The Calabar Bean and Its Alkaloids

Brian Robinson

The Calabar Bean and Its

Alkaloids
From Magic, via Miracle, to Memory
Brian Robinson MSc, PhD, DSc, FRSC
Eyam, Derbyshire, United Kingdom

ISBN 978-94-024-1190-4 ISBN 978-94-024-1191-1 (eBook)

https://doi.org/10.1007/978-94-024-1191-1

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My farewell to and with fond memories of
Sandra
and
for the Efik people of Old Calabar
Quotations

Semper aliquid novi Africam adferre (Plinii (the elder), Naturalis Historia, liber VIII)

I am afraid that the day is fast approaching when pygmies and bushmen accept Visa and
American Express (Neuwinger (1996))

The system of organic chemistry is one of the greatest achievements of the human mind (Sir
Frederick Gowland Hopkins as quoted by Sir Robert Robinson (Robinson 1976))

The highest teaching can never be that of him whose chief business is to teach (Roscoe
(1874))

If you can look into the seeds of time

And say which grain will grow and which will not (William Shakespeare, Macbeth, Act
1, Scene III)

Science without religion is lame, religion without science is blind (Einstein (1941))

Nature [she] is but the name

For an effect, whose cause is God (Anon, The Buxton Diamonds; or, Grateful Ellen.
For the amusement and instruction of children. William Darton, 58, Holborn Hill, London,
1834(?), p. 16)

Study to be quiet (I. Thessalonians 4,11 in the New Testament of the Holy Bible)

vii
Dr Brian Robinson (May 1936–February 2023)

The author Brian Robinson has died aged 86, just as this book was completed. He
will be remembered as a medicinal chemist who dedicated his career to the under-
standing of synthetic pathways involved in natural product chemistry and the
biosynthetic origins of plant constituents used for medicinal purposes.
Brian was born in Hathersage, Derbyshire in May 1936. He received his formal
undergraduate and postgraduate education between 1954 and 1960 in the

ix
x Dr Brian Robinson (May 1936–February 2023)

Department of Chemistry at the University of Manchester, during which he obtained

the degrees of BSc, MSc and PhD.
He subsequently held research fellowships at the Universities of St Andrews and
Nottingham, and in 1964 obtained a lectureship in pharmacy at the University of
Manchester, where he would spend the rest of his career. In 1970 he was appointed
senior lecturer and 10 years later was promoted to Reader in Pharmacy. In 1984 he
received a DSc from the university in recognition of his research into the chemistry
of indoles and indole alkaloids. During periods spent on leave-of-absence between
1963 and 1986, he held visiting professorships at the University of Illinois, Chicago,
USA, and at the University of Sassari, Sardinia, Italy.
An active researcher, Brian supervised and mentored many Master’s and PhD
students in his lab over the years, and will be remembered by thousands of Pharmacy
students as the imposing 60 300 , smartly-dressed, warm-hearted character who intro-
duced them to the wonders of organic chemistry. Those who majored in medicinal
chemistry in their final year were treated to a masterclass in heterocyclic chemistry,
and knew that an exam essential would be understanding the pivotal [3,3]-
sigmatropic rearrangement in the Fischer indole synthesis!
Brian was a prolific author in the scientific arena. In addition to well over 100
contributions to the literature, he also has several patents to his name, and published
a number of books, in particular his seminal treatise The Fischer Indole Synthesis
(1983). He also published The History of Pharmaceutical Education in Manchester
(1986). In his retirement Brian worked on this book, which would sadly be his last.
However – like all of Brian’s books – it is a characteristic blend of meticulous
research into the science and historical context, leavened with wry observations.
Outside of work, Brian possessed a deep interest in a variety of historical topics of
both local and national importance. He was a significant authority on the Royal
Maundy, publishing two books on its history: The Royal Maundy (1977) and Silver
Pennies & Linen Towels (1992). Other books that reveal the breadth of his interests
include Birchinlee: The Workmen's Village of the Derwent Valley Water Board
(1983); Walls Across the Valley: Building of the Howden and Derwent Dams
(1993); Seven Blunders of the Peak: Some Derbyshire Legends Reassessed (Editor)
(1994); and Howden and Derwent: The Building of the Upper Dams of the Derwent
Valley Water Board (2004).
Brian will be buried in St Michael and All Angels Church at Hathersage where
both parents are buried and where plaques commemorate the passing of his brother
and sister-in-law Roger and Barbara Robinson.

Paul Robinson
James Gavin
Brian Cox
Preface and Acknowledgements

l-Physostigmine (eserine), the [apparent (however, see Sect. 8.2)] major alkaloidal
component of the dried ripe seeds of Physostigma venenosum (Calabar beans), has
been for me a generous and stimulating companion throughout my professional
scientific life. It appears by both name and structure on the first page of my first
published work (Robinson 1958) and as the primary subject matter of what might be
my ultimate scientific research paper (Robinson 2002).
Following my tenure, from October 1960 to September 1961, as a Wellcome
Foundation postdoctoral research fellow in the Department of Pharmacology and
Therapeutics at the University of St Andrews – during which the project was
conceived – a further postdoctoral research fellowship, from October 1961 to
January 1964 at the University of Nottingham, afforded me the opportunity to direct
one of my main research endeavours toward an investigation of other alkaloids, the
isolation of which from Calabar beans – despite their total alkaloidal content being
quite low (circa 0.15–0.3%)1 – had already been reported. However, because of the
small quantities of material that had been available, published data on these was
usually confined to melting point and either empirical or molecular formulae (Henry
1949; Sumpter and Miller 1954a), but it had been observed that one of them, namely,
physovenine, “produces a powerful myotic effect on the pupil of the eye”
(Salway 1911).
To obviate the expense, if not the impossibility, of my acquisition of large
quantities of Calabar beans, Burroughs Wellcome and Co generously afforded me
a plentiful supply of the mixed base salicylate residues that remained after their then
commercial isolation of l-physostigmine (as its salicylate) from this botanical source.
From these residues, I liberated the corresponding mixture of the free bases which,
upon partial crystallisation followed by column chromatography, led to an organic
chemical Elysium – with crystals, crystals everywhere! Thus, one of the most
exciting and formative few hours (Robinson 1964a) of my fifty or so years of
scientific research ensued. At this juncture were laid the foundations of what was
to become an extremely fertile area of study, which I subsequently developed either
by myself or under my supervision and direction of some of the members of the

xi
xii Preface and Acknowledgements

extremely able research group that I ultimately assembled and had the privilege and
pleasure to lead within the Victoria University of Manchester.
The results from these investigations were not only published but were also
presented by me in seminars at various venues throughout Europe, North America
and the UK. However, an apposite and, for me, emotive venue for such an exposition
was the University of Calabar in Nigeria where, from 10th to 13th April 1988, on
what was to be my final (as yet?) of several visits over a period of some twenty-five
years to tropical equatorial West Africa, and at the invitation of the West African
Society for Pharmacology (Societe Ouest-Africaine de Pharmacologie), I made
presentations – to their 17th Annual Scientific Conference concerning “Alkaloids
in Medicine” – summarising the results from my by then effected research on the
chemistry and pharmacology of the alkaloids of the Calabar bean (Robinson 1988).
During this stay, I also visited the nearby tropical rain forest to inspect a massive
specimen of the P. venenosum vine that had been located – at the request of the
University of Calabar – by nearby villagers. This was, indeed, a rare opportunity
since it would appear that “In Calabar nowadays it is difficult to find anyone willing
to show the Physostigma plant to an interested party” (Neuwinger 1996), a circum-
stance that had been previously exemplified when a visitor to Calabar in 1963 “had a
good deal of difficulty finding anyone willing to show him the plant from which the
bean is obtained” (Holmstedt 1972).
The mystique surrounding the Calabar bean and its botanical parent had been
earlier manifest by the statements (Barger 1936) that “Before the seeds became an
object of commerce they were regarded by the natives with some mystery and were
only reluctantly parted with to Europeans” and that “In Old Calabar it was moreover
customary to destroy all plants whenever found, except a few which were reserved
for judicial purposes”.
Unlike many medicinally useful alkaloids which either are or were derived from
plants that either still are or were used in folkloric medical practice, l-physostig-
mine’s botanical source – the Calabar bean – was used to effect folkloric ritualistic
executions. Indeed, as noted some years ago (Neuwinger 1996), “Physostigma
venenosum seeds belong to one of the most notorious trial by ordeal poisons in
West Africa, although the area of use is relatively small. It is found mainly in
southeast Nigeria (Calabar Province, the Ibibio, Efik, Ekoi and Aro tribes) and
neighbouring Cameroon (Ossidinge District, the Ekoi, Keaka and Boki tribes)”.2
Moreover, it has been claimed (Neuwinger 1996) that “Although one expects a
medicinal plant to be as little toxic as possible, experience shows that poisonous
plants are the most important source of medicines in Africa” and that “Physostigma
is a prime example of the transformation of an extensively-used deadly African
poison into a beneficial healing medicine and was an important tool in experimental
pharmacology”.
In point of fact, if early investigation of the Calabar bean and its use in the ghastly
poison ordeal (Macinnis 2004) by the Efiks of Old Calabar (Chapter 1) had not been
undertaken, it may consequently have retarded our understanding of some of the
fundamental mechanisms occurring in physiology, pharmacology and biochemistry.
Undoubtedly, the bean that for centuries was responsible for bringing so much evil
Preface and Acknowledgements xiii

and death to the Efiks has, through its major alkaloidal component, l-physostigmine,
played a fundamental role in the elucidation of the mechanism of neurohumoral
transmission at the molecular level (Sect. 10.3); together with curare and atropine,
has been the focus of pioneering investigations into pharmacological antagonism
[(Henry 1949; Holmstedt 1972; Pal 1900) and (Sects. 10.5 and 10.13) (see footnote
17 of Chap. 1), respectively]; and has also provided a useful treatment – either actual
or potential (by providing a template and thereby acting as a “lead compound”) – for
a variety of neurological disorders associated with irregularities in cholinergic
transmission in which augmentation of cholinergic activity has proved to be bene-
ficial. This has thereby fulfilled the predictions that “We are entitled to infer that the
important physiological actions of the kernel of Physostigma may be employed with
the greatest advantage in the treatment of disease” (Fraser 1863) and that “The ordeal
bean will prove a most valuable addition to the Pharmacopoeia” (Harley 1863). In
regard to its above-mentioned use as a pharmacological “tool”, it has been asserted
(Flippen-Anderson et al. 2002) that “physostigmine was the first active agent to be
isolated as a natural product and used in medicine”. Moreover, it has been the
genesis – over the past century or thereabouts – of a sustained international research
effort, the progress of which can be traced through a series of reviews (Barger 1936;
Bentley 1957; Brossi 1990, 1992; Brossi et al. 1996; Cordell 1981; Coxworth 1965;
Dalziel 1948; Henry 1924, 1949; Holmstedt 1972; Hutchinson and Dalziel 1958;
Irvine 1961; Karczmar 1970; Longmore and Robinson 1973; Marion 1952; Muhtadi
and El-Hawary 1989; Neuwinger 1996; Robinson 1963a, 1964b, 1968, 1971, 1983,
1988, 2002; Saxton 1960; Schneck et al. 1989; Silver 1974b; Sumpter and Miller
1954a; Takano and Ogasawara 1989; Taylor 1966; Triggle et al. 1998; Witkop 1998;
Zhao et al. 2004), which was effected without resort to polemics until just over two
decades ago (Brossi 1989; Pomponi 1989). However, at this juncture, the situation
was superbly defused by the conclusion that “It is hoped that this work will
ultimately provide improved drugs for the treatment of geriatric disorders and will
continue in many places. After all, patients do not really care whether a drug is
developed in Switzerland, Italy or America” (Brossi 1989) – or in the UK?
The story of the Calabar bean and its alkaloidal components composes a classical
scientific journey throughout some one-and-a-half centuries. Travelling from anthro-
pological, ethnobotanical and ethnopharmacological fieldwork, via a considerable
input of innovative organic chemistry and synthesis  including structural elucida-
tion involving the early classical approach of degradation into products of known
structures with subsequent retrosynthetic interpretation  to state-of-the-art studies
in structural elucidation and in experimental therapeutics, the voyage has reached its
current destination where, for example, l-physostigmine is acting as a “lead com-
pound” to afford products that are showing promise (Brossi et al. 1996; Giacobini
2000b; Greig et al. 1995a, 2005a; Iversen et al. 1991; Klein 2007; Marta et al. 1988;
Muñoz-Ruiz et al. 2005; Pomponi et al. 1990, 1992; Thal 1991; Yu et al. 2003) for
the treatment of that most prevalent form of dementia, namely, of the Alzheimer’s
type (Alzheimer’s disease) (Sect. 10.7.2), and have applications (Aarsland et al.
2004) in Parkinson’s disease and dementia with Lewy bodies. It thus offers to the
scientific and medical researcher or historian a fascinating tale which not only
xiv Preface and Acknowledgements

thereby endorses a previous claim (Pomponi et al. 1992) that “The early pioneering
investigations on the biochemical and pharmacological action of Phy represent a
fascinating piece of medical history” but which is, moreover, still ongoing at the
forefront of chemical and medical discovery. As one who has participated in and
contributed to this journey for about five decades, it is now my privilege to have this
opportunity to present its history (so far, although apparently to the contrary, since l-
physostigmine can be considered as the prototype of the insecticidal carbamates
(Sect. 10.8) and consequent upon “the curious information (Heyndrickx 1960) that
modern pesticides have been used by African witch doctors3 as test poisons” it has
been concluded that “The circle of history of the Calabar bean can thereby be
considered as closed” (Holmstedt 1972)).
Finally, I offer my most sincere gratitude to the staff at the John Rylands
University Library of Manchester – especially the young ladies of its Document
Supply Unit and the staff of its Information Technology Service Desks – and to John
Blunden-Ellis, the faculty team librarian for chemistry in the Joule Library at the
University of Manchester, who have all for so long and so willingly put at my
disposal their considerable expertise and facilities; to the now late Drs Arnold Brossi
and Bernhard Witkop, both of the National Institutes of Health in Bethesda, Mary-
land, USA, for much stimulating help and encouragement through correspondence;
to Miss Panichakorn Jaiyong and Dr Waleed A Zalloum for their expertise in
effecting the data input relating to the chemical structures; and to Mrs Sandra
Baldwin who was – as she also has with many of my other published works  so
skilfully undertaking the long, daunting and arduous task of typing my manuscript
and producing it in the formats requested by my publisher but with whom, because
of her sudden and untimely death, I shall be unable to share in the ultimate
publication of this treatise.
Consequent upon Sandra’s grievous passing, I offer my appreciation and whole-
hearted thanks to Miss Angela Dermody who, after being introduced to me by Mrs
Helen Kreissl, has by her skill and hard work completed the monumental task of
preparing the data input from my manuscript.
Last but, of course, not least, I am mindful of and most sincerely grateful for the
help given to me by my publisher, Springer, and in particular that of Drs Jacco
Flipsen and Meran Owen and Mrs Ineke Ravesloot for their encouragement and
guidance of my study toward its target and for looking after me and my book so well.
This became increasingly difficult with the onset of the pandemic, at which
juncture Miss Angela Dermody applied her initiative by making available for
publication our book’s manuscript, which she had very carefully checked subse-
quent to my leaving it during my immediate pre-Christmas visit to meet her.
Consequently, we were joined by my nephew Paul, to whose late mother and father
I had already dedicated footnote 6 of Chap. 10 and to which Paul was to contribute
several facets of the crypt-church of St Mary in Lastingham and furthermore to make
possible Sects. 8.2 and 8.3 of Chap. 8. It was a pleasure to welcome him to our
“team,” to which he has made such a significant contribution.
Special thanks to Professor Brian Cox and Dr. James Gavin whose specialist
knowledge ensured that this book made it to publication.
Preface and Acknowledgements xv

Notes

1. From investigations at the Wellcome Chemical Research Laboratories in London, it was noted
(Salway 1911, 1912a) that “A representative sample of the Calabar beans . . ., when assayed by
the method of the United States Pharmacopoeia, yielded 0.091 per cent of alkaloid” (Salway
1911), but that “The method referred to, however, was found to give results which were much
too low, since the amount of physostigmine isolated when working on the large scale, . . ., was
equivalent to 0.179 per cent of the material employed” (Salway 1911). Indeed, the total
alkaloidal content in the Calabar bean was later (Mutadi and El-Hawary 1989) found to vary
between 0.15% and 0.3% and it has also been noted that “The low result by the above method of
assay has been ascertained to be due to the fact that three extractions with ether (as required by
the Pharmacopoeia) are quite insufficient to remove the alkaloid completely from a solution
which has been rendered alkaline with sodium hydrogen carbonate” (Salway 1911). It was found
that the alkaloid is more easily extracted from liquids rendered alkaline with sodium carbonate
than when sodium hydrogen carbonate is used (Salway 1912a) and the following assay process
was therefore suggested:-
Twenty grams of the drug finely powdered are agitated with 200 c.c. of ether,
10 c.c. of 10% aqueous sodium carbonate solution added, and the mixture well shaken
at intervals during four hours. One hundred c.c. of the clear ethereal solution are run
into a separator, and N/10-sulphuric acid added until the liquid is acid. The separator
is well shaken, the acid layer drawn off, and the treatment repeated twice, using
10 c.c. of N/10-acid each time. The acid liquids are combined, made alkaline with
10% sodium carbonate solution, and shaken with ten successive portions (20 c.c. each
time) of ether. The combined ethereal extracts are washed with 5 c.c. of distilled
water. The solvent is then distilled off, the residue dissolved in 5 c.c. N/10-sulphuric
acid, and the excess of acid titrated with N/50-alkali, using iodeosin as indicator
(Salway 1912a).
2. Like the Efiks, their Nigerian neighbours to their west, the Ibo (Igbo) have adopted the
Physostigma ordeal (Neuwinger 1996) and it has also been reported (Neuwinger 1996) that
“trial by ordeal with Physostigma appears to be known in the south of the CAR [Central African
Republic] too, from the Cameroon border to the Ubangui River”.
3. “The term ‘witch-doctor’ has been elaborated” (Watt 1956) as being “loosely used with a vague
connotation, indicating a type of practitioner who is not really regarded as being a doctor but at
the same time is credited with having occult powers. In fact this appellation is a misnomer for
there are distinct groups (a) the medicine-man, or herbalist (b) the diviner and (c) the magician.
The medicine-man or herbalist is usually a male, hereditary doctor and a dispenser of medicines
which are characterised by physical effects, although many of his medicines are ashed and thus
rendered inactive. The diviner, on the other hand, is often a woman and operates on a
background which assumes that all ills, misfortunes and deaths are due to some active external
agency. Although plants, parts of animals and minerals are used by the diviner, he in addition
always uses some aid to divination, most commonly the divination dice, “bones” or “delosse”.
These are often made of bone but sometimes wood. In addition, the divining bowl is used by the
Venda and by the Yao of Nyasaland. The function of the magician is to produce rain, to protect
against lightening, hail, flood and other natural calamities, to protect against witchcraft and to
vend charms to ward off any evil, including illness”.

Eyam, Derbyshire, UK Brian Robinson

September 2015
Chronology

1846 First European account of the use of the WF Daniell

Calabar bean in a native judicial ordeal
procedure
1855 Investigation of the toxicology of the Cal- R Christison
abar bean in the human (by self-
experimentation). Its use suggested for
humanely effecting capital punishment
1861 Physostigma venenosum botanically JH Balfour
described
1863 Investigation of the physiological actions TR Fraser
and therapeutical uses of the Calabar bean
1863 Potential clinical use of the Calabar bean TR Fraser
(miosis, antagonised by atropine) A von Graefe
recognised DMCLA Robertson
1863–1864 Reports of accidental poisoning with the TR Fraser
Calabar bean J Cameron and JH Evans
1864 Isolation of l-physostigmine (in an amor- J Jobst and O Hesse
phous state)
1865 l-Physostigmine crystallised and named A Vée
eserin [eserine]
1876 l-Physostigmine used to treat glaucoma by L Laqueur
lowering intraocular pressure A Weber (see Sect. 10.5)
1878 Use of the Calabar bean in the adminis-
tration of trial by ordeal made illegal
1893 Isolation of l-eseramine A Ehrenberg
1893 Chemical investigation of l-physostigmine A Ehrenberg
begins A Petit and M Polonovsky [sic]
1904 Chemical transmission in the autonomic TR Elliott
nervous system conceived
1906 Steroids isolated from the Calabar bean A Windaus and A Hauth
(continued)

xvii
xviii Chronology

1911 Isolation of l-physovenine. Non-alkaloidal AH Salway

components of the Calabar bean
investigated
1915 Isolation of l-geneserine M Polonovski and C Nitzberg
1921–1935 Discovery of Ach as neurohumoral HH Dale et al.
transmitter O Loewi et al.
(Dale and Loewi jointly awarded the
Nobel Prize in Medicine and Physi-
ology in 1936)
1925 Structure for l-physostigmine proposed G Barger, R Robinson and E Stedman
M Polonovski and
M Polonovski
1925 Structure for l-geneserine proposed M Polonovski and M Polonovski
1926 Recognition of l-physostigmine as an O Loewi and E Navratil
inhibitor of AchE W Feldberg (see Sect. 10.3)
H Fühner (in 1918) (see Sect. 10.3)
1934 l-Physostigmine employed in the treat- M Walker
ment of Myasthenia gravis (the “miracle”
at St Alfege’s)
1935 l-Physostigmine synthesised PL Julian and J Pikl
1938 AchE isolated and purified D Nachmansohn and E Lederer
1946 l-Physostigmine found to afford prophy- GB Koelle
lactic protection against intoxication by R Koster
organophosphates (including “nerve
gases”)
1952 Pest control with carbamate insecticides H Gysin et al.
1958 Isolation of l-N(8)-norphysostigmine J Maier
1960 Inhibition of AchE by transcarbamylation IB Wilson, MA Hatch and S
Ginsburg
1964 Structure of l-physovenine elucidated B Robinson
1964 Structure of l-eseramine elucidated B Robinson and G Spiteller
1964–1966 Structure of l-N(8)-norphysostigmine RB Longmore and B Robinson
elucidated G Spiteller and M Spiteller-
Friedmann
1965 dl-Physovenine synthesised RB Longmore and B Robinson
1965 dl-Eseramine synthesised B Robinson
1966 l-Physovenine synthesised RB Longmore and B Robinson
1968 Anti-AchE activities of the alkaloids of the B Robinson and JB Robinson
Calabar bean investigated in vitro. Pro-
posal of a new pharmacophore for l-
physostigmine
1969 Structure of l-geneserine revised C Hootelé
1969 Absolute configuration of l-physostigmine RK Hill and GR Newkome
established RB Longmore and B Robinson
(continued)
Chronology xix

1969 Absolute configurations of l-physovenine, RB Longmore and B Robinson

l-eseramine,
l-N(8)-norphysostigmine and l-geneserine
established
1970 Anti-AchE activities of d-physostigmine FJ Dale and B Robinson
and d-physovenine investigated in vitro
1970 Stereochemistry of l-geneserine verified FG Riddell et al.
B Robinson and D Moorcroft
1978 Antinociceptive activity of A and R Bartolini and A Galli et al.
l-eseroline
1983 Treatment of paraplegic anejaculation with P-A Chapelle et al.
l-physostigmine
1984–1998 Antiamnesic activity of l-physostigmine A Brossi et al.
and analogues in dementia of the EJ Glamkowski et al.
Alzheimer’s type M Pomponi et al.
1986 dl-Geneserine synthesised K Fukumoto et al.
1987–1989 Antinociceptive activity of:- (i) l-7- EJ Glamkowski et al.
Bromoeseroline (ii) 6-Hydroxyechibolines B Robinson et al.
1988 d-Physostigmine found to afford prophy- EX Albuquerque et al.
lactic protection against intoxication by
organophosphates (including “nerve
gases”)
1988 l-Eseramine synthesised A Brossi et al.
1989 Structure of l-geneserine further revised A Brossi et al.
1990 l-N(8)-Norphysostigmine synthesised S Takano et al.
1996 Cytotoxic activity of degradation products GA Cordell et al.
of l-physostigmine
1996–2006 Phenserine, in animal models of Greig et al.
Alzheimer’s disease, found to reduce the Haroutunian
production of β-amyloid precursor protein, Shaw et al.
the source of the disease’s toxin β-amyloid
Utsuki et al.
Yu et al.
2005 Phenserine subjected to clinical trial for
the treatment of dementia of the
Alzheimer’s type
Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2 l-Physostigmine (Eserine) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.1 Natural Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2 Structure Elucidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.3 Syntheses of:- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.3.1 By Early Approaches, Including the First to be
Successful . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.3.2 l-Physostigmine and the 3a–alkyl-1,2,3,3a,8,8a–
hexahydropyrrolo[2,3-b]Indole Ring System . . . . . . . 48
2.3.3 d-Physostigmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.4 Absolute Configuration, Together with that of the Other
Structurally-Established Alkaloids of the Calabar Bean . . . . . . 63
2.5 Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
2.6 Ultraviolet Absorption Spectrum and Reaction in an Acidic
Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2.7 Mass Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.8 Detection, Assay and Instability . . . . . . . . . . . . . . . . . . . . . . . 82
2.8.1 Qualitative and Quantitative Analysis . . . . . . . . . . . . 82
2.8.2 Rubreserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.8.3 Eserine Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.8.4 Eserine Brown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
3 l-Physovenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
3.1 Isolation and Structure Elucidation . . . . . . . . . . . . . . . . . . . . . 123
3.2 Synthesis of the . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.2.1 Racemate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.2.2 l- and d-Enantiomers . . . . . . . . . . . . . . . . . . . . . . . . 131
3.2.3 3a-alkyl-3,3a,8,8a-tetrahydro-2H-furo[2,3-b]indole
Ring System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
3.3 Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

xxi
xxii Contents

4 l-Eseramine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
4.1 Isolation and Structure Elucidation . . . . . . . . . . . . . . . . . . . . . 141
4.2 Synthesis of the Racemate and the l-enantiomer . . . . . . . . . . . 142
4.2.1 Racemate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4.2.2 l-enantiomer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
5 l-N(8)-Norphysostigmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
5.1 Isolation and Structure Elucidation . . . . . . . . . . . . . . . . . . . . . 145
5.2 Synthesis of the l-enantiomer . . . . . . . . . . . . . . . . . . . . . . . . . 147
6 l-Geneserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
6.1 Isolation and Structure Elucidation . . . . . . . . . . . . . . . . . . . . . 149
6.2 Synthesis of the l-enantiomer and the Racemate . . . . . . . . . . . 154
1
7 H-, 13C- and 15N–Nuclear Magnetic Resonance Spectra
of the Alkaloids of the Calabar Bean . . . . . . . . . . . . . . . . . . . . . . . . 155
8 Other Alkaloids That Have Been Isolated, or Allegedly So,
from the Calabar Bean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
8.1 Calabarine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
8.2 Eseridine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
8.3 Isophysostigmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
8.4 Calabatine and Calabacine . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
8.5 Investigations Still to Be Effected . . . . . . . . . . . . . . . . . . . . . . 163
9 Non–Alkaloidal Components of the Calabar Bean . . . . . . . . . . . . . 165
10 Biological Activities of the Alkaloids of the Calabar Bean . . . . . . . . 169
10.1 AntiAchE Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
10.1.1 AchE – Its Function in Neurohumoral Transmission,
Structure and Inhibition . . . . . . . . . . . . . . . . . . . . . . 169
10.2 Pharmacology of l-physostigmine . . . . . . . . . . . . . . . . . . . . . . 180
10.3 Role of l-physostigmine in the Discovery of the Mechanism
of Neurohumoral Transmission . . . . . . . . . . . . . . . . . . . . . . . 181
10.4 AntiAchE Activities of the Minor Alkaloids of the Calabar
Bean – The l-physostigmine Pharmacophore . . . . . . . . . . . . . . 186
10.5 Clinical use of l-physostigmine in Ophthalmology – Miotic
Activity and the Reduction of Intraocular Pressure
in Glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
10.6 Use of l-physostigmine in the Treatment of: - . . . . . . . . . . . . . 194
10.6.1 Myasthenia Gravis . . . . . . . . . . . . . . . . . . . . . . . . . . 194
10.6.2 Paraplegic Anejaculation . . . . . . . . . . . . . . . . . . . . . . 196
Contents xxiii

10.7 Use of l-physostigmine in the:- . . . . . . . . . . . . . . . . . . . . . . . . 198

10.7.1 Prophylactic Protection Against Intoxication by
Organophosphates (Including “Nerve Gases”) . . . . . . 198
10.7.2 Enhancement of Cognition and Memory (Including
Antiamnesic Activity in Dementia of the Alzheimer’s
Type – Alzheimer’s Disease) . . . . . . . . . . . . . . . . . . . 199
10.8 Bactericidal and Insecticidal Activities of l-Physostigmine . . . . 204
10.9 Prophylactic Protection with d-physostigmine
Against Intoxication by Organophosphates
(Including “Nerve Gases”) . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
10.10 Other Clinical Uses of l-physostigmine . . . . . . . . . . . . . . . . . . 207
10.11 Antinociceptive Activity of l-eseroline and Its Synthetic
Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
10.12 Cytotoxicities of Rubreserine and another Structurally-Related
Degradation Product of l-Physostigmine . . . . . . . . . . . . . . . . . 213
10.13 Poisonings (Either Accidental or Malicious) with the Calabar
Bean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Abbreviations

Å Angstrom unit(s)
Ach Acetylcholine
AchE Acetylcholinesterase
Bch Butyrylcholine
BchE Butyrylcholinesterase
bp Boiling point
cd Circular dichroism
chE Cholinesterase
cm.(cm) Centimetres
cps Cycles per second (in nmr)
(d) Doublet (in nmr)
DAT Dementia of the Alzheimer’s type
DMSO Dimethyl sulphoxide
ee Enantiomeric excess
Et Ethyl
ft. Feet
g Gram(s)
h Hour(s)
in. Inches
ir Infrared
J Coupling constant (in nmr)
kg Kilogram(s)
m Minute(s)
(m)L Multiplet (in nmr)
M Molecular ion (in mass spectrometry)
m/e Mass to charge ratio (in mass spectrometry)
Me Methyl
mg Milligram(s)
mm. Millimetres
mm Millimetre(s) (of mercury re bp)

xxv
xxvi Abbreviations

mμ Millimicron(s) (¼ nm and nm)

μ Microns
mp Melting point
nm Nanometre(s)
nmr Nuclear magnetic resonance
nPr Normal propyl
nOe Nuclear Overhauser effect
ord Optical rotatory dispersion
Ph Phenyl
ppm Parts per million (in nmr)
(s) Singlet (in nmr)
(t) Triplet (in nmr)
tBu Tertiary butyl
uv Ultraviolet