jr. and A. Demeter, J. Mugn. Res. Series A 115, 94 (1995). 90. Cs. Szantay, G. Blasko, M. Barczai-Beke, P. PCchy. G. Blaskb, and G. Dornyei, Tetrahedron Left 31, 3509 (1980). 91. Cs. Szantay, M. Barczai-Beke, P. PCchy, G. Blasko, and G. Dornyei, J. Org. Chem. 47, 594 (1982). 92. Cs. Szantay, G. Blask6, M. Barczai-Beke, G. Dornyei, and P. PCchy, Pluntu Medicu 48, 207 (1983). 93. Cs. Szantay, G. Dornyei, and G. Blasko, in “The Alkaloids” (G.A. Cordell and A. Brossi, eds.), Vol. 45, p. 128. Academic Press, New York, 1994. 94. G. Blasko, G. Dornyei, M. Barczai-Beke, P. PCchy, and Cs. Szantay, J. Org. Chem. 49, 1439 (1984). 95. Cs. Szantay and Zs. Kardos Balogh, in “The Alkaloids” ( G . A. Cordell, ed.), Vol. 46, p. 95. Academic Press, New York, 1995. 96. Cs. Szantay, Zs. Kardos-Balogh, I. Moldvai, Cs. Szintay, Jr., E. Temesvari-Major, and G. Blasko, Terruhedron Left. 35, 3171 (1994). 97. Cs. Szantay, Zs. Kardos-Balogh, I. Moldvai, Cs. Szantay, Jr., E. Temesviri-Major, and G. Blask6, Unpublished results.

-CHAPTER 11-

MONOTERPENOID INDOLE ALKALOID SYNTHESES UTILIZING BIOMIMETIC REACTIONS HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI Faculty of Pharmaceutical Sciences Chiba University Chiba, 263 Japan

I. Introduction

....................................................................................

415

11. Biomimetic Syntheses of Corynanthe-Related Alkaloids from Secologanin,

Strictosidine, and Their Analogs ...............

B. Aspidosperma to Melodinus Alkaloids

VII. Conclusions

..

...

I. Introduction

In 1917, Sir Robert Robinson brought to organic chemists the conc 0 of biomimetic synthesis (or biogeneticltype synthesis) of natural produ2s by the stunningly simple synthesis of tropinone ( I ) . Following the natural course (biosynthesis), which builds up a complex molecular architecture starting from simple components, by chemical means (reactions) we can discover a good model reaction of the natural substrate, develop new synthetic strategies, attain an overall efficiency of the synthetic scheme, and evaluate the mechanistic validity of a key step in a biogenetic route, etc. THE ALKALOIDS, VOL. 50 0099-95981YX $25.00

415

Copyright D 1998 by Academic Press All rights of reproduction in any form reserved.

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TAKAYAMA A N D SAKAI

Based on this idea, numerous efforts in biomimetic alkaloid syntheses have been carried out over the last 80 years. In this chapter, we review the significant accomplishments concerning the biomimetic synthesis of secologanin-derived monoterpenoid indole alkaloids, which have been reported in the past 10-15 years. This chapter consists of five areas of research in the field: (1) biomimetic syntheses of Corynanthe-related alkaloids from secologanin, strictosidine, and their analogues; (2) syntheses of Aspidosperma and Zboga alkaloids via secodine or dehydrosecodine intermediates; (3) biomimetic skeletal rearrangement and fragmentation; (4) biomimetic syntheses in the Sarpagine family; and ( 5 ) biomimetic bisindole alkaloid syntheses. Biogenetic numbering is used throughout.

11. Biomimetic Syntheses of Corynanthe-Related Alkaloids from

Secologanin, Stnctosidine, and Their Analogs The glucosidic indole alkaloid, strictosidine (3), which is formed by a Pictet-Spengler-type condensation of tryptamine (2) with the monoterpene secologanin (1) catalyzed by strictosidine synthase, is the pivotal intermediate in the early stage of the biosynthesis of almost 2000 monoterpenoid indole alkaloids. The biosynthesis of heteroyohimbine-type alkaloids, such as ajmalicine, as well as the ajmaline-sarpagine-type alkaloids have been studied in most detail (2). In the 1970s, Brown at Manchester reported many biomimetic conversions from secologanin (1) into the heteroyohimbine-type compounds (3,4), and this vein was recently extended to the synthesis of two indole alkaloids, cadambine (8) (5) and antirhine (14)(6). Secologanin tetraacetate was oxidized by Jones’ method, and the resulting secoxyloganin acetate (4)was subjected to iodolactonization to afford the key intermediate 5 in a highly chemo- and stereoselective manner. Treatment of iodolactone 5 with alkoxide gave the desired 3s-epoxide 6. Heating 6 with tryptamine in ethanol and subsequent acetylation gave the azepine lactam 7 via a regioselective nucleophilic attack of tryptamine on the C-4 position of the epoxide. Finally, Bishler-Napieralski cyclization of 7 followed by Zemplen deacetylation gave cadambine (8) in an overall yield of

11. MONOTERPENOID INDOLE

ALKALOID SYNTHESES

417

H

Tryptamine 2

""55 H\'.

t

Strictosidine 3

.,\OGlc

U

MeO2C

Secologanin 1

Many skeletal types of monoterpenoid indole alkaloids SCHEME 1.

48% from secologanin (1) (5). A synthesis of 3a-dihydrocadambine (9) from secologanin (1) has also been reported by McLean et al. (7). Enantiospecific synthesis of antirhine (14) from secologanin (1) utilizing an enzymatic reaction was also achieved (6). Glucoside hydrolysis and chemoselective reduction of secologanin ethylene acetal (10) with baker's

6

?O2c-"

OH

Cadambine 8

3tx-Dihydrocadambine9 SCHEME 2.

418

TAKAYAMA A N D SAKAl

yt

n

n

n0

0

11 OH

10

12

O

2

no

k

Antirhine 14

13

SCHEME 3.

yeast at pH 6.4 afforded a 60-80% yield of the aglucone 11. Saponification and deformylation of 11 by heating with sodium hydroxide gave on acidification a 76% yield of the lactone 12, which was then reacted with tryptamine to form the amide 13. Subsequent reduction with lithium aluminum hydride, followed by acid-catalyzed hydrolysis of the acetal and concomitant PictetSpengler condensation, afforded antirhine (14) as the only stereoisomer in 69% yield from 12. The Hoffmann-La Roche research group in Nutley succeeded in the stereoselective conversion of loganin aglucone (15)into a heteroyohimbinetype indole alkaloid, 19-epi-ajmalicine (16)(8). Enantioselective total syntheses of secologanin analogs or their equivalents were developed (9-I2), providing the synthesis of heteroyohimbine-type alkaloids via the condensation with tryptamine.

Loganin aglucone 16

19-Epi-ajmalicine 16 SCHEME 4.

11. MONOTERPENOID INDOLE ALKALOID

419

SYNTHESES

A new biomimetic access to indole alkaloid derivatives via strictosidine analogs has been developed by Tietze (Z3,Zd). For example, in the domino reaction consisting of a tandem Knoevenagel-hetero-Diels-Alderhydrogenation reaction, the three components, aldehyde 17, the 1,3carbonyl compound 18,and enol ether 20, gave the indole alkaloid derivative 22 having the normal type Corynanthe skeleton in 46% yield.

111. Biomimetic Syntheses of Aspidosperma and Iboga Alkaloids

Dehydrosecodine (26),which would be derived from geissoschizine (23) (Corynanthe alkaloid) via dehydropreakuammicine (24) (Srrychnos family) and stemmadenine (25),is widely accepted as the key intermediate in the biosynthesis of Aspidosperma- and Zboga-type of alkaloids, though it has not yet been found in Nature (Z5-Z7). Since the end of the 1960s,biomimetic conversion of natural alkaloids, i.e., of tabersonine (28) (Aspidosperma) into catharanthine (27)(Iboga) and pseudotabersonine (30),of stemrnadenine (25) (Strychnos) into 27, 28, and 30, of precondylocarpine acetate (31)or A18-tabersonine (29)into andranginine (32),were reported. These reactions could best be explained by the participation of dehydrosecodine (26)or its derivative. Afterwards, studies of the biomimetic total synthesis

OANA0

18

I

SCHEME 5.

1

420

TAKAYAMA A N D SAKAl

3

-* Dehydropreakuammicine 24

Geissoschizine 23

& l $ C02Me

Me02C

CH20H

Stemmadenine 25

Dehydrosecodine 26

"H C02Me

Catharanthine 27

Vincristine

/

Tabersonine 28 A"-Tabersonine 29

-N

\

Md2CfiCH~*C

Precondylocarpine acetate

Pseudotabersonine 30

/u

H Me02C

Andranginine 32

31

A Proposed Biogenetic Pathway of Indole Alkaloids SCHEME 6.

of Aspidospermu and Zbogu alkaloids utilizing fugitive dehydrosecodine or secodine derivatives in the crucial step of the synthetic scheme have been energetically carried out, as described below.

11. MONOTERPENOID INDOLE

ALKALOID SYNTHESES

421

A. VIASECODINE-TYPE INTERMEDIATES Kuehne’s methodology, which first appeared in 1978 (18),for the biomimetic total synthesis of dl-vincadifformine was very ingenious and efficient. Following the first accomplishment, Kuehne’s original strategy has been further improved, providing the biomimetic total synthesis of many indole alkaloids, such as racemic ervinceine (19),minovine (20),pseudovincadifformines (21),the pandolines (21),ibophyllidine, 20-epi-ibophyllidine (22), tabersonine (23),minovincine (24,25),coronaridine (26), and kopsine and several its related alkaloids (27).Because these achievements have already been reviewed (28,29),only a few recent examples will be introduced here. The biomimetic total synthesis of (-)- and (+)-vincadifformine (Ma, b) and of (-)-tabersonine (28) was attained in an enantioselective manner (30). Secodine itself is not chiral; therefore, Kuehne et al. utilized the enantiomeric secodine derivatives (36a, b) and (37a, b) for the chiral synthesis of the Aspidosperma alkaloids. Starting from D-mannitol,both enantiomers of chiral epichlorohydrin were prepared and then converted to the epoxyaldehydes 34a, b, in which the epoxide bearing a secondary carbon is enantiomerically pure. On condensation of the respective chiral aldehydes Ma, b with the indoloazepine 33, the bridged azepines 35a, b were formed. Through intramolecular N-alkylation and fragmentation reactions, the intermediates were rearranged primarily to the chiral (hydroxymethy1)norsecodines (36a, b). Spontaneous cyclization of these transient intermediates, in which the stereocenter at C-14 completely controlled the reaction course, provided the respective enantiomeric (hydroxymethy1)norvincadifformines (38a, b). In the intramolecular epoxide opening some 14-hydroxysecodines (37a, b) were also formed, and the resultant 14-hydroxyvincadifformines (39a, b) (minor product) could be readily removed from the major products (38). For completion of the enantioselective synthesis of (-)- and (+)vincadifformine (40a, b), the derivatives 38a, b were respectively converted to the related chloride, followed by reduction of the aziridinium intermediates. The enantiomeric purity of the crude products obtained in the final step was found to be >98% e.e. for (-)-vincadifformine (ma) and >97% e.e. for (+)-vincadifformine (40b). By dehydration of the axial alcohol in 14-hydroxyvincadifformine (39b) derived from the (S)-epoxide 34b, (-)tabersonine (28) could be obtained in >99% e.e. An improved biomimetic synthesis of both enantiomers of tabersonine using the chiral lactol chloride has been developed (31). Thus, condensation of the optically active lactols 42a, b with the indoloazepine esters 33,41 gave the bridged indoloazepines 43,44, which were allowed to undergo in situ N-alkylation and fragmentation, to generate the transient 14-hydroxysecodines. Intramolecular cyclization of 45, 46 would proceed

f

t

ZZP

IVXVS CINV VNVAVXVJ.

11. MONOTERPENOID

INDOLE ALKALOID SYNTHESES

423

by a stereoelectronically favored addition of the acrylate moiety to the hydroxypiperideine segment, which may take a preferred 14-hydroxy equatorial conformation. The hydroxy group in the products 39, 47 was removed to give tabersonine (28) and 11-methoxytabersonine (48), respectively. The subsequent oxidative elaboration of the E ring in 28, 48 by adopting Danieli's method (32) afforded vindorosine (49) and vindoline (50), which constituted the first enantioselective total synthesis of these alkaloids. The clinically useful anticancer agents, vinblastine and vincristine, are composed of two structurally very different types of monomeric indole alkaloids. However, these two units, i.e., the Zboga and Aspidosperma families, arise from a common precursor, dehydrosecodine (X), divergently in the biogenetic route (see Scheme 6). Following the biogenetic pathway, Kuehne et al. synthesized both classes of alkaloids, catharanthine (27) and tabersonine (28), via the same secodine derivative (52) (33).

R

CI

R=H, 33 R = OMe, 41

CGMe

42a CR) 42b ( S )

R = H , 43% b R=OMe, 44a,b

H

R=H, 46% b R=OMe, 46% b h m

R = H, 39a R = OMe, 47a

C4Me

a series

R = H, (->Tabersonine aS R=OMe, 48 SCHEME 8.

R = H, (-)-Vindorosine 49 R = OMe, (->Vindoline 50

424

TAKAYAMA AND SAKAI

15-0x0-secodine (52), which could be prepared by condensation of indoloazepine 33 and dienone 51, was synthetically ideal compound, because in an enolized form 54 was a highly reactive diene, providing the Zbogu skeleton by reaction with the acrylate moiety, while the ketone form 52, which was the stabilized enamide, was available for thermal cyclization with the indoloacrylate moiety, furnishing the Aspidosperma skeleton. Thus, on heating in toluene 52 was converted to 15-0x0-vincadifformine (53), which could then be transformed to tabersonine (28). The alternative desired cyclization of 52 could be achieved by spontaneous Diels-Alder reaction of the silyl enol ether derivative 54, providing the 15-((trialkylsilyl)oxy)catharanthine (55) in nearly quantitative yield. 15-0x0-coronaridine (56) obtained by cleavage of the silyl enol ether was converted to catharanthine (27) via reduction of the thio derivatives. Biogenetically, both the 21-nor Aspidospermu alkaloid, ibophyllidine (61), and the D-homo Aspidosperma alkaloid, iboxyphylline (59,) would be formed from peudovincadifformine group alkaloids, pandolines (57), through the ring opening and reconstruction of the D-ring, as shown in Scheme 10 (34). The syntheses of these alkaloids were performed by the biogenetically patterned D-ring transformation. Thus, the photooxidative

51

53

3

Tabersonine 28 Catharanthine 27

52

C02Me

C02Me

-

M-C

C02Me

56

SCHEME9.

55

11. MONOTERPENOID INDOLE

425

ALKALOID SYNTHESES

Stemmadenine 25 COzMe

C02Me

Pandolines 57

cC02Me

Ibophyllidine 61

Iboxyphylline 59

60

A Proposed Biogenetic Route of 59 and 61

7

steps

Versatiline 62

63

64

SCHEME 10.

cleavage of the enamine function in the D-ring of the compound 63, which was prepared from "versatiline" (62), and successive linear reaction sequences for the reconstruction of the five or seven membered D-ring afforded the desired alkaloids, 59 and 61 (26). The syntheses utilizing the intramolecular Diels-Alder reaction have been further extended to the total synthesis of Strychnos- and Aspidospermatan-type alkaloids (35,36). Kuehne's original methodology using the condensation of tetrahydro-0-carboline ester 65 with properly functionalized aldehydes was adopted by other researchers, leading to the total synthesis of (+ )-strempeliopine (37) and dl-cylindrocarines (66) (38). Das et al. reported that condensation of the indole-2-acrylate precursor 67 and the chiral amine 68 followed by acidic treatment generated a transient secodine-like intermediate, which spontaneously cyclized to (+)-and (-)20-epi-ibophyllidine (69) (39,40).

426

TAKAYAMA AND SAKAI

In place of the indoloazepine ester 33 in Kuehne’s procedure, secondary amine 70 was used in Szantay’s laboratory, resulting in the synthesis of many Aspidosperma alkaloids (41-43). Other approaches which feature the stepwise construction of secodine itself (44) or secodine-like intermediates for the synthesis of Aspidosperma skeleton were developed. For example, starting from 71 the N-oxide 72 was prepared by a multistep reaction and then subjected to the Polonovsky reaction using acetic anhydride to give dl-vincadifformine (40)and dl-pseudovincadifformine (45). A similar synthesis of dl-3-0x0-vincadifformine was reported by the same research group (46). By Danieli et al., dl -3-0x0-vincadifformine ethyl ester (74) was

@CH2C&Me Me0

n R

Cylindrocarine66

66

__

(+)-2O-Epi-ibophyllidine69

w:: C02Me

70

72

SCHEME 11.

11. MONOTERPENOID

INDOLE ALKALOID SYNTHESES

427

synthesized via cycloaddition of 3-oxo-secodine, which was prepared from enamide 73 by dehydrogenation with benzeneselenenic anhydride (47). Many other synthetic processes for the construction of Aspidosperma alkaloids via secodine-type intermediates were reported (48,49).

B. VIADEHYDROSECODINE-TYPE INTERMEDIATES Compared with the alkaloid synthesis via a secodine intermediate, fewer synthetic studies of Aspidosperma and Zboga alkaloids via a biogenetically postulated dehydrosecodine intermediate have been performed. Kutney el al. reported an attempt at the synthesis of Aspidosperma and Zboga alkaloids via fugitive dehydrosecodine-like intermediates (50). The masked dehydrosecodine derivative 76 was prepared by partial reduction of the pyridinium salt 75, followed by complexation of the resulting unstable dihydropyridine unit with tris(acetonitrile)tricarbonylchromium(O) and incorporation of the acrylate moiety with Eschenmoser’s salt. Removal of tricarbonylchromium with ethylenediamine and subsequent addition of acetic acid resulted in the formation of products 77 and 78 having the Aspidosperma and Zboga skeleta. Grieco et af. have developed a new strategy for the generation of a transient dehydrosecodine-type intermediate (51). Thus, the compound 79, which was prepared from the oxindole derivative via a tandem retro Diels-Alderhntramolecular aza-Diels-Alder sequence, was converted to the carbinolamine 80. Acidic treatment of 80 followed by heating at 80°C

3. WCHZ=NMez

76

76

Ph

78

77 SCHEME 12.

428

TAKAYAMA AND SAKAI

79

80

+--jl-j

Pseudo30

aq. acetone \ 2.1.p-TaOH. 8O"C, MeCN

Ph

CHO

82

Ph

81 SCHEME 13.

in the presence of triethylamine gave, through a dehydrosecodine-like intermediate 81, the pentacyclic pseudotabersonine skeleton 82, which was then elaborated to pseudotabersonine (30).

IV. Biomimetic Skeletal Rearrangements and Fragmentations Many classes of monoterpenoid indole alkaloids are considered biogenetically to be derived from other structurally dissimilar types of alkaloids by molecular rearrangement or fragmentation. These postulated pathways have been realized by chemical means to accomplish the syntheses of many skeletally unusual alkaloids, which will be introduced in this section. A. ASPIDOSPERMA TO VINCA ALKALOIDS

According to the biogenetic hypothesis (52), a pharmacologically important Vinca alkaloid, vincamine (84), would be formed by oxidation of the Aspidosperma alkaloid, vincadifformine (40), into the 16-hydroxyindolenine 83, followed by acid-catalyzed rearrangement. This hypothesis was first realized in vitro by Le Men et af. in 1972. Later, details of the conversion of tabersonine (28) into 14,15-dehydrovincamine and its 16-epimer were reported, which involved the oxidation of 28 with peracid, phosphine-

11.

MONOTERPENOID INDOLE ALKALOID SYNTHESES

429

induced reduction of the &-oxide, and subsequent treatment with acetic acid to give the vincamine derivatives (53). The minor product in this reaction had the B/C ring cleaved structure 85, which was transformed to a natural product, rhazinilam (86), by sequential hydrogenation, decarboxylation, and reduction. Further studies of the oxidationhearrangement of vincadifformine (40) or tabersonine (28) into vincamines have been carried out, and many procedures for this purpose have been developed (54-59). Among them, a "one-pot'' method found by Danieli et af. was very efficient, in which vincadifformine (40) was ozonized at 60°C in dilute sulfuric acidmethanol solution to furnish in 74% yield a 7 :3 mixture of vincamine (234) and its 16-epimer (54). Dye-sensitized photo-oxygenation of vincadifformine (40) and tabersonine (28) was investigated by the same group (55). Thus, irradiation of 40 in a solution of Rose Bengal in aqueous methanol in the presence of sodium thiosulphate afforded 16-hydroxyindolenine

(-)-VincadZformine40 A", GTabersonine 28

Vincamine 84

83

0

H

Rhazinilam 86

86

87 88

NHSG-

89 90 A14

A14

SCHEME14.

430

TAKAYAMA AND SAKAI

derivatives, which were then treated with acetic acid to give 84 and its 16epimer in 46% and 30% yields, respectively. The thermal rearrangement of the Aspidosperma framework to Vinca derivatives was also studied (59). The 16-nitroindolenine derivative prepared from vincadifformine (40) was converted to vincamone via the unsaturated nitro compound (60). Oxidation of vincadifformine and tabersonine by Fremy’s salt has been investigated. The resulting zwitterionic compounds 87 and 88 were rearranged to isooxazolines 89, 90. Reductive cleavage of the N-0 bond in 90 and subsequent diazotization gave the 14,15-dehydrovincamines (62). Oxidation of vindoline with active manganese oxide yielded a new rearrangement product having the vincine skeleton in 7% yield, together with other oxidized products (62).

B. ASPIDOSPERMA TO MELODINUS ALKALOIDS Biogenetically, the Melodinus alkaloids 92,93which feature the tetrahydroquinolone framework would be formed from Aspidosperma alkaloids through oxidation at C-16 and subsequent pinacol-type rearrangement. Early attempts at the skeletal transformation of Aspidosperma alkaloids resulted in the formation of the isomeric quinolone derivatives (63,64). In 1984, two research groups succeeded in the biomimetic conversion of vincadifformine (40) into alkaloids having the scandine-meloscine skeleton. Hugel and Lkvy (65) utilized the flow thermolysis of aziridine ester 95, which was prepared by sodium cyanoborohydride reduction of the 16chloroindolenine 94,providing the rearranged dihydroquinoline derivative %. Oxidation of the imine function in 96 gave the tetrahydroscandine 97, which was further transformed to tetrahydromeloscine (98)by the usual decarbomethoxylation. The other approach by Palmisano involved a crucial step that was the stereoelectronically controlled a-ketol rearrangement from 100 to the tetrahydroquinolone 101. The key intermediate 100 was prepared from vincadifformine (40)through the N,-methylation and introduction of a 2P-hydroxy group onto the 16-ketoindoline 99. The anionic rearrangement of 100 using potassium hydride-crown ether in DME or sodium hydride in THF gave a desired rearranged compound 101 as a single product in good yield. Removal of the 16-hydroxy group from 101 was achieved by a two-step process; Barton reduction of the xanthate derivative and successive reduction of the resulting unsaturated lactam using magnesium in ethanol to yield N,-methyl-tetrahydromeloscine (102) (66). Afterward, Hugel and Levy reported the first biomimetic synthesis of two natural products, scandine (92) and meloscine (93), by adopting their original aziridine method (67).

11. MONOTERPENOID

AB-Tabersonine 29

INDOLE ALKALOID SYNTHESES

91

431

R=a-COzMe, Scandine 92 R=fJ-H,Meloscine 93

A Possible Biogenetic Route for Melodinus Alkaloids

96

94

C02Me

flow thermolyais

H

96

99

c

100

R=a-COzMe,97 R=fJ-H,98

c

R=OH, 101 R=H, 102

SCHEME15.

C. ASPIDOSPERMA TO GONIOMITINE SKELETON Goniomitine (106),isolated from Goniorna rnalagasy by Husson et al., has an unusual structural type of indole alkaloid, and a biogenetic scheme was proposed (68). Thus, 106 may be formed from vincadifformine (40)

432

TAKAYAMA AND SAKAI

by oxidative fission of the Nb-C-5 bond, followed by decarboxylation, retroMannich reaction, and finally formation of a new C ring by reaction between the N , and C-21 positions. A biomimetic approach to the goniomitine skeleton from vincadifformine was reported by Lewin et al. (69).The crucial Nb-C-5 bond cleavage in 40 was achieved by: (1) introduction of the methoxy function onto C-5 via a modified Polonovsky reaction of the 16-chloroindolenine derivative 94; and then (2) oxidation with rn-chloroperbenzoic acid followed by methanolysis to give the hemiacetal 107.

Treatment of 107 with trifluoroacetic acid for 48 h provided the rearranged product 108 having a goniomitine skeleton.

CqMe

CGMe

(+)-Vincadifformine40

103

J

Goniomitine 106

105

A Proposed Biopnetic Route of Goniomitine

107 SCHEME 16.

108

104

11. MONOTERPENOID

INDOLE ALKALOID SYNTHESES

433

D. STRYCHNOS TO CALEBASSININE SKELETON Calebassinine-1 (114) isolated from Strychnos solimreana has a unique molecular framework. Palmisano et al. adopted the a-hydroxyketone rearrangement strategy, found in the Aspidosperma to Melodinus transformation, to the biomimetic conversion from the Strychnos alkaloid to a calebassinine skeleton (70). The Wieland-Gumlich aldehyde (109) prepared from strychnine was first converted to the ketone 110 by a five-step reaction. Regio- and stereoselective hydroxylation at C-2 of 110 was achieved by oxidation with m-chloroperbenzoic acid. The 2-hydroxy-Nb-oxide 111 thus obtained was treated with potassium hydride in dimethoxyethane in the presence of crown ether, furnishing the anionic rearrangement product 112 in 89% yield. The 3-hydroxyquinolone derivative 112 was transformed into N,-methyl-calebassinine (113). The biogenetic hypothesis of 114 first proposed by Hesse involved the heterolytic C-2-C-7 cleavage of the 2-perhydroxylated Strychnos precursor and successive B/C ring reconstruction, providing the core tetrahydro-2-quinolone skeleton in 114 (71). However, the synthetic result of Palmisano suggested a possible alternative biogenetic pathway for 114 via an a-hydroxyketone rearrangement process. E. REARRANGEMENT USINGTHEMODIFIED POLONOVSKY-POTIER REACTION

The biomimetic alkaloid transformation utilizing the modified Polonovsky reaction discovered by Potier et al. in the early 1970s was an impressive

P

-&-* Me

HO

109

Calebassine-1 114

‘H

0

OSiTBS

de? H 0 111 “H

110

113

SCHEME17.

112

OSiTBS

434

TAKAYAMA AND SAKAI

study in this research area (72,73). The skeletal change from vobasine alkaloids to the ervatamine group (74) and from stemmadenines to vallesamines (75) was accomplished, and the former transformation was again realized enzymatically using rat liver microsomes in the presence of NADPH and O2 (76). The result supports the hypothesis of the Polonovsky-Potier reaction being “biomimetic.” The methodology has been applied to the successful biomimetic synthesis of bisindole alkaloids of the vinblastine group (see Section VI).

F. FRAGMENTATION Several chemical transformations of indole alkaloids have been reported, which supported the hypothetical biogenesis that the usual monoterpenoid indole alkaloids would be the precursors of several naturally occurring, relatively simple indole alkaloids. Flavopereirine

3,14-Dehydrogeissoschizine was proposed earlier to be a biogenetic precursor of flavopereirine (73, which lacked the three carbon unit at C-15 of the Corynanthe-type alkaloids. Kan and Husson have developed a biomimetic chemical conversion of Nb,21-dehydrogeissoschizine(115) to 5,6dihydroflavopereirine (117),which involved a retro-Mannich reaction resulting in the loss of the p-hydroxyacrylate moiety and subsequent double bond migration (78). Harman

Aimi has proposed a new mechanism for harman formation in plants based on an in vitro experiment in which enzymatic cleavage of the glucoside bond in some p-carboline-type monoterpenoid glucoindole alkaloids, such as lyaloside (118),lyalosidic acid (119),or 10-hydroxylyalosidicacid (UO), afforded harman (121)or 6-hydroxyharman (12)via a fragmentation reaction. The formation of simple p-carbolines in Rubiaceae plants is considered to occur secondarily through monoterpenoid glucoindole alkaloids or their equivalents (79). Nauclefidine

The first proposed chemical structure of nauclefidine was revised to the formula 125 by total synthesis, and based on the new structure, its biogenesis was considered to be that, by fragmentation in the aglycone of strictosamide or vincoside lactam (123), the C-4 unit was eliminated and subsequent oxidation (aromatization) of the D-ring would produce nauclefidine (125).

11. MONOTERPENOID

INDOLE ALKALOID SYNTHESES

435

Along with this biogenetic speculation, vincoside lactam aglucone (124), corresponding to a plausible biogenetic precursor of 125, was heated with aqueous sulfuric acid in dioxane. Through the elimination of the crotonaldehyde unit and subsequent auto-oxidation of the Bring, nauclefidine (125) was produced (80).

OH

115

116

R1=H,&=Me, Lyaloside 118 R1=& = H,Lyalosidic acid 119 R1= OH, & =H, 120

5,6-Dihydroflavopereirine 117

J

autmxid.

Nauclefidine 125 R=Glc, Vincoside lactam 123 R=H,124

SCHEME18.

436

TAKAYAMA AND SAKAl

G. CAMPTOTHECIN

Camptothecin is an important anticancer natural quinoline alkaloid which is derived from strictosidine (81). Recently, two plausible intermediates of camptothecin biosynthesis were isolated (82,83);pumiloside having a quinolone nucleus and a quinoline alkaloid, deoxypumiloside. The total synthesis of camptothecine, which involved a biogenetically patterned aromatic functional group conversion, i.e., the indole-quinolone-quinoline ring, was developed by Winterfeldt and Kametani in the 1970s (84).

V. Biomimetic Synthesis in the Sarpagine Family Sarpagine-type indole alkaloids feature bonding between the C-5 and C16 positions in the Corynanthe-type compounds. Following this biogenetically crucial step by chemical means, ajmaline (151)was synthesized by van Tamelen et al. in the 1970s (85). The simple sarpagine-type alkaloids are biogenetically transformed into various structural types of indole alkaloids, such as macroline-type alkaloids, which would be formed by Nb-C-21 bond fission in the sarpagine alkaloids. For many years, biomimetic transformations among macroline-related alkaloids have been studied by Le Quesne et al. (86,87). More recently, new macroline-type alkaloids were found from cell suspension cultures of Rauwoljia serpentina after feeding ajmaline, and these alkaloids were synthesized from ajmaline based on biogenetic considerations (88). Recent extensive efforts in the chemical investigation of the Gelsemium plants by Cordell, Chinese groups, and ourselves have resulted in finding many various types of new alkaloids (89,90). More than 40 Gelsemium alkaloids were classified into five groups, i.e., simple sarpagine-, koumine-, humantenine-, gelsedine-, and gelsemine-types, based on their chemical structures. The biogenetic pathway of these alkaloids was proposed (92,92) and based on this speculation, the biomimetic synthesis of many Gelsemium alkaloids has been performed (93).The details of these studies were already described in “The Alkaloids” (90). However, some representative biomimetic transformations from simple alkaloids to the Gelsemium alkaloids will be reviewed in this section. The biosynthetic route to the cage structure of koumine (129)would be formed from a simple sarpagine alkaloid, 19(Z)-anhydrovobasinediol(127) (94). Oxidation of the allylic C-18 position in 127 would give an unnatural 18-hydroxy-l9(2)-anhydrovobasinediol(l28),and subsequent intramolec-

11. MONOTERPENOID

INDOLE ALKALOID SYNTHESES

437

ular coupling between the C-7 and C-20 positions would produce koumine (129) (Scheme 19). Based on the biogenetic hypothesis (9.9, the partial synthesis of koumine (129) was attained by two groups independently. Liu et af. realized the biogenetic concept using a sarpagine-type alkaloid, vobasine (130). Allylic oxidation of 131, which was prepared by reduction of 130, with Se02/H202gave koumine (129) in a modest (25%) yield (96).

Koumidine 126

19(Z)-Anhydrovobasinediol 127

Koumine 129 Proposed Biogenetic Route of Koumine

q L q H O

18

Vobasine 130

Anhydrovobasinediol 131 Koumime 129

c

R=OMe, 132 R = H , 133

‘OH

R = H , 134 R=Ac, 136 SCHEME 19.

‘OR

438

TAKAYAMA AND SAKAI

Using a Gardneria alkaloid, 18-hydroxygardnerine (132),an unnatural 11methoxykoumine was initially prepared by Pd(0) mediated transannular SN2' cyclization (97). Later, natural koumine was prepared from the same Gardneriu alkaloid 132 (98). Removing the methoxy group from the indole nucleus in 132 was achieved by reductive deoxygenation of the aryl triflate derivative assisted by a palladium catalyst. The 11-demethoxy derivative 133 thus obtained was converted to 18-hydroxy-anhydrovobasinediol(134) by C/D ring-opening with methyl chloroformate followed by reduction of the carbamate with LiAlH4. Koumine (129) was obtained in 80% yield, when the indole anion prepared from the 18-0-acetate 135 was treated with 0.1 eq. of Pd(OAc):! and 0.5 eq of triphenylphosphine at 80-90°C. Magnus et al. have developed an efficient synthetic route from (S)-(-)tryptophan to chiral sarpagine-type alkaloids. The synthetic intermediates in this series were further extended to the first total synthesis of antipodal koumine (99,100). In the final stage, both 19(Z)- and 19(E)(+)-18-hydroxy-anhydrovobasinediolwere respectively subjected to the modified Mitsunobu reaction, affording (+)-koumine in 40% and 34% yields, respectively. Biogenetically, the humantenine-type oxindole alkaloids represented by 136, 137 would be generated from the sarpagine-type compounds such as 19(Z)-anhydrovobasinediol(127) through rearrangement to the oxindoles and introduction of a methoxy function on the indole nitrogen. Based on this consideration, synthesis of humantenine-type alkaloids was studied as follows. Initially, transformation of sarpagine-type indole alkaloids into the corresponding oxindoles was investigated using the C/D ring-cleaved derivative of gardnerine (138).Oxidation of the indole 139 by the conventional method with t-butylhypochlorite gave chloroindolenine 140, which was directly treated with aq. acetic acid in methanol to afford two oxindoles 141 and 142 in 9% and 37% yields, respectively. The minor product 141 has the same stereochemistry at C-7 as that of natural humantenine-type alkaloids. On the other hand, treatment of 139 with 2.0 eq of Os04 in pyridine-THF afforded the oxindole 144 as a sole product in 77% yield, presumably through the spontaneous pinacol-type rearrangement of the C-2-C-7 di-a-hydroxy intermediate 143 (101). Oxidative rearrangement of the indole alkaloids into the oxindole derivatives in the Gelsemiurn plant may occur enzymatically via ,an intermediate similar to that of the osmylation process. Utilizing this rearrangement reaction, two minor Gelsemiurn alkaloids, Na-demethoxy-rankinidine and Na-demethoxy-humantenine, were synthesized from koumidine (126) (102). By employing newly developed methods, i.e., stereoselective conversion of indoles to the oxindole derivatives with Os04 and the transformation

11. MONOTERPENOID

439

INDOLE ALKALOID SYNTHESES

R =Me, Humantenine 136 R =H, Rankinidine 137

19(Z>Anhydrovobasinediol 127

A Possible Biogenetic Route of Humantenine-Type Alkaloids

Me0

Me0

139

Gardnerine 138

140

&OH, aq. MeOH

J

144

141

142

SCHEME20.

of oxindoles into the corresponding N,-methoxyoxindoles via sodium tungstate-catalyzed oxidation of indoline derivatives (203,104),humantenirine (149), a representative humantenine-type Gelsemiurn alkaloid, was synthesized from a sarpagine-type indole alkaloid (105).The oxindole derivative 144 prepared from gardnerine (138)was used for further transformation (Scheme 21). Because humantenines have a 19(Z)-configuration, the olefin inversion utilizing the vicinal diol function in 144 was needed. The

440

TAKAYAMA AND SAKAI

Humantenirine 149

11-Methoxy-gelsemamide150

Me OMe

Ajmaline 151

20-Hydroxy-dihydrorankinidine162 SCHEME 21.

configuration at C-19 in 144 was inverted by the oxidation-reduction sequence. After protection of .a vicinal diol with 2,2-dimethoxypropane, the lactam residue of the acetonide 145 was reduced with the BH3 * SMe2 complex to yield the secondary amine 146 in quantitative yield. Treatment of the amine 146 with urea hydrogen peroxide complex (H202 H2NCONH2) and a catalytic amount of sodium tungstate (Na2W04. 2H20) in aq. MeOH gave the hydroxamic acid 147,which was methylated with diazomethane to yield the N,-methoxyoxindole 148 in 31% overall

-

11. MONOTERPENOID

INDOLE ALKALOID SYNTHESES

441

yield from 146. Next, a vicinal diol function in the humantenine skeleton was converted to the 19(Z)-ethylidene double bond, and then the Nbprotecting group was removed with activated zinc in AcOH to furnish humantenirine (149).A new seco indole alkaloid, 1l-methoxy-gelsemamide (97)(206),might be formed from the humantenine-type oxindole alkaloid, humantenirine (149),by bond cleavage between the N , and C-2 and bond formation between the Nb and C-2 positions. To create the gelsemamide skeleton, humantenirine (149)was treated with NaOMe in dry MeOH to yield the target natural product, ll-methoxy-gelsemamide (150)in 78% yield (205). 20-Hydroxy-dihydrorankinidine(152),a new humantenine-type alkaloid isolated in 1991 (207), is the only one that has a hydroxy group at the C-20 position. Alkaloid 152 was prepared from ajmaline (151)in 22 steps utilizing a biogenetically patterned synthesis (208). Gelsedine-type alkaloids have a novel oxindole skeleton missing the C21 carbon of the humantenines. The appearance of a new Gelsemiurn alkaloid gelselegine (154) (209) suggested the possibility of a biogenetic pathway for gelsedine-type alkaloids. Thus, oxidation of sarpagine-type indole alkaloids would first provide the humantenine-type oxindole alkaloids. An aziridinium intermediate (153)would then be generated from 20hydroxydihydrorankinidine (152)or from rankinidine (137).Ring-opening by the attack of water at the C-21 position in 153 would produce gelselegine (154).Furthermore, gelsenicine (155)and gelsedine (156)would arise from 154 by loss of the C-21 carbon. Based on this biogenetic speculation, chemical synthesis of gelsedinetype alkaloids was studied as follows. A sarpagine-type alkaloid, gardnerine (138),was again chosen as the starting material ( Z Z O ) , and the methoxy group on the indole ring of 138 was initially removed by a six-step sequence. The resulting 19(E)-koumidine (160)was converted in 94% yield to the C/ D ring-opened derivative 161,which was then treated with Os04 to afford the humantenine-type alkaloid 162. Attempts at the preparation of an aziridine compound like 153 from 162 or 20-hydroxy-dihydrorankinidine (152) were unsuccsessful. As a clue to the construction of the gelsedine skeleton, double bond migration from the C-19-C-20 to the C-20-C-21 positions was then conducted using NaI and TMSCl in MeCN to provide the enamine 163. The enamine 163 was successively treated with Os04 and then NaBH4 to produce the diol 164 stereoselectively. At this stage, the N,-methoxyoxindole function was introduced. The lactam of 164 was reduced in 77% yield with the BH3 * SMe2 complex, and the resultant amine was oxidized with H 2 0 2 H2NCONH2,in the presence of a catalytic amount of Na2W04 . 2H20, followed by O-methylation with CH2N2 to yield the N,-methoxyoxindole 165 in 61% overall yield from 164. Treatment of 165 with N,N,N',N'-tetramethylazodicarboxamide and n-Bu3P in DMF gave the epoxide 166 in 63% yield. Removal of the &-carbarnate (Zn, AcOH)

442

TAKAYAMA AND SAKAI

R=OMe,Humantenirine 149 R=H,Rankinidine 137

L

167

-.

1-

O -*H

N m

M d

Gelselegine 154

J. Mad

+

Gelsenicine 166 Mad

OH

11-Methoxy-19(R)-hydroxy-gelselegine159

Q-& Mad

Gelsedine 156 A Proposed Biogenetic Route of Gelsedine-type Alkaloids

SCHEME22.

gave the primary amine, which gradually transformed into the natural product, gelselegine (154), in 50% yield, upon standing for 5 days at room temperature. It appears that the primary amine regioselectively attacked the

11. MONOTERPENOID

443

INDOLE ALKALOID SYNTHESES

C-20 position with complete inversion. In keeping with the above biogenetic speculation, the C-21 carbon of 154 was oxidatively cleaved with NaI04 in aqueous MeOH to yield gelsenicine (155) in 64% yield. Furthermore, catalytic reduction of the imine function of 155 furnished gelsedine (156) in quantitative yield ( 2 2 1 ) . Using a process similar to the transformation from gardnerine to gelsedine, a structurally similar alkaloid, gelsemicine was synthesized from 138 by a biomimetic route (222). Another member of the gelselegine-type compounds having a 19-hydroxy group was also prepared from gardnerine in a biomimetic manner (223J24). A biogenetic-route for ll-methoxy-19(R)-hydroxy-gelselegine(159) could be viewed as follows (Scheme 22). The double bond at the C-19, -20 positions in a humantenine-type oxindole alkaloid would be oxidized to form the epoxy derivative 157, and by the subsequent attack of the nitrogen (Nb)on the C-20 epoxy carbon, an aziridinium intermediate 158 would be generated. Furthermore, a new alkaloid skeletal type 159, possessing a hydroxymethyl group at the C-20 position, would arise from 158 by ring opening between the C-21 and Nb positions using water (Scheme 22).

163

166

X=H 164 X=OMe 165

zn

y

ACOH

Mad

Gelselegine 154

M d

Gelsenicine 155 SCHEME 23.

0

MeO

Gelsedine 156

444

TAKAYAMA AND SAKAI

In order to realize the above-mentioned biogenetic hypothesis using chemical means, gardnerine (138)was again chosen as the starting material. The C/D-ring cleavage in 138 and stereoselective rearrangement to the oxindole derivative 144 were carried out according to the method described above. Next, the lactam in 144 was chemoselectively reduced with a boranedimethylsulfide complex to give the corresponding indoline derivative in quantitative yield. The secondary amine was then oxidized with H 2 0 2 * H2NCONH2in the presence of Na2W04 2 H 2 0 followed by treatment of the resulting hydroxamic acid with ethereal CH2N2 to produce the N,methoxyoxindole derivative 167 in 40% overall yield. The diol on the side chain in 167 was converted to the epoxide 168 by a conventional method, i.e., mesylation of the secondary alcohol with mesyl chloride followed by treatment with potassium carbonate in methanol. By removal of the Nb protecting group in 168 with zinc in AcOH, the secondary amine 169 was obtained. The amine-epoxide 169 was then heated in dioxane at 150°C for 6 h to produce the aziridine derivative 170 in 61% yield, which corresponded to the biogenetic key intermediate. Finally, the aziridine 170 was refluxed in THF with CF3C02H for 0.5 h to furnish ll-methoxy-19(R)-hydroxygelselegine (159)in 77% yield.

-

VI. Biomimetic Bisindole Alkaloid Syntheses

By coupling between two different monoterpenoid indole alkaloids, many bisindole alkaloids have been prepared. Among them, the biomimetic synthesis of the antitumor alkaloids of the vinblastine group, via the coupling between catharanthine N-oxide and vindoline (50)using the PolonovskyPotier reaction was an outstanding study in the 1970s (215-217). Following this success, some new biomimetic processes for preparation of the vinblastine group were developed (228-121). Kutney el al. discovered that ferric ion mediated the coupling between catharanthine (27) itself, not its Noxide derivative, and vindoline (50)in aqueous acidic media, followed by a sodium borohydride work up to produce anhydrovinblastine (172)in 77% yield (222). Anhydrovinblastine is transformed into vinblastine (173)by employing flavine coenzyme-mediated photo-oxidation and reduced nicotinamide-adenine dinucleotide as a reactant (223).Furthermore, a highly efficient “one-pot’’ operation for the synthesis of vinblastine (173) and leurosidine from catharanthine and vindoline was investigated, which involved a five-step operation, i.e., a modified Polonovsky-mediated coupling of two monomeric units under careful reaction conditions, subsequent re-

11. MONOTERPENOID INDOLE

-

Gardnerine 138

Md

x

c

R=Troc

ALKALOID SYNTHESES

-

X=H, 144 X=OMe, 167

170

445

OM0

R=Troc, 168 R=H, 169

11-Methoxy-19(R)hydroxy-gelselegine 169

SCHEME24.

gioselective reduction by the NADH model substances, oxidation of the enarnine function by air-FeC13, and finally reduction of the resultant iminium by sodium borohydride (124). Most recently, the coupling of two units was performed by means of electrochemical oxidation at a controlled potential to yield anhydrovinblastine (172)and its 16-epimer in 53% and 12% yields, respectively, after sodium borohydride reduction of the iminium intermediate (125). Although a monomeric indole alkaloid, macroline, has not yet been found in Nature, it is considered to be a biogenetic precursor of some bisindole alkaloids. The biomimetic condensation of macroline and other types of indole alkaloids was well studied by Le Quesne in the 1970s (126). Recently, an Alstonia bisindole alkaloid, villalstonine (176),was again synthesized by coupling the macroline equivalent (174)with pleiocarpamine (175)in 0.2 N aqueous hydrochloric acid in the presence of fluoride ion (127).

Many examples of the condensation of two monomeric units, one of which has a nucleophilic center in the molecule and the other counterpart has an electrophilic position, have been reported (118,121,126). Some recent accomplishments will be introduced here. 16-Epi-deformoundulatin (180) was prepared by coupling, under acidic conditions, cabucraline (177),acting as an electron rich partner, and the 6-hydroxypericyclivine derivative 179,

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CQMe

MeO

Catharanthine27 .

Ir

Vindoline M)

Vinblastine 173

A15*20 172

9

HN

0.2NHCl

174

Me

W

+ N

F,

Pleiocarpamine 175 Villatstonine 176 SCHEME 25.

which was prepared by DDQ oxidation (228). Condensation of vobasinol with 3-oxo-coronaridine in methanol-hydrochloric acid yielded ervahaimine-A (181) and -B (182) (229).Vobasinol provided a bis-alkaloid, vobparicine, by coupling with apparicine (230).The Aspidosperma-Eburnea-type bisindole alkaloids, kopsoffine (231) and norpleiomutine (132), were syn-

11. MONOTERPENOID

INDOLE ALKALOID SYNTHESES

447

Cabucraline 177

16-Epi-deformoundulatin 180

a &,, 11

'

Me0

I

COzMe

conuectionat 11: connectionat 1 0

'

CoaW

Ervahaimine A 181 E m h i m i n e B 182

Tenuicausine 183

SCHEME26.

thesized, respectively, by condensation of (+)-eburnamine or (-)eburnamine with (-)-kopsinine. Another Aspidosperma-Eburnea-type bisindole alkaloid, tenuicausine (183),was also prepared from AI4-eburnamine and 11-methoxytabersonine (133).

VII. Conclusions As described above, many successful results concerning monoterpenoid indole alkaloid syntheses have been performed in recent decades by utilizing a biomimetic reaction in a synthetically crucial step. Adopting this biomimetic strategy, a number of structurally complex and/or unusual alkaloids have been synthesized efficiently in a regio- and stereoselective manner.

448

TAKAYAMA AND SAKAI

Furthermore, following the biosynthesis by chemical means has led us to the discovery of new synthetic methodology and reactions. In some cases, biogenetically patterned synthesis supported (or provided proof of) the postulated biosynthetic pathway. However, more than 1000indole alkaloids possessing complex and challenging structures presently await the development of ingenious synthetic methodologies (more selective, milder, and high-yielding) based on concepts followed by Nature. Note Added in Proof After completion of the manuscript, the following relevant papers were published: 1. A biomimetic total synthesis of Strychnos skeleton via corynantheoid framework was reported by Martin and colleagues: S. F. Martin, C. W. Clark, M. Ito, and M. Mortimore, J. Am. Chem. Soc. 118,9804 (1996). 2. Isolation and biomimetically patterned partial synthesis of 3(R)- and 3(S)-deoxypumiloside, which are the plausible biogenetic intermediates to camptothecin was reported M. Kitajima, S. Masumoto, H. Takayama, and N. Aimi, Tetrahedron Lett. 38,4255 (1997).

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25, Indoles, Part 4; Monoterpenoid Indole Alkaloids” ( J . E. Saxton, ed.), p. 762. WileyInterscience, Chichester, 1983. 85. E. E. van Tamelen, and L. K. Oliver, J . Am. Chem. SOC.92,2136 (1970). 86. R. L. Garnick and P. W. Le Quesne, J. Am. Chem. Soc. 100,4213 (1978); and references cited therein. 87. R. W. Esmond and P. W. Le Quesne, J. Am. Chem. Soc. 102,7116 (1980). 88. S. Endrep, H. Takayama, S. Suda, M. Kitajima, N. Aimi, S. Sakai, and J. StBkigt, Phytochem. 32, 725 (1993). 89. Z. J. Liu and R. R. Lu, in “The Alkaloids” (A. Brossi, ed.), Vol. 33, p. 83. Academic Press, New York, 1988. 90. H. Takayama and S . Sakai, in “The Alkaloids” ( G . A. Cordell, ed.), Vol. 49, p. 1. Academic Press, New York. 91. D. Ponglux, S. Wongseripipatana, S. Subhadhirasakul, H. Takayama, M. Yokota, K. Ogata, C. Phisalaphong, N. Aimi, and S. Sakai, Tetrahedron 44, 5075 (1988). 92. H. Takayama and S . Sakai, in “Studies in Natural Products Chemistry, Structure and Chemistry (Part C)” (A. Rahman, ed.), Vol. 15. Elsevier, Amsterdam, 1995. 93. S. Sakai and H. Takayama. Pure & Appl. Chem. 66,2139 (1994). 94. H. Takayama, M. Kitajima, S. Wongseripipatana, and S . Sakai, J. Chem. Soc., Perkin Trans. 1 , 1075 (1989). 95. M. Lounasmaa and A. Koskinen, Planta Medica 44, 120 (1982). 96. Z. J. Liu and Q. S. Yu, Youji Huaxu 1,36 (1986). 97. S. Sakai, E. Yamanaka, M. Kitajima, M. Yokota, N. Aimi, S. Wongseripipatana, and D. Ponglux, Tetrahedron Lett 27, 4585 (1986). 98. H. Takayama, M. Kitajima, and S. Sakai. Heterocycles 30, 325 (1990). 99. P. Magnus, B. Mugrage, M. DeLuca, and G. A. Cain,J. Am. Chem. SOC.112,5220 (1990). 100. P. Magnus, B. Mugrage, M. DeLuca, and G. A. Cain,J. Am. Chem. Soc. 111,786 (1989). 101. H. Takayama, K. Masubuchi, M. Kitajima, N. Aimi, and S . Sakai, Tetrahedron 45, 1327 (1989). 102. M. Kitajima, H. Takayama, and S. Sakai, J. Chem. SOC.,Perkin Trans. 1, 1773 (1991). 103. H. Takayama, N. Seki, M. Kitajima, N. Aimi. H. Seki, and S . Sakai, Heterocycles 33, 121 (1992). 104. H. Takayama, N. Seki, M. Kitajima, N. Aimi, and S . Sakai, Nut. Prod. Lett. 2,271 (1993). 105. H. Takayama, M. Kitajima, and S. Sakai, Tetrahedron 50,8363 (1994). 106. L. Z. Lin, G . A. Cordell, C. Z. Ni, and J . Clardy, Tetrahedron Lett. 30,1177 and 3354 (1989). 107. L. Z . Lin, G . A. Cordell, C. Z . Ni, and J. Clardy, Phytochemistry 30, 1311 (1991). 108. C. Phisalaphong, H. Takayama, and S . Sakai, Tetrahedron Lett. 34,4035 (1993). 109. L. Z. Lin, G . A. Cordell, C. Z. Ni, and J. Clardy, Phytochemistry 29, 3013 (1990). 110. H. Takayama, H. Odaka, N. Aimi, and S. Sakai, Tetrahedron Lett. 31,5483 (1990). 111. H. Takayama, Y. Tominaga, M. Kitajima, N. Aimi, and S. Sakai, J. Org. Chem. 59, 4381 (1994). 112. M. Kitajima, H. Takayama, and S. Sakai, J. Chem. SOC.,Pzrkin Trans. 1, 1573 (1994). 113. H. Takayama, M. Kitajima, and S. Sakai, 1. Org. Chem. 57, 4583 (1992). 114. H. Takayama, M. Kitajima, and S . Sakia, Tetrahedron 50, 11813 (1994). 115. P. Potier, N. Langlois, Y. Langlois, and F. F. Gueritte, J . Chem. Soc., Chem. Comrnun. 670 (1975). 116. P. Mangeney. R. Z . Andriamialisoa, N. Langlois, Y. Langlois, and P. Potier, J. Am. Chem. SOC.101, 2243 (1979). 117. J . P. Kutney, A. H. Ratcliffe, A. M. Treasurywala, and S. Wunderly, Heterocycles, 3, 639 (1975).

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118. G. A. Cordell and J. E. Saxton, in “The Alkaloids” (R. G. A. Rodrigo, ed.), Vol. 20, Chapter 1. Academic Press, New York, 1981. 119. G. Blasko and G. A. Cordell, in “The Alkaloids” (A. Brossi and M. Suffness, eds.), Vol. 37, Chapter 1. Academic Press, New York, 1990. 120. M. E. Kuehne and I. Marko, in “The Alkaloids” (A. Brossi and M. Suffness, eds.), Vol. 37, Chapter 2. Academic Press, New York, 1990. 121. J. Sapi and G. Massiot, in “The Chemistry of Heterocyclic Compounds, Supplement to Vol25, Part 4; Monoterpenoid Indole Alkaloids” ( J . E. Saxton, ed.), Chapter 11. WileyInterscience, Chichester, 1994. 122. J. Vukovic, A. E. Goodbody, J. P. Kutney, and M. Misawa, Tetrahedron 44,325 (1988). 123. J. P. Kutney, L. S. L. Choi, J. Nakano, and H. Tsukamoto, Heterocycles 27,1837 (1988). 124. J. P. Kutney, L. S. L. Choi, J. Nakano, H. Tsukamoto, M. McHugh, and C. A. Boulet, Heterocycles 27, 1845 (1988). 125. E. Gunic, I. Tabakovic, and M. J. Gasic, J. Chem. SOC.,Chem. Commun. 1496 (1993). 126. G. A. Cordell, in “The Chemistry of Heterocyclic Compounds, Vol. 25, Indoles, Part 4; Monoterpenoid Indole Alkaloids” (J. E. Saxton, ed.), p. 577. Wiley-Interscience, Chichester, 1983. 127. Y. Bi, J. M. Cook, and P. W. Le Quesne, Tetrahedron Lett. 35,3877 (1994). 128. G. Massiot, J. M. Nuzillard, B. Richard, and L. Le Men-Olivier, Tetrahedron Lett. 31, 2883 (1990). 129. X. 2. Feng, G. Liu, C. Kan, P. Potier, and S. K. Kan, J. Nut. Prod. 52,928 (1989). 130. T. A. van Beek, R. Verpoorte, and A. B. Svendsen, Tetruhedron Lett. 25,2057 (1984). 131. X. 2. Feng, C. Kan, H. P. Husson, P. Potier, S. K. Kan, and M. Lounasmaa, J. Nut. Prod. 47, 117 (1984). 132. P. Magnus and P. Brown, J. Chem. Soc., Chem. Commun. 184 (1985). 133. Y. L. Zhou, J. H. Ye, Z. M. Li, and 2. H. Huang, Pluntu Medicu 54,316 (1988).

-CHAPTER L

PLANT BIOTECHNOLOGY AND THE PRODUCTION OF ALKALOIDS: PROSPECTS OF METABOLIC ENGINEERING ROBERT VERPOORTE,' ROBERT VAN DER HEIJDEN~ AND J. MEMELINK' Division of Pharmacognosy LeidedAmsterdarn ,Center for Drug Research Leiden University 2300RA Leiden, The Nelherlands Instilute of Molecular Plant Sciences Leiden University 2.?OORA Leiden, The Netherlands

I. Introduction ......................... 11. Plant Cell Cultures for the Prod

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B. Selection .........................

A. Molecular Genetic M

IV. Transcriptional Regulation and V. Conclusions ........................................................... VI. Future Prospects ................... References .......................................................................................

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I. Introduction

Plant biotechnology has progressed greatly during the past two decades. The development of methodologies for plant cell and tissue culture and, subsequently, for genetic transformation of plants is largely responsible for this progress. Today, there exists renewed interest in the use of the THE ALKALOIDS, VOL. SO 0099.9598198 $25.00

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large-scale culture of plant cells as sources of commercially important secondary metabolites, among others because of the large screening programs for new biologically active natural compounds. A few years ago we reviewed the production of alkaloids by cultured plant cells (I), and concluded that it is feasible to grow plant cells on a large scale in bioreactors. The price calculations we made showed that for the more expensive natural products this could even be economically feasible. For a production level of 0.3 g of ajmalicine per liter in a bioreactor after a 2 week growth and production cycle a price of $1500 per kilogram was calculated. If productivity is increased tenfold to 3 g/l, the price drops to $430 per kilogram. Such productivities are realistic. The lower yield represents the optimum production of ajmalicine in Cutharunthus roseus cell cultures (for a review see (2). The higher yield can easily be realized for berberine (Fig. 1) in Coptis juponicu cell cultures, for which a production of 7 g/l, the highest productivity ever reported in plant cell culture, was achieved (3). However, this level is still far below the productivity in cultures of micro-organisms for antibiotics such as penicillin, which can be as high as 30-50 g/l. There is no theoretical reason why plant cells should produce a lower level of secondary metabolites. The fact that under certain conditions up to about 20-60% of the dry weight of a plant tissue or plant cells can consist of secondary metabolites, e.g., tannins and proanthocyanidins in callus cultures of Pseudorsugu menziesii ( 4 ) or anthraquinones in, among others, Rubiu fruticosu cell cultures (3, shows that plant cells are also capable of diverting a large part of their metabolic flux into secondary metabolism. However, as concluded in our previous review (I) it is also clear that the typical productivity of alkaloids in the mg/l range, and in a some cases virtually zero, is too low to allow commercial production. The various efforts to improve productivity for the type of alkaloids €or which a biotechnological production process has been attempted were also extensively discussed in the previous chapter. In the present chapter we will assess the recent progress. However, only those approaches resulting in considerable improvements, or representing new ideas, will be discussed here. We will address, in particular, the pros-

FIG.1. Berberine.

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pects of metabolic engineering for the production of alkaloids, both in transgenic cell cultures and in plants. Based on our present knowledge, we will discuss the strategies which may be followed to improve alkaloid production by means of metabolic engineering and will present our expectations of future developments. To understand the regulation of alkaloid biosynthesis by environmental signals, knowledge of the signal transduction chains is important. Therefore this will be reviewed with regard to alkaloid generation.

11. Plant Cell Cultures for the Production of Alkaloids

The main obstacle to the economically feasible production of alkaloids using bioreactor-cultured plant cells is the low productivity of the cultures. Consequently, research in the past years has focussed primarily on improving the yields of alkaloids in cell cultures; for which the following approaches have been used: 0 0 0 0 0 0

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screening for high-producing cell lines; selection of high-producing cell lines; optimization of growth and production media; culture of differentiated cells; elicitation of secondary metabolism; bioconversions of added precursors; and metabolic engineering.

These approaches, their results, as well as their limitations, will be discussed in more detail below. A. SCREENING Screening for high-producing cell lines is a well-established approach applied to optimize the production of antibiotics by micro-organisms. It has also been widely used, with quite variable results, for the optimization of secondary metabolite production by plant cell cultures. An extensive screening program resulted in high-producing cell cultures of Lithospermum erythroxylon that produced quantities of the naphthoquinone shikonin large enough for a commercially viable process by means of large-scale plant cell cultures (6). The clearest success with regard to the optimization of alkaloid production was achieved with the production of berberine (Fig. 1) by cell cultures of Coptis japonica. Yamada and co-workers (7-9) were

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able to select stable cell lines which had a high production of berberine. To obtain a stable high-producing cell line four to five screening steps were necessary. Interestingly, there was no clear relationship between the productivity of the starting plants and cell lines and the cell lines selected therefrom. In fact, the highest producing Coptis japonica cell line was obtained from a strain which produced only moderate amounts of berberine. In combination with optimization of growth and production media, production levels of 3.5 g/1 were obtained (20,21). Even a level of 7 g/l of berberine has been achieved for cell cultures of this plant (3). Screening efforts to obtain Catharanthus roseus cell lines producing large amounts of ajmalicine and serpentine, have met with completely opposite results. Zenk et al. (12) showed that it is possible to obtain cell lines which produce about 0.34 g/l of these alkaloids. However, those cell lines rapidly lost the trait; after several subcultures alkaloid production returned to the low, prescreening level (13). Among the root cultures of Duboisia rnyoporoides obtained by either medium manipulation or transformation with Agrobacteriurn rhizogenes, only the latter showed an increased scopolamine production after repeated screening (24). The scopolamine level improved from 0.15% of dry weight (DW) in the parent line to 3.2% of DW. The ratio of scopolamine to hyoscyamine also increased, whereas growth decreased. A high-density culture system of this root culture reaching 120 g DW/l was described, which allowed a scopolamine production of 1.35 g/1 in a 3 week growth cycle (25). Lack of phenotypic stability is a recurrent problem facing the establishment of high-producing cell lines. What causes the instability in cell cultures is not well understood, a number of hypotheses have been postulated to account for this problem (16).DNA methylation and repeat-induced point mutations connected with cytosine methylation ( I 7) have been mentioned, as possible mechanisms for instability which might occur in cell cultures because stability control mechanisms found in the plant do not function. The wide variation in chromosome number occurring among individual cells in cell suspension cultures, such as has been reported for Coptis japonica (18) and Nicotiana rustica (19), may also be a source of instability. On the other hand, it was shown that hairy root cultures of the latter species remained diploid, a situation also found in hairy root cultures of some other species. Since screening for high-producing cell lines is quite laborious, the process is mostly carried out when a commercial application becomes within sight. Because of the limited academic interest this work generates, published data on extensive screening programs is scarce. In the case of Taxus cell cultures, for example, such studies have certainly been and continue to be performed, but their outcome has not been published.

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Nevertheless, looking at the results of the various published screening programs ( I ) , we can roughly conclude that relative to the average, nonoptimized production level of a cell culture, an increase of 10- to 20-fold is feasible.

B. SELECTION Several examples of the use of selective media to obtain high-producing cell cultures have been reported. Berlin and coworkers (20) used media containing the toxic compound 4-methyltryptophan to select cell cultures of Cutharunthus roseus having an increased activity of tryptophan decarboxylase (TDC). Indeed, cultures showing high TDC activity were obtained; as a result these cultures accumulated more tryptamine, but they did not produce more alkaloid. Similar observations were reported for Pegunum hurmulu cell cultures (22-23). These results are in agreement with those obtained by genetic engineering (see below), which showed that increasing the level of TDC activity did not result in increased levels of alkaloids (22-26). Hairy root cultures of Nicotiunu rusticu were grown on media containing nicotinic acid as the selective agent. These cultures showed a 2- to 3fold increase in nicotine production and a 10-fold increase in anatabine production. The difference was attributed to differences in the availability of the two other precursors of these alkaloids (27). Efforts to select high quinine or quinidine producing strains of Cinchona were not successful as none of the intermediate metabolites used as a selective agent (the nonmethoxylated alkaloids cinchonine and cinchonidine, and the keto-forms cinchoninone and quinidinone) was sufficiently toxic for such a selection procedure (28). One of the reasons for the limited success of using selective media (see also Ref. ( I ) ) might be the fact that the target enzyme is not the (only) limiting step. C. OPTIMIZATION OF GROWTH AND PRODUCTION MEDIA Numerous papers have been published reporting new media suited for increasing growth and production of a certain secondary metabolite. The value of these data is limited, because in many cases they concern one specific cell line, and the data cannot always be extrapolated to other cultures of the same plant species. Moreover, many of these studies only look at the change of growth and/or production in the first subculture period. It was shown for Tubernuemontunadivuricufu cell cultures, that the effect of a change of growth hormones had only stabilized after about 10 subcultures (29). If such long periods occur between cause and effect, it

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becomes difficult to draw general conclusions, the more so as even the production levels of cell cultures may vary through time. This might be due to external factors. For example, Schripsema and Verpoorte (30) showed that two, genetically identical, cell lines cultured in parallel over a period of about 1 year showed similar variation in alkaloid levels during this period. Of course, the development of a production medium in a fedbatch type of process, is not hampered by long-term effects over a series of subcultures, and thus can be more fruitful. Due to these findings research now focusses more on the regulation of the biosynthesis rather than on extensive screening and optimization programs. The empirical data on the influence of media changes on production are useful to identify signals that induce the secondary metabolite pathways of interest. In connection with the various efforts to enhance yields of alkaloids, we can also mention efforts to improve production by the immobilization of cells (e.g., see Ref. ( I ) for a review), or by using two-phase cultures, in which the desired product is accumulated in a second, nonmiscible phase, which can be a solid phase (e.g., see Ref. (1)for a review) or a liquid phase (31). Both approaches have yielded interesting results. However, in our opinion they are less suitable for a large-scale production process, as both require much larger bioreactor volumes than for a conventional process of growing biomass containing the desired product. Since the product needs to be released to the medium, the ratio of medium to cells (i.e., the biomass density) is much less favorable than in a normal fedbatch mode of operation. As a consequence the production costs will be much higher (1,32).

D. CULTURES OF DIFFERENTIATED CELLS Various types of differentiated cell cultures have been reported: shoot, root, and embryoid cultures. Also cell aggregates, having some sort of differentiation, have been mentioned as production systems for alkaloids (1,33).In particular, hairy roots have been studied extensively (14,15,3335). These cultures, obtained by transformation with Agrobacterium rhizogenes, are capable of producing similar secondary metabolite profiles as the plant roots. By further selection, we might obtain cell lines which have a much higher production than the plant roots. This is nicely illustrated by the work of Yukimune et al. (14,15, see above), who have reported the selection of Duboisia myoporoides hairy root cultures and the further optimization of the growth in a special vessel, resulting in a scopolamine production of 1.35 g/l. Of course, similar results might be obtained by normal root cultures, but hairy roots have an additional advantage; along with the A . rhizogenes T-

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DNA, genes can be introduced which modify certain steps of the secondary metabolic process, e.g., increasing the yield of certain desired compounds, or leading to new compounds (see below). Some A. tumefaciensstrains give rise to shooty teratomas. Such transgenic organ cultures will have similar biosynthetic capacity as the aerial parts of the plant. For example, tobacco shooty teratomas are not capable of nicotine biosynthesis, but can convert nicotine into nornicotine. Similarly, Atropa belladonna shooty teratomas could not produce hyoscyamine, but were capable of the storage of this alkaloid and could convert it into scopolamine (36).

E. ELICITATION An approach that has shown some interesting results for improving the production of secondary metabolites in plant cell cultures is elicitation. Elicitors are compounds which induce a defense response in the plant (37). This response involves the production of phytoalexins, low molecular weight compounds which are synthesized and accumulated by plants after microbial infection (38). In other words, these are plant secondary metabolites usually not found in a healthy plant, but of which the biosynthesis is induced after wounding. Knowledge of the signal transduction pathway(s) involved is still limited (see below). Besides signal molecules derived from microorganisms and plant cells (e.g., form the cell walls), such as peptides, oligosaccharides, glycopeptides, and lipophilic substances (39-42), as well as UV light, heavy metals (abiotic elicitors), and some compounds such as jasmonate, are capable of inducing phytoalexin biosynthesis (see below). Plant cell cultures are obtained from callus cultures growing on explants as a wound tissue, obviously such cells are an excellent model system for studying the effect of elicitors (42,43).Also, some alkaloid pathways are induced by elicitors. A well-described example illustrating induction by elicitation is the production of sanguinarine (Fig. 2) in cell cultures of Pupuver somniferum (44). Poppy cell cultures do not produce the morphinan type of isoquinoline alkaloids (see, for review, ( I ) ) . However, using a preparation of the

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FIG.2. Sanguinarine.

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phytopathogenic fungus Botrytis as an elicitor, sanguinarine, a benzophenanthridine type of isoquinoline alkaloid, is produced at levels of 2.9% of biomass dry weight 79 h after treatment. More than 50% of the alkaloid was recovered from the medium. The combination of the undiminished, high viability of the cells after treatment, and the excretion of the alkaloid into the medium, formed the basis for the design of an industrial process. A semicontinuous process, comprised of a repeating sequence of elicitation and medium replenishment, yielded 50, 125, and 200 mg total alkaloid per liter medium, as collected after the first, second, and third successive elicitation, respectively (45-47).Also, in Eschscholtzia californica, the production of sanguinarine is inducible by fungal elicitors (48-50). Sanguinarine might thus be considered a phytoalexin since its accumulation is inducible by biotic elicitors and it has strong antimicrobial properties (52,52). Further examples are berberine accumulation in Thalictrum rugosum cultures by a yeast-derived elicitor (54)and the accumulation of acridone and furanoquinoline alkaloids in Ruta graveolens cultures by a Rhodotorula homogenate (55). Although the terpenoid indole alkaloids of C. roseus are not regarded as phytoalexins, the formation of ajmalicine could be induced by elicitors such as vanadyl sulfate (56) and Pythium aphanidermatum (53). However, the induction was not very strong and depended largely on the cell line used (see below). Not all alkaloids are phytoalexins. In fact, only for a limited number of alkaloid pathways has an appropriate elicitor been found. In several studies using elicitors to improve alkaloid production, different, nonalkaloid pathways were found to be induced, e.g., the biosynthesis of 2,3-dihydroxybenzoicacid in C. roseus (57-59). In Tabernaemontana divaricata (60,61)the terpenoid pathway leading to triterpenoids is induced and alkaloid biosynthesis is inhibited. Cinchona robusta cell cultures react to elicitation with the production of anthraquinones, the biosynthesis of which involves both chorismate and IPP also in precursors, the Cinchona alkaloid biosynthesis (62,63). The addition of jasmonate produces similar effects as that of fungal elicitors. Jasmonate has been proposed to form part of the signal transduction pathway of the elicitor signal, and itself acts as a signal molecule (see below). Jasmonate was shown to induce the accumulation of a wide range of secondary metabolites when added to cell cultures of a number of unrelated plant species. For example, the addition of methyljasmonate to a culture of Rauvolfia canescens resulted in an almost 30-fold increase in the accumulation of the alkaloid raucaffricine (Fig. 3 ) (64). In Eschscholtzia californica cell cultures both jasmonate and a yeast elicitor result in the induction of sanguinarine biosynthesis; alkaloid levels in the treated cells

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OAc

FIG.3. Raucaffricine.

reached 265 mg/l, while the control had only 18 mg/l. After treatment with methyljasmonate the alkaloid content of C. roseus seedlings doubled (65), the induction of some of the enzymes in the alkaloid pathway differed from the reaction upon treatment with biotic elicitors (see also below) (66,67). Salicylic acid also plays a role in the plant’s response to infection (68-70). However, few data are available on the effects of salicylic acid on the accumulation of alkaloids in cell cultures. It was found that 8-24 h after addition of 0.1 mM salicylic acid to C. roseus cultures, the steady-state mRNA levels of the strictosidine synthase and tryptophan decarboxylase genes were weakly induced; no mention was made regarding the accumulation of alkaloids (71). The use of jasmonate may overcome one of the problems encountered with elicitors, namely, their specificity. For each plant cell culture the optimal elicitor has to be selected, often molecules derived from a pathogen of the plant studied are quite effective, whereas in general with yeast elicitor preparations, cellulase or pectinase, some induction of the phytoalexin pathways can be observed as well. To avoid such optimization studies and the problem of an undefined crude elicitor preparation which might have multiple effects, induction by jasmonate is an interesting approach for increasing the yields of alkaloids. To enhance the production of secondary metabolites in plant cell cultures, the use of elicitors is an empirical approach, that has been demonstrated to be effective for a limited group of alkaloids. In most cases, the production of the alkaloids concerned could also be increased to similar levels by means of medium manipulation, the major advantage of elicitation thus being the possibility of the exact timing of the production. Moreover, elicitors are valuable tools in studying regulatory mechanisms in plant secondary metabolism (e.g., 39-43).

F. BIOCONVERSION A completely different approach to enhancing alkaloid production is the use of plant cells, or enzymes therefrom, for certain distinct enzymatic

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reactions. Several successful bioconversions have been reported (for a review, see ref. (72)). In the case of single steps, cloning of the gene encoding the enzyme responsible for the bioconversion opens the way for using micro-organisms, or isolated (immobilized) enzymes for this purpose. Some successful examples on a laboratory scale of production using immobilized enzymes have been reported already, e.g., strictosidine synthase (73,74) and (S )-tetrahydroprotoberberine oxidase (75). cDNAs encoding strictosidine synthase have been cloned and heterologuously expressed in E. coli (76,77) and insect cells (78), thus enabling the production of larger amounts of this enzyme for further studies, or using the enzyme to produce strictosidine. Also, the cDNA encoding berberine bridge enzyme ((S)-reticuline :oxygen oxidoreductase, EC 1.5.3.9) (78,79) was expressed in insect cells with the baculovirus expression system; 4 mg/l of the active enzyme could be obtained in this system. The ongoing studies on the enzymes involved in the biosynthesis of alkaloids will certainly result in the isolation of further enzymes capable of interesting bioconversions, e.g., the isolation of stereospecific oxidases or reductases, such as the poppy codeine: NADP oxidoreductase or related enzymes which stereospecifically reduce codeinone and morphinone (80-84).

HI. Metabolic Engineering In the past years, methods for the introduction of new genes into plants have been developed and are now routinely used. Two methods have evolved as the most successful: biolistic transfer of naked DNA using the particle gun, and the transfer of T-DNA using Agrobucterium turnefuciens and A. rhizogenes. This opens the way for modifying metabolic pathways. We see the following perspectives for the engineering of secondary metabolism in plant cell cultures, as well as in plants: 0 0

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increase in the production of certain compounds; introduction of the pathway to a desired product in a heterologous system more suitable for cultivation; and production of completely new compounds (“recombinatorial biochemistry”).

These all require a knowledge of the pathway involved and the cloning of the necessary genes. It is not realistic at this point to transfer complex pathways to other plants, but only one or two steps in the pathway; converting precursors already available in the target plant to the desired product is, however, feasible.

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Presently, most applications concern the introduction of new traits encoded by single foreign genes in plants, aiming, in particular, at an increased resistance against pests and diseases. Examples are the introduction of insecticidal toxins, such as Bacillus thuringiensis toxins into the plant and increased resistance against diseases, e.g., by introduction of genes encoding viral coat proteins (for a review, see, e.g., (85,236)).Herbicide resistance is another trait achieved by genetic engineering (87). Pharmaceutically important proteins such as human serum albumin have also been produced in plants (e.g., (88)). The production of antibodies in plants is another interesting application (e.g., (89-92).This can either be as source of antibodies for, e.g., diagnostics, or serve the plant in its resistance. Vaccines may also be produced in plants (e.g., (91-94)). In addition, plant metabolic processes have been successfully modified, e.g., starch biosynthesis, flower color formation, and fruit ripening (for reviews, see e.g., (91,95-97). This clearly shows that the time has come to consider also the possibilities of improving the production of economically important secondary metabolites, such as pharmaceuticals, flavors, and fragrances (98-100). Thus, metabolic engineering also offers interesting perspectives for the production of alkaloids in plants or in plant cell cultures. Metabolic engineering requires a knowledge of the individual steps in the pathway and their regulation. In addition, biosynthetic genes, promoter sequences for the desired spatio-temporal expression, targeting signals to direct proteins to their cellular destination, and transformation and selection methods are needed to be able to apply molecular genetic methods. In the following paragraphs, we will briefly outline how molecular genetic techniques can assist in various steps in the procedure leading to metabolic engineering.

A. MOLECULAR GENETIC METHODS Identification of Biosynthetic Steps in a Pathway

Instead of a biochemical approach to characterize a biosynthetic pathway step by step at the level of intermediates, enzymes, and genes, a genetic approach, that may include molecular techniques to identify the genes involved can be followed. Using appropriate screening methods, mutants can be selected, that show modifications in the amounts of certain secondary metabolites. Mutations can be introduced chemically, or by insertion of a transposon or T-DNA element. Further biochemical analysis of mutants can yield information about intermediates and enzymatic steps in a biosynthetic pathway. Such mutant screens using Arubidopsis thaliana have, for example, resulted in the identification of steps in the tryptophan biosynthetic pathway (101). Alterations in anthocyanin composition are easily scored by changes

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in flower color and mutants in the anthocyanin biosynthetic pathway have been isolated for a number of plant species (95,96). A large number of Arabidopsis mutants in lipid metabolism have been isolated and have shown to be very useful in studying the regulation of lipid biosynthesis (102). Isolation of Biosynthetic Genes

After mutants have been characterized, the mutated genes can be cloned, either by map-based cloning for plant species for which genetic maps exist, or using DNA sequence tags for mutants obtained by transposon or TDNA insertion. Genes that are evolutionary conserved in other organisms can be cloned by screening with heterologous DNA probes using hybridization or polymerase chain reaction (PCR) approaches. Alternatively, conserved genes can be cloned in Escherichia coli or yeast cells by complementation of the corresponding mutant. Micro-organisms can also be employed to clone genes by selection for a new phenotype, such as resistance against toxic pathway intermediates. In addition, the yeast two-hybrid protein-protein interaction system (for a review, see (103)) allows cloning of proteins that are suspected or known to act in a complex with an already available protein. In the case of unique enzymatic steps, as often occurs in secondary metabolite biosynthetic pathways, a straightforward approach is to purify the enzyme concerned. Subsequently, the corresponding gene can be cloned by screening a cDNA expression library with antibodies, or using a DNA hybridization or PCR approach if protein sequence information is available. Genes for biosynthetic enzymes that are only found in certain developmental stages or under certain environmental conditions can be isolated by differential screening of cDNA libraries, or using differential display PCR or RNA fingerprinting. (0ver)Expression of (Modified) Genes

To modify plant metabolism, genes from other organisms or plant species encoding the desired enzyme activity can be expressed. If the homologous plant gene is available, it can be constitutively expressed in the corresponding plant species to increase the amount of a rate-limiting enzyme. Increased levels of gene or enzyme activity can also be achieved by mutagenesis, either chemically or by transposon or T-DNA insertion. A number of examples from plant metabolism are discussed elsewhere in this chapter. A very illustrative example of the power of genetic engineering is provided by the studies on carbohydrate metabolism by Willmitzer, Sonnewald and co-workers (for a review, see (104)).These studies made elegant use of sense overexpression or antisense suppression of genes from plants or micro-organisms, and show the importance of using tissue-specific pro-

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moters to express genes in the correct tissues and using targeting signals to direct proteins to the correct cellular compartments. Knocking Out Expression of Genes in Branching Pathways

The expression of genes in undesired branch pathways that compete for common precursors can be reduced by a variety of methods. A very successful method for plants to reduce gene expression is the use of cosuppression (205)or antisense technology. A reduction in lignin content has been obtained by the expression of an antisense caffeic acid O-methyltransferase gene (106).A very succesful method in yeast and animals uses homologous recombination to knock out the expression of a gene. For plants, this method needs considerable further improvement to become a standard technique (107),but it has been shown that homologous recombination between introduced DNA and an endogenous plant gene is possible (108). Genes can be knocked out by mutagenesis, either chemically or using T-DNA or transposon insertions, if an appropriate selection procedure is available. For a Petunia line containing a high copy-number transposon, a general method was described to select plants with a transposon in a gene for which DNA sequence information exists (209).Biosynthetic steps can also be blocked after transcription of the gene, either by degradation of specific mRNAs using ribozymes (ZZO),or by inhibiting enzymatic activity by expressing a gene encoding enzyme-specific antibodies (222,222).In addition, undesired metabolites can be sequestered by expressing antibodies, as described for abscisic acid (123). Determination of Rate-Determining Steps in a Biosynthetic Pathway

To determine whether an enzymatic step is rate-limiting in a biosynthetic pathway, the expression of the corresponding gene can either be knocked out or increased, and effects on the metabolic flux can be determined in the resulting transgenic plants or cell lines. In this way, the role of phenylalanine ammonia lyase (PAL) in phenylpropanoid metabolism in tobacco was studied via reduction of the level of PAL using antisense expression (214). Hamster HMG-CoA reductase (HMGR) (225)and rubber tree HMGR (216)were overexpressed in tobacco to study the effect on isoprenoid biosynthesis. Determination of Unknown Gene Function

When a gene of unknown function has been cloned, which is suspected to participate in a certain metabolic pathway, its expression can be modified to study the effect on the accumulation of intermediates and endproducts in that pathway. For example, antisense expression of the ripening-related gene pTOM5 established its role in carotenoid biosynthesis (127).

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Transformation

The possibility to apply molecular genetic techniques depends largely on the ability to transform and regenerate the plant species of interest. In principle, every plant species can be efficiently transformed using the particle gun. Transformation using Agrobacteriurn tumefaciens is limited by the host range of the bacterium and strains are being improved to expand the host range. Some plant species, on the other hand, are extremely difficult to regenerate. A distinction should be made between the dominant or recessive effects of genetic engineering techniques. Gene knock-outs by mutation or homologous recombination are generally recessive, and a phenotype will only be observed in a plant that is homozygous for the introduced change. In contrast, antisense or overexpression techniques generally result in dominant phenotypes. This distinction is important, because for plants that are difficult to regenerate or have a long generation time, it can be an advantage to be able to score phenotypes in the primary transformants. Generation and analysis of mutants is facilitated by a short generation time, and the ability to grow and analyze large numbers of plants. For gene isolation, genetic maps can be helpful. Most efforts aimed at metabolic engineering have been directed at altering gene transcription and/or protein targeting. Metabolic fluxes could also be altered by increasing mRNA or protein stability, or by creating more active enzymes. This is much more difficult, because there is no clear-cut method to obtain the desired result. In addition, extensive knowledge about protein structure and activity is required.

B. STRATEGIES TO IMPROVE PRODUCTION In general, the following strategies can be thought of to increase alkaloid production (or any other secondary metabolite) using molecular genetic methods: 0 0 0 0

increase the flux through the pathway to the desired product; decrease the catabolism of the desired product; increase the percentage of producing cells; and random mutation/selection approach.

These approaches will be discussed below in more detail. Increase the Flux Through the Pathway

First of all, we can try to increase the total flow in the pathway toward the desired product. This approach requires a knowledge of the flux limiting

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Transport Induption

..... t Feedback inhibition

..... FIG.4. Schematic biosynthetic pathway.

step(s). Once these are known, the possibilities to overcome these steps need to be assessed. The cause of a limitation in the flux could be (Fig. 4): 0 0

0

relatively low, or no, activity of an enzyme involved; feedback inhibition of certain enzymes in the pathway by an intermediate or by the endproduct; and by competition with other pathways for certain intermediates.

Rate-Limiting Enzyme. To increase the activity of the rate-limiting enzyme, the amount of that protein in the plant can be increased, or the specific activity of the enzyme can be altered via protein engineering. To increase the enzyme amount, we can use the gene from the plant itself in combination with a strong promoter, or a gene from another plant or organism encoding an enzyme with a similar function. Several examples of this approach have been reported in the past years (see the following). An increased stability of the enzyme might be a further aim for protein engineering, in cases where the low activity of an enzyme is due to rapid turnover. Feedback Inhibition. When the plant enzyme is inhibited by an intermediate or an endproduct of the pathway, we have to find an enzyme from another source which is not sensitive to feedback inhibition. Alternatively, such an enzyme could be engineered based on the knowledge of the site involved in the interaction with the inhibitor. For anthranilate synthase, which is inhibited by tryptophan (for reviews, see (228-120)), tryptophan resistant isoenzymes have been found, among others, in Nicotiuna tubucum (221) and Solunum ruberosum cell cultures after selection for resistance to 5-methyltryptophan (222). Mutant Arubidopsis plants containing tryptophan-feedback resistant anthranilate synthase have also been reported (220,223).

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Competitive Pathways. If biosynthetic pathways compete for a common precursor, the nature of the mechanism controlling the flux into the competitive pathways must be established. Is competition due to the relative amounts of the competing enzymes, or to the relative affinities of the enzymes for the substrate? In the first case, the amount of protein can be increased. For the second possibility, the protein engineering approach holds more promise. Recently, catalytic antibodies (abzymes) have been reported. These antibodies bind unstable intermediates and thus catalyze steps in biosynthetic routes (124). Such a catalytic antibody was raised against the first intermediate in the conversion of chorismate into prephenate and the gene for this antibody was shown to be able to complement a yeast mutant blocked in chorismate mutase activity. A totally different approach is the suppression or even blocking of the competitive pathway by the introduction of an antisense gene(s) for the enzyme(s) competing for the same substrate. As mentioned above, expression of genes encoding ribozymes (220) or antibodies against the target enzyme (111,112) could be another approach to cut off competitive pathways. Decrease the Catabolism

Several studies have shown that metabolites thought to be endproducts are actually catabolized; for example, ajmalicine in C. roseus cell cultures is catabolized at almost the same rate as the biosynthetic rate at the end of the growth cycle (225). Similar results were found for the alkaloids in Tabernaemontana cell cultures (126-129). Catabolism can be due to chemical instability, or to enzymatic degradation. In the latter case, the enzymes involved in catabolism have to be identified, and subsequently the genes need to be cloned to enable reduction of their activity by the antisense gene approach discussed above. Alternatively the enzyme(s) can be blocked by means of the expression of antibodies (111,222). Increase the Percentage of Producing Cells

In plants, the different tissues typically produce different secondary metabolites, i.e., only a small part of the total biomass is involved in the production of certain secondary metabolites. Also, in cell cultures, there are examples known in which not all cells produce the desired product. For C. roseus cell cultures, it was found that the production of anthocyanidins is determined by the percentage of producing cells (230,232). Stafford et al. (232) reported that in C. roseus cell cultures less than 50% of the cells accumulated alkaloids. Also in the case of berberine it was found that the level of production in Coptis japonica cell cultures was correlated with the

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number of accumulating cells, the berberine content in an accumulating cell being similar in low- and high-producing strains (233-235). If we would be able, by genetic modification, to increase the percentage of producing cells, the total yield of the desired product would increase. Unfortunately, very little is known about the processes which make a cell produce a secondary metabolite, i.e., differentiate in a certain direction. Even the possibility that the production is dependent on the occurrence of different types of cells, each doing a discrete part of the biosynthetic process, cannot be excluded.

Random MutatiodSelection Approach Another way to improve metabolite yield is to select the plants or cell lines with the desired properties from a population of mutants. This strategy requires little or no prior knowledge about rate-limiting steps in the pathway of interest, but depends on the availability of an appropriate screening or selection method. Mutations can be introduced chemically, or by the insertion of a transposon or T-DNA. This approach was taken to increase the amount of tryptophan in Arabidopsis (220).A generally applicable method for mutagenesis is activation tagging using T-DNA (136).This method has resulted in increased levels of polyamines in tobacco (237).

C. RESULTS The feasibility of engineering secondary metabolism in plants was first visualized by modifying flower color. A white flowering Petunia was transformed with a gene encoding dihydroflavonol reductase; the concomittant channeling of anthocyanidin biosynthesis in the direction of the red-colored pelargonidin glycosides caused the flowers to become red (138).Following this result, numerous examples of the modification of flower color have been reported (for reviews, see (95,96,239)).The use of antisense genes was also shown to be useful for flower color modification; an antisense gene encoding chalcone synthase was used to modify Petunia flower color (95,240-243). In recent years, the first results of efforts to modify alkaloid production have been reported. Here we will review these results.

Terpenoid Indole Alkaloids Terpenoid indole alkaloids have one intermediate in common, strictosidine (Fig. 5). This glyco-alkaloid is formed through the stereospecific condensation of tryptamine and secologanin, catalyzed by the enzyme strictosidine synthase (Str). Secologanin is an iridoid formed in a number of steps from geraniol, which is first hydroxylated by the enzyme geraniol

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CHzOH

Tryptophan

44 1

H Tryptophan decarboxylase

V

N H2

Geraniol- 10- hydroxylase

Tryptomine

Secologonin

H

Strictosidine synthase

**’

0-Glucose

Strictosidine

11 Glucosidase 1 ca. 3000 lndole alkaloids, e.g.,:

FIG.5. Early steps in terpenoid indole alkaloid biosynthesis.

10-hydroxylase (GlOH). Tryptamine is formed from tryptophan by the enzyme tryptophan decarboxylase (TDC). These three enzymes have extensively been studied by several groups (for reviews, see (2,244-246). The gene encoding strictosidine synthase was first cloned from Rauvof!a serpentina (247). McKnight and co-workers (248) used part of this sequence to clone the cDNA from C. roseus. The cDNA encoding tryptophan decarboxylase was first described by De Luca et af. (249).

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Independently within our Biotechnological Sciences Delft Leiden (BSDL)-working group, assays were developed for all three enzymes and the enzymes were subsequently purified from C. roseus. Antibodies were then raised against the purified TDC and Str, and some amino acid sequences were obtained, which led to the cloning of the genes (71,150,151). For GlOH, this approach has so far not been successful, because a number of other closely related cytochrome P-450 enzymes occur in the plant. Both the cytochrome P-450 protein and the NADPH-cytochrome P-450 reductase were purified (152). But only for the latter protein has the gene been cloned (see following) (253). With the Tdc- and Str-genes a series of experiments have been performed in several laboratories. The introduction of the Tdc-gene driven by the strong CaMV35S promoter into C. roseus cells, using Agrobacterium tumefaciens, resulted in a clear increase of tryptamine levels (154). However, the indole alkaloid production was not significantly affected. Probably the availability of secologanin is a rate limiting step, other experiments have produced similar results (155). An antisense Tdc-gene introduced in C. roseus cells blocked the alkaloid biosynthesis (154). The introduction of the Tdc-gene driven by the cauliflower mosaic virus (CaMV) 35s promoter into tobacco plants gave rise to the production of about 1%of tryptamine (156,257). The activity of anthranilate synthase, the first enzyme in the tryptophan pathway, did not show any increase in these transgenic tobacco plants (157). These plants could apparently make 1% of their dry weight of a new compound, without affecting their normal growth and metabolism. Since plants that produce indole alkaloids accumulate about 1%of the total biomass in the form of alkaloids, a separate tryptophan pathway is probably not required for alkaloid biosynthesis. Interestingly, the transgenic tobacco plants caused a 97% decrease in the reproduction of whitefly feeding on these plants. As whitefly is a major pest for tobacco, this finding may be exploited as a possibility to protect plants against such pests (158). The Tdc-gene has also been used to lower the production of tryptophanderived glucosinolates in canola (Brassica napus). The level of these compounds, which limit the use of canola as animal feed, was reduced to 3% of that of the control (159). The Tdc-gene driven by the CaMV 35s promoter, has also been expressed in potato. The level of expression in potato, as well as in canola, was lower than in tobacco. The level of TDC activity found in a series of transgenic tobacco plants was 3- to 10-fold higher than in series of transformed potato and canola plants, and the tryptamine levels were 12- to 50-fold higher. Different plant species may thus react differently to the introduction of transgenes (160). In potato tubers, the introduction of the Tdc-gene resulted in a 40% reduction in the level of tryptophan and in the accumulation of tryptamine

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Metabolic interlock

lsochorismate

a

ICS Chorismate

Prephenate

. 1 I I I

\ . \

Phenylalanineltyrosine

1\ I ‘I

\

Anthranilate

Q

Tryptophan

FIG.6. Regulation of plastidial biosynthesis of aromatic amino acids.

(about 10 pg/g). Elicitation resulted in an even further decrease of the tryptophan pool to only 6% of the control. Also, the phenylalanine pool was decreased to about 50% of the control. This might be due to the reduced tryptophan level, as tryptophan induces chorismate mutase, the first enzyme in the phenylalanine pathway (Fig. 6). The levels of lignin and phenolic compounds, such as chlorogenic acid in the transgenic tubers were much lower. These tubers were found to be more susceptible to fungal infections (262). The Tdc-cDNA behind the CaMV 35s promoter was also constitutively expressed in Peganum harmala hairy roots and cells. With the Agrobacterium turnefaciens strain LBA4404 cell suspensions were obtained, whereas the strain C58CI pRi44 gave rise to root cultures. Considerable increase of TDC activity was found in the transgenic cell lines, but tryptamine levels were similar to those of controls, whereas an up to 10-fold increase of serotonin levels was observed. Apparently, tryptamine is rapidly converted into serotonin. However, the production of the desired harman-type of alkaloids was not increased (Fig. 7). As serotonin levels were always about 2% of the dry weight of the cells in the various transgenic strains, and independent of the TDC activity, a limitation in the tryptophan supply was hypothesized. Indeed, feeding of tryptophan resulted in a significant increase of the serotonin levels in cell lines with high TDC activity. This shows that by overcoming one limiting step, new steps can become rate limiting for the total flow through the pathway. In this particular example, even an excess of the necessary intermediate does not lead to the production of the desired harman-type of alkaloids, instead a competitive pathway converts the intermediate into another product (22-27).

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TDC

473

Serotonin

--I>

H

H L-tryptophan

Tryptamine

Harmalol

FIG.7. Alkaloid biosynthesis in transgenic Peganurn cell cultures.

The C. roseus Tdc-gene has also been patented as a selection marker for transformation; TDC is capable of detoxifying 4-methyl-tryptophan and transformed cells will thus survive in a medium containing this toxic tryptophan analog (262). The Str-gene from RauvolJa has been expressed in E. coli and insect cells (76-78). In both systems high levels of the active enzyme were produced. Enzymes produced in this way can be immobilized and used for the production of strictosidine from tryptamine and secologanin (73,74).The C. roseus enzyme has also been expressed in E. coli (77) and in tobacco (163).The enzyme was found to be active and was stored in the vacuole of the transgenic tobacco plants. The total activity found was 3-22 times higher than in C. roseus. Introduction of the Sfr-gene into C. roseus cell cultures and Tabernaemontana pandacaqui plants, as expected, did not result in an increased alkaloid production, despite a 3- to 20-fold increase of enzyme activity as compared with controls. This is probably due to the limited availability of secologanin (M. I. Lopes Cardoso (264) BSDL, unpublished results). Recently, we have introduced two genes encoding two consecutive steps of terpenoid indole alkaloid biosynthesis into various plants. With the A . tumefuciens binary vector system, the Tdc- and Str-genes driven by the CaMV 35s promoter were introduced together into tobacco. Upon feeding secologanin to cell cultures of these plants, strictosidine is formed (165). In contrast to the situation in C. roseus where strictosidine is stored in the vacuole, in this instance the alkaloid is excreted into the medium of a suspension culture of the transgenic tobacco cells. Also, A. rhizogenes has been used to transform plants with the Tdc- and Str-genes. Hairy roots of Cinchona ledgeriana, among other species, were obtained, using an A. rhizogenes harboring both the Tdc- and Str-genes

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(266). The C. ledgeriana cultures contained high levels of tryptamine and strictosidine. As the strictosidine synthase of this cell line did not accept 10-methoxytryptamine as a substrate, the Str activity is probably due to the expression of the C. roseus Str-gene, since the enzyme from the latter plant only accepts tryptamine, whereas the Cinchona enzyme also accepts the corresponding methoxy derivative (166,167). The level of quinoline alkaloids in these cultures were similar to those previously reported for a Cinchona ledgeriana hairy root culture (268),pointing to a limitation of the secologanin, as also was found in the transgenic C. roseus callus cultures containing the Tdc-gene (254). Cytochrome P-450 enzymes play a major role in terpenoid biosynthesis. The hydroxylation of geraniol, a crucial step in the biosynthesis of terpenoid indole alkaloids, is catalyzed by a cytochrome P-450 enzyme (252,269-272). The cytochrome P-450 enzymes form a complex with a NADPH :cytochrome P-450 reductase (EC 1.6.2.4), which is involved in the electron transfer from NADPH to the P-450 heme group. The reductase coupled with GlOH was first purified by Madyastha and Coscia (173). Meijer et al. (153) were the first to clone the gene encoding the reductase from a plant. They could detect only one gene encoding this enzyme in C. roseus. Expression of the cDNA in E. coli resulted in a functional protein. It has clear homology with reductases from other organisms. Expression of the cDNA in tobacco plants or C. roseus cell cultures did not lead to an increased activity of the enzymes, and an antisense gene also did not affect activity also (164). Thus considerable progress has been made in unraveling the biosynthesis of terpenoid indole alkaloids. First results of metabolic engineering show that apparently the terpenoid-iridoid pathway is a limiting factor in alkaloid biosynthesis. Further studies on the regulation may lead to the cloning of regulatory genes, controlling at least part of the pathway. This will eventually open the way for manipulating these genes to improve the production of terpenoid indole alkaloids. Isoquinoline Alkaloids In the biosynthesis of isoquinoline alkaloids tyrosine- and dopa-decarboxylase (TyDC, DoDC, EC 4.1.1.25) are at the beginning of the pathway (274). In contrast to indole alkaloid biosynthesis, where only one Tdc-gene is present, in plants producing isoquinoline alkaloids several genes encoding decarboxylases are present (175-178). Using PCR, Facchini and De Luca (175) picked up a DNA fragment in poppy (Papaver somniferum) seedlings with degenerate primers for conserved areas of aromatic amino acid decarboxylases (TDC from C. roseus and DoDC from fruit fly and mammalians). With this fragment, a TyDC cDNA was isolated from a cDNA library. By heterologous screening with the tryptophan decarboxylase probe two further TyDC cDNAs were picked up. A fourth TyDC4-gene as well as

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the TyDCZ-gene were obtained from a genomic library through screening with the TyDCZ cDNA. By genomic DNA blotting, six to eight genes homologous to the TyDCZ and four to six genes homologous to the TyDC2 cDNAs were detected (175). Within these subsets a homology of more than 90% exists on the nucleotide level; between the subsets there is somewhat less than 75% homology. Both types have been expressed in bacteria (176) and shown to be able to decarboxylate both tyrosine and dopamine, with a higher activity for dopamine. TyDCS, which is slightly different from both the TyDCl and TyDC2 subfamilies (respectively, 86% and 75% homology at the amino acid level), has a higher activity for tyrosine (177). t-Phenylalanine and L-tryptophan were not accepted as substrates. The various TyDC genes showed different patterns of expression in the plant, and the occurrence of alkaloids in the various tissues suggests different roles of these genes for the various alkaloid biosynthetic pathways (178). TyDCZ-genes were thought to be connected with sanguinarine biosynthesis and the TyDC2 genes with morphinan alkaloid biosynthesis (178). For several isoquinoline alkaloids, such as the morphinans, berberine and sanguinarine, the pathways have been elucidated and the enzymes involved have been identified and characterized (99,Z79-185). Moreover, the regulation of the berberine and sanguinarine pathways has been studied extensively. For some of the steps of these pathways the genes have been cloned, two of which encode enzymes involved in the oxidative phenol coupling, an important mechanism in isoquinoline alkaloid biosynthesis, that is responsible for the formation of the various basic skeletons of isoquinoline alkaloids, Both are cytochrome P-450enzymes. These enzymes are berbamunine synthase (EC 1.1.3.34) involved in bisbenzylisoquinoline formation in Berberis srolonifera (Fig. 8) (186-188), and salutaridine synthase catalyzing the formation of the morphinan skeleton from (R)-reticuline (Fig. 9)

R = H U H = (S)-coclaurine R = H p H = (R)-coclaurine R = CH3, uH = (S)-N-methylcoclaurine R I CH3, p H = (R)-N-methylcoclaurine

R = H (I H = 2'-norberbamunine R = CH3. p H = guattegaumerine R = CH3. (I H = berbamunine

FIG.8. Formation of the bisbenzylisoquinoline alkaloid berbamunine cytochrome P-450 enzyme.

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CH3O HO

OH OCH3 R-reticuline

c

Salutaridine

FIG.9. Formation of morphinan-skeleton catalyzed by a cytochrome P-450 enzyme

(286,289).The enzyme (S)-tetrahydroberberine oxidase, catalyzing the last step in the biosynthesis of berberine, is also a cytochrome P-450 enzyme (Fig. 10) (290). The gene encoding (S)-tetrahydroberberine oxidase was cloned from Coprisjuponicu and expressed in E. coli. It had clear homology with mammalian cytochrome P-450 enzymes. Although a protein was ob-

RO

--D

OR

OR

OR

OR

OR

OR

OR

OR

RO

FIG.10. Reactions catalyzed by the cytochrome P-450 enzyme (S)-tetrahydroberberine oxidase.

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tained which reacted with antibodies against the enzyme, no activity could be measured in E. coli. Similarly, Schroeder and co-workers were not able to detect enzyme activity for a cytochrome P-450 cDNA expressed in E. coli, tobacco, or Arabidopsis which was thought to encode geraniol-10hydroxylase (292,292). The berbamunine synthase cDNA was cloned from Berberis sfofonifera and overexpressed in an active form with the aid of a baculovirus based expression system in insect cells (288).By means of a two-step purification procedure the enzyme was obtained in pure form; the insect cell system proved efficient, as 5 mg of the pure enzyme was obtained from about 1 liter of cultured cells. The production of this enzyme offers an opportunity for the semisynthetic production of bisbenzylisoquinoline alkaloids. The cDNA encoding berberine bridge enzyme ((S)-reticuline : oxygen oxidoreductase (EC 1.5.3.9), which catalyzes the reaction from ( S ) reticuline to (S)-scoulerine (Fig. 11) has been cloned from Eschscholtzia californica (78,79).The plant seemed to have a single gene encoding this enzyme. Upon elicitation, its transcription was rapidly and transiently induced. The gene was expressed in yeast and in insect cells using the baculovirus system, both resulting in an active enzyme. Although several pathways leading to different types of isoquinoline alkaloids have been completely elucidated, including the identification of all the enzymes involved, our knowledge about the regulation of these pathways is still limited. Several of the isolated enzymes offer interesting perspectives for application in bioconversions. Tobacco Alkaloids

Amino acid decarboxylases catalyze the first committed step in a number of alkaloid pathways; consequently, several attempts have been made to increase the activities of these enzymes by means of genetic engineering using microbial genes.

(S)-reticuline

(S)-scoulerine

FIG. 11. Berberine bridge enzyme catalyzes the reaction leading to the protoberberine skeleton.

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Hamill et al. (193,294) expressed ornithine decarboxylase (ODC) from yeast in hairy roots of Nicofiana rustica with the aim of increasing nicotine levels (Fig. 12). Indeed, higher levels of putrescine, the product of decarboxylation of ornithine, and nicotine were found in these cultures. The nicotine levels were increased twofold if compared with control cultures, suggesting that further factors are involved in regulating the flux through the pathway. Also, a mammalian ornithine decarboxylase ( O D C ) cDNA (from mouse) has been expressed into tobacco, resulting in a 4-12 times increased level of putrescine in callus cultures, and a 2- to 4-fold increase of this compound in leaves of the transgenic tobacco plants (195). Berlin and co-workers (196)cloned a gene encoding lysine decarboxylase (LDC) from Hafkia alvei and expressed it in tobacco leaves using a rbcS

n Lysine

HOOC

Ornithine

J z

NH2 NH2

HOOC

t

t

.

Putrescine

<<

C'I

NH2 NH2

NH2 NH2

t N-methylputrescine

t H

NH2 NH2

NH2 NHCH3

0

v n I

NH2

,A L

t

0 N

Nicotinic acid CHq

Anabasine

Nicotine

FIG.12. Biosynthesis of tobacco alkaloids.

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promoter and the rbcS transit peptide sequence from potato (26). Indeed cadaverine was formed in the chloroplasts, the site of lysine biosynthesis. However, no increased anabasine production was observed, as the alkaloid biosynthesis occurs in the roots. Using vectors with the CaMV35S promoter and the rbcS transit peptide sequence, some LDC activity could be obtained in root cultures of Nicotiana glauca. Although the LDC activity was very low, about 100-fold lower than in the leaves of the transgenic tobacco plants, the levels of the product of lysine decarboxylation, cadaverine, were increased 10-fold, whereas anabasine levels were about doubled. The use of a vector containing a double 35s-ldc expression cassette did not result in higher LDC activity levels. Also, in N. tabacum root cultures, best results were obtained when the Zdc-gene was combined with the rbcS transit peptide, targeting the enzyme to leucoplasts. These cell lines showed 2-10 times higher levels of LDC activity if compared with transgenic root cultures in which no signal peptide was present in the vector. In the cell line with the highest LDC activity, a considerable increase in cadaverine and anabasine levels was observed. Moreover, the ratio of nicotine : anabasine had changed to 4 : 1, compared to 75 : 1 for the control cultures. Feeding lysine to this root culture resulted in about a doubling of cadaverine and anabasine levels, whereas feeding of cadaverine did not lead to a higher level of this alkaloid if compared to lysine feeding. The availability of lysine might thus be a limiting factor. Also, in plants regenerated from the transgenic tobacco roots, increased cadaverine and anabasine levels were observed. Particularly in leaves, cadaverine levels were high, about 40-fold higher than in a control plant; in stems, about 20-fold higher levels were found. Anabasine in stem and leaves were about four-fold higher than in control plants (23-25). These studies once more show the importance of the correct targeting of an enzyme to the site of alkaloid biosynthesis. Moreover, it shows that overexpression of one single enzyme will reveal the next limiting step, in this case, the availability of lysine. Hibi et aZ. (297) identified two nuclear genes which regulate nicotine levels in tobacco. These genes reduce the level of several enzymes of the nicotine pathway and increase the polyamine levels in cultured roots.

Tropane Alkaloids Tropane alkaloids are important pharmaceutical compounds, consequently their biosynthesis has been studied extensively, and a number of steps in a the pathway are now well established (for reviews, see, e.g., (284,298)). Considering metabolic engineering for their production, a quite interesting and successful approach was followed by Hashimoto and Yamada (299). They identified the enzyme responsible for the conversion of I-hyoscyamine

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into the more valuable scopolamine. This enzyme, hyoscyamine 60hydroxylase (H6H, EC 1.14.11.11), is a 2-oxoglutarate-dependentdioxygenase, which first catalyzes the conversion of 1-hyoscyamine into the 6-hydroxy derivative, and then converts this intermediate into scopolamine, which has an epoxide function (Fig. 13) (200,202). Subsequently, the cDNA clone encoding H6H was obtained from Hyoscyarnus niger (202). The H6H cDNA was combined with the cauliflower mosaic virus (CaMV) 3.5s promoter introduced into tobacco plants with the aid of A. turnefaciens and A. rhizogenes (203). The transgenic plants had H6H activity, and upon feeding 1-hyoscyamine or its 6-hydroxy derivative to these plants, scopolamine could be detected in the leaves. In Atropa belladonna the H6H harboring construct was introduced (204,205). Plants and hairy roots were both obtained. The H6H activity levels increased about five-fold in the hairy roots, if compared with control hairy roots obtained by transformation with a wild type A. rhizogenes, and the scopolamine levels were two to five times higher. In the control plants, I-hyoscyamine represents 90% of the alkaloid content; in the transgenic plants scopolamine was almost the sole alkaloid in the aerial parts and the main roots, occurring at levels similar as those of total alkaloid in the control plants. However, in the root branches of the transgenic plants, I-hyoscyamine, 6-hydroxy-hyoscyamine and scopolamine were present in about equal amounts. This confirms earlier findings that scopolamine is formed upon translocation of hyoscyamine from its site of biosynthesis in the roots to the aerial parts, i.e., two different types of cells are involved in the biosynthesis of scopolamine. In the biosynthetic pathway of f-hyoscyamine there is a major branching point at the intermediate tropinone (Fig. 14). Two different tropinone reductases compete for the same precursor (206). One reductase (TR-I,

p

3

NBH

CH3

H

I -Hyoscyamtne

0 -3

0

1‘

0 CHzOH

6p -Hydrox yhyosc yam1ne

Scopolamine

I Hyoscyomine 6p-hydroxylase

II

6~-Hydroxyhyascyom1ne epoxldose

FIG.13. Biosynthesis of scopolamine from I-hyoscyamine.

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Tropinone R,=H. R2=OHTropine

R1= OH Rp =H Pseudolropine

FIG.14. Reduction of tropinone.

EC 1.1.1.206) yields tropine (3cr-hydroxy), the precursor for, e.g., 1hyoscyamine, the other (TR-11, EC 1.1.1.236) yields pseudo-tropine (3phydroxy), the precursor for, e.g., the calystegins. From Darura srramonium the cDNAs for both reductases were cloned and expressed in E. coli. The recombinant proteins showed the same strict stereochemistry for the products of the reduction. The two genes have 64% homology at the amino acid level. The genes could also be detected in some other tropane alkaloid producing plants. These proteins have clear homology to short-chain dehydrogenases. By making a series of chimeric enzymes, it was found that the stereochemistry is ruled by the orientation of tropinone at the binding site. Proteins could be constructed that had both activities (207). Because of the close similarity of the two genes, it might be difficult to block just one of these by means of an antisense gene. The results obtained with scopolamine production, clearly demonstrated the feasibility of secondary pathway engineering. For commercial applications it seems that the final steps in biosynthetic routes, resulting in highvalued products are interesting targets. D. ISSUESTO

BE

RESOLVED

The results as discussed above show that basic studies on the biosynthesis of secondary metabolites may lead to a broad range of applications, some of which are not directly related to the pathway studied. Moreover, genetic engineering allows the production of compounds not normally produced in the target plant, which might even be novel compounds. However, despite the first promising results, these studies have also brought up some issues that need to be solved eventually in order to develop the metabolic engineering of secondary metabolism to its full potential. Cloning the Genes Involved in Secondary Metabolism

Metabolic engineering requires the availability of genes from secondary metabolism. A number of relatively rapid methods have been developed in the past years to clone a certain desired gene, e.g., transposon tagging,

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complementation of mutant microorganisms, and differential screening (see preceding text). However, for secondary metabolism these approaches have not been very successful, partly due to the complexity of the secondary metabolite pathways, which consist of a large number of steps, and thus genes. Extensive series of mutants are only available for very few plant species and pathways. Moreover, in many cases, our knowledge about the secondary metabolite pathways is very limited. Only a few secondary metabolite pathways are known at the level of (some of) the enzymes involved, e.g., the tropane alkaloids (94,99),indole alkaloids from C. roseus (99,246,283)and Rauvolfia (209),morphinan alkaloids, protoberberine alkaloids, and benzophenanthridine alkaloids (99J 79-285). Only the pathway leading to flavonoids has extensively been studied at the level of the genes, and metabolic engineering has been applied to this pathway, with the aim of modifying flower colors (95,96,239),or to better understand the plant’s response to various forms of stress (220). Since the secondary metabolite pathways are not well known, the use of probes based on genes from other organisms or plants is also not a feasible way to clone genes. Consequently, for the cloning of secondary metabolite biosynthetic genes, researchers have mainly followed the elaborate approach from product, via enzyme, to the gene. After purification of the enzyme to homogeneity, antibodies are raised against the enzyme, and the protein is partly sequenced for amino acid composition. The antibodies can be used to screen a cDNA expression library of the plant. The amino acid sequence can then be used to make probes which can be used to screen genomic or cDNA libraries for that specific sequence. These sequences can also be used for a PCR-approach to clone the desired gene. The most important constraint in this approach is that many of the target enzymes only occur at low levels, requiring extensive purification, which, in combination with the instability of proteins, often results in low yields of the protein. Antibodies obtained against plant enzymes may lack specificity because of the common occurrence of glycosylation. Also, the presence of closelyrelated proteins constitutes a problem in gene cloning strategies based on the use of antibodies. The latter we experienced, for example, in our efforts to clone the gene for geraniol-10-hydroxylase (GlOH), a cytochrome P-450 dependent enzyme, which catalyzes the first step in the biosynthesis of secologanin, one of the precursors of strictosidine (Fig. 5). A plant contains a large number of cytochrome P-450 enzymes, and thus a correspondingly large number of P-450 genes. Polyclonal antibodies raised against purified GlOH did not lead to the isolation of a clone encoding GlOH. Using a PCR approach, in which degenerate primers were used corresponding to conserved areas of cytochrome P-450 genes, 16 highly similar genes were detected. We did not attempt to connect any of these with GlOH activity

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(222).Differential screening using an induced and noninduced cell culture of C. roseus resulted in the isolation of a cDNA encoding a cytochrome P-450 enzyme, but no GlOH activity could be confirmed for the protein expressed by this cDNA in various organisms (292,293).Probably, internal amino acid sequences might eventually lead to the cloning of the gene encoding GlOH. Finally, it has to be noted that not all of the steps of secondary metabolite pathways are due to enzymatic reactions, some can proceed chemically, e.g., the conversion of neopine to codeinone in the morphinan biosynthetic pathway (222). Gene Expression

Presently, techniques to introduce new genes into plants do not allow a prediction about the site of integration and the level of gene activity, even when a strong promoter is used. To obtain a plant line with a high expression level, it is necessary to screen a large number of transformants. Therefore, in drawing general conclusions about the effect of genetic engineering, we need to make sure that the observed phenotype is correlated with the expression of the introduced construct, and that it is reproducible. From the first results of the various studies, it is clear that the efficiency of expressing a certain gene in different plant species may vary considerably (e.g., see above for Tdc-gene, (160)). Even with the use of a constitutive promoter, the level of the resulting enzyme activity may vary in different plant parts (23). The 35s promoter is a viral promoter, which uses the transcription factors of the host for its activity. Possibly, not all plants contain the required transcription factors. The same applies for heterologous plant promoters. This might, in some cases, limit the application of any heterologous promoters. The heterologous expression of a stilbene synthase genomic clone from peanut (including its own promoter), which is inducible by elicitors, showed a similar behavior in the aerial parts of transgenic tobacco, but in roots it is constitutively expressed (213).The levels of resveratrol, the phytoalexin formed by this enzyme, are about 100-fold higher in peanut callus after elicitation than in tobacco (223). Using a grapevine genomic gene, about 400 pglg fresh weight (FW) could be reached in the leaves of tobacco, considerably higher than in the transgenic tobacco calli containing the peanut gene (50 ng/g FW) (224). The expression of a gene does not always result in the accumulation of a functional protein, as was observed for example, after expressing a bacterial gene encoding lysine decarboxylase in tobacco (23,26).The introduction of the C. roseus gene encoding TDC into tobacco resulted not only in the accumulation of tryptamine, but also of tyramine in some of the transgenic plants. In vitro, however, the enzyme is only able to decarboxylate

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tryptophan (225). Although this is just one single observation, the message is two-fold. First, because the results observed in transgenic plants often concern a single individual plant, we should be very careful in making general conclusions from such an observation, since it may involve a mutation not connected with the introduction of the transgene. Second, the activities of enzymes measured in vitro, both quantitatively and qualitatively, do not necessarily reflect the real activity in the cell. Stability

Another unknown aspect of genetic transformation is how stable a transgenic trait will be. It has been reported that in subsequent generations of transgenic plants, the transgenes are gradually silenced (226).The mechanisms of gene silencing are not well understood, but in many cases silencing is associated with DNA methylation (227). Reliable methods to prevent transgene inactivation are not available at the moment, although some general measures can be taken to minimize the problem (22 7). Transgenes can be inactivated by mechanisms resembling position effect variegation in Drosophila. A possible solution for this problem is the presence of matrix-attachment regions and/or insulator elements on the construct (228).Alternatively, we could try to target the construct to a chromosomal region that provides a favorable environment for stable transgene expression. This can be achieved by homologous recombination to an endogenous site or to a previously introduced recombination site for a sequence-specific recombination system, such as the Cre-loxP system. Transgenes can also be inactivated by co-suppression (205,229). Since cosuppression seems to be associated with multiple copies of rearranged transgenes, a possible solution is the selection of transformants with a single intact copy of the transgene. Particularly in the case of plant cell cultures, where a continuous selection for rapid cell division and growth occurs, studies on the stability of transgenic cells will be necessary to be able to assess the feasibility of using transgenic cell cultures for the high production of secondary metabolites. Some metabolites may be toxic, and their continuous production may not be compatible with cell division. This problem can be solved by placing transgenes under the control of inducible promoters. For plants, a number of inducible promoter systems have been described, that are based on regulation by tetracyclin (220), steroid hormones (222), or copper (222). Inefficient uptake of the inducer by plants or plant cells can restrict the usefulness of inducible promoters. Alternatively, toxic metabolites or their biosynthetic enzymes could be targeted to cellular compartments that are insensitive to the toxic effects. A specific problem in plant cell suspensions is the instability of the ploidy level of the genome (see above). This causes

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an inherent genetic variability, and also interferes with the ability to regenerate plants. Compartmentation Two of the strategies presented for metabolic engineering are based on the assumption that every cell has the complete biosynthetic machinery for the desired product. In fact, this is certainly not the rule. Compartmentation of biosynthetic pathways does occur, both at the cellular and subcellular level. Cellular Compartmentation. In some cell cultures only a certain percentage of the cells contain the desired compounds: if the concentration of the compounds is similar in all the producing cells, the productivity of the culture is determined by their relative abundance, the concentration of the compounds being about similar in the accumulating cells. Thus, important questions to be solved are, for example: Is the whole pathway downregulated in nonproducing cells, or only one or a few key steps? In the latter case, the above-mentioned approach of increasing constitutively the activity of such enzyme(s) could be followed. In the case of downregulation of the complete pathway, we need to identify the regulatory mechanism in order to be able to switch on the pathway (see above). Another question is whether (part of) the biosynthetic pathway might occur in other cells than storage. Hashimoto and co-workers reported that the final step in the biosynthesis of scopolamine, the epoxidation of 1-hyoscyamine, occurs in pericycle cells of young roots, i.e., the outermost cells around the young vascular cylinders, and subsequently they are translocated to the aerial parts where storage occurs (223,224).It might be possible that 1-hyoscyamine or earlier precursors are produced in other root cells from where they are then moved to the pericycle cells. In any case, this example clearly shows that production and storage can occur in different cells. Subcellular Compartmentation. Secondary metabolite pathways are not just a series of consecutive reactions, but steps may occur in different subcellular compartments. Compartmentation of biosynthetic pathways has been studied quite extensively for both terpenoid indole (for a review, see (246)) and isoquinoline alkaloids (for a review, see (284)). In the case of indole alkaloid biosynthesis in C. roseus, the early steps of the terpenoid pathway (leading to geraniol) and the tryptophan pathway are, by analogy to what is known from other plants, expected to occur in plastids (for reviews, see (63,225-234). However, the possibility of a cytosolic tryptophan pathway cannot yet be excluded (for reviews, see (228220)). The site of the secologanin pathway remains unknown. Tryptophan

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decarboxylase is known to be cytosolic (72,235-240), whereas strictosidine synthase is a vacuolar enzyme (235,238,240-243). The next enzyme in the indole alkaloid biosynthesis is a specificglucosidase, which is located outside the vacuole on the tonoplast and in the cytosol (235,238). The step from ajmalicine to serpentine is catalyzed by vacuolar peroxidases (244),whereas other steps leading to catharanthine are in the cytosol, and one of the steps leading to vindoline is localized in the thylakoids (237). Subcellular compartmentation has also been shown to play an important role in the biosynthesis of berberine (for a review, see (184)). Several of the enzymes involved in the biosynthesis of this alkaloid and related compounds in Berberis wilsoniae are found in distinct vesicles. These enzymes include among others: the berberine bridge enzyme, which is responsible for the oxidation of the N-methyl group in (S)-reticuline, the first step for the formation of the tetrahydroprotoberberine skeleton; and (S)-tetrahydroprotoberberine oxidase, which converts the tetrahydro derivative into the quaternary protoberberine compound (245).The subcellular compartmentation of the various steps requires that for metabolic engineering, it might be necessary to target a protein to the appropriate compartment, otherwise the substrate might not be available for the enzyme. This was, for example, clearly shown for lysine decarboxylase (see above, (23)). Compartmentation requires transport from one compartment to the other. Such transport can be active or passive (diffusion). Active transport means that energy requiring carriers are responsible for the transport of a molecule through subcellular membranes. In the case of indole alkaloids, selective, active transport of ajmalicine and vindoline into vacuoles of C. rosem cells was postulated by Deus-Neumann and Zenk (246).They found that the transport is ATP-ase dependent; other alkaloids, such as morphine, codeine, scopolamine, and nicotine were not taken up by these vacuoles. Others have presented ample evidence for the involvement of an ion-trap mechanism for the accumulation of ajmalicine in the vacuole (244,247). Blom ef al. (248) showed similar results for the quinoline alkaloids in Cinchona cells. In the ion-trap model, transport is driven by the pH difference between cytosol and vacuole. The larger the pH gradient between the acidic vacuole and the cytosol, the stronger the accumulation of the alkaloids. Transport through cell membranes occurs in the neutral form of the alkaloid; in the acidic vacuole it will be protonated. Accumulation is thus governed by the ATP-dependent proton pumps responsible for acidifying vacuoles. Tryptamine is produced in C. roseus cells by decarboxylation of tryptophan in the cytosol. The enzyme catalyzing the next step of the biosynthesis of terpenoid indole alkaloids is located in the vacuole. The transport of the strongly basic tryptamine into the vacuoles is probably also driven by the pH gradient. This example clearly shows that transport

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might play an important role in the regulation of secondary metabolism, as well as pH-controlling genes. Enzymes from a metabolic pathway might be arranged in a sequential manner in metabolic channels or metabolons as postulated by several authors (e.g., 234,249-252). Such hypothetical metabolic channels are thought, for example, to occur in the endoplasmic reticulum for the biosynthesis of sesquiterpenes, and a separate one for sterols (234,251).Also, the pathway from phenylalanine to flavonoids is thought to occur in the endoplasmic reticulum catalyzed by a multienzyme complex, with the first (PAL) and the last step (a flavonoid glucosyltransferase) located in the lumen of the membranes (250). Probably, this requires specific targeting information in the proteins of such a metabolon, and also their expression is expected to be coordinately regulated. For metabolic engineering, the occurrence of such metabolons would imply that the possibilities of increasing a single step in such a metabolon are limited. The regulatory genes should therefore be the targets to try to increase productivity in case of such metabolons. Regulation

In case we want to modify the secondary metabolite production by manipulating the regulatory genes, it should be noticed that the regulation of a similar pathway may be quite different for various plant species. Anthraquinone biosynthesis in some Rubiaceae plants might serve as an illustration (for a review, see (252)).In Rubia tinctoriurn the anthraquinones are found in the roots, in Morinda citrifolia in the bark, and in Cinchona species they are only found after infection with micro-organisms (253).This means that genes having similar protein-encoding regions are regulated in different ways in these plants, i.e., have different promoter regions. In the case of the flavonoid and anthocyanin biosynthesis this has been studied in more detail (see below). Certain enzyme activities measured in plants may result from different isoenzymes encoded by gene families, e.g., as described for tyrosine decarboxylase in poppy (see above, (175-178)) and for HMG-CoA reductase (254),which plays a role in terpenoid indole alkaloid biosynthesis (255,256). To unravel the regulation of such systems and relate them to secondary metabolite pathways and eventually modify these to increase secondary metabolite production will be a major challenge. Moreover, the formation of a certain product can follow different pathways in different plants. This can be nicely illustrated by the sequence of the steps in the biosynthesis of berberine. In Berberis stolonifera, the quaternary alkaloid columbamine is first formed from tetrahydrocolumbamine, subsequently the methylenedioxy bridge is formed. In C o p h juponica these two steps are reversed (Fig. 15) (257).

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CH3O

fl

HO-

\

OCH3 OCH3

c

H

3

0

m

.

Colurnbarnine

\

h

fl


OCH3

OCH3 Berberine OCHi

YX---

H” (s)-Canadine

OCH3

FIG. 15. Last steps in berberine biosynthesis in Berberis stolonifera (above) and Coptis japonica (below).

Even in one plant, different products can be formed from a certain intermediate in different parts of the plant. Plants producing terpenoid indole alkaloids are an example. From the intermediate strictosidine (Fig. 5 ) about 3000 different indole alkaloids are formed, and the pathway is diverging somewhere after the formation of this glycoalkaloid. In C. roseus, the bisindole alkaloids vinblastine and vincristine are only found in the leaves. The biosynthetic pathway leading to one of the precursors, vindoline, includes a step performed by an enzyme localized in chloroplasts (237). On the other hand, ajmalicine and serpentine are the main alkaloids in the roots, and do not require the presence of chloroplasts for the pathway leading from strictosidine. Another example is the Cinchona tree (258-262). It was shown that in young seedlings, directly after the radicle breaks the seedskin (day 0), quinoline alkaloid production is rapidly switched on, reaching a steady-state level at day 5 . Tryptophan decarboxylase and strictosidine synthase (Str) activity, not present in the seeds, rapidly increases at day 0, resulting in the formation of tryptamine and subsequently quinoline alkaloids. The level of quinoline alkaloids was shown to be such that complete feeding deterrence for slugs occurred. In 6-month old Cinchona plants, the highest level of Str activity is found in the young leaves, but no or very low levels of quinoline alkaloids are present. Instead, cinchophylline-type

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alkaloids are found, also derived from strictosidine, but following another pathway (Fig. 16). These alkaloids were shown to have strong antifeedant activity against larvae of Spodoptera exigua, but were not active against slugs. The opposite holds true for the quinoline alkaloids. Apparently, in different parts of the plants, the terpenoid indole alkaloid pathway is regulated differently, serving the plant in its resistance against various predators by producing different, variously biologically active compounds. In this connection, it is noteworthy that plants usually produce a broad range of similar compounds, rather than one specific compound. This might be an advantage from the evolutionary point of view, as resistance against such a mixture of compounds might be more difficult to develop, than against a single compound. In the case of phytoalexins, such as the anthraquinones in Cinchona species, complex mixtures are also found. This may be a strategy to follow in the development of antibiotics. On the other hand, when aiming at producing a single compound in a plant or plant cell culture, the number of related compounds needs to be reduced in order to lower the costs associated with the purification process. So far we have only discussed the regulation at the level of the genes; regulation at the enzyme level is also quite important. This includes regulation by post-translational modifications, enzyme turnover, availability of cofactors and feedback inhibition or activation. Catabolism can be illustrated with tryptophan decarboxylase (TDC) in C. roseus. Fernandez et af. (263) reported that TDC is stabilized by Mn2+ or Mg2+,whereas ATP increased the rate of inactivation of TDC. The monomer of which TDC is built up was found to be ubiquinated, and thus inactivated (264). Increasing the stability of an enzyme might thus also be an approach for enhancing the activity. Even pH might be a regulatory factor, as was described for the hydroxylase converting coumaryl CoA into caffeoyl CoA in parsley. This cytosolic enzyme is only active in a very narrow pH range, having a maximum activity at pH 6.5, whereas at pH 7.5 and at pH 4.5 it is almost inactive (the activity being restored by adjusting the pH to 6.5). Only after elicitation, when the intracellular pH drops, does the enzyme become active (265). Stephanopoulos and Vallino (266) extensively discussed the problems of metabolic engineering in primary metabolism. They postulated that because of the rigidity of the metabolic networks, it is difficult to redirect a larger part of the carbon flux toward certain desired products. Redirection means that at certain branching points the carbon flux has to be redirected. We might distinguish three types of such branching points (nodes). Nodes in which the enzymes at the branching point are not regulated. Those at which one branch is dominating, either because one enzyme has a higher level of activity or higher affinity and is not regulated by feedback inhibition, as

t-

Strictosidme

'

CH3O

-.& Cinchonamine

Cinchonominol

Cinchophyllines

i

Ouinornine

I R = H Cinchonidinone R = OCH3

R=H

R = H Cinchonidine R = OCH3 Ouinine

R H Cinchonice R = OCH3 Qvinidine

R = OCH3 Ouinidinone

FIG.16. Biosynthesis of Cinchona alkaloids.

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the minor branch might be. In the third type of node the split ratio is tightly regulated, usually by feedback from the products of each of the branches, and sometimes trans-activation between the branches by their respective products. Obviously in the first case, there will be no difficulty in redirecting the flux. However, in the other two it becomes increasingly difficult with increased interaction between the branches. Overproduction in one branch will cause feedback inhibition of that branch and possibly induction of the other. The regulation of anthranilate synthase and chorismate mutase in plastids are an example of a rigidly controlled branching point (Fig. 6). Anthranilate synthase is feedback inhibited by tryptophan, the final product of this pathway. At the same time, tryptophan activates the plastidial chorismate mutase isoenzyme, the first enzyme of the pathway leading to phenylalanine and tyrosine. These two amino acids inhibit chorismate mutase, an inhibition which is reversible by tryptophan activation (228-220,267). Such an interlock might cause the clear decrease in the phenylalanine levels, as well as of lignin and phenolic compounds in transgenic potato tubers, in which the tryptophan levels are reduced by the introduction of the Tdc-gene (262). In developing a strategy for redirecting fluxes by metabolic engineering we have thus to take such metabolic interlocks into account. Particularly in networks, such as is common in primary metabolism, such regulation makes it difficult to engineer major changes in fluxes. Fortunately, most secondary metabolite pathways are not networks, but directing more carbon from primary metabolism into secondary metabolism might be hampered by the above-mentioned type of regulation. Not only increasing levels of enzyme activity, but also overcoming the feedback inhibitions and transactivations, will be important in increasing the flux of carbon into secondary metabolite pathways.

IV. Transcriptional Regulation and Signal Transduction Pathways Metabolic pathways often branch out in the direction of several endproducts. Since metabolic engineering aims at channeling a pathway toward the product of interest, knowledge about the regulation of metabolic flows is an important topic. Moreover, some secondary metabolites are produced in distinct developmental stages and in specific tissues of the plant. This is nicely illustrated by the alkaloid production in Cinchona plants and seedlings (see above). Knowledge about the signal transduction pathways controlling these processes is thus of crucial importance.

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Plant growth hormones such as cytokinins and auxins play an important role in the developmental regulation of the metabolic processes in the cell, including secondary metabolism. Merillon and co-workers have shown that cytokinins may induce alkaloid biosynthesis in 2,4-D depleted C. roseus cells, a process involving CaZ+as a second messenger (268) and calmodulin (269-271). Calmodulin has been isolated from C. roseus cultured cells (272). Auxins also affect alkaloid biosynthesis in C. roseus cells. In cell suspension cultures the Tdc- and Str-genes are downregulated by auxins (71,150). However, in seedlings TDC-activity is enhanced by auxins (273). In addition, many secondary metabolite pathways are inducible by exogenous signals. Several signal molecules, termed elicitors, that trigger the cell to produce a certain product are now known. Elicitors are molecules that evoke a defense response, and can originate from the plant, or can have other biotic or even abiotic origins. Fungal cell wall preparations were found to affect various secondary metabolite pathways leading to phytoalexins, antimicrobial compounds of low molecular weight. The induction of sanguinarine biosynthesis in poppy cell cultures is an example of the effect of elicitors on alkaloid biosynthesis (44).The chemical structures of several elicitor molecules have been identified. They include oligosaccharides, peptides, glycopeptides, or lipophilic substances (for a review, see (274)).Evidence exists that elicitors are recognized by specific receptors (39-41), that activate downstream processes, including increased secondary metabolism. Rapid responses to elicitation include the oxidative burst, plasma membrane depolarization, cross-linking of cell wall proteins, ion fluxes, protein (de)phosphorylation, jasmonate release, and induction of gene expression (39,274).Many elicitor responses have been found to be sensitive to protein kinase inhibitors. A tobacco serine-threonine kinase has been detected, that is activated by phosphorylation on tyrosine residues in response to fungal elicitor (275). For the alkaloid pathways affected by elicitors, so far no fungal elicitor molecule has been structurally identified. In C. roseus, fungal elicitors induce enzyme activities (276) and genes (72,153,277) active in terpenoid indole alkaloid biosynthesis. Preliminary results from partial purification of the elicitor molecule from yeast extract indicate that a small peptide is responsible for the induction ((278), and BSDL, unpublished results). Evidence was obtained that the phosphatidylinositol cycle is involved in the signal transduction pathway of the elicitor signal. Lithium, a known inhibitor of myo-inositol-recycling enzymes in phosphatidylinositol metabolism, blocks elicitation with yeast extract, whereas myo-inositol overcomes the inhibitory effect of lithium (279). A bacterial toxin, coronatine, was shown to induce alkaloid biosynthesis in Rauvolfia serpentina and Eschscholtzia californica (280). Coronatine is

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structurally similar to precursors of jasmonic acid, and was postulated to mimic the activity of the octadecanoid molecules discussed below (280). Jasmonates are signal molecules of plant origin. The many-fold biological functions of jasmonate have been extensively reviewed (281-284). Farmer and Ryan (283-285) proposed a model in which jasmonate is involved in the induction of defense systems in plants. The regulation of gene expression in plants by jasmonate was discussed extensively by Reinbothe et al. (286). The octadecanoic pathway leading to jasmonate, and the regulatory role of this compound in alkaloid biosynthesis, was studied in some detail (287,288). Addition of rnethyljasmonate to Rauvolfia canescens cultured cells resulted in a 30-fold increase of the accumulation of the indole alkaloid raucaffricine (Fig. 3) (64). In Eschscholtzia californica cell cultures, jasmonate induces sanguinarine biosynthesis. Alkaloid levels reached 265 mg/l, whereas control cultures contained only 18 mg/l. The seven cytosolic enzymes catalyzing the conversion from tyrosine to (S)-reticuline were not induced by a yeast elicitor or methyljasmonate, whereas the activities of five membrane-bound enzymes specific for the route from (S)-reticuline to sanguinarine, including four specific cytochrome P-450 enzymes, were all induced 6.5- to 16-fold (288). The biosynthesis of jasmonic acid itself is induced by fungal elicitors. About 20 min after elicitation by a fungal elicitor, cis-jasmonic acid synthesis starts, and reaches a transient maximum at 90-240 min depending on the plant species. Similar kinetics are found for 12-oxophytodienoic acid (oxoPDA), a precursor for jasmonate. There is evidence that oxoPDA itself also acts as a signalling molecule (288). In E. californica cell cultures y-linolenic acid, which is thought to be a precursor for oxoPDA, is formed after elicitation. Thirty minutes after elicitation it becomes detectable and it reaches its maximum level after 130-180 min, whereafter its level remains constant. Based partly on the kinetics of jasmonate formation and the dose-response effect of the elicitor, it has been proposed that cis-jasmonate acts as an intermediate signal in elicitorinduced gene expression responses (288). Since induction of terpenoid indole alkaloid biosynthetic genes by fungal elicitors occurs within 15 min and does not require de novo protein synthesis (71), the proposed role of jasmonates as signalling intermediates would require an extremely rapid synthesis of jasmonate by pre-existing enzymes in a biosynthetic pathway that is activated by the elicitor signal. A likely candidate for such a signalregulated enzyme is the postulated lipase that generates y-linolenic acid. Aerts et al, (65)reported that methyl jasmonate vapors increased alkaloid biosynthesis in seedlings of C. roseus. An about twofold increase of total alkaloids was observed, with some differences in the effect for the individual alkaloids. Also, some of the enzymes of the pathway (e.g., Str) were induced. Interestingly, the activities of the last two enzymes in the biosynthesis of

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vindoline, desacetoxyvindoline 17-hydroxylase(D17H) and acetyl-CoA :170-deacetylvindoline 17-0-acetyltransferase (DAT) were induced, a phenomenon not observed after treatment with biotic elicitors (66,67).In Cinchona ledgeriana a twofold increase of alkaloids was observed (65). Salicylic acid is another signal molecule which plays a role in plant defense (68-70). Salicylic acid has an inducing effect on the Tdc- and Str-genes in the terpenoid indole alkaloid pathway in C. roseus (71). Although it has no effect on isoquinoline alkaloid biosynthesis, which is strongly regulated by jasmonate (288). From these results, it is obvious that alkaloid biosynthesis in plants can be influenced by external signal molecules. The type of metabolic response and the nature of the signaling molecules involved are probably related to the specific role of that class of alkaloids for the plant concerned. For improving the production of alkaloids, knowledge of the signal transduction pathways regulating alkaloid biosynthesis is of great value. However, knowledge of proteins active in these signal transduction pathways, such as receptors, protein kinases, and transcription factors, is limited. There is accumulating evidence that plants contain many of the signal transduction components that have been characterized in animals and yeast. Genes encoding G proteins, 14-3-3 proteins (289), enzymes involved in phospholipid metabolism, calmodulin, various classes of protein kinases (290), and phosphatases, have been cloned. Most of these were cloned in random sequencing projects, by homology or by complementation of yeast mutants. Only in very few cases is the signal transduction pathway in which the cloned genes are active, known, and in even fewer cases are other signaling components in the pathway known. For a number of signalling pathways, biochemical studies using (ant)agonists of signal transduction steps, have led to some insight about the nature of the signalling steps. However, these studies often do not allow the determination of the sequence of the different steps. A good example of the biochemical analysis of a pathway is provided by studies using the micro-injection of signal transduction (ant)agonists into cells of a tomato light-receptor mutant to analyze initial steps in light perception (reviewed in (292)).In addition, an increasing number of genes that are in some way involved in light perception have been cloned using Arabidopsis mutant screens (292). Arabidopsis genetics have also led to some insight into ethylene signaling, and to the cloning of genes encoding the receptor and downstream kinases (for a review, see (292,293)).Genetic analysis of disease resistance genes has led to the cloning of receptors and kinases acting in a cascade to transduce bacterial recognition (294-296). Most or all genes involved in metabolic pathways are highly regulated at the transcriptional level. Generally, genes that are active in a certain

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pathway are coordinately regulated. This indicates that they are controlled by a common set of transcriptional regulators. There is relatively little known about plant transcription factors (for a review, see (297)).Most of our knowledge is derived from studies on genes active in anthocyanin biosynthesis in the plant species maize, snapdragon, petunia, and Arabidopsis (96,298,299).This is, among others, due to the fact that the activity of anthocyanin biosynthetic genes can be easily monitored by looking at flower color. A large number of structural genes in the anthocyanin pathway are controlled by transcriptional activators belonging to the C-1 and R families. Members of the C-1 class bear resemblance to the Myb proto-oncogene, while members of the R class are helix-loop-helix proteins that show homology to the Myc transcription activator. In maize, C-1 and R transcription factors control all of the structural genes required for anthocyanin biosynthesis, while in petunia and snapdragon these regulators control mainly the genes acting late in the pathway (109). This indicates that plant species differ in the regulatory networks that control expression of the structural genes. Nevertheless, the function of C-1 and R family members is highly conserved between plant species. Introduction of maize C-1 and R in Arabidopsis and tobacco switches on anthocyanin biosynthesis in tissues that normally d o not produce these compounds (300).Whenever C-1 and R are expressed in a certain tissue, they activate their target genes. This implies that there are no additional regulatory mechanisms such as phosphorylation that control their activity. It is not known how the tissue-specific expression of C-1 and R is regulated, but possibly transcription factors that determine cell identity, such as homeodomain or MADS-box proteins, are involved. Koes et al. (299)proposed a model for the evolution of the regulatory network controlling flavonoid biosynthesis. Besides being constitutively expressed in certain plant organs, a large number of genes in phenylpropanoid metabolism are also induced by stress conditions (210,301). These include UV light, pathogens, and pathogenrelated signals. Relatively little is known about the nature of transcription factors controlling these processes. It is not known, for example, whether stress-induced expression of genes in phenylpropanoid metabolism is controlled by members of the C-1 and R families of transcription factors. A protein termed BPF-1 that binds to an element found in PAL and transcinnamate 4-hydroxylase (4CL) gene promoters was cloned from parsley (302). BPF-1 represents a novel class of DNA-binding proteins, however, its function in transcription is yet unknown. The corresponding gene is itself transcriptionally activated following elicitation. Three bZIP transcription factors, CPRF1-3, binding to a light-responsive element from the parsley CHS promoter were cloned from parsley. CPRFl was shown to be transcrip-

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tionally induced by light, but unexpectedly it was found to act as a repressor of CHS transcription (303,304). Five DNA-binding proteins containing a zinc finger motif that bind to the promoter of the 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) gene were cloned from petunia (305,306). The tissue-specific expression patterns of several of these zinc finger proteins are similar to that of the EPSPS gene. For other metabolic pathways, little is known about transcription factors that control the expression of the biosynthetic genes. But we may expect a rapidly increasing knowledge in this field, which may be of great help to better understand the regulation of secondary metabolism.

V. Conclusions

The rapid development of molecular tools for the cloning of genes and the transformation of plants that has occurred in the last few years provides new opportunities to study the regulation of plant secondary metabolism and to manipulate the production of plant secondary metabolites. Metabolic engineering has the potential to generate superior plant varieties, i.e., ones that are more resistant to pests and diseases, or plants and cell cultures that produce more of a commercially important compound. Recombinatorial biochemistry, the process of enabling the plant to produce compounds normally not found in it or totally new compounds, is also a clear possibility. Secondary metabolite pathways need to be understood at the level of the products, enzymes and genes in order to be able to identify possible limiting steps. Regulation occurs at all these levels. This means that increasing the activity of a single step does not necessarily result in the desired improvement; another step will always become limiting. Therefore, the isolation of regulatory genes might be more useful to increase the flux in a pathway. Blocking pathways by antisense genes is a more easily achieved goal, which also might have applications, such as turning off competitive pathways or the biosynthesis of toxic compounds. A more thorough understanding of the biosynthesis of secondary metabolites will certainly result in the identification of novel enzymes, which may find use in bioconversions. Despite the enormous diversity found among secondary metabolites, most are derived from just a few intermediates, the C5-(isoprenoid-)unit, phenylalanine and tyrosine (phenylgropanoids), acetate units, and some amino acids. In fact, related enzymes catalyze the early steps of many different secondary metabolite pathways; amino acid decarboxylases (see above), chalcone synthases (307),and terpenoid cyclases (308) are a few

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examples. Genes encoding these enzymes have been cloned from different plant species, and show a high degree of homology. Cloning strategies targetting genes that encode enzymes of the early steps of a secondary metabolite pathway can thus make use of probes or primers based on the conserved regions of these genes. However, in some cases, the occurrence of a series of closely related enzymes encoded by a family of closely related genes might be a complicating factor. Further down the pathway more specific enzymes are found, and often belonging to a larger class of similar enzymes. In those cases, the route involving the isolation of the enzyme is probably the most suitable approach to clone the gene. The cytochrome P-450 enzymes are an example of this (see above). Although it is clear that great progress has been made in elucidating the biosynthetic pathways of alkaloids, we can still easily count on our fingers the number of cloned genes encoding enzymatic conversions in alkaloid biosynthetic pathways. Some of these have already found quite interesting commercial applications (see above). The Tdc-gene, for instance, has been patented as a selection marker; it has also been used to decrease glucosinolate production, and causes considerable reduction in the reproduction of whitefly feeding on transgenic tobacco plants containing the Tdc-gene. The gene encoding H6H, which is used to enhance scopolamine production, is another very successful example. Considering the first results of modifying alkaloid biosynthetic pathways, it is obvious that we might expect quite some interesting results along this approach, both for plants and plant cell cultures. First commercial applications will probably be with single gene traits, e.g., encoding enzymes at the end of a pathway, or antisense approaches in the case of inhibiting pathways. Having the tools of molecular biology now well developed, studies on the biosynthetic pathways, i.e., phytochemistry and enzymology, as well as on the role of secondary metabolites, are now needed to eventually exploit the full potential of plant biotechnology.

VI. Future Prospects

What will the impact of plant biotechnology be on alkaloid production by plants and plant cell cultures? Certainly, current efforts to apply metabolic engineering to increase the production of economically important alkaloids will not only continue, but will also expand to include more target plant species and compounds.

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In the case of the terpenoid indole alkaloids, for example, we can use iridoid producing plants as a target. The introduction of the Tdc- and Strgenes in such plants might result in the production of strictosidine, which might be further processed by the plant biochemical machinery into (novel) alkaloids. We can envisage the biosynthesis of the quinoline alkaloids quinine and quinidine in other strictosidine-producing plants, provided that the knowledge of the enzymes involved in converting strictosidine to these quinoline alkaloids is acquired. We could then choose a target plant which can be grown on an annual basis rather than on a 7-10 years growth period. For plants, the quality of which is influenced by their alkaloid content, it is of interest to block alkaloid biosynthesis, such as is the case for nicotine in tobacco and caffeine in Coffea. Similarly,plants containing toxic alkaloids (e.g., hepatotoxic pyrrolizidine alkaloids) may be detoxified by blocking a step in the biosynthesis by means of antisense genes. The production of novel secondary metabolites, including new alkaloids, may result from the introduction of genes encoding enzymes that are capable of transforming a broad range of secondary metabolites, e.g., mammalian cytochrome P-450enzymes, into plants. Such recombinatorial biochemistry offers interesting perspectives for finding new biologically active compounds and for improving resistance against pests and diseases. In the future, this series might include chapters devoted to new transgenic alkaloids. The identification of regulatory genes will make it possible to switch on complete pathways, rather than just improving single steps. It may still be necessary to combine both approaches to obtain the optimal results. For the identification of regulatory genes it will be necessary to clone at least some genes from the pathway of interest. Cloned transcription factors may also be used to knock out entire coordinately regulated pathways by expressing dominant-negative versions of transcriptional regulators (309,320), or by mutation of a transcriptional regulator via homologous recombination (208). Activation tagging (136) is a promising method to clone structural and regulatory genes that are active in metabolic pathways. This method requires relatively little prior knowledge about the pathway, and is applicable to plant species that are genetically poorly characterized. Completely new approaches might be developed on the basis of further knowledge on the role of alkaloids in the plant. A number of alkaloids have antimicrobial (52,52,322,322),antiviral (312), or antifeedant activity (322);these properties can be put to work to improve the resistance of plants against microbes, virus, and herbivores. When using alkaloid biosynthesis to afford this sort of resistance in edible plants, it will be very important to ascertain that these compounds do not represent a health hazard.

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The enormous worldwide effort to screen plants for new biologically active compounds, is expected to bring new drugs, some of which will probably be alkaloids, to the market. In some cases, this might require the production by means of plant cell cultures or genetically engineering plants to obtain high levels of the desired compounds. In the coming years, considerable interest may thus be expected in the field of plant cell biotechnology and metabolic engineering of secondary metabolism by the pharmaceutical industry. For all of us working on alkaloids, the future looks very exciting. In close collaboration with molecular biology on the one hand, and plant ecology on the other, we will be able to much better understand the role and regulation of alkaloid biosynthesis. Eventually, our work will lead to a number of interesting applications in the field of plant breeding, both for agriculture and horticulture, and in the production of specialty chemicals and pharmaceuticals.

Acknowledgment The authors are grateful to Dr. C. Canel for critical reading and for helpful suggestions.

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-CHAPTER 1-

HISTORY AND FUTURE PROSPECTS OF CAMPTOTHECIN AND TAXOL MONROE E. WALLA N D MANSUKH C. WAN] Research Triangle Institute Research Triangle Park, North Carolina 27709

B. Background .. C. Novel Mode o

11. Taxol ........................ A. Introduction ..................

.............. ..............

I. Camptothecin A. INTRODUCTION

The isolation and structural elucidation of camptothecin (CPT) (1) was reported in 1966 in a communication from our laboratories ( 2 ) . Full reports have been subsequently published (2,3). This announcement generated considerable excitement, both because of the unique heterocyclic ring system and because of the excellent activity in the resistant L1210 mouse leukemia assay (2-3). Because 1 was water insoluble, it was tested in the form of the water soluble sodium salt 2;the clinical results were disappointing, with leukopenia and hemorrhagic cystitis being the primary complications of treatment, and interest in 1 decreased (3). The discovery in 1985 that 1 uniquely inhibited the activity of the enzyme topoisomerase I (T-I) ( 4 ) resulted in a rapid resurgence of interest, not only in 1, but also in THE ALKALOIDS, VOL. 50 0099-9598/98 $25.00

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Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved.

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more potent synthetic and/or semisynthetic analogs (5). At present, CPT and five analogs are in clinical trials (Fig. l), and other analogs have clinical potentialities ( 6 ) . Table I summarizes the development of camptothecins over the past 38 years. This chapter gives a brief history of the discovery of camptothecin and provides an overview of the current chemistry, biology, and clinical trials of camptothecins. B.

BACKGROUND

1. Antitumor Activity in Extracts of Camptotheca acuminata

During the period 1950-1959, a group of scientists, at the Eastern Regional Research Laboratory (ERRL), the United States Department of Agriculture (USDA), under the direction of one of us (M.E.W), was involved in a search for sources of steroidal sapogenins that could be converted to cortisones. Thousands of plants were screened in this program. These plant collections consisted of samples from many of the plant introduction stations, including the one in Chico, California, which supplied leaves of C. acuminata, family Nyssaceae. The history of how this plant became available for screening has been described by Perdue et al. (7). Qualitative analysis of this plant showed the presence of flavonoids, tannins, and sterols, but gave negative tests for the sapogenins and alkaloids. Although the ethanolic extracts of various plants including C. acuminata did not contain the desired steroidal sapogenins, these extracts were not discarded by the ERRL and USDA. In 1957,the late Dr. Jonathan Hartwell, who initiated the program for the screening of plants for antitumor agents

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FIG.1. Camptothecin, carnptothecin sodium, and analogs.

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13. CAMPTOTHECIN AND DEVELOPMENT OF CPT 1958 1966 1970-1972 1985 1986-1 991 1989 1989 1991- 1996

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TABLE I ITS ANALOGS AS ANTICANCER AGENTS

Extracts from Carnptotheca acicminata display antitumor activity (3). Active agent 20(S)-CPT isolated and its structure established ( I ) . Phase 1/11 clinical trials of CPT sodium salt (8-10) 20(S)-CPT inhibits DNA topoisomerase I ( 4 ) Analogs 9-amino-20(S)-CPT (9AC) ( 4 2 ) .CPT-11 (.?3,47,48),and topotecan (34.60,61)synthesized and tested. DNA topoisomerase I is elevated in several types of human malignancies ( I 5,16). Unprecedented effectiveness of Y-amino-20(S)-CPT (9AC), 2O(S)-CPT, and other analogs against human cancer xenografts (15). Two water soluble analogs, topotecan (60,61) and CPT-I 1 (Irinotecan) (51-53) are currently in advanced clinical trials in the United States. The latter is approved for clinical use in Japan (1994) and France (1995). Another water soluble analog. GG-211. is entered in Phase I clinical trials in Europe (65,66).Water insoluble CPT itself (38) and two analogs, 9-nitro-20(S)-CPT (9NC) (44,46) and 9-amino-20(S)-CPT (9AC) ( 4 / , 4 2 ) are in Phase 1 clinical trials.

at the National Cancer Institute (NCI) requested Dr. Wall to provide plant extracts for antitumor screening. Of the several thousand plant extracts which were evaluated, only the extract of C. acuminata showed a notable antitumor response. 2. Isolation and Structure Determination of Camptothecin

M. E. Wall left ERRL in 1960 and established a natural products group at the Research Triangle Institute (RTI) with support from the NCI. By 1963, a large sample (20 kg) of the wood and bark of the C. acuminata tree was supplied to our group by the NCI for bioassay-directed fractionation (1-3). In brief, the plant material was defatted by treatment with hot heptane and the insoluble residue was extracted with hot 95% ethanol. The residue from the aqueous ethanol was extracted with chloroform. Only the chloroform extract was found to be highly active in the in vivo L1210 mouse life prolongation assay. Pure CPT (1)was isolated from the chloroform extract by an 11-tube Craig Countercurrent Distribution (CCD) procedure. The molecular composition of 1 was found to be C20H16N204 by highresolution mass spectrometry, and the structure was established by a combination of chemical and spectroscopic methods, including single crystal Xray crystallography (1-3). CPT has a highly conjugated pentacyclic ring structure with one asymmetric center in ring E with a 20(S)-configuration. Another notable feature is the presence of the a-hydroxy lactone moiety

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in ring E which, on treatment with alkali, is readily cleaved to form a water soluble sodium salt (2) ( 2 ) . 3. Early Preclinical and Clinical Testing

CPT (1) showed remarkable activity in the life prolongation of mice bearing the L1210 leukemia, It demonstrated antileukemic activity at 0.2 mg to 3 mg/kg with T/C values frequently in excess of 200% (T/C = survival time of treated animals + survival time of control animals X 100). The compound was also very active in the inhibition of the growth of solid tumors in rodents. In view of the promising anticancer activity of CPT (l),the NCI decided to go to clinical trial with the water soluble sodium salt 2. Compound 2 was preferred over the insoluble parent 1because of the ease of formulation for i.v. administration. In Phase I trial by Gottlieb and Luce (8) involving eighteen patients, five partial responses were observed. These responses, which were primarily in gastrointestinal tumors, were short lived. Doselimiting hematological depression was the main toxicity, along with some vomiting and diarrhea. Because of the somewhat encouraging results obtained in the Phase I study by Gottlieb and Luce (8),a Phase I1 study was hastily undertaken in 61 patients with adenocarcinomas of the gastrointestinal tract, but only two patients showed objective partial responses (9). In another Phase I trial, only two partial responses were found in ten evaluable patients (20).Because of these poor responses and unpredictable toxicities, clinical trials with the sodium salt 2 were halted. The lack of activity of the sodium salt 2 in these early trials could be explained by the later finding from our laboratory that the sodium salt 2 is only one-tenth as active as CPT (1)in the P388 assay ( 2 2 ) . C. NOVELMODEOF ACTION:TOPOISOMERASE I AS THE CELLULAR TARGET OF CPTs In the early 1970s, CPT (1)was shown to inhibit macromolecular synthesis. It was shown to induce a reversible RNA inhibition (22) and a partially reversible DNA inhibition in mammalian cells (23,14). In isolated DNA, however, the binding of 1was either nonexistent or at best very weak (23), and it showed no inhibitory effects in studies employing purified DNA or RNA polymerase (13). In 1985, almost 15 years later, Hsiang et al. ( 4 ) discovered the novel mechanism of action of 1.It was demonstrated that 1is a potent inhibitor of mammalian enzyme topoisomerase I (T-I). This enzyme has been implicated in various DNA functions including transcription and replication. CPT (1) and its analogs bind to a complex formed by DNA and T-I. Furthermore, it has been found that an overexpressed T-I exists in advanced

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stages of human colon adenocarcinoma (15) and other malignancies (16) compared to their normal counterparts. This opened the possibility for clinical use of CPT and analogs by virtue of their potent inhibition of T-I.

D. CHEMISTRY 1. Early Total Synthesis of Camptothecin (CPT, 1)

After our publication of the structure of 1, many total syntheses of this exciting new structure were reported. However, all these early syntheses, including one from our own laboratory, proceeded in poor yields and, more importantly, were not flexible enough to permit analog development. These syntheses have been reviewed in detail by Cai and Hutchinson (17). 2. Improved Synthesis of CPT (1) Suitable for Analog Development

Initially, the improved synthesis developed at RTI terminated at the desoxy synthon 3 (Fig. 2) which on reaction with an appropriate o-aminobenzaldehyde under Friedlander conditions yielded 20-deoxy-CPT or analogs (Scheme l(a)). The latter required a difficult hydroxylation step, mainly due to solubility problems, to give 20(RS)-CPT or analogs (Scheme l(a)) (11). This synthesis was then improved considerably by a procedure which yielded the hydroxylated tricyclic 20(RS) (CPT numbering) synthon 4 (5,18,19).Another major improvement in the total synthesis of CPT or analogs involved the resolution of the 20(RS) synthon 5 to give the 20(S)and 20(R)-analogs 6 and 7, respectively (20). After deketalization, the corresponding tricyclic ketones, 20(RS) 4,20(S) 8, and 20(R) 9, required for the Friedlander condensation could be obtained (Scheme l(b)).

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FIG.2. Tricyclic intermediates.

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SCHEME 1. Synthetic construction of CPT and analogs.

Two alternate syntheses of the 20(S) tricyclic ketone 8 have recently been reported. In 1990, Ejima and co-workers (22) described an enantioselective synthesis of 8 via a novel diastereoselective ethylation. Yet another enantioselective synthetic route to 8, reported by Jew and co-workers (22), involves Sharpless asymmetric dihydroxylation of the olefin 10 as the key reaction. Both these routes employ intermediates common to our procedure and d o not offer much overall improvement to our procedure. Comins and co-workers (23) reported an eight-step asymmetric synthesis of the key bicyclic synthon 11 which could be converted to 1 in two steps by reaction with an o-disubstituted quinoline 12 (Scheme l(c)). Recently, Fang and co-workers have accomplished a more efficient, high yield, enantioselective synthesis of 11 using as the key steps a tandem intramolecular Heck reaction-olefin isomerization process and Sharpless asymmetric dihydroxylation reaction (24).

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By employing an appropriately substituted quinoline derivative, this approach could possibly be used to generate CPT analogs substituted in rings A and B. E. STRUCTURE-ACTIVITY RELATIONSHIPS (SARs)

Our work on the development of CPT (1)as a clinically useful anticancer agent virtually ceased by the late 1960s with the preparation (50 g) of the water soluble sodium salt 2. However, our interest in the isolation and/or synthesis of CPT analogs did not disappear, particularly with regard to SARs. A few years after the report on 1, the isolation of 10-hydroxy- (13) and 10-methoxy-CPT (14) was reported from our laboratory (25). The many CPT analogs synthesized predominantly in our laboratory over more than 25 years have afforded extensive structure-activity correlations. A detailed discussion of SARs of CPT analogs is beyond the scope of this chapter. Moreover, the relationship between the structure of CPT analogs and in vitro and in vivo activity has been reported in detail (5,26,27).The salient aspects of SARs are summarized below (refer to structure 1 for numbering and labeling of rings): 1. The pentacyclic structure of CPT is required for activity. Tetracyclic analogs lacking ring A, tricyclic analogs lacking rings A and B, and bicyclic analogs lacking rings A, B, and C are inactive (5). 2. Analogs without the a-hydroxy lactone moiety in ring E are inactive ( I J ) . 3. The 20(S) configuration is absolutely essential for activity. In general, 20(RS)-CPT or analogs are less active and the corresponding 20(R) compounds are inactive (5,20). 4. Substitution of NH2 for O H or nitrogen for lactone oxygen in ring E leads to loss of activity (28,29). 5. Replacement of the pyridone D ring by a benzene ring leads to inactivation (28). 6. The 20-ethyl substituent is required for activity. However, there is some flexibility; for example, replacement of the ethyl group by an ally1 group improved activity, whereas replacement by a methyl group resulted in loss of activity (30). 7. Certain substituents in ring A (e.g., 10-OH, NH2, or C1; 9-NH2 or C1) give compounds with improved activity (5,19,31). 8. Disubstitution in the 10- and 11-positions (e.g., dimethoxy) led to compounds with reduced activity. However, appending a methylenedioxy or an oxazole ring at the same positions considerably enhanced activity ( I 9,3I,32). 9. Substitution in the 11- and 12-positions led to CPT analogs with reduced or no activity (5,18,19,31).

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F. RECENT PRECLINICAL AND CLINICAL STUDIES As indicated in the introductory section, CPT itself and five of its analogs are now in active clinical trial (Fig. 1). CPT (l), 9-nitro-CPT (9-NC, 15), and 9-amino-CPT (9-AC, 16), all first discovered at RTI (1,31) are water insoluble. Two water soluble analogs of 10-hydroxy-CPT (13),which was also first reported by us (25), are in very intense clinical evaluation. These are CPT-11 (Irinotecan, 17) (33),a product of the Japanese Pharmaceutical Company, Daiichi, and topotecan (18) ( 3 4 , a product of the American pharmaceutical company, SmithKline Beecham. Finally, the third latest water soluble synthetic analog, GG-211(19) (35),which originated at Glaxo, is in Phase I clinical trials. 1. CPT (1)

In Section B.3, we have described the early preclinical studies on the sodium salt 2. More recently, there is a renewed interest in 1 because it has been found that treatment of human xenograft tumor-bearing mice by 1 resulted in complete remissions in 11 of 14 lines, such as lung, breast, ovary, pancreas, and stomach cancer (36,37).CPT (1)has also shown activity against melanoma and lung adenocarcinoma xenograft lines in a central nervous system model of metastasis (37). The above preclinical findings prompted a Phase I clinical trial of CPT administered orally in a gelatin capsule (38). In this study, involving 52 patients with a variety of tumors, there were partial responses in two patients with breast cancer, two patients with melanoma, and one with prostrate cancer. In three additional patients with lung and breast cancers and melanoma, the disease was stable for an extended period while on CPT (1). One patient with a therapy-resistant non-Hodgkin’s lymphoma remained completely free from the disease for 1 year while being treated with 1. Diarrhea and cystitis were dose-limiting toxicities. 2. 9-Arnino-20(S)-CPT (9-AC, 16) As discussed in the preceding Section E, in connection with the SAR studies, various CPT analogs were synthesized in our laboratory either by semisynthesis or total synthesis (5,18-20). A number of these analogs including 9-AC (16)showed topoisomerase I-mediated DNA cleavage and cytotoxicity (39,40).From these analogs, the most promising, 9-AC (16), was selected for additional preclinical testing and possible clinical development. It was further evaluated in several other xenograft models in which it showed remarkable activity (37,41,42).In some tumor lines, single treatment induced complete remissions which lasted over the life-span of the experimental animals (42).

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Encouraged by the very promising preclinical results, the NCI selected 16 for further evaluation. Because it is insoluble in water, the NCI developed a formulation consisting of dimethylacetamide, polyethylene glycol, and phosphoric acid suitable for Phase I clinical trials. Currently the drug is in Phase I and I1 clinical trials under the auspices of the NCI. Commercial development of this analog, including an alternate CD (colloidal dispersion) formulation, will be carried out by a pharmaceutical company, Pharmacia/ Farmitalia Carlo Erba under a contract with the NCI. 3. 9-Nitro-20(S)-CPT (9-NC, 15)

This analog is readily obtained in one step from 1 (31). Early studies from our laboratory had already established that 15, like 9-AC (16), also showed very high activity in murine L1210 leukemia assay, albeit at a considerably higher dose (31).Although at that time no experimental proof was available, it was surmized that the nitro compound 15 may be a prodrug and its activity may be due to its in vivo reduction to 16 (31). Since then, our prediction has been confirmed by Hinz et al. (43). Because of a simpler semisynthesis compared to that of 16, 9-NC (15) was considered to be an attractive candidate for development as an anticancer agent. It was therefore evaluated further in tissue culture using normal and malignant cell lines and in human cancer xenografts in nude mice. In resistant human cancer xenografts, such as colon adenocarcinoma or malignant melanoma, 15 was found to be more active than 1, but less so than 16 (37,42).In tissue culture experiments, it stopped the proliferating cells at the S- or G2-phase of the cell cycle (44-46). O n the basis of the above preclinical findings, protocols for clinical studies of this analog by the oral route were prepared by the group at the Stehlin Foundation for Cancer Research (SFCR) and an investigational new drug (IND) application was approved by the Food and Drug Administration (FDA) in March 1995 (IND #45952). Currently, it is in clinical trials involving previously treated metastatic cancer patients at SFCR. It is being administered orally packaged in a gelatin capsule. 4. CPT-11 (Irinotecan, 7-Ethyl-lO-[4-(l-piperidino)-l-

PiperidinoICarbonyloxy-CPT,17) CPT-11 (17)is a semisynthetic, water-soluble analog which is the most advanced of the CPTs in clinical investigations. It demonstrated good activity against solid mouse tumors when administered by different routes, such as i.p., i.v., or oral (47,48).CPT-11 (17)also showed good activity against a variety of human tumor xenografts in nude mice, including colon adenocarcinoma Co-4, mammary carcinoma MX-1, and squamous cell lung

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carcinoma QG-56 (49).It was also active against human cancer xenografts resistant to topotecan (18),vincristine, or melphalan (50). Phase I studies on 17 were carried out initially in Japan (51),and subsequently in the United States (52) and France (53). Despite the use of different schedules of drug administration ranging from a 30 min infusion (CIV) every week to a 120 h continuous intravenous infusion every 3 weeks, the dose limiting toxicities for 17 have consistently been diarrhea and neutropenia. Other types of toxicities were minor. Objective responses observed during Phase I trials included colorectal cancer, nonsmall cell lung cancer (NSCL), uterine cervix cancer, head and neck cancer, breast cancer, and mesothelioma. Because of the wide range of activity observed in Phase I trials, many Phase I1 trials of 17 in different tumor types have been performed in Japan (51).It exhibited activity against almost all tumor types in which it was evaluated. Partial or complete remissions have been reported in 32% of patients with colorectal cancer; 24% of patients with ovarian cancer; 24% of patients with cervical cancer; 34% of chemotherapy naive patients with nonsmall cell lung cancer; and 50% of untreated and 33% of previously treated refractory small-cell lung cancer patients. By the use of granulocyte colony-stimulating factor (GCSF), the dose of 17 could be escalated by 33% for the treatment of nonsmall lung cancer (54). The use of 17 along with other anticancer agents has also been evaluated. For example, a combination of 17 and 5-fluorouracil for the treatment of metastatic colorectal cancer gave a 33% response (55). Toxic effects observed during Phase I1 trials have been similar to those observed during Phase I trials, with one notable addition of pulmonary toxicity observed in studies involving patients with nonsmall and small cell lung cancers (56). CPT-11 (17)has been approved for clinical use in Japan since 1994 for the treatment of nonsmall lung, ovarian, and cervical cancers. In 1995, it has been approved for clinical use in France for the treatment of colorectal cancer. Extensive advanced Phase I1 trials are in progress in the United States, and FDA approval in the near future is anticipated. 5. Topotecan (9-Dimethylaminomethyl-ZO-Hydroxy-2O(S)-CPT, 18)

This semisynthetic water soluble analog of CPT (l),is the most extensively studied compound in the United States. Unlike CPT-11 (17),topotecan (18)is not a pro-drug and does not require metabolic activation for its activity. It exhibits in vivo activity in a variety of animal tumor models, including the P388 and L1210 leukemias in vivo. Topotecan (18)was found to be superior to both CPT (1)and 9-AC (16)against Lewis

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lung carcinoma and B16 melanoma (57,58).In more recent studies, it exhibited impressive activity against a panel of human colon cancer, rhabdomyosarcoma, and osteogenic sarcoma xenografts when administered either orally or intraperitoneally on a continuous i.v. infusion schedule (59). As a result of proven activities in preclinical studies, Phase I trials of topotecan (18) were initiated using several dosing schedules in the United States (60) and Europe (62). In these studies, complete responses were observed in patients with nonsmall cell lung cancer and leukemia, and minor responses were seen in many other cancers including small cell lung cancer, ovarian cancer, esophageal cancer, renal cancer, and prostate cancer. The dose-limiting toxicity was neutropenia and thromocytopenia, the latter occurring more commonly with continuous infusion schedules. Fatigue, fever, vomiting, diarrhea, and alopecia were relatively infrequent. In Phase I1 trials, only partial responses were observed in patients with colorectal, ovarian, renal cell, and prostate cancers. It is possible that longterm infusion may offer a better response rate in these tumors. In Phase I1 studies, dose escalation has been achieved by the administration of GCSF. Topotecan (18) is currently under evaluation in combination with other antitumor drugs such as cisplatin, etoposide, and taxol (62-64). 6. GG-21I (7-N-Methylpiperizomethylene-l0,lI - Ethylenedioxy-20(S)CPT, 19)

This newest, totally synthetic, water soluble analog of 1 was found to be five to ten times more potent than topotecan (18) in human tumor cell cytotoxicity assays using five different cell lines, ovarian (SKV03), ovarian with upregulated MDRp-glycoprotein (SKVLB), melanoma (LOX), breast (T470), and colon (HT29) (35).It also induced tumor regressions in established HT29 and SW-48 human colon xenografts (65). In a recent Phase I study, 22 patients were given doses ranging from 0.25-2 mg/m2/day. Under these conditions, the drug is well tolerated with reversible myelosuppression as the dose-limiting toxicity (66). 7. DX-8952 (20)

This is yet another, totally synthetic (25 steps), water-soluble analog of CPT (1) reported by the Japanese workers (67).It is one of the most potent (in vivo,rodent tumors) CPT analog ever reported. It is likely to enter into Phase I clinical trials in Japan in the near future. (Dr. A. Ejima, Daiichi Pharmaceutical Company, Private communication).

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G. FUTURE PROSPECTS After a rapid rise and fall in clinical utility of camptothecin in the early 1970s, it is gratifying to note that two decades later, the camptothecins have re-emerged as promising anticancer agents now in clinical trials in the United States, Europe, and Japan. The renewed interest in these compounds is primarily due to the promising results of in vitro and in vivo studies on new CPT analogs synthesized in our laboratory and elsewhere. Encouraging results have been obtained in Phase I and Phase I1 clinical trials with CPT-11 (17),topotecan (18), and 9-AC (16) in patients with therapy-resistant tumors such as colon and nonsmall cell lung cancers. Further studies to confirm these findings are continuing. Although the CPT analogs have been in clinical trials for the past several years, the optimum schedule/route of application has not been determined. Several strategies are being explored currently. These include: (a) a low dose continuous intravenous (CIV) infusion over a period of 21 days; (b) a tapered-off CIV providing tapered-off plasma levels of the active lactone form; and (c) oral administration (68). In the case of the two most advanced drugs, 17 and 18, there is a need to evaluate these compounds in combination with other chemotherapeutic agents, radiation therapy, and biological response modifiers. As pointed out earlier, combination treatments with cisplatin followed by topotecan (18) for patients with extensive small cell and nonsmall cell lung cancers have been initiated (62). Initial studies involving a combination of 18 with taxol and topoisomerase I1 inhibitor etoposide have also been reported (63,64). While the clinical studies to define the role of CPT analogs in cancer chemotherapy are in progress, it is important that the simultaneous investigation of the biochemistry of these agents should also be carried out. For example, the determination of the three-dimensional structure of ternary complex between CPT analog, topoisomerase I enzyme, and DNA by Xray crystallography should provide an insight into the biochemistry of the drug-enzyme-DNA interaction. This information may also be useful in the rational design and synthesis of this class of compounds with less toxicity and more potency. In conclusion, topoisomerase I inhibitors are going to be a valuable addition to the medical oncologist’s armamentarium against cancer. However, there are still many unsolved problems, and continued basic and clinical research on this novel class of compounds is warranted.

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11. Taxol

A. INTRODUCTION This section of the overall review of outstanding developments occurring with camptothecin and taxol during the period 1980-1995 will be concerned with taxol’ (21) (paclitaxel).

I . Brief Review of Major Events Prior to 1980 Prior to presenting the major developments occurring during 1980-1995, the events leading to the discovery, structure, and mechanism of action of taxol will be briefly reviewed.

a. Early Collection of Taxus brevifolia. In 1962 a USDA botanist, the late Arthur Barclay, in the course of collecting flora in the Pacific northwest for antitumor screening by the National Cancer Institute, obtained samples of the bark and wood of Taxus brevifolia, a slow-growing member of the yew family (69,70). The samples were found to be cytotoxic. One of us (M.E.W.) had previously noted a good relationship between cytotoxicity and in vivo activity. At his request, a number of cytotoxic plants, including T. brevifofia, were assigned to his program for further study by the National Cancer Institute (NCI). b. Isolation and Structure Elucidation. The isolation and structure elucidation of taxol was reported many years ago (72,72) and has been presented in several recent reviews (69,70,73). In brief, by 1967, after purification by sequential Craig Countercurrent Procedures, taxol (21) was isolated (72). Structure elucidation required low temperature alkaline methanolysis of 21 to give the a-hydroxy ester 22 and the tetraol(10-deacetyl baccatin 111) 23. These were each converted to halogenated analogs, and the structures were determined by X-ray analysis (72). The structures of 21, 22, and 23 are shown in Fig. 3.

c. Bioactivity and Mechanism of Action. Because of the low yield, water insolubility, and modest activity in certain “in vivo” rodent antitumor assays such as mouse L1210 leukemia, taxol (21) was for several years relegated to comparative obscurity. Due to the efforts of the NCI staff officer, the late Dr. Matthew Suffness, who noted that 21 had sufficient activity in

’

The name “taxol” has been trademarked by Bristol-Myers Squibb. Since it was first named by us (72). long before it was trademarked, we prefer to continue to use the term “taxol.”

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22 Methyl ester

23 Tetra01 (1 0- deacetyl baccath 111, DAB)

FIG.3. Structure of taxol, taxotere, and methanolysis products.

B-16 melanoma to meet the NCI development criteria (70,74), interest in

21 increased. In rapid succession, papers appeared in 1979 and 1980 showing that 21 was an antimitotic poison (75) and that it had a unique mechanism of action involving microtubule assembly (76-78). As a consequence of these findings, interest in 21 by the NCI was firmly established, and the stage was set for developments during the period 1980-1995 which resulted in taxol (21)and its closely-related analog, taxotere2 (24),becoming leading cancer chemotherapeutic agents. Table I1 summarizes the chronology of the discovery and development of taxol as a clinical agent.

* The name “taxotere” has also been trademarked by Rhone-Poulenc-Rorer. The generic term is docetaxel. For simplicity, we will continue to use “taxotere,” which was in use for a number of years before it was trademarked.

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TABLE I1 THEDISCOVERY AND DEVELOPMENT OF TAXOL AS

AN

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Collection of Tuxus brevifoliu in northwest coastal area (Washington State). Shipment of large bark sample to M. Wall at RTI. October 1964 First isolation of pure taxol guided by bioassay in KB, P1534, and October 1966 Walker systems Report of taxol isolation to American Chemical Society. 1967 Chemical structure of taxol published. May 1971 B16 melanoma activity observed by M. Suffness. April 1974 Activity in B16 confirmed, meets NCI development criteria. June 1975 Publication of taxol as antimitotic drug. August 1978 Publication of taxol as promoter of microtubule assembly. February 1979 September 1983 NDA application filed. NDA application approved. April 1984 April 1984 Phase I clinical trials begin. Activity in advanced ovarian cancer, published by Johns Hopkins group. August 1989 November 1989 Selection of Bristol-Myers Squibb as CRADA partner by NCI. Large-scale production of taxol by HauserlBMS. 1990-1993 December 1992 NDA approved for refractory ovarian cancer. Efficient procedures developed for semisynthesis of taxol from 1990- 1994 10-DAB. Total syntheses of taxol published by Nicolaou and Holton. 1994 Supplemental approval of taxol for metastatic breast cancer. April 1994 Taxol now being- tested clinically in combination with other cancer 1994chemotherapeutic agents, particularly cisplatinum in breast cancer. August 1962

B. TAXOL SUPPLIES AND SOURCES 1. Bark of T.brevifolia

The initial source of taxol was the bark of T. brevifoliu, in which it was first discovered. However, during the development of 21 as an investigational new drug, supply problems arose. It was soon noted that the availability of this promising new drug was severely limited by the low concentrations found in the bark. Moreover, T. brevifolia is a very slow-growing tree, present in relatively low density, and is destroyed by the process of bark removal. The possibility of the extinction of this species and the fact that the spotted owl, a threatened bird, nested in this tree, engendered great controversy between environmental groups and lumber groups. Eventually, an environmental impact statement was promulgated by the Forest Service Bureau of Land Management (79). An excellent review concerned with

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T. brevifolia and related “yew” species and the issues described above has been prepared by Croom (80). As a consequence of the environmental problems, there has been an intensive search for alternative sources during the last 10 years (1985-1995) culminating in a decision by Bristol-Myers Squibb (BMS) to discontinue the use of the bark of T. brevifolia by August, 1994 (80).

2. Sources for Semisynthesis Taxol(21) and its closely related analog, taxotere (24),structurally consist of two moieties. One, the central nucleus contains many asymmetric carbon atoms. The other is a much simpler side chain with only two asymmetric carbons which, in the case of 21, is the N-benzoyl derivative of (2R,3S)-3phenylisoserine, 25,and in the case of 24 is the N-t-butoxycarbonyl derivative, 26. Several taxanes, notably baccatin I11 (27)and 10-deacetyl-baccatin I11 (23,DAB) have been found in certain Taxus species in much higher concentration than 21. DAB was found in yields of 0.1% or higher in leaves of cultivated T. baccata, a European yew, more than five times the best yield for taxol(80-82). T. wallichiana, a Himalayan yew, is another promising source of 23. Renewable sources of 23 and 27,such as twigs and needles, will soon replace the bark of T. brevifolia as sources for 21 (80). The chemistry involved in the semisynthesis of 21 and 24 will be discussed in Section II.C.l. 3. Taxol and Taxanes from Endophytic Fungus, Taxomyces andreanae

Recently a group from Montana State University have made the rather startling announcement that 21 and, to a lesser extent, 27 have been found in a new fungus, Taxomyces andreana, isolated from the bark of T. brevifolia (83-85). The fungus can be grown in semisynthetic medium and produces both taxol and taxanes (83).At this time it is unknown whether large scale production of 21 or related taxanes 23 and 27 can be achieved. 4. Taxol by Plant Cell Culture

Taxol can be produced by plant cell cultures (86).A detailed discussion of this procedure, its current status, and future prospects has recently been presented by Gibson et al. (86).Although several biotechnology companies are pursuing this area, increases in productivity and yield will be required for commercialization of this route. C. CHEMISTRY As interest in taxol increased in the early 1980s and, as a consequence of the financial research support by the NCI and pharmaceutical companies,

13. CAMPTOTHECIN AND

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TAXOL

a large number of semisyntheses of 21 and 24 were developed. This review, which deals with highlights of research on 21 and 24, can include only a small fraction of the large volume of published research. During the early 1980s, it became evident that 21 had potent chemotherapeutic efficacy. Concern now increasingly arose whether sufficient supplies of 21 would be available from T. brevifofia. At the same time, information became available that baccatin I11 (27) and 10-deacetylbaccatin I11 (23) were available in other Taxus species in considerably higher concentration than 21 (cf. Section II.B.2). Both compounds 27 (Fig. 4) and 23 are quite similar to 21 and 24 in regard to the structure of the central nucleus, which contains most of the asymmetric carbon atoms in these compounds. Consequently, it remained only to devise syntheses of the much simpler side chains of 21 and 24 and unite them with 27 and 23, respectively. A number of excellent reviews are available (87-91). In this section, we will discuss only a few of the very large number of partial syntheses of 21 and 24 which have appeared in the literature. The limited number of the semisynthetic procedures which will be presented include brief discussions of methods with historical importance and those which currently seem to be the most important for the semisynthesis of 21 and 24. Only the immediate

-- n

ococ6H5

33 Protected taxotere analogue

27 Baccatin 111, R=H, R,=Ac 28 7-Triethylrilyl baccatinlll, R=TES, &=Ac 31 7.10-bis-Trichloroethoxy(Troe).10-dcacclyl baccatin 111, R=&=Troc

t-BOCN C6HHs"

OR 25 R = H , R ' = C ~ H ~ C O 2 6 R=H,R'=t-BuOCO 29 R=EE,Ri=C&CO 32 R=Ba,RI = t - B u m 0 35 R=R'=H

'OR

34 R=H

36 R=TIPS 37 R=TBS

FIG.4. Semisynthesis of taxol and taxotere.

38

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WALL A N D WAN1

precursors of the various side-chain analogs along with the respective central taxane nucleus will be shown. Literature references for the various procedures discussed will be presented. 1. Esterification of 27 or 23 with Protected N-Benzoyl-3-Phenylserines

The potential of 27 and 23 for the semisynthesis of 21 and 24 was first recognized by Potier and co-workers (92,93). a. Greene-Potier Procedure (94,95). The final steps of this procedure are shown in Fig. 4. Esterification of 7-triethylsilyl-baccatin I11 (28)with the 2’-ethoxyethyl (EE) sidechain analog 29 gave the protected taxol analog 30, which was converted to 21 [92] by removal of the protective groups under acidic conditions.

b. Greene Synthesis of Taxotere (96). A considerably improved version of the procedure shown in Section C.1.a is shown in Fig. 4. Esterification of the 7,1O-bis-trichloroethoxycarbonyl(troc) analog of DAB (31)with the N-t-butoxycarbonyloxy side-chain analog 32 gave the protected taxotere analog 33. Taxotere (24)was obtained by removal of the protective groups of 33. 2. Semisynthesis of 21 and 24 from 27 and 23 Utilizing Improved Side Chain Acylating Agents The procedures described in Sections C.1.a and b above have limitations. These include, amongst others, harsh reaction conditions, low conversion, loss of the expensive baccatin I11 or 10-deacetyl baccatin I11 derivatives, and formation of C-2’-epimerization products. As a consequence, much effort has been expended in the synthesis both of the side chain of 21 and 24 and the conversion of these to acylating agents which can be esterified with 27 or 23. This review, which deals only with the “high-spots’’ of taxol research during 1980-1995, cannot present the many interesting procedures for the synthesis of the taxol side chain. This topic has been reviewed in depth by Holton et al. (91). a. Synthesis of P-Lactams. Georg was one of the first researchers on side-chain synthesis to recognize that the P-lactam, (3R, 4S)-3-hydroxy-4phenyl-Zazetidinone (34) (97,98) could serve as a practical precursor for (2R,3S)-3-phenylisoserine (35). Almost simultaneously, Georg and Ojima applied the ester enolate-imine cyclocondensation to the synthesis of 25, resulting in the asymmetric synthesis of the P-lactams 36 and 37 (97-202). Holton has stated that 36 and 37 are probably the quickest and most efficient access to chiral reactants that can be directly converted to 21 (91).

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527

b. Synthesis of Taxol via N-Acyl-P-Lactams. The Holton group has perfected the utilization of N-acyl-P-lactams for the esterification of 27, thus opening the route to the efficient syntheses of taxol (103). Holton’s initial procedures involved esterification of 7-triethylsilyl (TES)-baccatin III,28, with P-lactams similar to 36 and 37 (104).However, a much improved procedure involving reaction of P-lactams with structures similar to 36 and 37 with the C-13 lithium alkoxide salt of 28, gave excellent results with many variants of these P-lactams (105). Moreover, and of considerable practical importance, the lithium alkoxide was found to react diastereoselectively with racemic P-lactams. Hence in most cases there was no longer a need to prepare the P-lactams in optically active form (91). c. Oxazolidines. Other acylating agents such as oxazolidines 38 have been used as acylating agents for the syntheses of taxotere (24) by Commercon et af. (106-108). Reaction of 37 or similar analogs with 7,lO-bis-troc10-deacetyl baccatin I11 (31) yielded protected esters which could then be converted to 24 by removal of protective groups (104-105) (cf. also (89) for a review).

d. Summary. Holton has presented an excellent review of the current (1995) status of the semisynthesis of taxol and taxotere (91). There are now available a large number of synthetic routes. The semisyntheses of taxol via N-acyl P-lactams has been scaled up to a highly efficient industrial process, and multikilogram quantities of taxol have been prepared in this way. Industrial quantities of 10-deacetylbaccatin I11 are also available. As a consequence, Bristol-Myers Squibb Company has announced that it will no longer harvest yew bark for taxol. According to Holton, the semisynthesis of 24 from oxazolidine also promises to supply adequate quantities of taxotere. 3. Total Synthesis of Taxol, 21

In 1994,groups from the laboratories of R. A. Holton and K. C. Nicholaou simultaneously announced the total synthesis of taxol(21) (109-112). The announcement of the two syntheses represented an epochal event in the synthesis of complex natural products. The total syntheses of 21 had been a major challenge for many outstanding organic chemists for over 20 years. This review will not present the synthetic details, which are described in Ref. (109-112). In brief, the Holton synthesis (109-111) was based on camphor, readily available in either enantiomeric form. The various rings A, B, C, D of baccatin I11 were constructed in a linear fashion utilizing conformational control to enable functionalization of the eight-membered B-ring. The side

528

WALL AND WAN1

chain was synthesized by Holton’s azetidine procedure and joined to a baccatin I11 analog by procedures described in Section C.2. The Nicolaou procedure (112,113) was, for the most part, completely different. Initially, an appropriately derivatized A-ring was prepared. Then ring C was constructed and connected to ring A. Intramolecular cyclization gave the ABC ring system of 21. Finally, the oxirane ring D was added. At the end, a baccatin I11 analog was prepared and joined to the side chain in the manner discussed above. Neither procedure will challenge the preparation of taxol from yew or the semisynthetic procedure described in Section C.2. In conclusion, both the Holton and Nicolaou groups achieved remarkable total syntheses of taxol. Each group overcame innumerable technical problems. Although neither synthesis is practical for large-scale operations, many new and potentially valuable analogs are sure to come from this work. 4. Structure-Activity Relationships (SAR) of Taxol and Analogs

Over the last 10 years, and with increasing frequency in recent years, many studies have been made of the SAR of taxol (21), particularly by Kingston and Georg. A number of comprehensive recent reviews are available (87,114-117). A review on the SAR of taxotere (24)has also appeared recently (118). Because of the similarity of the SAR data for 21 and 24, only the former will be considered in this review. SAR studies of taxol have always been accompanied by comparative in vitro assays for cytotoxicity and tubulin binding. Although both sets of assays usually show similar trends, this is not always the case. Of the two, the tubulin binding is the most important feature. The SAR structure of taxol is shown in Fig. 5. For SAR discussion, the compound can be divided in three regions using Kingston’s nomenclature: side chain and the northern and southern hemispheres of the central taxol nucleus comprising, respectively, the region from C-12 to C-6 and from C1 to c-5. a. Side Chain. Referring to Fig. 5 , the requirement for the entire side chain of 21 for activity (cytotoxicity measurements) was determined at the time of the discovery of taxol (69,72). There is an absolute requirement for the presence of the free 2’-hydroxyl group or hydrolyzable esters thereof (114-127). A number of 2’-water soluble esters which are readily hydrolyzable have been prepared (117). Taxanes with the 2R,3S stereochemistry are much more active than those with different stereochemistry. The C-3’ phenyl group or a close analog is required (114-117). The N-acyl group is required, but considerable structural modification is still consistent with activity (114-117). Taxanes with various acyl groups are all active, e.g.,

13. CAMPTOTHECIN

AND TAXOL

529

Northern hemimhere

RlCONH c6Hs4

L

0 ,0

.

OH Sidechain

1

L

Southem hemisphere

R=Ac, Baccatin 111, Taxol R=H, 10-Deacetyl baccatin 111, Taxotere Rl=C&Is. Tax01 RI=r-BuO; Taxotere FIG.5. SAR of taxol.

taxol, N-benzoyl; taxotere, N-t-butoxycarbonyloxy; cephalomannine (Ntigloyl). b. Northern Hemisphere. In general, considerable flexibility has been noted with substituents in this area. Thus the 7P-hydroxyl group can be esterified, epimerized, or removed without significant loss of activity (114). At C-10, esters other than acetate are active, and the des-acetyl-10-hydroxy moiety is active (114-117). The 9-carbonyl group can be reduced with no loss of activity (114). c. Southern Hemisphere. This region is considerably less open to modification without loss of activity. The 2-benzoyloxy group is essential; however, meta substitution on the aromatic moiety greatly increased activity (114). The 4,5-oxetane ring is absolutely required, as may be the 4-acetyl moiety (114-117). It is difficult, however, to remove the 4-acetate substituent without affecting the oxetane ring. Surprisingly, there is at present no available information on whether the C-1-hydroxyl moiety is required for activity.

D. CLINICAL STUDIES It is now recognized that taxol is an important new cancer chemotherapeutic agent. Its clinical development was initially slow due to the limited supply of the drug, poor solubility, and life-threatening hypersensitivity reactions. A comprehensive review of clinical studies with both taxol and taxotere has recently been published (119). A Cooperative Research and

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Development Agreement (CRADA) between NCI and BMS was established in January 1991. In December 1992, the FDA approved taxol for treatment of previously treated ovarian cancer, and in April 1994 limited approval for previously treated breast cancer patients was obtained. Taxol also has activity in many other tumor types (119). 1. Toxicity

Taxol has been reported to cause neurotoxicity (120). Leukopenia and neuropathy are the most frequent side effects associated with dose limiting toxicity. Initially, hypersensitivity was encountered due, to some extent, to the use of Cremophor EL in the vehicle for taxol. These problems have largely been overcome (119). 2. Responses in Various Tumors

Ovary. Objective responses, PR and CR, vary from 20-48%, the majority being partial responses (PR). Nonsmall cell lung, 3-24%. Small-cell lung, 5 1 6 % . Breast, 23-62%. At the present time, taxol is clearly the best available drug for ovarian cancer.

3. Formulation Because of concern that Cremophor EL contributes to taxol toxicity, liposome formulations and water-soluble pro-drugs are under evaluation (121-122).

4. Combination Therapy

As an increased supply of 21 has become available, combination clinical trials with many standard anticancer agents, including cyclophosphamide, doxorubicin, and cisplatin, have been initiated [cf. (116) for a full review].

5. Taxotere Taxotere (24) is a semisynthetic product introduced by Rhone-PoulencRorer. Toxicity and general uses are similar to 21. The drug may be somewhat superior to taxol against breast cancer. As high as 40-60% objective responses have been observed in Phase I1 trials (116). Phase I11 trials in breast and lung cancer are underway. E. FUTURE PROSPECTS

Taxol is one of the most promising anticancer drugs developed in recent years. There is, however, still a need for much additional research. In the

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531

area of taxol supply, genetic manipulations may lead to development of rapid-growing, high-yield Tuxus species. Similar genetic manipulation can led to the development of plant cell culture, or bacterial and fungal sources which may yield taxol in large quantities similar to antibiotic production. In the chemical synthesis area, many studies will continue to be conducted on the semisyntheses of the side chain, an area of much commercial importance. Indeed, recently, Sharpless and co-workers have developed an attractive enantiomeric aminohydroxylation process by which the taxol side chain with correct stereochemistry was prepared in only three steps (123). Undoubtedly, new total syntheses will be forthcoming. However, few, if any, of these will replace semisynthetic methodology. SAR studies will continue with taxol and taxotere. Although some of the structural modifications, both in the side chain and nucleus, show increased potency viz-d-viz both 21 and 24 (ZZ4-ZZ7), it is unlikely that most of these analogs will receive the necessary great expenditure required to obtain FDA approval for clinical trials and marketing because of the long time lead possessed by the pharmaceutical companies already marketing 21 and near FDA approval for 24. Clinical studies with both taxol and taxotere will continue with emphasis on combination therapy against ovarian, breast, and many other forms of cancer.

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87. D. G. 1. Kingston, in “Taxane Anticancer Agents” (G. I. Georg, T. T. Chen, I. Ojima, and D. M. Vyas, eds.), ACS Symposium, Series 583, pp. 203-216. American Chemical Society, Washington, DC, 1995. 88. G. I. Georg, G. C. B. Harriman. D. G. Vander Velde, T. C. Boge, Z. S. Cheruvallath, A. Datta, M. Hepperle, H. Park, R. H. Himes, and L. Jayasinghe, in “Taxane Anticancer Agents” (G. I. Georg, T. T. Chen, I. Ojima, and D. M. Vyas. eds.), ACS Symposium, Series 583, pp. 217-232. American Chemical Society, Washington, DC, 1995. 89. F. Gueritte-Voeglein, E. Tuenard, J. Dubois, A. Wahl, R. Marder, R. Muller, M. Lund, L. Bricard, and P. Potier, in “Taxane Anticancer Agents” (G. 1. Georg, T. T. Chen, I. Ojima, and D. M. Vyas, eds.), ACS Symposium, Series 583, pp. 189-202. American Chemical Society, Washington, DC, 1995. 90. A. Commercon, J. D. Bourzat, E. Didier, and F. Lavelle, in “Taxane Anticancer Agents” (G. I. Georg, T. T. Chen, I. Ojima, and D. M. Vyas, eds.), ACS Symposium, Series 583, pp. 233-246. American Chemical Society, Washington, DC, 1995. 91. R. A. Holton, R. J. Biediger, and P. D. Boatman, in “Taxol: Science and Applications” (M. Suffness, ed.), pp. 97-122. CRC Press, Boca Raton, FL, 1995. 92. G. Chauviere, D. GuCnard, F. Picot, V. Senilh, and P. Potier, C. R. Acad. Sci. Parts, Ser. 11, 293, 501 (1981). 93. V. SCnilh, F. GuCritte, D. Guenard, M. Colin, and P. Potier, C. R. Acad. Sci. Paris, Ser. 11, 299, 1039 (1984). 94. J.-N. Denis, A. E. Greene, D. GuCnard, F. GuCritte-Voegelein, L. Mangatal, and P. Potier, J. Am. Chem. SOC.110, 5917 (1988). 95. J.-N. Denis, A. E. Greene, A. A. Serra, and M.-J. Luche, J . Org. Chem. 51,46 (1986). 96. A. M. Kanazawa, J.-N. Denis, and A. E. Greene, J. Org. Chem. 59, 1238 (1994). 97. G. I. Georg, Tetrahedron Lett. 25, 3779 (1984). 98. G. I. Georg, J. Kant, and H. J. Gill, J. Am. Chem. Soc. 109,1129 (1987). 99. I. Ojima, I. Habus, M. Zhao, G. I. Georg, and L. Jayasinghe,J. Org. Chem. 56,1681 (1991). 100. G. I. Georg, Z. S. Cheruvallath, R. H. Himes, M. R. Mejillano, and C. T. Burke, J. Med. Chem. 35,4230 (1992). 101. I. Ojima, I. Habus, M. Zhao, M. Zucco, Y. H. Park, C. M. Sun, and T. Brigaud, Tetrahedron, 48, 6985 (1992). 102. G. I. Goerg, Z. S. Cheruvallath, G. C. B. Harriman, M. Hepperle, and H. Park, Bioorg. Med. Chem. Lett. 3, 2467 (1993). 103. R. A. Holton, U.S. Patent No. 5,175,315 (1992). 104. R. A. Holton, R. J. Biediger, and P. D. Boatman, in “Taxol: Science and Applications” (M. Suffness, ed.), pp. 112-113. CRC Press, Boca Raton, FL, 1995. 105. R. A. Holton, U.S. Patent No. 5,229,526;5,274,124 (1993). 106. J. D. Bourzat and A. Commercon, Tetrahedron Lett. 34,6049 (1993). 107. E. Didier, E. Fouque, I. Taillepied, and A. Commercon, Tetrahedron Lett. 35,2349 (1994). 108. E. Didier, E. Fouque, and A. Commercon, Tetrahedron Len. 35,3063 (1994). 109. R. A. Holton, C. Somoza, H. B. Kim, F. Liang, R. J. Biediger, P. D. Boatman, M. Shindo, C. C. Smith, S. Kim, H. Nadizadeh, Y. Suzuki, C. Tao, P. Vu, S. Tang, P. Zhang, K. K. Murthi, L. N. Gentile, and J. H. Liu, J. Am. Chem. Soc. 116, 1597 (1994). 110. R. A. Holton, H. B. Kim, C. Somoza, F. Liang, R. J. Biediger, P. D. Boatman, M. Shindo, C. C. Smith, S. Kim, H. Nadizadeh, Y. Suzuki, C. Tao, P. Vu, S. Tang, P. Zhang, K. K. Murthi, L. N. Gentile, and J. H. Liu, J. Am. Chem. Soc. 116, 1599 (1994). 111. R. A. Holton, C. Somoza, H.-B. Kim, F. Liang, R. J. Biediger, P. D. Boatman, M. Shindo, C. C. Smith, S. Kim, H. Nadizadeh, Y. Suzuki, C. Tao, P. Vu, S. Tang, P. Zhang, K. K. Murthi, L. N. Gentile, and J. H. Liu, in “Taxane Anticancer Agents” (G. 1. Georg. T. T. Chen, I. Ojima, and D. M. Vyas, eds.), ACS Symposium, Series 583, pp. 288301. American Chemical Society, Washington, DC, 1995.

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112. C. Nicolaou, Z. Yang, J. J. Liu, H. Ueno, P. G. Nantermet, R. K. Guy, C. F. Claiborne, J. Renaud, E. A. Couladouros, K. Paulvannan, and E. J. Sorensen, Nature 367,630 (1994). 113. K. C. Nicolaou and R. K. Guy, in “Taxane Anticancer Agents” (G. I. Georg, T. T. Chen, 1. Ojima, and D. M. Vyas, eds.), ACS Symposium, Series 583, pp. 302-311. American Chemical Society, Washington, DC, 1995. 114. D. G. I. Kingston, in “Human Medicinal Agents from Plants” (A. D. Kinghorn and M. F. Balandrin, eds.), Vol. 534, pp. 138-148. American Chemical Society, Washington, DC, 1993. 115. D. G. I. Kingston, in “Taxane Anticancer Agents” (G. I. Georg, T. T. Chen, I. Ojima, and D. M. Vyas, eds.), ACS Symposium, Series 583, pp. 203-216. American Chemical Society, Washington, DC, 1995. 116. G. I. Georg, G. C. B. Harriman, D. G. Vander Velde, T. C. Boge, A. S. Cheruvallath, A. Datta, M. Hepperle, H. Park, R. H. Himes, and L. Jayasinghe, in “Taxane Anticancer Agents” (G. I. Georg, T. T. Chen, I. Ojima, and D. M. Vyas, eds.), ACS Symposium, Series 583, pp. 217-231. American Chemical Society, Washington, DC, 1995. 117. G. I. Georg, T. C. Boge, A. S. Cheruvallath, J. S. Clowers, G. C. B. Harriman, M. Hepperle, and H. Park, in “Taxol: Science and Applications” (M. Suffness, ed.), pp. 317-378. CRC Press, Boca Raton, FL, 1995. 118. F. Gueritte-Voegelein, D. GuCnard, J. Dubois, A. Wahl, R. Marder, R. Muller, M. Lund, L. Bricard, and P. Potier, in “Taxane Anticancer Agents” (G. I. Georg, T. T. Chen, I. Ojima, and D. M. Vyas, eds.), ACS Symposium, Series 583, pp. 189-202. American Chemical Society, Washington, DC, 1995. 119. S. G. Arbuck and B. A. Blaylock, in “Taxol: Science and Applications” (M. Suffness, ed.), pp. 379-415. CRC Press, Boca Raton, FL, 1995. 120. E. K. Rowinsky, V. Chaundhry, D. R. Cornblath, and R. C. Donehower, Monogr. Natl. Cancer Inst. 15, 107 (1993). 121. R. Weiss, R. C. Donehower, P. H. Wiemik, T. Ohnuma, R. J. Gralla, D. L. Trump, J. R. Baker, Jr., D. A. Van Echo, D. D. Von Hoff, and B. Leyland-Jones, J. Clin. Oncol. 8,1263 (1990). 122. K. C. Nicolaou, C. Riemer, M. A. Kerr, D. Rideout, and W. Wrasidlo, Nature 364, 464 (1993). 123. V. Sharpless, G. Li, and H.-T. Chang, Angew. Chem. Inr. Ed. 35,451 (1996).

-CHAPTER 1

6

ALKALOID CHEMOSYSTEMATICS PETERG. WATERMAN Phytochemistry Research Laboratories Department of Pharmaceutical Sciences University of Strathclyde Glasgow GI IXW, Scotland, UK

1. Introduction ........................................................................ 537 Systematics: Laying Do 11. Alkaloids in 111. The Evolution of Alkaloids .................................. A. The Chemical Mechanism .................... B. Alkaloids as Evolutionary Events ........... ............................... 541 C. Evolutionary Origins ...................................... D. Driving Forces Mediating Production? .............................................. 543 544 ............................... IV. Handling Alkaloid Data in Systematic Studies . Higher Plant Taxa .... 548 V. Systematically Significant Distributions of Alkal 548 A. Major Tyrosine/Phenylalanine-DerivedAlkaloids ................................ ............................... 553 B. Major Tryptophan-Derived Alkaloids ...... C. The Betalains ............................................... D. Anthranilate-Derived Alkaloids of the Rut E. Alkaloids Originating from Ornithine and Lysine (Tropanes, Pyrrolizidines, and Quinolizidines) .................... ............................... 559 563 VI. Concluding Comments ............................................................ ........................................................................ 564 References

I. Introduction

The “dawn” of chemical systematics, as far as alkaloids are concerned, can probably be associated with Alston and Turner’s Biochemical Systematics ( I ) , and the chapter by Robert Hegnauer in Swain’s Chemical Plant Taxonomy (2).Both of these were published in 1963 and contain contributions which can still be considered as seminal in alkaloid chemical systematics today. Gibbs (2a),in reviewing the history of chemical taxonomy prior to 1963, reflected on the already-established value of a number of very THE ALKALOIDS, VOL. 50 0099-9598/98 $25.00

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simple chemical characters (lapechol, silica, calcium oxalate, cyanogenesis, etc.) and on the early work with discrete monoterpenoid secondary metabolites in Eucalyptus and Pinus. He pointed to the burgeoning number of phytochemical surveys that were adding comparative data to knowledge of distribution of compounds at an accelerating rate and gave some examples of where this information already seemed to show systematic promise. Looking to the future he made the following observations: In our own case we may be sure: that the pace will accelerate; that more and more plants will be investigated as travel becomes quicker and easier; that more and more chemicals will be discovered as techniques for recognition, isolation, and characterization improve; and that automation will be necessary to process the vast bulk of information resulting from all that activity. Will it be a better world for the chemo-taxonomist?

The 1960s were indeed an exciting time to be involved in alkaloid chemistry. It saw the beginnings of chromatography and spectroscopy which, collectively, were going to raise the speed of discovery and the potential for comparative analysis to new heights. This was also the time during which most of the major discoveries delineating alkaloid biosynthetic pathways were being made, producing a framework within which it was possible to distinguish biosynthetic relationships as opposed to following sometimes misleading structural relationships. Thus, Gibbs was certainly correct in anticipating that our capacity to isolate and identify alkaloids would greatly improve and that there would be an appreciable advancement in our capacity to perform comparative analysis. So did it become a better world for the alkaloid chemical taxonomist? Some 21 years after Gibbs posed the question, Harborne and Turner (3) were still far from certain of the true worth of alkaloids in systematics, summarizing their discussion of these metabolites as follows: . . . the various alkaloid classes have a rather variable distribution, family by family, within the flowering plants and their occurrence, as yet, offer only limited insight into familial and ordinal relationships. Nonetheless, the situation is one of considerable potential and undoubtedly alkaloids will become of greater systematic interest as more information accrues.

While Harborne and Turner were still striking an optimistic note it has to be acknowledged that many of the examples of potential value being cited by them were those already recognized at the “launch” of the subject 21 years previously! On the basis of their discussion a somewhat less optimistic view of the potential of alkaloids would have been just as arguable. In this chapter, I will review the situation again, with the benefit of some further 10 years of data.

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11. Alkaloids in Chemical Systematics: Laying Down the Rules

Hegnauer (2) was the first to confront the issue of just what was included, in a systematic sense, under the umbrella term “alkaloid.” Traditionally, the term “alkaloid” had been broadly employed to encompass basic nitrogencontaining compounds of natural origin; with “alkaloids proper” being a subgroup where the nitrogen was heterocyclic, where distribution was restricted (to within the plant kingdom), and where the compounds were associated with pharmacological activity. Hegnauer recognized that this definition would not be satisfactory for taxonomic purposes and proposed the following: Alkaloids are more or less toxic substances which act primarily on the central nervous system. They have a basic character, contain heterocyclic nitrogen, and are synthesized in plants from amino acids or their immediate derivatives. In most cases they are of limited distribution in the plant kingdom.

This more strict systematic definition does not permit the inclusion of many compounds that had traditionally been regarded as alkaloids and because of this two further alkaloid-related groups were recognized by Hegnauer:

The Protoalkaloids. Substances which d o not contain their nitrogen in a heterocyclic ring, but which otherwise fulfill the defined requirements of the systematic definition of an alkaloid. The protoalkaloid “concept” does create a problem in that a small, but significant, number of compounds such as colchicine (1)and stephenanthrine (2) give the appearance of being protoalkaloids. However, they are actually the products of fission of the

1

2

heterocyclic ring of true alkaloids and must themselves be treated as true alkaloids in systematic arguments, and differentiated from the true protoalkaloid.

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The Pseudoalkaloids (Alkaloids Irnperfecta). Substances where the nitrogen is not amino acid-derived and where the primary biosynthetic origin is from a nonnitrogenous precursor, usually either the mevalonate or acetate pathways. Steroidal alkaloids and diterpene-based alkaloids are both groups with wide occurrence; steroidal alkaloids are notable in the Apocynaceae, Solanaceae, Buxaceae, and Liliaceae, and in some reptiles, while diterpene alkaloids are abundant in some parts of the Ranunculaceae and in the Garryaceae. The pseudoalkaloids will not be further considered in this review.

Hegnauer’s definitions remain at the heart of alkaloid chemical systematics today. However, while it remains true that alkaloids are, as a group, generally bioactive, that part of the description that equated the definition with biological activity in the central nervous system is certainly no longer valid. Indeed, it is questionable whether any reference to biological activity is relevant to a taxonomic definition. The implied restriction in occurrence to higher plants (which it should be remembered was made in the context of a symposium on plant taxonomy) is, of course, not true and systematic value is certainly not to be considered as restricted to angiosperms. One point that was never satisfactorily resolved from Hegnauer’s original definitions was the position of glucosinolates and cyanogenic compounds. Given that the nitrogen in both these groups originates from amino acids it seems perfectly sensible that they should be treated as protoalkaloids. Another group of compounds which are often not considered as part of alkaloid systematics are the nonprotein amino acids. Again I can see no reason why they should not be considered as protoalkaloids. All three of these groups show distributions that are of interest to systematic analyses, notably the glucosinolates of the Cruciferae and the nonprotein amino acids of the Leguminosae. Unfortunately, space limitations mean that these groups will not be considered further here.

111. The Evolution of Alkaloids

A. THECHEMICAL MECHANISM The biosynthetic development of all of the major groups of true alkaloids are linked together by a common theme consisting of (a) the formation of a C-N bond through the interaction of an amine (usually primary) with a ketone (usually an aldehyde); and

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(b) cyclization of the resulting imino intermediate to give the heterocyclic system of a true alkaloid.

An example of this, for the formation of the l-benzyltetrahydroisoquinoline (1-btiq) system, is shown in Scheme 1. The amino acid and ketone donors that give rise to the major groups of alkaloids are listed in Table I. The essentials of the chemistry involved remains the same for each major alkaloid class.

B. ALKALOIDS AS EVOLUTIONARY EVENTS The repetitive nature of the initial stages of alkaloid biosynthesis from one major group of alkaloids to another was noted by McKey ( 4 ) , who made some interesting observations regarding the possible evolutionary significance of this thematic uniformity. The thrust of his argument was that the change from formation of 1-btiq alkaloids (Scheme 1) through the condensation of dopamine and 3,4-dihydroxyphenylacetaldehyde;to the formation of ipecoside (3) from dopamine and secologanin acid (the ketone donor), and to the formation of complex indole alkaloids like strictosidine (4) from tryptamine and secologanin, were changes relating to substrate, and not to differences in the fundamental chemistry or the biosynthetic mechanisms involved in the formation of each alkaloid group. This being the case, he posed the question of whether the degree of genetic evolution necessary to “jump” from one major alkaloid class to another was necessarily a major evolutionary event? Indeed, is the difficulty in changing substrate any greater than that required to produce structural

Tyrosine

SCHEME 1. Condensation of amine and ketone to form a “true” alkaloid.

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TABLE I PRECURSORS OF SOMEOF THE MAJOR CLASSES OF ALKALOIDS Alkaloid Type

N-Source

C = 0 Source

Amaryllidaceae

Tyrosine/phenylalanine

1-Benzylisoquinoline

Tyrosine/phenylalanine

Emetine type Indole-monoterpene Betalains Tropane Pyrrolizidine Quinolizidine

Tyrosine Tryptophan Tyrosine or proline Ornithine Ornithine Lysine

Phenylbenzaldehyde from tyrosine/ phenylalanine Phenylacetaldehyde from tyrosine/ phenylalanine Secologanin Secologanin Tyrosine (betalamic acid) Ornithine (same molecule) Ornithine Lysine

~

~

rearrangements within a class of alkaloids? For example, what is the relative evolutionary difficulty of the jump from forming the skeleton of ipecoside (3) to forming that of strictosidine (4), where tyrosine is replaced by tryptophan as the N-donating component, in comparison with that of the conversion of the tetrahydroprotoberberine ( 5 ) into the benzophenanthridine (6), a process that requires fission of the C-6-N bond in 5 and a recyclization of C-6 to C-12? Traditionally, we have always tended to think of the formation of each of the major skeletal classes of alkaloids (as noted in Table I) as being highly significant evolutionary events, while the structural diversification that has gone on within alkaloid types has been viewed as having significance at a relatively lower evolutionary or taxonomic level. To put it simply, we assume that the generation of the 1-btiq nucleus (Scheme 1) is a more weighty event than the conversion of protoberberine ( 5 ) to benzophenanthridine (6).McKey’s observations are a warning that we should keep an open mind on this point. Cell cultures of species from a wide range of families with no record of quinolizidine alkaloid expression could be induced to synthesize quinolizidine alkaloids, implying the presence of dormant genes for their synthesis in these species (5). How widespread are such dormant genes and do they occur for other classes of alkaloids? If they do occur, and are widespread, then it has ramifications for the taxonomic use of alkaloid distribution as we are confronted with species able to “store” biosynthetic information and “switch” it back on, at a point evolutionarily distant from where it was “switched” off. Disappointingly, to date, this work does not appear to have been pursued.

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C. EVOLUTIONARY ORIGINS

There would be great benefit to establishing an evolutionary origin for alkaloids. However, while it does seem safe to say that a major radiation of true alkaloids has occurred in the Angiospermae, there are very significant secondary areas of production found in the fungi ( 6 ) ,in marine organisms (7), and in animals (8).A greater proportion of the alkaloids found in these other sources appear to be, biosynthetically, pseudoalkaloids or compounds not formed through the mediation of the classical Schiff base, Mannich condensation route of the major Angiosperm classes (Scheme 1). It can now be safely assumed that the alkaloid and alkaloid-like compounds found today in living organisms are polyphyletic in origin, and that, accordingly, we can consider the alkaloids of higher plants in isolation from other sources. D. DRIVING FORCES MEDIATING PRODUCTION?

It is now common to think of alkaloids in terms of defensive agents against herbivores or other potentially detrimental organisms (9,10), which gives them an evolutionary ruison d'2tre. However, other roles cannot be ruled out for many alkaloids which may also involve interactions with extrinsic factors (e.g., the betalains as flower pigments for pollinator attraction) or some as yet unrecognized physiological role. The fact that we

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assume a usefulness for alkaloids that relates to the external environment is another complicating factor for systematic use, as it implies that external forces will be able to influence the expression of alkaloids. If this occurs, and the alkaloid-production capacity of plant species is as plastic as is suggested by the observations of McKey ( 4 ) and Wink and Witte ( 5 ) , then the likelihood of production of a heterogeneous array of final products seems rather high. Gottlieb ( 2 2 ) and Kubitzki and Gottlieb (22,23) have suggested that, in higher plants, the evolution of different classes of metabolites has been linked to the occurrence of an abundance or overabundance of the precursor metabolites. Metabolites of the shikimate pathway were assumed to predominate in putatively more ancient lineages of higher plants, and this has led to the idea that the 1-btiq alkaloids were the first group to arise, with tyrosine and phenylalanine as the first superabundant nitrogenous substrates. This same shikimate pathway could also give excesses of anthranilic acid and tryptophan (Scheme 2), while, more recently, other amino acids not originating from the shikimate route, notably lysine and ornithine, became available as nitrogen sources. This is an interesting concept which, if credence is given to the proposals made by McKey, suggest that the occurrence of excess precursor metabolites would be a key feature in governing alkaloid production and distribution. If this were so, then the truely important systematic biochemical markers would be at the primary metabolic level and would be concerned with how metabolic excesses manifested themselves as different substances in different taxa. As things stand, the unpalatable truth is that while we may now understand a great deal about the mechanisms of alkaloid formation, we are still struggling to understand their evolution and distribution. The fact that we are clearly able to associate certain families with the occurrence of particular skeletal types of alkaloids seems to support the normally held view that substrate changes represent the “quantum leaps.” However, if the reader remains in doubt that our understanding of alkaloid evolution is still at best rudimentary, it is recommended that they read the short discussion of this problem given by Robinson ( 9 ) . This may now be some 18 years old, but the problems more-or-less all remain the same!

IV. Handling Alkaloid Data in Systematic Studies Interpreting the systematic value in a series of data on the distribution of alkaloids, or any other class of secondary metabolite, depends primarily on an understanding of the biosynthetic mechanisms through which those

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Carbohydrate

Shikimic acid

n Chorismic acid

Phenylpyruvic acid

Anthranilic acid

Tyrosinel phenylalanine

Tryptophan

Cinnamic acid

SCHEME 2. The shikimic acid pathway: tyrosine/phenylalanine, anthranilic acid, and tryptophan are each the starting point for major groups of true alkaloids.

alkaloids have been formed. While our understanding of alkaloid biosynthesis is still imperfect, we are fortunate today in that there is sufficient substantiated information to allow the systematically vital biosynthetic steps of formation to be assumed for most, newly isolated compounds. However, those assumptions will only reflect the chemical mechanisms involved in the biosynthetic process and need not reflect comparability in the enzymes responsible for catalyzing those processes. At the enzyme level, the amount of information available is still very limited and this represents a severe impediment to the systematic use of alkaloids in a number of important cases.

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Major problems that have to be confronted in interpreting alkaloid distribution information were first explicitely enumerated by Hegnauer (24). These include: Parallelism. The occurrence of structurally closely allied alkaloids in disjunct parts of the plant kingdom (biosynthetic origins not resolved). Convergence. Structurally allied alkaloids, elaborated from the same biogenetic pathway, occurring in seemingly unrelated parts of the plant kingdom. Divergence. Taxa which are regarded on other criteria as being closely related, but which accummulate alkaloids with obviously different biosynthetic origins. Homology. Related taxa using the same biosynthetic pathway, but with the expressed products being chemically very different. In addition, two other important points must be recognized. First, that the biosynthetic process is reticulate, that is a product, or even an intermediate, can often be generated by more than one sequence of steps. Second, that evolutionary advance can, sometimes, be measured on the basis of the appearance of new metabolites, leading either to increasing complexity or to the production of new skeletal types. However, it is equally possible for an evolutionary advance to be manifested by loss of some part of a biosynthetic pathway, which will be revealed in a simplification of the metabolic profile. Thus, while the structure of a compound is known, and the biosynthetic process whereby it is formed is understood, its systematic value will still be ambiguous unless it happens to be the climax product of a pathway. This is not a phenomenon that is unique to secondary metabolites, but is a problem that also afflicts interpretation of morphological, anatomical, cytological, and enzymological data. In systematic studies the ambition is, of course, to find the patterns that occur in what at first sight may appear a chaotic jumble of data and interpret them so as to throw light on phylogenetic relationships and evolutionary strategies. In the optimal organization of a set of chemical structures for systematic use, decisions need to be made on which of the criteria which could be used are most appropriate and relevant. Gottlieb and his co-workers have devoted an enormous amount of time and effort to the development of procedures for the standardization of chemical data for use in taxonomic analysis. This has resulted in their formulation of a system of micromolecular systematics which they have applied to the investigation of many groups of alkaloids (15). Two particular criteria have been used. (1) To attempt to identify the “most highly advanced skeleta in biosynthetic sequences” that occur within a taxon and link these to an analysis of the relative probability of the occurrence of each skeletal type in a group of taxa (the result being expressed numerically and referred to as RPOx); and

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(2) To produce a computed value for the degree of complexity found within key steletal types (“quantification of structural and substitutional characteristics of mapped compounds”), by assigning points to specific modifications of the skeleton (number of oxidations, reductions, etc.). This is usually referred to as RPOy. The RPOx and RPOy scores are plotted against one another and taxonomic significance is attributed to the placing of individual taxa on that plot. The methods adopted by Gottlieb have not been well received. The problem that arises is that the rigid rules used in generating the two RPO values are generally unable to cope with the reticulation of. steps that certainly occur in the building of a series of biosynthetically complex molecules. In particular, RPOy is incapable of coping with different oxidation/ reduction patterns within a skeletal type that happen to take the same number of steps (they finish up with the same numerical score). A further problem is that the methodology fails to cope with the bipolar nature of a chemical marker; that is, it does not answer the question posed previously: Is the expression of compound A indicating the evolution of a new biosynthetic step not present in the progenitor, or does its expression occur because of the loss of part of the biosynthetic mechanism of a progenitor with a more extensive biosythetic matrix in which compound A was an intermediate? The failure to give such an insight is not a specific criticism of the micromolecular systematics approach of Gottlieb, it is a general problem that covers any discussion of evolutionary relationships based on expressed secondary metabolites. Currently, it seems to the author, that the type of system adopted by Gottlieb requires unacceptable assumptions on the systematic value of the expressed metabolite. Chemical markers in systematics suffer from all of the problems of their predecesors and it must be recognized that the interpretation of their phylogentic significance must be attempted with the same philosophy. Chemical systematics remains as much an art as a science. Modern numerical methods (cladistics) do offer an option that reduces personal bias, but to work effectively, cladistic analyses need a degree of completeness in the information used that is still rarely satisfied with secondary metabolites. Currently then, as in the past, the most appropriate use of chemical data appears to be to test phylogenies that have arisen from the interpretation of more complete nonchemical data sets. In this context, the pictorial systems generated, most notably by Dahlgren (16) (Figure 1) and Huber (19, offer a very useful framework around which to examine the distribution of alkaloids and assess their systematic significance. Examples of the use of Dahlgren’s “bubble diagrams” will be shown below. The alternate

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FIG.1. Diagrammatic representation of the Angiospermae (after Dahlgren (16)). Named super orders are major sources of “true” alkaloids.

approach of constructing phylogenies, which rest primarily on alkaloids or other metabolites without taking into account other insights (18), and inevitably with an incomplete knowledge of distribution of secondary metabolites, does nothing but harm to the perceived usefulness of such characters among the taxonomic community (29).

V. Systematically Significant Distributions of Alkaloids in Higher Plant Taxa A. MAJOR TYROSINE/PHENYLALANINE-DERIVED ALKALOIDS Two main groups will be considered here, those which are considered to have arisen from a C6-C2-N-C2-C6 precursor (the classical 1-btiq alkaloids), and those arising from a C6-C2-N-C1-C6 precursor. It is now firmly established that 1-btiq alkaloids are formed from an amine (C6C2N)derived from tyrosine (or phenylalanine?) and a phenylacetaldehyde (C6G) which originates from one or other of these amino acids (cf. Scheme 1).The subsequent further dimerization and/or cyclizations of the tricyclic 1-btiq with oxidation of the resulting skeleta, then bond fissions and rearrangements generate a huge diversity of final structures. The most

549

14. ALKALOID CHEMOSYSTEMATICS

recent of a series of reviews of just one subset of 1-btiq alkaloids, the aporphinoids, reveals that well over 600 discrete structures are now known (20). Scheme 3, which has been taken from an early systematic study made by Rezende et af., (21) shows the relationships between some major subclasses of alkaloids that arise from the 1-btiq precursor. There has long been widespread agreement among systematicists, based on nonchemical data, that most of the families now known to be rich in 1btiq alkaloids (Annonaceae, Aristolochiaceae, Berberidaceae, Hernandia-

Dimerlc 1-ETIQ

-

1-BTIQ

Oxoapmphmes

Anstolochic acids

Aporphines

+ isopawnes culannes etc

Benzophenanthndine

Tetrahydroprdoberberines

0 Morphinan

Hasuban

Erylhrinane

Dibenzazonines

SCHEME3. Major structural classes within the 1-benzyltetrahydroisoquinoline“family” of alkaloids. (Reprinted with modifications from Biological Systematics and Ecology, vol. 3, by C. M. A. d. M. Rezende, 0. R. Gottlieb, and M. C. Mary, p. 63, Copyright (1975), with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington OX5 lGB, UK (21).

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ceae, Lauraceae, Magnoliaceae, Menispermaceae, Monomiaceae, Nymphacaceae, and Ranunculaceae) are closely phylogenetically associated. These were traditionally placed in large suprafamilial taxa, such as the Polycarpicae or Ranales, which many regard as among the most “primitive” extant angiosperms. Hegnauer (2) and Kubitzi (22) were perhaps the first to recognize the 1-btiq alkaloids as markers for the Polycarpicae. It is noteworthy that from the outset Hegnauer was well aware of the limitations of the data that he was interpreting (2). He recognized that while these alkaloids were an obvious metabolic feature of the Polycarpicae, their distribution was patchy and that other types of alkaloids also occurred as well as nonalkaloidal metabolites of systematic value, such as the neolignans. He suggested that four metabolic profiles could be recognized among the families of the Polycarpicae: (a) those containing only 1-btiq alkaloids; (b) those containing 1-btiq and other types of alkaloids; (c) those containing only other alkaloid types; and (d) those that were essentially alkaloid free. On the basis of these profiles, Hegnauer proposed that their were two different possible evolutionary scenarios for the Polycarpicae that would explain the observed distribution of secondary metabolites. The first, in which the progenitor families were 1-btiq producing and that the ability to produce these alkaloids was then lost, sometimes to be replaced by the production of other alkaloids. The second would be where the progenitor families were alkaloid-free and alkaloid production was evolved. In order to use the alkaloid data to assess these two possibilities we had to wait, he considered, until we were unequivocally able to place a family in a given group. This is still not possible. However, the significance of the 1-btiq alkaloids as markers delineating taxa has been recognized by a number of modern systematicists such as Thorne (23) (families of his superorder Annoniflorae) and Dahlgren (24) (superorders Magnoliiflorae, Nymphaeiflorae, and Ranunculiflorae). The distribution of 1-btiq alkaloids as it relates to Dahlgren’s classification is shown in Fig. 2. In considering the impact of 1-btiq alkaloids on currently accepted phylogenic relationships, it must not be overlooked that they were instrumental in the transfer of the Papaveraceae and Fumariaceae from the Rhoeadales, where they had historically been situated, and in which they were the only alkaloid-producing families, into an association with the Polycarpicae and, in particular, with the Berberidaceae (a position adopted in all modern phylogenetic schemes, Ranunculiflorae, Fig. 2) (2). The Papaveraceae are perhaps the most prolific of all the 1-btiq alkaloid-producing families and are noteworthy for the capacity of most species to elaborate derivatives of

14. ALKALOID CHEMOSYSTEMATICS

55 1

FIG.2. The occurrence of 1-benzyltetrahydroisoquinolinealkaloids among orders in Dahlgren’s system of classification. 1 = tricyclic 1-btiq alkaloids and dimers, 2 = proaporphines, 3 = aporphines and derivatives, 4 = protoberberines, 5 = protopines, 6 = benzophenanthridines, 7 = morphinans and derivatives, 8 = dibenzazones, 9 = rhoedines, 10 = pavines and isopavines.

tetrahydroprotoberberines, such as benzophenanthridines and protopines, which require fission of N-C bonds (see 5 to 6 and Scheme 3). This realignment should be regarded as a major success of alkaloid systematics. There has been no such resolution for the Rutaceae, which Hegnauer (2) also noted as a source of 1-btiq alkaloids, many of which were shared with the Papaveraceae. Extensive further studies on the family (see Waterman (25),and references cited therein) has shown that these alkaloids are ubiquitous in a small number of genera ( 5 out of about 100 which have been studied to date) and appear to be totally absent from others. Other genera of Rutaceae produce a diverse range of alkaloids based on anthranilic acid as the nitrogen source (25).These anthranilate alkaloids remain as strong systematic markers for the family, and, together with the highly oxidized tetranortriterpenes (limonoids and quassinoids), make the Rutales one of the chemically most well defined of orders. Clearly, the situation in the Rutaceae is different from that of the Papaveraceae. A recent survey (26) revealed that outside the Annoniflorae and Nymphaeiflorae of Thorne (Magnoliiflorae, Nymphaeiflorae, and Ranunculiflorae of Dahlgren), what appear to be structurally normal 1-btiq-derived alkaloids have been recorded from Alangiaceae, Araceae, Buxaceae,

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Caprifoliaceae, Euphorbiaceae, Leguminosae, Liliaceae, Rhamnaceae, Rutaceae, Sapindaceae, and Umbelliferae! Their presence, albeit in most cases as isolated and minor components of the overall secondary metabolite profile, in such a diverse range of taxa (Figure 2) is a clear illustration of the capacity of many different taxa with diverse phylogenetic affinities to express a major structural class of secondary metabolites. If the significance of the presence of 1-btiq alkaloids in the Rutaceae to the relationships of that family remains unanswered, it is possible to assert that 1-btiq alkaloids are valuable in intrafamilial systematics. The large Old World genus Euodia was recently split up by Hartley (27),who recognized it was polyphyletic. A subset of species were placed in the genus Tetradium, and were cited as being closely allied to Phellodendron and Zanthoxylum, which are two of the 1-btiq producing genera of the family. Useful support for Hartley’s revision would come from the identification of 1-btiq alkaloids in Tetradium species. This was duly achieved, with the isolation of a benzophenanthridine from T.glabrifolium (28) and a protopine from T. trichotomum (29). The presence of aporphine alkaloids in the small family Eupomatiaceae (30)proved to be valuable in confirming the close link between that family and the Annonaceae. However, an attempt to detect systematically useful patterns in the common 1-btiq alkaloids of the Annonaceae relating to intrafamilial classification failed to yield significant results (32). The (C6-C2-N) part of the C6-C2-N-C1-C6 alkaloids are formed from tyrosine, but with no additional oxidation occurring on the aromatic ring, while the C6-C1 moiety appears to arise from phenylalanine which suffers side-chain reduction and double oxidation of the aromatic ring (32).Norbel-’ lidine (7) is considered to be the bicyclic progenitor that gives rise to more complex tri- and tetracyclic alkaloid types, such as lycorine (8) and galanthamine (9); the oxidative coupling driven cyclization reactions being

7

8

9

analogous to those seen in the ring-closure associated with the 1-btiq alkaloids. The occurrence of these alkaloids (33) is restricted to the family Amaryllidaceae, which forms part of the Liliiflorae. Dahlgren’s “bubble

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diagram” of higher plant taxonomy (Fig. 2) places the 1-btiq rich orders of the Dicotyledonae (Magniiflorae, Ranunculiflorae) close to the interface with the Monocotyledonae, and cites evidence for links between the Magnoliiflorae and monocotyledons (24). The presence of these two groups of biosynthetically analogous alkaloids in such close proximity across the monocot/dicot divide is clearly supportive of that proposed link. This is given further credence by the occurrence of colchicine (1) in the Liliaceae. Colchicine can be thought of as a C6-C2-N-C3-C6 structure, based on tyrosine as the source of the nitrogen (as C6-C2-N) with cinnamic acid (C3-C6) arising from phenylalanine (32).

B. MAJORTRYPTOPHAN-DERIVED ALKALOIDS The metabolites formed by the initial combination of tryptophan and the monoterpene secologanin are numerically the largest single group of true alkaloids and also have the greatest structural complexity. There are also a wide range of simpler indole alkaloids such as the 0-carbolines, most of which are more widespread in higher plants (34). One notable group are the canthinones (e.g., 10)which are found most commonly in the closely allied families, the Simaroubaceae and Rutaceae (Rutiflorae, Fig. 2) (25,35).

10

Here we will concentrate on the indole-monoterpene group for which the major sources are the Loganiaceae, Apocynaceae and, in part, the Rubiaceae, which all form part of the Gentianales (Gentianiflorae, Fig. 2) of Dahlgren (26). There is a much less extensive second proliferation of this alkaloid type in the Corniflorae (Cornales-Alangiaceae, Nyssaceae, and Icacinaceae). These two superorders stand side-by-side in Dahlgren’s system, and the Alangiaceae is noteworthy as also being a minor source of 1-btiq alkaloids, while the Rubiaceae also combines secologanin with tyrosine in the ipecoside-type alkaloids (e.g., 3). Compared to the 1-btiq alkaloids, the structural complexity achieved by the indole-monoterpenoids is quite staggering (34). Making biosynthetic sense of these compounds has been a major achievement in alkaloid chemistry to which many eminent scientists have contributed and which has been reviewed many times in “The Alkaloids” series. Particularly important has been the recognition of a series of modifications of the secologanin moiety

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after its initial bonding to the tryptophan acyclic nitrogen (36). Based on these skeletal modifications Hesse and co-workers (36-39) have evolved a biogenetic classification recognizing a wide range of subgroups formed on these different secondary modifications. Some examples of this classification are shown in Scheme 4. What has become clear as a result of the accumulation of data and its biosynthetic arrangement is that the Apocynaceae is particularly versatile in its capacity to evolve new modifications of the monoterpene nucleus, with the relatively simple corynanthean and highly modified plumeran skeletons being most common. In the Loganiaceae, the capacity for modification of the monoterpene was much diminished and the strychnan skeleton has become the most widespread. The Rubiaceae provided the least diversification, with the corynanthean type again predominating, but showed unique versatility in the capacity to combine secologanin with tyramine rather than tryptamine (emetine alkaloids), and to modify the indolemonoterpene into quinoline alkaloids such as quinine.

Plumeran type

Corynanthean type

z J

Quinine type

Seco-loganin

Strychnan type

SCHEME4. Incorporation of seco-loganin with tryptophan to give the indole-monoterpene alkaloids. Some examples of modification of the seco-loganin skeleton.

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14. ALKALOID CHEMOSYSTEMATICS

The intimate knowledge of the biosynthetic processes involved allowed Hesse et af.to analyze the occurrence of specific steps within those biosynthetic processes. A particularly good example is their analysis of modifications arising from the formation of an extra bond originating from either C-16 or C-17 of the corynanthean skeleton, and the distribution of such compounds across various taxa within the three families (37). This is reproduced here as Table 11. The studies undertaken by Hesse et af. culminated in a review published in 1983 (39). Their findings were instrumental in a taxonomic revision of the genus Tabernaemontana which saw many of the genera previously recognized as distinct in the subfamily Plumeroideae being submerged in Tabernaemontana. More recently, Hesse et al. (40) have suggested that the further data now available is perhaps more in sympathy with the “old” classification of the Plumeroideae. Interestingly, they reflect on how some of the species examined appear to have alkaloid content that shows “extraordinary sensitivity to environmental influences such as soil, light intensity, etc.” This distribution of indole-monoterpene alkaloids is clearly of value in confirming the relationship between these three families and can also give useful indicators of the phylogenies of major intrafamilial taxa (see Table 11). However, the interpretation (37) of alkaloid data as supportive of the phylogeny expressed in Fig. 3 is arguable. What is interesting is the apparent absence of the indole-monoterpene alkaloids in the proposed climax family, the Asclepiadaceae, where cardenolides and pregnane-based steroids predominate (41). These compounds are also found in the Apocynaceae, where they seem to replace the typical alkaloids in some taxa (38). TABLE I1 ABUNDANCE OF T W O MODIFICATIONS OF THE CORYNANTHEAN LOGANIACEAE, APOCYNACEAE, A N D RUBIACEAE (TAKEN FROM KISAKUREK AND HESSE)(37)

COMPARISON OF THE RELATIVE

NUCLEUS IN

THE

Family Subfamily C-16% C-17%

J 17J

16

17

LOG Gel Str APO Car Tab Als Rau RUB

78 0 82

55 87 95 68 29 0

22 100 18 44 13

5 32 71 100

LOG = Loganiaceae, APO = Apocynaceae, RUB = Rubiaceae, Gel = Gelsemieae, Str = Strychneae, Car = Carisseae, Tab = Tabernaemontaneae. Als = Alstonieae, Rau = Rauvolfieae; Arrows point to positions of linkage for C-16 and (2-17.

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Asclepiadaceae

t

Apocynaceae

Rubiaceae Loganiaceae

Fic. 3. A proposed phylogeny for the Loganiaceae and allied families based on the occurrence of indole alkaloids (37).

C. THEBETALAINS The betalains are probably the single best example of the value of alkaloids, or any secondary metabolites, as taxonomic markers. Early reviewers were confused as to what they were; for example, in Alston and Turner ( I ) they were included in a chapter on miscellaneous compounds, while in Swain (2) they were mentioned only in the chapter on anthocyanins. The confusion occurs in that they fail Hegnauer’s original definition of an alkaloid on the grounds of their lack of biological activity. Yet in a biosynthetic sense they are clearly alkaloids in that they originate entirely from amino acid precursors. The red-colored pigment betanidin (11) is derived from dopa, complete with the carboxylic acid carbon, and betalamic acid (12), which is itself the product of a complex rearrangement of dopa (Scheme 5). The combination of dopa and 12 to form 11 employs the “standard” condensation and cyclization reactions that typify all of the major groups of true alkaloids. A second group of mainly yellow compounds, the betaxanthins, are derived by the linking of 12 with proline rather than tyrosine. Certainly Cordell (32) regarded them as alkaloids, and I can see absolutely no reason not to do so. The systematic significance of the betalains was first reviewed in depth by Mabry (42). Nearly 30 years ago he noted that their distribution was restricted to a number of families belonging to the order Centrospermae, these being the Chenopodiaceae, Amarantaceae, Portulacaceae, Nyctaginaceae, Phytolaccaceae, Stegnospermaceae, Aizoaceae, Basellaceae, Cactaceae, and Didieraceae. Two other families generally considered to be part of the Centrospermae, the Caryophyllaceae and Molluginaceae, did not yield betalains. This situation remains unchanged. The presence of betalains has been used to decide the affinity of difficult and ambiguous taxa (43), and has been used to support an argument for the recognition of two suprafamilial taxa, the betalain-containing Chenopodiineae, and the betalainfree Caryophyllinae (44).

14. ALKALOID CHEMOSYSTEMATICS

557

COOH

H2

a

HOOC

/

A

l

H

O

/

,

HOOC

COOH

Lo

11 fission

l2

-

\ p

;

H

$fH HOOC

HOOC

Y

COOH

COOH

H

HOOC

N Y

OH N

Y

SCHEME 5. The origin of the red betalain alkaloid betanidin (11) and the formation of betalamic acid (12) from 3,4-dihydroxyphenylalanine.

The great success of the betalains has come from the restriction of their distribution in higher plants to this one group, apparently without exception, although similar substances do occur in fungi (45). It has been stated ( 3 ) that “probably no group of secondary compounds has provided so much taxonomic impact at the family level (and phyletic controversy) as have the betalains” ( 2 2 ) . The controversy arises from the attempts (44) to produce a phylogeny that reflects the betalains supplanting the widely distributed anthocyanin pigments in the Caryophylliflorae or Centrospermae. This argument continues. Within the order, the distribution of the betalains has proved only of limited value in resolving relationships between and within families (45). Dahlgren (26) retains both betalain-containing and betalain-free families in his Caryophylliflorae (Fig. l), which are placed close to the 1-btiq producing taxa Ranunculiflorae and Magnoliiflorae. Treating the betalains as alkaloids that derive from tyrosine raises the possibility of a link between the Caryophylliflorae with the 1-btiq producing families. This has been commented upon by Waterman and Gray (46), who drew attention to the ability to “split” the aromatic ring of tyrosine in betalain production with

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the capacity of some species of Annonaceae to "split" the aromatic A-ring of aporphines to form aza-anthracenes (20). Given the existence of other circumstantial evidence suggesting an affinity between these taxa (flavonoid profiles, similarities in plastids of sieve elements) ( 4 3 , this does seem to be an hypothesis worthy of further investigation. D. ANTHRANILATE-DERIVED ALKALOIDS OF THE RUTACEAE

That the Rutaceae were prolific in producing alkaloids was recognized by Price (in Swain (2)).The most widespread group of alkaloids, and one which can be regarded as characteristic of the Rutaceae, are those based directly on the combination of anthranilic acid with other substrates; most commonly those which are polyketide in nature. The resulting skeleta, such as the 2- and 4-quinolones and acridones, are often further elaborated by the addition of mevalonate units with the subsequent formation of furan or pyran ring systems (i.e., furoquinolines, 2- and 4-pyranoquinolones). The formation and distribution of these alkaloids has been considered in a number of reviews, (25,4830) and will not be dealt with here. While these are not the only alkaloids found in the Rutaceae, others include the 1-btiq group discussed above and tryptamine-derived canthinones such as 10 (shared with the Simaroubaceae), they are the only group that are good family markers. Their occurrence in taxa of uncertain affinity, such as the

I F'yrrolizidine

14

Amino-pyrrolizidine

SCHEME6. The route to tropane and pyrrolizidine nuclei from the amino acid ornithine.

14. ALKALOID

559

CHEMOSYSTEMATICS

Spathelioideae and Flindersoideae, has been important in confirming these taxa as part of the family (48).

E. ALKALOIDS ORIGINATING FROM ORNITHINE AND LYSINE (TROPANES, PYRROLIZIDINES, AND QUINOLIZIDINES) These three large groups of alkaloids are treated together here as there are shared features in their biosynthesis which both draw them together and distinguish them from the alkaloid types discussed previously. The “common” route to the pyrrolidine nucleus, shared by both the tropane and the pyrrolizidine alkaloids, is demonstrated in Scheme 6. It proceeds through the conversion of L-ornithine to putrescine and then via specific deamination/oxidation to the aminoaldehyde 13 which, through the juxtaposition of aldehyde and amine produces the system required for an intramolecular condensation to the pyrrolidine ring of tropanes. Alternatively, a second molecule of putrescine can be incorporated to yield an imine

Lupinine

Cytisine

Pohakuline

(2 x lysine)

(3 x lysine)

(3 x lysine)

Matrine

Sparteine

Ormosanine

(3 x lysine)

(3 x lysine)

(4 x lysine)

FIG.4. Combinations of two to four lysine units to yield a range of quinolizidine alkaloids.

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intermediate such as 14 which can, in turn, cyclize to give the pyrrolizidine nucleus. Most pyrrolizidines are characterized by the presence of a hydroxymethyl group at C-1; a few have this replaced with an amino group. The generation of the piperidine ring, and of the quinolizidine nucleus, commences with L-lysine rather than L-ornithine and seems to parallel exactly the formation of the pyrrolizidine. However, the addition of further lysine-derived units to give tri- and tetracyclic quinolizidines and allied compounds (Fig. 4) is confined to the quinolizidines. All three major alkaloid types (tropanes, pyrrolizidines, and quinolizidines) exhibit distribution patterns that include a number of disjunct taxa in the Angiospermae. The taxonomic groups in which each type occurs are listed in Table I11 and are plotted on a Dahlgren “bubble diagram” in Fig. 5. The tropane alkaloids show a range of different structural features including family-based variation in stereochemistry at the point of esterification, the presence or absence of the carboxylic acid on the tropane and

DISTRIBUTION

OF

TABLE 111 TROPANE, PYRROLlZlDlNE A N D QUlNOLlZlDlNE ALKALOIDS IN THE ANGIOSPERMAE

Taxonomic group Commeliniflorae-Poaceae Liliiflorae-Orchidaceae Asteriflorae- Asteraceae Fabiflorae-Leguminosae Gentianiflorae- Apocynaceae Gentianiflorae-Rubiaceae Malviflorae-Euphorbiaceae Malviflorae-Elaeocarpaceae Myrtiflorae-Rhizophoraceae Primuliflorae-Sapotaceae Proteiflorae-Proteaceae Ranunculiflorae-Ranunculaceae Ranunculiflorae-Berberidaceae Rutiflorae-Erythroxylaceae Rutiflorae-Linaceae Santaliflorae-Celastraceae Solaniflorae-Boraginaceae Solaniflorae-Convolvulaceae Solaniflorae-Ehretiaceae Solaniflorae-Solanaceae Violiflorae-Cruciferae a

=

Tropanes (5132)

+

+ + + +

Pyrrolizidines (53,541

Quinolizidines (55.56)

+C

+b +a,b + a,c +b

+

++ +

+b +C

+b +a

+

+ +

+C

++

++ +

+b +a,b

+b

macrocyclic ester subgroup; b = aryl and/or aliphatic esters; c = 1-amino type

+

14. ALKALOID

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561

FIG.5. Distribution of ornithine and lysine derived alkaloids in the orders of Dahlgren’s classification. A = tropanes, B1 = pyrrolizidines with macrocyclic di-esters, B2 = pyrrolizidines with simple esterification, B3 = I-amino-pyrrolizidines, C = quinolizidines. Large lettering denotes major sources.

FIG.6. Distribution of shikimate-derived and ornithinellysine-derived alkaloids in the Angiospermae. Dark dotted super orders are main sources of shikimate alkaloids, darkish horizontal stripes are lesser sources of shikimate alkaloids, grey dotted are major sources of lysine/ ornithine alkaloids, vertical lines are lesser sources of lysine/ornithine alkaloids. Intermediate intensity hatched super orders contain both alkaloid types.

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a range of different esterifying acids, but these are not differentiated here. Normal 1-hydroxymethylpyrrolizidines and amino pyrrolizidines are treated as two groups, and the former are further differentiated by the presence or absence of a macrocyclic diester or simple esterification at C1. Quinolizidines are treated as a single group. It seems unsound to treat the three types of product independently of one another. The Solaniflorae (Solanales and Boraginales) illustrate this point perfectly. While the Solanales is obviously a major site for proliferation of tropane alkaloids, it also produces quinolizidines. The Boraginales, by contrast, are one of the major sources of pyrrolizidines. The conjunction of Asterales and Boraginales (Fig. 5) is clearly supported by the cooccurrence of the pyrrolizidines in both. The Fabales (Leguminosae) produces both pyrrolizidines and quinolizidines, while in the Ranunculales, quinolizidines are reported from both the Berberidaceae and the Ranunculaceae, and pyrrolizidines also occur in the latter family. Both tropanes and pyrrolizidines are also recorded from the Orchidaceae in the Liliiflorae. It has been suggested that the genetic apparatus needed for their production is widespread, but generally dormant (5). Given this possibility, and their sporadic, but widespread occurrence, it seems impossible to produce any single unifying taxonomic hypothesis for the producers based on the distribution of these alkaloids. It is highly probable that the pyrrolizidines at least have evolved more than once, as is graphically illustrated by the similarities among the alkaloids produced by the Orchidales and the Boraginales. Likewise the tropanes, where the Solanales and Geraniales are major

FIG.7. Diagrammaticrepresentation of the putative evolution of shikimate based alkaloids in the Angiospermae from an origin in the Magnoliiflorae. A = formation of the Amaryllidaceae alkaloids, B = betalains, C = involvement of anthranilic acid, D = major developments from tryptophan. The “thickness” of the lines associated with each precursor (bottom of diagram) indicates their contribution at that point.

14. ALKALOID CHEMOSYSTEMATICS

563

centers of production that seem only very distantly related. By contrast, the quinolizidines have only one center of proliferation, in the Fabales, and where they occur elsewhere, as in the Berberidaceae and Ranunculaceae, the isolated alkaloids are also present in the Rutales. However, even here there are reports of quinolizidines from the Solanaceae, a taxon distant from both the Fabales and Ranunculales.

VI. Concluding Comments In Figure 6, the distribution of all of the true alkaloid groups discussed in this review have been plotted using the criterion that the nitrogen source is either: (a) derived from an amino acid originating from shikimic acid (tyrosine, phenylalanine, tryptophan, and anthranilic acid); or (b) derived from lysine or ornithine which originates from the tricarboxylic acid cycle. While not entirely convincing, it is possible to imagine a progression in the shikimate-based alkaloids which sees a development from the tyrosineand phenylalanine-based products, which undergo three distinct evolutionary developments (normal 1-btiq, Amaryllidaceae alkaloids, and betalains), through a short-lived use of anthranilic acid, into the use of tryptophan with the major development of the indole-secologanin group in the Gentianales. We have made an attempt to express this diagrammatically in Scheme 7, which should be viewed in conjunction with Fig. 6. As noted previously it does not seem possible to generate such an evolutionary continuity for the lysinelornithine based alkaloids. The basic problem we face now is that alkaloid chemical systematics has not changed greatly from that of 30 years ago; that is we still have so imperfect a knowledge of these substances that we can still only make rather imprecise predictions about their distribution. Moreover, we are also confronted with several other major sources of concern. We realize that extrinsic factors (environmental, ecological) can have a considerable impact on what we observe. There is the possibility that rather than being lost, the genes responsible for alkaloid biosynthesis become temporarily “silent” and can, in evolutionary time, be switched back on and off, perhaps repeatedly. There is also the possibility that seemingly major biosynthetic events in alkaloid formation, like substrate switching, may actually be no more unusual, in evolutionary terms, than new modifications within a single pathway.

564

WATERMAN

Thirty years ago, Gibbs pondered on what the future held for the chemical taxonomist. Clearly, we have not reached the promised land, and what is more, this may just be as good as it gets!

References

1. R. E. Alston and B. L. Turner, “Biochemical Systematics.” Prentice-Hall, Englewood Cliffs, NJ, 1963. 2. T. Swain (ed.), “Chemical Plant Taxonomy.” Academic Press, London, 1963. 2a. R. D. Gibbs, “Chemotaxonomy of Flowering Plants.” McGill-Queens University Press, Montreal. 3. J. B. Harborne and B. L. Turner, “Plant Chemosystematics.” Academic Press, London, 1984. 4. D. B. McKey, Am. Nut. 115,754 (1980). 5. M. Wink and L. Witte, FEBS Len. 159, 196 (1983). 6. R. Antkowiak and W. Z. Antkowiak, in “The Alkaloids” (A. Brossi, ed.), Vol. 40, p. 189. Academic Press, New York, 1991. 7. W. Fenical, in “Alkaloids: Chemical and Biological Perspectives” (S. W. Pelletier, ed.), Vol. 4, p. 276. Wiley, New York, 1986. 8. T. H. Jones and M. S. Blum, in “Alkaloids: Chemical and Biological Perspectives” (S. W. Pelletier, ed.), Vol. 1, p. 33. Wiley, New York, 1983. 9. T. Robinson, in “Herbivores: Their Interactions with Secondary Plant Metabolites” (G. A. Rosenthal and D. H. Janzen, eds.), p. 413. Academic Press, New York, 1979. 10. K. S. Brown and J. R. Trigo, in “The Alkaloids” (G. A. Cordell, ed.), Vol. 47, p. 227. Academic Press, San Diego, 1995. 11. 0. R. Gottlieb, An. Acad. Bras. Cienc. 56,43 (1984). 12. K. Kubitzki and 0. R. Gottlieb, Taxon 33, 375 (1984). 13. K. Kubitzki and 0. R. Gottlieb, Acta Bot. Neerl. 33, 457 (1984). 14. R. Hegnauer, in “Comparative Phytochemistry” (T. Swain, ed.), p. 211. Academic Press, London, 1966. 15. 0. R. Gottlieb, in “Chemosystematics: Principles and Practice” (F. A. Bisby, J. G. Vaughan, and C. A. Wright, eds.), p. 329. Academic Press, London, 1980. 16. R. M. T. Dahlgren, Bof. J. Linn. Soc. SO, 91 (1980). 17. H. Huber, Miff.Bot. Munchen 18,59 (1982). 18. M. F. d. G. F da Silva, 0. R. Gottlieb, and F. Ehrendorfer, Plant. Syst. Evol. 161,97 (1988). 19. P. G. Waterman, Plant Sysf. Evol. 173, 39 (1990). 20. H. Guinaudeau, M. Leboeuf, and A. Cave, J. Nat. Prod. 57,1033 (1994). 21. C. M. A. d. M. Rezende, 0.R. Gottlieb, and M. C. Marx, Biochem. Sysf. Ecol. 3,63 (1975). 22. K. Kubitzki Taxon 18,360 (1969). 23. R. F. Thorne, Aliso 6, 57 (1968). 24. R. M. T. Dahlgren, Nord. J. Bot. 3, 119 (1983). 25. P. G. Waterman, Rec. Adv. Phyfochem. 27, 203 (1993). 26. H. Guinaudeau and J. Bruneton, in “Methods in Plant Biochemistry” (P. G. Waterman, ed.), Vol. 8, p. 373. Academic Press, London, 1993. 27. T. G. Hartley, Garden Bull. Singapore 34,91 (1981).

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28. K. M. Ng, P. P.-H. But, A. I. Gray, T. G. Hartley, Y.-C. Kong, and P. G. Waterman, Biochem. Syst. Ecol. 15,587 (1987). 29. A. Quader, P. P.-H. But, A. I. Gray, T. G. Hartley, Y.-J. Hu, and P. G. Waterman, Biochem. Syst. Ecol. 18, 251 (1990). 30. B. F. Bowden, K. Picker, E. Ritchie, and W. C. Taylor, Aust. J. Chem. 28, 2681 (1975). 31. A. CavC, M. Leboeuf, and P. G. Waterman. in “Alkaloids: Chemical and Biological Perspectives” (S. W. Pelletier, ed.), Vol. 5, p. 133. Wiley, New York, 1987. 32. G. A. Cordell, “Introduction to Alkaloids: A Biogenetic Approach.” Wiley, New York, 1981. 33. S. F. Martin, in “The Alkaloids” (A. Brossi, ed.), Vol. 30, p. 251. Academic Press, New York, 1987. 34. F. Tillequin, S. Michel, and E. Seguin, in “Methods in Plant Biochemistry” (P. G. Waterman, ed.), Vol. 8, p. 309. Academic Press, London, 1993. 35. T. Ohmoto and K. Koike, in “The Alkaloids” (A. Brossi, ed.), Vol. 36, p. 135. Academic Press, New York, 1989. 36. I. Kompis, M. Hesse, and H. Schmid, Lloydia 34, 269 (1971). 37. M. V. Kisakurek and M. Hesse, in “Indole and Biogenetically Related Alkaloids” (J. D. Phillipson and M. H. Zenk, eds.), p. 11. Academic Press, London, 1980. 38. D. Ganzinger and M. Hesse, Lloydia 39, 326 (1976). 39. M. V. Kisakurek, A. J. M. Leeuwenberg, and M. Hesse, in “Alkaloids: Chemical and Biological Perspectives” (S. W. Pelletier, ed.), Vol. 1, p. 211. Wiley, New York, 1983. 40. J.-P. Zhu, A. Guggisberg, M. Kalt-Hadamowsky, and M. Hesse, Plant. Syst. Evol. 172, 13 (1990). 41. R. Hegnauer, “Chemotaxonomie der Pflanzen,” Vol. 9. Birkhauser Verlag, Basel, 1989. 42. T. J. Mabry, in “Comparative Phytochemistry” (T. Swain, ed.), p. 231. Academic Press, London, 1966. 43. H. Reznik, in “Pigments in Plants” (F.-C. Czygan, ed.). p. 370. Gustav Fischer Verlag, Stuttgart, 1980. 44. T. J. Mabry, An. Mo. Bot. Card. 64,210 (1977). 45. D. Strack, W. Steglich, and V. Wray, in “Methods in Plant Biochemistry” (P. G. Waterman, ed.), Vol. 8, p. 421. Academic Press, London, 1993. 46. P. G. Waterman and A. I. Gray. Nat. Prod. Rep. 4, 175 (1987). 47. R. J. Gornell, B. A. Bohm, and R. Dahlgren, Bot. Notiser. 132, 1 (1979). 48. P. G. Waterman, Biochem. Syst. Ecol. 3, 149 (1975). 49. P. G. Waterman and M. F. Grundon (eds.), “The Chemistry and Chemical Taxonomy of the Rutales.” Academic Press, London, 1983. 50. M. F. Grundon, in “The Alkaloids” (A. Brossi, ed.), Vol. 32, p. 341. Academic Press, New York, 1988. 51. A. Romeike, Bot. Notiser 131, 85 (1978). 52. J. G. Wooley, in “Methods in Plant Biochemistry” (P. G. Waterman, ed.), Vol. 8, p. 133. Academic Press, London, 1993. 53. C. C. J. Culvenor, Bot. Notiser. 131,473 (1978). 54. D. J. Robbins, in “Methods in Plant Biochemistry” (P. G. Waterman, ed.), Vol. 8, p. 175. Academic Press, London, 1993. 55. A. D. Kinghorn and M. F. Balandrin, in “Alkaloids: Chemical and Biological Perspectives” (S. W. Pelletier, ed.), Vol. 2, p. 105. Wiley, New York, 1984. 56. M. Wink, in “Methods in Plant Biochemistry” (P. G. Waterman, ed.), Vol. 8, p. 197. Academic Press, London, 1993.

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CUMULATIVE INDEX OF TITLES

Aconitum alkaloids, 4,275 (1954), 7,473 (1960), 34,95 (1988) CI9diterpenes, 12, 2 (1970) Cz0 diterpenes, 12, 136 (1970) Acridine alkaloids, 2, 353 (1952) Acridone alkaloids, experimental antitumor activity of acronycine, 21, 1 (1983) N-Acyliminium ions as intermediates in alkaloid synthesis, 32,271 (1988) Ajmaline-Sarpagine alkaloids, 8, 789 (1965), 11, 41 (1968) enzymes in biosynthesis of, 47, 116 (1995) Alkaloid chemistry, synthetic studies, 50, 377 (1998) Alkaloid production, plant biotechnology of, 40, 1 (1991) Alkaloid structures spectral methods, study, 24, 287 (1985) unknown structure, 5,301 (1955), 7,509 (1960), 10, 545 (1967), 12,455 (1970), 13,397 (1971), 14,507 (1973), 15,263 (1975), 16,511 (1977) X-ray diffraction, 22, 51 (1983) Alkaloids biosynthesis, regulation of, 49, 222 (1997) containing a quinolinequinone unit, 49, 79 (1997) containing a quinolinequinoneimine unit, 49,79 (1997) ecological activity of, 47,227 (1995) forensic chemistry of, 32, 1 (1988) histochemistry of, 39, 1 (1990) in the plant, 1, 15 (1950), 6, 1 (1960) plant biotechnology, production of, 50,453 (1998) Alkaloids from amphibians, 21,139 (1983), 43,185 (1993) ants and insects, 31, 193 (1987) Chinese traditional medicinal plants, 32, 241 (1988) mammals, 21,329 (1983), 43,119 (1993) marine organisms, 24,25 (1985), 41,41 (1992) medicinal plants of New Caledonia, 48, 1 (1996) plants, 49,301 (1997) plants of Thailand, 41, 1 (1992) Allelochemical properties or the raison d’Ctre of alkaloids, 43, 1 (1993) 567

568

CUMULATIVE INDEX OF TITLES

A110 congeners, and tropolonic Colchicum alkaloids, 41, 125 (1992) Alsronia alkaloids, 8, 159 (1965), 12,207 (1970), 14, 157 (1973) Amaryllidaceae alkaloids, 2,331 (1952), 6,289 (1960), 11,307 (1968), 15, 83 (1975), 30,251 (1987) Amphibian alkaloids, 21, 139 (1983), 43,185 (1983) nature and origin, 50, 141 (1998) Analgesic alkaloids, 5, 1 (1955) Anesthetics, local, 5,211 (1955) Anthranilic acid derived alkaloids, 17, 105 (1979), 32, 341 (1988), 39, 63 (1990) Antifungal alkaloids, 42, 117 (1992) Antimalarial alkaloids, 5, 141 (1955) Antitumor alkaloids, 25, 1 (1985) Apocynaceae alkaloids, steroids, 9, 305 (1967) Aporphine alkaloids, 4, 119 (1954), 9, 1 (1967), 24, 153 (1985) Aristofochia alkaloids, 31, 29 (1987) Aristotelia alkaloids, 24, 113 (1985), 48, 249 (1996) Aspergillus alkaloids, 29, 185 (1986) Aspidosperma alkaloids, 8,336 (1965), 11,205 (1968), 17, 199 (1979) synthesis of, 50, 343 (1998) Azafluoranthene alkaloids, 23,301 (1984) Bases simple, 3, 313 (1953), 8, 1 (1965) simple indole, 10, 491 (1967) simple isoquinoline, 4, 7 (1954), 21, 255 (1983) Benzodiazepine alkaloids, 39, 63 (1990) Benzophenanthridine alkaloids, 26, 185 (1985) Benzylisoquinoline alkaloids, 4, 29 (1954), 10, 402 (1967) Betalains, 39, 1 (1990) Biosynthesis in Catharanthus roseus, 49,222 (1997) isoquinoline alkaloids, 4, 1 (1954) pyrrolizidine alkaloids, 46, 1 (1995) quinolizidine alkaloids, 46, 1 (1995) tropane alkaloids, 44, 116 (1993) in Rauwolfia serpentina, 47,116 (1995) Bisbenzylisoquinoline alkaloids, 4, 199 (1954), 7, 429 (1960), 9, 133 (1967), 13,303 (1971), 16,249 (1977), 30,l (1987) synthesis, 16,319 (1977) Bisindole alkaloids, 20, 1 (1981) noniridoid, 47, 173 (1995)

CUMULATIVE INDEX OF TITLES

569

Bisindole alkaloids of Catharanthus C-20’ position as a functional hot spot in, 37, 133 (1990) isolation, structure elucidation and biosynthesis, 37, 1 (1990) medicinal chemistry of, 37, 145 (1990) pharmacology of, 37,205 (1990) synthesis of, 37,77 (1990) therapeutic use of, 37,229 (1990) Buxus alkaloids, steroids, 9, 305 (1967), 1 4 , l (1973), 32,79 (1988) Cactus alkaloids, 4, 23 (1954) Calabar bean alkaloids, 8,27 (1965), 10,383 (1967), 13,213 (1971), 36, 225 (1989) Calabash curare alkaloids, 8, 515 (1965), 11, 189 (1968) Calycanthaceae alkaloids, 8,581 (1965) Camptothecine, 21, 101 (1983), 50,509 (1998) Cancentrine alkaloids, 14, 407 (1973) Cannabis sativa alkaloids, 34,77 (1988) Canthin-6-one alkaloids, 36, 135 (1989) Capsicum alkaloids, 23,227 (1984) Carbazole alkaloids, 13, 273 (1971), 26, 1 (1985) chemistry and biology of, 44,257 (1993) Carboline alkaloids, 8,47 (1965), 26, 1 (1985) P-Carboline congeners and Ipecac alkaloids, 22, 1 (1983) Cardioactive alkaloids, 5, 79 (1955) Catharanthus roseus biosynthesis of terpenoid indole alkaloids in, 49, 222 (1997) Celastraceae alkaloids, 16, 215 (1977) Cephalotaxus alkaloids, 23, 157 (1984) Cevane group of Veratrum alkaloids, 41, 177 (1992) Chemotaxonomy of Papaveraceae and Fumaridaceae, 29, 1 (1986) Chemosystematics, 50, 537 (1998) Chinese medicinal plants, alkaloids from, 32, 241 (1988) Chromone alkaloids, 31, 67 (1988) Cinchona alkaloids, 3, 1 (1953), 14, 181 (1973), 34,332 (1988) Colchicine, 2,261 (1952), 6,247 (1960), 11,407 (1968), 23, 1 (1984) Colchicum alkaloids and all0 congeners, 41, 125 (1992) Configuration and conformation, elucidation by X-ray diffraction, 22,51 (1983) Corynantheine, yohimbine, and related alkaloids, 27, 131 (1986) Cularine alkaloids, 4,249 (1954), 10,463 (1967), 29,287 (1986) Curare-like effects, 5, 259 (1955) Cyclic tautomers of tryptamine and tryptophan, 34, 1 (1988) Cyclopeptide alkaloids, 15, 165 (1975)

570

CUMULATIVE INDEX OF TITLES

Daphniphyllum alkaloids, 15, 41 (1975), 29, 265 (1986) Delphinium alkaloids, 4, 275 (1954), 7, 473 (1960) Clo-diterpenes, 12, 2 (1970) Czo-diterpenes,12, 136 (1970) Dibenzazonine alkaloids, 35, 177 (1989) Dibenzopyrrocoline alkaloids, 31, 101 (1987) Diplorrhyncus alkaloids, 8,336 (1965) Diterpenoid alkaloids Aconifum, 7,473 (1960), 12,2 (1970), 12, 136 (1970), 34,95 (1988) Delphinium, 7,473 (1960), 12,2 (1970), 12,136 (1970) Garrya, 7,473 (1960), 12,2 (1960), 12, 136 (1970) chemistry, 18,99 (1981), 42, 151 (1992) general introduction, 12, xv (1970) structure, 17, 1 (1970) synthesis, 17, 1 (1979)

Eburnamine-vincamine alkaloids, 8,250 (1965), 11,125 (1968), 20, 297 (1981), 42, 1 (1992) Ecological activity of alkaloids, 47, 227 (1995) Elaeocarpus alkaloids, 6, 325 (1960) Ellipticine and related alkaloids, 39, 239 (1990) Enamide cyclizations in alkaloid synthesis, 22, 189 (1983) Enzymatic transformation of alkaloids, microbial and in v i m , 18, 323 (1981) Ephedra alkaloids, 3, 339 (1953) Epibatidine, 46,95 (1995) Ergot alkaloids, 8, 726 (1965), 15, 1 (1975), 38 1 (1990) biochemistry of, 50, 171 (1998) Erythrina alkaloids, 2,499 (1952), 7, 201 (1960), 9,483 (1967), 18, 1 (1981), 48,249 (1996) Erythrophleum alkaloids, 4, 265 (1954), 10,287 (1967) Eupomafia alkaloids, 24, 1 (1985) Forensic chemistry, alkaloids, 12, 514 (1970) by chromatographic methods, 32, 1 (1988) Galbulimima alkaloids, 9, 529 (1967), 13, 227 (1971) Gardneria alkaloids, 36, 1 (1989) Garrya alkaloids, 7,473 (1960), l 2 , 2 (1970), 12, 136 (1970) Geissospermum alkaloids, 8, 679 (1965) Gelsemium alkaloids, 8, 93 (1965), 33,84 (1988), 49, 1 (1997) Glycosides, monoterpene alkaloids, 17,545 (1979) Guafferiaalkaloids, 35, 1 (1989)

CUMULATIVE INDEX OF TITLES

571

Haplophyton cirnicidurn alkaloids, 8, 673 (1965) Hasubanan alkaloids, 16,393 (1977), 33,307 (1988) Histochemistry of alkaloids, 39, 165 (1990) Holarrhena group, steroid alkaloids, 7, 319 (1960) Hunteria alkaloids, 8, 250 (1965) Zboga alkaloids, 8,203 (1965), 11,79 (1968) Imidazole alkaloids, 3,201 (1953), 22,281 (1983) Indole alkaloids, 2,369 (1952), 7, 1 (1960), 26, 1 (1985) biosynthesis in Catharanthus roseus, 49,222 (1997) biosynthesis in Rauwolfia serpentina, 47, 116 (1995) distribution in plants, 11, 1 (1968) simple, 10,491 (1967), 26, 1 (1985) Reissert synthesis of, 31, 1 (1987) Indolizidine alkaloids, 28, 183 (1986), 44, 189 (1993) In vitro and microbial enzymatic transformation of alkaloids, 18, 323 (1981) 2,2’-Indolylquinuclidinealkaloids, chemistry, 8, 238 (1963, 11, 73 (1968) Ipecac alkaloids, 3, 363 (1953), 7,419 (1960), 13,189 (1971), 22,l (1983) Isolation of alkaloids, 1, 1 (1950) Isoquinoline alkaloids, 7,423 (1960) biosynthesis, 4 , l (1954) I3C-NMR spectra, 18,217 (1981) simple isoquinoline alkaloids, 4, 7 (1954), 21, 255 (1983) Reissert synthesis of, 31, 1 (1987) Isoquinolinequinones, from Actinomycetes and sponges, 21, 55 (1983) Khat (Catha edulis) alkaloids, 39, 139 (1990) Kopsia alkaloids, 8,336 (1965) Lead tetraacetate oxidation in alkaloid synthesis, 36, 70 (1989) Local anesthetics, 5,211 (1955) Localization in the plant, 1, 15 (1950), 6, 1 (1960) Lupine alkaloids, 3, 119 (1953), 7,253 (1960), 9, 175 (1967), 31, 16 (1987), 47,2 (1995) Lycopodiurn alkaloids, 5,265 (1955), 7,505 (1960), 10,306 (1967), 14, 347 (1973), 26,241 (1985), 45,233 (1994) Lythraceae alkaloids, 18,263 (1981), 35, 155 (1989) Macrocyclic peptide alkaloids from plants, 26,299 (1985) 49, 301 (1997) Mammalian alkaloids, 21, 329 (1983), 43, 119 (1993) Manske, R. H. F., 50,3 (1998) Marine alkaloids, 24,25 (1985), 41,41 (1992) Maytansinoids, 23,71 (1984) Melanins, 36,254 (1989)

572

CUMULATIVE INDEX OF TITLES

Melodinus alkaloids, 11, 205 (1968) Mesembrine alkaloids, 9,467 (1967) Metabolic transformation of alkaloids, 27, 323 (1986) Microbial and in vitro enzymatic transformation of alkaloids, 18, 323 (1981) Mitrugyna alkaloids, 8,59 (1965), 10,521 (1967), 14, 123 (1973) Monoterpene alkaloids, 16,431 (1977) glycosides, 17, 545 (1979) Monoterpenoid indole alkaloid syntheses utilizing biomimetic reactions, 50,415 (1998) Morphine alkaloids, 2, 1 (part 1, 1952), 2, 161 (part 2, 1952), 6,219 (1960), 13, 1 (1971), 45, 127 (1994) Muscarine alkaloids, 23, 327 (1984) Mushrooms, alkaloids from, 40, 190 (1991) Mydriatic alkaloids, 5, 243 (1955) a-Naphthophenanthridine alkaloids, 4, 253 (1954), 10,485 (1967) Naphthylisoquinoline alkaloids, 29, 141 (1986), 46, 127 (1995) Narcotics, 5, 1 (1955) New Caledonia, alkaloids from the medicinal plants of, 48, 1 (1996) Nuphar alkaloids, 9,441 (1967), 16, 181 (1977), 35,215 (1989) Ochrosia alkaloids, 8, 336 (1965), 11,205 (1968) Ouroupuriu alkaloids, 8, 59 (1965), 10,521 (1967) Oxaporphine alkaloids, 14,225 (1973) Oxazole alkaloids, 35,259 (1989) Oxindole alkaloids, 14, 83 (1973) Papaveraceae alkaloids, 19,467 (1967), 12, 333 (1970), 17,385 (1979) pharmacology, 15,207 (1975) toxicology, 15,207 (1975) Pauridiunthu alkaloids, 30,223 (1987) Pavine and isopavine alkaloids, 31, 317 (1987) Pentaceras alkaloids, 8, 250 (1965) Peptide alkaloids, 26,299 (1985), 49, 301 (1997) Phenanthrene alkaloids, 39, 99 (1990) Phenanthroindolizidine alkaloids, 19, 193 (1981) Phenanthroquinolizidine alkaloids, 19,193 (1981) P-Phenethylamines, 3,313 (1953), 35,77 (1989) Phenethylisoquinoline alkaloids, 14, 265 (1973), 36, 172 (1989) Phthalideisoquinoline alkaloids, 4, 167 (1954), 7, 433 (1960), 9, 117 (1967), 24,253 (1985) Picrulima alkaloids, 8, 119 (1965), 10, 501 (1967), 14, 157 (1973) Piperidine alkaloids, 26, 89 (1985)

CUMULATIVE INDEX OF TITLES

573

Plant alkaloid biosynthesis, molecular genetics of, 50, 257 (1998) Plant biotechnology, for alkaloid production, 40, 1 (1991) Plant systematics, 16, 1 (1977) Pleiocarpa alkaloids, 8, 336 (1965), 11, 205 (1968) Polyamine alkaloids, 22, 85 (1983) Polyamine derivatives, 50,219 (1998) Polyamine toxins, 45, 1 (1994), 46,63 (1995) Pressor alkaloids, 5,229 (1955) Protoberberine alkaloids, 4,77 (1954), 9,41 (1967), 28, 95 (1986) biotransformation of, 46,273 (1995) transformation reactions of, 33, 141 (1988) Protopine alkaloids, 4, 147 (1954), 34, 181 (1988) Pseudocinchoma alkaloids, 8,694 (1965) Pseudodistomins, 50,317 (1998) Purine alkaloids, 38,226 (1990) Pyridine alkaloids, 1, 165 (1950), 6, 123 (1960), 11, 459 (1968), 26, 89 (1985) Pyrrolidine alkaloids, 1,91 (1950), 6,31 (1960), 27, 270 (1986) Pyrrolizidine alkaloids, 1, 107 (1950), 6, 35 (1960), 12,246 (1970), 26,327 (1985) biosynthesis of, 46, 1 (1995) Quinazolidine alkaloids, see Indolizidine alkaloids Quinazoline alkaloids, 3, 101 (1953), 7, 247 (1960), 29, 99 (1986) Quinazolinocarbolines, 8, 55 (1965), 21,29 (1983) Quinoline alkaloids related to anthranilic acid, 3,65 (1953), 7,229 (1960), 17, 105 (1979), 32, 341 (1988) Quinolinequinone alkaloids, 49, 79 (1997) Quinolinequinoneimine alkaloids, 49, 79 (1997) Quinolizidine alkaloids, 28, 183 (1985), 47, 1 (995) biosynthesis of, 46, 1 (1995) Rauwolfia alkaloids, 8, 287 (1965) biosynthesis of, 47, 116 (1995) Reissert synthesis of isoquinoline and indole alkaloids, 31, 1 (1987) Reserpine, chemistry, 8, 287 (1965) Respiratory stimulants, 5, 109 (1955) Rhoeadine alkaloids, 28, 1 (1986) Salamandra group, steroids, 9, 427 (1967) Sarpagine-type alkaloids, 49, 1 (1997) Sceletium alkaloids, 19, 1 (1981) Secoisoquinoline alkaloids, 33,231 (1988)

574

CUMULATIVE INDEX OF TITLES

Securinegu alkaloids, 14, 425 (1973) Senecio alkaloids, see Pyrrolizidine alkaloids Simple indole alkaloids, 10,491 (1967) Simple indolizidine alkaloids, 28, 183 (1986), 44, 189 (1993) Sinomenine, 2,219 (1952) Solunum alkaloids chemistry, 3,247 (1953) steroids, 7, 343 (1960), 1 0 , l (1967), 19, 81 (1981) Sources of alkaloids, 1, 1 (1950) Spectral methods, alkaloid structures, 24,287 (1985) Spermidine and related polyamine alkaloids, 22, 85 (1983) Spermine and related polyamine alkaloids, 22, 85 (1983) Spider toxin alkaloids, 45, 1 (1994), 46,63 (1995) Spirobenzylisoquinoline alkaloids, 13,165 (1971), 38, 157 (1990) Sponges, isoquinolinequinone alkaloids from, 21, 55 (1983) Stemonu alkaloids, 9,545 (1967) Steroid alkaloids Apocynaceae, 9,305 (1967), 32,79 (1988) Buxus group, 9,305 (1967), 14,l (1973), 32,79 (1988) chemistry and biology, 50, 61 (1998) Holurrhenu group, 7,319 (1960) Sulumundru group, 9,427 (1967) Solunum group, 7,343 (1960), 10, 1 (1967), 19,81 (1981) Verutrum group, 7,363 (1960), 10,193 (1967), 14, 1 (1973), 41, 177 (1992) Stimulants respiratory, 5,109 (1955) uterine, 5, 163 (1955) Structure elucidation, by X-ray diffraction, 22, 51 (1983) Strychnos alkaloids, 1, 375 (part 1, 1950), 2, 513 (part 2, 1952), 6, 179 (1960), 8, 515, 592 (1965), 11, 189 (1968), 34,211 (1988), 36,1 (1989), 48,75 (1996) Sulfur-containing alkaloids, 26, 53 (1985), 42, 249 (1992) Synthesis of alkaloids, Enamide cyclizations for, 22, 189 (1983) Lead tetraacetate oxidation in, 36, 70 (1989) Tubernuemontuna alkaloids, 27, 1 (1983) Taxol, 50,509 (1998) Taxus alkaloids, 10,597 (1967), 39, 195 (1990) Terpenoid indole akaloids, 49,222 (1997) Thailand, alkaloids from the plants of, 41, 1 (1992) Toxicology, Papaveraceae alkaloids, 15,207 (1975)

CUMULATIVE INDEX OF TITLES

575

Transformation of alkaloids, enzymatic microbial and in vitro, 18, 323 (1981) Tropane alkaloids biosynthesis of, 44,115 (1993) chemistry, 1,271 (1950), 6, 145 (1960), 9,269 (1967), 13,351 (1971), 16, 83 (1977), 33,2 (1988), 44, 1 (1993) Tropoloisoquinoline alkaloids, 23, 301 (1984) Tropolonic Colchicum alkaloids, 23, 1 (1984), 41, 125 (1992) Tylophoru alkaloids, 9, 517 (1967) Unnatural alkaloid enantiomers, biological activity of, 50, 109 (1998) Uterine stimulants, 5, 163 (1955) Verutrum alkaloids cevane group of, 41, 177 (1992) chemistry, 3,247 (1952) steroids, 7, 363 (1960), 10, 193 (1967), 14, 1 (1973) Vincu alkaloids, 8,272 (1965), 11, 99 (1968), 20,297 (1981) Voucungu alkaloids, 8,203 (1965), 11,79 (1968) Wasp toxin alkaloids, 45, 1 (1994), 46,63 (1995) X-ray diffraction of alkaloids, 22, 51 (1983) Yohimbe alkaloids, 8, 694 (1965), 11, 145 (1968), 27, 131 (1986)

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INDEX

Acetylcholine, physostigmine, structure analysis with, 124 Acridone alkaloids plant alkaloid biosynthesis, molecular genetics, 304-309 acridone synthase, molecular genetics, 307-309 anthranilate synthase, molecular genetics, 307 applications, 304-305 furofoline-I, enzymatic synthesis, 305-307 Acridone synthase, molecular genetics, 307-309 Agroclavine, and elymoclavine hydroxylase, ergoline alkaloid formation, enzymology, 199-200 Ajmaline, enzymatic synthesis, 259-263 Alkaloid biosynthesis, see Plant alkaloid biosynthesis, molecular genetics Alkaloid chemistry, synthetic studies, 377-414 Aspidosperma alkaloids, synthesis, 399-400 berbanes, synthesis, 385 Catharanthus roseus alkaloids, synthesis, 400-405 corynantheidine alkaloids, synthesis, 383 epibatidine, 407-410 introduction, 377-378 ipecacuanha alkaloids, 379-380 morphine, synthesis, 405-407 Rauwolfia alkaloids, synthesis, 384-385 vincamine and structurally related alkaloids, synthesis, 386-398 cuanzine, synthesis, 397-398 interconversions, 391-396 tacamine, synthesis, 396-397 (+)-vincamine and (-)-vincamone, synthesis, 386-391 yohimbine alkaloids, 380-382

577

Alkaloid chemosystematics, 537-565 data in systematic studies, handling, 544-548 distributions of alkaloids in higher plant taxa, 548-563 betalains, 556-558 ornithine- and lysine-derived alkaloids, 559-563 Rutaceae, anthranilate-derived alkaloids, 558-559 tryptophan-derived alkaloids, 553-555 t yrosine/phenylalanine-derived alkaloids, 548-553 evolution of alkaloids, 540-544 chemical mechanism, 540-541 evolutionary events, 541-542 forces mediating production, 543-544 setting rules, 539-540 Alkaloid enantiomers, unnatural, biological activity, 109-139 analytical criteria, 110-1 12 1-benzyltetrahydroisoquinolines,113-1 14 norarmepavine, 113 norcoclaurine, 113, 114 norreticuline, 113, 114 reticuline, 113-114 tetrahydropapaverine, 113, 114 tetrahydropapaveroline, 113, 114 colchicine, 128-132 2,3-dehydroemetine, 116-1 17 antiamebic effect, 116-117 dihydroquinine, 117 diverse structures, 118 (R)-cherylline, 118 coralydine, 118 (R)-l,2-dihydroxyapomorphine, 118 (lR)-a-

hydroxybenzyltetro~~~o~e, 118 0-methylcorytenchirine, 118 (R)-1,2-methylenedioxyapomorphine, 118

578

INDEX

Alkaloid enantiomers (continued) perhydrohistrionicotoxin,118 (S)-tetrahydroharmine, 118 emetine, 116-117 introduction, 109-1 10 mefloquine morphine, 118-123 Rice total synthesis, 119-120 nicotine, 133-135 1-phenethyltetrahydrokoquinohes,114-1 16 methopholine, 115-1 16 phenopropylamine, 115-116 physostigmine, 123-128 Julian total synthesis, 125;127-128 Robinson synthesis, 124-125 tetrahydroisoquinolines, simple, 112-1 13 carnegine, 112 isosalsoline, 112 N-methylisosalsoline, 112 N-methylsalsoline, 112 salsolidine, 112 salsoline, 112 salsolinol, 112 Alkaloid evolution, 540-544 chemical mechanism, 540-541 chemosystematics, 540-544 evolutionary events, 541-542 forces mediating production, 543-544 origins, 543 Alkaloids, see specific type Allopumiliotoxins and pumiliotoxins, 146-148 extracts from Dendrobates, 147 Dendrobates pumilio, 146 Mantella, 147 Minyobates, 147 Pseudophyrene, 147 Amphibian alkaloids, 141-169 batrachotoxins, 143-145 bicyclic izidine alkaloids, 159-164 3,5-disubstituted indolizidines, 160-161 5,8-disubstituted indolizidines, 161-162 pyrrolizidines, 159 quinolizidines, 163-164 5,6,8-trisubstituted indolizidines, 163 coccinellines, 158-159 cyclopenta[b]quinolizidines, 154 decahydroquinolines, 152-153

epibatidine, 155-156 gephyrotoxins, 151-152 histrionicotoxins, 149-151 monocyclic, 164-165 pseudophrynamines, 156-157 pumiliotoxin-class, 145-149 homopumiliotoxins, 148-149 other alkaloids, 149 pumiliotoxins and allopumiliotoxins, 146-148 pyrrolizidine oximes, 157-158 samandarines, 142-143 Amphibian skins, alkaloids from, 141-142 Anthranilate synthase, molecular genetics, 307 Ants consumption by pyrrolizidine-containing frogs, 159 source of alkaloids for frogs, 141-142 Aromatic-L-amino-acid decarboxylase, molecular genetics, 265-267, 285-287 Aspidofractinine alkaloids, synthesis, 366-369 Aspidosperma alkaloids, synthesis, 343-376, 399-400 aspidofractinine group, 366-369 aspidospermine group, 344-346 biomimetic synthesis to goniomitine skeleton, 431-432 Melodinus alkaloids, 430-431 Vinca alkaloids, 428-430 kopsine group, 369-374 meloscine group, 366 vincadifformine group, 355-361 vindolinine group, 361-365 vindorosine and vindoline, 346-354 Aspidospermine group, Aspidosperma alkaloids. synthesis, 344-346

Batrachotoxinin, isolation and structure history, 143 Batrachotoxins, 143-145 from Phyllobafes aurotaenia, 143-144 from Phyllobates bicolor, 143-144 from Phyllobates lugubris, 144 from Phyllobates terribilis, 144 Beetles coccinellines in, 158 source of alkaloids for frogs, 141-142

INDEX

1-Benzyltetrahydroisoquinolines, unnatural alkaloid enantiomer, 113 Berbamunine, enzymatic synthesis, 291-292 Berbamunine synthase, molecular genetics, 292-295 Berbanes, synthesis, 385 Berberine bridge enzyme, molecular genetics, 287-290 enzymatic synthesis, 272-277 Betalains, alkaloid chemosystematics, 556-558 Bicyclic izidine alkaloids amphibian alkaloids, 159-164 3,5-disubstituted indolizidines, 160-161 5,8-disubstituted indolizidines, 161-162 pyrrolizidines, 159 quinolizidines, 163-164 5,6,8-trisubstituted indolizidines, 163 Bioconversion. plant cell cultures for alkaloid production, 461-462 Biogenesis, pseudodistomins: structure, synthesis, and pharmacology, 338 Biomimetic reactions, monoterpenoid indole alkaloids, syntheses utilizing, 415-452 Biomimetic syntheses Aspidosperma and Ibogu alkaloids, 419-428 via dehydrosecodine-type intermediates, 427-428 via secodine-type intermediates, 421-427 bisindole alkaloids, 444-447 in sarpagine family, 436-444 skeletal arrangements and fragmentations, 428-436 Aspidosperma to goniomitine skeleton, 431-432 Melodinus alkaloids, 430-431 Vinca alkaloids, 428-430 fragmentation, 434-436 carnptothecin, 436 flavopereirine, 434 harman, 434 nauclefidine, 434-435 modified Polonovsky-Potier reaction, rearrangement with, 433-434 Strychnos to calebassinine skeleton, 433

579

Biotechnology production ergot alkaloids, biochemistry, 201-204 bioconversion, 202-204 fermentation, directed, 201-202 Bisbenzylisoquinoline alkaloids biosynthesis, molecular genetics, 290-295 berbamunine enzymatic synthesis, 29 1-292 berbamunine synthase, molecular genetics, 292-295 Bisindole alkaloids, biomimetic synthesis, 444-447 Buchnerine, synthesis, 246-247 Buxaceae alkaloids biogenesis, 90-92 isolation and structure elucidation, 63-67 Buxus alkaloids mass spectra, 82-83 NMR spectra, 75-79

Calebassinine skeleton, Strychnos biomimetic synthesis to, 433 Calycanthine, research of R.H.F. Manske, 18 Cumptothecu acuminata, extracts, antitumor activity, 510-511 Camptothecin, 509-536 background, 510-512 Camptothecu ucuminuta extracts, antitumor activity, 510-511 early preclinical and clinical testing, 512 isolation and structure determination, 511-512 chemistry, 5 13-5 15 early synthesis, 513 improved synthesis, 513-515 preclinical and clinical studies, recent, 516-519 9-amino-20(S)-camptothecin, 5 16-5 17 9-nitro-20(S)-camptothecin,517 camptothecin, 516 DX-8951,519-520 GG-211, 519 structure-activity relationships, 515 topoisomerase I as cellular target, 512-513

580

INDEX

9-Amino-20(S)-camptothecin,preclinical and clinical studies, 516-517 9-Nitro-20(S)-camptothecin,preclinical and clinical studies, 517 R-Canadine, enzymatic synthesis, 277 Carnegine, unnatural alkaloid enantiomer, biological activity, 112 Catharanthus roseus alkaloids, synthesis, 400-405 Celacinnine class alkaloids with spermidine skeleton, 229-238 loesenerines, 229-233 mayfoline, 233-238 Cerveratrum-type alkaloids mass spectra, 83-84 NMR spectra, 79 Cevine-type alkaloids, mass spectra, 83-84 Chanoclavine-I cyclase, ergoline alkaloid formation, 199 (R)-Cherylline, unnatural alkaloid enantiomer, biological activity, 118 Clavine alkaloid biosynthesis, cis-trans isomerizations, ergoline ring system, 188 Clavine alkaloids, and secoergolines, 176-177 Coccinellines alkaloids in beetles, 158-159 amphibian alkaloids, 158-159 from Dendrobates pumilio, 158-159 Colchicine unnatural alkaloid enantiomer, biological activity, 128-132 antitumor agents from, 130 preparation, 130- 131 tubulin binding, 128-129 X-ray analysis, 129 Conanine-type alkaloids mass spectra, 84 NMR spectra, 79 Coralydine, unnatural alkaloid enantiomer, biological activity, 118 Corydaline, enzymatic synthesis, 277 Corynantheidine alkaloids, synthesis, 383 Corynanthe-related alkaloids, biomimetic syntheses, 416-419 Cuanzine, synthesis, 397-398

Cyclopenta[b]quinolizidines from amphibians, 154 Minyobates bombetes, 154 Decahydroquinolines, 152-153 from Dendrobates auratus, 153 Mantella, 153 Melanophryiniscus, 153 amphibian alkaloids, 152-153 Fourier-transform infrared spectroscopy, 152 Dehydroelymoclavine, ergot alkaloid, biochemistry, 178-179 2,3-Dehydroemetine, see Emetine Dehydrohomopumiliotoxins, pumiliotoxinclass amphibian alkaloids, 149 Dendrobates, pumiliotoxin and allopumiliotoxin extracts in, 147 Dendrobates auratus, pyrrolizidine oximes from, 158 Dendrobates auratus, decahydroquinolines from, 153 Dendrobates histrionicus 3.5-disubstituted indolizidines in, 160-161 gephyrotoxin detection in, 151-152 monocyclic alkaloids in, 164 Dendrobates pumilio coccinellines from, 158-159 pumiliotoxin-class extracts in, 145-146 pyrrolizidine oximes from, 157-158 Dendrobates speciosus 3.5-disubstituted indolizidines in, 160 5,8-disubstituted indolizidines in, 162 monocyclic alkaloids in, 164 Dendrobatidae alkaloids from, 141-169 histrionicotoxins detection in, 150 8-Deoxypumiliotoxins, 149 Dihydroperiphylline, 238-243 6,10-Dihydropumiliotoxins, 149 Dihydroquinine, see Hydroquinine (R)-1,2-Dihydroxyapomorphine, unnatural alkaloid enantiomer, biological activity, 118 Dimethylallyltryptophan synthase, ergoline alkaloid formation, 198-199

INDEX

Dopa decarboxylase, see Aromatic+ amino-acid decarboxylase DX-8951, preclinical and clinical studies, recent, 519-520

Elymoclavine hydroxylase, and agroclavine, ergoline alkaloid formation, enzymology, 199-200

Elymoclavine-0-P-D-fructofuranoside, biochemistry, 179 Emetine, unnatural alkaloid enantiomer, biological activity, 116-117 Epibatidine, 155-156 amphibian alkaloids, 155-156 analgesic properties, 155 from Epipedobares, 155-156 from Epipedobates tricolor, 155-156 synthetic studies, 407-410 Epipedobares epibatidine from, 155-156 pumiliotoxin and allopumiliotoxin extracts in, 147 Epipedobates tricolor, epibatidine from, 155-156 Ergobalansine, ergot alkaloid, biochemistry, 181 Ergobine, ergot alkaloid, biochemistry, 181 Ergogaline, ergot alkaloid, biochemistry, 181 Ergoline alkaloid formation enzymology, ergot alkaloids, biochemistry, 198-201 agroclavine hydroxylase, 199-200 chanoclavine-I cyclase, 199 dimethylallyltryptophan synthase, 198-199 elymoclavine hydroxylase, 199-200 N-methyltransferase, 199 Ergoline ring system clavine alkaloid biosynthesis, cis-trans isomerizations, 188 N-methylation, 190-192 ring C formation: modification of isoprene unit, 188-190 ring D formation, 192-193 tryptophan isoprenylation, 185-186 Ergolines pharmacological properties, 204-207 antitumor and antimicrobial properties, 207

581

neurotransmitter receptor mediation, 206-207 Ergot alkaloids, biochemistry, 171-218 biosynthesis, 183-201 ergoline alkaloid formation, enzymology, 198-201 agroclavine hydroxylase, 199-200 chanoclavine-I cyclase, 199 dimethylallytryptophan synthase, 198-199 elymoclavine hydroxylase, 199-200 enzymes related to, 200-201 N-methyltransferase, 199 ergoline ring system, 184-193 clavine alkaloid biosynthesis, cis-trans isomerizations, 188 clavine interrelationships, 187-188 N-methylation, 190-1 92 ring C formation: modification of isoprene unit, 188-190 ring D formation, 192-193 tryptophan isoprenylation, 185-186 lysergic acid derivatives, 193-198 peptide moiety, 196-198 biotechnological production, 201-204 bioconversion, 202-204 fermentation, directed, 201-202 ergolines, pharmacological properties, 204-207 antitumor and antimicrobial properties, 207 neurotransmitter receptor mediation, 206-207 future challenges, 208-211 enzymology and molecular genetics, 208-209 evolutionary aspects, 210-211 regulation, 210 historical background, 172-173 natural, 173-181 clavine alkaloids and secoergolines, 176-1 77 lysergic acid derivatives, 174-176 peptide alkaloids, 174-175 ergopeptam alkaloids, 175 simple, 176 new alkaloids, 178-181 dehydroelymoclavine, 178-179 elymoclavine-0-0-ofructofuranoside, 179 ergobalansine, 181

582

INDEX

Ergot alkaloids (continued) ergobine, 181 ergogaline, 181 10-hydroxy-cis-paspalicacid amide, 180 8-hydroxyergine, 179 8-hydroxyerginine, 179 10-hydroxy-trans-paspalic acid amide, 180 12'-U-methylergocornine,180 12'-U-methyl-a-ergokryptine, 180 structural types, 174 producing organisms, 182-183 ergot fungi biology, 182 fungi, other types, 182-183 higher plants, 183

Frogs, see also Dendrobatidae; specific genus pyrrolizidine-containing,consumption of ants, 159 Furofoline-I, enzymatic synthesis, 305-307

Gephyrotoxins amphibian alkaloids, 151-152 configuration questions, 151-152 from Dendrobates histrionicus, 151-1 52 GG-211, preclinical and clinical studies, recent, 519 Goniomitine skeleton, Aspidosperma biomimetic synthesis to, 431-432

Heterocyclic chemistry, research of R.H.F. Manske, 37-40 Histrionicotoxins amphibian alkaloids, 149-151 Phyllobate aurotaenia, 149-151 Homobatrachotoxins, found in skin and feathers of New Guinean birds, 145 Homopumiliotoxins, 148-149 detection in dendrobatid species, 148 Mantella species, 148 Melanophryniscus species, 148 structural similarity to pumiliotoxins, 148 Hydroquinine, alkaloid enantiomer, unnatural, biological activity, 117

(1R)-w Hydroxybenzyltetrahydroisoquinoline, biological activity, 118 10-Hydroxy-cis-paspalicacid amide, biochemistry, 180 8-Hydroxyergine, biochemistry, 179 8-Hydroxyerginine, biochemistry, 179 10-Hydroxy-trans-paspalicacid amide biochemistry, 180 Hyoscyamine 6P-hydroxylase,in tropane and nicotine alkaloids biosynthesis, 302-304 Iboga alkaloids biomimetic syntheses, 419-428 via dehydrosecodine-type intermediates, 427-428 via secodine-type intermediates, 421-427 Indolizidines, 160-163 3.5-disubstituted in Dendrobates speciosus, 160 structures in Dendrobates histrionicus, 160-161 5,8-disubstituted, in Dendrobates speciosus, 162 5,6,8-trisubstituted, structural analysis, 163 Insects, see Ants; Millipedes; Spiders; Wasps Ipecac alkaloids, synthesis, 379-380 Ipecacuanha alkaloids, see Ipecac alkaloids Isoquinoline alkaloids plant biotechnology, 474-477 research of R.H.F. Manske, 20-35 established ring systems, 21-29 cancetrine alkaloids, 34-35 synthesis and alkaloid transformations, 34-35 cularine alkaloids, 29-31 spirobenzylisoquinoline alkaloids, 32-33 Isosalsoline, unnatural alkaloid enantiomer, biological activity, 112 Jerveratrum-type alkaloids mass spectra, 85 NMR spectra, 79-80

IN1>EX

Julian total synthesis, physostigmine, 125;127-128 Knapp’s first asymmetric synthesis, tetrahydro-pseudodistomin, 326-328 Kobayashi’s synthesis, key intermediate for pseudodistomin C, tetrahydropseudodistomin, 331 Kopsine alkaloids, synthesis, 369-374 P-Lactams, in synthesis of taxol, 526-527 N-Acyl-0-lactams, in synthesis of taxol, 527 Liliaceae, isolation of alkaloids from, 67-69 Loesenerines, celacinnine class, alkaloids with spermidine skeleton, 229-233 Lycopodium alkaloids, research of R.H.F Manske, 35 Lysergic acid derivatives ergot alkaloids, biochemistry, 174-176, 193-1 96, 193- 198 peptide moiety, 196-198 peptide alkaloids, 174-175 ergopeptam alkaloids, 175 ergopeptide alkaloids, 175 simple, 176 Lysine, alkaloids derived from, chemosystematics, 559-563 Macarpine. enzymatic synthesis, 277-281 Manske, R.H.F. fifty years of alkaloid chemistry, 3-59 awards and honors, 49 childhood and formative years, 7-8 concluding remarks, 47-48 curriculum vitae, 48-49 editorship, 40-42 higher education and early employment, 8-18 General Motors Corporation (1926-1927) and Yale University (1927-1929), 15-18 Manchester University (1924-1926), 9-15 Queen’s University (1919-1924) 8-9 introduction, 3-7 National Research Council of Canada (1930-1943). 18-39 calycanthine, 18

583

heterocyclic chemistry, 37-40 isoquinoline alkaloids, 20-35 established ring systems, 21-29 cancetrine alkaloids, 34-35 cularine alkaloids, 29-31 spirobenzylisoquinoline alkaloids, 32-33 Lycopodium alkaloids, 35 miscellany, 36-37 Senecio alkaloids, 20 naturalist, orchidist, musician, and cuisinier, 45-47 scientist and society, 42-44 Mantella decahydroquinolines from, 153 homopumiliotoxin detection in, 148 pumiliotoxin and allopumiliotoxin extracts in, 147 Marine sponges, polyamine derivatives, natural, 249-254 Mayfoline, celacinnine class, alkaloids with spermidine skeleton, 233-238 Mefloquine, unnatural alkaloid enantiomer, biological activity, 117 Melanophryniscus decahydroquinolines from, 153 homopumiliotoxin detection in, 148 quinolizidines in, 164 Melodinus alkaloids, biomimetic synthesis from Aspidosperma, 430-431 Meloscine group, alkaloids, synthesis, 366 Metabolic engineering plant biotechnology, 462-491 issues for resolution, 481-491 cloning genes in secondary metabolism, 481-483 compartmentation, 485 cellular, 485 subcellular, 485-487 gene expression, 483-484 regulation, 491 stability, 484-485 molecular genetic methods, 463-466 biosynthetic genes, isolation, 464 biosynthetic steps in pathway, identification, 463-464 expression of genes in branching pathways, knocking out, 465 overexpression of modified genes, 464-465

584

INDEX

Metabolic engineering (continued) rate-determining steps in biosynthetic pathways, determination, 465 transformation, 466 unknown gene function, determination, 465 production, strategies for improving,

466-469 catabolism decrease, 468 competitive pathways, 468 enzymes, rate-limiting, 467 feedback inhibition, 467 increase of flux through pathway, 466 producing cells, increasing percentage, 468-469 random mutations/selection approach, 469 results, 469-481 isoquinoline alkaloids, 474-477 terpenoid indole alkaloids, 469-474 tobacco alkaloids, 477-479 tropane alkaloids, 479-481 Methopholine, see Metofoline p-Methoxycinnamoyl-buchnerine,synthesis,

246-247 N-Methylation, ergoline ring system,

190-192 0-Methylcorytenchirine, unnatural alkaloid enantiomer, biological activity, 118

(R)-1,2-Methylenedioxyapomorphine, unnatural alkaloid enantiomer, biological activity, 118 12'-0-Methylergocornine,biochemistry, 180 12'-O-Methyl-a-ergokryptine, biochemistry,

180 N-Methylisosalsoline, unnatural alkaloid enantiomer, biological activity, 112 N-Methylsalsoline, unnatural alkaloid enantiomer, biological activity, 112 N-Methyltransferase, in ergoline alkaloid formation, 199 Metofoline, unnatural alkaloid enantiomer, biological activity, 115-116 Millipedes, source of alkaloids for frogs,

141-142 Minyobates, pumiliotoxin and allopumiliotoxin in extracts of, 147 Minyobates bombetes, cyclopenta[b]quinoliidines from, 154

Molecular genetics plant alkaloid biosynthesis, 258-316 acridone alkaloids, 304-309 acridone synthase, molecular genetics, 307-309 anthranilate synthase, molecular genetics, 307 furofoline-I, enzymatic synthesis,

305-307 bisbenzylisoquinoline alkaloids,

290-295 berbamunine enzymatic synthesis,

291-292 berbamunine synthase, molecular genetics, 292-295 monoterpenoid indole alkaloids, 259-271 ajmaline enzymatic synthesis, 259-263 strictosidine synthase, molecular genetics, 267-271 tryptophan decarboxylase, molecular genetics, 265-267 vindoline enzymatic synthesis, 263-265 tetrahydrobenzylisoquinoline alkaloids,

272-290 berberine bridge enzyme, molecular genetics,

287-290 enzymatic synthesis, 272-277 R-canadine, enzymatic synthesis,

277 corydaline, enzymatic synthesis, 277 dopa decarboxylase, molecular genetics, 285-287 macarpine, enzymatic synthesis, 277-281 morphine, enzymatic synthesis,

281-284 tyrosine decarboxylase, molecular genetics, 285-287 tropane and nicotine alkaloids,

295-304 hyoscyamine 6P-hydroxylase,

302-304 putrescine N-methyltransferase,

299-300 scopolamine, enzymatic synthesis,

296-299 tropinone reductase-I, molecular genetics, 300-302 putrescine N-methyltransferase, 299-300 Monocyclic amphibian alkaloids, 164-165

INDEX

Monoterpenoid indole alkaloids biomimetic syntheses Aspidosperma and Iboga alkaloids, 419-428 via dehydrosecodine-type intermediates, 427-428 via secodine-type intermediates, 421-427 bisindole alkaloids, 444-447 corynanthe-related alkaloids from secologanin and strictosidine, 416-419 in sarpagine family, 436-444 skeletal arrangements and fragmentations, 428-436 Aspidosperma to goniomitine skeleton, 431-432 Melodinus alkaloids, 430-431 Vinca alkaloids, 428-430 fragmentation, 434-436 camptothecin, 436 flavopereirine, 434 harman, 434 nauclefidine, 434-435 modified Polonovsky-Potier reaction, rearrangement with, 433-434 Sfrychnos to calebassinine skeleton, 433 plant alkaloid biosynthesis, molecular genetics, 259-271 ajmaline enzymatic synthesis, 259-263 strictosidine synthase, molecular genetics, 267-271 tryptophan decarboxylase, molecular genetics, 265-267 vindoline enzymatic synthesis, 263-265 syntheses utilizing biomimetic reactions, 4 15-452 Morphine enzymatic synthesis, 281-284 synthesis, 405-407 unnatural alkaloid enantiomer, analgesic properties, 118-123 pharmacological investigations, 120 Rice total synthesis, 119-120

National Research Council of Canada, R.H.F. Manske’s work, 18-39

585

Natsume’s synthesis, tetrahydropseudodistomin, 323-324 Nicotine and tropane alkaloids plant alkaloid biosynthesis, molecular genetics, 295-304 hyoscyamine 6fl-hydroxylase. 302-304 putrescine N-methyltransferase, 299-300 scopolamine, enzymatic synthesis, 296-299 tropinone reductase-I, molecular genetics, 300-302 unnatural alkaloid enantiomer, binding properties, 135 inhibition, 135 synthesis, 133 Ninomiya’s second synthesis, by 1,3-~ycloadditionof nitrone, tetrahydro-pseudodistomin, 328-331 Ninomiya’s synthesis, by enamide photocyclization, tetrahydropseudodistomin, 324-326 Norarmepavine, unnatural alkaloid enantiomer, biological activity, 113 Norcoclaurine, unnatural alkaloid enantiomer, biological activity, 113 Norreticuline, unnatural alkaloid enantiomer, biological activity, 113

Oncinofis alkaloids with spermidine skeleton, 221-229 Optimization, growth and production media in plant biotechnology, 457-458 Ornithine, alkaloids derived from, chemosystematics, 559-563 Oxazolidines, in synthesis of taxol, 527

Perhydrohistrionicotoxin, unnatural alkaloid enantiomer, biological activity, 118 Pharmacology, pseudodistomins, 340 1 -Phenethyltetrahydroisoquinolines, unnatural alkaloid enantiomers, biological activity, 114-116 Phenpropylamine, unnatural alkaloid enantiomer, biological activity, 115-116

586

INDEX

Phenylalanine, alkaloids derived from, chemosystematics, 548-553 3-Phenylpropenoyl, derivatives of spermine and spermidine, 247-249 Phyllobates aurotaenia, batrachotoxins from, 143-144 Phyllobates bicolor, batrachotoxins from, 143-144 Phyllobates lugubris, batrachotoxins from, 144 Phyllobates terribilis, batrachotoxins from, 144 Phyllobates aurotaenia, histrionicotoxins in, 149-151 Ph ysostigmine unnatural alkaloid enantiomer, biological activity, 123- 128 acetylcholine structure analysis, 124 Julian total synthesis, 125;127-128 Robinson synthesis, 125-126 Plant alkaloid biosynthesis, molecular genetics, 258-316 acridone alkaloids, 304-309 acridone synthase, molecular genetics, 307-309 anthranilate synthase, molecular genetics, 307 furofoline-I, enzymatic synthesis, 305-307 bisbenzylisoquinoline alkaloids, 290-295 berbamunine enzymatic synthesis, 291-292 berbamunine synthase, molecular genetics, 292-295 monoterpenoid indole alkaloids, 259-271 ajmaline enzymatic synthesis, 259-263 strictosidine synthase, molecular genetics, 267-271 trytophan decarboxylase, molecular genetics, 265-267 vindoline enzymatic synthesis, 263-265 tetrahydrobenzylisoquinoline alkaloids, 272-290 berberine bridge enzyme, molecular genetics, 287-290 enzymatic synthesis, 272-277 R-canadine, enzymatic synthesis, 277 corydaline, enzymatic synthesis, 277

dopa decarboxylase, molecular genetics, 285-287 macarpine, enzymatic synthesis, 277-281 morphine, enzymatic synthesis, 281-284 tyrosine decarboxylase, molecular genetics, 285-287 tyrosine/dopa decarboxylases, molecular genetics, 285-287 tropane and nicotine alkaloids, 295-304 hyoscyamine 6P-hydroxylase, 302-304 putrescine N-methyltransferase, 299-300 scopolamine, enzymatic synthesis, 296-299 tropinone reductase-I, molecular genetics, 300-302 Plant biotechnology, 453-508 cell cultures for alkaloid production, 455-462 bioconversion, 461-462 differentiated cells, cultures, 458-459 elicitation, 459-461 optimization of growth and production media, 457-458 screening, 455-457 selection, 457 metabolic engineering, 462-491 issues for resolution, 481-491 cloning genes in secondary metabolism, 481-483 compartmentation, 485-487 gene expression, 483-484 regulation, 491 stability, 484-485 molecular genetic methods, 463-466 biosynthetic genes, isolation, 464 biosynthetic steps in pathway, identification, 463-464 expression genes in branching pathways, knocking out, 465 overexpression of modified genes, 464-465 rate-determining steps in biosynthetic pathways, determination, 465 transformation, 466 unknown gene function, determination, 465

INDEX

production, strategies for improving, 466-469 catabolism decrease, 468 competitive pathways, 468 enzymes, rate-limiting, 467 feedback inhibition, 467 increase of flux through pathway, 466 producing cells, increasing percentage, 468-469 random mutations/selection approach, 469 results, 469-481 isoquinoline alkaloids, 474-477 terpenoid indole alkaloids, 469-474 tobacco alkaloids, 477-479 tropane alkaloids, 479-481 transcription regulation and signal transduction pathways, 491-496 Plant cell cultures, alkaloid production, 455-462 bioconversion, 461-462 differentiated cells, cultures, 458-459 elicitation, 459-461 optimization of growth and production media, 457-458 screening, 455-457 selection, 457 Polonovsky-Potier reaction, skeletal biomimetic syntheses, 433-434 Polyamine derivatives, natural, 219-256 alkaloids with spermidine skeleton, 221-243 celacinnine class spermidine alkaloids, 229-238 loesenerines, 229-233 mayfoline, 233-238 dihydroperiphylline class spermidine alkaloids, 238-243 Oncinotis species, 221-229 spermine alkaloids, 243-247 biogenetic considerations, 243-246 buchnerine, synthesis, 246-247 p-methoxycinnamoyl-buchnerine, synthesis, 246-247 verbacine, synthesis, 246-247 verballocine, synthesis, 246-247 verbascenine, synthesis, 246-247 spermine and spermidine, 3phenylpropenoyl derivatives, 247-249

587

from spiders, wasps, and marine sponges, 249-254 Pregnane-type alkaloids, mass spectra, 86 Pseudodistomin A isolation and structure, 320-321 total synthesis, 335-338 Pseudodistomin B isolation and structure, 319-320 total synthesis, 333-335 Pseudodistomin C, isolation and structure, 318, 321-322 Pseudodistomins structure, synthesis, and pharmacology, 317-342 biogenesis, 338 isolation and structure, 318-322 pseudodistomin A, structure, 318, 320-321 pseudodistomin B, structure,318,319-320 pseudodistomin C, structure, 318, 321-322 tetrahydro-pseudodistomin, structure, 319 pharmacology, 340 synthesis, 322-338 pseudodistomins and analogs, total synthesis, 331-338 pseudodistomin A, total synthesis, 335-338 proposed structure, 335-336 revised structure: pseudodistomin A total synthesis, 337-338 pseudodistomin B, total synthesis, 333-335 revised structure: pseudodistomin B total synthesis, 334-335 tetrahydro-pseudodistomin, synthesis, 322-331 Knapp’s first asymmetric synthesis, 326-328 Kobayashi’s synthesis of key intermediate for pseudodistomin C, 331 Natsume’s synthesis, 323-324 Ninomiya’s second synthesis by 1,3-cycloaddition of nitrone, 328-331

588

INDEX

Pseudodistomins (continued) Ninomiya’s synthesis by enamide photocyclization, 324-326 synthesis, 322-338 Pseudophrynamines from Pseudophryne, 157 Pseudophryne semimarmorata, 156 amphibian alkaloids, 156-157 Pseudophryne pseudophrynamines from, 156-157 pumiliotoxin and allopumiliotoxin extracts in, 147 Pseudophryne semimarmorata pseudophrynamines from, 156 Pumiliotoxins and allopumiliotoxins, 146-148 extracts from Dendroba tes, 147 Dendrobates pumilio, 146 Epipedobates, 147 Mantella, 147 Minyoba tes, 147 Pseudophyrene, 147 amphibian alkaloids, 145-149 allopumiliotoxins, 146-148 dehydrohomopumiliotoxins, 149 8-deoxypumiliotoxins, 149 6,10-dihydropumiliotoxins, 149 homopumiliotoxins, 148-149 extracts from Dendrobates pumilio, 145-146 Putrescine N-methyltransferase, molecular genetics, 299-300 Pyrrolizidine oximes amphibian alkaloids, 157-158 from Dendrobates auratus, 158 from Dendrobates pumilio, 157-158 Pyrrolizidines, in ant-consuming dendrobatid frogs, 159

Quinolizidines Fourier-transform infrared spectra, 164 in Melanophryniscus, 164

Rauwolfia alkaloids, synthesis, 384-385 Reticuline, unnatural alkaloid enantiomer, biological activity, 113

Ring C formation, modification of isoprene unit, ergoline ring system, 188-190 Ring D formation, ergoline ring system, 192-193 Robinson synthesis, physostigmine, 125-126 Rutaceae, anthranilate-derived alkaloids, chemosystematics, 558-559

Salamandra salamandra, samandarine synthesized by, 142-143 Salamandra-type alkaloids, mass spectra, 86 Salsolidine, unnatural alkaloid enantiomer, biologicai activity, 112 Salsoline, unnatural alkaloid enantiomer, biological activity, 112 Salsolinol, unnatural alkaloid enantiomer, biological activity, 112 Samandarines, amphibian alkaloids, 142-143 Sarpagine, biomimetic syntheses, 436-444 Scopolamine, enzymatic synthesis, 296-299 Secoergolines, and clavine alkaloids, 176-177 Secosolanidine-type alkaloids, mass spectra, 87 Senecio alkaloids, research of R.H.F. Manske, 20 Skeletal arrangements and fragmentations, biomimetic syntheses, 428-436 Aspidosperma to goniomitine skeleton, 431-432 Melodinus alkaloids, 430-431 Vinca alkaloids, 428-430 modified Polonovsky-Potier reaction, rearrangement with, 433-434 Strychnos to calebassinine skeleton, 433 Solanaceae, isolation and structure elucidation, 69-72 Solanidine-type alkaloids mass spectra, 87-88 NMR spectra, 80 Spermidine and spermine alkaloids, 3phenylpropenoyl derivatives, 247-249 Spermidine skeleton alkaloids, 221-243 Celacinnine class, 229-238 loesenerines, 229-233 mayfoline, 233-238

INDEX

dihydroperiphylline, 238-243 Oncinotis species, 221-229 Spermine alkaloids biogenetic considerations, 243-246 polyamine derivatives, natural, 243-247 buchnerine, synthesis, 246-247 p-me thoxycinnamoyl-buchnerine, synthesis, 246-247 verbacine, synthesis, 246-247 verballocine, synthesis, 246-247 verbascenine, synthesis, 246-247 Spiders, natural polyamine derivatives from, 249-254 Spirosolane-type alkaloids mass spectra, 89 NMR spectra, 80 Steroidal alkaloids, chemistry and biology, 61-139 biogenesis, 90-92 Apocynaceae and Buxaceae, 90-92 Liliaceae and Solanaceae, 92 isolation and structure elucidation, 63-75 Apocynaceae, 63 Buxaceae, 63-67 Liliaceae, 67-69 from marine organisms, 74-75 Solanaceae, 69-72 from terrestrial animals, 72-74 pharmacology, 98-103 Apocynaceae, 98-99 Buxaceae, 99-100 Liliaceae, 101 from marine organisms, 103 Solanaceae, 101-102 from terrestrial animals, 102-103 physical properties, 75-90 mass spectra, 81-89 Buxus alkaloids, 82-83 cerveratrum- and cevine-type alkaloids, 83-84 conanine-type alkaloids, 84 jerveratrum-type alkaloids, 85 pregnane-type alkaloids, 86 salamandra-type alkaloids, 86 secosolanidine-type alkaloids, 87 solanidine-type alkaloids, 87-88 spirosolane-type alkaloids, 89 NMR spectra, 75-81 Buxus alkaloids, 75-79 cerveratrum-type alkaloids, 79

589

conanine-type alkaloids, 79 jerveratrum-type alkaloids, 79-80 solanidine-type alkaloids, 80 spirosolane-type alkaloids, 80 X-ray crystallography, 89-90 synthetic studies and chemical transformations, 92-97 Strictosidine synthase, molecular genetics, 267-271 Strychnos, biomimetic synthesis to calebassinine skeleton. 433

Tacamine, synthesis, 396-397 Taxol, 521-536 bioactivity and mechanism of action, 52 1-522 chemistry, 524-531 esterification Greene-Potier procedure for synthesis of taxotere, 526 semisynthesis utilizing improved side chain acylating agents, 526-527 clinical studies, 529-530 combination therapy, 530 formulation. 530 taxotere, 530 toxicity, 530 tumors, responses in, 530 p-lactams, in synthesis, 526-527 N-acyl-P-lactams, in synthesis, 527 oxazolidines, 527 side chain, 528-529 taxol and analogs, structure-activity relationships, 528 total synthesis, 527-528 isolation and structure elucidation, 521 major events prior to 1980, brief review, 521 supplies and sources, 523-524 Taxus brevifolia, early collection, 521 Taxotere, clinical studies, 530 T a u s brevifolia early collection, 521 supplies and sources, 523-524 Terpenoid indole alkaloids, plant biotechnology, 469-474

590

INDEX

Tetrahydrobenzylisoquinolinealkaloids plant alkaloid biosynthesis, molecular genetics, 272-290 berberine, enzymatic synthesis, 272-277 berberine bridge enzyme, molecular genetics, 287-290 R-canadine, enzymatic synthesis, 277 corydaline, enzymatic synthesis, 277 macarpine, enzymatic synthesis, 277-281 morphine, enzymatic synthesis, 281-284 tyrosine/dopa decarboxylases, molecular genetics, 285-287 @)-Tetrahydroharmhe, unnatural alkaloid enantiomer, biological activity, 113 Tetrahydroisoquinolines. unnatural alkaloid enantiomers, biological activity, 112-113 Tetrahydropapaverine, unnatural alkaloid enantiomer, biological activity, 113 Tetrahydropapaveroline, unnatural alkaloid enantiomer, biological activity, 113 Tetrahydro-pseudodistomin isolation and structure, 319 Knapp’s first asymmetric synthesis, 326-328 Kobayashi’s synthesis of key intermediate for pseudodistomin C, 33 1 Natsume’s synthesis, 323-324 Ninomiya’s second synthesis by 1,3cycloaddition of nitrone, 328-331 Ninomiya’s synthesis by enamide photocyclization, 324-326 synthesis, 322-331 Tobacco alkaloids, plant biotechnology, 477-479 Transcription regulation, and signal transduction pathways, plant biotechnology, 491-496 Tropane and nicotine alkaloids plant alkaloid biosynthesis, molecular genetics, 295-304 hyoscyamine 6fl-hydroxylase. 302-304 putrescine N-methyltransferase, 299-300

scopolamine, enzymatic synthesis, 296-299 tropinone reductase-I, molecular genetics, 300-302 Tropane alkaloids, plant biotechnology, 479-481 Tropinone reductase-I, molecular genetics, 300-302 Tryptophan alkaloids derived from, chemosystematics, 553-555 isoprenylation, ergoline ring system, 185-1 86 Tryptophan decarboxylase, see Aromatic-Lamino-acid decarboxylase Tyrosine, alkaloids derived from, chemosystematics, 548-553 Tyrosine decarboxylase, molecular genetics, 285-287

Verbacine, synthesis, 246-247 Verballocine, synthesis, 246-247 Verbascenine, synthesis, 246-247 Vincu alkaloids, biomimetic synthesis from Aspidospermu alkaloids, 428-430 Vincadifformine alkaloids, synthesis, 355-361 Vincamine, and structurally related alkaloids, synthesis, 386-398 cuanzine, 397-398 tacamine, 396-397 (+)-vincamine and (-)-vincamone, 386-391 Vincamone, synthesis, 386-391 Vindoline enzymatic synthesis, 263-265 and vindorosine, synthesis, 346-354 Vindolinine group alkaloids, synthesis, 361-365 Vindorosine, synthesis, 346-354

Wasps, natural polyamine derivatives from, 249-254

Yohimbine alkaloids, synthesis, 380-382

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