Alkaloids

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THE ALKALOIDS: Chemistry and Pharmacology VOLUME 50

THE ALKALOIDS Chemistry and Biology

This Page Intentionally Left Blank

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

This book is printed on acid-free paper.

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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

Academic Press a division of Harcourt Brace & Companv

525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NW 1 7DX, UK http://www.hbuk.co.uklap/ International Standard Book Number: 0- 12-469550-7 PRINTED IN THE UNITED STATES OF AMERICA 97 98 9 9 0 0 01 0 2 Q W 9 8 7 6

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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

vii

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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CONTENTS

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

ix

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

548 563 564

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

561 517


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

xvi

PREFACE

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

-CHAPTER 1-

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

5

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

6


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


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

1. R.


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


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


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


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.


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


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.


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


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


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

24


(+)-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


(+)-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.


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


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.


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


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.


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


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.


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


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


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


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.


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


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.


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


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


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.

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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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59

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).


-CHAPTER 2--

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

IQBAL

CHOUDHARY

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

111.

IV.

V. VI.

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

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

61

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

101 102

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

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

61

Copyright 6 1998 by Academic Press All rights of reproduction in any form reserved.

62

AITA-UR-RAHMAN A N D CHOUDHARY

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.

2.

CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS

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.


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.


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


(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.


(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.


/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.


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.


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


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


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.


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

89


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


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


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


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.


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.


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


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

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(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

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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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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

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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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115

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

3.


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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121

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

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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.


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.

126

BROSSI A N D PEI

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-

3. BIOLOGICAL ACTIVITY

OF UNNATURAL ALKALOID ENANTIOMERS

127

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,

3.


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.

130

BROSSI A N D PEI

: : : 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 -


N
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


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


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


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


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


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

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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


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


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


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,

446

TAKAYAMA AND SAKAI

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


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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W. A. Carroll and P. A. Grieco, J. Am. Chem. Soc. 115, 1164 (1993). E. Wenkert and B. Wickberg, J. Am. Chem. Soc. 87, 1580 (1965). G. Hugel, B. Gourdier, J. Levy, and J. Le Men, Tetrahedron 36,511 (1980). B. Danieli, G. Lesma, and G. Palmisano, J. Chem. Soc., Chem. Commun. 908 (1981). L. Calabi, B. Danieli, G . Lesma, and G. Palmisano, J. Chem. Soc., Perkin Trans. I, 1371 (1982). 56. G. Hugel, J. Y. Laronze, J. Laronze, and J. Levy, Heterocycles 16, 581 (1981). 57. G. Hugel and J. Levy, Tetrahedron 39,1539 (1983). 58. G. Lewin and J. Poisson, Tetrahedron Lett. 25, 3813 (1984). 59. G. Hugel and J. Levy, Tetrahedron 40, 1067 (1984). 60. G. Lewin and J. Poisson, Tetrahedron 43,493 (1987). 61. G. Palmisano, B. Danieli, G. Lesma, F. Trupiano, and T. Pilati, J. Org. Chem. 53, 1056 (1988). 62. H. BBlcskei, E. Gacs-Baitz, and C. Szantay, Tetrahdron Lett. 30, 7245 (1989). 63. Omunim Chimique Societe Anonyme, Belg. Patent 826,066 (Cl. C07D); Chem. Abstr. 84, 135917j (1976). 64. N. Aimi, S. Tanabe, Y. Asada, Y. Watanabe, K. Yamaguchi, and S. Sakai, Chem. Pharm. Bull. 30,3427 (1982). 65. G . Hugel and J. LCvy, J. Org. Chem. 49,3275 (1984). 66. G. Palmisano, B. Danieli, G. Lesma, R. Riva, S. Riva, F. Demartin, and N. Masciocchi, J. Org. Chem. 49,4138 (1984). 67. G. Hugel and J. Levy, J. Org. Chem. 51, 1594 (1986). 68. L. Randriambola, J. C. Quirion, C. Kan-Fan, and H. P. Husson, Tetrahedron Lett. 28, 2123 (1987). 69. G . Lewin, C. Schaeffer, and P. H. Lambert, J. Org. Chem. 60, 3282 (1995). 70. G. Palmisano, B. Danieli, G. Lesma, and M. Mauro, J. Chem. Soc., Chem. Commun. 1564 (1986). 71. A. Guggisberg, R. Prewo, J. H. Bieri, and M. Hesse, Helv. Chim.Acta 65,2578 (1982). 72. P. Potier, in “Stereoselective Synthesis of Natural Products” (W. Bartmann and E. Winterfeldt, eds.), p. 19. Excerpta Medica, Amsterdam, 1979. 73. P. Potier, in “Indole and Biogenetically Related Alkaloids” (J. D. Phillipson and M. H. Zenk, eds.), Chapter 8. Academic Press, London, 1980. 74. A. Husson, Y. Langlois, C. Riche, H. P. Husson, and P. Potier, Tetrahedron 29, 3095 (1973). 75. A. I. Scott, C. L. Yeh, and D. Greenslade, J. Chem. Soc., Chem. Commun. 947 (1978). 76. C. Thal, M. Dufour, P. Potier, M. Jaouen, and D. Mansuy, J. Am. Chem. Soc. 103, 4956 (1981). 77. A. I. Scott, Bioorg. Chem. 3,398 (1974). 78. C. Kan Fan and H. P. Husson, Tetrahedron Lett. 21,4265 (1980). 79. N. Aimi, H. Murakami. T. Tsuyuki, T. Nishiyama, S. Sakai, and J. Haginiwa, Chem. Pharm. Bull. 34,3064 (1986). 80. H. Takayama, R. Yamamoto, M. Kurihara, M. Kitajima, N. Aimi, L. Mao, and S. Sakai, Tetrahedron Lett. 35, 8813 (1994). 81. J. C. Cai and C. R. Hutchinson, in “The Alkaloids” (A. Brossi, ed.), Vol. 21. p. 101. Academic Press, New York, 1983. 82. N. Aimi, M. Nishimura, A. Miwa, H. Hoshino, S. Sakai, and J. Haginiwa, Tetrahedron Lett. 30,4991 (1989). 83. B. K. Carte, C. DeBrosse, D. Eggleston, M. Hemling, M. Mentzer, B. Poehland, N. Troupe, J. W. Westley, and S. M. Hecht, Tetrahedron 46, 2747 (1990). 51. 52. 53. 54. 55.

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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

0

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.

476


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

N@

z2

n

a

o

Me

18 R=9-CH2NMe20HCI, 10-OH 19 R=7-CH2-N

N

/

N-Me*HCI; 10,11qCH2)&

NHpHCI

0

20

U

FIG.1. Camptothecin, carnptothecin sodium, and analogs.

m

13. CAMPTOTHECIN AND DEVELOPMENT OF CPT 1958 1966 1970-1972 1985 1986-1 991 1989 1989 1991- 1996

AND

TAXOL

511

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)).

"*.do co

0

0

3 R=O,R'=-H 4 R=O, R1=-OH

10

5 6 7 8 9

R=-O(CH2)20-,R1=wOH R=-O(CH2)20-.R1=-OH R=-O(CH2)20-,R1=---OH R=O, R'=-OH R=O. R1=---OH

FIG.2. Tricyclic intermediates.

514

a

WALL A N D WAN1

R

aNb +

H+,

20-deoxy-CPT analogs

CHO

0

3 hydroxy'ation*

c

20(RS)-CPT or analogs

aBr

R \

+

/

,

,

,

$

$

2) l)BuaBu* Heck

ZO(S)-CPT, (1) or analogs

reaction

12 R=H

O 11

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).

13. CAMFTOTHECIN

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515

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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517

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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519

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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TAXOL

TABLE I1 THEDISCOVERY AND DEVELOPMENT OF TAXOL AS

AN

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ANTICANCER AGENT

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,

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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).

13. CAMITOTHECIN

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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

14. ALKALOID

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547

(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


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

14. ALKALOID

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553

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.

555


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).


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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WATERMAN

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

CHEMOSYSTEMATICS

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.


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

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CHEMOSYSTEMATICS

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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.


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


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)


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


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)


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


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)


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


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)


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)


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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