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THE ALKALOIDS: Chemistry and Pharmacology
VOLUME 50
THE ALKALOIDS
Chemistry and Biology
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THE ALKALOIDS: Chemistry and Pharmacology
VOLUME 50
THE ALKALOIDS
Chemistry and Biology
Edited by
Geoffrey A. Cordell
College of Pharmacy
University of Illinois at Chicago
Chicago, Illinois
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CONTENTS
CONTRIBUTORS. ........................................................................... xi
PREFACE ................................................................................. 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. Scientific Career and Research ............................................
V. Editorship.. ......... .................................................
VI. The Scientist and SOC
VII. Naturalist. Orchidist,
VIII. Concluding Remarks .................................................
Publications of R. H. . . . . . . . . . . . . . . .
....................................
. . . . . . . . . . . . . . .
3
7
8
18
40
42
45
47
51
Chemistry and Biology of Steroidal Alkaloids
ATTA-UR-RAHMAN A N D M. IQBAL CHOUDHARY
I. Introduction. ......................................... . . . . . . . . . . . 61
11. Isolation and Structure Elucidation ........................................... 63
111. Physical Properties . . . . . ..................................... 75
IV. Biogenesis.. .................... 90
VI. Pharmacology.. ................ 98
V. Some Synthetic Studies and Chemical Transformations.. .................... 92
References ........................................... 103
Biological Activity of Unnatural Alkaloid Enantiomers
ARNOLD BROSSI A N D XUE-FENG PEI
1. Introduction ............... ....................... 109
11. Analytical Criteria. ....... ........................... 110
111. Unnatural Alkaloid Enan 112
IV. (+)-Morphine.. ............................................................. 118
V. (+)-Physostigmine.. .. ................................................. 123
VII. (+)-Nicotine ................................................................ 133
VI. (+)-Colchicine ............................. 128
V
vi CONTENTS
VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Nature and Origin of Amphibian Alkaloids
JOHN W. DALY
1. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
...................................................
.................
V. Histrionicotoxins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Gephyrotoxins . . . . . . . . . .
IX. Epibatidine.. . . . . . . . . . . . .
XI. Pyrrolizidine Oximes . . .
XII. Coccinellines.. . . . . . . . . . . .................................. ....
.......................................
VII. Decahydroquinolines.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VIII. Cyclopenta[b]quinolizidines.. .. .. . .. ...... .. .. ..... ... .. ... .. . . . .. ... . . .. ... .. .
.......................................
X. Pseudophrynamines . . , .
..................................
XIII. Bicyclic “Izidine” Alkal
XIV. Monocyclic Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XV. Summary and Prospects .......................................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biochemistry of Ergot Alkaloids-Achievements and Challenges
DETLEF GROCER AND HEINZ G. FLOSS
I. Introduction.. . . ........................................................
11. Historical Background.. ......................
IV. Producing Organisms.. . ......................
V. Biosynthesis.. . . . . . . . . . . .
111. The Natural Ergot Alka
......................
VI. Biotechnologica
VII. Pharmacologica
VIII. Future Challenges . . . . . . . . . . . . . . . . . . . . . . . . . ......................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Natural Polyamine Derivatives-New Aspects of Their Isolation,
Structure Elucidation, and Synthesis
ARMIN GUGGISBERC A N D MANFRED HESSE
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
135
136
141
142
142
145
149
151
152
154
155
156
157
158
159
164
165
167
172
172
173
182
183
20 1
204
208
212
219
22 1
243
247
249
254
1. Introduction.. . . . . . . . . . . .
11. Sarnandarines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111. Batrachotoxins., .. ... ... .. .. . . . . .. . .. .. , .. . . . .. . .. . _ ._ . _ _ . . . .. .. . .. .. . . . .. . .. ...
IV. The Purniliotoxin Class. ......................
V. Histrionicotoxins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.............
VI. Gephyrotoxins . . . . . . . . . .
IX. Epibatidine.. . . . . . . . . . . . .
X. Pseudophrynamines . . . .
.......................................
VII. Decahydroquinolines.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VIII. Cyclopenta[b]quinolizidines.. .. .. . .. ...... .. .. ..... ... .. .
......................
......................
XI.Pyrrolizidine Oxirnes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XII. Coccinellines ........................................................
XIV. Monocyclic Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XV. Summary and Prospects .......................................
References . . . . . . . . . . . . . .
141
142
142
145
149
1.51
152
1.54
155
156
157
158
159
164
16.5
167
CONTENTS vii
Molecular Genetics of Plant Alkaloid Biosynthesis
TONI M. KUTCHAN
I . Introduction.. .......................................................... 258
259
111. Tetrahydrobcnzylisoquinoline Alkaloids ...................................... 272
IV. Bisbenzylisoquinoline Alkaloids ........................... 290
295
VI. Acridone Alkaloids ................................ 304
VII. Conclusions and Fu .................................. 309
References ...................................................................... 311
11. Monoterpenoid lndole Alkaloids.. ............................................
V. Tropane and Nicotine Alkaloids.. .............................................
Pseudodistomins: Structure, Synthesis, and Pharmacology
ICHIYA NINOMIYA. TOSHIKO KIGUCHI. A N D TAKEAKI NAITO
I . Introduction. .................................................................... 317
11. Isolation and Structure.. . . . ................. 318
111. Synthesis ........................................................................ 322
IV. Biogenesis.. ..................................................................... 338
V. Pharmacology 340
References ...................................................................... 341
Synthesis of the Aspidosperma Alkaloids
J. EDWIN SAXTON
I . Introduction.. ..................................................
11. The Aspidospermine Group ...................................................
111. Vindorosine and Vindoline ... ...................................
IV. The Vincadifformine Group .................................
V. The Vindolinine Group ........................................................
VI. The Meloscine Group . . . . . . . . . .
.....
..................................
VII. The Aspidofractinine Group. . ..............................
VIII. The Kopsine Group ............................................................
References ...................... .................................
Synthetic Studies in Alkaloid Chemistry
CSABA SZANTAY
1. Introduction. ....................................................................
11. Synthesis of Ipecacuanha Alkaloids ...........................................
111. Synthesis of Yohimbine Alkaloids. ........................................
IV. Synthesis of Corynantheidine Alkaloids.. .....................................
V. Synthesis of Rauwolfia Alkaloids.. ............................................
VI. Synthesis of Berbane
VII. Synthesis of Vincamine and Structurally Related Alkaloids .................
VIII. Synthesis of Aspicfospenna Alkaloids .........................................
343
344
346
355
361
366
366
369
374
377
379
380
383
384
385
386
399
...
V l l l CONTENTS
IX. Synthesis of Alkaloids from Catharanthus roseus.. ...........................
X. Synthesis of Morphine.. ........................................................
XI. Synthesis of Epibatidine.. ......................................................
400
405
407
References ...................................................................... 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.
111. Biomimetic Syntheses of Aspidospernia and fboga Alkaloids ...............
IV. Biomimetic Skeletal Rearrangements and Fragmentations .........
V. Biomimetic Synthesis in the Sarpagine Family.. ..............................
Strictosidine. and Their Analogs. ..............................................
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
RoeERr VERPOORTE. ROBERT VAN DER HEIJDEN. A N D J. MEMELINK
I. Introduction ...................... 453
455 11. Plant Cell Cultures for the Production of Alkaloids .........................
462 111. Metabolic Engineering ............................
IV. Transcriptional Regulati ansduction Pathways .............. 491
........ 496 V. Conclusions ..................... ................
497 VI. Future Prospects.. ................................. ................
499 References .............. .......................................
History and Future Prospects of Camptothecin and Taxol
MONROE E. WALL A N D MANSUKH c. WAN1
I. Camptothecin ................................................................... 509
11. Taxol ............................................................................ 521
References ...................................................................... 531
Alkaloid Chemosystematics
PETER G. WATERMAN
I. Introduction.. ................................................................... 537
539
111. The Evolution of Alkaloids.. .................................................. 540
544
11. Alkaloids in Chemical Systematics: Laying Down the Rules ................
IV. Handling Alkaloid Data in Systematic Studies ...............................
CONTENTS ix
V. Systematically Significant Distributions of Alkaloids
in Higher Plant Taxa ........................................................... 548
References ...................................................................... 564
VI. Concluding Comments ......................................................... 563
CUMULATIVE INDEX OF TITLES.. 561
INDEX 517
.......................................................
..................................................................................
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CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors' contributions begin.
AITA-UR-RAHMAN (61), H. E. J. Research Institute of Chemistry, Univer-
sity of Karachi, Karachi-75270, Pakistan
ARNOLD BROSSI (109), School of Pharmacy, University of North Carolina,
Chapel Hill, North Carolina 27599
M. IQBAL CHOUDHARY (61), H. E. J. Research Institute of Chemistry,
University of Karachi, Karachi-75270, Pakistan
JOHN W. DALY (141), Laboratory of Bioorganic Chemistry, National Insti-
tute of Diabetes and Digestive and Kidney Diseases, National Institutes
of Health, Bethesda, Maryland 20892
HEINZ G. FLOSS (171), Department of Chemistry, University of Washing-
ton, Seattle, Washington 98195
DETLEF GROCER (171), Institute for Plant Biochemistry, Halle (Saale),
Germany
ARMIN GUCCISBERG (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
TONI M. KUTCHAN (257), Laboratorium fur Molekulare Biologie, Univer-
sitat 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 Univer-
sity, 2300RA Leiden, The Netherlands
TAKEAKI NAITO (317), Kobe Pharmaceutical University, Higashinada,
Kobe 658, Japan
xi
xii CONTRIBUTORS
ICHIYA NINOMIYA (317),Kobe Pharmaceutical University, Higashinada,
Kobe 658, Japan
XUE-FENG PEI (109), Laboratory of Bioorganic Chemistry, National Insti-
tutes of Health, Bethesda, Maryland 20892
SHIN-ICHIRO SAKAI (415), Faculty of Pharmaceutical Sciences, Chiba Uni-
versity, Chiba 263, Japan
J. EDWIN SAXTON (343), Department of Chemistry, The University of
Leeds, Leeds LS2 9JT, United Kingdom
V. SNIECKUS (3), Guelph-Waterloo Center for Graduate Work in Chemis-
try, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
CSABA SZANTAY (377), Institute of Organic Chemistry, Technical Univer-
sity, 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/Amster-
dam 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
PETER G. WATERMAN (537), Phytochemistry Research Laboratories, De-
partment 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 chemis-
try 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 data-
base 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 Alka-
loids.’’ 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 pro-
duction.
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 ener-
getic 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
...
X l l l
xiv 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 spec-
trum. As a result, this unique perspective of alkaloid chemistry offers a
wonderful historical overview of life as an alkaloid chemist in the mid-
1920s 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 intri-
guing 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 re-
search 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 tremen-
dous impact that .enzyme isolation and molecular genetics are having, and
will continue to have, in the future strategies for understanding the forma-
tion and availability of important alkaloids is reviewed in detail byKutchan.
Tunicates of the genus Pseudostoma have yielded a number of novel metab-
olites whose structure elucidation and synthesis have been engaging several
Japanese research groups. Ninomiya, Kiguchi, and Naito clarify the confu-
sion that has surrounded the structures of these particular alkaloids.
The past 18 years have seen some remarkable developments in the effi-
cient 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 Chem-
istry 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 monoterpen-
oid 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. How-
ever, many of them have been evaluated, successfully, through chemical
incitement, and these efforts are reviewed by Takayama and Sakai. Substan-
tial 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 ap-
proved 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 “dor-
mant” 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 sur-
mounted. 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 com-
plex 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 be-
come 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 dedica-
tion, 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 ..................
A. Queen's University (1919-1924) .................................................
C. General Motors Corporation (1926-1927) and Yale University
IV. Scientific Career and Research ..................
111. Higher Education and Early Employment ............................................... 8
B. Manchester University (1924-1926) ................................................... 9
(1927-1929) .................................................................
A. Calycanthine ...
C. The Isoquinoline Alkaloids ............................................................ 20
D. The Lycopodiurn Alkaloids ...
E. Miscellany .................................................................................. 36
F. Heterocyclic Chemistry .........
V. Editorship ...............................................
VI. The Scientist and Society ...........
VII. Naturalist, Orchidist, Musician, and Cuisinier .......................................... 45
VIII. Concluding Remarks .....................................................................
Publications of R. H. F. Manske ........................................................... 51
I. Introduction
My mother discovered that tincture of laudanum relieved my insomnia. . . . I slept
-R. H. F. Manske commented on his first acquaintance with alkaloids at the age
long and peacefully and became a model child.
of 18 months. [2]
THE ALKALOIDS, VOL. 50
0099-Y5Y8/YX $25.00
3 Copyright 8 IYYX hy Academic Press
All rights of reproduction in any form reserved.
4 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 molecu-
lar weight determinations of the alkaloids and their derivatives and func-
tional 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 reas-
sembled to arrive at a tentative structure in accord with the molecular
composition. The ultimate proof of structure was the synthesis, by unambig-
uous 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 enan-
tiomeric relationships were readily resolved, the establishment of absolute
stereochemistry was not possible. Diastereomeric relationships were recog-
nizable, 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 man-
power 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 BI2 were conquered and retrosyn-
thetic 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 Man-
Chester, 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 isoquino-
line 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 MACLEAN AND SNIECKUS
alkaloids from Corydulis species, hence the name [8]. Also during this
period he was made a Fellow of the Royal Society of Canada (1935) and
was awarded the D.Sc. degree from Manchester University (1937).
Staff and equipment were difficultly accessible in 1943 but we lit our first Bunsen
In 1943, given carte blanche by the then President, Paul C. Jones, Manske
assumed the challenging position of Director of Research, Dominion Rub-
ber 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 contin-
ued 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.
burner on June first of that year. It has burned ever since. [l]
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 several1. 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 alka-
loids 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 Septem-
ber 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 AND 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 Fahren-
heit”) 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 (Battle-
ford), 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 agrar-
ian 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
A. QUEEN’S UNIVERSITY (1919-1924)
It was a cold and clammy evening early in September of 1919 when 1 said good-
bye 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 do 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 Pro-
fessors 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 contro-
versy 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 cyclohepta-
none. [l] (See also Fig. 2.)
Richard Manske entered the Ph.D. program at Manchester where “the
smog . . . did nothing to lessen” hisenthusiasm for learning. In the first
year, he carried out research under the supervision of Arthur Lapworth
while his second year was spent on a problem set by Robert Robinson
although “In actual fact it was my second year with Robinson. He was also
interested in mechanism and had paid me frequent visits, . . . and donated
many rare carbonyl compounds.”
10 MACLEAN AND SNIECKUS
FIG. 1. First page of the M.Sc. Thesis of R. H. F. Manske submitted to Queen’s Univer-
sity, 1924.
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 11
FIG. 2. A page from the Ph.D. Thesis of R. H. F. Manske submitted to Manchester
University, 1926, depicting an aldol condensation and describing the equilibrium constants
for cyanohydrin formation for carbonyl compounds which laid the foundations of modem
mechanistic organic chemistry.
12 MACLEAN AND SNIECKUS
Manske’s Ph.D. thesis is comprised of three parts. Part I, under the
supervision of Lapworth, was entitled “The Influence of Groups on the
Reactivity of Organic Compounds. Cyanohydrin Formation,” and led to
three publications (5,7,23) (Fig. 2). The first paper (5) showed that the
previously proposed structures of menthone cyanohydrin and camphor
cyanohydrin were untenable. This conclusion was based on the results
reported in the second and third publications (7,23) in which the dissociation
constants of a large number of ketone cyanohydrins were measured. Fur-
thermore, an examination of 0-, m-, and p- substituent effects on the dissoci-
ation 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].
expect a third-year student to complete in six afternoons. [2]
aware that diazonium salts can be made to react with acetoacetic esters. . . . [l]
It took me six months to synthesize harmaline-an achievement which I now would
The sequence of reactions was scribbled in a hurry. He (Robinson) was vaguely
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 rutaecar-
pine (4). The former was a benchmark achievement of synthetic confirma-
tion 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 dor-
mant for a year before the puzzle was clarified and the results were pub-
lished. The authors called it “an unexpectedly simple synthesis” (4) [2].
. . . it was desirable to have the hydrazide of benzylphthalamic acid and this was
to be prepared by heating the acid with hydrazine. Unexpectedly at that time but later
perfectly obvious the result was benzylamine and phthalylhydrazide. [l]
Part I11 of his thesis, “A Modification of the Gabriel Synthesis of Primary
Amines,” also stemmed from an accidental discovery [2]. In an attempt to
prepare the hydrazide (5) of benzylphthalamic acid (4) by treatment with
hydrazine, followed by acid hydrolysis, Manske observed the formation
1. R. H. F. MANSKE: FIFIY YEARS OF ALKALOID CHEMISTRY 13
FIG. 3. A page from the Ph.D. Thesis of R. H. F. Manske which shows, in part, rationalization
of the effect of benzaldehyde substituents on cyanohydrin formation.
14 MACLEAN AND SNIECKUS
FIG. 4. A page from the Ph.D. Thesis of R. H. F. Manske delineating the biogenesis of
harman from tryptophan as suggested by Perkin and Robinson.
1. R. H. F. MANSKE: FIFIY YEARS OF ALKALOID CHEMISTRY 15
1 2 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 [ 111 which
represented a major improvement over prior practice because the tradi-
tional 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)
AND YALE UNIVERSITY (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
H2NNHz
4 Co2H 1
CONHCHpPh <!: + H2NCH2Ph
CONHNH,
0
TI- Ct
HPNNH~
a
5 6 7
SCHEME 2. Experiment which led to the Manske-Ing modification of the Gabriel synthesis
of primary amines.
16 MACLEAN A N D SNIECKUS
FIG. 5. A page from the Ph.D. thesis of R. H. F. Manske describing the experiments which
led to the development of the Manske-Ing modification of the Gabriel synthesis of amines.
1. R. H. F. MANSKE: F I F W YEARS OF ALKALOID CHEMISTRY 17
The carbonyl of propiophenone was less reactive than that of phenylacetone by several
orders of magnitude. 1 inferred therefore that the carbonyl adjacent to the methyl
phenyl diketone would react with methylamine to yield a monoketimine with the other
carbonyl intact. [ I ]
In 1926, having completed his Ph.D. and with dubious prospects for a
job, Manske returned to North America and was fortunate to obtain a
position as a research chemist with General Motors Corp. in Detroit. His
tenure as an industrial chemist was brief for, in the following year, he was
offered an Eli Lilly Research Fellowship by Professor T. B. Johnson at
Yale University, having been recommended by Dr. Elizabeth Gatewood,
a former student of Johnson and Manske's former laboratory partner at
Manchester [l]. As a Lilly Fellow (1927-1929), he developed a new synthe-
sis 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-propanedione and other a-
diketones was reported (10).
Following observations made at Manchester, Manske showed that ure-
thanes and ureas, by-productsformed 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 prue-
In work apparently not linked with the Lilly Fellowship, Manske collabo-
rated with the biochemist R. W. Jackson on the synthesis of 3-indolyl butyric
cox (12).
(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 metabo-
lism (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 spon-
sored 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 (1 7J9). Further degradation studies,
jointly with L. Marion and M. Kulka (45,54,90), some of which were carried
out at Dominion Rubber Co., led to the isolation of substituted indoles,
quinolines, and P-carbolines. They were probably misled by the prevalence
of indoles and P-carbolines and failed to deduce the correct structure of
the alkaloid [e.g., see (U)] or of a key degradation product, calycanine (13).
It remained for the groups of Woodward at Harvard and Harley-Mason at
Cambridge to propose the correct structure in 1 9 0 , which was verified, in
an accompanying paper by Robertson and co-workers at Glasgow, through
an X-ray analysis [12].
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 19
10 (Kalycanthine)
' N'
12 (Manske's partial structure of calycanthine) 13 (Calycanine)
SCHEME 4. Structure of I-calycanthine: Manske's ncmesis.
B. THE SENECIO ALKALOIDS
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, respec-
tively. 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 struc-
tures of the Senecio alkaloids were eventually determined by Roger Adams
and others [13].
20 MACLEAN A N D SNIECKUS
C . THE ISOQUINOLINE ALKALOIDS
1. Introduction
At an early date I involved myself with plants belonging to the Fumariaceae family
and to my surprise I found one or more new alkaloids in virtually all of the thirty
species that I examined. Only about four of these species were native to eastern Canada.
Several of the others were obtained from collectors but most of them were grown in
my own garden. [l]
The first of the series of papers, “Alkaloids of Fumariaceous Plants,”
(21), appeared in 1932; the last, the fifty-seventh communication, in 1969
(239) [14]. Systematically and without spectral information, Manske made
his major mark in science through these contributions. Manske developed
a method of separation based on the solubility properties of hydrochlorides
in chloroform (24). This procedure simplified the separation process and
it was not uncommon for him to isolate and characterize eight or more
alkaloids from a single plant extract by fractional crystallization without
the modern-day benefit of chromatography. By application of this technique
to new and previously examined species, many new members of established
ring systems, as well as many alkaloids which defied structural categorization
at that time, were discovered. The latter were carefully preserved for further
study in Manske’s celebrated little brown bottles (Fig. 6) which, in turn,
FIG. 6. Manske’s little brown bottles: left: 3-indolyl-propionic acid (see Section 1V.F); right:
bicuculline (Dicentru cuculluriu).
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 21
were stored in pipe tobacco cans at least until 1952 when he quit smoking
in protest to an increase in the federal tax on tobacco [15]. Among these
were alkaloids of the cularine, spirobenzylisoquinoline, and cancentrine
families, whose structures, representing new ring systems, were resolved
many years after their isolation. In the beginning, Manske designated each
new or uncharacterized alkaloid by a Greek letter; soon, however, he
exhausted the Greek alphabet and devised a new open-ended system (42),
e.g., bicuculline, originally Alkaloid-a, became F1; the last alkaloid of the
series, F64, is now known as fumariline.
The accumulation of aporphine and protoberberine alkaloid types led
Manske to speculate on their biogenesis. These speculations were based
primarily on structural relationships among the various types and are sum-
marized 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 classifica-
tion (Fig. 7).
The ensuing account of Manske’s contribution to the isoquinoline alka-
loids 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, pro-
toberberine, and protopine families in Fumaria species was already recog-
nizedat 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 f in-
gosa (24,25) and the structure of the former established by oxidative deg-
radation (25). Capnoidine, a new alkaloid isolated from Corydalis sempervi-
rens (26) proved to be the enantiomer of adlumidine and diastereomeric
22 MACLEAN AND SNIECKUS
FIG. 7. Lecture delivered by R. H. F. Manske on the subject of alkaloids as an aid to
plant taxonomy.
with bicuculline (202). Corlumine (34), found in C. scouleri (35), C. sibiricu
(36), C. nobifis (60), and D. cucuffariu (34), was shown to be a diastereomer
of adlumine (34). Corlumidine, found only in C. scouleri (35), was converted
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 23
TABLE I
PHTHALIDEISOQUINOLINES AND SECOPHTHALIDEISOQUINOLINES
Alkaloid Source" Referencesh
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
" Plant source in which it was first reported.
' Hydrolysis product of bicuculline.
" Secophthalideisoquinolines.
References given for isolation and structural elucidation
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 References OH OMe OCH,O
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
1
1
1,10
2.1 1
2 9
1.11
1
13R
2
1
2,3,9,10
2,3,9,10
2,3,9
3,10
3,10
2,3,9,10
2,10
2,10,11
9,10 2.3
3 9.10
2.9.10
24 MACLEAN AND SNIECKUS
(+)-Bicuculline (1 s) (1 'R)
(+)-Adlumidine (1s) (1 's)
(-)-Capnoidine (1 R) (1 'R)
(- )-Bicucine (Lactone hydrolysis
product of Bicuculline)
Me
(+)-Corlumine R = Me (1s) (1 'R)
Cordrastine-1 Bicucullinine 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 communica-
tion (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 R = H
Allocryptopine R = Me
SCHEME 6. Protopine alkaloids.
1. R. H. F. MANSKE: F I R Y YEARS OF ALKALOID CHEMISTRY 25
Me0
MeO
Capaurine R = Me
Capauridine = (t)-Capaurine
OMe Capaurimine R = H
Caseamine
Caseadine
R’ = R4 = Me; R2 = R3 = H
R’ = R3 = R4 = Me; R2 = H
Caseanidine
OH
Coreximine
OMe
OMe
(+)-Tetrahydropalmitine R’ = R2 = R3 = R4 = Me
Cheilanthifoline
Ophiocarpine
R’ = Me; R2 = H; R3 + R4 = CH2
(3)-Aurotensine R‘ = R3= Me; R2 = R4= H
SCHEME 7. Tetrahydroprotoberberine alkaloids isolated by Manske (see Table 11).
and oxidative degradation. The alkaloids are listed in Table I and their
currently accepted structures are shown in Scheme 5.
Their structures were determined largely by intuition while making up for liquid
and salt losses by temperate imbibition. In one unfortunate case this process failed
and resort to experiment was necessary. The structure arrived at unfortunately was
compounded of a series of errors and the alkaloid turned out to be a specially pure
sample of cryptopine. Be it remembered though in extenuation that we had no IR
machine and no lithium aluminum hydride. [2]
26 MACLEAN AND SNIECKUS
(+)-Thalictrifoline
(2 )-Cavidine
(t)-/\pocavidine
R’ = R2 = R3 = Me; R4 = H
R’ = R2 = R4= Me; R3= H
R‘ = R4 = Me; R2 = R3 = H
‘%OR’ ‘ OR2
(+)-Thalictricavine
Epiapavidine
R’ = R2 = R4 = Me; R3 = H
R’ = R4 = Me; R2 = R3 = H
Me0
OMe
OMe
Solidaline
SCHEME 8. 13-Methyltetrahydroprotoberberine alkaloids 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-pentasubstituted tetrahydroprotober-
berine 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 C-
3 and C-10 methoxy groups (104,109), a “wrong” substitution pattern. It
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 27
was the first example of a norcoralydine in Nature. Aurotensine (F18), a
constituent of C. aurea (42), was later shown to be comprised mainly of
(?)-scoulerine (62). d,f-Tetrahydropalmitine was also isolated from C.
aurea (42).
Corydafis caseana (43) afforded two new tetrahydroprotoberberine alka-
loids, 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 tetrame-
thoxy compound. They were established to be tetrahydroprotoberberines
with a novel 1,2,10,11-substitution pattern (136). Although the single OH
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, ophiocar-
pine (F39), a tetrahydroprotoberberine substituted with an alcoholic OH
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 quantityin 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 diazo-
methane (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-methyltetrahydroprotoberberines were isolated and char-
acterized (Scheme 8). Examination of C. thafictrifolia (76) afforded four
new bases, namely thalictrifoline, a quaternary base from which (+)-thalic-
trifoline 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 thalictrifo-
line in which the positions of the substituents are transposed. Epiapocavi-
dine, also found in C. tuberosa, is des-0-methylthalictricavine carrying the
phenolic group at C-10 (153).
28 MACLEAN AND SNIECKUS
Based on spectroscopic examination of solidaline, a minor alkaloid of
C. solida (166), it has been proposed that the alkaloid is a protoberberine
with methoxyl groups at C-2, C-3, C-9, and C-10, a methyl and an OH
group at C-13, and an intriguing C-8-C-14 methylenedioxy bridge.
Corypalline (Scheme 9), was isolated from C. pallida and C. aurea (38).
Its 0-ethyl ether was identical with a synthetic sample of 7-ethoxy-6-
methoxy-2-methyl-l,2,3,4-tetrahydroisoquinoline thereby establishing its
structure. The bisbenzylisoquinoline, dauricine, was isolated from Meni-
spermum 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(ll-
hydrochelerythriny1)acetone (A), a previously unknown compound, dihy-
drosanguinarine (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-methyllauro-
tetanine for which they suggested the trivial name, lauroscholtzine. Two
apparently new alkaloids (Scheme lo), eschscholtzine, and a small amount
Corypalline
bH
Analobine
Dihydrochelerythrine 1 I-Oxosanguinarine
SCHEME 9. Miscellaneous alkaloids isolated by Manske.
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 29
0 0 OMe
(-)-Eschscholtzine (-)-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 fuma-
riaceous 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 bot-
tles which appear not to have been investigated further. These include F53,
F54, and F55 from C. nobilis (60), F56 and F57 from C. montana (65), F58
from H. fumariaefolia (71), and F60 from C. thalictrifolia (76).
3. Alkaloids with New Ring Systems
Cularine Alkaloids
I had worked on cularine for a number of years and though my intuition had given
me a satisfactory structure I was not able to confirm it experimentally. One day I was
looking up a paper on the vapor phase methylation of aniline but what really got my
attention was the following one in which the authors said that metallic sodium dissolved
in ammonia will quantitively hydrogenolyze diary1 ethers. After reading it a second
time and re-checking with my secretary, who also reads English, I confirmed the
structure of cularine while my assistant made some dimethylaniline. [2]
30 MACLEAN AND SNIECKUS
In 1938, Manske reported the isolation of cularine (Scheme 11) and
cularidine from D. cuculluriu and of cularine and cularimine from D. eximiu
(42). The fourth member of this group, cularicine (125) was separated
subsequently as a minor component of a mixture of phenolic bases obtained
from D. cluviculutu (58), of which the major component was cularidine. It
was only in 1950, after Manske had become Director of Research at Domin-
ion Rubber Co., that the structures of cularine and cularimine (des-N-
methylcularine) 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 dimeth-
ine (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
might yield a 1-benzyl-1,2,3,4-tetrahydroisoquinoline, a substance more
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 prod-
uct in which the ether oxygen was retained as a phenolic group on the
aromatic nucleus of the benzyl group. The 0-Me 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-trimeth-
oxybenzoic acid). These degradation experiments defined the structure of
Cularine R’ = R2 = R3 = R4= Me
R ’ O T R4 Cularimine R’ = R2 = R3 = Me; R4 = H
Cularidine R’ = H; R2 = R3 = R4 = Me
Cularicine R‘ = H; R2 + R3 = CH2; R4 = Me
-
\ /
R ~ O OR^
SCHEME 11. Manske’s cularine alkaloids.
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 31
Two-stage
Hofmann
degradation
14 (Cularine)
16 (Tricarboxylic acid) 17 (Xanthone)
SCHEME 12. The structure of cularine. Hofmann degradation and oxidation of the result-
ing dimethine.
the alkaloid. The terminus of the ether linkage was locatedat C-8 of the
isoquinoline nucleus on the basis of steric considerations and the observa-
tion 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 af-
ford cularine.
Cularidine underwent 0-methylation to afford cularine, thereby estab-
lishing 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 sub-
jected to ether cleavage with Na/NH3 and the product subsequently oxidized
with permanganate (130).
15 (Dimethine)
32 MACLEAN AND SNIECKUS
-
Meo? Me0 \ / OMe Me
Me0 W N .Me
Me0 OMe
14 (Gularine)
Na I liq NH3 -
18
I 1. OMethylation 2. Two-stage Hofmann
+ .
OMe
a C 0 2 H COpH HOpC)J+ OMe OH-I MnO; % / Me0 OMe \ Me0
OMe OMe
19 20 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 Alkaloid-
i 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 subse-
quently proved to be of the spirobenzylisoquinoline type include ochrobi-
rine (F14), initially obtained from C. sibirica (36) and subsequently from
C. lutea (52) and C. ochroleuca (53), fumaricine (44), and the related fumari-
tine and fumariline (139), from F. oficinalis, and sibiricine from C. sibi-
rica (139).
Thirty years after its detection, the brown bottles of ochotensine and
ochotensimine were reopened and their structures were elucidated in col-
laboration with S. McLean (132) [20]. The tool of the 1960s, NMR spectros-
copy, rather than chemical degradation, played the key role in the structural
elucidation and verification was achieved by X-ray crystallographic analysis
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 33
Ochotensine
Ochotensimine R' = = ~e Fumaritine R = H
R' = H; R2 = Me Fumaricine R = Me
Fumariline Sibiricine
Ochrobirine Fumarofine
SCHEME 14. 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 exo-
methylene 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 spiro-
benzylisoquinoline class and was later shown to have an interesting benza-
zepine structure by Shamma and co-workers [21].
34 MACLEAN AND SNIECKUS
The Cancentrine Alkaloids
Then there was M2 later called cancentrine. 111
From Dicentra canadensis, a species which holds the distinction of being
the first of the fumariaceous plants which he examined (21), Manske isolated
a base, as orange needles, which was not named at that time but later
designated F22 (42). This alkaloid was named cancentrine in 1970, some
30 years after its isolation, when it yielded its secrets (22, Scheme 15) to
X-ray crystallographic analysis of a degradation product (145). A single
stage Hofmann followed by hydrogenation and treatment with diazometh-
ane 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-methyl-
ether, its 0-acetate, and its methine, the structure of cancentrine was de-
duced. Two dehydro derivatives of cancentrine were subsequently charac-
terized (150) and its reactions were studied (149,156). The dimeric structure
of cancentrine and its dehydro derivatives is unique among the isosquiono-
line 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
MeO
1. Hofmann -
2. H 2 I R
3. CH2N2
Me0
Me0
7 Me
Me
Y
22 (Cancentrine) 23 (Dihydromethine-Omethylether)
S C H E M E 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 benza-
zepine alkaloid, rhoeadine (160), and the benzilic alkaloid, cryptopleu-
rospermine (165), were achieved.
Furthermore, methods were developed for the conversion of the proto-
berberine ring system into spirobenzylisoquinolines (157), into protopines
(158), and into rhoeadines (161).
D. THE LYCOPODIUM 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 chemistsworldwide with a challenging playground.
The resulting ingenious achievements in total synthesis have enriched the
field of organic chemistry and are an appropriate measure of the impact
of the initial work by Manske and Marion; equally appropriately, they have
36 MACLEAN AND SNIECKUS
Lycopodine Annotinine Annotine
p-Obscurine
Cemuine
Fawcettimine Me
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 ochofensis with 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 rea-
sons. . . . 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 alka-
loids, 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 37
Lobinaline 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 trypt-
amine. . . . (71
The scientific contributions of Manske were not confined to alkaloids
but extended into several areas of general heterocyclic chemistry. He wrote
review articles on the synthesis and reactions of quinolines (RZ) and isoqui-
nolines (R2). Together with M. Kulka, his collaborator at Uniroyal, he
contributed a chapter, “The Skraup Synthesis of Quinolines” to Organic
Reactions (R4). These were comprehensive and definitive contributions
to the literature at that time and still provide valuable background for
contemporary workers in these areas of heterocyclic chemistry. Aside from
these reviews, Manske reported on modifications of the Skraup quinoline
synthesis (64,99) and, in a definitive paper, described the preparation and
characterization of seven monomethyl and twenty-one dimethylquinolines
(69) which proved to be useful reference materials in the commonly used
vigorous S and Se degradation studies of alkaloids.
In a series of investigations, Kulka and Manske synthesized the twelve
isomeric pyridocarbazoles (92,97,102,206,222,223) using quinolines (112),
N-substituted tetrahydroquinolines and hydrazino isoquinolines (96) as
key intermediates (e.g., Scheme 18). Still others were obtained from
38 MACLEAN AND SNIECKUS
3-carbethoxy-4-hydroxy-l l Kpyrido[2,3-a]carbazole
SCHEME 18. 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 pro-
duced in these syntheses. The pyridocarbazoles proved to be valuable refer-
ence compounds in the later structural elucidation of the antitumor alka-
loids, 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 dedi-
cated to his memory [25] (Figs. 9-12).
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 39
FIG. 8. A page from laboratory book of R. H. F. Manske showing an account of 3-indolyl
w-substituted carboxylic acids prepared and sold to nurseries and agricultural research centers.
40 MACLEAN AND SNIECKUS
FIG. 9. R. H. F. Manske in Dominion Rubber Co. laboratories, Guelph, Ontario. ca 1944-
1945. 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. He himself wrote on alkaloids with which he was intimately
familiar and, in the later years, instituted a continuing chapter on miscellane-
ous alkaloids (see Publications Lists, Section XI). Of the Volumes 1-20
with Manske on the spine, Volumes 1-4 were coedited with H. L. Holmes
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 41
FIG. 10. Gathering for a seminar at the Dominion Rubber Co. laboratories, 1965. Front
row, left to right: A. Harrison, R. H. F. Manske: second row, head above Manske: M. Kulka.
(Courtesy D. Brewer.)
and Volumes 17-20 bear his name, posthumously, with R. G. A. Rodrigo.
Rereading the Prefaces of Volumes 1-16 offers, in addition to the Manske
mastery of crisp and correct English, an instructive portrait of alkaloid
chemistry over a 25-year period.
As an editor, Manske was known to be precise, demanding, critical but
ultimately objective and impartial. From experience (V. S.), we know that
there had to be a very good reason for a tardy manuscript and that the
English grammar and syntax was carefully evaluated (at times, with the help
of his son-in-law, H. MacCallum, a Professor of English at the University of
Toronto). There were not a few manuscripts which were entirely rewritten
by Manske. On the other hand, we also know that he listened carefully to
counterargument and admitted openly his errors when proven wrong. These
characteristics, clearly evident in Volumes 1-16 of the series, contributed
not insignificantly to making The Alkaloids unsurpassable as a compre-
hensiveaccount of alkaloid research. He also served as Associate Editor
42 MACLEAN AND SNIECKUS
FIG. 11. R. H. F. Manske (third from right) with his research group, Dominion Rubber
Co.. December 1963. From left to right: M. Kulka, A. E. Ledingham, W. Boos. R. H. F.
Manske, K. McPhee, and G. Rozentals. (Courtesy D. Brewer.)
(1939-1948) of the Journal of the American Chemical Society and of Organic
Reactions (Vol. 7 , 1953) with equal diligence.
VI. The Scientist and Society
In summary the burden of my message has been that he who professes science is
truly a scientist only if he strives to achieve an awareness of his place in society as a
whole. If the buries himself in the confines of his discipline and neither knows nor
cares about the broad vista of the world about him he has failed as a man; and if he
does not apply the objective, that is scientific, method to matters other than to those
of his narrow discipline he has failed as a man. Indeed the scientist has failed as a man
if he has not made it a sine qua non of his life to question authority, be it of Mohamet
or of Darwin. [26b]
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 43
FIG. 12. Doris and Richard Manske at his retirement party from the Dominion Rubber
Co. (Uniroyal Ltd.), September 1966. (Courtesy D. Brewer.)
In 1963, Manske was invited to assume the office of Vice-president of
the Chemical Institute of Canada (CIC) (now Canadian Society of Chemis-
try, 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], thought-
provoking messages regarding the role of science and scientists in society.
His thesis, that “objectivity and objectivity alone should guide them in
making decisions and that such a modus vivendi should be applicable to
matters other than scientific,” provoked a great deal of discussion at a time
when the social responsibility of the scientist was a widely debated topic.
More than 10 years later and shortly before the end, in a lecture entitled
“Science, Society, and Survival or Time is Running Out” he attempted
again “. . . to have scientific society expend some of its expertise on the
44 MACLEAN AND SNIECKUS
FIG. 13. Note by R. H. F. Manske concerning the autobiographical handwritten manuscript
[ I ] . “Dear Victor-This is hurried. crude, and perhaps not legible. Please suffer it and treat
it firmly and not necessarily kindly. Unless I get a stop order I will continue to scribble.
Sincerely Dick M” shows the highly individual style and flair of the Editor of The Alkaloids.
socio-political issues of our times.” [9]. Although disappointed (“To my
regret nothing came of it.”), Manske maintained strong and outspoken
principles in this matter, as the following quotes clearly indicate.
I . . . maintain that a society that functions only on subjectivity and emotions is an
anachronism at a time when so much factual knowledge is available. [ I ]
If we leave the decisions to politicians and theologians we will inherit a society
which scientists will not like and we will have only ourselves to blame. [ l )
Not only politicians and theologians received Manske’s wisdom and wit.
Whenever he believed that his scientific training would allow a knowledge-
able contribution, he attempted to establish a reasoned dialogue. Thus he
participated in a lengthy debate on the question of the sale of Canadian
wheat to Red China and, in the editorial pages of his home town newspaper,
entered into a lively discussion on science and religion [15]. On the last
topic, he delivered a series of lectures at the University of Waterloo.
Throughout his life, he maintained an alert interest in the world around
him and a concern for human relations.
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 45
VII. Naturalist, Orchidist, Musician, and Cuisinier
The beautiful yellow buffalo bean proved thirty years later to be Thermopsis rhombi-
foliu 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. He 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 expedi-
tions 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 experi-
mented 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. He 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.
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 47
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
witha 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
48 MACLEAN AND SNIECKUS
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
1927-1929, Eli Lilly Research Fellow, Yale University, New Haven,
1929-1930, Sterling Fellow, Yale University.
1930-1943, Associate Research Chemist and then Head, Organic Chem-
Corp., Detroit, Michigan, USA.
CT, USA.
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 49
istry Section, National Research Council of Canada, Ottawa, On-
tario, 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, On-
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 So-
1975, A. C. Neish Lecturer, NRC of Canada, Halifax, Nova Scotia,
Publications: 167 papers on the structural elucidation and synthesis of al-
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.
tario, Canada.
ciety.
Canada.
kaloids.
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 manu-
scripts (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.
50 MACLEAN AND SNIECKUS
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), accord-
ing to family legend. Dicentra species were also under intensive study at that time. Bur-
gener, 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 undergrad-
uates 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,
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 ) ,
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 ,
20. S. McLean, and M.-S. Lin, Tetrahedron Lett., 1964, 3819; S. McLean, M.4. Lin, A. C.
21. G. Blask6, N. Murugesan, S. F. Hussain, R. D. Minard, M. Shamma. B. Sener, and M.
22. D. B. MacLean, The Alkaloids, 1985,26, 241; W. A. Ayer, and L. S. Trifinov, ibid., 1994,
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 Water-
loo a month before his first meeting with Manske.
p. 155.
1971,2541.
1973, 1226.
MacDonald, and J. Trotter, ibid, 1966, 185.
Tanker, Tetrahedron Lett., 1981,22, 3135.
45, 233.
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.,
27. M. H. Kreish, “I Cook as I Please.” Exposition Press, Hicksville, NY, 1978, 47 pp.
28. D. B. MacLean, The Alkaloids, 1979, 17, xi.
March 1964, p. 12.
Reviewed V. Snieckus, J . Chem. Educ., 1979,56, A182.
1. R. H. F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 51
Publications of R. H. F. Manske
1. A Modification of the Gabriel Synthesis of Amines. H. R. Ing and R. H. F. Manske,
2. Harmine and Harmaline. Part IX. A Synthesis of Harmaline. R. H. F. Manske, W. H.
3. The Decomposition of P-3-Indolylpropionic Azide. R. H. F. Manske and R. Robinson,
4. A Synthesis of Rutaecarpine. Y. Asahina, R. H. F. Manske, and R. Robinson, J. Chem.
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, 2269-
2272 (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 D-
Mannose. 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 .
20. The Alkaloids of Senecio Species. I. The Necines and Neck Acids of S. refroisus and
21. Alkaloids of Fumariaceous Plants. I. Dicentra canadensis Walp. R. H. F. Manske, Can.
J . Chem. Soc. 2348-2351 (1926).
Perkin, Jr., and R. Robinson, J. Chem. Soc. 1-14 (1927).
J. Chem. Soc. 240-242 (1927).
SOC. 1708-1710 (1927).
Res. 5,592-600 (1931).
S. jacobaea. R. H. F. Manske, Can. J. Res. 5,651-659 (1931).
J. Res. 7,258-264 (1932).
52 MACLEAN AND SNIECKUS
22. Alkaloids of Fumariaceous Plants. 11. Dicentra cucullaria (L.) Bernh. R. H. F. Manske,
23. Alkaloids of Fumariaceous Plants. 111. A New Alkaloid, Bicuculline, and its Constitution.
24. Alkaloids of Fumariaceous Plants. IV. Adlumina fungosa Greene. R. H. F. Manske,
25. Alkaloids of Fumariaceous Plants. V. The Constitution of Adlumine. R. H. F. Manske,
26. Alkaloids of Fumariaceous Plants. VI. Corydalissempervirens (L.) Pers. R. H. F. Manske,
27. Alkaloids of Fumariaceous Plants. VII. Dicentra eximia (Ker) Torr. R. H. F. Manske,
28. Alkaloids of Fumariaceous Plants. VIII. Corydalis aurea, Willd. and the Constitution of
29. Alkaloids of Fumariaceous Plants. IX. Dicentra formosa Walp. R. H. F. Manske, Can.
30. Alkaloids of Fumariaceous Plants. X. Dicentra oregana Eastwood. R. H. F. Manske,
31. Reaction Products of Indoles with Diazoesters. R. W. Jackson and R. H. F. Manske,
32. A Synthesis of Indolyl-Valeric Acid and the Effects of Some Indole Acids on Plants.
33. The Alkaloids of Senecio Species. 11. Some Miscellaneous Observations. R. H. F. Manske,
34. Alkaloids of Fumariaceous Plants. XI. Two New Alkaloids, Corlumine, and Corlumidine
35. Alkaloids of Fumariaceous Plants. XII. Corydalis scouleri Hk. R. H. F. Manske, Can.
36. Alkaloids of Fumariaceous Plants. XIII. Corydalis sibirica Pers. R. H. F. Manske, Can.
37. The Natural Occurrence of Acetylornithine. R. H. F. Manske, Can. J. Res. 15B,
38. Alkaloids of Fumariaceous Plants. XIV. Corypalline, Corlumidine, and Their Constitu-
39. Alkaloids of Fumariaceous Plants. XV. Dicentra chrysantha Walp. and D. ochroleuca
40. An Alkaloid from Delphinium brownii Rydb. R. H. F. Manske, Can. J. Res. 16B,
41. Anolobine, an Alkaloid from Asimina triloba Dunal. R. H. F. Manske, Can. J. Res. 16B,
42. The Alkaloids of Fumariaceous Plants. XVI. Some Miscellaneous Observations.
43. The Alkaloids of Fumariaceous Plants. XVII. Corydalis caseana A. Gray. R. H. F.
44. The Alkaloids of Fumariaceous Plants. XVIII. Fumaria officinalis L. R. H. F. Manske,
45. Calycanthine. 111. Some Degradation Experiments. L. Marion and R. H. F. Manske,
46. Lobinaline, an Alkaloid from Lobelia cardinalis L. R. H. F. Manske, Can. J. Res. 168,
Can. J . Res. 7,265-269 (1932).
R. H. F. Manske, Can. J . Res. 8, 142-146 (1933).
Can. J . Res. 8,210-216 (1933).
Can. J . Res. 8,404-406 (1933).
Can. J . Res. 8,407-411 (1933).
Can. J . Res. 8,592-599 (1933).
Bicucine. R. H. F. Manske, Can. J. Res. 9,436-442 (1933).
J. Res. 10, 521-526 (1934).
Can. J . Res. 10,165-770 (1934).
Can. J . Res. 13B, 170-174 (1935).
R. H. F. Manske and L. C. Leitch, Can. J. Res. 14B, 1-5 (1936).
Can. J . Res. 14B, 6-11 (1936).
and Their Constitutions. R. H. F. Manske, Can. J. Res. 14B, 325-327 (1936).
J . Res. 14B, 347-353 (1936).
J. Res. 148,354-359 (1936).
84-87 (1937).
tions. R. H. F. Manske, Can. J. Res. 15B, 159-167 (1937).
Engelm. R. H. F. Manske, Can. J. Res. 15B, 274-277 (1937).
57-60 (1938).
76-80 (1938).
R. H. F. Manske, Can. J. Res. 16B, 81-90 (1938).
Manske and M. R. Miller, Can. J. Res. 16B, 153-157 (1938).
Can. J . Res. 16B, 438-444 (1938).
Can. J . Res. 16B, 432-437 (1938).
445-448 (1938).
1. R. H. F. MANSKE: FIlTY YEARS OF ALKALOID CHEMISTRY 53
47. The Alkaloids ofSenecio Species. 111. Senecio integerrimus, S. longilobus, S. spartioides,
48. The Alkaloids of Senecio Species. IV. Erechtites hieracifolia (L.) Raf. R. H. F. Manske,
49. A Synthesis of a-Naphthyl-Acetic Acid and Some Homologues. R. H. F. Manske and
50. The Alkaloids of Fumariaceous Plants. XIX. Corydalis ophiocarpa Hook. F. et Thorns.
51. The Alkaloids of Fumariaceous Plants. XX. Corydalis micrantha (Engelm.) Gray and
52. The Alkaloids of Fumariaceous Plants. XXI. Corydalis lutea (L.) DC. R. H. F. Manske,
53. The Alkaloids of Fumariaceous Plants. XXII. Corydalis ochroleuca Koch. R. H. F.
54. Calycanthine. IV. A Structural Formula. R. H. F. Manske and L. Marion, Can. J. Res.
55. The Alkaloids of Papaveraceous Plants. XXIII. Clauciumflavum Crantz. R. H. F. Man-
56. The Alkaloids of Fumariaceous Plants. XXIV. Corydalis ochotensis Turcz. R. H. F.
57. The Alkaloids of Fumariaceous Plants. XXV. Corydalis pallida Pers. R. H. F. Manske,
58. The Alkaloids of Fumariaceous Plants. XXVI. Corydalis claviculata (L). DC. R. H. F.
59. The Alkaloids of Fumariaceous Plants. XXVII. A New Alkaloid, Cheilanthifoline, and
60. The Alkaloids of Fumariaceous Plants. XXVIII. Corydalis nobilis Pers. R. H. F. Manske,
61. The Alkaloids of Fumariaceous Plants. XXIX. The Constitution of Cryptocavine.
62. The Alkaloids of Fumariaceous Plants. XXX. Aurotensine. R. H. F. Manske, Can. J.
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 Lycopod-
ine. 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).
and S. ridellii. R. H. F. Manske, Can. J. Res. 17B, 1-7 (1939).
Can. J . Res. 178, 8-9 (1939).
A. E. Ledingham, Can. J. Res. 178, 14-20 (1939).
R. H. F. Manske, Can. J. Res. 17B, 51-56 (1939).
Corydalis crystalha Engelm. R. H. F. Manske, Can. J. Res. 178, 57-60 (1939).
Can. J . Res. 178, 89-94 (1939).
Manske, Can. J. Res. 178, 95-98 (1939).
178, 293-301 (1939).
ske, Can. J. Res. 18B, 75-79 (1940).
Manske, Can. J . Res. 18B, 75-79 (1940).
Can. J . Res. 18B, 80-83 (1940).
Manske, Can. J. Res. 18B, 97-99 (1940).
Its Constitution. R. H. F. Manske, Can. J. Res. 18B, 100-102 (1940).
Can. J . Res. 188,288-292 (1940).
R. H. F. Manske and L. Marion, J. Am. Chem. SOC. 62,2042-2044 (1940).
Res. 18B, 414-417 (1940).
54 MACLEAN AND SNIECKUS
72. The Natural Occurrence of 3-Methoxy-pyridine. R. H. F. Manske, Can. 1. Res. 2oB,
73. The Alkaloids of Fumariaceous Plants. XXXV. Corydalis platycarpa Makino. R. H. F.
74. An Alkaloid from Menispermum canadense L. R. H. F. Manske, Can. J. Res. 21B,
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,
77. The Alkaloids of Fumariaceous Plants. XXXVII. Dactylicupnos macrocapnos Hutchin-
son. 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 Lycocton-
ine. 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. (Urosta-
chys 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.
94. The Alkaloids of Lycopodium Species. X. Lycopodium cernuum. L. L. Marion and
95. Some Anomalous Reactions of Phenylmagnesium Chloride. R. H. F. Manske and A. E.
265-267 (1942).
Manske, Can. J. Res. 21B, 13-16 (1943).
17-20 (1943).
111-116 (1943).
69,2126-2129 (1947).
R. H. F. Manske, Can. J. Res. 26B, 1-2 (1948).
Ledingham, Can. J. Res. 27B, 158-160 (1949).
1. R. H. F. MANSKE: F I F W YEARS OF ALKALOID CHEMISTRY 55
96. The Synthesis of Some Isoquinolines. R. H. F. Manske and M. Kulka, Can. J. Res. 278,
97. The Synthesis of Some Pyridocarbazoles. R. H. F. Manske and M. Kulka, Can. J. Res.
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 Culari-
mine. 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,
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 Coraly-
dine. 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.
107. 3-Bromometameconine. R. H. F. Manske, J. A. McRae, and R. Y. Moir, Can. J. Chem.
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,
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, 1501-
1504 (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).
161-167 (1949).
278, 291-296 (1949).
3207-3208 (1950).
72,4997-4999 (1950).
29,526-535 (1951).
711-719 (1952).
56 MACLEAN AND SNIECKUS
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).
121. 1,6-Bis(y-Carbethoxypropyl)-2,3,7,8-Dibenzopyrocoll. A By-product in the Preparation
of y-(3-Indolyl)butyric Acid R. H. F. Manske and W. R. Boos, Can. J. Chem. 38, 620-
621 (1960).
122. The Genus Oceanopapaver, R. H. F. Manske, Nature 200,1123 (1963).
123. Diphenylmethane-3,3’-Dicarboxylic Acid. 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 Menisper-
mum 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 Alka-
loids. R. P. K. Chan, J. Cymerman Craig, R. H. F. Manske, and T. 0. Soine, Tetrahedron
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, 1951-
1956 (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).
23,4209-4214 (1967).
1. R. H. F. MANSKE: FIFIY YEARS OF ALKALOID CHEMISTRY 57
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 Tetrahydroprotoberberine from Corydalis tuber-
osa. 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. %, 1944-
1945 (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.
164. 3,4-Methylenedioxyphthalide-a-carboxylic Acid; Its Use in the Total Synthesis of Isoqui-
54,471-472 (1976).
58 MACLEAN AND SNIECKUS
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 pleu-
rosperrna. 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 12-
0x0-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,
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).
59-98 (1953).
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
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
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
R21. The Alkaloids of Pseudocinchona and Yohimbe. R. H. F. Manske, The Alkaloids 8,
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-Naphthaphenanthridine Alkaloids. R. H. F. Manske, The Alkaloids 10,485-489 (1965).
R25. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 10,
R26. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids U,
R27. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 13,
(1955).
(1960).
8, 679-691 (1965).
694-723 (1965).
545-595 (1965).
455-512 (1970).
397-430 (1971).
1. R. H . F. MANSKE: FIFTY YEARS OF ALKALOID CHEMISTRY 59
R28. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 14,
R29. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 15,
R30. Alkaloids of Dendrobium. Symp. Sci. Aspects of Orchids (H. H. Szmant and J. Wemple,
R31. Alkaloids Unclassified and of Unknown Structure. R. H. F. Manske, The Alkaloids 16,
507-573 (1973).
263-306 (1975).
eds.), pp. 122-125, Chemistry Department, University of Detroit, 1974.
511-556 (1977).
MISCELLANEOUS
M1. The Isoquinoline Alkaloids, Centenary Lecture. R. H. F. Manske, J. Chem. SOC., 2987-
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. 871-
2990 (1954).
884, (1985).
This Page Intentionally Left Blank
-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- 752 70, Pakistan
I. Introduction ........................ .......................................... 61
B. Steroidal Alkaloids of the Buxaceae .......................................... 63
C. Steroidal Alkaloids of the Liliaceae .................................
D. Steroidal Alkaloids of the Solanaceae ................................................. 69
E. Steroidal Alkaloids from Terrestrial Animals ................... 72
F. Steroidal Alkaloids from Marine Organisms ......................
111. Physical Properties ...........................................................
A. NMR Spectra ...................................... ................ 75
................................................................. 81
.......................................... 89
A. Steroidal Alkaloids of the Apocynaceae and Buxaceae ........................... 90
B. Steroidal Alkaloids of the Liliaceae and Solanaceae ............................... 92
IV. Biogenesis ...................................................
V. Some Synthetic Studies and Chemical Transformations ............
VI. Pharmacology ... .................. ................... 98
A. Steroidal Alk ................... 98
D. Steroidal Alkaloids of the Solanaceae ........ ...................... 101
E. Steroidal Alkaloids from Terrestrial Animals ..................................... 102
References .................................................................
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 1998by 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 “steroi-
dal 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 ob-
tained by the chemical conversion of structurally related steroidal alka-
loids.
With the advent of new and more sensitive spectroscopic, bioassay, and
isolation techniques, the field of steroidal alkaloids has witnessed a renais-
sance 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 amphibi-
ans 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 pres-
ent 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 Apocyna-
ceae, 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 1 and 2, respectively.
Conessine (3) was the first, and the most common, member of conanine-
type 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 ,4-
trienin-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
(7) Regholarrhenine C
(41 Holonamlne (5) Regholarrhenlne A: R = Me
(6) Regholarrhenlne B; R = H
.... NHMe
(8) R1 = H. Rz = OH (10) PO-epi-Klbatallne H l C \ H (11) Cyclobuxine-D
(9) R' = Rz =OH ,
30 31 1121
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. har-
landi, 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. sempervir-
ens), 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. micro-
phylla and B. koreana), Goutarel (B . balearica, B. rolfei, and B. malayana),
Doepke, and more recently, by our research group in Pakistan (B. papil-
losa 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-trimet hyl-5a-pregnane) and 13 [deriva-
tives of abeo-9 (10 + 19)-4,4,14a-trimethyl-5a-pregnane]. In each skeleton,
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 tetrahydrooxa-
zine 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. papil-
losa (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
doublebond 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-buxafuran-
amine (18) have been isolated by us from B. hildebrandtii of Ethiopian
origin (28).
A number of reviews containing spectral generalizations of Buxus alka-
loids 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 13C NMR spectroscopy, UV and IR spectropho-
tometry, 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
(14) Harappamine. R = H
(151 Moenjodaramine. R = CH,
(171 06-Buxafuranamlne
30 31
(191 Buxane
y NMe2
MelN 9 H
/ R
H
MeHN
(16) Papllamine
(181 O'o-Buxafuranamlne
(21) Saracoclne: A5.6
(221 Saracodine
_.
(20) Pachysamine-A
67 2. CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS
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. Structur-
ally, 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 cervera-
trum 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 CZ7 skeleton with six rings that are often highly oxygen-
ated. 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
21
26
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
69 2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS
occurring acids such as benzoic, 2-methylbutanoic, acetic, 2‘methylbuten-
oic, 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 alka-
loids 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 com-
mon example of this structural class, isolated from various species of Fritil-
laria (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 CZ7 cholestane 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 C27 nor-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 produc-
tion of hormonal steroids because of their closely related pregnane struc-
tures. This was demonstrated 30 years ago when Sat0 et al. announced the
chemical transformation of the spirosolane alkaloids solasodine (34) and
tomatidine (35) into 3/3-acetoxypregna-5,16-dien-20-one and its 5,6-dihydro
derivative (4,38).
Solasodine (34) is an important member of this class isolated from many
Solanum species (26), and has been receiving increased interest as a starting
material for the commerical production of steroidal drugs. It has also been
regarded as the “diosgenin of the next decade” (4 ) .
70 A’ITA-UR-RAHMAN AND CHOUDHARY
(311 Solacongeslldlne-type
18
2
3
1321 Solanocapsine-type (331 Jurbidlne-type
21
18 i k !
Solasodine (34) has remained an important target of synthetic studies
over the past 10 years. A number of derivatives of 34, such as N-cyano and
A-nor-3-aza derivatives and degradative products, were prepared in order
to obtain “new physiologically active steroids.” The related steroidal alka-
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 71
*..*.*
",*. .....
(381 Etiolinine, R = Glc(4 + 1)Clc
OH (401 Solanocapslne
HO Y
O H
(41) R' = OH: Rz = Me: R3 = Et. AZ2lN)
(42) R' = Me: R2 = R3 = H
139) 3-O~-Lycotrlaoslde
HN &YR ..A 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, solash-
banine, solaradine, robustine, and ravifoline, have been isolated from vari-
ous species of the genus Solanurn (16,20).72 ATTA-UR-RAHMAN AND 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
(I 690).
Leaves, roots, and stems of Solanum havanense Jacq. have furnished P-
solamarine 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 (agly-
cone) and glycoalkaioids, are known. Solanidine (29) is the most important
member of this class, being isolated from many plants of the genera Rhino-
petalum, Veratrum, Solanum, and Fritillaria. Stems of S. lyratum have
yielded a mixture of new steroidal glycoalkaloids, including 3-0-p-lycotriao-
side (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,22pH-
cholestane-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. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 73
(46) Bufotallin a: R = H
(47) Bufotallin b: R = Me
0
(491 Zoanthamine
(48) Batrachotoxin
0
( 50) Zoanthenamine
%o \ N
0
( 52) 28-Deoxy-zoanthenamine
Biogenetically, these alkaloids are derived from mevalonate via choles-
terol. 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), zoanthen-
amine (50), zoanthamide (51) (51), 28-deoxy-zoanthenamine (52), 22-epi-
28-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 deter-
mined by X-ray diffraction analysis (54). Fifteen members of this series,
cephalostatins 1-15, have been isolated by Pettit et al. (55,56). Cephalo-
statins apparently arise in nature by the condensation of two 2-amino-3-
oxosteroid units to yield dimeric steroidal molecules connected by a pyr-
azine ring.
Recently, another class of highly cytotoxic, dimeric and steroidal alkaloids
structurally related to the cephalostatins has been isolated from the lipo-
philic extract of the tunicate Ritterella tokioka Kott (Polyclinidae). Thirteen
members of the series, designated ritterazines (ix. , ritterazines A-M) (56-
a), 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, lokyster-
olamine A (71) and lokysterolamine B (72), from an unidentified species
2. CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS 75
(53) 22-epi-28-Deoxy-zoanihenamine (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 = H; 22R
IQI Ritternrine n P = u. m e
27 (60) Ritterazine E, R = Me: 22s
1 y
OH
(57) Ritterazine B, R1 = OH. R2 = H, R3 = H; 22R
(61) Ritterazine F, R 1 = OH, RZ = H, R3 = H; 22s
(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: 22s
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 alka-
loids confers certain characteristic spectral properties. In the case of an
unsubstituted triterpenoid skeleton, the C-19 cyclopropyl methylenic pro-
tons appear strikingly upfield in the region of 6 0.1-0.5 as AB doublets
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 77
I /Plw+"- H
0 X I I n
.-ft\ft'/"'W H ,*
(66) Ritterazine K, R1 = H.R2 = OH: 22R
(67) Ritterazine L, R' = H, R2 = H: 22R
(68) Ritterazine M, R1 = H, R2 = H; 22s
(69) Plakinamine A; R, = H. R, =
(70) Plakinamlne 8; R, = Me, R, =
6H
(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 H
\ ,...%me, \ .,.,*me2
21 21
(73) N-Benzoyl-0-acetylcyclobuxoline-F (74) Cyclobuxapaline-C
Med
(75) Verabenzoamine
H
(76) Noriatifoline (77) Malouetafrine
them to the region between 6 0.9 and 1.5. Compounds bearing C-1, C-2
and C-11, C-12 double bonds or C-1 and C-11 hydroxy groups exhibit only
one-half of the AB doublets at about 6 0.6, the other proton being shifted
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 79
downfield (13). Compounds with C-6, C-7 double bonds such as 74 exhibit
a pronounced upfield shift of the cyclopropyl protons to -6 0.4-0.1. This
is due to the P-oriented cyclopropyl methylene protons lying in the shielding
region of the 6,7-double bond, as in cyclobuxapaline-C (66). The 'H NMR
spectra of Bums bases generally show five methyl signals, one of which
(C-21 methyl) appears as a doublet at 6 1.0. The C-31 methyl is often
oxygenated as CH20H, 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 pro-
tons 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 satu-
rated, 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 skele-
ton 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
(801 Solanogantamine
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 reso-
nates at 6 3.7 if a C-5, C-6 double bond is present. An upfield shift of about
-6 0.5 is observed if ring B is completely saturated, as in the case of
hupehenisine (79) (74).
5. Solanidine- Type Alkaloids
The solanidine-type steroidal alkaloids generally show four methyl signals
in their 'H NMR spectra, two of which are secondary (C-27 and C-21) and
appear as doublets; the doublet for the C-21 methyl generally resonates
upfield of the C-27 methyl doublet. The multiplet for the C-3 methine
proton geminal to the hydroxyl or amino group appears at -6 3.7 or 2.8,
respectively. The presence of another downfield signal at 6 3.8 is generally
due to the C-23 methine proton when a hydroxyl group is present at C-23,
as in solanogantamine (80) (75).
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 81
6. Spirosolane- Type Alkaloids
The 'H NMR spectra of spirosolane-type steroidal alkaloids contain
doublets for two secondary methyls (C-21 and C-27) resonating between
S 0.8-0.96 and singlets for two tertiary methyls (C-19 and C-18) which
appear at -6 1.1 and -0.8, respectively. Spirosolane-type alkaloids possess
spiro-linked piperidine and tetrahydrofuran moieties which give them a
characteristic spectral pattern. A downfield multiplet at -8 4.2 is due to
the C-16 methine proton geminal to the oxygen of the tetrahydrofuran
ring as in solasodine (34) (76,77). The C-26 methylene protons geminal to
nitrogen resonate in the range of S 2.6-2.8. The C-22 quaternary carbon,
resonating at 6 -99.0 in the 13C NMR spectra, is characteristic of all spiro-
solane alkaloids (62).
B. MASS SPECTRA
The mass fragmentation pattern of steroidal alkamines is characteristi-
cally 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 compo-
sitions 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 Buxa-
ceae) classes, while compounds with only one nitrogen atom may be mem-
bers 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 unsatura-
tion in different compounds.
Mass spectrometric studies of a large number of steroidal alkaloids have
provided some important generalizations that are extremely useful in struc-
ture elucidation. Some of these are summarized below. A number of review
articles on the mass spectrometry of steroidal alkaloids have been pub-
lished (71,78-8I).
82 AITA-UR-RAHMAN AND CHOUDHARY
1. BUMS Alkaloids
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 fragmentsresulting 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 f l 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 character-
istic peaks in their mass spectra. For example, the mass spectrum of buxanol-
idine (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
f Q l 1
/ m/z 72
SCHEME 1 .
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 83
R = Me, m / z 71 (lOoO/o)
R = H. m / z 57 (100%)
SCHEME 2.
attached to C-15 or C-16. The ion at d z 85 (CSHIIN) with no oxygen arose
by the cleavage of the N-containing group of ring D along with C-17, and,
since it was devoid of oxygen, indicated the presence of the O H group at
C-16. Alkaloids bearing OAc instead of -OH on ring D at C-16 such as
16a-acetoxybuxabenzamidenine (83) isolated from B. papillosa show peaks
at m/z 157 and 171 (Scheme 3) (22,23,83).
2. Cerveratrum- or Cevine-Type Alkaloids
The presence of a C-27 carbon skeleton with six fused rings, one nitrogen,
and at least one oxygen is a characteristic common to all cevine-type steroi-
dal bases. Many other cevine-type bases also contain an ether bridge be-
tween C-9 and C-4. The mass spectra of cerveratrum alkaloids contain a
significant peak at d z 112 or 11 1 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
-C-
33) 1 6a-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 frag-
ment at d z 112 of zygacine (&I), a 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).
184) Zygacine
OH
m/z 112
SCHEME 4.
2. CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS 85
4 m/z 71
Me
I
m/z 300 (85) 5a-Conanine
SCHEME 5.
4. Jerveratrum- Type Alkaloids
The jeveratrum-type steroidal alkaloids generally contain only one nitro-
gen 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 (Lilia-
ceae), 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 tetrahy-
drofuran 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 stenan-
zine (78) isolated from Rhinopetalurn stenantherurn, show the most abun-
dant ion at d z 114, again resulting from the a-cleavage of the piperidine
(C-22-C-20) side chain (Scheme 8) (73).
+.
(86) Holanamine
SCHEME 6.
86 ATTA-UR-RAHMAN A N D CHOUDHARY
I (26) Jervine
H
SCHEME 7.
5. Pregnane- Type Alkaloids
It is well known that the fragmentation in nitrogen-bearing substances
is preferentially initiated by the cleavage of the bond between the carbons
a and P to the nitrogen atom. For example, the mass spectrum of 3-
dimethylaminopregnane shows characteristic ions at mfz 84 and 110, result-
ing 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 mfz 58 or 72, respec-
tively, 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).
H
I + ' _ HoQ
m/z 429 m/z 114 HO
(781 Stenanzine
0
SCHEME 8
87 2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS
m/z 84
R = N(Me)COMe
SCHEME 9.
7. Secosolanidine- Type Alkaloids
The mass spectra of verarine (SS), hosukinidine, veralinine, and verami-
line contain several characteristic fragment ions that facilitate the identifi-
cation 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 consid-
erably 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
+.
SCHEME 10.
88 A'ITA-UR-RAHMAN AND CHOUDHARY
m / z 98
SCHEME 11.
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 rearrange-
ment from C-12 to C-14. The base peak at m / z 150 is formed by the cleavageof 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.
(29) Solanidine
rn/z 204 m/z 150
Ho & 4
(29) Solanidine
rn/z 204 m/z 150
SCHEME 12.
2. CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS 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-RAY CRYSTALLOGRAPHY
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 cylobux-
idine F (99), shinonomenine (ZOO), tortifoline (F. tortifofiu) (ZOZ), ussurie-
nine (F. ussuriensis Maxim) (Z02), veratrenone (Z03), veratridine perchlo-
rate (ZO4), verticine N-oxide (ZO5), verticinone hydrochloride (ZO6),
verticinone methobromide (F . verticiffutu Wild.) (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 struc-
tural similarities of the two groups of secondary metabolites. This assump-
tion 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 pre-
cursor of the Buxus alkamines (Scheme 14).
A. STEROIDAL ALKALOIDS OF THE APOCYNACEAE AND BUXACEAE
Alkaloids of the plant families Apocynaceae and Buxaceae form very
large groups of steroidal bases. Although some biosynthetic studies have
been conducted on other classes of steroidal bases, little experimental work
has been done on these classes of bases.
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 91
H
HO HO
(90) Cycloartenol
Monoamino
and
diamino steroids
SCHEME 14.
Khuong-Huu et al. in 1972 suggested that the reactive ll-keto-9fl,19-
cyclo system encountered in some Buxus alkaloids might be the biogenetic
precursor of bases containing the conjugated transoid 9(10 += 19) abeo-
diene sysem, as, for example, in buxaquamarine (91) (122).
MeN
I
HZC, ,CHz
(91) B uxaquamarine
0
MeN
H
(92) R = H
(93) R = OH
(94) Paravallarine, R = H
(95) Paravallaridine, R = OH
92 ATTA-UR-RAHMAN AND CHOUDHARY
It was also proposed earlier that the biosynthesis of these alkaloids
involves the intermediacy of C-3 or C-20 ketones bearing the steroidal
skeleton. The occurrence of C-3 steroidal ketones such as 92 and 93 in
Pnravalfaris microphyffa having the characteristic 18 20 lactone function
of paravallarine (94) and paravallaridine (95) further supported this hypoth-
esis (113).
B. STEROIDAL ALKALOIDS OF THE LILIACEAE A N D SOLANACEAE
Mitsuhashi and co-workers proposed Schemes 15 and 16 for the biosyn-
thesis 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 solasapo-
genin (97), have been isolated from Solanum and Veratrum species contain-
ing 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 spirosta-
nols (115-127).
In other biosynthetic investigations, radioactive labeled (25R)-26-amino-
cholesterol administered to Solanum laciniatum was found to be incorpo-
rated 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 alka-
loids, 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 BIOLOGY OF STEROIDAL ALKALOIDS
OH
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS
OH
(96) Dormantinol HO
Cholesterol
I
Cholesterol
I
93
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
‘ H H
.’. 20
f 22
/ HO
‘* ’ 16 25
\ Veratramine HO \ ’
HO
(26) Jervine [Jerveratrum-type]
SCHEME 16.
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 95
Me,N
H
AcO AcO
' (98) (991
H
I
AcO
AcO ( 100)
(102) R = H
(103)R = NO,
96 ATFA-UR-RAHMAN A N D CHOUDHARY & .....,,,H
..... 1,111 {fip
A
H2N d'
(107) Funturnine
k I1091 R = H
k (108) (110) R = I
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 synthe-
sized, e.g., 3~-acetoxy-16a(l-nitro-l-methoxycarbonylmethyl)-20-0~0-pregn-5-ene (98), (24S)-3~-acetoxy-22-aza-23-oxo-24-nitro-l6,24-~yclocho-
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 97
la-5,17-diene (~),3~-acetoxy-22-aza-23-hydroxy-24-nitro-l6,24-cyclochola-
5,17,22,24-tetra-ene (loo), and 3~-acetoxy-24-amino-22-aza-23-oxo-16,14-
cyclocholaJ,17,24-triene (101) (11 9) .
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-nitroso-
oxyd-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-20-
one has been achieved (122).
The steroidal amide 108 yielded a mixture of products, such as the spiro-
solane (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 ste-
roidal 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 NaBH4 to give the pyrrolidinospirosolane (115) (125).
3-Hydroxy-4-keto-steroidal alkaloids isolated from various species of
Solanum exhibit interesting pharmacological properties. To obtain these
4-keto-steroidal alkaloids from solasodine (a), the following two routes
were attempted:
(a) allylic acetoxylation of (22S,25R)-22,26-N-Cbz-epi-iminocholest-5-
(b) hydroboration of (22S,25R)-16P-acetyl-22,26-N-Cbz-epi-imino-
The first route yielded (22S,22R)-3P-hydroxy-16P-acetoxy-22,26-N,N-
Cbz-epi-iminocholestan-5,6-oxido-4-one (118), while the second one af-
forded two products, i.e., (22S,25R)-3/3-hydroxy-l6P-ethoxy-22,26-N-Cbz-
epi-imino-5a-cholestan-4-one (119) and its 16P-acetoxy homologue (120)
(126).
en-3PJ6P-diol-acetate (116); and
cholest-4-en-3-one (117).
98 A'lTA-UR-RAHMAN AND 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
99 2. CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS
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 mi-
crobial 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-ene-
3,17-dione (230). Conessine (l23), the major alkaloid of H. antidysenterica,
is reported to be active against both intestinal and extra-intestinal amoe-
biasis (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 tuber-
culosis. 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 sup-
pressed 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
0 HO
k 1128) Solanine
OH
GlcO
6 H
(127) Tomatine (129) Capsirnine-3-OP- D-glucoside
2. CHEMISTRY A N D BIOLOGY OF STEROIDAL ALKALOIDS 101
C. STEROIDAL ALKALOIDS OF THE LILIACEAE
Plants of the genus Veratrum have been noted for their pharmacological
activity for centuries. The powder of the root of V. viride is used for the
treatment of toothache. The boiled, thin root slices in vinegar are considered
to be useful against Herpes milliaris. This plant was used by several Native
American Indian tribes to kill lice, as a catarrah remedy, against rheuma-
tism, 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 protov-
eratrine 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 sensiti-
zation 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. Imperi-
alone 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 blockadeof 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 ana-
logues. 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 iso-
lated 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 patho-
genic Actinomyces (81).
Solanine (128), an alkaloid isolated from several different Solanum spe-
cies, is found to be a mitotic poison and inhibits human plasma cholinester-
ase. Solasodine (34) and its glycosides exhibit bradycardiac activity similar
to that of veratramine (81).
Capsimine-3-O-P-~-glucoside (129), a new steroidal alkaloid isolated
from the root bark of S. capsicasfrurn, exhibited strong activity against
102 AlTA-UR-RAHMAN AND CHOUDHARY
(130) Solamargine
CCI4-induced liver damage in ICR male mice (0.1 mg/kg). The steroidal
alkaloids etioline, capsimine, capsicastrine, and naringgenin showed pro-
nounced 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 intraperitone-
ally, 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
Batrachotoxin (a), an alkaloid isolated from several species of frogs
(Phyflobates genus), is the most potent cardiotoxin known and produces
2. CHEMISTRY AND BIOLOGY OF STEROIDAL ALKALOIDS 103
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 IC50 values
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 adenocar-
cinoma (HT-29), and human melanoma (MEL-28) assays. In addition, it
showed medium immunomodulatory activity (LcVIMLR > 187) and anti-
microbial 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 ...................................................................................
11. Analytical Criteria ...................................................
B. 1-Benzyltetrahydroisoquinolines .............. .............................
C. I-Phenethyltetrahydroisoquinolines ..........
D. (+)-Emetine and (+)-2.3-Dehydr
E. (+)-Dihydroquinine ................. ........................
F. Unnatural Alkaloid Enantiomers s ......................
IV. (+)-Morphine .. ...................................................
VII. (+)-Nicotine .... ...................................................
111. Unnatural Alkaloid
A. Simple Tetrahydroisoquinolines .....................................................
V. (+)-Physostigminc ..................................................
VI. (+)-Colchicine ...............................................................................
VIII. Conclusions ..........................................................
109
110
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
109 Copyright 0 IYYX by Academic Press
All rights of reproduction in any form reserved.
110 BROSSI AND PEI
revealed that proper stereochemistry and proper absolute configuration
(enantioselectivity) are most often required to provide the desired pharma-
cological effect which is essential for their various medical uses. Enantiosel-
ectivity 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 re-
viewed here, are generally in line with the view that the pharmacological
effect of natural isomers is enantioselective. However, unnatural enantio-
mers 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 care-
fully 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 re-
placed by CD and ORD 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-demethoxy-
colchicine) 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 thiocol-
chicine, was not active (3) . Morphinans of the unnatural (+)-series, in
contrast to their enantiomers of the (-)-series which are chemically con-
nected with natural morphine, were found to be inactive as analgesics in vivo
(4 ) . The compounds of the (+)-series, however, possess useful antitussive
properties and, when optically pure, are free of the side effects of their
3. BIOLOGICAL ACTIVITY OF UNNATURALALKALOID ENANTIOMERS 111
(-) -0ripavine
1
(-)-Thebaine (-)-Neopinone
(-)-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).
112 BROSSI A N D PEI
(-)-enantiomen. The synthesis of alkaloid enantiomers is now well ad-
vanced 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 isosalso-
line, 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 anti-
Parkinson activity in the reserpine-reversal assay in mice and were devoid
of significant antihypertensive activity. Stereoselective competitive inhibi-
tion of MAO-A was observed with the (R)-enantiomers of salsolinol, salso-
line, 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 N ~ ~ ALKALOIDS
Alkaloids R' R? R3 Refs
Salsolinol H H H (6,8b)
Salsoline H H CH3 (7,8a)
Isosalsoline H CH1 H (7.8a.Rb)
Carnegine CH3 CH3 CH3 (7,8a)
N-Me thylsalsoline CHI H CH? (7.8a)
N-Methylisosalsoline CH3 CH3 H (7)
Salsolidine H CH3 CH3 (8a.9)
3. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 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 notewor-
thy (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). Tetrahy-
dropapaaeroline (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 enan-
tiomeric form by synthesis ( ] I ) , and the reticulines obtained on N-methyla-
tion 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 anti-
nociceptive activity in the hot-plate assay in mice, showed significant
differences (14). Enantioselectivity appeared to be less apparent for the
TABLE I 1
ALKALOIDS OF NATURAL A N D UNNATURAL CONFIGURATION"
~ 3 0 R2q.Rl
$H2
OR4
Alkaloids R' R2 R7 R" RS Refs.
Tetrahydropapaveroline H
Tetrahydropapaverine H
Reticuline CH3
Norreticuline H
Norarmepavine H
Norcoclaurine H
"The natural alkaloids of plant origin usually have the (S)-configuration.
114 BROSSI AND PEI
a-adrenergic or dopamine receptors. (S)-Tetrahydropapaveroline inhibited
the binding of a radiolabeled ligand to beta-adrenergic receptors one hun-
dred 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-isoquino-
line-1-carboxylic acids, and of several 1-benzyltetrahydro-isoquinolines of
importance in the biosynthesis of morphine (see Frg. l ) , with (S)-adenosyl-
L-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-carboxylic acid (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-isoquinolines shown in Table 111.
The 0-methylation of (S)-norcoclaurine in the presence of the mamma-
lian 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). Biocon-
version of (S)-3’-hydroxy-N-methylcoclaurine into (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-methylcoclaurine yielded 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 (race-
mic 1 pchlorophent hyl-2-methyl-6,7-dimet hoxy- 1,2,3,4-tetrahydroisoquin-
oline), 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
3. BIOLOGICAL AChVITY OF UNNATURAL ALKALOID ENANTIOMERS 115
TABLE 111
ENZYMATIC OMETHYLATION OF OFTICALLY ACTIVE COCLAURINES WITH RADIOLABELED
SADENOSYLMETHIONINE I N THE PRESENCE OF MAMMALIAN COMT
OH
(9
Q
OH
(R)
Norcoclaurine
80% 6-OMe 24% 6-OMe
20% 7-OMe 76% 7-OMe
OH
(9
HO
QH 4'
OH
(R)
3'-Hydroxy-N-Nor- and N-methylcoclaurines
R = H 31% 4'-OMe 32% 4'-OMe
45% 3'-OMe 27% 3'-OMe
R = CH3 14% 4'-OMe 44% 4'-OMe
86% 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 inac-
tive. These phenpropylamines can freely rotate around the C-C bonds
giving rise to a multitude of conformers. It is interesting to note that the
116 BROSSI A N D PEI
CH30
6
(Rplethopholine, active
I
CI
(R)-Phenpropylamine, not active
61
(S)-Methopholine, not active
1) CH#Acetone
2) AgZOlHfl, 150-1 70 OC
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
aminoacid enantiomers.
D. ( +)-EMETINE A N D (+)-2 ,3-DEHYDROEMETlNE
The alkaloid emetine is an active ingredient of Ipecac (Cephaelis ipecacu-
anha) used by South American Indians for the treatment of amoebic dysen-
tery. The chemistry of natural emetine, now available by total synthesis,
has been reviewed (26). In both the emetine series and the synthetic 2,3-
dehydroemetine 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. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 117
CH3O CH3O
OCH3
NH H # L O C H 3
:H3
(+)-Ernetine (+)-2,3-Dehydroernetine
FIG. 3. Structures of (+)-emetine and (+)-2.3-dehydroemetine.
form showed the antiamebic effect to be highly enantioselective, and re-
stricted 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.
CH3O &FH5 0 8
\ / CF3
CF3
(+)-Dihydroquinine W-Mefloquine
FIG. 4. Structures of antimalarial aminoalcohols.
118 BROSSI A N D PEI
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 dia-
stereomers 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 perhydrohistrionico-
toxin (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 synthe-
sis of (+)-morphine from (-)-sinomenine was later reported by an NIH
3. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 119
(-)-Sinornenine (+)-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 chemi-
cal 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 (+)-Dihydrocodeinone (+)-Brornonordihydrothebainone
(+)-Codeine. R'=H. R2=CH3
(+)-Heroine. R'=R2=Ac
FIG. 6. Key compounds in the Rice total synthesis of (+)-morphine and (+)-codeine.
120 BROSSI AND PEI
(S)-tetrahydroisoquinoline was obtained by Bischler-Napieralski cycliza-
tion of an appropriate amide, reduction of the 3,4-dihydroisoquinoline, and
chemical resolution of the tetrahydroisoquinoline with tartaric acid. Birch-
reduction of the (S)-enantiomer and formylation of the product gave the
desired (S)-hexahydroisoquinolineformamide. Transketalization and bro-
mination followed by deketalization gave the (S)-octahydro-isoquinolin-6-
one which was brominated at the orfho-position of the phenethyl substitu-
ent. Bromo-directed Grewe cyclization was accomplished with triflic acid to
give the desired, optically active, (+)-bromo-N-formyl-nordihydrothebain-
one 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 (+)-bromonordihydro-
codeinone 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 possi-
ble 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
3. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 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 homoge-
nates, (+)-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 recep-
tors involved possess a high degree of stereoselectivity. They are blocked
by naloxone, mediate analgesia and the endogenous ligands for these recep-
tors are the endorphins and the encephalins (52).
In the in vivo assays, (+)-morphine was microinjected into the periaque-
ductal gray region (a site known to mediate morphine analgesia) of drug-
naive 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-D-
aspartate (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 phe-
nolic 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
122 BROSSI AND PEI
demonstrates that there is an inherent substrate stereoselectivity for mor-
phine metabolism in rat hepatic uridine-5’-diphosphate-glucuronyltransfer-
ase, 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 condition-
ing (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 combina-
tion 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 hyperexcit-
ability, 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-
3. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 123
ined in three assay systems was found to have no more than 1/1000-
1/10,00Oth 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 analge-
sics 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 Holm-
stedt 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 calledeserine, 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 understand-
ing of basic cholinergic mechanisms without studying physostigmine, al-
though 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 acetyl-
choline by cholinesterases are called anticholinesterases, or acetylcholine-
blocking agents. One of the first compounds to be recognized as an acetyl-
choline-blocking agent was physostigmine. It is used clinically as its soluble
salicylate in the treatment of glaucoma by reducing intraocular tension.
Because of its miotic properties it is employed after atropine to return the
pupil to its normal size. These pharmacological effects are produced by
the competitive inhibition of cholinesterases by blocking the active site on
124 BROSSI AND PEI
the enzyme by carbamoylation. Physostigmine similarly inhibits in vitro
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the former
is present in red blood cells. in the brain, and in nerve tissues, and the
latter is present in blood serum, pancreas, and liver.
To help in the further elucidation of the structural requirements of the
acetylcholine active centers, Robinson et al. have prepared the enantiomer
of the natural alkaloids, namely (+)-physostigmine and (+)-physovenine,
the latter being the antipodal isomer of the ether alkaloid physovenine
which is also occurring in Physosfigma venenosiim (62). The Robinson
synthesis of unnatural (+)-physostigmine, shown in Fig. 7, is grosso mod0
I I
CH3 CH3
(+)-Eserethole, R = Et
(+)-Esermethole, R = CH3
CH3 x
CH3
(+)-Eseroline: R = H X = l
(+)-Physostigrnine: R = CH3NHCO
FIG. 7. Syntheses of (+)-physostigrnine.
3. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 125
identical with that of the natural alkaloid reported by Julian and Pikl (63a),
and greatly improved later (63b-d). A chemical resolution was executed
at the stage of eserethole, with (+)-tartaric acid to give the (+)-enantiomer
following work reported by Kobayashi (64). Reaction of (+)-eserethole
with aluminum chloride gave (+)-eseroline which was reacted with methyl-
isocyanate 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 alter-
nate 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, ex-
plained 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 enantio-
meric separation also was achieved with the corresponding amides, origi-
nally prepared from an oxindole-3-acetic acid of proper configuration (69).
-* M e O e
+
I
' N O
I I
CH3 CH3 CH3
R = CN, CONHCH3. CONHBn
CH3 R
R = H, CH3, En
FIG. 8. Practical synthesis of unnatural (-t )-physostigmine.
126 BROSSI AND 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 biologi-
cal activities were (+)-physostigmine (62,70,73), (+)-phenserine (67,68),
(+)-N'-norphenserine (68), and (+)-physovenine (72). The phen-
serines 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 consid-
erably 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
VALUES OF NATURAL AND UNNATURAL PHYSOSTTGMINE, PHENSERINE, N'-
NORPHYSOSTIGMINE, NI-NORPHENSERINE, AND PHYSOVENINE VERSUS
HUMAN ERYTHROCYTE ACHE AND HUMAN ASM MA BCHE
1% (nM)
Compound AChE BChE Refs.
(-)-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
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 measuringthe
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 repre-
sented 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 ob-
served with the tricyclic compounds.
The NIH-modification of the Julian total synthesis of natural (-)-physo-
stigmine 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 mecha-
nisms not related to cholinesterase carbamoylation (78). It was found that
unnatural (+)-physostigmine, which had a much lower AChE inhibitory
128 BROSSI AND PEI
H2/R02/CF3COOH 1 NaBHmeOH 1
PhNHCOO 0
PhNHCOO 6 0 NHCH3 6 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 unnat-
ural 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 acetylcholines-
receptor 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 pharmacol-
ogy of colchicine has repeatedly been reviewed (80). The colchicine binding
to tubulin is highly selective €or the conformational states of colchicine,
3. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 129
and requires the phenyl-tropolone system to be (as)-configured (a]), as
evidenced by the presence of strong negative Cotton effects at 260 nm and
at 340-350 nm in the CD spectra of the natural colchicinoids (82-84).
The arrangement of the two aromatic moieties in a counterclockwise
helicity in natural colchicine (Fig. 10) and derived allo-congeners has been
confirmed by X-ray analysis of several representative compounds (80).
The importance of the (as)-configuration of the phenyl-tropolonic unit for
interaction with tubulin and bovine serum albumin (85) has been confirmed
by a Swedish group (86). It was shown that only (-)-(as)-deacetamidocol-
chicine, lacking the chiral acetamido group at C-7, and obtained by chro-
matographic optical resolution on a chiral column, did inhibit tubulin poly-
merization, 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 stereo-
selectivity in the interaction of (-)-colchicine with tubulin and other pro-
teins. 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 AND PEI
b H
Optically Active Schiff Base
of Deacetylcolchiceine
OH
Racemic Deacetylcolchiceine
: : : : q = H * - p h CH3O
-
OH
Ketimine
b H
1 0-Demethylcolchicone
CH30
Colchicone
FIG. 1 1 . 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 re-
solved with (+)-10-camphorsulfonic acid, and the antipodal isomers con-
verted after 0-methylation, separation of the ether isomers, and N-acetyla-
tion into (-)- and (+)-colchicine, and (-)- and (+)-isocolchicine. It was
later found that 0x0-deacetamido-colchiceine (the enol of colchicone), re-
sulting 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. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 131
*
CH3O cH30q N<
CH30 - OAc
CH30 CH30
1). 0.1 N HCVAcOH
2). 20 % H2SO4
(- )-Colchicine
4
2). CH2N2
CH3O OH
W-Trifluoroacetyldeacetylcolchicine u-Deacetylcolchiceine
Aq. K2C03/(CH&CO I ::::qNH2 CH30
1 ) dCarnphorsulfonic Acid CH30 c H 3 q N H A c
2). Ac20 CH30
- 0
- 0
CH30
CH3O
0- Deacetylcilchicine (+)-Cobhicine
FIG. 12. Improved procedure for the preparation of unnatural (+)-colchicine.
a triacetate which has lost the chirality at C-7, and on hydrolysis with 0.1
N HCl gave (+)-colchiceine. Heating the racemate with 20% sulfuric acid
in acetic acid yielded racemic deacetylcolchiceine which, on treatment with
trifluoroacetic anhydride in the presence of sodium carbonate, afforded the
racemic trifluoroacetamide. 0-Methylation with diazomethane in methanol
gave, after workup and chromatography, the desired ether isomer as the
faster running compound, followed by the iso-isomer (90). Hydrolysis of
the trifluoroacetamide with aqueous potassium carbonate gave racemic
deacetylcolchicine which was resolved with (+)-10-camphorsulfonic acid
in methanol. The less soluble salt, on treatment with ammonia, gave (+)-
deacetylcolchicine, and unnatural (+)-colchicine on treatment with acetic
anhydride (50).
132 BROSSI AND PEI
In agreement with earlier findings (87), (+)-colchicine crystallized from
chloroform, whereas the enantiomeric (- )-colchicine could be obtained
crystalline only from ethyl acetate. The CD spectra of these solvated enanti-
omers do not fully conform in solution (91), but they did after the samples
were dried at 70°C in high vacuum (80).
Optically pure (+ )-colchicine prepared by Corrodi and Hardegger, when
assayed in vitro as an inhibitor of mitosis, was found to be only 1/100th as
potent as the natural alkaloid (92). It was recognized at that time that these
alkaloids not only express chirality at C-7, but at the same time through
their molecular asymmetry (93), an important detail investigated and fully
confirmed later (82). Unnatural (+)-colchicine prepared from (+)-deacetyl-
colchicine, when assayed for inhibition of tubulin, showed a low potency
(32% versus 90% for the (-)-enantiomer), and it was much less toxicin mice
when given i.m. (123 mg/kg versus 3.6 mg/kg for the (-)-enantiomer) (94).
The lower potency of (+)-colchicine on comparison with the (-)-
enantiomer was also noted in its affinity for three antisera prepared by
coupling deacetylcolchicine to bovine serum albumin (85). There is great
promise that the elegant total synthesis of natural (-)-colchicine by Banwell
which introduced chirality by an optically active reducing agent followed
by SN2 replacement of the (7R)-configured alcohol by an azide ion will
offer a new route to colchicinoids of unnatural configuration (95).
Secothiocolchicinoids, shown in Fig. 13, with a six-membered ring B were
obtained from deacetylthiocolchicine by a Demjanov rearrangement and
they have the phenyltropolone moiety in an (aR)-arrangement (96). The
alcohol, and the derived methylene compound which is optically inactive,
are believed to equilibrate in solution and to interact with tubulin as the
(as)-atropisomers (96). Continuation of such efforts with the inclusion of
both enantiomers will improve our understanding of the effects which
conformational isomers of colchicinoids and their chemical analogs exert
in their binding to tubulin. Such information is highly desirable to develop
antitumor agents belonging to this class of spindle-toxins.
FIG. 13. Secothiocolchicine and 6-methylene analogue.
3. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 133
VII. (+)-Nicotine
(-)-Nicotine is the natural enantiomer of nicotine, and (+)-nicotine the
synthetic, nonnatural enantiomer. Nornicotine is a minor tobacco alkaloid
in most species of Nicotiana (97), and may also be a metabolite of nicotine
(Fig. 14) (98).
Nicotine has long played an important role in furthering our understand-
ing of the cholinergic system. There are several early reports in which the
effects of unnatural (+)-nicotine on the nicotinic receptors and toxicities
were compared to those of natural (-)-nicotine (99-105). Although the
relative potency of the (+)-enantiomer varied with investigators and with
the purity of the agents used, (+)-nicotine is qualitatively less potent than
the natural (-)-nicotine on the stimulation andlor blockade of the nicotinic
receptors in the peripheral nervous system. It should be noted that nicotine
free base is hygroscopic and subject to autoxidation and absorption of COz.
(+)-Nicotine di-d-tartrate and (-)-nicotine di-l-tartrate, however, are
anhydrous and stable salts suitable for biological studies (103). The optically
pure (+)-nicotine can be prepared by resolution of (+)-nicotine (103),
which can be obtained by racemizing natural nicotine (106), or by synthetic
methods. Numerous total syntheses of racemic nicotine have been reported,
but the early synthesis, described in Fig. 15 (103, still seems most practical.
3-Cyanopyridine, prepared by sulfonation of pyridine followed by treat-
ment with potassium cyanide, reacted with a Grignard reagent to give p-
pyridyl-y-ethoxypropyl ketone. The ketone ws converted to the oxime, and
the oxime reduced to an amine. After the cleavage of the ethyl ether with
48% HBr, the aminoalcohol was cyclized by treatment with NaOH to
give racemic nornicotine. The racemic nicotine was obtained from the
nornicotine on N-methylation with MeI.
Resolution of racemic nicotine has proved tedious because of the unsta-
bility of nicotine free base. Complete resolution was achieved not long ago
with d-tartaric acid combined with di-p-toluoyl-l-tartaric acid, leading to
optically pure (+)-nicotine (103).
R = CH3, (-)-Nicotine
R = H, (-)-Nornicotine
R = CH3, (+)-Nicotine
R = H. (+)-Nornicotine
FIG. 14. Nicotines and nornicotines.
134 BROSSI AND PEI
R = H, (&Nornicotine
R =CH3, (+)-Nicotine
FIG. 15. Synthesis of racemic nicotine and racemic nornicotine.
The nornicotine enantiomers have been resolved recently via HPLC on
a chiral column (108). Optically pure (+)-nicotine could also be prepared
from (+)-nornicotine by N-methylation.
It was reported that (+)-nicotine is less toxic than its natural antipode.
The intravenous acute LDS0 value in mice is 2.75 mg/kg for the (+)-nicotine
compared with 0.38 mg/kg for the (-)-nicotine (203). (+)-Nicotine is much
less potent than (-)-nicotine in raising blood pressure in anesthetized rats,
and in the isolated guinea-pig ileum, with a potency ratio of 0.06, and 0.019,
respectively (203). For the ganglionic nicotine receptor on cat superior
cervical ganglion (stimulation and blockade), the relative potency of (+)-
nicotine is 0.2 of that of (-)-nicotine. Both (+)- and (-)-nicotine, however,
had the same blocking effect for the muscle-type nicotinic receptor on the
neuromuscular junction of rat diaphragm (105). In the adrenergic nerve
terminals of the isolated rabbit pulmonary artery, (-)-nicotine produced
sympathomimetic effects by releasing norepinephrine from those terminals.
(+)-Nicotine, on the other hand, did not produce such effects, but instead
inhibited the effects due to (-)-nicotine (105). Because (+)-nicotine had
no effect on the response to exogenously applied norepinephrine and
blocked the 3H-efflux induced by (-)-nicotine, the inhibitory effect of (+)-
nicotine was attributed to its presynaptic action. Such an inhibitory effect
of (+)-nicotine was noncompetitive, on the basis of the shift of the concen-
tration-contraction curve obtained with (-)-nicotine. Simultaneous appli-
cation of both enantiomers produced no inhibition of the response to (-)-
nicotine, irrespective of the concentration of (+)-nicotine. Occurrence of
the inhibitory effect of (+)-nicotine was not prevented by hexamethonium,
a competitive nicotinic antagonist. (+)-Nicotine did not inhibit the re-
sponses of the artery to electrical transmural stimulation. These results
3. BIOLOGICAL ACTIVITY OF UNNATURAL ALKALOID ENANTIOMERS 135
TABLE VI
EFFECTS OF SOLUBlLlZATION ON DISPLACEMENT OF ( -)-[3H]NICOTINE
BINDING BY NlCOTlNES AND NORNlCOTlNES IN RAT BRAIN
K , (nM)
Cortex Hippocampus Cerebellum
(+)-Nicotine 31.6 47.1 97.5
(-)-Nicotine 1.44 1.87 3.83
(+ )-Nornicotine 41.4 37.6 30.6
(-) -Nornicotine 56.4 46.3 33.0
~
K, = affinity constant.
indicate that (+)-nicotine inhibits the response to (-)-nicotine by acting
neither on the nicotinic receptor nor on excitation-secretion coupling mech-
anisms (105). (+)-Nicotine may possibly act at a site other than the nicotinic
receptors, and induce desensitization of nicotinic receptors.
The binding properties of the enantiomers of nicotine and nornicotine
were studied in solubilized preparations of rat cortex, hippocampus, and
cerebellum (109). The (-)-nicotine was more potent than (+)-nicotine in
the assays, but (-)-nornicotine is less potent than (-)-nicotine. It is interest-
ing to note that the nornicotines show no enantioselectivity in the binding
assay (Table VI).
VIII. Conclusions
Investigating the antipodal isomers of biologically active alkaloids is
challenging and useful for several reasons: it will require a practical resolu-
tion of racemic intermediates, or an efficient asymmetric synthesis; it will
show whether the natural alkaloid is enantioselective in its pharmacological
action; it will signal whether the unnatural enantiomer has a pharmacologi-
cal quality of its own which may be potentially useful; and it will give
qualitative information whether the toxicities of the two enantiomers are
significantly different. It is obvious that such information has to be based
on proper analytical data, and data allowing for the quantitation of the
optical purity of the two enantiomers. Should the antipodal isomer have
potentially valuable pharmacological properties of its own, not covered by
its natural enantiomer, it is suggested that this be further evaluated with
an appropriate and acceptable formulation, such as a pharmacologically
acceptable salt (HCl, H2S04, H3P04, fumarate, tartrate, maleate, etc.).
136 BROSSI AND PEIFurther study of (+)-physostigmine, or analogues of the (+)-series may
ultimately lead to valuable information regarding the nicotinic acetyl-
choline-receptor-channel, or even to a new drug for treating cholinergic
disorders, or organophosphate poisoning. Developing an analog of (+)-
morphine as an antitussive agent has to show improvements over dextro-
methorphan which is widely accepted. Antipodal isomers of alkaloids hav-
ing the same pharmacological effect and practically identical toxicities,
as observed with antimalarial aminoalcohols, may well set the stage for
developing a racemic drug. It is hoped that this review of unnatural alkaloid
enantiomers will stimulate further research in this area, and will support the
conviction that chiral is better based on experimental data from unnatural
antibiotics, sugars, peptides, steroids, and amino acids, to mention a few.
Acknowledgments
The authors wish to thank Ms. Sheng Bi for her considerable help in the preparation of
the manuscript. We would also like to thank Dr. B. Witkop, Institute Scholar of the National
Institutes of Health, for his most valuable comments and suggestions.
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-CHAPTER A
THE NATURE AND ORIGIN
OF AMPHIBIAN ALKALOIDS
JOHN W. DALY
Laboratory of Bioorganic Chemistry
National Institute of Diabetes and Digestive and Kidney Diseases
National Institutes of Health
Bethesda, Maryland 20892
I. Introduction ................................................................................... 141
11. Samandarines
111. Batrachotoxin
IV. The Pumilioto
A. Pumiliotoxins and Allopumiliotoxins
C. Other Pumiliotoxin-Class Alkaloids ................................................ 149
V. Histrionicotoxins
VI. Gephyrotoxins ...
VII. Decahydroquinolines ....................................................................... 152
VIII. Cyclopenta[b]quinolizidines
IX. Epibatidine .....................
X. Pseudophrynamines . .
XI. Pyrrolizidine Oximes
XII. Coccinellines ...........
XIII. Bicyclic “Izidine” Alkaloids .............................................................. 159
A. Pyrrolizidines 159
B. 3,5-Disubstitute ines ...................................................... 160
C. 5.8-Disubstituted Indolizidines ...................................................... 161
D. 5,6,8-Trisubstituted Indolizidines 163
E. Quinolizidines ........................................................................... 163
XV. Summary and Prospects 165
References .................................................................................... 167
XIV. Monocyclic Alkaloids ... 164
I. Introduction
Alkaloids are normally thought of as nitrogenous secondary metabolites
produced by and stored in plants. However, amphibian skins have in the
past few decades proven to be a source of a diverse array of alkaloids
THE ALKALOIDS. VOL. SO
Ix)w-959x/Yx 525.00
141
142 DALY
unprecedented in the plant kingdom. Such skin alkaloids apparently serve
the amphibians in chemical defense against predators. Structures of amphib-
ian alkaloids have been reviewed in detail, most recently in 1993 (I). The
distribution of various alkaloids among some 40 species of frogs of the
neotropical family Dendrobatidae was presented in 1987 (2). Synthetic
efforts leading to amphibian alkaloids have been reviewed, most recently
in 1986 (3). Since the 1993 review, many more alkaloids have been detected
in amphibian skin extracts using gas chromatographic (GC) mass spectral
and GC-Fourier-transform infrared (FTIR) spectral analyses; further struc-
tural classes have been defined, and most importantly evidence has been
obtained that indicates that alkaloid-bearing amphibians, with the exception
of the European fire salamander, probably do not synthesize their skin
alkaloids, but instead rely on dietary sources and merely efficiently seques-
ter and store for extended periods alkaloids that they obtain from ants,
beetles, millipedes, and probably other small arthropods, whose identities
remain shrouded in mystery. The amphibian skin alkaloids, thus, would
represent a remarkable instance of a chemical ecology, wherein the amphib-
ian is wholly dependent on dietary arthropods as a source of the alkaloids
that comprise the active principles in its defensive skin secretions. The
identities of the ultimate source of the unique so-called “dendrobatid alka-
loids,” a term coined from the family name Dendrobatidae of the frogs
from which they were discovered, remain a challenge for future research.Such “dendrobatid alkaloids” include the batrachotoxins, the pumiliotoxins
and related congeners, the histrionicotoxins, the gephyrotoxins, the decahy-
droquinolines, the cyclopentaquinolizidines, and epibatidine.
11. Samandarines
The poisonous nature of the striking black and yellow European fire
salamander (Salamandra salamandra) has been known since ancient times.
The active principles were discovered to be highly toxic alkaloids in the
1860s, but it was not until the pioneering studies of Clemens Schopf, begun
in the early 1930s, that the steroidal structures were realized and elucidated.
Such structure elucidation was completed prior to the emergence of mass
spectrometry and nuclear magnetic resonance (NMR) spectroscopy as pow-
erful analytical techniques, and, thus, was dependent on classical methods,
involving chemical degradation and UV and IR spectral analysis. X-Ray
crystallographic analysis played a significant role in the later stages of
this research. Fortunately, relatively large quantities of alkaloids could be
4. THE NATURE A N D ORIGIN OF AMPHIBIAN ALKALOIDS 143
obtained from the parotid glands of the salamanders. The major alkaloid
from the salamander was samandarine (1). By 1961 the structures of saman-
darine and several congeners had been determined and were reviewed in
detail (4) . A total of nine samandarine-class alkaloids have been isolated;
all but three have the oxazolidine ring system of samandarine and all have
a seven-membered nitrogen-containing steroidal A-ring (ZJ).
Samandarines are known in Nature only from the European fire salaman-
der and are apparently synthesized by the salamander (personal communi-
cation, G. Habermehl, 1988). The samandarines represent the first example
of an “animal alkaloid.” Limited pharmacological studies on these ex-
tremely toxic substances indicate that they are powerful local anesthetics
(see Ref. I ) .
Ill. Batrachotoxins
Two brightly colored dendrobatid frogs (Phyllobates aurotaenia and Phyl-
lobates bicolor) of the rain forests of the Pacific coast in Colombia were
known to have extremely toxic skin secretions based on the use of such
secretions to poison blow-darts by native peoples of that region (see Ref.
6). The nature of the active principles was unknown until studies were
initiated at NIH in 1962, involving field collection of such frogs, and leading
ultimately to the isolation and structure elucidation of the steroidal alkaloids
batrachotoxin (2), homobatrachotoxin (3), batrachotoxinin A (4), and some
minor congeners (7-9). Efforts at structure elucidation (see Ref. 3) relied
heavily on mass spectrometry and NMR spectroscopy, but ultimately it was
the X-ray analysis of ap-bromobenzoate of batrachotoxinin A that revealed
the steroidal moiety of these alkaloids (7). The nature and site of the
Ehrlich-positive pyrrole moiety of 2 and 3 were deduced from spectral
properties and confirmed through the synthesis of 2 by esterification of (4)
with 2,4-dimethylpyrrole carboxylic acid (8). Extracts of 5000 skins had
144 DALY
yielded only 46 mg of batrachotoxinin A, 11 mg of batrachotoxin, and 16
mg of homobatrachotoxin.
0
R =
R =
H3C N
I
H I
H
4 R = H
Batrachotoxin proved to be a specific and potent activator of voltage-
dependent sodium channels in nerve and muscle and, as such, both it and
a radioactive batrachotoxinin-A benzoate have become widely used as
pharmacological research tools (see Ref. 1). Fortunately, a new species
of dendrobatid frog containing much higher levels of batrachotoxins was
discovered in a remote river drainage in western Colombia in the early
1970s. The frog was named Phyllobates terribilis in view of its extraordinary
toxicity. Batrachotoxins isolated from this frog ( 9 ) represent the sole source
of these valuable research tools to the present time, since the syntheses of
such alkaloids are multistep and impractical for large quantities.
Batrachotoxins are known to occur at high levels only in the skins of the
three true poison-dart frogs (P . aurotueniu, P. bicolor, P. terribilis), which
are to this day still used to poison blow-darts by the indigenous peoples
of western Colombia. Much lower levels of batrachotoxins are found in
the skin of the other two species (P. lugubris, P. vittatus) of the genus.
Batrachotoxins have not been detected in other dendrobatid frogs and,
indeed, their presence in the skin was one taxonomic character leading
to the definition of Phyllobates as a monophyletic genus. Such frogs are
insensitive to batrachotoxin, due to an altered sodium channel that does
not respond to batrachotoxin by opening (10). Remarkably, Phyllobates
frogs raised in terraria on a diet of fruit flies and crickets had no trace of
batrachotoxin in their skin (10). The lack of alkaloids in these captive-
raised frogs was the first indication that skin alkaloids in dendrobatid frogs
might have a dietary origin, but this was not fully appreciated until years
later, in part because the wild-caught frogs maintained significant skin levels
4. THE NATURE A N D ORIGIN OF AMPHIBIAN ALKALOIDS 145
of batrachotoxins for up to 6 years in captivity (20). It is now realized that
dendrobatid frogs have the ability to sequester dietary alkaloids into the
skin and to retain such alkaloids for extended periods ( I I ) , probably be-
cause frogs eat their skin during shedding, thus “recycling” any skin alka-
loids. The insensitivity of frogs of the genus Phyllobates to the action
of batrachotoxins would permit them to ingest putative batrachotoxin-
containing arthropods with impunity.
Remarkably, one of the batrachotoxins, namely homobatrachotoxin (3)
has now been discovered to be present in the skin and feathers of New
Guinean birds of the genus Pitohui (22). Such birds are recognized as being
toxic by the natives of Papua New Guinea. Whether there is a requisite
dietary source, or whether the bird has its own biosynthetic pathway of
homobatrachotoxin is unknown.
IV. The Pumiliotoxin Class
The initial studies on batrachotoxin from the Colombian poison-dart
frogs attracted the attention of a herpetologist, Charles W. Myers, who was
interested in a brightly colored and extremely variable dendrobatid frog,
Dendrobates pumilio, in Panama. Thirty years of collaborative field work
by Myers and Daly on dendrobatid frogs in the rain forests of Central and
South America ensued (23). The collaboration has resulted in the detection
of over 400 alkaloids in amphibian skin extracts and the discovery of over
a dozen new species of dendrobatid frogs. It began with the investigation of
the levels and nature of toxic alkaloids in skin extracts from the Panamanian
dendrobatid species Dendrobates pumilio, and the possible correlation of
toxicity with brightness of coloration for populations of this extremely
variable frog. There was no correlation and the skin alkaloids did not
include batrachotoxins, but were instead much simpler substances (14). In
this initial study three major skin alkaloids were isolated in quantities of
less than 2 mg each. Mass spectra of the more toxic pumiliotoxins A and
B indicated formulas of C19H33N02 and C19H33N03r respectively. NMR
spectral analyses were not definitive and the compounds were incorrectly
suspected to be steroidal in nature, like the samandarines and batrachotox-
ins. Further quantities were later isolated from some 250 skins and analysis
indicated that pumiliotoxins A and B were closely related bicyclic alkaloids
with two double bonds, differing only in the presence of one or two hydroxyl
groups, respectively, in a side chain (25). Instability and limited supplies
thwarted efforts to prepare a crystalline salt for X-ray analysis and modern
146 DALY
2D-NMR techniques had not yet been developed, so that a decade after
the initial studies, the structures of pumiliotoxins A and B were still only
partially defined.
During this 10-yearperiod, many further “dendrobatid alkaloids” were
discovered and alkaloid profiles were delineated for many species and
populations of dendrobatid frogs, using gas chromatography and mass spec-
tral analysis. A code system, employing in boldface the nominal molecular
weight and a letter, when necessary, for each alkaloid was introduced
in an attempt to cope with the hundreds of alkaloids being detected in
dendrobatid frog skin extracts.
A. PUMILIOTOXINS AND ALLOPUMILIOTOXINS
A number of the “dendrobatid alkaloids” appeared to be related in
structure to pumiliotoxins A and B in exhibiting diagnostic prominent mass
spectral fragment ions at d z 166 (C&16NO+) and d z 70 (C4H8N+).
These alkaloids, now totalling almost thirty, were grouped into a subclass
called pumiliotoxins. A further set of alkaloids exhibited diagnostic promi-
nent mass spectral fragment ions at d z 182 ( C I O H ~ ~ N O ~ ) and d z 70,
indicating, in consort with other data, the presence of an additional hydroxyl
group in the bicyclic ring system of the pumiliotoxins. These alkaloids, now
totalling almost twenty, were grouped into a subclass called allopumiliotox-
ins. Finally in 1978, extracts from 750 skins of an Ecuadoran dendrobatid
frog, Epipedobates tricolor, yielded some 21 mg of pumiliotoxin 251D. This
was unexpected, the extracts having been obtained in hopes of isolating
and analyzing a trace alkaloid with analgetic activity, detected in early
extracts from seven of these frogs (see Epibatidine below). Most of the
pumiliotoxin 251D had apparently been lost during evaporations in the
earlier fractionation. The hydrochloride of pumiliotoxin 251D was obtained
crystalline and X-ray analysis revealed the structure 5 (16). A reinterpreta-
tion of the mass and NMR spectra in light of the structure and spectra of
251D allowed structures to be advanced for many of the pumiliotoxins,
which have been confirmed and refined by derivatization, degradation, and
synthesis (see Ref 2). NMR analyses on allopumiliotoxins isolated from
extracts of 1080 skins of Dendrobates pumilio defined structures of several
alkaloids of this subclass (I 7). Structures for pumiliotoxin 251D, pumilio-
toxin A (307A), pumiliotoxin B (323A), allopumiliotoxin 267A, and the
allopumiliotoxin 339A are shown in 5-9 respectively. All are relatively
common in dendrobatid frogs. Structures of many of the other 50 pumilio-
toxins and allopumiliotoxins are based only on GC-mass spectral and GC-
lT IR spectral data. The latter technique has proven an invaluable comple-
ment to GC-mass spectrometry in defining the structures of alkaloids that
4. THE NATURE A N D ORIGIN OF AMPHIBIAN ALKALOIDS 147
are often found in skin extracts in small amounts in complex mixtures
consisting of dozens of alkaloids. Pumiliotoxins/allopumiliotoxins have
characteristic Bohlmann bands and “fingerprint” regions in F l J R spectra.
Pumiliotoxins and allopumiliotoxins are the most widely distributed of
all alkaloids found in amphibian skin. Most are CI6- or C19-compounds and
have isoprene units in their structures. They have cardiotonic and myotonic
activity apparently due to enhancing sodium channel function (see Ref. 2 ) .
cH3 “‘OH
OH
251 D 307A
323A 267A
CH,
339A 267C
10
In frogs of the neotropical family Dendrobatidae, the pumiliotoxins and/
or allopumiliotoxins are major alkaloids in most species from the genera
Dendrobates, Epipedobates, and Minyobates, while being absent or trace
alkaloids in species of the genus Phyllobares. They are major alkaloids
in a dendrobatid frog. Dendrobates auratus, introduced into Hawaii in
1932 (18).
In 1984, the first report of pumiliotoxins/allopumiliotoxins in skin extracts
from nondendrobatid frogs and toads appeared (29). Indeed, this report
was the first to demonstrate that “dendrobatid alkaloids” occurred in non-
dendrobatid amphibians. Pumiliotoxins/allopumiliotoxins occur in all spe-
cies of frogs as yet examined of the genus Pseudophryne of the endemic
Australian family Myobatrachidae (20,21) and in all species of frogs as yet
examined from the genus Manteffa of the endemic Madagascan subfamily
Mantellinae (22,23). In toads of the South American genus Melanophrynis-
cus of the family Bufonidae, a pumiliotoxin 267C (lo), which was at that
time unknown in dendrobatid species, was discovered and its structure
defined by NMR spectroscopy (19). It also occurred in the Australian
148 DALY
Pseudophryne and the Madagascan Mantella. Pumiliotoxins/allopumilio-
toxins also occur in the two species of toads of the genus Melanophryniscus
that have been examined (24). Pumiliotoxins/allopumiliotoxins have not
been detected in skin extracts of some 70 other amphibian genera, nor for
that matter have any alkaloids been detected in any of these other genera.
The wide distribution of pumiliotoxins/allopumiliotoxins, coupled with
their absence in captive-raised dendrobatid frogs (11,18,25), suggests that
any putative dietary source for such alkaloids must be widely distributed
over the world in both tropical and subtropical regions. The nature of such
a small dietary arthropod is unknown. Pumiliotoxins/allopumiliotoxins are
as yet unknown in Nature, save in amphibian skin from four of the six genera
of neotropical dendrobatid frogs, in skins from one genus of Madagascan
mantelline frogs, in skins from one genus of Australian myobatrachid frogs,
and in skins from one genus of bufonid toads. As yet only dendrobatid
frogs have been demonstrated to have the ability to accumulate pumiliotox-
ins (and other alkaloids), as provided in their diet, into their skin (11).
B. HOMOPUMILIOTOXINS
Homopumiliotoxins are closely related in structure to the pumiliotoxins,
but have a quinolizidine ring system rather than an indolizidine system. Only
one, namely homopumiliotoxin 2236 (ll), has been isolated in sufficient
quantities of NMR spectral analysis (26), since, unlike the pumiliotoxins/
allopumiliotoxins, the homopumiliotoxins have been minor or trace alka-
loids when detected in skin extracts. Thus, for the homopumiliotoxins, as
for many amphibian alkaloids present as minor or trace components in
complex mixtures of alkaloids, diagnostic features of GC-mass spectra and
GC-FTIR spectra have been critical to the classification and postulation
of structures. The homopumiliotoxins exhibit diagnostic prominent mass
spectral ions at d z 180 (CI1Hl8NO+) and d z 84 (C5HloN+) and have a
characteristic FTIR pattern, particularly in the Bohlmann band region (24).
There are now about 15 alkaloids that can be assigned to the homopumilio-
toxin subclass.
223G U
11
Homopumiliotoxins, like the pumiliotoxins/allopumiliotoxins, are known
in nature only from amphibian skin, but, unlike the pumiliotoxins/allopumi-
4. THE NATURE A N D ORIGIN OF AMPHIBIAN ALKALOIDS 149
liotoxins, they occur only sporadically and at low levels. Homopumiliotoxins
have been detected in certain dendrobatid species and in some species of
Mantella and Melanophryniscus, but not in Pseudophryne.
C. OTHER PUMILIOTOXIN-CLASS ALKALOIDS
The existence of three other subclasses of pumiliotoxin alkaloids have
been proposed, namely 6,10-dihydropumiliotoxins (22), dehydrohomopum-
iliotoxins (22), and 8-deoxypumiliotoxins (27). Alkaloids of the first two
putative subclasses have been detected only in Madagascan frogs of the
genus Mantella, and the proposed structures must be considered very tenta-
tive until sufficient material is obtained for NMR spectral analysis. The 8-
deoxypumiliotoxin 251H (12) was recently isolated from extracts obtained
in 1976 in quantities (ca. 1 mg) sufficient for NMR spectral analysis (27).
Such 8-deoxypumiliotoxins exhibit diagnostic prominent mass spectral ions
at d z 150 (CloH16N+) and d z 70 (C4HsN+). As yet, 8-deoxypumiliotoxins
have been detected only in neotropical dendrobatid frogs and in Madagas-
can frogs of the genus Mantella.
12Another apparent pumiliotoxin subclass has recently been detected as
trace alkaloids in extracts of Mantella (23). The prominent mass spectral
ions are at d z 166 (CloHI6NO+) and d z 84 (C5H10N+). Tentative struc-
tures have not been proposed and for convenience this group of alkaloids
was referred to as “isopumiliotoxins” (23).
V. Histrionicotoxins
During the initial fieldwork in the 1960s on the dendrobatid poison-dart
frog Phyllobates aurotaenia, analysis of extracts from a few skins of a
microsympatric dendrobatid frog, Dendrobates histrionicus, had revealed
the presence of C19-alkaloids with prominent fragment ions at d z 218
(Cl4HZ0NO+) and d z 96 (C6HloN+). In search of a source for such alkaloids,
150 DALY
Myers and Daly in 1970 targeted a population of Dendrobates histrionicus
in southwestern Colombia known to be extremely abundant. Extracts from
400 skins afforded two major alkaloids, histrionicotoxin (13) and isodihy-
drohistrionicotoxin (14), which were crystallized as hydrochloride salts and
the novel structures revealed by X-ray analyses (28). Subsequently, from
additional extracts, further histrionicotoxins were isolated and their struc-
tures determined by NMR spectral analysis. Most were C19-compounds,
differing only in the degree and nature of unsaturation in the side chains,
but some C17-compounds, such as 259A (15), and some CI5-compounds,
such as 235A (16), were also found. All show a major fragment ion at
d z 96 (C6HION+) and most show a significant fragment ion corresponding
to a-cleavage of the side chain next to the nitrogen. GC-FTIR spectra of
histrionicotoxins and their phenylboronate derivatives provided valuable
data, particularly with respect to the nature of the unsaturation in side
chains (29). A total of 16 histrionicotoxins have been detected from dendro-
batid frogs. Unlike the pumiliotoxins/allopumiliotoxins, the histrionicotox-
ins could arise from a precursor with a linear carbon skeleton,
The toxin designation proved inappropriate, since histrionicotoxins have
relatively low toxicity. They are potent noncompetitive blockers of nicotinic
receptor-channels and as such have proved to be useful tools, both in
natural and radiolabeled form (see Ref. I). Many of the other amphibian
alkaloids, including the gephyrotoxins, the decahydroquinolines, indolizid-
ines, pyrrolidines, and piperidines also are noncompetitive blockers of nico-
tinic receptor-channels (see Ref. I).
Histrionicotoxins have been detected in Nature only in dendrobatid frogs.
They have not been detected in the tiny dendrobatid frogs of the genus
Minyobates, nor in alkaloid-containing frogs of the nondendrobatid genera
4. THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS 151
Pseudophryne, Mantella, and Melanophryniscus. Interestingly, histrionico-
toxins do not occur in the dendrobatid frog Dendrbbates auratus introduced
into Hawaii in 1932, even though the founding population of that frog
in Panama contains significant levels of histrionicotoxins (18). In some
dendrobatid frogs, a set of highly unsaturated C19-histrionicotoxins occur
together, while others contain nearly exclusively the C19-alkaloid octahy-
drohistrionicotoxin, and in a few the C19-histrionicotoxins are replaced by
CIS- and CI7-histrionicotoxins. The lack of any histrionicotoxins was one
consideration in defining the Colombian species Dendrobates lehmanni as
a new species, since all populations of Dendrobates histrionicus, among
which Dendrobates lehmanni was at that time included, had histrionicotox-
ins as prominent alkaloids (30).
Dendrobatid frogs have the ability to accumulate into their skin histrio-
nicotoxins provided to them in the diet (11). Feeding leaf litter insects from
a Panamanian site at which the dendrobatid frog Dendrobates auratus
occurs did result in low levels of histrionicotoxins in some of the frogs
raised in terraria on such insects (25). It seems likely that an arthropod
source of the histrionicotoxins occurs only in the New World tropics, based
on the absence of histrionicotoxins in Old World alkaloid-containing frogs,
in New World semitropical toads, and in the dendrobatid frog introduced
into Hawaii.
VI. Gephyrotoxins
Another alkaloid isolated along with the histrionicotoxins from the Co-
lombian dendrobatid frog Dendrobates histrionicus proved on X-ray analy-
sis of a crystal of the hydrobromide salt to be a tricyclic alkaloid, which
was named gephyrotoxin (17) (31). There are questions concerning the
absolute configuration of gephyrotoxin (see Refs. 1,3). Only two gephyro-
toxins have been detected.
/
HO
17
152 DALY
Gephyrotoxins are known in Nature only from a very few dendrobatid
species of the genus Dendrobates, where they always occur along with a
set of CI9-histrionicotoxins. It seems likely that whatever small arthropods
are the source of the histrionicotoxins, they will also prove to be the source
of gephyrotoxins.
VII. Decahydroquinolines
The initial studies on Dendrobates purnilio in the late 1960s had resulted
in the isolation of three alkaloids, two of which, pumilotoxins A and B,
were the first members of the pumiliotoxin class to be characterized. The
third was a decahydroquinoline cis-195A (18), whose structure was deter-
mined by X-ray crystallography (32). This alkaloid was at that time referred
to as pumiliotoxin C, but that name proved unsatisfactory on two accounts;
first, the alkaloid is relatively nontoxic, and second, the name made for
confusion with pumiliotoxins A and B, which are toxic and which are
structurally unrelated to the decahydroquinoline class of amphibian alka-
loids.
H
cis-195A
18
The mass spectra of such 2,5-disubstituted decahydroquinolines are domi-
nated by a-cleavage resulting in loss of the 2-substituent. In some cases, a
loss of 43 amu, corresponding to loss of carbons 6,7, and 8 of the alicyclic
ring is significant (unpublished results): Mass spectral data, often in conjunc-
tion with FTIR spectral analysis, suggests the presence of 30-40 decahydro-
quinolines to be present in amphibian skin extracts. All could be derived
from a precursor with a linear carbon skeleton. The FTIR spectra provide
information as to the relative configurations at carbons 2, 4a, and 8a, but
not at carbon 5 (33,34). The structures of four representative decahydroqui-
nolines isolated from extracts of dendrobatid frog skin and analyzed by
NMR are shown in 19-22. Both cis- and trans-fused decahydroquinolines
4. THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS 153
occur. The absolute configurations of cis-195A (18) and trans-219A (20)
are as shown, as determined by X-ray analyses (32,35).
19 21
I&& N ,r&JJ H y
I
H H
cis-243A trans-269AB
20 22
The major decahydroquinolines isolated from dendrobatid frogs include
CI3- (cis-l95A), C15- (cis- and trans-219A), c 1 7 - (cis- and trans-243A), and
C19- (trans-269AB) compounds. The CI5-, c17-, and C19-decahydroquino-
lines, like the C15-, C17-, and C19-histrionicotoxins, have highly unsaturated
side chains.
2,5-Disubstituted decahydroquinolines, such as cis-l95A, are as yet unre-
ported in Nature except from amphibian skin. Decahydroquinolines occur
in a wide range of dendrobatid frogs, often together with histrionicotoxins.
Like the histrionicotoxins, decahydroquinolines appear to be absent or
virtually absent in the tiny dendrobatid frogs of the genus Minyobates.
Decahydroquinolines do occur in frogs of the Madagascan genus Mantella
and in bufonid toads of the genus Melanophryniscus. Neither of these
genera have histrionicotoxins. Decahydroquinolines are not present in myo-
batrachid frogs of the genus Pseudophryne. Decahydroquinolines are
readily taken up into the skin of the dendrobatid frog Dendrobates auratus
when provided in the diet (IZ). Low levels of the C19-decahydroquinoline
269AB were found in dendrobatid frogs raised on leaf-litter insects in
Panama (25). Interestingly,the decahydroquinoline cis-195A was a major
alkaloid in skin extracts from frogs at the leaf-litter site, but was not detected
in frogs raised on insects collected using Berlese funnels from leaf-litter at
the same site.
154 DALY
VIII. Cyclopenta[b]quinoliidines
Structure elucidation of the batrachotoxins, pumiliotoxins, histrionico-
toxins, gephyrotoxins, and decahydroquinolines were facilitated by the ease
with which large numbers of frog skins could be obtained in the 1960s,
1970s, and early 1980s. Since that time, international conservation efforts
have made it impossible in most cases for scientists to obtain permits to
collect more than a limited number of frogs, in spite of the fact that many
dendrobatid frogs are incredibly abundant. Thus, the isolation of quantities
of the remaining minor and trace alkaloids for NMR spectral analysis
became difficult in the 1980s and characterization of such alkaloids had to
rely almost wholly on mass, FTIR spectral, and microchemical (perhydroge-
nation, methylation, acylation, and boronate formation) analyses. Fortu-
nately, the sensitivity and analytical potential of NMR increased remarkably
during the 1980s, and submilligram quantities of alkaloids have now become
amenable to structure elucidation. A tricyclic alkaloid, detected in a new
species of a Colombian dendrobatid frog, Minyobates bornbetes, was one
such alkaloid, whose structure elucidation awaited the advent of more
sensitive and more powerful NMR instrumentation and techniques. The
alkaloid and its congeners were unusual in exhibiting base peaks of an odd
mass, for example, at d z 111 (Cl7Hl3N+) for the major alkaloid 251F.
From 100 skins of frogs collected in 1983, sufficient alkaloid 251F (ca
300 pg) was isolated in 1990 for NMR spectral analyses (36). The major
tricyclic alkaloid 251F (23) proved to be a cyclopenta[b]quinolizidine. Ten-
tative structures for eight other such alkaloids were deduced from mass
and FTIR spectral data. The proposed structure of 251F has now been
confirmed by synthesis (37).
251 F
23
Such cyclopenta[b]quinolizidines are known in Nature from only two
populations of the tiny dendrobatid frog Minyobates bornbetes. If cyclopen-
ta[b]quinolizidines come from a dietary source, then the lack of such alka-
loids in other dendrobatid frogs is remarkable.
4. THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS 155
IX. Epibatidine
An interesting alkaloid, present in trace amounts in skin extracts of the
Ecuadoran dendrobatid frog Epipedobates tricolor, was discovered in the
late 1970s because of its striking biological effects. Thus, injection of small
amounts of extract obtained in 1974 from seven frogs elicited a Straub-tail
reaction in mice, a reaction normally diagnostic for an opioid-class alkaloid,
such as morphine. A further 750 skins were obtained in 1976 and using a
Straub-tail assay with chromatographic fractionation, the alkaloid responsi-
ble proved to be a trace constituent containing a chlorine atom and having
a probable empirical formula of CllCI3N2C1. The quantity of this alkaloid,
20W210, isolated (ca 700 pg) was insufficient in the early 1980s to obtain
definitive NMR data. The purified alkaloid, later to be named epibatidine,
was shown to have analgetic activity in mice some 200-fold greater than
that of morphine, but, unlike morphine, the activity was not blocked by
naloxone. By serendipity these extracts provided sufficient pumiliotoxin
251D and 8-deoxypumiliotoxin 25lH to define structures for two other
classes of “dendrobatid alkaloids” (16,27). Further extracts of the Ecua-
doran frog obtained in 1979 and in 1987 had much lower amounts of the
analgetic alkaloid; a major disappointment. International restrictions placed
late in 1987 prevented further large-scale collecting of any dendrobatid
frogs in spite of the incredible abundance of many of the species. Another
disappointment was the discovery that the frog Epipedobates tricolor raised
in captivity had no skin alkaloids. Thus the remaining sample of purified
epibatidine represented the only means to ever discover the structure of
the analgetic alkaloid. In 1990, it was determined to undertake the NMR
analysis. In order to avoid further chromatographic losses, the combined
impure sample of epibatidine was converted to an N-acetyl derivative and
small amounts of contaminating tertiary amines (pumiliotoxins) were re-
moved by acid extraction. NMR analysis of the N-acetyl derivative revealed
the structure of epibatidine to be that of the chloropyridyl azabicyclohep-
tane 24 (38). Synthetic material was later used to elucidate the basis for the
analgetic activity. Epibatidine proved to be an extremely potent nicotinic
agonist (39) and is now the focus of active investigation in many labora-
tories.
24
156 DALY
Epibatidine is unknown in Nature except from Ecuadoran dendrobatid
frogs of the genus Epipedobates. The levels vary greatly among the four
species in which it has been detected and even in different populations of
the source species Epipedobates tricolor. A dietary source is suspected, but
there is no clue as to what small arthropod might be involved. It is possible
that epibatidine, like its structural relative, nicotine, has a plant origin.
X. Pseudophrynamines
The GC-mass spectral analysis of a single skin of an Australian myoba-
trachid frog, Pseudophryne semimarmoruta, in the early 1980s revealed the
presence of two alkaloids, one of which was a pumiliotoxin and the other
an allopumiliotoxin (29). This result prompted the collection of additional
specimens and species of the genus Pseudophryne in 1987. Such extracts
yielded pumiliotoxins/allopumiliotoxins, and also a new class of alkaloids
not seen in dendrobatid frogs. NMR analysis revealed the structures of
two major alkaloids as pseudophrynamine A (25) and pseudophrynaminol
(26) (20,21). The former could be converted by methanolysis into pseudo-
phrynaminol and an ester, which was identical to the alkaloid 286A (27),
also isolated from the Pseudophryne frogs.
0
I I I t
H CH3 CH3 H
25 H37cH20H
I I
H CH3 H CH3
27
286A
26
4. THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS 157
Pseudophrynamines are as yet unknown in Nature except in the skin of
nocturnal frogs of the Australian myobatrachid genus Pseudophryne, and
have not been detected in any of the alkaloid-containing frogs that are
diurnal, namely, the dendrobatid frogs, the mantelline frogs, and the bu-
fonid toads of the genus Melunophryniscus. Whether the presence of pseu-
dophrynamines is associated with a nocturnal prey item or whether the
source is endemic to Australia remain unsolved questions.
XI. Pyrrolizidine Oximes
The Panamanian dendrobatid frog Dendrobutes pumilio provided not
only pumiliotoxins A and B and decahydroquinoline cis-l95A, but from
extracts obtained in 1983, a set of three new tricyclic alkaloids. The originally
proposed tentative amidine structures of these new alkaloids (26) were
later concluded to be incorrect based upon GC-FTIR spectral analysis. At
that point further NMR spectral analyses delineated the structures as being
those of pyrrolizidine oximes (40), of which the predominate member in
dendrobatid skin extracts is the 0-methyloxime 236 (28). The corresponding
oxime 222 and two hydroxy analogs of 236 also occur in dendrobatid frogs.
The structure of 236 has been confirmed by synthesis (41).
236
28
Pyrrolizidine oximes are known in Nature only from amphibian skin.
They are major alkaloids in recent extracts from several dendrobatid spe-
cies. Minor or trace amounts of pyrrolizidine oximes have been detected
in a myobatrachid frog, in mantelline frogs, and in a bufonid toad. Although
oximes are unknown in arthropods, a close relative of the pyrrolizidine
oximes, nitropolyzonamine (29), is a constituent in defensive secretions
of a millipede (42). Nitropolyzonamine and another millipede alkaloid
polyzonimine(30) have been detected as trace alkaloids in some dendro-
batid extracts (unpublished results).
158 DALY
29 30
It seems highly likely that the pyrrolizidine oximes in dendrobatid frog
skin originate from small neotropical millipedes. Indeed, after being raised
on Panamanian leaf-litter arthropods, the major alkaloid in the dendrobatid
frog Dendrobates auratus was the pyrrolizidine oxime 236 (25). The emer-
gence of pyrrolizidine oximes as significant skin alkaloids in certain popula-
tions of Panamanian Dendrobates pumilio and Dendrobates auratus may
reflect an increased availability of alkaloid-containing millipedes as the
habitat has changed from 1970 to the present time.
XII. Coccinellines
The tricyclic alkaloid precoccinelline (31) has now been identified as a
minor component by GC-mass and GC-FTIR spectral analyses in extracts
from a number of species of dendrobatid frogs, where it has been given
the code number 193C (I). Coccinellines, including precoccinelline, are
well known as alkaloids in ladybug and other beetles (43), where they
presumably serve as defensive substances.
31
Some of the other tricyclic alkaloids, detected in extracts of amphibian
skin, are probably related in structure to the coccinellines. One of these,
alkaloid 205B, was isolated from extracts of the Panamanian dendrobatid
frog Dendrobates pumilio and a tentative structure was proposed (26).
Further NMR and FTIR spectral analyses of 205B are in progress.
4. THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS 159
Precoccinelline occurs in several dendrobatid frogs and in a bufonid toad
of the genus Mefanophryniscus (24). In a population of the dendrobatid
frog Dendrobares auratus introduced into Hawaii, it is a major alkaloid
(18). It seems almost certain that precoccinelline and perhaps other similar
tricyclic alkaloids have their origin in dendrobatid frog skin from small
dietary beetles. Indeed, precoccinelline was a significant alkaloid in dendro-
batid frogs raised on arthropods from leaf-litter in Panama (25).
XIII. Bicyclic “Izidine” Alkaloids
A wide range of simple bicyclic alkaloids, for which we might use the
term “izidine” alkaloids, have been found in skin extracts from dendrobatid
frogs, mantelline frogs, and bufonid toads of the genus Melanophryniscus
( I ) . They are absent in myobatrachid frogs of the genus Pseudophryne.
The “izidine” alkaloids detected in amphibian skin include 3,5-disubstituted
pyrrolizidines, 3,5-disubstituted indolizidines, 5,8-disubstituted indolizid-
ines, 5,6,8-trisubstituted indolizidines, and 1,4-disubstituted quinolizidines.
Many probably originate from dietary ants.
A. PYRROLIZIDINES
A variety of 3,5-disubstituted pyrrolizidines have been identified in ex-
tracts from’ dendrobatid and mantellid frogs and in bufonid toads of the
genus Mefanophryniscus ( I ) . Identification has been based on GC-mass and
GC-ITIR spectral analyses and in some instances comparison to synthetic
material. Major mass spectral fragment ions are due to a-cleavage and the
FTIR spectra show virtually no Bohlmann bands. Structures of pyrrolizidine
cis-223H (32) and cis- and rrans-251K (33) and (34) are shown. The absolute
configurations are not known. About 15 alkaloids detected in extracts from
amphibian skin, appear to be 3,5-disubstituted pyrrolizidines; some are
present as two diastereomers.
3,5-Disubstituted pyrrolizidines are present as venom constituents in
myrmicine ants, and indeed cis-223H proved identical on GC analysis with
a (5Z,8E)-3-heptyl-5-methylpyrrolizidine from the thief ant (Solenopsis
sp.) (44), whereas trans-25lK was identical on GC analysis with a (5E,8E)-
3-butyl-5-hexylpyrrolizidine from a Venezuelan ant (Megalomyrmex mo-
destus) (45). It seems highly likely that all of the 3,5-disubstituted pyrroliz-
idine alkaloids in frog skin owe their presence to a diet of ants containing
such alkaloids. Dendrobatid frogs, in particular, are known to consume
large numbers of ants and have been referred to as ant specialists (46).
160 DALY
cis-223H R/
32
cis-251 K
33
trans-251 K
34
B. 3,5-DISUBSTITUTED INDOLIZIDINES
In 1978,3,5-disubstituted indolizidine structures were proposed for three
alkaloids found in the dendrobatid frog Dendrobates histrionicus (15). The
structures were based on mass spectral data and biosynthetic speculation.
The structure of one, namely 223AB (39, was subsequently confirmed by
demonstrating its identity with synthetic (5E,9E)-3-butyl-5-propylindolizid-
ine (47). Since that time, three of the four diastereomers of indolizidine
223AB have been detected in amphibian skin (see Ref. I). Remarkably,
while the sole diastereomer of 223AB in a Colombian Dendrobates histrion-
icus was the 5E,9E isomer 35 (48), the sole diastereomer in Panamanian
Dendrobates speciosus proved to be the 52,92 isomer 36 (I).
qJ qJ
woH @J CH3 WJ 6H3 6
5E,9E-223AB v: 52,92-223AB [oH5E.9E;!9AB v=
35 36
5E,9E-239CD 5E,9E-l95B 5Z, 9Z-195 B
v=
38 39 40
About 15 alkaloids detected in extracts of amphibian skin appear to be
3,5-disubstituted indolizidines, some of which are represented by as many
4. THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS 161
as four diastereomers. Structures of many are based only on diagnostic
features of the mass and FTIR spectra. The major fragment ions are due
to loss of either one or the other side chain. Loss of methyl is a relatively
minor event. A fragment ion at d z 124 is often present due to a McLafferty
rearrangement during cleavage of the second side chain. Bohlmann bands
in the FTIR spectra are relatively weak and broad for the 5 2 , 9 2 diastereo-
mer, and are even weaker for the 5E,9E and other diastereomers (24).
Certain members of this class of amphibian alkaloids, such as the 223AB
isomers (see previous discussion), 5 E,9E-239AB (37) 5 E,9E-239CD (38)
and 5E,9E-195B (39) have been isolated and their relative configuration
defined by NMR spectral analysis (35,48). The absolute configurations of
35, 37, 38, and 39 are also known based on comparison with synthetic
enantiomers (see Ref. I).
The presence of simple 3- or 5-substituted indolizidines in alkaloid frac-
tions from amphibian skin has been proposed, based solely on mass spectral
analysis (1). However, the compounds could instead be 3,5-disubstituted
pyrrolizidines with one substituent being a methyl group. Unfortunately,
the original alkaloid samples have proven to be insufficient in amount for
GC-FTIR spectral analysis.
3,5-Disubstituted indolizidines represent another alkaloid class present
as venom constituents in myrmicine ants. Indeed, monomorine I from ants
of the genera Monornoriurn and Solenopsis (49,50) is diastereomeric to the
indolizidine 5E,9E-l95B (39) found widely in dendrobatid frogs. All four
diastereomers of 195B, including the diastereomer 52,92-195B (40) identi-
cal by GC analysis with monomorine I, were present in extracts from a
bufonid toad of the genus Melanophryniscus (24). Recently, both monomor-
ine I and the amphibian diastereomer 5E,9E-l95B were found in a Puerto
Rican myrmicine ant (51). Dendrobatid frogs (Dendrobates auratus) fed
Pharoah’s ant (Monornorium pharaonis) efficiently accumulated the ant
alkaloid monomorine I and two minor 3,5-disubstituted indolizidines into
their skin (12). Indolizidine 5E,9E-l95B was a major alkaloid in a dendro-
batid frog (Dendrobates aurutus) raised in outside cages in Hawaii (18).
It therefore seems likely that the 3,5-disubstituted indolizidines found in
amphibian skin are the result of sequestration from myrmicine ants. 3 3 -
Disubstituted indolizidines occur in dendrobatid frogs of the genera Den-
drobates and Phyllobates, but apparently not in the genera Epipedobates
and Minyobates ( I ) . They occur in mantelline frogs and in bufonid toads
of the genus Melanophryniscus.
c . 5,8-DISUBSTITUTED INDOLIZIDINES
Another new class of amphibian alkaloids was established on thebasis
of NMR analysis of a minor alkaloid 207A isolated in the mid-1980s from
162 DALY
258 skins of the Panamanian montane dendrobatid frog Dendrobates specio-
sus (52). The structure of the 5-substituted-8-methyl indolizidine 207A (41)
is shown, as are the structures of other members of this class, namely
203A, 205A, 235B', and 235B" (42-45) that have been isolated in quantities
sufficient for NMR analysis (33,35,52, see also Ref. I ) . Absolute configura-
tions of 41-44 are known, based on comparison to synthetic enantiomers
(see Ref. I ) . About 40 alkaloids detected in extracts of amphibian skin
appear to be 5,8-disubstituted indolizidines, based on diagnostic features
of the mass and FTIR spectra. The 5-substituted-8-methylindolizidines have
a base peak at m l z 138 (GHI6N+) and a diagnostic fragment at m l z 96
(C6HloNt), arising from the m/z 138 ion by a retro-Diels-Alder process.
Another group of 5,8-disubstituted indolizidines appears to have 8-substitu-
ents other than methyl and, therefore, yield base peaks of d z 152 or higher,
dependent on the 8-substituent. All yield the diagnostic retro-Diels-Alder
ion at m l z 96. One such alkaloid is the relatively widely occurring indolizid-
ine 217B (46). A sharp and intense band at about 2785 cm-' in the FUR
is diagnostic for the 5,8-disubstituted indolizidine class, virtually all of which
have H-5 and H-9 in a cis-relationship (22,2433).
The 5,8-disubstituted indolizidines and the 5,6,8-trisubstituted indolizid-
ines (see following section) appear to be unique in Nature to amphibian skin
and have not been reported from an arthropod. They are very widespread in
dendrobatid frogs and have also been found in mantelline frogs and bufonid
toads of the genus Melanophryniscus (I).
207A 203A 205A
41 42 43
2358'
44
2358"
45
21 78
46
4. THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS 163
D. 5,6,8-TRISUBSTITUTED INDOLIZIDINES
There now appear to be a class of 5,6,8-trisubstituted indolizidines exem-
plified by the relatively widespread dendrobatid alkaloid 223A (47). NMR
spectral analysis indicates the structure shown (53). The mass spectral base
peak, due to a-cleavage, is at d z 180 (C12H22N+) and there is a further
retro-Diels-Alder fragment ion at m/z 124 (C8HI4N+). The FTIR spectrum
exhibits, as in the case of the 5,8-disubstituted indolizidines, a sharp, intense
Bohlmann band at about 2785 cm-'. There are some half-dozen other
bicyclic alkaloids from amphibian skin that appear likely to be 5,6,8-trisub-
stituted indolizidines, since they exhibit a base peak plus the diagnostic
retro-Diels-Alder fragment ion at d z 124.
223A
47
E. QUINOLIZIDINES
The existence of a quinolizidine class of alkaloids in extracts of amphibian
skin has been.proposed based on mass and FTIR spectral data (22,24). The
simplest members were proposed to be 4-substituted l-methylquinolizi-
dines, as exemplified by 217A (48) and 231A (49), which show a mass
spectral base peak corresponding to a-cleavage at d z 152 (CloH18N+) for
217A, and at d z 166 (ClIH20N+) for 231A, along with a retro-Diels-Alder
fragment at d z 110 (C7HI2N+) that was proposed to be diagnostic for this
class of amphibian alkaloids. Alkaloids of this proposed 1,4-disubstituted
quinolizidine class show a somewhat broader and less intense Bohlmann
band (22,24) than do the 5,8-disubstituted and 5,6,8-trisubstituted indolizid-
ines. Over 20 alkaloids detected in extracts from amphibian skin were
tentatively assigned to a 1,4-disubstituted quinolizidine class, based on mass
spectra and in a few cases on mass and FTIR spectra (I). However, it now
seems likely that some may prove to be 4,6-disubstituted quinolizidines,
some may prove to be 5,6,8-trisubstituted indolizidines, and some may even
prove to contain another bicyclic ring system. Isolation and NMR analysis
of alkaloids that have been tentatively assigned to the quinolizidine class
have a high priority in this research area.
The alkaloids that are tentatively proposed as 1,4-disubstituted quinolizi-
dines are relatively common in dendrobatid frogs, in mantelline frogs, where
164 DALY
21 7A
48
231 A
49
217A and 231A are major alkaloids in one species (22,23), and in bufonid
toads of the genus Melanophryniscus (24). Such alkaloids have not been
reported in any arthropod.
XIV. Monocyclic Alkaloids
2J-Disubstituted pyrrolidines and 2,6-disubstituted piperidines occur in
amphibian skin, but usually only as trace constituents (I). However, a
pyrrolidine 197B (50) was a major alkaloid component in skin extracts of
a Colombian dendrobatid frog, Dendrobates histrionicus and was identified
as a trans-2-butyl-5-pentylpyrrolidine (48). Identification and a later deter-
mination of absolute configuration were by comparison to synthetic samples
(see Ref. I). The piperidine 241D (51) was a major alkaloid component in
skins extracts of the Panamanian dendrobatid frog, Dendrobates speciosus
(52). The structure was defined by NMR spectral analysis and later con-
firmed by synthesis (54). The other 2,5-disubstituted pyrrolidines, about
five in total, and the other 2,6-disubstituted piperidines, about 18 in total,
in skin extracts have been defined based only on diagnostic features of
mass and FTIR spectra. In the case of the piperidines, both cis- and trans-
isomers are often present together in skin extracts from dendrobatid frogs,
and can be distinguished by Bohlmann bands in the FTIR spectra (34).
Cis-and trans-pyrrolidines are easily distinguished by FIYR spectra after
N-methylation (34).
trans-1978
50
241 D
51
4. THE NATURE A N D ORIGIN OF AMPHIBIAN ALKALOIDS 165
2,5-Disubstituted pyrrolidines and 2,6-disubstituted piperidines are well
known as venom constituents in myrmicine ants (55,56). The ant pyrroli-
dines are all trans, as is the case for the pyrrolidine 197B from amphibian
skin, while the ant piperidines are usually cis/trans mixtures, as often is the
case of piperdines detected in amphibian skin extracts. Many of the ant
piperidines have a 2-methyl substituent and many of the piperidines de-
tected in amphibian skin appear to have a 2-methyl substituent. The pyrroli-
dinedpiperidines seem somewhat restricted in their amphibian distribution,
being most common in dendrobatid species of the genus Dendrobates, and
are rare or absent in other dendrobatid genera and in other alkaloid-bearing
amphibians (Mantella, Melanophryniscus, Pseudophryne). While it seems
highly likely that such pyrrolidines and piperidines, when found in amphib-
ian skin, originate from dietary ants, it should be noted that a Costa Rican
dendrobatid frog (Dendrobates auratus) did not accumulate trans-2-heptyl-
5-hexenylpyrrolidine when fed Pharoah’s ants containing that pyrrolidine,
nor did Dendrobates auratus accumulate a piperidine in a mixture of alka-
loids provided on dusted fruit flies (22 ) .
XV. Summary and Prospects
During the past 30 years, a coordinated program of field work, isolation,
structure elucidation, synthesis, and pharmacological evaluation has led to
detection of a total of over 400 alkaloids from amphibian skin, several of
which have become valuable pharmacological tools. The research has led
to the discovery of nearly a dozen new species of frogs, to the introduction of
unique new alkaloid structures, and most recently to evidence that probably
none of these alkaloids are produced by the frogs themselves, but instead
are taken up from dietary arthropods, including ants, beetles, and milli-
pedes. Such dietary alkaloids appear to be sequestered unchanged into
secretory skin glands of the frog, where they then serve as a chemical
defense for their new host. Ironically, such alkaloids apparently failed to
protect the arthropods, and, indeed, may cause the frogs to target as prey
such alkaloid-containing arthropods.
The research has changed over the years; initially large-scale collections
of amphibianswere permitted, and chromatographic isolation of major
alkaloid constituents on the 10-50 mg scale was followed by NMR analysis,
and often by crystallization and X-ray crystallography. With such para-
digms, structures for the major classes of amphibian skin alkaloids, namely
the batrachotoxins, the pumiliotoxins, allopumiliotoxins, homopumilio-
toxins, histrionicotoxins, gephyrotoxins, and decahydroquinolines were
166 DALY
lished during the 1970s. The challenge remained for the 1980s of the minor
and trace alkaloids, where the amounts that could be isolated even from
hundreds of frog skins often was in the submilligram scale. But during the
1980s, the sensitivity and power of 2D-NMR spectroscopy greatly increased
and structures of 5,8-disubstituted indolizidines, epibatidine, a cyclopentyl
[blquinolizidine, the pyrrolizidine oximes, and pseudophrynamines were
established with quantities insufficient to attempt crystallization. During
the late 1980s and early 1990s, sensitive GC-FTIR analysis was used to
complement the information gleaned from mass spectral analysis, the latter
also providing deuterium exchange data. Microchemical perhydrogenation,
methylation, acylation, and phenylboronation provided further information
on the nature and position of various structural entities. With such compos-
ite data almost three-quarters of the over 400 alkaloids detected in amphib-
ian skin extracts can be assigned or tentatively assigned to one of some
nearly 20 structural classes. HPLC separations and NMR analyses of such
alkaloids on the submilligram scale continue in order to verify structures.
Syntheses have in the past confirmed some structures and undoubtedly will
continue to confirm or dismiss further structures. A current major problem
is that permission to collect more than a token few frogs is now nearly
impossible. Most of the blame for this lies with the International Commis-
sion for Trade in Endangered Species, which in 1987, in violation of its
own guidelines, placed all of the dendrobatid frogs on a threatened list in
spite of evidence to the contrary for most of the dendrobatid species. Thus,
many of the trace alkaloids, which might be isolated in sufficient quantity
for NMR analysis from 100 or more skins, will never be available at that
level because of limitations imposed on scientific collecting.
The demonstration that dendrobatid frogs raised in captivity on fruit
flies and crickets have no alkaloids in the skin, and that such frogs readily
sequester alkaloids provided to them in their diet unchanged into skin,
strongly suggests that, with the exception of the samandarines, the more
than 400 alkaloids that have been demonstrated in amphibian skin come
from dietary sources. A challenge for further research is the identification
of what tiny arthropods are the source of the batrachotoxins, the pumiliotox-
ins, homopumiliotoxins and related congeners, the histrionicotoxins, the
gephyrotoxins, the decahydroquinolines, the cyclopenta[b]quinoliidines,
epibatidine, and the literally hundreds of trace alkaloids whose structures
are unknown or tentative. Such tiny arthropods might be expected to be the
source of a treasure-trove of alkaloids that presumably could be obtained in
quantities sufficient for structure elucidation and pharmacological evalua-
tion. The amphibians in question eat only small mobile creatures, being
cued to feed by movement, but there is the possibility that the dietary trail
will lead through small arthropods to alkaloid-containing plants eaten by
4. THE NATURE AND ORIGIN OF AMPHIBIAN ALKALOIDS 167
such arthropods. Plant alkaloids have been identified as minor alkaloid
components in extracts from 1000 skins of the dendrobatid frog Phyllobates
terribilis (9). These were the indole alkaloids chimonanthine and calycan-
thine and a dipyridylpiperidine, noranabasamine. Morphine has been de-
tected as a trace alkaloid in the skin of a bufonid toad, Bufo marinus (57).
Of the over 400 alkaloids detected in amphibian skin, about 60 are likely
to be ant-derived alkaloids, namely the 3,5-disubstituted pyrrolizidines, the
3,5-disubstituted indolizidines, the 2,5-disubstituted pyrrolidines, and the
2,6-disubstituted piperidines. Precoccinelline undoubtedly originates from
small beetles, as probably do some dozen other tricyclic alkaloids. The
pyrrolizidine oximes, nitropolyzonamine, and polyzonimine undoubtedly
come from small millipedes. This leaves over 300 alkaloids of amphibian
skin with an unknown biological source, certainly a major challenge for
further research.
Acknowledgments
The author acknowledges his great debt to all who have contributed so much to the past
three decades of research on “amphibian alkaloids.” In particular, I wish to express gratitude
to my biologist colleague and mentor in field work, Dr. Charles W. Myers, to the chemists,
Drs. Takashi Tokuyama, Thomas F. Spande, and H. Martin Garraffo, who have contributed
so much over, the years, to the X-ray crystallographer, Dr. Isabella Karle, who revealed
structures of some of these alkaloids, to the pharmacologists, Drs. Edson X. Albuquerque
and Fabian Gusovsky, who were instrumental in defining sites of action of many of these
alkaloids, and to Dr. Bernard Witkop who started me on this long journey.
References
1. J. W. Daly, H. M. Garraffo, and T. F. Spande, in “The Alkaloids” (G. A. Cordell, ed.),
2. J. W. Daly, C. W. Myers, and N. Whittaker, Toxicon 25, 1023 (1987).
3. J. W. Daly and T. F. Spande, in “Alkaloids: Chemical and Biological Perspectives”
4. CI. Schopf, Experientia 17, 285 (1961).
5. G. Habermehl in “The Alkaloids” (R. H. F. Manske, ed.), Vol. 9, pp. 427-439. Academic
6. F. Marki and B. Witkop, Experientia 19, 329 (1963).
7. T. Tokuyama, J. Daly, B. Witkop, I. L. Karle, and J. Karle, J. Am. Chem. Soc. 90,
Vol. 43, pp. 185-288. Academic Press, New York, 1993.
(S. W. Pelletier, ed.), Vol. 4, pp. 1-274, Wiley, New York, 1986.
Press, New York, 1967.
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168 DALY
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-CHAPTER L
BIOCHEMISTRY OF ERGOT
ALKALOIDS-ACHIEVEMENTS AND CHALLENGES*
DETLEF GROCER
Institute for Plant Biochemistry
Halle (Saale), Germany
HEINZ G. FLOSS
Department of Chemistry
University of Washington
Seattle, Washington 981 95
I. Introduction
111. The Natural Ergot Alkaloids ............................................................. 173
A. Structural Types ........................... .......................................... 174
B. Lysergic Acid Derivatives ............................................................ 174
C. Clavine Alkaloids and Secoergolines . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
D. New Alkaloids . . . ,. . .. . .. . .. . . . . . .. . .. . ... . .. . .. . ... . ..
IV. Producing Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Biology of Ergot Fungi ................................................................ 182
B. Other Fungi ........................
C. Higher Plants ...................... .................................................. 183
V. Biosynthesis ................................................................................... 183
B. Biosynthesis of Lysergic Acid Derivatives ....................................... 193
C. Enzymology of Ergoline Alkaloid Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
VI. Biotechnological Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 201
A. Directed Fermentation . .. . .. . .. . . . . . ... . ... . ... ... . ... . .. ....................... 201
B. Bioconversion of Ergot Alkaloids ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
VII. Pharmacological Properties of Ergolines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
A. Biological Activities Mediated by Neurotransmitter Receptors . . . . . . . . . . . . . 206
B. Ergolines with Antitumor and Antimicrobial Properties . . . . . . . . . . . . . . . . . . . . . 207
..................... 208
A. Enzymology and Molecular Genetics .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
B. Regulation ................................................................................ 210
C. Evolutionary Aspects .................................................................. 210
References . . . . . .. . .. . .. . .. . . ... ... .. ... . ... . ... ... . . . .. . . ... ... . .. . ... . ... . .. . . ... . .. . . .. . . . .. 212
.... , ............... , .............. ,.
A. Biosynthesis of the Ergoline Ring System ........... 184
VIII. Future Challenges .... . .. . . .. . ... . . . . .
* Dedicated to Dr. Dr. h.c.mult. Albert Hofmann, the great pioneer of ergot research, on
the occasion of his 90th birthday.
THE ALKALOIDS, VOL. 50
0099-9598/98 $25.00
171 Copyright 0 1998 by Academic Press
All rights of reproduction in any form reserved.
172 GROGER AND FLOSS
I. Introduction
Ergot alkaloids comprise a group of indole alkaloids which are predomi-
nantly found in various species of the ascomycete Claviceps. In pharmacope-
ias, the sclerotia of Claviceps purpurea (Fr.) Tulasne parasitizing on rye,
Secale cereale L., are designated as ergot or Secale cornutum. Now, the
term ergot is used in a broader sense to describe the sclerotia of various
Claviceps species growing on different host plants or their saprophytic my-
celia.
Ergot fungi are the oldest known producers of mycotoxins. In contrast
to other mycotoxicoses, ergotism is today practically eliminated. Due to
their many fascinating features, there is a continuing and extensive interest
in these secondary metabolites. Thus the chemistry of ergot alkaloids has
presented many challenges to organic chemists. A number of natural alka-
loids and semisynthetic ergolines are important drugs which are widely
used in clinical medicine. Moreover, ergot alkaloids have been an important
stimulus in the development of new drugs by providing structural prototypes
of molecules with pronounced pharmacological activities.
The chemistry of ergot alkaloids, including newly detected alkaloids, has
been described in Volumes VIII ( 1 ) and XV (2) of this treatise. A recent
review in Volume 38 (3 ) covered the major synthetic work in the ergoline
field. In the present review, a picture of our current knowledge of the
formation of ergot alkaloids in Nature will be given. Extensive work has
been done on this subject, which proved to be unexpectedly complex and
full of surprises. Biotechnological aspects and some current trends in ergot
alkaloid pharmacology will also be covered. Another purpose is to draw
attention to unsolved questions which merit further investigation.11. Historical Background
In classic antiquity, ergot was apparently not known, although there are
some hints in the old literature. In the Middle Ages, however, severe
epidemics occurred in Central and Western Europe in both man and ani-
mals. The animal poisonings resulted from ingestion of ergot-infected
grasses, while in man the toxic effects were caused by bread made from
rye contaminated with ergot. The symptoms in man were known as “ignis
sacer” or “holy fire.” Ergot was first mentioned as a remedy in 1582 in the
Kreuterbuch of Adam Lonicer (4 ) , and in 1808 the American physician J.
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 173
Stearns introduced ergot into official medicine (5). The life cycle of Clavi-
ceps purpurea was described in 1853 in a classical paper by Tulasne (6) ,
and the first chemically pure, homogeneous ergot alkaloid, ergotamine, was
isolated in 1918 by Stoll (7). In 1934 lysergic and isolysergic acid were
obtained as degradation products of ergot alkaloids by Jacobs and Craig
(8). Lysergic acid diethylamide (LSD) was first prepared in 1938 and its
extremely potent hallucinogenic activity was reported in 1943 (9) . The basic
principles of the parasitic cultivation of rye ergot were established by von
BCkCsy in 1935 (10) and by Hecht in 1941 (12). After World War 11, in
Japan the first representative of a new class of natural ergoline derivatives,
agroclavine, was isolated from, most remarkably, saprophytic cultures of
grass ergot (22,23). The first total synthesis of lysergic acid was accomplished
by Kornfield et al. in 1954 (24) and the total synthesis of the cyclol-type
alkaloid ergotamine was described in 1961 by Hofmann and his colleagues
(25). A phytochemical sensation was the structure elucidation of the active
hallucinogenic principles of “ololiuqui,” the Aztec name for the seeds of
Morning Glory (Zpornoea sp.), an old magic Mexican drug, which proved
to be amides of lysergic acid and other ergolines (26). The large-scale
production of simple lysergic acid derivatives in submerged culture was
described by Arcamone et al. in 1961 (27). A hypothesis on the biosynthetic
origin of the ergoline ring system by condensation of tryptophan with an
isoprenoid C-5 unit was proposed by the groups of Mothes and Weygand
in 1958 (28). Subsequently, the first radioactive feeding experiments with
saprophytic cultures of grass ergot were performed, demonstrating the
incorporation of [D-l4C]tryptophan into elymoclavine (29). For further his-
torical information the reader is referred to several comprehensive re-
views (20-25).
111. The Natural Ergot Alkaloids
The first pharmaceutical-chemical investigation of ergot was published
in 1816 by the French pharmacist Vauquelin (26). A crystalline alkaloid
preparation, “ergotinine cristallisee,” was obtained by Tanret in 1875 (27).
A special landmark in ergot alkaloid chemistry, as mentioned, was the
isolation of ergotamine by A. Stoll in 1918, a pioneer in this field (7).
Now quite a number of natural ergolines are known which have been
isolated from different sources. Some general information on ergot alkaloids
and “new alkaloids” which have been isolated since 1989 are summarized
below. For a detailed description of individual alkaloids some earlier compi-
lations should be consulted (2,3,28).
174 GRdGER AND FLOSS
A. STRUCTURAL TYPES
Ergot alkaloids are 3,4-substituted indole derivatives. An essential struc-
tural element of ergot alkaloids is the tetracyclic ergoline ring system (1)
(29) or slight modifications thereof. Most of the naturally occurring ergot
alkaloids are derivatives of 8-substituted 6-meth~l-A~.~- or A9."-ergolene.
On the basis of their structures they can be divided in two major classes:
(a) Amide derivatives of lysergic acid (2) and the stereoisomeric isolyser-
gic acid (3). The amide portion can be a small peptide or a simple alkylam-
ide. A structural isomer of lysergic acid is paspalic acid (4). In this As-
ergolene the hydrogen atom at C-10 has the a-configuration, trans to 5-H.
(b) The clavine alkaloids, or clavines, are hydroxy- and dehydro-deriva-
tives of 6,8-dimethyl-ergolenes and the corresponding ergolines. In the
stereoisomeric chanoclavines the D-ring is open between N-6 and C-7.
1
'H
2 3 4
B. LYSERGIC ACID DERIVATIVES
1. Peptide Alkaloids
Peptide ergot alkaloids are composed of lysergic acid and a peptide
moiety. They are divided into two major groups. The "classic" ergot alka-
loids possessing a cyclol structure are called ergopeptines (30). The ergopep-
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 175
tams (31) contain a noncyclol lactam and, in contrast to the ergopeptines,
a D-proline.
a. Ergopeptine Alkaloids. These alkaloids are characterized by a modi-
fied tripeptide containing proline and an a-hydroxy-a-amino acid which
has undergone cyclol formation with the carboxyl carbon of L-proline. The
amino acids present in the cyclol peptide portion characterize the different
ergopeptines. In Table I the cyclol-type alkaloids are grouped in some form
of a “periodic table” (32).
b. Ergopeptam Alkaloids. The first member of a new group of peptide
ergot alkaloids, N-[N-( d-lysergyl-L-valy1)-L-phenylalanyl-D-proline lactam,
later designated as ergocristam, was isolated in the 1970s (33,34). Ergot
alkaloids of this noncyclol type are called ergopeptams (Table 11). They
occur only in traces in sclerotia and saprophytic cultures. Their biochemical
relevance has still to be elucidated. Members of the ergotamam and er-
goxam group, corresponding to the ergotamine and ergoxine group, respec-
tively, have not yet been found in Nature.
TABLE I
ERCOPEP~INE ALKALOIDS“
Ergotamine group Ergoxine group Ergotoxine group
R’ R = CH3 R = CzHS R = CH(CH3)2
CHZ-C~HS Ergotamine (5) Ergostine (10) Ergocristine (15)
CHz-CH(CH3)z a-Ergosine (6) a-Ergoptine (11) a-Ergokryptine (16)
CHCHS-C~HS P-Ergosine (7) P-Ergoptineb (12) P-Ergokryptine (17)
CH(CH3)2 Ergovaline (8) Ergonine (13) Ergocornine (18)
CH2CH3 Ergobine (9) Ergobutine (14) Ergobutyrine (19)
The derivatives of isolysergic acid characterized by the ending -kine are not listed here, e.g., ergotam-
Not yet found in nature.
inine.
176
H-
GROGER AND FLOSS
TABLE I1
ERGOPEPTAM ALKALOIDS“
Ergoannam group
RI Ergotoxam group R = CH(CH& R = CH(CH3)CzHs
CHz- GHs Ergocristam (20)
CHz-CH(CH3)z a-Ergokryptam (21) a,P-Ergoannam” (25)
CH(CH3)CzHs P-Ergokryptam (22) P,P-Ergoannam (26)
CWCHdz Ergocornam (23)
CHzCH? Ergobutyram”
“The derivatives of isolysergic acid characterized by the ending -inam are not listed here.
Not yet found in nature.
2. Simple Lysergic Acid Derivatives
From the “water soluble fraction” of rye ergot the propanolamide of
lysergic acid (27) was isolated in 1935 (35-38) which exhibited a pronounced
oxytoxic activity. It was variously designated as ergometrine, ergobasine
or ergonovine. Abe et al. obtained a compound from ergot which gave
upon hydrolysis lysergic acid, pyruvic acid, and valine. The name ergoseca-
line and, tentatively, the structure 28 were assigned to this compound (39).
Saprophytic cultures of Cluviceps paspali were the source for the isolation
of lysergic acid a-hydroxyethylamide (29), lysergic acid amide (ergine) (30),
and isolysergic acid amide by Arcamone el al. in 1961 (17) (Table 111).
C. CLAVINE ALKALOIDS AND SECOERGOLINES
Agroclavine (31) was found in sclerotia and saprophytic cultures of ergot
parasitizing on Agropyrurn sernicostaturn Nees in Japan (12,Z3). It was the
first member of a new class of ergot alkaloids called clavine alkaloids or
clavines. In the clavines C-17 has a lower oxidation state than in the lysergic
acid derivatives, and the double bond in the D-ring may be in the 8,9- or
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 177
TABLE 111
SIMPLE LYSERGIC ACID DERIVATIVES
R Compound
CH3
I
-NH - C -H
I
CH,OH
Ergonovine (27)
(Ergobasine, Ergometrine)
CH3 I Lysergicacid a-hydroxyethylamide (29)
-NH - CH -OH
- NH, Lysergic acid amide (30)
(Ergine)
9,lO-position or may be lacking altogether. In the tricyclic 6,7-secoergolines
ring D is not closed. Prominent members of this group are the chanoclavines.
Chanoclavine-I, originally designated as chanoclavine, was discovered by
Hofmann et al. in 1957 (41). Later it was found to be one of several
stereoisomers (42). In chanoclavines-I and -11 the hydrogens at positions
5 and 10 are in trans and cis arrangements, respectively. Chanoclavine-I
(37) is an essential intermediate in the biosynthesis of tetracyclic ergolines.
Clavines are found also in fungi outside the genus Claviceps and in higher
plants. Some representatives of the major types of the more than 30 known
clavine alkaloids are depicted in Fig. 1.
178 GROCER AND FLOSS
8-Ergolenes Ergolines
CH20H COzH
&H.cH3 / &2H3 / dCCH3 / gH. / c H3
H0 H' H' H'
31 Agroclavhe 32 Elymoclavine 33 Festuclavine 34 Fumigaclavine
9-Ergolenes 6,7-Secoergolenes
CHzOH CHzOH & HO, CHzOH 8, HO, CH3 &$CH3 &$H3
"CH3 CH3
/ / / 0
N N N N
H0 H' H0 H'
35 Penniclavine 36 Setoclavine 37 Chanoclavine-I 38 ChanoclavineLl
FIG. 1 . Various types of clavine alkaloids.
D. NEW ALKALOIDS
Ergot fungi, endophytes of grasses, and some species of higher plants
are the sources of new ergot alkaloids. The structures of ergolines which
were elucidated mostly during the period of 1989-1994 are summarized
as follows.
1. Dehydroelymocluvine (39)
From the roots of an African plant Securiducu longipedunculutu (Polyga-
laceae) an alkaloid fraction was isolated. The natives of Guinea Bissau use
extracts of this plant in religious rites, due to their psychotropic effects.
The structures of two alkaloids were determined by electron ionization
(EI) and fast atom bombardment (FAB) mass spectrometric measurements
5. BIOCHEMlSTRY OF ERGOT ALKALOIDS 179
as elymoclavine (32), and dehydroelymoclavine (39). Other evidence sup-
porting these identifications is lacking (43).
2. Elymoclavine-O-fi-~-fructofuranosyl-(2 4 I)-O-fi-o-
fructofuranoside (40)
Elymoclavine fructosides have been isolated (44) from saprophytic cul-
tures of Claviceps purpurea and grass ergot cultivated in sucrose media.
Besides the known (45) elymoclavine-0-0-D-fructofuranoside, another gly-
coside was also isolated. Acidic hydrolysis gave elymoclavine, and the UV
spectra showed the presence of a ASv9 double bond. From these results
and interpretation of MS and ‘H and I3C NMR spectra the structure 40
was deduced.
3. 8-Hydroxyergine (41) and 8-Hydroxyerginine (42a)
8-Hydroxyergine (41) and 8-hydroxyerginine (41s) were the main alka-
loids found in the culture broth of a Clavicepspaspali strain after a fermenta-
tion period of 28 days (46). The structures of 41 and 41a have been proposed
based mainly on physical data (UV, MS, ‘H and I3C NMR).
C q O H
I
H‘
39 Dehydmelymclavine 40 Elymoclavine-O-~-D-fructofuranosyl-(2+1)0-
p-D-fructofuranoside Q$ “CH,
H‘
41 @=OH, Ri=CONH2)
%-HY&OX-
41. (R=CONHz, Rl=OH)
S-HY~IQXY-
180 GROCER AND FLOSS
4. 10-Hydroxy-cis- and 10-hydroxy-trans-paspalic acid amides
(42) and (43)
During the post-production phase of a Claviceps paspali strain cis- and
trans-10-hydroxypaspalic acid amides (42 and 43) accumulated. Based on
their mass spectra, 42 and 43 were recognized as isomers of 8-hydroxyer-
gines. Facile elimination of H 2 0 from the molecular ion and abundant
peaks at d z 170 and 171 support the presence of an OH group at C-10.
Comparison of the I3C NMR spectra of the A8y9-ergines bearing a 10-
hydroxy substituent with those of the corresponding cis/truns pairs of di-
hydrolysergic acid derivatives and agroclavine/agroclavine-I allowed the
assignment of the C/D ring stereochemistry of 42 and 43 (47).
5. 0-12’-Methylergocornine (44) and 0-12’-methyL-u-ergokryptine (45)
Some minor alkaloids isolated from the saprophytic culture of a Claviceps
purpurea strain (48) have been characterized as the first naturally occurring
ergopeptines possessing an OCH3 group at C-12‘, namely O-l2’-methyler-
gocornine (44) and 0-12’-methyI-a-ergokryptine (45). The structure eluci-
dation was based mainly on physical data (MS, M’, d z 575 for 44,589 for
45; I3C-NMR: 12’-OCH3 at S 49.2 ppm in 44,48.8 ppm in 45).
CONH2
I
42 (R=-OH)
IO-Hydroxy4s-paspaIic acid arnide
10-Hydroxy-trans-paspspalic acid arnide
OH) 43 (R= ... w
H‘
44 (R=CH(CH3h)
45 (R=CH&H(CH3)2)
0-12’ Methylergocomine
0- 12’ Methylergokqptine
H’
46 Ergobalansine.
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 181
6. Ergobafansine (46)
The grass species, Cenchrus echinatus L., is native to the tropics and
subtropics. Very often it is infected by fungal endophytes, e.g., Bafansia
species. Powell et af. (49) isolated from Bafansia-infected C. echinatus an
ergot alkaloid, ergobalansine (46), and its C-8 epimer, ergobalansinine. The
same alkaloids were also produced by saprophytic cultures of Bafansia
obtecta and B. cyperi. Ergobalansine proved to be a peptide derivative
of lysergic acid, but differs from other known ergopeptines in that the
characteristic proline residue has been replaced by an alanine residue. The
structure has been elucidated by analysis of the mass spectra (M+, d z 521,
prominent ions at d z 267,221,207,192,180,167, and 128) and by 'H and
I3C NMR spectroscopy. Most surprisingly, the same alkaloids have also
been found, in addition to other ergolines, in the seeds and epigeal parts
of Ipornoea piurensis, a South American morning glory species (50).
7. Ergobine (9)
Ergobine (9) was isolated in trace amounts from submerged cultures of
a Cfaviceps purpurea strain (51). The structure was established through
chemical degradation, UV, 'H NMR, and mass spectrometry, and amino
acid analysis. Its isolation completes the series of natural ergopeptines
having a-aminobutyric acid as the second amino acid of the peptide moiety.
8. Ergogaline (47)
A minor alkaloid was recently isolated from sclerotia of a particular
Cfaviceps purpurea strain (52). The structure was primarily established by
X-ray crystallography and other physical methods (IR, MS, 'H, and I3C
NMR). Ergogaline (47; C33 H43N505; m.p. 182") is a new naturally occurring
member of the "ergotoxine" family containing L-homoisoleucine in the
peptide moiety. Apparently 47 is the first natural product containing this
unique amino acid.
47 Ergogaline 48 Cycloclavine
182 GROCER AND FLOSS
IV. Producing Organisms
A. BIOLOGY OF ERGOT FUNGI
Ergot alkaloids have been known for a long time as constituents of fungi
of the genus Claviceps, which belongs to the order Clavicipitales (53) and
to the class of the Ascomycetes. About 40 species of the genus Claviceps,
which are plant parasites, have been described (5435). In the compilation
of Brady (56), host plants belonging to the families Juncaceae, Cyperaceae,
and Gramineae are listed. Within the various Claviceps species different
biochemical “races” have been distinguished, based on their alkaloid con-
tent. The genus Claviceps may be divided biochemically into three
groups (57):
1. Claviceps species parasitizing on Agropyron and Pennisetum host
plants. They form only clavine alkaloids.
2. Ergot fungi of the Claviceps paspali type, which produce clavines and
simple lysergic acid derivatives.
3. Claviceps purpurea and related fungi, which are able to synthesize
clavines, simple lysergic acid derivatives, and peptide ergot alkaloids.
The life cycle of the most prominent Claviceps species, C. purpurea was
described more than 100 years ago (6,58). Several reviews on the biology
of Claviceps have been published (32,59,60,62).
B. OTHER FUNGI
Spilsbury and Wilkinson (62) discovered the first ergolines in fungi out-
side the genus Claviceps. They isolated fumigaclavine A and B and fes-
tuclavine from Aspergillus &migatus Fres. Fumigaclavine was also found
in Rhizopus nigricans. Later,various clavines were isolated from different
Aspergillus and Penicillium species. Of special interest are the rugulovasines
from Pencillium species which feature a benz[c,d]indole skeleton with a
spirobutanolide side chain (63-65). Relevant literature on this topic has
been compiled by Narayan and Ra6 (66).
Toxicoses caused by endophyte-infected grasses are a serious problem
for animal breeding in many parts of the world. The loline- and ergot-
type alkaloids produced by these fungal endophytes are responsible for
substantial losses to cattle and sheep producers. Balansia species and Epi-
chloe typhina from toxic pasture grasses produce clavine alkaloids, inter
alia 6,7-secoagroclavine. Moreover, E. typhina was found to synthesize
ergovaline (8) and its stereoisomer ergovalinine (67-70). Tall fescue (Fes-
183 5. BIOCHEMISTRY OF ERGOT ALKALOIDS
tuca arundinacea Schreb) infected with Sphacelia typhina (Acremonium
coenophialum) contained ergopeptine alkaloids, predominantly ergovaline
(8) (71,72). Ergobalansine (46) is synthesized by Balansia obtecta, an endo-
phyte infecting the annual grass species, Cenchrus echinatus L. (49). The
perennial grass, Stipa robusta, which is indigenous to the Southwestern
United States is often contaminated with Acremonium. Extraction of in-
fected grass yielded chanoclavine-I, ergonovine, 8-hydroxylysergic acid am-
ide and, in a remarkably high concentration, lysergic acid amide and isoly-
sergic acid amide (73).
C. HIGHER PLANTS
From the seeds of various Convolvulaceae, including Zpomoea violacea
L. and Rivea corymbosa (L.) Hall, Hofmann and Tscherter isolated ergine
(30), isoergine and chanoclavine (37) (16), followed later by elymoclavine
(32), lysergol and ergometrine (27) (7475). Since then, a number of authors
have demonstrated the occurrence of various ergolines in different species
of the family of twining plants (Convolvulaceae), mostly in Zpomoea, Argyr-
eia, and Strictocardia. Interestingly, cycloclavine (48), representing a novel
type of clavine, was isolated from Zpomoea hildebrandtii Vatke (76). Even
peptide ergot alkaloids were obtained from Zpomoea. Ergosine (7) and its
epimer ergosinine are constituents of Zpomoea argyrophylla Vatke (73,
and ergobalansine (46) has recently been isolated from Zpomoea piurense
(50). The latter and cycloclavine (48) have so far not been detected in
Claviceps species. Some compilations of ergolines found in Argyreia, Zpo-
moea, and Rivea have been published (78-80). The first and only report
on the occurrence of clavines in a plant not belonging to the Convolvulaceae
family appeared in 1992 (43). From a chemotaxonomical point of view an
independent confirmation of this finding would be extremely desirable.
V. Biosynthesis
The biosynthesis of ergot alkaloids has been studied for nearly 40 years.
A number of hypotheses on the origin of lysergic acid were published in
the 1950s, (85). Finally, in 1958 Mothes et al. (18), as well as Birch (81),
proposed that the ergoline ring is built up from tryptophan, a C5 isoprene
unit and a methyl group (Scheme l ) , and this proposal was quickly con-
firmed experimentally. Most remarkable, at the time, was the finding that
mevalonic acid (50) is not only a precursor of typical isoprenoids, but also
184 GROGER AND FLOSS
49
SCHEME 1.
participates in the formation of other secondary metabolites, like alkaloids.
The very first experiments in ergot alkaloid biosynthesis were done with
sclerotia growing parasitically on rye plants (28J9). Since the early 1960s,
when saprophytic alkaloid-producing strains became available, practically
all laboratories have used fermentation procedures for such biosynthetic
studies.
In this chapter, an outline of the general picture of ergot alkaloid biosyn-
thesis will be presented. Emphasis will be placed on some complex and
unexpected reactions uncovered by the experimental work and on results
obtained during the last decade.
A. BIOSYNTHESIS OF THE ERCOLINE RING SYSTEM
By the use of isotope techniques it was established in the late 1950s and
in the 1960s that ergot fungi synthesize the ergoline ring system from three
major precursors, tryptophan (49), an isoprenoid C5-unit (52), ultimately
derived from mevalonic acid (SO), and a methyl group provided by methio-
nine. Formation of the rings C and D of the ergoline system was studied
with multiple labeled mevalonic acid samples and specific potential interme-
diates. The biogenetic interrelationships of clavine and lysergic acid alka-
loids were also studied intensely. Since 1970 crude and purified enzyme
preparations catalyzing individual steps of ergot alkaloid formation have
become available. Summarizing all the results, the ergoline biosynthetic
pathway can now be formulated as depicted in Scheme 2. It starts with
the isoprenylation of tryptophan (49) to 4-(y,y-dimethylallyl)tryptophan
(DMAT) (53) which is subsequently methylated to 54. In a reaction which
is not yet completely understood, the tricyclic chanoclavine-I (37) is formed,
which in turn is oxidized to the corresponding aldehyde (55). In the next
step, the first tetracyclic clavine, agroclavine (31), is synthesized which can
be oxidized at C-17 to elymoclavine (32) and further to lysergic acid (2).
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 185
54 62 37
CH3 -
H'
31
*
7H20H
@ / H3
H'
32
- LysergicAcid(2) * Isotopic Labels
SCHEME 2.
The experimental evidence for various steps of the ergoline alkaloid path-
way and mechanistic aspects will be discussed in detail in the following.
1. Isoprenylation of Tryptophan
The formation of the ergoline ring system requires decarboxylation, N-
methylation and isoprenylation of 49. Tryptamine and Nw-methyltrypt-
186 GROCER AND FLOSS
amine were not incorporated into ergot alkaloids (88,89). This rules out
decarboxylation as the first step.
Feeding experiments (90) indicated that L-tryptophan is a more immedi-
ate precursor than the D-isomer, and that it is incorporated with retention
of the a-hydrogen and the amino nitrogen. In the course of the reaction
at the a-carbon of the tryptophan side chain an inversion of configuration
takes place. Since it was ruled out (92) that tryptophan is activated for the
condensation with dimethylallyl pyrophosphate (52) by hydroxylation at
the 4-position, a direct isoprenylation of 49 was indicated. The initial con-
nection of the isoprene unit could be either the a-position of 49 or at the
4-position of the indole ring (Scheme 3). The two potential precursors were
synthesized in labeled form, 57 by Weygand et af. (92) and DMAT (53) by
Plieninger and Liede (93). DMAT (53) was always incorporated much
better than 57, suggesting the intermediacy of 53 in ergoline ring biosynthe-
sis (94,95). Moreover DMAT was isolated from ergot cultures incubated
in the absence of oxygen (96) or in the presence of ethionine (97). Finally,
an enzyme preparation was isolated catalyzing the condensation of trypto-
phan and dimethylallyl pyrophosphate to give 53 (98).
52 49
A d 2 ($iH
N N
H' 53
H'
57
SCHEME 3.
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 187
2. Interrelationships of Clavines
The oxidative reaction sequence chanoclavine + agroclavine + ely-
moclavine + lysergic acid (Scheme 4) was proposed in 1958 (99). Experi-
mental support for the irreversible conversion of agroclavine into ely-
moclavine (32) was provided by Agurell and Ramstad (200-102) and by
Baxter et al. (203). The latter alkaloid in turn was shown by Mothes et al.
(104) to be a progenitor of lysergic acid derivatives. The conversion of
chanoclavine-I (37) into these tetracyclic alkaloids was first demonstrated
HO CH3 HO CH#H
$\CH3 / $\CH3 /
N N
H' H'
36 35
CHzOH d:cH3 \
31
HMN
FH3 t
"CH3 @
H'
31
CH2OH
I
"CH3 99 H'
32
H'
SCHEME 4.
188 GROCER AND FLOSS
in 1966 (105,106). Hydroxylation of 31 and 32 leads to setoclavine (36) and
penniclavine(39, respectively, and their C-8 epimers. These reactions
involve hydroxylation at C-8 with a shift of the double bond into the 9,lO-
position and are catalyzed by peroxidases. Besides Claviceps, a number of
other fungi, as well as plant homogenates, can catalyze these hydroxylations
(107). An alternative route of clavine transformations was proposed by
Abe’s group (108,109), but their results have never been confirmed in
other laboratories.
3. Cis- Trans Zsomerizations in Clavine Alkaloid Biosynthesis
It has been clearly established (106) that [2-’4C]mevalonate (SO) specifi-
cally labels the CH3 group at the 8,9-double bond of chanoclavine-I (37)
which occupies the trans position relative to the vinyl hydrogen, whereas
in the tetracyclic agroclavine (31) and elymoclavine (32) it labels C-17,
which is located cis to this hydrogen (103,110) (Scheme 2). These results
suggested the occurrence of cis-trans isomerizations during the formation
of tetracyclic ergolines from mevalonic acid. The labeling pattern of
agroclavine and elymoclavine obtained after feeding [ 17-14C]- or [7-14C]cha-
noclavine-I showed that the hydroxymethyl group of 37 becomes C-7 of
31 and the C-methyl group of 37 becomes C-17 of 31 (106,111). These
findings demonstrate the occurrence of one cis-trans isomerization between
chanoclavine-I and agroclavine.
It was suggested that a second cis-trans isomerization occurs during the
formation of chanoclavine-I from mevalonate (111). This was deduced from
the fate of the two diastereotopic hydrogens at C-4 of mevalonate, the
pro4R hydrogen is retained during elymoclavine formation (Scheme 2),
suggesting that the isopentenyl pyrophosphate isomerase reaction takes
the “normal” steric course in ergot. This means that 52 and 53 should carry
the label from C-2 of mevalonate in the methyl group that is located cis
to the vinyl hydrogen at the allylic double bond. This was subsequently
proven by feeding [Z-I4CH3]-53 and showing by degradation of 37,31, and
32 that the label was located in the hydroxymethyl group of chanoclavine-
I, but at C-7 of agroclavine and elymoclavine (112,113). Therefore, two cis-
trans isomerizations must occur in ergoline biosynthesis, the first between
DMAT and chanoclavine-I, and the second between chanoclavine-I and
agroclavine.
4. Formation of Ring C: Modification of the Isoprene Unit
The first alkaloid of the ergoline pathway starting from DMAT is the
tricyclic chanoclavine-I (37). Despite many experimental efforts, the exact
mechanism by which .ring C is closed is still not completely understood. It
is known (114) that H-5 is retained, that the configuration at C-5 is inverted,
that one hydrogen is lost from C-10, and that 37 is the product of the ring
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 189
closure. There is no correlation between the stereochemistry of hydrogen
abstraction from C-10 (ergoline numbering) and the stereochemistry of
the chanoclavine isomer resulting from the cyclization; all of the different
clavines and chanoclavine isomers are formed with retention of the pro-
5s and loss of pro-% hydrogen of mevalonate (225-227).
Desoxychanoclavine-I, its N-nor-derivative, and its N-methyl derivative
are not precursors of tetracyclic clavines and paspalic acid (228), suggesting
that oxygenation of one of the allylic methyl group precedes ring C closure.
Therefore, Plieninger’s group (229) synthesized (E)-4-(4’-hydroxy-3’-
methyl-2’-butenyl)-tryptophan (56) labeled in the hydroxymethyl group
and fed this promising precursor to a Claviceps culture. They reported
incorporation into both agroclavine and elymoclavine. However, later (222)
it was found that only elymoclavine is labeled and, surprisingly, that the
label is located at C-17, not at C-7 as expected. Subsequently, Arigoni’s
group (122,223) synthesized both the E and 2 isomers of 4’-hydroxy-
DMAT, [14CH3]-56 and [‘4CH3]-58. Both compounds labeled elymoclavine
at C-7, but did not label agroclavine. This labeling pattern would result
if 56 were processed as if the hydroxy group were not present, undergo-
ing the two cis-trans isomerizations and eventually generating 32 directly
in the cyclization which normally produces 31 (Scheme 5) . The mode of
I
@ / :H
N
H’ H‘
56 E-OH-DMAT 58 Z-OH-DMAT
CH20H
I
56 59
SCHEME 5 .
32
190 GROGER AND FLOSS
incorporation of the 2 isomer, [14CH3]-58, has never been adequately ex-
plained; it may involve initial isomerization to [14CH3]-56. In any case, the
results demonstrated that the incorporations of 56 and 58 are artefacts of
feeding compounds that are not intermediates on the normal biosyn-
thetic pathway.
Searching for a potential intermediate between DMAT and chanoclav-
ine-I, Kozikowski et af. (120) synthesized both diastereomers of the diol
60, deuterium-labeled in the N-methyl group, but found no incorporation
into elymoclavine by Claviceps strain SD58 (Scheme 6). Subsequently, the
same group (121) synthesized the monohydroxylated DMAT derivative
[N-CD3]-61 which clearly labeled elymoclavine (32) when fed to Claviceps
strain SD58. Trapping experiments, however, failed to identify 61 as a
genuine constituent in the ergot fungus but, surprisingly, instead revealed
the presence of the diene 62 and its ready formation from 61. Later, Kozi-
kowski’s group (122) succeeded in the synthesis of 62. Deuterium-labeled
62 was efficiently incorporated into elymoclavine, and its natural occurrence
in Claviceps was confirmed in a trapping experiment and by its detection
in the culture medium of strain SD58. All of the data indicate that 61 is
not on the normal biosynthetic pathway to the ergot alkaloids. Rather, it
is channeled into the pathway by, probably nonenzymatic, dehydration to
the true intermediate 62. A plausible pathway for ring C formation can now
be formulated as shown in Scheme 6: 62 is probably formed from 54 by hy-
droxylation at the benzylic position of the isoprenoid moiety to give 64, fol-
lowed by a 1,Cdehydration to 62. The diene 62 could then be epoxidized
by cytochrome P-450 to the vinyl oxirane 63 which has been proposed (113)
to undergo decarboxylative ring closure via an SN2’ process to give chano-
clavine-I. The benzylic alcohol 64 is apparently a rather labile compound.
It has not yet been found in nature, and attempts to synthesize it chemic-
ally have so far been unsuccessful. Circumstantial evidence for its likely for-
mation may, however, be seen in the isolation of the clavicipitic acids (64a),
a pair of diastereomeric shunt products of ergoline biosynthesis (122a-d).
Another feature of ergoline formation was investigated by Kobayashi
and Floss (123). They demonstrated unequivocally that the oxygen atoms of
both chanoclavine-I and elymoclavine are derived from molecular oxygen.
These findings and other results support the view that the formation of
ring C of the ergot alkaloids proceeds by a mechanism involving a potential
carbocation at the benzylic carbon (C-10, ergoline numbering) and a poten-
tial carbanion at C-a of the amino acid side chain, e.g., in a reactive species
generated from 63.
5. The N-Methylation Step
The N-methylation step must occur between DMAT (53) and chanoclav-
ine-I. Originally, methylation after ring C formation seemed attractive,
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 191
H'
63
H'
37
CHzOH &
H'
SCHEME 6.
because this would have allowed the involvement of pyridoxal5'-phosphate
catalysis in the decarboxylation/C ring closure reaction. Norchanoclavine-
I and -11 were detected in Cluviceps cultures (1249, but feeding experiments
192 GROGER AND FLOSS
with both labeled compounds gave no incorporation into tetracyclic cla-
vines. Therefore the methylation step must occur before or simultaneously
with closure of ring C. This idea was supported by the detection of N -
methyl-DMAT (54) in ergot cultures and from some preliminary feeding
experiments with 54 (225). Clear-cutevidence came from experiments with
double labeled ['5N-CD3]-54, which was efficiently incorporated into ely-
moclavine without cleavage of the bond between the nitrogen and methyl
group (226). No incorporation was observed with the corresponding trypt-
amine derivative (87). Summarizing these results we may conclude that
methylation of the amino group of 53 is the second step in ergoline biosyn-
thesis and, by implication, that the decarboxylation and C ring closure do
not involve pyridoxal phosphate catalysis (87).
6. Formation of Ring D
A number of mechanisms have been proposed for the closure of ring D
in ergoline biosynthesis. A potential candidate as an intermediate in the
conversion of 37 into 31 seemed to be paliclavine (65), an alkaloid isolated
in 1974 (227). Subsequent feeding experiments with [N-rnethyl-'4C]cha-
noclavine-I and [N-rnethyl-'4C]paliclavine showed no incorporation of 65
into paspalic acid or the tetracyclic clavines (228). These results rule out
an SN2' reaction of paliclavine as the mechanism for closure of ring D.
Instead, there is substantial evidence that chanoclavine-I is converted
into 31 via chanoclavine-I aldehyde (55). Feeding experiments revealed
that 55 is a more efficient precursor of tetracyclic ergolines than is cha-
noclavine-I. [17-3H,4-'4C]Chanoclavine-I was converted by Claviceps into
elymoclavine with complete, stereospecific loss of one of the labeled hydro-
gens, HR, from C-17 (229,230). Finally, a blocked mutant of Claviceps was
isolated which accumulates 55 (232). All this evidence leaves little doubt
that chanoclavine-I aldehyde is a true intermediate in ergoline formation
(Scheme 2).
Mechanistic aspects of the closure of ring D were investigated extensively
(222,229). Incorporation experiments with [7-'4C,9-3H]chanoclavine-I and
(4R)-[2-'4C,4-3H]mevalonate yielded labeled elymoclavine and lysergic acid
hydroxyethylamide with only 70% tritium retention. A mechanism was
proposed to account for the partial loss of tritium, which envisioned an
intermolecular recycling of the vinyl hydrogen in the tricyclic substrate.
Substantial evidence supports such a process. The most conclusive evidence
comes from double labeling experiments in which a mixture of [2-13C]- and
[4-D2]mevalonic acid was fed to ergot cultures. The appearance in the
tetracyclic alkaloids, but not in chanoclavine-I, of molecules containing
both I3C and deuterium, according to the mass spectra, demonstrates clearly
the intermolecular transfer of the hydrogen from the 9-position. The results
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 193
on the cyclization/isomerization of chanoclavine-I to agroclavine can be
summarized as follows (87):
The hydrogen at C-10 is completely retained;
the hydrogen at C-9 is partly eliminated; low rates of alkaloid produc-
tion correlate with low tritium retention;
(c) the hydrogen at C-9 seems to undergo an intermolecular transfer
during the reaction; and
(d) the original pro-R hydrogen from C-17 of chanoclavine-I is elimi-
nated.
Further experimental data and comprehensive discussions and mechanis-
tic interpretations have been presented (87,129).
B. BIOSYNTHESIS OF LYSERGIC ACID DERIVATIVES
A number of lysergic acid derivatives are pharmacologically very active
compounds which are widely used in medicine. It is therefore somewhat
surprising that our knowledge of the exact mechanism of their biosynthesis
is still fragmentary. The earlier work on this topic has been reviewed
(85-87); some of it and more recent results are discussed in the following.
1. Lysergic Acid and Its Derivatives
Agroclavine (31) and elymoclavine (32) are precursors of lysergic acid
(2). However, the exact sequence of the steps from 32 to 2 is not yet known.
Lysergene, lysergol and penniclavine are not intermediates on this pathway
(131,132). Labeled paspalic acid (4) was incorporated into lysergic acid
amides (133,134), but it is not clear if 4 is a natural intermediate because
it can isomerize spontaneously in aqueous solution very easily. Another
possibility is the isomerization of the double bond from A8q9 to A9*l0 at the
aldehyde stage. Lysergaldehyde has not yet been synthesized, but its enol
acetate, 6-methyl-8-acetoxymethylene-9-ergoline, could be prepared (135).
This compound was incorporated into lysergic acid amide alkaloids. While
this observation does not constitute proof, it is at least consistent with the
assumption that lysergaldehyde is a true intermediate. In this context it
was proposed that lysergaldehyde, rather than lysergic acid, is converted
to the CoA ester en route to the lysergic acid amide alkaloids (Scheme 7).
This speculation explains the observation that no A8q9-amide alkaloids are
found in nature and that 4, but not lysergic acid, accumulates in appropriate
Cluviceps purpurea strains (87,132). The idea is supported by experiments
(136) in which an ergotamine (5)-producing strain was grown in an atmo-
sphere of The lysergyl fragment of 5 showed the same "0 enrichment
as the cyclol oxygen, favoring a pathway involving direct formation of an
194 GROGER AND FLOSS
H'
64a
H'
65
SCHEME 7.
activated derivative of lysergic acid from an aldehyde intermediate without
further dilution of the l80 of elymoclavine. On the other hand, results
obtained by Keller's group (137,138) support the alternative idea that D-
lysergic acid is a free intermediate in the biosynthesis of ergot peptide alka-
loids.
Lysergic acid a-hydroxyethylamide (29) is a typical constituent of C h i -
ceps paspali strains. Ergonovine (27) is accumulated in sclerotia and sapro-
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 195
phytic cultures of various Cfuviceps species, although the biosynthesis of
these simple lysergic acid amides is not yet well understood. A number
of potential precursors were not incorporated into 29 (102,139), but [U-
I4C]alanine labeled the carbinolamide moiety of 29 (102). Radioactivity
from [2-14C]alanine was incorporated primarily into the carbinolamide car-
bon and I5N from ~-[U- '~C, '~N]alanine into the amide nitrogen (140,242).
Alanine was also incorporated into the L-alaninol part of ergonovine, but
feeding experiments with alaninol gave ambiguous results (242,143). Lyser-
gylalanine (66) was suggested by Agurell (102) to play a key role in the
biosynthesis of lysergic acid amides. However, lysergyl-~-[2-'~C]alanine did
not label 29 significantly and showed a small, albeit specific, incorporation
into ergonovine (27) (97.4% of radioactivity at C-2 and C-3 of the alaninol
side chain) (244,145). The low incorporation rate and the lack of proof of
formation of lysergylalanine in Cfuviceps puspuli makes it questionable
whether 66 is a normal intermediate in ergonovine biosynthesis (245)
(Scheme 8) .
H' 66
CH3
CH20H
/ CH3 OH #+\ 0. ,NH-CH\ /
& \CH3 cs;'- Ergotarmne (5) /
N
H' H'
29 27
SCHEME 8.
196 GROGER AND FLOSS
2. Biosynthesis of the Peptide Moiety of Ergot Alkaloids
Knowledge about the biosynthesis of the cyclol part of the classical ergot
alkaloids, e.g., ergotamine and ergotoxines, is rather fragmentary and it is a
particular challenge to biochemists to solve this intriguing problem. Several
reviews on this topic have been published (85,87,246, 247).
Biogenetically, the cyclol alkaloids may be viewed as modifications
of linear peptides, e.g., ergotamine: d-lysergyl-alanyl-L-phenylalany1-L-
proline, ergocornine: d-lysergyl-valyl-L-valyl-L-proline. Numerous feeding
experiments (142,148-152) revealed that lysergic acid and the cyclol-specific
amino acids, valine, leucine, phenylalanine, and proline are specifically
incorporated into the appropriate parts of the corresponding peptide alka-
loids. Also, the a-hydroxy-a-amino acid moiety is derived from the corres-
ponding a-amino acid, valine in the case of ergotoxines and alanine in the
case of ergotamine. The mechanism of this formal hydroxylation reaction
wasclarified in Floss' laboratory. A 2,3-dehydroamino acid intermediate
was ruled out by deuterium labeling experiments (253). The two other
alternatives are
( a ) dehydrogenation to the imine followed by the addition of water; or
(b) direct hydroxylation at the a-position.
It was shown that the oxygen in the cyclol ring is derived from molecular
oxygen, favoring alternative (b) (236).
Many mechanistic possibilities may be considered for the assembly of
the peptide portion of the cyclol alkaloids. Chain elongation may start from
the lysergic acid or from the proline end. Are individual amino acids added
successively, or are intermediates (di- and tripeptides) assembled and then
attached to a starter molecule? How is a linear peptide intermediate modi-
fied to give the diketopiperazine and finally the cyclol structure? What
types of enzymes are involved in this reaction sequence, and do some
steps occur spontaneously? Results answering some of these questions are
summarized below.
As proposed by Agurell(202), lysergylalanine (66) should be the precur-
sor for ergotamine and ergosine and, by analogy, lysergylvaline for the
ergotoxine alkaloids (254). However, labeled lysergylalanine and lysergyl-
valine were not incorporated intact into ergopeptines, but only after hydro-
lytic breakdown (245,252). Similarly, labeled dipeptides, diketopiperazines,
and tripeptides fed to various Claviceps strains were not incorporated intact.
For example, labeled L valyl-L-proline, L-leucyl-L-proline lactam, L-valyl-
L-proline lactam (255), ~-valyl-~-valyl-~-[U-~~C]proline (156), and L-valyl-L-
leucyl-~-[U'~C]proline (257) labeled the alkaloids regardless of whether they
had the right sequence or not. The isotope distribution in the alkaloids clearly
indicated cleavage of the precursors prior to incorporation. Furthermore, ra-
5. BIOCHEMISTRY OF ERGOT ALKALOIDS 197
dioactivity was also found in the protein fraction and in the free amino acid
pool. It was also demonstrated (257) that washed mycelia of Cluwiceps are
capable of hydrolyzing the added peptides, e.g., leucyl-proline lactam.
Although all these negative results do not prove the absence of discrete
peptide intermediates, they did lead to the suggestion (256) that peptide
chain assembly and elaboration of the cyclol structure takes place in a
concerted fashion on a multienzyme complex in analogy to peptide antibi-
otic formation (258). Chain growth could start at the C-terminal end, as
suggested by the higher specific activity in the a-hydroxyvaline moiety,
compared to the valine moiety of ergocornine (250,252). Taking ergocris-
tine synthesis as an example (Scheme 9) successive activation and transfer
reactions would form a lysergyltripeptide (67) covalently linked to an SH-
EIU-SH
L-proline, L-phenylalanine,
L-valine, d-lysergic acid, ATF’
----
EIU-SH I;’ I 0, H 3 c . C H * c H 6 p 9 1 0 ’
2 Lys-N-CH C 1 . Hydroxylation Lys-N-C’ ‘c
I I 2. CYCIOI fomaEon I I
C-N,COC==o C-N,COCao
H‘ \CH2
0”
H‘ \CHI
0”
I I
68
I
Epimerization I
c-N, / L O
C
H‘ ‘FH2
0”
Ergocristine (15) 0
Lys = d-Lysergx Acid
SCHEME 9.
198 GROGER AND FLOSS
group through the carboxyl end of proline. Release from the multienzyme
complex by internal displacement of the sulfur would lead simultaneously
to the formation of the lactam ring as in 68. Hydroxylation of the a-carbon
of the valine adjacent to lysergic acid, followed by cyclol formation are the
final steps in the synthesis of 15. Favoring this proposal is the isolation of
the D-proline analogue of 68, which might arise by the facile nonenzymatic
epimerization of the L-prolyl-L-phenylalanyl lactam 68 -+ 69 (33). The
multienzyme hypothesis is also supported by inhibitor experiments which
indicate that ergopeptine formation is a nonribosomal process (159). The
mechanism of peptide bond formation is still unknown, and no “cyclol-
synthetase complex” has been characterized from ergot fungi.
C. ENZYMOLOGY OF ERGOLINE ALKALOID FORMATION
Although tracer studies with radioactive and stable isotopes are a valu-
able tool, they are only a prelude for the further elucidation of a biosynthetic
pathway by characterization of the enzymes catalyzing individual steps in
the reaction sequence. Studies at the enzyme level are also necessary for
detailed studies of the reaction mechanisms involved. Our knowledge of
the enzymes involved in ergoline alkaloid formation is unfortunately still
very fragmentary.
1. Dimethylallyltryptophan Synthase
The first ergoline pathway-specific enzyme was detected in Claviceps
strain SD58 in 1971 by Heinstein et af . (98). The enzyme was also isolated
and partially characterized from two other alkaloid-producing Claviceps
strains (260). Later, dimethylallyl pyrophosphate: L-tryptophan dimethylal-
lyltransferase (DMAT synthase) was purified to apparent homogeneity
(161) and was described as a monomeric protein with a molecular mass of
70-73 kDa. Cress er af . (162) used the same Claviceps strain and obtained
DMAT synthase in homogenous crystalline form, showing that the enzyme
contains two similar or identical subunits of 34,000 molecular weight.
DMAT synthase was active in the absence of divalent metal ions. Ca2+ seems
to be an allosteric effector which deregulated the enzyme at concentrations
above 20 mM. Recently, Gebler and Poulter (163) purified DMAT synthase
to apparent homogeneity and came to the conclusion that this enzyme is
an a2 dimer with an Mr of 105 kDa.
The chemical mechanism of the reaction catalyzed by DMAT synthase
was clarified by Shibuya et af. (264) in extensive studies. The isoprenylation
of tryptophan catalyzed by DMAT synthase involves displacement of the
allylic pyrophosphate moiety by C-4 of the indole ring with inversion of
configuration at C-1 of dimethylallyl pyrophosphate (DMAPP). The geome-
try of the allylic double bond is retained and no scrambling of labeled
199 5. BIOCHEMISTRY OF ERGOT ALKALOIDS
hydrogens between the two methyl groups was observed. The results are
fully consistent with a mechanism for DMAT synthase involving direct
attack of DMAPP on C-4 of the indole apparently through a stabilized
allylic carbocation or ion pair as intermediate. Furthermore, the results
support earlier conclusions of Arigoni's group (212,113) that the conversion
of mevalonate into DMAT in Claviceps is not completely stereospecific,
apparently due to some stereochemical infidelity in the isopentenyl pyro-
phosphate isomerase reaction. The mechanism of the prenyl transfer reac-
tion catalyzed by DMAT synthase was also studied with analogs of both
substrates (265). The authors came to the conclusion that the prenyl transfer
reaction catalyzed by DMAT synthase is an electrophilic aromatic substitu-
tion and is mechanistically similar to the electrophilic alkylation catalyzed
by farnesyl diphosphate synthase.
2. N-Methyltransferase
The second pathway-specific step in ergoline biosynthesis is catalyzed
by S-adenosylmethionine:dimethylallyltryptophan N-methyltransferase,
which was detected in crude cell-free extracts of a clavine producing strain
(266). DMAT N-methyltransferase has a sharp pH maximum at 8.0-8.5,
and its activity is related to the age and alkaloid production of Claviceps
cultures. Using the chiral methyl group methodology (267,268) it was found
that the methylation step in ergoline formation proceeds with net inversion
of methyl group configuration. The process most likely involves a direct
migration of the methyl group of SAM to the amino nitrogen of DMAT
in a ternary enzyme complex via an SN2 transition state (269).
3. Chanoclavine-I cyclase
Three groups ( 2 70-2 74) have obtained crude enzyme preparations from
different Claviceps strains which catalyze the conversion of chanoclavine-
I (37) into agroclavine (31) and/or elymoclavine (32). The reaction required
ATP, NADH or NADPH, and Mg2'. One group (273) found that theMais conteúdos dessa disciplina
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