Synthesis of novel phenserine-based-selective inhibitors of butyrylcholinesterase for Alzheimer's disease

Article abstract of DOI:10.1021/jm980459s

Four novel analogues (8-11) of cymserine (2) were synthesized by methods similar to those recently developed for the total syntheses of N⁸- norphenserine (Yu, Q. S.; et al. J. Med. Chem. 1997, 40, 2895-2901) and N¹,N⁸-bisn

Full text of DOI:10.1021/jm980459s

J . Med. Chem. 1999, 42, 1855-1861
1855
Syn th esis of Novel P h en ser in e-Ba sed -Selective In h ibitor s of
Bu tyr ylch olin ester a se for Alzh eim er ’s Disea se†
Qian-sheng Yu,‡ Harold W. Holloway,‡ Tadanobu Utsuki,‡ Arnold Brossi,§ and Nigel H. Greig*,‡
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, and School of Pharmacy, University of North Carolina at Chapel Hill,
North Carolina 27599-7361
Received August 7, 1998
Four novel analogues (8-11) of cymserine (2) were synthesized by methods similar to those
recently developed for the total syntheses of N8-norphenserine (Yu, Q. S.; et al. J . Med. Chem.
1997, 40, 2895-2901) and N1,N8-bisnorphenserine (Yu, Q. S.; et al. J . Med. Chem. 1998, 41,
2371-2379). As our structure-activity studies predicted, these compounds are highly potent
and selective inhibitors of human butyrylcholinesterase (BChE) and will test the novel
hypothesis that BChE inhibitors are useful in the treatment of Alzheimer’s disease. In a similar
manner, the same modifications that provided BChE selectivity were applied to the acetyl-
cholinesterase (AChE)-selective inhibitor, tolserine (5), to provide the novel tolserine analogues
12-15. As predicted, these modifications altered the AChE-selective action of tolserine (5) to
favor a lack of cholinesterase enzyme subtype selectivity.
In tr od u ction
tors of BChE, except in the agricultural industry where
toxic irreversible BChE inhibitors have long been used
Alzheimer’s disease (AD) is the most common pro-
gressive dementia associated with aging, with â-amyloid
plaques, neurofibrillary tangles, and synaptic loss being
the major neuropathological hallmarks of the disease.1
The cholinergic system is the earliest and most pro-
foundly affected neurotransmitter system in AD, with
substantial losses in the forebrain, cortex, and hippoc-
ampus.2 This neurotransmitter together with these
brain regions are critical in the acquisition, processing,
and storage of memories and have supported the use of
cholinomimetics in the treatment of AD.3,4 Thus far, the
agents that have demonstrated the greatest activity in
AD therapy are cholinesterase inhibitors.3 Two forms
of cholinesterase coexist ubiquitously throughout the
body, acetylcholinesterase (AChE; EC 3.1.1.7) and bu-
tyrylcholinesterase (BChE; EC 3.1.1.8), and although
highly homologous, >65%, they are products of different
genes on chromosomes 7 and 3 in humans, respectively.5
Both subtype unselective cholinesterase and AChE-
selective inhibitors have been used in AD to amplify the
action of acetylcholine (ACh) at remaining cholinergic
synapses within the AD brain,3,4 and this has promoted
the synthesis and development of novel inhibitors of
AChE with favorable characteristics for in vivo use by
the pharmaceutical industry.3 The role of BChE in
normal, aging, and diseased brain remains largely
unknown, and there has been minimal interest in the
design, synthesis, and development of selective inhibi-
as insecticides.5
Interestingly, recent research has shown that over-
expression of BChE occurs in neuritic Aâ plaques in the
AD brain6 and, furthermore, that the presence of BChE
with Aâ dramatically amplifies the toxicity of Aâ in
vitro.7 Additionally, BChE is now known to be elevated
in the AD brain, and a specific mutation in the gene
encoding BChE, producing a functioning K variant form
of BChE, together with APOE 4 results in an increase
in the susceptibility of sporadic AD by 30-fold.8 These
facts suggest that inappropriate BChE activity increases
the risk and/or progression of AD. It is hence possible
that well-tolerated inhibitors of BChE may have utility
in the treatment of AD.
To test this hypothesis, we initiated studies to design
and synthesize novel, potent, and highly selective
reversible inhibitors of BChE to provide a candidate for
therapeutic development. Our prior quantitative struc-
ture-activity studies identified that specific 4′-substitu-
tions in the phenylcarbamates of eseroline,9 N1-norese-
roline,9 and C-ring heterocongeners10-12 provided the
resulting compounds with a selectivity that favored
BChE versus AChE. In this regard, the unsubstituted
phenylcarbamate of eseroline, phenserine (1) (Chart 1),
is a potent and 65-fold selective inhibitor of AChE
versus BChE, whereas cymserine (2) has a 15-fold
selectivity for BChE.9,11,12 In contrast, 2′-substitutions
in the phenylcarbamates of eseroline and derivatives
provide compounds such as tolserine (5) that favor
AChE-selective inhibitory activity.9,11,12 In light of our
former studies which demonstrated that alterations in
the N1 position of physostigmine and carbamates alter
the relative selectivity of the resulting compounds for
AChE and BChE,13 we modified the N1 and N8 positions
of cymserine (2), both separately and together, to
provide analogues with improved BChE selectivity. We,
† This paper is dedicated to Dr. Arnold Brossi, Scientist Emeritus
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, and Department of Chemistry, School
of Pharmacy, University of North Carolina, on the occasion of his 75th
birthday and with whom we look forward to collaborating for many
years more.
* To whom correspondence should be addressed. Phone: 410-558-
8278. Fax: 410-558-8323. E-mail: GreigN@vax.grc.nia.nih.gov.
‡ NIH.
§ University of North Carolina at Chapel Hill.
10.1021/jm980459s CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/04/1999

1856 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Ch a r t 1. Chemical Structures of Compounds 1-7
Brief Articles
additionally, undertook parallel syntheses to apply
successful modifications to provide BChE selectivity to
the AChE-selective inhibitor tolserine (5) as a negative
control to assess whether such modifications would
reduce ensuing AChE inhibitory action, as would be
predicted.
We synthesized cymserine (2), tolserine (5),9,13 and
their analogues N1-norcymserine (3),13 N1-phenethyl-
norcymserine (4),13 N1-nortolserine (6),13 N1-pheneth-
ylnoryolserine (7),13 and we reconfirmed their AChE and
BChE inhibitory action. Additionally, we undertook
total syntheses of the novel cymserine analogues
N8-norcymserine (9) and N1,N8-bisnorcymserine (11)
and the novel tolserine analogues N8-nortolserine (13)
and N1,N8-bisnortolserine (15). Their potency and se-
lectivity, together with those of their intermediates,
N8-benzylnorcymserine (8), N1,N8-bisbenzylnorcym-
serine (10), N8-benzylnortolserine (12), and N1,N8-
bisbenzylnortolserine (14), are reported herein.
Biologica l Eva lu a tion . Table 1 illustrates the an-
ticholinesterase activities of compounds 8-15 and of the
previously synthesized compounds 3, 4, 6, and 7 against
human AChE and BChE, compared to those of phen-
serine (1), cymserine (2), and tolserine (5).
In accord with our previous reports,9,11,12,14,16 phen-
serine (1) and tolserine (5) possessed potent anticho-
linesterase action with a high selectivity for AChE. In
contrast, cymserine (2) possessed potent and selective
BChE inhibitory action. This BChE selectivity was
heightened in N1-norcymserine (3) and dramatically
further heightened by lipophilic N1 substitution, as in
N1-phenethylnorcymserine (4)13 (as compared to cym-
serine (2), p < 0.05). Interestingly, compounds 3 and 4
were 6- and 9-fold more potent as BChE inhibitors than
was cymserine (2). Substitutions in the N8 position alone
resulted in compounds 8 and 9. These, likewise, pos-
sessed a greater selectivity for BChE than did cymserine
(2), but this occurred as a consequence of their reduced
AChE inhibitory activity (p < 0.05 versus 2) as their
potency for BChE inhibition was in the same order of
magnitude as that of cymserine (2). Of greatest interest,
combined N1,N8 substitution conferred a high BChE
selectivity to compounds 10 and 11, with the activity of
the latter being 50-fold more potent as a BChE inhibitor
than cymserine (2).
Resu lts
Ch em istr y. The important starting material, opti-
cally pure (3aS)-N8-benzylnoresermethole (17), was
made from commercially available 5-methoxytryptamine
(16) by five steps and with a final chemical resolution
which was described in our previous publication (Scheme
1).14 Demethylation of compound 17 gave N8-benzyl-
noreseroline (18) by treatment with BBr3 in CH2Cl2.
Reaction of either 4-isopropylphenyl isocyanate or o-tolyl
isocyanate with compound 18 afforded N8-benzylnor-
cymserine (8) or N8-benzylnortolserine (12), respec-
tively, according to the known procedure.15 N-Debenz-
ylation of compounds 8 and 12 was accomplished by
catalytic hydrogenation in an acidic medium using Pd-
(OH)2/C as a catalyst to give N8-norcymserine (9) and
N8-nortolserine (13),14 respectively. Thereafter, the N1-
benzylnoresermethole (17) was transformed to N1,N8-
bisbenzylnoresermethole (19) by the carbinolamine
route, as described in our recent publication.16 N1,N8-
Bisbenzylnoreseroline (20), the demethyl product of
compound 19, then was reacted with either 4-isopropyl
isocyanate or o-tolyl isocyanate to give N1,N8-bisben-
zylnorcymserine (10) and N1,N8-bisbenzylnortolserine
(14), respectively. Finally, catalytic debenzylation of
compounds 10 and 14 in a neutral medium (2-propanol)
gave the desired products N1,N8-bisnorcymserine (11)
and N1,N8-bisnortolserine (15), respectively.
In contrast to analogues of cymserine (2), replacement
of a methyl by a hydrogen in the N1 position of tolserine
(5) resulted in compound 6 that maintained potent
AChE inhibitory action which was slightly reduced
compared to that of 5 (p < 0.05). Lipophilic N1 substitu-
tion, however, rendered compound 7 largely inactive
against both AChE and BChE. Similarly, replacement
of a methyl by a hydrogen in the N8 position of tolserine
(5) resulted in compound 13 that maintained potent
AChE inhibitory action but whose activity and hence
AChE selectivity were reduced compared to that of 5
(p < 0.05). Lipophilic N8 substitution, rendering com-
pound 12, further reduced potency against AChE to
provide a moderately potent but unselective cholinest-
erase inhibitor. Combined N1,N8 substitution, as in 14
and 15, further reduced AChE inhibitory potency com-
pared to that of their monosubstituted analogues (6, 13,
7, 12), to provide 15 with moderate anticholinesterase
activity devoid of enzyme subtype selectivity.

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1857
Sch em e 1a
a
Reagents: (a) BBr3, CH2Cl2; (b) 4-isopropylphenyl isocyanate, Na, Et2O; (c) o-tolyl isocyanate, Na, Et2O; (d) Pd(OH)2/C, H2O, CH3OH,
TFA; (e) Pd(OH)2/C, i-PrOH.
Ta ble 1. IC50 Values (nM) of Compounds versus Human Erythrocyte AChE and Plasma BChEa
IC50 (nm) ( SEM
selectivityb
no.
compound
phenserine
AChE
BChE
AChE
65
BChE
1
2
3
4
8
24 ( 6.0
1560 ( 45
50 ( 1.0
cymserine
758 ( 21
324 ( 4.0
>30000
15
39
>5000
152
26
N1-norcymserine
8.3 ( 1.0
6.0 ( 1.0
85 ( 11
N1-phenethylnorcymserine
N8-benzylnorcymserine
N8-norcymserine
13000 ( 1600
2020 ( 284
>30000
110 ( 15
10.3 ( 1.6
17.0 ( 1.0
550 ( 55
100 ( 3
9
78 ( 17
10
11
5
6
7
12
13
14
15
N1,N8-bisbenzylnorcymserine
N1,N8-bisnorcymserine
tolserine
132 ( 8.0
1.0 ( 0.1
1950 ( 240
2165 ( 85
1790 ( 180
600 ( 205
1430 ( 310
641 ( 175
396 ( 100
>227
110
189
127
3
6
36
N1-nortolserine
N1-phenethylnortolserine
N8-benzylnortolserine
N8-nortolserine
40 ( 15
1760 ( 126
285 ( 21
N1,N8-bisbenzylnortolserine
N1,N8-bisnortolserine
3
1
a
b
Minimum of four measurements per compound. Because a smaller IC50 represents a higher activity, the selectivity is defined as:
selectivity for AChE ) IC50(BChE)/IC50(AChE), and selectivity for BChE ) IC50(AChE)/IC50(BChE).
Discu ssion
(11, 15). As predicted, the described novel analogues of
cymserine (2), and in particular 10 and 11, proved to
be highly potent and selective inhibitors of BChE.
Indeed, they were dramatically more potent than the
known highly toxic irreversible BChE inhibitor, Iso-
OMPA (IC50: BChE, 980 nM; AChE, 340 000 nM; BChE
We report, herein, the first synthesis and initial
pharmacological evaluation of N8-benzylnorcymserine
and -tolserine (8, 12), of N8-norcymserine and -tolserine
(9, 13), of N1,N8-bisbenzylnorcymserine and -tolserine
(10, 14), and of N1,N8-bisnorcymserine and -tolserine

1858 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Brief Articles
selectivity 346), a widely used standard for selective
BChE inhibition.5,17 The same analogues (14, 15) of our
potent and AChE-selective inhibitor, tolserine (5), ren-
dered the compound unselective and dramatically less
potent, reiterating the selectivity of these chemical
manipulations for differential BChE activity.
ditionally, lipophilic groups present at positions N1 and
N8 of the compounds would interact with hydrophobic
regions reported close to the choline subsite of BChE,
which are not present within AChE.5,18-22 The potent
biological activity of compounds 2, 4, 8, and 10 suggests
that these predictions may have been correct, and we
are planning to undertake X-ray crystallography studies
to confirm them.
Extensive biochemical analyses5,18-22 have demon-
strated that distinct regions within AChE and BChE,
close to the bottom of a narrow 20 Å deep gorge that is
invaginated into the surface of the enzymes, constitute
binding domains for ACh and inhibitory ligands and
provide the structural basis for specificity differences
between the two enzyme subtypes. Three primary
domains exist. These include (i) an acyl pocket that
defines an active center involved in the catalysis of ACh
and substrates through an active serine residue. This
residue, similar to serine proteases, causes substrate
hydrolysis through electron transfer in a catalytic triad.
(ii) An active center choline subsite exists that is
involved in the attraction and binding of the choline
moiety of choline esters. (iii) A peripheral anionic site
also exists which is uninvolved with ACh hydrolysis and
lies closer to the entrance of the gorge.23 Biochemical
and structural data confirm that choline ester catalysis
involves a nucleophilic attack on the substrate carbonyl
by the activated active site serine (Ser203 and Ser198 in
mammalian AChE and BChE and Ser200 in torpedo
AChE, respectively).5,18-22 In the proposed catalytic
triad, the serine hydroxyl group is rendered nucleophilic
via a charge relay involving the glutamate hydroxyl of
close-by Glu334 (mammalian AChE) and the histidine
imidazole of His447. The deprotonation of serine allows
the rapid subsequent attack on the substrate, causing
substrate hydrolysis and subsequent release of the
choline moiety in the case of ACh and choline esters.
The resulting acetyl enzyme is highly labile to hydroly-
sis and rapidly reactivates.
Of particular interest, and unpredicted from our prior
studies of inducing BChE selectivity by providing lipo-
philicity at the N1 and N8 positions,13 was the dramatic
potency of N1,N8-bisnorcymserine (11) to inhibit BChE.
Compared to cymserine (2), 11 was 50-fold more potent
against BChE and only 7-fold more potent against
AChE, providing a BChE selectivity of 110-fold versus
15-fold for 2. Changing the N1 and N8 methyl groups
within 2 to a H within 11 would increase the basic
nature of the N1 and N8 nitrogen groups,24 both sepa-
rately as in 3 and 9 and combined as in 11. This would
also increase the hydrogen-bonding potential of these
compounds, which may provide improved attraction to
the proposed active center choline binding site. This
modification would also reduce the lipophilicity of 3, 9,
and 11 compared to 2 but nevertheless render them
sufficiently lipid-soluble for blood-brain barrier pen-
etration.25 The mechanism by which this minor modi-
fication so dramatically alters the differential selectivity
to favor BChE inhibitory action remains to be eluci-
dated. It is possible, however, that conformational
changes induced by drug-enzyme interactions,26 which
have not been defined in X-ray crystallographic analyses
of the static enzyme, may account for the unpredicted
selective potency of 11 for BChE and may additionally
account for the inactivity of other compounds27 that
were designed to optimally fit the enzyme.
Preliminary studies, which will be published sepa-
rately, demonstrate that compounds 2, 4, and 11,
similar to our experimental AD therapeutic phenserine
(1), are well-tolerated in vivo, enter the brain, and
improve cognitive performance in rodents. Of significant
interest, however, is that whereas phenserine (1) pro-
duces cognitive improvement by selectively inhibiting
AChE, the enzyme that degrades ACh, 2, and particu-
larly 4 and 11 appear to do so through BChE inhibitory
action alone. Compared to tacrine (IC50 values: AChE,
190 nM; BChE, 47 nM; 4-fold BChE selectivity),11,17 one
of the only two drugs approved for the treatment of AD
in the United States, compounds 4 and 11 are highly
potent and selective. Recent studies have reported that
subtype-selective cholinesterase inhibitors are not as-
sociated with the unfavorable side effect profile of
unselective ones.28 The present studies have thus
provided us with biologically active and interesting
compounds whose further pharmacological assessment
will allow us to select a candidate for development to
define the role of BChE in (i) the brain in development,
health, and aging and (ii) the value of its inhibition in
AD as a novel therapeutic strategy. These compounds
represent the first available potent, reversible, and
selective inhibitors of BChE to undertake this task.
Drugs that have a carbamoyl ester linkage, such as
physostigmine and phenserine (1), act as alternative
substrates and bind to the active centers in a similar
orientation as ACh and choline esters. Nucleophilic
attack by the active site serine, however, gives rise to a
carbamoylated enzyme, with the carbamoyl moiety
residing within the boundary of the acyl pocket. The
carbamylated enzyme is far more stable than is the
acetylated form, precluding further enzyme-catalyzed
hydrolysis of ACh for a time that is dependent on the
carbamate’s structure.23
The limit of the acyl pocket and base of the gorge
within AChE is delineated by two phenylalanines
(Phe295 and Phe297), which are replaced by valine and
leucine within BChE. Our structure-activity studies
predicted that the 4′ substitution in the phenylcarbam-
ate moiety of cymserine (2) and analogues (3, 4, 8-11)
would fit into the larger acyl pocket present in BChE,
created by virtue of the smaller side chains associated
with valine and leucine compared to phenylalanine,
close to its active serine (Ser198) binding site. This larger
pocket allows the binding of the synthetic substrate
butyrylcholine in BChE but not in AChE5 and thus
provides a means to design agents to differentially
inhibit BChE. We predicted that not only would the 4′-
isopropyl substitution in the phenylcarbamate of 2-4
and 8-11 fit into this extended pocket but that, ad-
Exp er im en ta l Section
Ch em ist r y. Gen er a l. Melting points (uncorrected) were
measured with a Fisher-J ohns apparatus; 1H NMR were
recorded on a Bruker (Billerica, MASS) AC-300 spectrometer;

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1859
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 ASO, model DIP-370 (J apan,
Spectroscopic Co., Ltd.).
compound 8 (14 mg, 51.0%) as a gum: [R]20 -71.1° (c ) 0.3,
D
CHCl3); 1H NMR (CDCl3) δ 7.29 (d, J ) 8.5 Hz, 2H, C2′-H and
C6′-H), 7.10 (d, J ) 8.5 Hz, 2H, C3′-H and C5′-H), 6.80 (m,
2H, C4-H and C6-H), 6.55 (d, J ) 8.5 Hz, C7-H), 5.20 (s,
1H, C8a-H), 2.90 (m, 1H, Ph-CH<), 2.80 (m, 2H, C2-H2), 2.13
(m, 2H, C3-H2), 1.45 (s, 3H, C3a-CH3), 1.18 (d, J ) 7.0 Hz,
>CMe2); EI-MS m/z (relative intensity) 190 (MH+ - ArNHCO,
98), 174 (10), 160 (70), 146 (100), 133 (11), 117 (15), 103 (5.0),
91 (14); HR-MS m/z calcd for C21H25N3O2 351.1948, found
351.1941.
(-)-(3a S)-8-Ben zyl-1,3a -d im 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-yl N-4′-Isop r op ylp h en ylca r ba m -
ate (8). (-)-(3aS)-8-Benzyl-1,3a-dimethyl-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indol-5-ol (18) (33 mg, 0.112 mmol), made
from compound 17,14 was dissolved in ether (2 mL), and a piece
of Na metal (approximately 1 mg) was added. The mixture
was stirred at room temperature for 1 min; then 4-isopropy-
lphenyl isocyanate (18.1 mg, 0.112 mmol) was added. The
mixture was stirred at room temperature for 5 min. After the
removal of solvent, the residue was chromatographed (CH2-
Cl2/MeOH ) 20/1) to give 8 (40 mg, 80.0%) as a foam: [R]D
-60.0° (c ) 0.2, CHCl3); 1H NMR (CDCl3) δ 7.40-7.08 (m, 9H,
Ar-H), 6.80 (d, J ) 2.2 Hz, 1H, C4-H), 6.70 (dd, J ) 2.2, 8.5
Hz, 1H, C6-H), 6.15 (d, J ) 8.5 Hz, 1H, C7-H), 4.45 and 4.35
(AB, J ) 16.6 Hz, 2H, Ph-CH2), 4.25 (s, 1H, C8a-H), 2.80 (m,
1H, Ph-CH<), 2.68 (m, 2H, C2-H2), 2.32 (s, 3H, N1-CH3),
1.90 (m, 2H, C3-H2), 1.35 (s, 3H, C3a-CH3), 1.15 (d, J ) 7.0
(-)-(3a S)-8-Ben zyl-1,3a -d im 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-yl N-2′-Meth ylp h en ylca r ba m a te
(12). (-)-(3aS)-8-Benzyl-1,3a-dimethyl-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indol-5-ol (18) (36 mg, 0.122 mmol), made
from compound 17,14 was dissolved in ether (2 mL), and Na (1
mg) was added. The mixture was stirred at room temperature
for 1 min; then 2-methylphenyl isocyanate (16.3 mg, 0.122
mmol) was added. The mixture was stirred at room temper-
ature for 5 min. After the removal of solvent, the residue was
chromatographed (CH2Cl2/MeOH ) 20/1) to give 12 (33 mg,
63.4%) as a foam: [R]D -59.7° (c ) 0.3, CHCl3); 1H NMR
(CDCl3) δ 7.30-6.90 (m, 9H, Ar-H), 6.80 (d, J ) 2.2 Hz, 1H,
C4-H), 6.78 (dd, J ) 2.2, 8.5 Hz, 1H, C6-H), 6.15(d, J ) 8.5
Hz, 1H, C7-H), 4.45 and 4.35 (AB, J ) 16.6 Hz, 2H, Ph-CH2),
4.28 (s, 1H, C8a-H), 2.68 (m, 2H, C2-H2), 2.35 (s, 3H, N1-
CH3), 2.25 (s, CH3, Ph-CH3), 1.90 (m, 2H, C3-H2), 1.37 (s, 3H,
C3a-CH3); EI-MS m/z (relative intensity) 294 (MH+ - ArN-
HCO, 90), 280 (1.0), 264 (2.0), 250 (20), 203 (9.2), 160 (33), 91
Hz, 6H, >CMe2); EI-MS m/z (relative intensity) 294 (MH+
-
ArNHCO, 65), 280 (2.2), 265 (3.6), 237 (75), 207 (58), 160 (34),
91 (100); HR-MS m/z calcd for C29H33N3O2 455.2573, found
455.2569.
(-)-(3aS)-1,3a-Dim eth yl-1,2,3,3a,8,8a-h exah ydr opyr r olo-
[2,3-b]in dol-5-yl N-4′-Isopr opylph en ylcar bam ate (9). Com-
pound 8 (22 mg, 0.048 mmol) was dissolved in a mixture of
MeOH (1 mL), H2O (1 mL), and TFA (0.5 mL). Palladium
hydroxide on carbon (5 mg) was added. The reaction mixture
was stirred under hydrogen at atmospheric pressure and room
temperature for 1 h, and then the catalyst was filtered. The
filtrate was evaporated in vacuo to provide a residue which
was dissolved in H2O, basified by Na2CO3, extracted with
ether, and then dried over Na2SO4. After removal of solvent
the residue was chromatographed on preparative TLC (silica
(100); HR-MS m/z calcd for
C27H29N3O2 427.2262, found
427.2258.
(-)-(3aS)-1,3a-Dim eth yl-1,2,3,3a,8,8a-h exah ydr opyr r olo-
[2,3-b]in d ol-5-yl N-2′-Meth ylp h en ylca r ba m a te (13). Com-
pound 12 (27 mg, 0.062 mmol) was dissolved in a solution
containing MeOH (1 mL), H2O (1 mL), and TFA (0.5 mL), and
then palladium hydroxide on carbon (4 mg) was added. The
reaction mixture was stirred under hydrogen at atmospheric
pressure and room temperature for 1 h, and then the catalyst
was filtered. The filtrate was evaporated to give a residue
which was dissolved in H2O, basified by Na2CO3, and extracted
by ether. The ether solution then was dried over Na2SO4. After
the removal of solvent, the residue was chromatographed on
preparative TLC (silica gel) (CH2CI2/MeOH ) 10/1) to give
gel) (CH2CI2 ) 10/1) to give product 6 (12 mg, 65.7%) as a
1
gum: [R]20 -73.8° (c ) 0.2, CHCl3); H NMR (CDCl3) δ 7.30
D
(d, J ) 8.5 Hz, 2H, C2′-H and C6′-H), 7.10 (d, J ) 8.5 Hz, 2H,
C3′-H and C5′-H), 6.80-6.70 (m, 2H, C4-H and C6-H), 6.50
(d, J ) 8.5 Hz, C7-H), 4.65 (s, 1H, C8a-H), 2. 85 (m, 2H,
C2-H2), 2.64 (m, 1H, -HC<), 2.48 (s, 3H, N1-CH3), 2.00-
1.90 (m, 2H, C3-H2), 1.42 (s, 3H, C3a-CH3), 1.20 (d, J ) 7.0
product 13 (13 mg, 60.1%) as a gum: [R]20 -75.5° (c ) 0.2,
D
Hz, 6H, >CMe2); EI-MS m/z (relative intensity) 204 (MH+
-
CHCl3) EtOH); 1H NMR (CDCl3) δ 7.20-6.90 (m, 4H, Ar-H),
6.70 (m, 2H, C4-H and C6-H), 6.50 (d, J ) 8.5 Hz, 1H, C7-
H), 4.65 (s, 1H, C8a-H), 2.90-2.55 (m, 2H, C2-H2), 2.48 (m,
3H, N1-CH3), 2.35 (s, 3H, Ph-CH3), 2.05 (m, 2H, C3-H2), 1.45
ArNHCO, 99), 189 (25), 174 (8.3), 117 (10); HR-MS m/z calcd
for C22H27N3O2 365.2105, found 365.2100.
(-)-(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-yl N-4′-Isop r op ylp h en ylca r ba m -
ate (10). (-)-(3aS)-1,8-Dibenzyl-3a-methyl-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indol-5-ol (20) (68 mg, 0.18 mmol), made from
compound 19,16 was dissolved in anhydrous ether (2 mL), and
a piece of Na metal (approximately 1 mg) was added. The
mixture was stirred at room temperature for 1 min; then
4-isopropylphenyl isocyanate (30 mg, 0.18 mmol) was added
and stirred for 5 min. Evaporation of solvent gave a crude
product which was directly chromatographed to give 10 (89
mg, 93.1%) as a gum: [R]20D -44.7° (c ) 0.5, CHCl3); 1H NMR
(CDCl3) δ 7.50-7.10 (m, 14H, Ar-H), 6.78 (d, J ) 2.2 Hz, 1H,
C4-H), 6.70 (dd, J ) 2.5, 8.5 Hz, 1H, C6-H), 6.15 (d, J ) 8.5
Hz, 1H, C7-H), 4.48 (s, 1H, C8a-H), 4.30-4.15 (AB, J ) 16.6
Hz, 2H, Ph-CH2-N8), 3.73 (s, 2H, Ph-CH2-N1), 2.80 (m, 1H,
-HC<), 2.70 (m, 2H, C2-H2), 1.90 (m, 2H, C3-H2), 1.40 (s,
3H, C3a-CH3), 1.15 (d, J ) 7.0 Hz, 6H, >CMe2); EI-MS m/z
(relative intensity) 370 (MH+ - ArNHCO-, 1.0), 294 (90), 279
(10), 237 (8.0), 174 (95), 160 (92), 132 (60), 104 (55), 91 (100);
HR-MS m/z calcd for C35H37N3O2 531.2888, found 531.2907.
(-)-(3a S)-3a -Met h yl-1,2,3,3a ,8,8a -h exa h yd r op yr r olo-
[2,3-b]in dol-5-yl N-4′-Isopr opylph en ylcar bam ate (11). Com-
pound 10 (42 mg, 0.078 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 60 h; then the catalyst was filtered. Evapora-
tion of solvent gave a residue which was chromatographed
(CH2Cl2/MeOH ) 10/1) to give as the most polar component
(s, 3H, C3a-CH3); EI-MS m/z (relative intensity) 204 (MH+
-
ArNHCO, 95), 189 (25), 174 (8.0), 160 (100), 146 (87), 131 (8.0),
117 (9.0); HR-MS m/z calcd for C20H23N3O2 337.1792, found
337.1789.
(-)-(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-yl N-2′-Meth ylp h en ylca r ba m a te
(14). (-)-(3aS)-1,8-Dibenzyl-3a-methyl-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indol-5-ol (20) (64 mg, 0.17 mmol), made from
compound 19,16 was dissolved in anhydrous ether (2 mL), and
a piece of Na metal (approximately 1 mg) was added. The
mixture was stirred at room temperature for 1 min, and then
o-tolyl isocyanate (23 mg, 0.17 mmol) was added and stirred
for 5 min. Evaporation of solvent gave a crude product which
was directly chromatographed to give 14 (60 mg, 70.0%) as a
gum: [R]20D -47.9° (c ) 0.2, CHCl3); 1H NMR (CDCl3) δ 7.30-
6.90 (m, 14H, Ar-H), 6.83-6.65 (m, 2H, C4-H and C6-H),
6.23 (d, J ) 8.5 Hz, 1H, C7-H), 4.58 (s, 1H, C8a-H), 4.35-
4.25 (AB, J ) 16.6 Hz, 2H, Ph-CH2-N8), 3.85 (s, 2H, Ph-CH2-
N1), 2.80 (m, 2H, C2-H2), 2.30 (s, 3H, Ph-CH3), 2.05 (m, 2H,
C3-H2), 1.40 (s, 3H, C3a-CH3); EI-MS m/z (relative intensity)
370 (MH+ - ArNHCO-, 31), 294 (80), 280 (30), 237 (8.0), 207
(90), 174 (82), 160 (81), 132 (50), 104 (30), 91 (100); HR-MS
m/z calcd for C33H37N3O2 503.2566, found 503.2568.
(-)-(3a S)-3a -Met h yl-1,2,3,3a ,8,8a -h exa h yd r op yr r olo-
[2,3-b]in d ol-5-yl N-2′-Meth ylp h en ylca r ba m a te (15). Com-
pound 14 (28 mg, 0.05 mmol) was dissolved in 2-propanol (1
mL), and Pd(OH)2/C (5 mg) was added. The reaction mixture

1860 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Brief Articles
(2) Terry, R. D.; Masliah, E.; Salmon, D. P. Physical Basis of
Cognitive Alterations in Alzheimer’s Disease: Synapse Loss is
the Major Correlate of Cognitive Impairment. Ann. Neurol. 1991,
30, 572-580.
(3) Giacobini, E. Cholinesterase Inhibitors do More than Inhibit
Cholinesterase. In Alzheimer’s Disease: Molecular Biology to
Therapy; Becker, R., Giacobini, E., Eds.; Birkha¨user: Boston,
1997; pp 188-204.
(4) Becker, R. E.; Moriearty, P.; Unni, L.; Vicari, S. Cholinesterase
Inhibitors as Therapy in Alzheimer’s Disease: Benefit to Risk
Considerations in Clinical Application. In Alzheimer’s Disease:
from Molecular Biology to Therapy; Becker, R., Giacobini, E.,
Eds.; Birkha¨user: Boston, 1997; pp 257-266.
(5) Soreq, H.; Zakut, H. Human Cholinesterases and Anticholinest-
erases; Academic Press: New York, 1993.
(6) Guillozet, A. L.; Smiley, J . F.; Mash, D. C.; Mesulam, M. M.
Butyrylcholinesterase in the Life Cycle of Amyloid Plaques. Ann.
Neurol. 1997, 42, 909-918.
(7) Barber, K. L.; Mesulam, M. M.; Kraft, G. A.; Klein, W. L.
Butyrylcholinesterase Alters the Aggregation of Aâ Amyloid.
Proc. Soc. Neurosci. 1996, 22, 1172.
(8) Lehman, D. J .; J ohnston, C.; Smith, A. D. Synergy Between
Genes for Butyrylcholinesterase K Variant and Apolipoprotein
E4 in Late-Onset Confirmed Alzheimer’s Disease. Hum. Mol.
Genet. 1997, 6, 1933-1936.
was stirred under hydrogen at atmospheric pressure and room
temperature for 60 h; then the catalyst was filtered. Evapora-
tion of solvent gave a residue which was chromatographed
(CH2Cl2/MeOH ) 10/1) to give, as the most polar component,
compound 15 (8 mg, 50.0%) as a gum: [R]20 -78.5° (c ) 0.2,
D
1
CHCl3); H NMR (CD3OD) δ 7.70-6.85 (m, 7H, Ar-H), 4.15
(s, 1H, C8a-H), 2.70-2.50 (m, 2H, C2-H2), 2.30 (s, 3H, Ph-
CH3), 1.65 (m, 2H, C3-H2), 1.30 (s, 3H, C3a-CH3); EI-MS m/z
(relative intensity) 232 (M+ - tolyl, 100), 217 (70), 188 (94),
174 (92), 160 (38), 133 (10), 91 (13); HR-MS (NH3) m/z calcd
for C19H21N3O2 323.1635, found 323.1630.
P h a r m a cology. Qu a n tita tion of An tich olin ester a se
Activity. The action of compounds 1-15 to inhibit the ability
of freshly prepared human AChE and BChE, derived from
plasma and erythrocytes, respectively, to enzymatically de-
grade the specific substrates acetyl-(â-methyl)thiocholine and
s-butyrylthiocholine (each 0.5 mmol/L) (Sigma Chemical Co.,
St. Louis, MO) was 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 concentra-
tions to provide a final concentration range that spanned from
0.3 nM to 30 mM. Tween 80/EtOH was diluted to in excess of
1 in 5000 and possessed no inhibitory action on either AChE
or BChE.
(9) Brzostowska, M.; He, X. S.; Greig, N. H.; Brossi, A. Selective
Inhibition of Acetyl- and Butyrylcholinesterases by Phenylcar-
bamates of (-)-Eseroline, (-)-(N1)-Noreseroline and Physovenol.
Med. Chem. Res. 1992, 2, 238-246.
Freshly collected blood was centrifuged (10000g, 10 min, 4
°C), and the plasma was removed and diluted 1:125 with 0.1
M Na3PO4 buffer (pH 7.4). Erythrocytes 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 Na3-
PO4 buffer (0.75 mL total volume). Compounds were preincu-
bated 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 coincubated under conditions of absolute enzyme
inhibition (by the addition of 1 × 10-5 M physostigmine) and
the associated alteration in absorbance was subtracted from
that observed through the concentration range of each test
compound. Each compound was analyzed on four occasions and
assayed alongside (-)-physostigmine, as a control and external
standard whose activity we have previously reported.9-17,23
(10) He, X. S.; Greig, N. H.; Rapoport, S. I.; Brossi, A.; Li, Y. Q.; Yu,
Q. S. Thiaphysovenine and Carbamate Analogues: A New Class
of Potent Inhibitors of Cholinesterases. Med. Chem. Res. 1992,
2, 229-237.
(11) Greig, N. H.; Pei, X. F.; Soncrant, T.; Ingram, D. K.; Brossi, A.
Phenserine and Ring C Hetero-Analogues: Drug Candidates for
Treatment of Alzheimer’s Disease. Med. Res. Rev. 1995, 15,
3-31.
(12) Brossi, A.; Pei, X. F.; Greig, N. H. Invited Review. Phenserine,
a Novel Anticholinesterase Related to Physostigmine: Total
Synthesis and Biological Properties. Aust. J . Chem. 1996, 49,
171-181.
(13) Pei, X. F.; Greig, N. H.; Brossi, A. Preparation and Selective
Inhibition of Human Butyrylcholinesterase by N1-Phenylethyl-
norphysostigmine analogues. Med. Chem. Res. 1995, 5, 455-
461.
(14) Yu, Q. S.; Pei, X. F.; Holloway, H. W.; Greig, N. H.; Brossi, A.
Total Syntheses and Anticholinesterase Activities of (3aS)-N8-
Norphysostigmine, (3aS)-N8-Norphenserine, Their Antipodal
Isomers, and Other N8-Substituted Analogues. J . Med. Chem.
1997, 40, 2895-2901.
(15) Yu, Q. S.; Brossi, A. Practical Synthesis of Unnautral (+)-
Physostigmine and Carbamate Analogues. Heterocycles 1988, 27,
745-750.
(16) Yu, Q. S.; Greig, N. H.; Holloway, H. W.; Brossi, A. Total
Synthesis and Anticholinesterase Activities of (3aS)-N8-Norphy-
sostigmine, (3aS)-N8-Norphenserine, their Antipodal Isomers,
and Other N8-Substituted Analogues. J . Med. Chem. 1998, 41,
2371-2379.
(17) Atack, J . R.; Yu, Q. S.; Soncrant, T. T.; Brossi, A. Comparative
Inhibitory Effects of Various Physostigmine Analogues Against
Acetyl- and Butyrylcholinesterase. J . Pharmacol. Exp. Ther.
1989, 249, 194-202.
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. Enzyme 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.
A two-tailed Student’s t-test was carried out to compare
between the values of two means. When more than two means
were compared, one-way analysis of variance and the Bonfer-
roni multiple test were used.29
(18) Quinn, D. M. Acetylcholinesterase: Enyme Structure, Reaction
Dynamics and Virtual Tansition States. Chem. Rev. 1987, 87,
955-979.
(19) Soreq, H.; Zakut, H. Amplification of Butyrylcholinesterase and
Acetylcholinesterase Genes in Normal and Tumor Tisuues:
Putative Relationship to Organophosphorous Poisoning. Pharm.
Res. 1990, 7, 1-7.
(20) Taylor, P. The Cholinesterases. J . Biol. Chem. 1991, 266, 4025-
4028.
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.
(21) Sussman, J . L.; Harel, M.; Farlow, F.; Oefner, C.; Goldman, A.;
Toker, L.; Silman, I. Atomic Structure of Acetylcholinesterase
from Torpedo Californica: A Prototypic Acetylcholine-Binding
Protein. Science 1991, 253, 872-879.
(22) Taylor, P.; Radic, Z. The Cholinesterases: From Genes to
Proteins. Annu. Rev. Pharmacol. Toxicol. 1994, 34, 281-320.
(23) Yu, Q. S.; Greig, N. H.; Holloway, H. W.; Utsuki, T.; Pei, X. F.;
Brossi, A. (-)-(3aS)-Eseroline Carbamate, a Close Analogue of
Physostigmine. Med. Chem. Res. 1997, 7, 116-120.
(24) March, J . Advanced Organic Chemistry: Reactions, Mechanisms
and Structure; J ohn Wiley & Sons: New York, 1992; pp 248-
272.
(25) Greig, N. H. Drug Entry to the Brain and its Pharmacologic
Manipulation. In Handbook of Experimental Pharmacology;
Bradbury, M. W. B., Ed.; Springer-Verlag: Heidelberg, Germany,
1992; Vol. 103, pp 487-523.
Refer en ces
(1) Khachaturian, Z. S. Diagnosis of Alzheimer’s Disease. Arch.
Neurol. 1985, 42, 1097-1105.

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1861
(26) Kozikowski, A. P.; Campiani, G.; Sun, L. Q.; Wang, S. M.;
Saxena, A.; Doctor, B. P. The Identification of a More Potent
Analogue of the Naturally Occurring Alkaloid Huperzine A.
Predictive Molecular Modeling of its Interaction with AChE. J .
Am. Chem. Soc. 1996, 118, 11357-11362.
(27) Campiani, G.; Kozikowski, A. P.; Wang, S. M.; Ming, L.; Nacci,
V.; Saxena, A.; Doctor, B. P. Synthesis and Anticholinesterase
Activity of Huperzine A Analogues Containing Phenol and
Catechol Replacements for the Pyridone Ring. BioOrg. Med.
Chem. Lett. 1998, 8, 1413-1418.
(28) Liston, D.; Nielson, J . A.; Villalobos, A.; Chapin, D.; J ones, S.
B.; Hubbard, S.; Shalaby, I.; White, W. F. CP-118, 954: A potent
and selective AChE inhibitor. The importance of selective AChE
inhibition in vivo. Abstr. Soc. Neurosci. 1994, 20, 606.
(29) Miller, R. Simultaneous Statistical Inferences; McGraw-Hill: New
York, 1966; pp 76-81.
J M980459S