Syntheses and anticholinesterase activities of (3aS)-N1,N8- bisnorphenserine, (3aS)-N1,N8-bisnorphysostigmine, their antipodal isomers, and other potential metabolites of phenserine

Article abstract of DOI:10.1021/jm9800494

Hydrolysis of the carbamate side chains in phenserine [(-)1] and physostigmine [(-)2] yields the metabolite (-)-eseroline (3), and the red dye rubreserine (4) on air oxidation of the former compound. Both compounds lacked anticholinesterase activity in co

Full text of DOI:10.1021/jm9800494

J . Med. Chem. 1998, 41, 2371-2379
2371
Syn th eses a n d An tich olin ester a se Activities of (3a S)-N1,N8-Bisn or p h en ser in e,
(3a S)-N1,N8-Bisn or p h ysostigm in e, Th eir An tip od a l Isom er s, a n d Oth er P oten tia l
Meta bolites of P h en ser in e†
Qian-sheng Yu, Nigel H. Greig,* and Harold W. Holloway
Drug Design & Development, Laboratory of Cellular and Molecular Biology, Gerontology Research Center (4E02),
National Institute on Aging, Intramural Research Program, National Institutes of Health, 5600 Nathan Shock Drive,
Baltimore, Maryland 21224-6825
Arnold Brossi
School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina 27599-7361
Received J anuary 22, 1998
Hydrolysis of the carbamate side chains in phenserine [(-)1] and physostigmine [(-)2] yields
the metabolite (-)-eseroline (3), and the red dye rubreserine (4) on air oxidation of the former
compound. Both compounds lacked anticholinesterase activity in concentrations up to 30 mM,
which would be unachievable in vivo. A second group of potential metabolites of 1 and 2 are
the N1,N8-bisnorcarbamates (-)9 and (-)10, prepared from (3aS)-N8-benzylnoresermethole
(-)12 by the carbinolamine route. These entirely novel compounds proved to be highly potent
inhibitors of acetylcholinesterase [(-)9] and of acetyl- and butyrylcholinesterase (AChE and
BChE) [(-)10], respectively. To elucidate further the structure/anticholinesterase activity
relationship of the described compounds, the antipodal isomers (3aR)-N1,N8-bisnorcarbamates
(+)9 and (+)10 were likewise synthesized from (3aR)-N8-benzylnoresermethole (+)12 and
assessed. The compounds possessed moderate but less potent anticholinesterase activity, with
the same selectivity as their 3aS enantiomers. Finally, the anticholinesterase activities of
intermediates N1,N8-bisnorbenzylcarbamates (-)18, (-) 19, (+)18, and (+)19, also novel
compounds, were additionally measured. The 3aS enantiomers proved to be potent and selective
inhibitors of BChE, particularly (-)19, whereas the antipodal isomers lacked activity.
Phenserine [(-)1], the phenyl carbamate analogue of
physostigmine [(-)2], is a new and selective inhibitor
of acetylcholinesterase (AChE) with minimal butyryl-
cholinesterase (BChE) action which is about to enter
clinical trials for the treatment of Alzheimer’s disease.1,2
The compound is sufficiently long acting for once daily
administration and preferentially enters the brain, the
site of Alzheimer’s disease, reaching and maintaining
a brain/plasma ratio of 10:1.1,3 As a consequence of
these incorporated design features, the compound has
an unusually wide therapeutic window, a highly favor-
able toxicological profile in dogs and rodents, and
dramatic action in animal cognitive models.1,3 Ad-
ditionally, it modulates the molecular events involved
in the neuropathology of Alzheimer’s disease, reducing
the synthesis and secretion of â-amyloid precursor
protein, the source of the Alzheimer neurotoxic peptide
â-amyloid, both in in vitro and in vivo studies.3,4
key tricyclic compounds derived from phenserine [(-)1]-
and physostigmine [(-)2] by such action and evaluate
their anticholinesterase activities (Figure 1).
On the basis of our recent successful syntheses of the
N1-nor series7 and N8-nor series8 of both phenserine and
physostigmine, we accomplished the novel syntheses of
(3aS)-N1,N8-bisnorphenserine [(-)9] and (3aS)-N1,N8-
bisnorphysostigmine [(-)10]. As our earlier studies
proved that the 3aS enantioselectivity is clearly main-
tained in the N8-nor series8 but not in the N1-nor series,9
we additionally synthesized the (3aR)-N1,N8-bisnor
compounds (+)9 and (+)10 to assess their anticholinest-
erase activities, and report on them herein. Finally, no
report on the synthesis, and chemical and biological
evaluation of potential metabolites of phenserine and
physostigmine would be complete without the inclusion
of (-)-eseroline (3), the hydrolysis product of (-)1 and
(-)2, and the red dye rubreserine (4), which is readily
formed on air oxidation of 3. The evaluation of these
compounds is likewise reported herein and was assessed
against freshly obtained and prepared human AChE
and BChE in vitro, alongside and compared to the
activities of (-)- and (+)-phenserine (1) and physostig-
mine (2).
An attractive and pivotal point in the development
of phenserine as a therapeutic is the assessment and
synthesis of its potential metabolites and elucidation of
their biological properties. Since hydrolysis of carbamyl
esters and N-demethylation are well-established meta-
bolic pathways,5,6 we thought it prudent to prepare the
Resu lts
* To whom correspondence should be addressed. Phone: 410-558-
8278. Fax: 410-558-8323. E-mail: GreigN@vax.grc.nia.nih.gov.
† This paper is dedicated to Dr. Nelson J . Leonard, Faculty Associate
in Chemistry, California Institute of Technology, Pasadena, CA, on
the occasion of his 80th birthday.
Ch em istr y. Rubreserine (4) was obtained on air
oxidation of eseroline (3),10 instead of by its isolation
from a mixture of degradation products from physo-
S0022-2623(98)00049-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/29/1998

2372 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Yu et al.
F igu r e 1.
Sch em e 1a
This route uses the known (-)-N8-benzylnoresermet-
hole [(-)12], which was made from 11 in six steps
including its optical resolution, as has been reported.8
For introducing a benzyl group into the N1-position of
precious optically pure compound (-)12, we adopted,
step by step, a procedure reported in the literature.16
Starting with (-)12, it was quaternized with CH3I, ring-
C-opened under basic conditions, and again quaternized
with CH3I, leading via (-)13 to (-)14 and (-)15.
Reaction of (-)15 with BnNH2 directly provided N1,N8-
bisbenzylnoresermethole [(-)16] in a total of four steps
with a final yield of 74%. Demethylation of (-)16 gave
the phenol [(-)17] which, when reacted with either
phenyl isocyanate or methyl isocyanate, afforded (-)-
N1,N8-bisbenzylnorphenserine [(-)18] and (-)-N1,N8-
bisbenzylnorphysostigmine [(-)19], respectively.
a
Reagents: (a) C4H9OH, Na; (b) H2O, Na2CO3, O2, CHCl3.
stigmine [(-)2].11-14 Rubreserine (4) was initially iso-
lated and identified by Auterhoff.11 Later, Scho¨nen-
berger et al. characterized it by X-ray diffraction analy-
sis13 and, more recently, Cordell provided its H and 13C
1
NMR.14 The synthesis of compound 4 is described in
this paper (Scheme 1).
The catalytic debenzylation of (-)18 and (-)19 to give
the desired bisnor compound (-)9 and (-)10, respec-
tively, was accomplished over Pd(OH)2/C using i-PrOH
as a solvent. The (3aR)-N1,N8-bisnorphenserine [(+)9]
and (3aR)-N1,N8-bisnorphysostigmine [(+)10] were pro-
duced from (3aR)-N8-benzylnoresermethole [(+)12] in
the same manner, as shown in Scheme 2. This deben-
zylation proved unexpectedly difficult and is sum-
marized in Scheme 3. First, we tried the conditions
used in our previous preparation of the N8-nor series.8
The debenzylation of (-)18 was initially attempted in
an acidic medium of CF3COOH/CH3OH/H2O over the
catalyst Pd(OH)2/C. After 1.5 h of hydrogenation at
atmospheric pressure, only one product, the ring-open
â-2′-aminoethyl indole, compound 20, could be isolated
from the reaction mixture. This suggests that ring C
The major task in this project, which had not previ-
ously been accomplished, was the synthesis of N1,N8-
bisnorphenserine [(-)9] and its methylcarbamate ana-
logue (-)10, both obtainable from (-)1 and (-)2 by
N-demethylation. We suspected that (3aS)-N1,N8-bis-
noresermethole (not shown), obtained from 5-methoxy-
tryptamine (11),15 was not a good substrate to complete
our task as the corresponding phenol would be exquis-
itely sensitive to air oxidation and hence difficult to
handle. Furthermore, the lack of selectivity of O-H
over N-H for reaction with isocyanate would have
resulted in undesired products and hence make the
synthesis far more complex than necessary. We have
therefore developed an alternative route to prepare the
compounds of the N1,N8-bisnor series, and this is shown
in Scheme 2.

Syntheses and Activities of Metabolites of Phenserine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2373
Sch em e 2a
a
Reagents: (a) CH3I, ether; (b) EtOH, NaOH (50%); (c) CH3I, Et2O; (d) BnNH2; (e) BBr3, CH2Cl2; (f) phenyl isocyanate, Et2O, Na; (g)
methyl isocyanate, Et2O, Na; (h) Pd(OH)2/C, i-PrOH.
opening of the N1,N8-bisnor compounds formed during
hydrogenation occurs much more readily than does that
of the N8-nor compounds.
alcohol. We tried numerous solvents other than CH3OH
to avoid N1-methylation, but these proved unsuccessful.
Finally, we utilized i-PrOH or t-BuOH as the reaction
solvents, and both provided positive results (Scheme 3).
Biologica l Eva lu a tion . Table 1 illustrates the
anticholinesterase activity of compounds 3, 4, (-)- and
(+)9, 10, 18, and 19 against human AChE and BChE,
compared to those of (-)- and (+)-phenserine (1) and
physostigmine (2).
Extensive experimentation culminated in our use of
a neutral solvent, initially CH3OH, which, after 60 h of
hydrogenation, provided (-)-N8-norphenserine 7, the
only product that we were able to isolate from the
reaction mixture. This suggests that during the long
period of catalytic hydrogenation the initially formed
N1-nor compound was N-methylated, leaving the N8-
benzyl group attached to the aniline nitrogen before
finally giving the N8-nor compound 7. This result is in
accord with studies by Baiker and Richarz17 and by He
and Brossi.18 They described the alkylation of second-
ary amines on catalytic hydrogenation using a primary
alcohol as the solvent at high temperature and proposed
that the key intermediate of this reaction is the alde-
hyde formed from dehydrogenation of the primary
In accord with previous reports, neither (-)-eseroline
(30) nor (-)-rubreserine (4) possessed anticholinesterase
action. In contrast, the N1,N8-bisnorcarbamates (-)9
and (-)10 were highly potent in this regard.
(-)-N1,N8-Bisnorphenserine [(-)9] demonstrated po-
tent and selective AChE inhibitory action with an IC50
value similar to that of (-)-phenserine [(-)1]. Like (-)-
phenserine, the compound lacked BChE inhibitory
potency, and had a selectivity of AChE action of some

2374 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Yu et al.
Sch em e 3a
a
Reagents: (a) Pd(OH)2/C, H2O, CH3OH, TFA, 1.5 h; (b) Pd(OH)2/C, i-PrOH, 60 h; (c) Pd(OH)2/C, MeOH, 60 h.
Ta ble 1. 50% Inhibitory Concentration (IC50, NM) + SEMa of Compounds versus Human Erythrocyte AChE and Plasma BChE
compound
IC50 AChE (nM)
IC50 BChE (nM)
(-)1, (-)-phenserine
24.0 ( 6.0a
3500 ( 55
27.9 ( 2.4
9890 ( 6
NDAb
1300 ( 85a
23500 ( 300
16.0 ( 2.9
2490 ( 290
NDA
(+)1, (+)-phenserine
(-)2, (-)-physostigmine
(+)2, (+)-physostigmine
3, (-)-eseroline
4, (-)-rubreserine
NDA
NDA
(-)9, (-)-N1,N8-bisnorphenserine
(+)9, (+)-N1,N8-bisnorphenserine
(-)10, (-)-N1,N8-bisnorphysostigmine
(+)10, (+)-N1,N8-bisnorphysostigmine
(-)18, (-)-N1,N8-bisbenzylnorphenserine
(+)18, (+)-N1,N8-bisbenzylnorphenserine
(-)19, (-)-N1,N8-bisbenzylnorphysostigmine
(+)19, (+)-N1,N8-bisbenzylnorphysostigmine
5, (-)-N1-norphenserine
22.1 ( 2.3
231 ( 23
10.9 ( 1.2
1490 ( 120
41130 ( 11760
NDA
1090 ( 123
3560 ( 400
13.8 ( 0.7
21.0 ( 1.0
40.8 ( 3.4
56.7 ( 4.4
330 ( 100
161 ( 41
897 ( 104
952 ( 107
2.4 ( 0.8
237 ( 55
513 ( 311
NDA
8.1 ( 2.1
776 ( 312
612 ( 1
2.0 ( 1.0
516 ( 601
6.8 ( 2.2
10.4 ( 4.0
21.8 ( 10.9
6, (-)-N1-norphysostigmine
7, (-)-N8-norphenserine
8, (-)-N8-norphysostigmine
21, (-)-N8-benzylnorphysostigmine
22, (-)-N1-benzylnorphysostigmine
a
b
Standard error of the mean. NDA: no detectable activity (at 3 × 10-5 M).
40-fold. In stark contrast to (+)-phenserine [(+)1] which
lacks anticholinesterase activity, (+)-N1,N8-bisnor-
phenserine [(+)9] possessed a low but nevertheless
significant potency of AChE inhibition. The IC50 value
of this was 10-fold lower than that of (-)9 for AChE,
whereas the BChE inhibitory action of (+)and (-)9 were
similar and low.
[(+)2] which entirely lacked anticholinesterase action,
(+)-N1,N8-bisnorphenserine [(+)9] proved inactive against
AChE but possessed low BChE inhibitory action, quan-
titatively similar to that of (-)9.
The AChE inhibitory activity of the intermediates (-)-
N1,N8-bisbenzylnorphenserine [(-)18] and (-)-N1,N8-
bisbenzylnorphysostigmine [(-)19] were dramatically
reduced compared to (-)9 and (-)10, respectively.
However, their respective BChE inhibitory action was
maintained, with (-)19 being particularly potent. In
contrast, the (+)-enantiomers were dramatically less
active, with (+)-N1,N8-bisbenzylnorphenserine [(+)18]
(-)-N1,N8-Bisnorphysostigmine [(-)10] possessed a
high potency of inhibitory action against both AChE and
BChE, with activity some 3- and 6-fold greater than that
of (-)-physostigmine [(-)2] against the two enzyme
subtypes, respectively. In contrast to (+)-physostigmine

Syntheses and Activities of Metabolites of Phenserine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2375
Ta ble 2. Relative Selectivity of Compounds versus Human
Erythrocyte AChE and Plasma BChE
entirely lacking demonstrable activity in concentrations
up to 30 mM.
AChE
BChE
compound
selectivity selectivity
Discu ssion
(-)1, (-)-phenserine
54-fold
2-fold
We report, herein, the first synthesis and initial
pharmacological evaluation of both natural and un-
natural N1,N8-bisnorphenserine (9) and of N1,N8-bis-
norphysostigmine (10). Their structures and absolute
configurations are secured with the chemical reactions
described. Not only were the 3aS enantiomers of these
novel compounds highly potent as anticholinesterases,
but additionally they are potential metabolites of
phenserine [(-)9] and physostigmine [(-)10].
(-)2, (-)-physostigmine
5, (-)-N1-norphenserine
44-fold
11-fold
32-fold
13-fold
8-fold
7-fold
41-fold
5-fold
6, (-)-N1-norphysostigmine
21, (-)-N1-benzylnorphysostigmine
7, (-)-N8-norphenserine
8, (-)-N8-norphysostigmine
22, (-)-N8-benzylnorphysostigmine
(-)9, (-)-N1,N8-bisnorphenserine
(-)10, (-)-N1,N8-bisnorphysostigmine
(-)19, (-)-N1,N8-bisbenzylnorphysostigmine
134-fold
The classical anticholinesterase physostigmine [(-)2]
has been utilized widely in clinical studies over the last
several decades, and it as well as the quaternary
carbamates such as neostigmine and pyridostigmine
have demonstrated utility and acceptability in treating
a variety of disorders, primarily involving an awry
cholinergic system. These include paralytic ileus and
atony of the bladder, glaucoma, myasthenia gravis,
termination of the actions of competitive neuromuscular
blocking agents, and overcoming intoxication with
atropine, tricyclic antidepressants, or phenothiazines.
Physostigmine is available for injection (antilirium), as
an ointment, and, more recently, for experimental use
in Alzheimer’s disease as a slow-release formulation
(synapton) that is in phase III clinical trials. As a first-
generation drug, its major drawbacks in clinical use are
its low therapeutic window and short duration of
unselective action on acetyl- and butyrylcholinesterase,
with pharmacokinetic and pharmacodynamic half-lives
of 30-90 min, respectively.1
In contrast, phenserine [(-)1] and analogues over-
come these disadvantages and possess a long duration
of action (half-life 8-10 h) and an unusually wide
therapeutic window which results in dramatic cognitive
effects.1,3 Additionally, unlike physostigmine, phenserine
possesses the rare characteristic of modulating the
processing of â-amyloid precursor protein to limit for-
mation of the Alzheimer toxic peptide, â-amyloid.3,4 The
agent therefore represents a promising drug candidate
for the treatment of Alzheimer’s disease as it has actions
to potentially improve cognition and slow the disease
course, through its interaction with the molecular
events involved in the disease process, and its true value
is about to be assessed in forthcoming clinical trials.
(-)-N1,N8-Bisnorphenserine [(-)9] and (-)-N1,N8-
bisnorphysostigmine [(-)10] both possessed potent an-
ticholinesterase action and hence, if produced in vivo
as a consequence of metabolism by N-demethylation,
likely would potentiate or extend the action of (-)1 or
(-)2, respectively. Indeed, whereas (-)9 is equipotent
to (-)1 versus AChE and BChE, with a slightly more
modest AChE selectivity of 41-fold compared to 54-fold
for (-)-phenserine [(-)1], (-)-N1,N8-bisnorphysostig-
mine [(-)10] proved to be more potent than (-)-
physostigmine [(-)2]. Indeed, it was 6-fold more active
in inhibiting BChE and 3-fold more potent against
AChE, providing it a selectivity for BChE activity of
some 5-fold, versus 2-fold for (-)-physostigmine [(-)2].
Thus, similar to (-)-N1 and (-)-N8 analogues, also
potential metabolites of (-)1 and (-)2, N-demethylation
at both the N1 and N8 positions, separately and simul-
taneously, results in highly potent anticholinesterases
with a modest altered selectivity away from AChE
(Table 2).
We have previously demonstrated that replacement
of the methyl carbamate moiety of (-)-physostigmine
[(-)-2] with a substituted or unsubstituted phenylcar-
bamate,1,2,3,19 or with a longer alky carbamate, such as
with a butyl, heptyl, or octyl carbamate, incrementally
extends the duration of potent anticholinesterase action
of the resulting compound.1,20,21 However, their longer
duration of action likely results as a consequence of their
higher rates of carbamylation of the cholinesterase
enzyme and lower rates of decarbamylation,1,22,23 com-
pared to (-)-physostigmine [(-)2], rather than as a
consequence of their in vivo disappearance rates.1 For
example, as discussed, (-)-physostigmine [(-)2] has a
duration of action of some 30-90 min and an in vivo
disappearance half-life of 15 min.24 The heptyl carbam-
ate (Eptylstigmine, Mediolanum, Italy) in phase II
clinical trials for the treatment of Alzheimer’s disease,
has a duration of action of in excess of 8 h and an in
vivo disappearance half-life of 5 h.20,25 In contrast, (-)-
phenserine [(-)1], as discussed, likewise has a duration
of cholinesterase inhibition of in excess of 8 h, but an
in vivo disappearance half-life of only 12 min.1,3 It is
therefore probable that in the event that (-)-N1,N8-
bisnorphenserine [(-)9] and (-)-N1,N8-bisnorphysostig-
mine [(-)10] are generated as metabolic products of (-)-
phenserine [(-)1] and (-)-physostigmine [(-)2]
administration in clinical studies, such active metabo-
lites would have a greater affect to potentiate the action
of (-)-2 as its rate of enzyme decarbamylation is far
more rapid than for (-)-1. Although (-)-eseroline (3)
has been reported to be pharmacologically active in
antinociceptive assays,13 we reconfirm the lack of anti-
cholinesterase activity of this known metabolite of (-)-
physostigmine [(-)2] and of (-)-rubreserine (4), easily
obtained from 3 on air oxidation and synthesized via a
novel route.
We have previously demonstrated that inhibition of
both AChE and BChE by (-)1, (-)2, and other N1-
methyl-substituted carbamates is highly enantioselec-
tive, resting entirely on the 3aS enantiomers (Table 1).1,9
This, however, does not hold for the N1-nor series9 or
for ring C heterocongeners,1,2,25,26 but does hold for the
N8-nor series.8 Similar to the N1-nor series,9 (+)-N(1,8)-
bisnorphenserine [(+)9] and (+)-N(1,8)-bisnorphyso-
stigmine [(+)10] retained anticholinesterase action,
which was some 10-fold reduced compared to their 3aS
enantiomers. Nevertheless, their activity is of the same
magnitude as several anticholinesterases presently in

2376 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Yu et al.
mine [(-)19], which demonstrated a surprising potency
and selectivity for BChE. Whereas long-acting and
nontoxic selective AChE inhibitors hold potential to
augment memory processing and reduce â-amyloid
synthesis in the treatment of Alzheimer’s disease and
age-associated memory deficits,24,33 recent studies have
implicated BChE in the seeding and formation of
â-amyloid plaques and its toxicity in Alzheimer’s dis-
ease,34,35 providing a potential clinical use for interesting
selective nontoxic BChE inhibitors.
F igu r e 2.
clinical use: tacrine (Cognex, Warner-Lambert, MI) IC50
AChE 190 nM, BChE 47 nM, and galanthamine (Johnson
& J ohnson, NJ ) IC50 AChE 800 nM, BChE 7300 nM.
We predicted from our previous studies involving
modification of the N1- and N8-positions of natural
physostigmine [(-)2]1,2,8,27 that the intermediate (-)-
N(1,8)-bisbenzylnorphysostigmine [(-)19] would possess
anticholinesterase action with a selectivity for BChE,
as 21 and 22 (Figure 2) proved to be BChE inhibitors
equipotent to (-)2 but with a 32-fold and 7-fold selectiv-
ity (Tables 1 and 2). We were, however, surprised by
the remarkable potency and 134-fold BChE selectivity
of (-)19. Our previous studies have demonstrated that
the incorporation of a lipophilic group, such as a
phenylethyl group,28 in the N1-position imparts a dra-
matic selectivity that favors BChE inhibitory action. The
present studies clearly indicate that BChE inhibitory
potency and selectivity can be increased by simulta-
neous lipophilic substitution in the N1- and N8-positions;
comparing the activity of (-)19 to 21 and 22. Addition-
ally, they further suggest that BChE selectivity and,
likely, duration of inhibition could be yet further
increased by changing the methyl carbamate of (-)19
to a 4′-substituted phenyl carbamate. We have previ-
ously demonstrated that such carbamates provide an
extended duration of action, similar to that of (-)1, and
a BChE selectivity.1,2,3,27
In contrast, (-)-N(1,8)-bisbenzylnorphenserine [(-)18],
which incorporates components to both favor BChE
selectivity, in its lipophilic N(1,8)-bisbenzyl groups, and
favor AChE selectivity, in its unsubstituted phenyl
carbamate, possessed a minimal potency compared with
(-)1 and (-)19 against both AChE and BChE. Like-
wise, the enantiomers (+)18 and (+)19 dramatically
lacked activity, clearly indicating that whereas reduced
steric factors associated with N1-methyl substitution in
the nor compounds,9 physovenols,25 and thiaphys-
ovenols26 allow potent activity in the antipodal isomers,
bulky substitutions do not.
A significant body of work has been published that
describes the structures of AChE and BChE and their
catalysis of acetylcholine (ACh) and related sub-
strates.29,30,31 Both proteins have been fully sequenced,
and although they are products of different genes on
different chromosomes they share a remarkable degree
of amino acid homology, particularly around the site
involved in ACh hydrolysis.32 As we have described
previously,8 it is clearly possible to design and synthe-
size analogues with specific substitutions that exploit
critical differences around the active catalytic site that
differentiate AChE from BChE to provide cholinesterase
subtype inhibitory action for the resulting compounds.
In summary, we have synthesized and characterized
the N(1,8)-bisnor analogues of both natural, (-)-3aS,
and unnatural, (+)-3aR, physostigmine and phenserine.
The 3aS enantiomers, potential metabolites of (-)1 and
(-)2, proved to be highly active anticholinesterases, as
did the intermediate (-)-N(1,8)-bisbenzylnorphysostig-
Exp er im en ta l Section
Ch em istr y. Melting points (uncorrected) were measured
1
with a Fisher-J ohns apparatus; H NMR were recorded on a
Bruker (Bellevica, MA) AC-300 spectrometer; MS (m/z) were
recorded on a Hewlett-Packard 5890 GC-MS (EI) and on a
Finnigan-1015D mass spectrometer. Optical rotations were
measured by J ASCO, model DIP-370 (J apan, Spectroscopic
Co., Ltd.); elemental analyses were performed by Atlantic
Microlab, Inc. Unless otherwise indicated, all separations
were carried out using flash column chromatography (silica
gel 60, 230-400 mesh) with the described solvents. All
reactions involving nonaqueous solutions were performed
under an inert atmosphere.
Ru br eser in e (4). The fumarate salt of eserine (3)10 (300
mg, 0.90 mmol) was dissolved in H2O (10 mL). The solution
was mixed with sodium carbonate (95 mg, 0.90 mmol) and
CHCl3 (20 mL). The reaction mixture was stirred under
constant aeration, by filtering air flow through a small pipet
for 20 h, and CHCl3 was occasionally added to maintain a
constant volume of CHCl3. The separated CHCl3 solution was
washed with brine and dried over NaSO4. After evaporation
of solvent, the residue was chromatographed (CH2Cl2/MeOH
) 20/1) and the visual red fraction was collected. Evaporation
of solvent gave a residue which was crystallized from ether to
give 4 (44 mg, 21.0%) as red crystals: mp 150-151 °C (lit.9
mp 152 °C; lit.12 mp 98-99 °C); [R]20D -256.3° (c ) 0.2, CHCl3);
the 1H NMR is identical with that reported in the literature
(ref 14).
Meth iod id e of (-)-(3a S)-8-Ben zyl-1,3a -d im eth yl-1,2,3,
3a,8,8a- h exah ydr o-5-m eth oxypyr r olo[2,3-b]in dole [(-)13].
Compound [(-)12]8 (180 mg, 0.58 mmol) was dissolved in ether
(5 mL), and then CH3I (0.5 mL) was added. The methiodide
salt gradually formed and precipitated from the solution. The
mixture was left overnight and filtered to give crystalline
(-)13 (240 mg, 92.0%): mp 140 °C; [R]20 -109.0° (c ) 0.5,
D
EtOH). Anal. (C19H21N3O2) C, H, N.
(-)-(3s)-1-Ben zyl-3-m eth yl-3-[2′-(dim eth ylam in o)eth yl]-
5-m eth oxy-2,3-d ih yd r o-1H-in d ol-2-ol [(-)14]. To a solu-
tion of (-)13 (240 mg, 0.53 mmol) in H2O (5 mL) and EtOH (1
mL) were added 50% NaOH (0.5 mL) and ether (5 mL). After
the mixture was stirred at room temperature for 5 h, the ether
layer was separated and the aqueous layer was washed with
ether (2 × 5 mL). The combined ether layer then was washed
with brine, dried over Na2SO4, and evaporated to give (-)14
1
(182 mg, 100%) as a gum: [R]20 -21.9° (c ) 0.5, CHCl3); H
D
NMR (CDCI3) δ 7.40-7.15 (m, 5 H, Ph-H), 6.50 (m, 2H, C4-H,
C6-H), 6.25 (d, J ) 8.0 Hz, 1H, C7-H), 4.60 (s, 1H, C2-H), 4.45
and 4.15 (AB, J ) 16.6 Hz, 2H, PhCH2N), 3.60 (s, 3H, OCH3),
2.15 (s, 6H, N-Me2), 2.00-2.10 (m, 2H, CH2N), 1.90-1.40 (m,
2H, C3-CH2), 1.25 (s, 3H, C3-CH3); EI-MS m/z (relative
intensity) 322 (M+ - H2O, 51), 307 (64), 264 (4.5), 91 (100).
Anal. (C33H33N3O3‚0.3H2O) C, H, N.
Met h iod id e of (-)-(3s)-1-Ben zyl-3-m et h yl-3-[2′-(d i-
m eth yla m in o)eth yl]-5-m eth oxy-2,3-d ih yd r o-1H-in d ol-2-
ol [(-)15]. Compound (-)14 (180 mg, 0.53 mmol) was
dissolved in ether (5 mL), and then CH3I (0.5 mL) was added.
The methiodide salt gradually formed and precipitated from
solution. The mixture was left overnight and filtered to obtain
crystalline (-)15 (243 mg, 95.0%): mp 172-173 °C; [R]20
D
-391.0° (c ) 0.2, EtOH). Anal. (C19H21N3O2‚0.2H2O) C, H,
N.

Syntheses and Activities of Metabolites of Phenserine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2377
(-)-(3a S)-1,8-Diben zyl-3a -m eth yl-1,2,3,3a ,8,8a -h exa h y-
d r o-5-m et h oxyp yr r olo[2,3-b]in d ole [(-)16]. Compound
(-)15 (230 mg, 0.48 mmol) was dissolved in CH3CN (5 mL),
and benzylamine (103 mg, 0.96 mmol) was added. The
reaction mixture was stirred and refluxed for 6 h. Evaporation
of solvent gave a residue which was partitioned between H2O
and Et2O. The ether layer was evaporated and chromato-
stirred under hydrogen at atmospheric pressure and room
temperature for 60 h, and then the catalyst was filtered.
Evaporation of solvent gave a residue which was chromato-
graphed (CH2Cl2/MeOH ) 10/1) to obtain the most polar
component and this was crystallized from petroleum ether to
provide compound (-)9 (12 mg, 64.7%) as crystals: mp 153-
155 °C; [R]20D -73.7° (c ) 0.2, EtOH); 1H NMR (CDCl3/CD3OD
) 8/2) δ 7.50-7.00 (m, 5H, Ph-H), 6.90-6.50 (m, 3H, Ar-H),
4.80 (s, 1H, C8a-H), 3.10-2.75 (m, 2H, C2-H2), 2.15-1.80 (m,
2H, C3-H2), 1.45 (s, 3H, C3a-CH3); EI-MS m/z (relative
intensity) 232 (M+ - phenyl, 67), 217 (38), 190 (11), 174 (100),
160 (73); CI-MS (NH3) m/z 310 (MH+). Anal. (C27H29N3O3)
C, H, N.
graphed (CH2Cl2/MeOH ) 20/1) to give (-)16 (157 mg, 85.0%)
1
as a gum: [R]20 -72.3° (c ) 0.4, CHCl3); H NMR (CDCI3) δ
D
7.30-7.20 (m, 10 H, Ar-H), 6.65 (d, J ) 2.5 Hz, 1H, C4-H),
6.55 (d, J ) 8.5 Hz, 1H, C6-H), 6.25 (d, 1H, J ) 8.5 Hz, C7-H),
4.45 (s, 1H, C8a-H), 4.40 and 4.20 (AB, J ) 16.6 Hz, 2H,
PhCH2-N8), 3.80 (m, 2H, PhCH2-N1), 3.60 (s, 3H, OCH3), 3.70
(s, 3H, CH3O), 2.75 (t, J ) 7 Hz, 2H, C2-H2), 1.95 (t, J ) 7 Hz,
2H, C3-H2), 1.40 (s, 3H, C3a-CH3); EI-MS m/z (relative
intensity) 294 (MH+ - benzyl, 48), 279 (8.9), 188 (23), 173 (58),
91 (100). Anal. (C26H28N2O) C, H, N.
(-)-(3a S)-3a -Meth yl-1,2,3,3a ,8,8a -h exa h yd r op yr r ol[2,3-
b]in d ol-5-yl N-Meth ylca r ba m a te [(-)10]. Compound (-)19
(34.0 mg, 0.08 mmol) was dissolved in 2-propanol (1 mL), and
Pd(OH)2/C (5 mg) was added. The reaction mixture was
stirred under hydrogen at atmospheric pressure and room
temperature for 48 h, and then the catalyst was filtered.
Evaporation of solvent gave a residue which was chromato-
graphed (CH2Cl2/MeOH ) 10/1) to obtain the most polar
(-)-(3a S)-1,8-Diben zyl-3a -m eth yl-1,2,3,3a ,8,8a -h exa h y-
d r op yr r olo[2,3-b]in d ol-5-ol [(-)17]. Compound (-)16 (140
mg, 0.36 mmol) was dissolved in CH2Cl2 (2 mL), and boron
tribromide (456 mg, 1.82 mmol) was added with stirring. The
reaction mixture was stirred at room temperature for 1 h,
MeOH (1 mL) was added dropwise, and the mixture was
stirred for 10 min. After evaporation of solvent in vacuo, the
residue was dissolved in H2O (2 mL), basified with NaHCO3,
and extracted with Et2O (3 × 5 mL). The ether extract was
dried over Na2SO4 overnight, evaporated, and chromato-
component, compound (-)10 (12 mg, 60.7%), as a gum: [R]20
D
-114.3° (c ) 0.2, EtOH); 1H NMR (CDCl3/CD3OD ) 8/2) δ
6.90-6.55 (m, 3H, Ar-H), 5.18 (s, 1H, C8a-H), 2.95-2.70 (m,
2H, C2-H2), 2.85 (d, J ) 4.0 Hz, 3H, NCH3), 2.10-1.80 (m,
2H, C3-H2), 1.50 (s, 3H, C3a-CH3); EI-MS m/z (relative
intensity) 232 (M+ - methyl, 65), 217 (38), 189 (8.3), 174 (100);
CI-MS (NH3) m/z 248 (MH+). Anal. (C27H29N3O3) C, H, N.
graphed (CH2Cl2/MeOH ) 10/1) to give (-)17 (120 mg, 90.0%)
1
as a foam: [R]20 -76.5° (c ) 0.2, CHCl3); H NMR (CDCl3) δ
D
(+)9 and (+)10 were synthesized from (+)12 according to
the same procedures used for making their antipodes. The
1H NMR and MS of (+)9 and (+)10 and the intermediates
(+)14, (+)16, (+)17, (+)18, and (+)19 were identical to those
of their respective antipodes. The optical rotation values of
the plus series compounds were equal and opposite to those
of their minus enantiomers.
7.40-7.00 (m, 10 H, Ar-H), 6.70-6.00 (m, 3H, C4-H, C6-H,
C7-H), 4.40-3.90 (m, 2H, PhCH2-N8), 3.80-3.60 (m, 2H,
PhCH2-N1), 2.65 (m, 2H, C2-H2), 1.89 (m, 2H, C3-CH2), 1.30
(s, 3H, C3a-CH3); EI-MS m/z (relative intensity) 294 (MH+
phenyl, 15), 380 (34), 265 (9.3), 250 (2.8), 91 (100). Anal.
-
(C33H33N3O3‚0.9 H2O ) C, H, N.
(-)-N8-Nor p h en ser in e (7) Ma d e fr om Com p ou n d (-)18.
Compound (-)18 (29.3 mg, 0.06 mmol) was dissolved in MeOH
(1 mL), and Pd(OH)2/C (5 mg) was added. The reaction
mixture was stirred under hydrogen at atmospheric pressure
and room temperature for 60 h, and then the catalyst was
filtered. Evaporation of solvent gave a residue which was
chromatographed (CH2Cl2/MeOH ) 10/1) to obtain the most
polar component and this was crysyallized from petroleum
ether to provide compound 7 (13.6 mg, 70.0%) as crystals: The
mp, optical rotation, 1H NMR, and MS were identical with
those reported in the literature.8
(-)-(3s)-3-Met h yl-3-[2′-(a m in o)et h yl]-2,3-d ih yd r o-1H -
in d ol-5-yl N-P h en ylca r ba m a te (20). Compound (-)18 (58.6
mg, 0.12 mmol) was dissolved in a mixture of MeOH (2 mL),
H2O (2 mL), and TFA (1 mL), and Pd(OH)2/C (10 mg) was
added. The reaction mixture then was stirred under hydrogen
at atmospheric pressure and room temperature for 1.5 h, and
thereafter the catalyst was filtered. The filtrate was evapo-
rated in vacuo to give a residue which was dissolved in H2O,
basified by Na2CO3, extracted with CH2Cl2, and then dried over
Na2SO4. After removal of solvent, the residue was chro-
matograghed on preparative TLC (silica gel) (CH2Cl2/MeOH
(-)-(3a S)-1,8-Diben zyl-3a -m eth yl-1,2,3,3a ,8,8a -h exa h y-
d r op yr r ol[2,3-b]in d ol-5-yl N-P h en ylca r b a m a t e [(-)18].
Compound (-)17 (40.7 mg, 0.11 mmol) was dissolved in
anhydrous ether (2 mL), and a piece of Na metal (ap-
proximately 1 mg) was added. The mixture was stirred at
room temperature for 1 min, phenyl isocyanate (13.1 mg, 0.11
mmol) was added, and the mixture was stirred for 5 min.
Evaporation of solvent gave a crude product which was directly
chromatographed to give (-)18 (48 mg, 89.1%) as a gum: [R]20
D
-59.1° (c ) 0.7, CHCl3); 1H NMR (CDCl3) δ 7.40-6.90 (m, 15H,
Ar-H), 6.75 (d, J ) 2.5 Hz, 1H, C4-H), 6.73 (d, J ) 8.5 Hz, 1H,
C6-H), 6.17 (d, J ) 8.5 Hz, 1H, C7-H), 4.45 (s, 1H, C8a-H),
4.30-4.20 (AB, J ) 16.6 Hz, 2H, PhCH2-N8), 3.72 (s, 2H,
PhCH2-N1), 2.70 (m, 2H, C2-H2), 1.90 (m, 2H, C3-H2), 1.38 (s,
3H, C3a-CH3); EI-MS m/z (relative intensity) 370 (MH+
PhNHCO, 31), 354 (1.0), 279 (8.0), 264 (2.0), 91 (100). Anal.
(C27H29N3O3) C, H, N.
-
(-)-(3a S)-1,8-Diben zyl-3a -m eth yl-1,2,3,3a ,8,8a -h exa h y-
d r op yr r ol[2,3-b]in d ol-5-yl N-Met h ylca r b a m a t e [(-)19].
Compound (-)16 (47.5 mg, 0.13 mmol) was dissolved in
anhydrous ether (2 mL), and a piece of Na metal (ap-
proximately 1 mg) was added. The mixture was stirred at
room temperature for 1 min, methyl isocyanate (14.6 mg, 0.26
mmol) was added, and the mixture stirred for 10 min.
Evaporation of solvent gave a crude product which was directly
) 10/1) to give the most polar and also major component 20
1
(7.4 mg, 20%) as a syrup: [R]20 -63.2° (c ) 0.1, CHCl3); H
D
NMR (CDCl3/CD3OD ) 8/2) δ 7.40-7.00 (m, 5H, Ph-H), 6.85-
6.70 (m, 2H, C4-H, C6-H), 6.50 (d, J ) 8.5 Hz, 1H, C7-H), 3.75
and 3.65 (AB, J ) 12 Hz, 2H, C2-H2), 3.40-3.20 (AB, J ) 12
Hz, CH2N), 1,65 (m, 2H, C3-CH2), 1.30 (s, 3H, C3-CH3); CI-
MS (NH3) m/z 312 (MH+). Anal. (C13H16N2O3) C, H, N.
chromatographed to give (-)19 (50.0 mg, 90.0%) as a gum:
1
[R]20 -58.2° (c ) 0.7, CHCl3); H NMR (CDCl3) δ 7.40-7.20
D
(m, 10H, Ar-H), 6.75 (d, J ) 2.2 Hz, 1H, C4-H), 6.64 (d, J )
8.5 Hz, 1H, C6-H), 6.24 (d, J ) 8.5 Hz, 1H, C7-H), 4.65 (s, 1H,
N-H), 4.40 (s, 1H, C8a-H), 4.35-4.20 (AB, J ) 16.6 Hz, 2H,
PhCH2-N8), 3.70 (s, 2H, PhCH2-N1), 2.80 (d. J ) 3.9 Hz, 3H,
NHCH3), 2.70 (m, 2H, C2-H2), 1.90 (m, 2H, C3-H2), 1.35 (s,
Qu a n tita tion of An tich olin ester a se Activity. The ac-
tion of compounds 3, 4, and (-)- and (+)9, 10, 18, and 19 to
inhibit the ability of freshly prepared human AChE and BChE,
derived from plasma and whole red blood cells, respectively,
to enzymatically degrade the specific substrates acetyl(â-
methyl)thiocholine and s-butyrylthiocholine (0.5 mmol/L) (Sigma
Chemical Co., St. Louis, MO) were quantified. Compounds
were dissolved in Tween 80/EtOH, 3:1 (v:v; <150 µL total
volume), and were diluted in 0.1 M Na3PO4 buffer (pH 8.0) in
half-log concentrations to provide a final range spanning 0.3
3H, C3a-CH3); EI-MS m/z (relative intensity) 370 (MH+
CH3NHCO, 33), 354 (1.5), 279 (8.5), 264 (3.0), 91 (100). Anal.
(C27H29N3O3) C, H, N.
-
(-)-(3a S)-3a -Meth yl-1,2,3,3a ,8,8a -h exa h yd r op yr r ol[2,3-
b]in d ol-5-yl N-P h en ylca r ba m a te [(-)9]. Compound (-)18
(29.3 mg, 0.06 mmol) was dissolved in 2-propanol (1 mL), and
Pd(OH)2/C (5 mg) was added. The reaction mixture was

2378 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Yu et al.
(9) Pei, X. F.; Greig, N. H.; Bi, S.; Brossi, A.; Toome, V. Inhibition
of Human Acetylcholinesterase by Unnatural (+)-(3aR)-N1-
Norphysostigmine and Arylcarbamate Analogues. Med. Chem.
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(10) Yu, Q. S.; Scho¨nenberger, B.; Brossi, A. Reactions of (-)-
Physostigmine and (-)-N-Methylphysostigmine in Refluxing
Butanol and at High Temprature: Facile Preparation of (-)-
Eseroline. Heterocycles 1987, 26, 1271-1275.
(11) Auterhoff, H.; Hamacher, H. Die Farbreaktion des Eserines.
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nM to 30 mM. Tween 80/EtOH was diluted in excess of 1 in
5000 and had no inhibitory action on either AChE or BChE.
Freshly collected blood was centrifuged (10000g, 10 min, 4
°C), and plasma was removed and diluted 1:125 with 0.1 M
Na3PO4 buffer (pH 7.4). Whole red blood cells were washed
five times in isotonic saline, lysed in 9 volumes of 0.1 M
Na3PO4 buffer (pH 7.4) containing 0.5% Triton-X (Sigma), and
then diluted with an additional 19 volumes of buffer to a final
dilution of 1:200. Analysis of anticholinesterase activity,
utilizing a 25 µL sample of each enzyme preparation, was
undertaken at their optimal working pH, 8.0, in 0.1 M Na3PO4
buffer (0.75 mL total volume). Compounds were preincubated
with enzymes (30 min, room temperature) and then were
incubated with their respective substrates and 5,5′-dithiobis-
2-nitrobenzoic acid (25 min, 37 °C). Production of a yellow
thionitrobenzoate anion was measured by spectrophotometer
at 412 nm λ. To correct for nonspecific substrate hydrolysis,
aliquots were co-incubated under conditions of absolute en-
zyme inhibition (by the addition of 1 × 10-5 M physostigmine
[(-)-2]), and the associated alteration in absorbance subtracted
from that observed through the concentration range of each
test compound. Each was analyzed on four separate occasions
and assayed alongside (-)-phenserine [(-)-1] and (-)-physo-
stigmine [(-)-2] as control and external standards whose
activity we have previously reported.1,2,7,8,18,21
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Some Degradation Products of Physostigmine. J . Pharm. Sci.
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The enzyme activity at each concentration of test compound
was expressed as a percent of activity in the absence of
compound, transformed into a logit format (logit ) %activity/
(100 - %activity)) and then plotted as a function of its log
concentration. Inhibitory activity was calculated as an IC50
,
defined as the concentration of compound (nM) required to
inhibit 50% of enzymatic activity, and determined from a
correlation between log concentration and logit activity.
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Ackn owledgm en t. The authors are extremely grate-
ful to Noel F. Whittaker, Laboratory of Bioorganic
Chemistry, National Institute of Diabetes and Digestive
and Kidney Diseases, NIH, for undertaking the MS
analysis of the compounds reported herein and are
indebted to Dr. Amy Newman, Medicinal Chemistry,
National Institute on Drug Abuse, NIH, for use of NMR
and optical rotation equipment.
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