Inhibition of human acetyl- and butyrylcholinesterase by novel carbamates of (-)- and (+)-tetrahydrofurobenzofuran and methanobenzodioxepine

Article abstract of DOI:10.1021/jm050578p

A new enantiomeric synthesis utilizing classical resolution provided two novel series of optically active inhibitors of cholinesterase: (-)- and (+)-O-carbamoyl phenols of tetrahydrofurobenzofuran and methanobenzodioxepine. An additional two series of (-)- and (+)-O-carbamoyl phenols of pyrroloindole and furoindole were obtained by known procedures, and their anticholinesterase actions were similarly quantified against freshly prepared human acetyl- (AChE) and butyrylcholinesterase (BChE). Both enantiomeric forms of each series demonstrated potent cholinesterase inhibitory activity (with IC₅₀ values as low as 10 nM for AChE and 3 nM for BChE), with the exception of the (+)-O-carbamoyl phenols of pyrroloindole, which lacked activity (IC₅₀ values > 1 μM). Based on the biological data of these four series, a structure-activity relationship (SAR) analysis was provided by molecular volume calculations. In addition, a probable transition-state model was established according to the known X-ray structure of a transition-state complex of Torpedo californica AChE-m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone (TcAChE-TMTFA). This model proved valuable in explaining the enantioselectivity and enzyme subtype selectivity of each series. These carbamates are more potent than, or similarly potent to, anticholinesterases in current clinical use, providing not only inhibitors of potential clinical relevance but also pharmacological tools to define drug-enzyme binding interactions within an enzyme crucial in the maintenance of cognition and numerous systemic physiological functions in health, aging, and disease.

Full text of DOI:10.1021/jm050578p

2174
J. Med. Chem. 2006, 49, 2174-2185
Inhibition of Human Acetyl- and Butyrylcholinesterase by Novel Carbamates of (-)- and
(+)-Tetrahydrofurobenzofuran and Methanobenzodioxepine
Weiming Luo,†,‡ Qian-sheng Yu,†,‡ Santosh S. Kulkarni,§ Damon A. Parrish,| Harold W. Holloway,‡ David Tweedie,‡
Avigdor Shafferman, Debomoy K. Lahiri,# Arnold Brossi,∞ and Nigel H. Greig*,‡
Drug Design & DeVelopment Section, Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, National
Institutes of Health, 5600 Nathan Shock DriVe, Baltimore, Maryland 21224, Medicinal Chemistry Section, Intramural Research Program,
National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock DriVe, Baltimore, Maryland 21224, Laboratory for the
Structure of Matter, Department of the NaVy, NaVal Research Laboratory, Washington, D.C. 20375, Department of Biochemistry and
Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100 Israel, Psychiatric Institute, Indiana UniVersity School of
Medicine, Indianapolis, Indiana 46202, and School of Pharmacy, UniVersity of North Carolina at Chapel Hill, North Carolina 27599
ReceiVed June 17, 2005
A new enantiomeric synthesis utilizing classical resolution provided two novel series of optically active
inhibitors of cholinesterase: (-)- and (+)-O-carbamoyl phenols of tetrahydrofurobenzofuran and methano-
benzodioxepine. An additional two series of (-)- and (+)-O-carbamoyl phenols of pyrroloindole and
furoindole were obtained by known procedures, and their anticholinesterase actions were similarly quantified
against freshly prepared human acetyl- (AChE) and butyrylcholinesterase (BChE). Both enantiomeric forms
of each series demonstrated potent cholinesterase inhibitory activity (with IC50 values as low as 10 nM for
AChE and 3 nM for BChE), with the exception of the (+)-O-carbamoyl phenols of pyrroloindole, which
lacked activity (IC50 values >1 µM). Based on the biological data of these four series, a structure-activity
relationship (SAR) analysis was provided by molecular volume calculations. In addition, a probable transition-
state model was established according to the known X-ray structure of a transition-state complex of Torpedo
californica AChE-m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone (TcAChE-TMTFA). This
model proved valuable in explaining the enantioselectivity and enzyme subtype selectivity of each series.
These carbamates are more potent than, or similarly potent to, anticholinesterases in current clinical use,
providing not only inhibitors of potential clinical relevance but also pharmacological tools to define drug-
enzyme binding interactions within an enzyme crucial in the maintenance of cognition and numerous systemic
physiological functions in health, aging, and disease.
Introduction
inhibitor interactions. Our research focus has been to undertake
both simultaneously.
Dementia of the Alzheimer’s type afflicts some 4.5 million
Americans and some 18 million people worldwide. On the basis
of the cholinergic hypothesis of memory, the depletion of its
neurotransmitter acetylcholine (ACh), and loss of associated
synapses during the progression of Alzheimer’s disease (AD),1,2
cholinesterase inhibitors (ChEIs) have been synthesized and
developed for clinical practice to ameliorate the associated
cognitive symptoms.3 This is achieved by reducing the rate of
hydrolysis of ACh in brain and thereby augmenting its cholin-
ergic activity. Although ChEIs and the recently FDA approved
drug memantine are the only ones to have consistently induced
improvements in cognitive function in mild to moderate AD,4
such improvements are, unfortunately, generally modest. Indeed,
recent clinical trials have triggered debate as to the relevance
of the benefit of this drug class.5,6 Such concern has prompted
two avenues of research: one to develop a new generation of
ChEIs with activity beyond the modest symptomatic levels
associated with initial agents, and another to optimize current
agents on the basis of a greater understanding of enzyme/
A classical ChEI, used to elucidate the original nature of
chemical transmission of nerve action and the cholinergic system
(Sir Henry Dale and Professor Otto Loewi, Nobel Prize 1936),
is the natural product (-)-physostigmine (15a). An alkaloid
isolated from the seeds of the Calabar bean, Physostigma
Venenosum,7-9 (-)-physostigmine (15a) has provided a template
in the development of several AD drugs, including rivastigmine
(Novartis, Basel, Switzerland), phenserine (National Institute
on Aging, Baltimore, MD, and Axonyx, New York, NY),
eptylstigmine (Mediolanum, Milan, Italy), and the parent
compound, itself, in a slow-release formulation (Synapton,
Forrest Laboratories, St Louis, MO).3,10,11 Extensive structure-
activity relationship studies of this class of ChEIs indicate that
(i) all derivatives of physostigmine with anticholinesterase
activity are levorotary, with a 3a-S configuration in their
pyrroloindole tricyclic ring system, and (ii) selectivity between
the two cholinesterase forms, acetyl- (AChE, EC 3.1.1.7) and
butyrylcholinesterase (BChE, EC 3.1.1.8), is primarily correlated
with the structure of the N-substituted moiety of the carbamoyl
side chain.10-12
* To whom correspondence should be addressed: phone 410-558-8278;
fax 410-558-8323; e-mail Greign@grc.nia.nih.gov.
† W.L. and Q.-S.Y. made equal contributions to this research and are
equal first authors.
An exception to the enantiomeric selectivity of physostigmine
derivatives is the N1-nor series of derivatives.13,14 (-)-Physo-
venine (20a), a congeneric alkaloid of physostigmine,9,10 also
lacks enantiomeric selectivity.10,15,16 The (+)-isomers of these
two series of compounds, interestingly, have unexplainable
anticholinesterase activity.13-16 In our continuing effort to design
new and improved ChEIs, we recently successfully synthesized
‡ National Institute on Aging, NIH.
§ National Institute on Drug Abuse, NIH.
| Naval Research Laboratory.
Israel Institute for Biological Research.
# Indiana University School of Medicine.
∞ University of North Carolina at Chapel Hill.
10.1021/jm050578p CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/11/2006

Inhibition of Acetyl- and Butyrylcholinesterase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2175
Scheme 1a
a (i) (a) (-)-Menthyl chloroformate, Et3N, benzene, N2, room temperature, 1.5 h; (b) recrystallized several times in hexane, then in ethanol. (ii) (a) NaOH,
MeOH, room temperature, 1.5 h; (b) room temperature, 1 N HCl.
two novel series of bioactive racemic carbamates with the tri-
cyclic skeletons furobenzofuran and methanobenzodioxepine.17
Herein, we describe the synthesis and activity of their optically
pure enantiomers with the dual purpose of (i) developing new
AD drug candidates and (ii) utilizing them as tools to elucidate
the molecular interactions required by the tricyclic skeleton of
this important class of ChEI to achieve enantioselectivity within
AChE and BChE.
Results
Chemistry. The starting material, 5-hydroxy-3-methyl-3-
(methoxycarbonylmethylene)benzofuran-2-one (1), was pro-
duced from C3-alkylation of the condensation product of 1,4-
cyclohexandione and pyruvic acid, according to a previously
reported procedure.17 First, compound 1 was reacted with (-)-
menthyl chloroformate to provide a mixture of the stereoisomers
of (3S)- and (3R)-menthyl carbonates of 5-hydroxy-3-methyl-
3-(methoxycarbonylmethylene)benzofuran-2-one 2 and 3 (Scheme
1).
Since it proved difficult to isolate stereoisomers 2 and 3 with
chromatography, we separated them by repeated crystallization.
This was performed from hexane, at first, and then from ethanol
to eventually yield two separate crystals with different crystalline
forms: needle (2) and flaky (3) (Experimental Section).
Figure 1. X-ray crystallographic picture and corresponding chemical
structure of 3.
been published.17,18 In our recent prior research, we reported
that the reductive cyclization of (()-5-hydroxy-3-methyl-3-
(methoxycarbonylmethylene)benzofuran-2-one by LiAlH4 gave
tetrahydrofurobenzofuran and methanobenzodioxipene in a yield
of 10%.17 In the present report, the reductive lithium-aluminum
complexes of compound 2 and 3 were acidified by 1 M HCl in
anhydrous Et2O, instead of oxalic acid, to elevate the yield to
approximately 30% (Scheme 2).
Phenols 6a and 7a, as well as 6b and 7b, were reacted with
different isocyanates to give six pairs of corresponding car-
bamates: 8a and 11a, 9a and 12a, and 10a and 13a, as well as
8b and 11b, 9b and 12b, and 10b and 13b, respectively.
Thereafter, each of these pairs of compounds was separated by
preparative thin-layer chromatography (TLC), utilizing a pro-
cedure reported previously.17
The absolute configuration of the C-3 position was determined
on the basis of the known configurations of C-19, C-21, and
C-24 from compound 3 by X-ray crystallography. Figure 1
shows that compound 3 has an R configuration at its 3-position.
As a consequence, compound 2 should possess an S configu-
ration at its 3-position.
The use of chiral HPLC to measure the optical purity of
compounds 2 and 3 proved unsuccessful. The hydrolysis of
stereoisomers 2 and 3 provided (-)-enantiomer 4, [R]D26 -2.6°
27
(c ) 0.62, CHCl3), and (+)-enantiomer 5, [R ]D +2.2° (c )
0.45, CHCl3) (Scheme 1), respectively. The enantiomeric excess
value (ee, %) of each was measured by chiral HPLC analysis
and is reported in the Experimental Section (Figure 2).
Compounds 4 and 5 were, for practical purposes, optically pure.
In the process of reduction, the stereochemistry at the 3-position
of compounds 2 and 3 was unchanged. Hence, 2 gave 6a (3aS
configuration) and 7a (5S configuration), whereas 3 gave 6b
(3aR configuration) and 7b (5R configuration). The probable
mechanism underpinning this reductive cyclization has recently
The syntheses of carbamates of the physostigmine and
physovenine series of analogues are shown in Scheme 3.
Compounds 15a,b, 17a, 18a, 20a,b, 22a, and 23a are known
compounds.15,16 Compounds 16a and 21a were synthesized from

2176 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Luo et al.
Scheme 2a
Figure 2. Chiral HPLC analysis of compounds 1, 4, and 5. Instrument,
HP1100; column, ChiraDex 5 µm; flow rate, 0.5 mL/min; detection,
UV 254 nm; Eluent, MeOH/H2O 45/55; temperature, room temperature.
Retention times: 1, 11.164 and 11.952 min; 4, 11.50 min; 5, 11.133
min.
a (i) (a) LAH/Et2O, room temperature, 1 h; (b) 1 M HCl in Et2O, room
temperature, 0.5 h. (ii) Na, RNCO/Et2O, room temperature, 0.5-1.0 h.
compounds 14a and 19a, which were obtained from natural
physostigmine according to a known procedure.17 Compounds
17b, 18b, 22b, and 23b were synthesized from (+)-3a(R)-
eseroline (14b) and (+)-3a(R)-physovenol (19b). These phenols,
14b and 19b, were obtained by asymmetric syntheses starting
from N-methylphenetidine.14
With the sole exception of the compound pair 17a,17b, which
possessed low and approximately similar BChE inhibitory
activity (R/S ) 0.6), all compounds with a S-configuration
proved to be more potent than their corresponding enantiomer
(R/S > 1).
X-ray Crystallography. The results of the X-ray studies of
compound 3 are illustrated in Figure 1. The chirality of the
asymmetric centers is as follows: C3-R, C19-R, C21-R, and
C24-S. The six-membered ring of the menthyl moiety has a
chair conformation; in contrast, the benzofuran system is flat.
Biological Evaluation. Table 1 shows the anticholinesterase
activity of enantiomers 8-13, 15-18, and 20-23 against freshly
prepared human AChE and BChE, derived from erythrocytes
and plasma, respectively. The concentration of compound
required to inhibit 50% enzyme activity (IC50 value) was
quantified by a modified Ellman technique,13-17,19-21 and values
for R-configuration versus S-configuration are represented by
the symbol R/S. A smaller IC50 is associated with a lower Ki
value and a higher affinity of inhibitor-enzyme binding.22-24
An R/S value of 1 is indicative of similar inhibitory activity for
the R- and S-configurations. For an R/S value >1, the compound
with R-configuration has a lower potency than its enantiomer.
This was most evident in the physostigmine series, where
AChE inhibitory activity was achieved with an R/S value g
353 and BChE inhibitory action was achieved with an R/S value
g 102. Hence, in the physostigmine series, carbamates of (+)-
physostigmine (15b, 17b, and 18b) possess minimal cholinergic
activity, quite the opposite of their potent (-)-enantiomers.
In complete contrast with the physostigmine series, both
enantiomers of the novel tetrahydrofurobenzofuran (8a,b, 9a,b,
and 10a,b) and dihydrobenzodioxepine (11a,b, 12a,b, and
13a,b) series possessed potent anticholinesterase action for
AChE, BChE, or both, with R/S values of e9.3. This activity
is in accord with the congener physovenine carbamates (20a,b,
21a,b, 22a,b, and 23a,b) that are similarly active in both
enantiomeric forms with a preference for the S-configuration.
As described, N-demethylation of physostigmine (15a) mini-
mizes the enantioselectivity of this series,14 which suggests that

Inhibition of Acetyl- and Butyrylcholinesterase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2177
Discussion
Scheme 3a
Molecular Comparison by Volume Calculations. As all of
the described carbamates possess close structural similarity as
well as a similar inhibitory mechanism, we can initially compare
them by means of superimposition to further elucidate the basis
of their enantioselectivity. This was undertaken for the four
AChE potent inhibitors with S-configuration (carbamates 9a,
12a, 17a, and 22a) by generating a molecular volume map′ of
each molecule that corresponds to their van der Waals sur-
face.25,26
As illustrated in Figure 3, for each compound, the relative
positions of their two phenyl moieties, on either side of the
carbonyl group in their optimized conformation, were almost
identical, closely overlaying one another. These four compounds
could be closely superimposed, except for disparity in their
tricyclic systems. The combined molecular volume map of
compounds 9a, 12a, 17a, and 22a provides an ”enzyme-
excluded map” (green area in Figure 3), within which all active
compounds should fit.
The molecular volume of the R-2′-methylphenylcarbamates
9b, 12b, 17b, and 22b minus the enzyme-excluded volume
provides an “estimate volume” that represents the additional
volume generated by that of each R-isomer (yellow meshed area
within Figure 3).
As shown in Table 3, the lowest estimate extra volume of
compound 9b (7.1 Å3) corresponds to the lowest enantioselec-
tivity (R/S ) 1.8). The highest estimate extra volume (24.8 Å3)
of compound 17b corresponds to the highest enantioselectivity
(R/S ) 551). Specifically, the larger estimate extra volume of
compound 17b (yellow meshed area in Figure 3) likely seriously
hinders the approach to and hence binding between the inhibitor
and enzyme. Molecular modeling studies were undertaken to
elucidate how this additional estimate extra volume hinders
binding.
Molecular Model of the Transition State. The wide
structural diversity of available ChEIs suggests that unlike types
of inhibitors bind with AChE in different ways via disparate
yet specific interactions between compound and enzyme.27-29
Most anticholinesterases, such as those in Table 2 as well as
the potent nonclinical tacrine-based triazoles and other hybrids,
are cationic at physiological pH, whereas some, including those
of the present study and arisugacin, are not.30-32 Human
cholinesterases are large complex molecules composed of
catalytic subunits that can clearly accommodate a variety of
specific binding interactions associated with these diverse
inhibitors. They contain up to 583 amino acids, have a molecular
mass of 70-80 kDa, and are variably glycosylated.29,33
Three-dimensional analyses of AChE and BChE, based on
X-ray crystallography, have provided structural information
regarding the positioning of the catalytically important amino
acid residues within these proteins.34-41 In synopsis, three major
binding domains have been described within AChE and two
within BChE in an internalized, primarily hydrophobic gorge
of some 20 Å length but as narrow as 0.5 Å wide. Deepest
within this gorge is a catalytic acyl binding domain, which
hydrolyzes choline esters through electron transfer within a
catalytic triad, termed a charge relay system. The triad includes
Ser200, the imidazole group of His440, and the carboxylic acid
moiety of Glu327 (TcAChE numbering). A choline binding
domain resides midway along the gorge, and a peripheral anionic
site exists at the gorge mouth for AChE but not BChE;30-41
this latter site may be involved in the complexing of AChE
with amyloid-â peptide in the Alzheimer brain.42 Notably, the
neurotoxicity associated with Aâ-AChE complexes has been
a (i) Na, RNCO/Et2O, room temperature.
the N1-substitution of the tricyclic ring is a key determinant
regarding enantioselectivity. Additionally, the development of
members of the tetrahydrofurobenzofuran and dihydro-
benzodioxepine series for clinical development could be under-
taken with either the potent S-(-)-enantiomers or the racemates,
whose synthesis would be far easier and cheaper (avoiding
optical resolution) and whose anticholinesterase activity is
almost equal. A comparison of Tables 1 and 2, the latter defining
the anticholinesterase activity of agents of current and recent
clinical interest against enzyme drawn from the same individual
and analyzed similarly, suggests that compounds 9a and 12a
and compounds 10a and 13a possess sufficient potency and
selectivity for AChE and BChE, respectively, to be of clinical
interest.
In each of the four series of compounds detailed in Table 1,
the differential selectivity for AChE or BChE is determined by
the structure of N-substituted moiety of the carbamate. Specif-
ically, the N-methylcarbamates provide minimal enzyme subtype
selectivity. The N-ethylcarbamates have a moderate BChE
preference. The N-2′-methylphenylcarbamates have high AChE
selectivity, and N-4′-isopropylphenylcarbamates reverse this and
demonstrate a high BChE preference. Of particular note, the
AChE and BChE selectivities of the novel tetrahydrofuro-
benzofuran and dihydrobenzodioxepine series are among the
greatest found of all ChEIs yet synthesized on the tricyclic
backbone of physostigmine.

2178 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Luo et al.
Table 1. 50% Inhibitory Concentrations of Compounds versus Freshly Prepared Human Erythrocyte AChE and Plasma BChE
no.
compounds
AChE (IC50, nM, ( SEM)
R/Sa
BChE (IC50, nM, ( SEM)
R/Sa
A/Bb
Carbamates of Tetrahydrofurobenzofuran
8a
8b
9a
9b
10a
10b
(-)-(3aS)-ethyl
100 ( 34
3 ( 1
28 ( 1
33 BChE
17 BChE
570 AChE
1000 AChE
>2300 BChE
167 BChE
(+)-(3aR)-ethyl
486 ( 63
20 ( 1
4.8
1.8
9.3
3.2
3.1
(-)-(3aS) -2′-methylphenyl
(+)-(3aR)-2′-methylphenyl
(-)-(3aS)-4′-isopropylphenyl
(+)-(3aR)-4′-isopropylphenyl
11 400 ( 1300
36 400 ( 4750
13 ( 5
36 ( 1
c
6700 ( 1750
40 ( 2
Carbamates of Benzodioxepine
610 ( 106
11a
11b
12a
12b
13a
13b
(-)-(5S)-ethyl
23 ( 5
120 ( 66
52 000 ( 10 000
c
27 BChE
20 BChE
1350 AChE
>310 AChE
>2000 BChE
64 BChE
(+)-(5R)-ethyl
2420 ( 213
38 ( 1.0
97 ( 4
4.0
5.3
(+)-(5S) -2′-methylphenyl
(-)-(5R)-2′-methylphenyl
(+)-(5S)-4′-isopropylphenyl
(-)-(5R)-4′-isopropylphenyl
2.6
c
15 ( 1.5
54 ( 11
3430 ( 145
3.5
Carbamates of Physostigmined
28 ( 2
15a
15b
16a
17a
17b
18a
18b
(-)-(3aS)-methyl
16 ( 3
2490 ( 290
4 ( 1
2 BChE
4 BChE
(+)-(3aR)-methyl
9890 ( 9
94 ( 12
10 ( 2
353
155
(-)-(3aS)-ethyle
24 BChE
195 AChE
5 BChE
15 BChE
>6 BChE
(-)-(3aS) -2′-methylphenyl
(+)-(3aR)-2′-methylphenyl
(-)-(3aS)-4′-isopropylphenyl
(+)-(3aR)-4′-isopropylphenyl
1950 ( 240
1180 ( 450
50 ( 1
5510 ( 190
760 ( 20
c
551
0.6
5100 ( 575
102
Carbamates of Physovenined
20a
20b
21a
22a
22b
23a
23b
(-)-(3aS)-methyl
27 ( 1
4 ( 1
56 ( 15
2 ( 0
7 BChE
none
(+)-(3aR)-methyl
56 ( 1
82 ( 4
2.1
14
(-)-(3aS)-ethylf
40 BChE
120 AChE
36 AChE
225 BChE
>1300 BChE
(-)-(3aS) -2′-methylphenyl
(+)-(3aR)-2′-methylphenyl
(-)-(3aS)-4′-isopropylphenyl
(+)-(3aR)-4′-isopropylphenyl
13 ( 1
1560 ( 120
5100 ( 1780
17 ( 2
142 ( 135
3860 ( 970
c
11
3.3
1.4
23 ( 2
a IC50 of R-configuration/IC50 of S-configuration. b Selectivity for AChE or BChE from IC50 values. c None: Insufficient activity in the range of 0.3
nM-30 µM to calculate an IC50 value, and hence considered to be inactive. d The IC50 data for compounds 15a and 15b are from ref 14; the data for
compounds 20a, 23a, and 20b are from ref 15; the data for compounds 17a, 22a, and 18a are from ref 16; and the data for compounds 16a and 21a are from
ref 17. In each case, however, enzyme was obtained from the same individual and assays were performed similarly. e The (+)-(3aR)-ethylcarbamate compound
was not synthesized. f The (+)-(3aR)-ethylcarbamate compound was not synthesized.
Table 2. Comparison of the IC50 Values (+ SEM) of Approved ChEIs
of Clinical Interest versus Freshly Prepared Human Erythrocyte AChE
and Plasma BChE (Figure 6)
catalytic triad on its carbonyl group. A quaternary transition
state between Ser200 and ACh momentarily exists that then
rapidly collapses into an acylated-enzyme intermediate and
released choline. Thereafter, prompt hydrolysis of the acetyl
ester reactivates the enzyme to allow efficient cleavage of as
IC50 valuea (nM ( SEM)
compound
tacrine
AChE
BChE
selectivity
many as 104 ACh molecules s-1 37,38
(-)-Physostigmine (15a)
.
190 ( 40
22 ( 8
4150 ( 160
800 ( 60
22 ( 1.4
47 ( 10
4150 ( 1700
37 ( 5
7300 ( 830
1560 ( 45
4 BChE
and analogues interact similarly with the same two binding
domains. However, nucleophilic attack of its carbonyl moiety
results in a transient (-)-physostigmine-AChE intermediate,
in a tetrahedral conformation, that then swiftly collapses to a
carbamylated drug-enzyme complex that is dramatically more
stable than the acetyl enzyme one associated with ACh; enzyme
inhibition occurs consequent to the slow rate of enzyme
decarbamylation. The inhibition process, hence, involves AChE-
catalyzed hydrolyses of inhibitors.37,38 In such a chemical
reaction, the highest energy transition state controls the overall
rate of the reaction. A catalytic enzyme can lower this energy
barrier by specific interaction with the inhibitor in the transition
state (24, 25).45 (Scheme 4). As shown in Table 1, each of the
six different carbamates with the same N-substituted side chain
(e.g., 8a,b, 11a,b, 16a, and 21a) should generate identical
carbamylated enzyme structures after their reaction with either
AChE or BChE. Hence their different IC50 values should be
related to their affinities and rates of carbamylation, and not to
decarbamylation, which would impact the time dependence of
the inhibition associated with each. As a consequence, we can
focus on carbamylation due to the short time frame of our
biological studies.
donepezil
188 AChE
122 BChE
9 AChE
rivastigmine
galanthamine
(-)-phenserine
70 AChE
(-)-(3aS)-Phenylcarbamate of Physostigmine
heptylstigmine 22 ( 2 5.0 ( 0.1
4 BChE
(-)-(3aS)-Heptylcarbamate of Physostigmine
huperzine A
BW284c51b
iso-OMPAb
47 ( 22
18 ( 8
>10 000
48 000
>212 AChE
2660 AChE
350 BChE
340 000
980 ( 550
a IC50 values were determined in duplicate on a minimum of four different
occasions from enzyme collected from the same individual as assays
described for Table 1. b BW284c51 [1,5-bis(4-allyldimethylammoniumphen-
yl)pentan-3-one dibromide] and iso-OMPA [tetra(monoisopropyl)pyrophos-
phortetramide] are classical selective inhibitors of AChE and BChE,
respectively.
shown to be greater than that induced by the Aâ peptide alone
in both cell culture and animal experiments.39
On the basis of calculations and electrooptical measurements,
it has been suggested that electrostatic charges associated with
seven negatively charged amino acids residues close to the
entrance of the gorge trap and steer charged ligands into its
mouth.43,44 In the case of ACh, the quaternary choline moiety
interacts with Trp84 of TcAChE and, to a lesser extent, with
Phe330 within the choline binding domain. This orientates the
compound to allow the approach and nucleophilic attack of the
A better insight into the molecular interactions of the enzyme
and inhibitor can be obtained at the transition state. Whereas it
is not currently possible to produce a crystal structure of an

Inhibition of Acetyl- and Butyrylcholinesterase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2179
Figure 3. Union of molecular volume maps for the four active, S-configuration compounds 9a, 12a, 17a, and 22a (green area) provides the
enzyme-excluded volume. The molecular volume map of compound 17b minus the enzyme-excluded volume is the estimate extra volume of 17b
(yellow meshed area).
Table 3. Estimate Extra Volumea and the Ratio of Anticholinesterase
Activity R/S
Scheme 4
estimate
extra
R/S
(AChE)
no.
9b
compounds
volume (Å3)
Tetrahydrofurobenzofuran Series
(+)-(3aR)-2′-methylphenylcarbamate
7.1
19.9
24.8
19.4
1.8
2.6
Dihydrobenzodioxepine Series
(-)-(5R)-2′-methylphenylcarbamate
12b
17b
22b
Physostigmine Series
(+)-(3aR)-2′-methylphenylcarbamate
Prior investigation of the X-ray crystallographic structure of
TcAChE complexed with the known physostigmine analogue
MF268 [8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline]
demonstrated the presence of a covalent bond between the
carbonyl carbon and γ-O of Ser200 and also that the N-substituted
side chain of the carbamoyl moiety is located inside the acyl-
binding pocket and extends to the rim of the active site
gorge.47,48 The X-ray crystallographic transition-state analogue
structure of TcAChE-TMTFA (code 1AMN) was obtained
from the RCSB protein databank. After extraction of TMTFA,
the (-)-(3aS)-N-2′-methylphenylcarbamoyl phenol of furo-
benzofuran (9a) was docked, in accord with knowledge gained
from the binding of MF268.47,48
551
Physovenine Series
(+)-(3aR)-2′-methylphenylcarbamate
11
a Estimate extra volume ) volume of R-configuration - enzyme-
excluded volume map (union of volumes of active S-configuration
compounds: 9a, 12a, 17a, and 22a).
enzyme complexed with its substrate in the transition state, due
to the short transition-state lifetime versus the time required
for X-ray data collection, one can exploit the high affinity of
transition-state analogues. In this regard, the analogue m-(N,N,N-
trimethylammonio)-2,2,2-trifluoroacetophenone (TMTFA), pos-
sessing a low Ki (15 fM) for inhibition of TcAChE,46 was
utilized to model the interaction of our compounds within the
active site of AChE.
As shown in Figure 4, six hydrogen bonds are apparent within
the model (thin yellow lines): the NH of Gly118, Gly119, and

2180 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Luo et al.
Figure 5.
shape. The pockets can be differentiated on the basis of two
amino acid residues located at the bottom of the acyl loop. These
residues are aromatic in AChE (Phe288 and Phe290) but aliphatic
in BChE (Leu286 and Val288).33,35,37,38,46 The latter pocket, hence,
is slightly larger due to the smaller protruding side chains
associated with Leu and Val.12
Figure 4. Close-up of compound 9a-TcAChE complex showing its
covalent bonding with Ser200. The thin yellow lines denote hydrogen
bonds to the residues in the oxyanion hole, that is, Gly118, Gly119, and
Ala201, and in the catalytic triad, Ser200, His440, and Glu327
.
Our previous studies have shown that the conformational
energy barrier of the N-phenyl group of compound 17a and its
analogues, rotated around the C1′-N bond, is correlated to the
IC50 of AChE. A higher energy barrier is associated with a lower
inhibitory activity (a larger IC50); although, inexplicably, the
Ala201 with the oxygen of the ligand carbonyl, the γ-O of Ser200
with a single H, the hydrogen with N of His440, and the NH of
His440 with the oxygen of Glu327. Together with the covalent
bond of the γ-O of Ser200 with the C of the ligand’s carbonyl
group, the illustrated hydrogen bonds comprise an acylation site.
This site is considered as a crucial part of the transition state.
A transition state model was developed by (i) keeping the
acylation site intact (by constraining the relative distances
between the described atoms) and (ii) minimizing the complex
energy, after automatic adjustment of the conformation of the
remaining complex.
This model resembles a beam balance. The central supporting
point is a covalent bond between the γ-O of Ser200 and the C
of the ligand’s carbonyl group. The π-π interaction system
between the N-phenyl group of carbamate (9a) and the phenyl
group of Phe288 of the enzyme comprises one side of this
balance. The π-π or lipophilic (C-H‚‚‚π) interaction system
of the furobenzofuran moiety of compound 9a and the indole
system of Trp84 of the enzyme forms the other side of the
balance. Such C-H‚‚‚π interactions have been observed in the
X-ray crystal structure of other neutral molecules with TcAChE.49
Any change on either side of this balance may adversely affect
the formation of the transition state. Utilizing this model, we
can revisit the basis of enantioselectivity and enzyme subtype
selectivity.
Enantioselectivity. As shown by molecular volume calcula-
tions (Figure 3, Table 3), for all carbamates with an R-
configuration, with the exception of the physostigmine series,
their molecular volumes are not greatly different. This would
support the lipophilic interaction between the tricyclic ring
system of the ligands and the indole system of Trp84 of the
enzyme. Hence, all of these compounds retain substantial
anticholinesterase activity compared to their S-isomers.
The only exception involves R-configuration compounds
having an N1-methyl group (the physostigmine series). The
model predicts that the N1-methyl group will become inserted
between the tricyclic ring system and the indole system of Trp84.
The lipophilic interaction will consequently disappear, and a
transition state between ligand and enzyme complex would not
be readily formed without this lipophilic interaction. As a result,
the (+)-physostigmine analogues 15b, 17b, and 18b would be
predicted to possess poor AChE and BChE inhibitory action,
in accord with their measured poor ChEI activity.
21
energy barrier is as low as 0-0.7 kcal/mol.
According to the described transition-state model (Figure 4),
the π-π interaction system between the N-phenyl group of the
carbamate (17a) and the phenyl group of Phe288 of the enzyme
comprises one side of this balance. Rotation of the N-phenyl
group will decrease this π-π interaction and directly influence
the stability of the transition state, consequently lowering the
affinity between ligand and enzyme.21
Utilizing the same model, we can now propose a basis for
the cholinesterase subtype selectivity of the different carbamate
moieties. (i) The small methyl group of N-methylcarbamates
has almost no lipophilic interaction with residues of the acyl
pocket; it thus has little influence on this balance and hence
there is minimal subtype selectivity between AChE and BChE
for N-methylcarbamates of all series. (ii) The ethyl group of
N-ethylcarbamates, likewise, has relatively meager but neverthe-
less greater lipophilic interaction than N-methylcarbamates. The
direction of this interaction is more favorable to form a stable
transition state within BChE than AChE. Hence, all of the
N-ethylcarbamates possess moderate BChE selectivity. (iii) The
phenyl group of N-phenylcarbamates provides a significant
lipophilic interaction with the aromatic ring of Phe288. This
interaction together with the lipophilic interaction between the
tricyclic ring system and indole moiety of Trp84 provides a
highly stable transition state. Thus, all N-phenyl- and N-
methylphenylcarbamates possess potent AChE activity. Within
BChE, however, Phe288 is replaced by an amino acid with an
aliphatic residue. A similar π-π interaction cannot be formed
and leads to an unfavorable stability in the transition state and
consequent weak BChE inhibitory activity. N-Phenyl- and
N-methylphenylcarbamates hence have high AChE selectivity.
Finally, (iv) the 4′-isopropylphenyl moiety of N-4′-isopropyl-
phenylcarbamates cannot achieve a π-π interaction within the
acyl-binding pocket of AChE consequent to the bulk and
hindering action of the isopropyl moiety. Within BChE,
however, the lipophilic interaction of the isopropylphenyl group
with residues of BChE in its larger acyl-binding pocket is
favored (consequent to replacement of AChE Phe288 and Phe290
by Val and Leu), which stabilizes the transition state. Thus all
of the N-4′-isopropylphenylcarbamate series have a potent and
selective BChE inhibitory action.
Enzyme Subtype Selectivity. The acyl-binding pockets
within AChE and BChE differ from one another in size and

Inhibition of Acetyl- and Butyrylcholinesterase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2181
oped a beam balance-like transition-state model of the TcAChE-
N-phenylcarbamate complex. Using this model, we can now
propose a basis to account for the enantioselectivity of physo-
stigmine and its congeners.
Experimental Section
Chemistry. Melting points (uncorrected) were measured with a
Fisher-Johns apparatus. 1H NMR and 13C NMR spectra were
recorded on a Bruker (Bellevica, MA) AC-300 spectrometer. MS
spectra (m/z) were recorded on a Hewlett-Packard 5973 gas
chromatograph-mass spectrometer with chemical ionization [GC-
MS (CI)]. High-resolution mass spectrometry (HRMS) was per-
formed by the UCR Mass Spectrometry Facility, Department of
Chemistry, University of California. Optical rotations were mea-
sured by Jasco, Model DIP-370 (Japan, Spectroscopic Co., Ltd.).
Elemental analyses were performed by Atlantic Microlab, Inc. The
ee % value of optically active compounds was determined by HPLC
analyses using a HP1100 instrument and a chiral column (ChiraDex
5 µm) (Agilent Technology), mobile phase MeOH/H2O ) 45/55,
elution rate 0.5 mL/min., UVD 254 nm, all at room temperature.
All reactions involving nonaqueous solutions were performed under
an inert atmosphere.
(3S)- and (3R)-Menthyl Carbonates of 5-Hydroxy-3-methyl-
3-(methoxycarbonylmethylene)benzofuran-2-ones (2 and 3).
Under a nitrogen atmosphere, a solution of 5-hydroxy-3-methyl-
3-methoxycarbonylmethylenebenzofuran-2-one (1) (116 mg, 0.491
mmol) in 0.06 mL of triethylamine and 1.5 mL of benzene was
dropwise added into (-)-menthyl chloroformate (118 mg, 0.539
mmol) at room temperature. The mixture was stirred for 1.5 h at
the same temperature. The crude product was subjected to chro-
matography on silica gel (EtOAc/hexane ) 1/3) to give 156.5 mg
of white crystalline product, yield 76.2%: 1H NMR (CDCl3) δ
7.20-7.02 (m, 3H, Ar-H), 4.68-4.52 (m, 1H, CH-OCdO), 3.53
(s, 3H, CH3O), 3.10, 2.96 (AB, Jgem)17.6 Hz, 2H, C3-CH2COO),
2.21-2.11 (m, 1H, CH-iPr), 2.10-1.98 (m, 1H, CH-Me), 1.80-
1.65 (m, 1H, CHMe2), 1.51 (s, 3H, C3-CH3), 1.59-1.41 (m, 2H,
CH2COCdO), 1.26-0.80 (m, 4H, CH2CH2), 0.97 (d, 6H, CH3CCH3)
and 0.85 (d, 3H, CH3CCCOCdO) ppm; CI-MS (CH4), m/z 419
(MH+), 418, 417, 371, 237, 181, 153 and 109; HR-MS m/z calcd
for C23H34NO7(MNH4+) 436.2335, found 436.2326. The above
white crystal product was, thereafter, recrystallized from both
hexane and ethanol, separately, several times, until the optical
rotations and melting points of the two different isolated crystalline
forms did not change. One, a needle crystalline form, was product
Figure 6. Chemical structures of clinically relevant cholinesterase
inhibitors (Table 2).
In accord with this, one of the most potent and selective BChE
inhibitors in the physostigmine series, (-)-N1-phenethyl-
norcymserine (26) (IC50 for AChE >30 000 nM, IC50 for BChE
6.0 ( 1.0 nM, selectivity >5000-fold BChE) (Figure 5),19
similarly can fit within the elongated acyl binding pocket
associated with BChE. However, as the N1-phenethyl moiety
is directed outside the lipophilic interaction system, activity is
not perturbed. Indeed, it is augmented, likely as a consequence
of hydrophobic interactions close to the gorge mouth. Interest-
ingly, the elongated (-)-4′-benzoxyphenylcarbamate of physo-
stigmine, 27 (Figure 5),50 which lacks a hindered 4′-isopropyl
group, can maintain the π-π interaction associated with potent
AChE binding but make use of the larger acyl pocket associated
with BChE to achieve BChE inhibitory potency (IC50 for AChE
58 ( 4 nM, IC50 for BChE 7.0 ( 1.0 nM, selectivity 7-fold
BChE).50
In synopsis, we report a new enantiomeric synthesis, through
classical resolution, of two novel series of potent anticholinest-
erases: the O-carbamoyl phenols of furobenzofuran and benzo-
dioxepine. These two series provide candidates to selectively
inhibit either AChE or BChE and, additionally, include some
that possess neurotrophic actions.17 Such actions may or may
not be cholinergically mediated and provide this class of drugs
actions beyond those that are classically considered to be only
symptomatic.51,52 Carbamates of these novel series are suf-
ficiently active to be of potential clinical interest, comparing
favorably with the inhibitory action of approved and recent
ChEIs (Table 2, Figure 6), and could be developed either as
their potent S-enantiomers or as their synthetically cheaper
racemates. In addition, their enzyme subtype selectivity com-
pares favorably with the classical selective anticholinesterases
BW284c51 and iso-OMPA. On the basis of the described
molecular volume calculations and known X-ray structure of a
transition-state complex of TcAChE-TMTFA, we have devel-
25
2: mp 106.8-107.4 °C; [R]D -60.8° (c ) 0.53, CHCl3). Anal.
(C23H30O7) C, H. The other, a flaky crystalline form, was product
26
3: mp 148.0-148.8 °C; [R]D -19.7° (c ) 0.74, CHCl3). Anal.
(C23H30O7) C, H.
(-)-(3S)-5-Hydroxy-3-methyl-3-(methoxycarbonylmethylene)-
benzofuran-2-one (4). Under a nitrogen atmosphere, a mixture of
reactant 2 (0.493 g, 1.178 mmol) and sodium hydroxide (0.2 g,
5.0 mmol) in 18 mL of methanol was stirred for 1.5 h at room
temperature. It was then neutralized with 1 N HCl. After removal
of solvent, the residue was chromatographed on silica gel (CH2Cl2/
MeOH ) 10/1) to give 168.2 mg of product 4, yield 60.4%: mp
26
157.0-159.0 °C; [R]D -2.6° (c ) 0.62, CHCl3); ee ) 100%
(HPLC); 1H NMR (CDCl3) δ 6.92-6.61 (m, 3H, Ar-H), 5.07 (s,
1H, OH), 3.44 (s, 3H, OCH3), 3.02, 2.84 (AB, Jgem ) 18.0 Hz, 2H,
C3-CH2CdO), and 1.41(s, 3H, C3-CH3) ppm; CI-MS (CH4) m/z
237(MH+), 205, 177, and 163.
(+)-(3R)-5-Hydroxy-3-methyl-3-(methoxycarbonylmethylene)-
benzofuran-2-one (5). Compound 5 was prepared as described
above, yield 66.0%: mp 156.0-158.6 °C; [R]D27 +2.2° (c ) 0.45,
CHCl3); ee ) 100%. The 1H NMR and CI-MS (CH4) are the same
as that of compound 4.
(3aS)-5-Hydroxy-3a-methyl-2,3,3a,8a-tetrahydrofuro[2,3-b]-
benzofuran (6a) and (5S)-7-Hydroxy-5-methyl-4,5-dihydro-2,5-
methano-1,3-benzodioxepine (7a). Under a nitrogen atmosphere,
a solution of compound 2 (177 mg, 0.423 mmol) in 2 mL of ether
was added dropwise to lithium aluminum hydride (32 mg, 0.843

2182 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Luo et al.
mmol) in ether at 0 °C. The mixture, after beomg stirred at 0 °C
for 0.5 h, was stirred for another 1 h at room temperature.
Thereafter, 1 M HCl ether solution was to provide a mixture of
pH 3-4, which then was stirred for 0.5 h at room temperature.
Next, the mixture was filtered and the filtrate was concentrated.
The residue was subjected to chromatography on silica gel (EtOAc/
hexane ) 1/3) to give 24.4 mg of a mixture of compounds 6a and
(s, 3H, C5-CH3), and 1.16 (t, 3H, C7-CH3CH2NHCOO) ppm;
CI-MS (CH4) m/z 264 (MH+), 246, 220, 193, 175, 149, and 72.
Anal. (C14H17NO4) C, H, N.
(-)-(3aS)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-b]benzo-
furan-5-yl N-(o-Tolyl)carbamate (9a) and (+)-(5S)-5-Methyl-
4,5-dihydro-2,5-methano-1,3-benzodioxepin-7-yl N-(o-Tolyl)-
carbamate (12a). Under a nitrogen atmosphere, two small pieces
of sodium (about 1 mg) were added into a solution of mixture of
6a and 7a (27.9 mg, 0.145 mmol) in 5 mL of anhydrous ether at
room temperature. The mixture was stirred for 2 min, and then
o-tolyl isocyanate (18.9 µL, 0.149 mmol) was added to the mixture
in one portion. The reaction was continued for 35 min at room
temperature, 4 mL of water then was added, and the ether layer
was separated. After drying over sodium sulfate and filtering, the
filtrate was evaporated to remove solvent. The residue was
chromatographed on a silica gel plate (EtOAc/hexane ) 1/3) to
1
7a. The GC-CI-MS and H NMR of this mixture were the same
as that of the racemic17 and showed an approximate 1:1 molar ratio
of (3aS)-5-hydroxy-3a-methyl-2,3,3a,8a-tetrahydrofuro[2,3-b]ben-
zofuran (6a) and (5S)-7-hydroxy-5-methyl-4,5-dihydro-2,5-methano-
1,3-benzodioxepine (7a). The yields were 30%, and the compounds
were directly used as starting material for the next reactions.
(3aR)-5-Hydroxy-3a-methyl-2,3,3a,8a-tetrahydrofuro[2,3-b]-
benzofuran (6b) and (5R)-7-Hydroxy-5-methyl-4,5-dihydro-2,5-
methano-1,3-benzodioxepine (7b). Compounds 6b and 7b were
prepared from compound 3, according to the procedure described
for compounds 6a and 7a.
27
afford products 9a and 12a. Product 9a (15.6 mg, 66.1%): [R]D
1
--103.0° (c ) 0.10, CHCl3); ee ) 100%; H NMR (CDCl3) δ
7.84 (br s, 1H, NH), 7.32-6.70 (m, 7H, Ar-H), 5.89 (s, 1H, C8a-
H), 4.18-3.68 (m, 2H, C2-H), 2.39-2.05 (m, 2H, C3-H), 2.31
(s, 3H, Ar-CH3), and 1.56 (s, 3H, C3a-CH3) ppm; CI-MS (CH4)
m/z 326 (MH+). Anal. (C19H19NO4) H; calcd C 70.14, N 4.25; found
C 69.18, N 3.71. Product 12a (15.0 mg, 63.5%): [R]D27 +30.4° (c
) 0.135, CHCl3), ee ) 100%; 1H NMR (CDCl3) δ 7.87 (br s, 1H,
C7-NHCOO), 7.33-6.70 (m, 7H, Ar-H), 5.79 (d, J ) 1.80 Hz,
1H, C2-H), 4.22, 3.79 (AB, Jgem ) 6.84 Hz, 2H, C4-H), 2.37-
2.01 (m, 2H, C10-H), 2.31 (s, 3H, Ar-CH3), and 1.51 (s, 3H,
C5-CH3) ppm; CI-MS (CH4) m/z 326 (MH+). Anal. (C19H19NO4)
C; H calcd 5.89, found 6.36; N calcd 4.31, found 3.57.
(-)-(3aS)-3a-Methyl-2,3,3a,8a-tetrahydrofuro[2,3-b]benzo-
furan-5-yl N-Ethylcarbamate (8a) and (-)-(5S)-5-Methyl-4,5-
dihydro-2,5-methano-1,3-benzodioxepin-7-yl N-Ethylcarbamate
(11a). Under a nitrogen atmosphere, two small pieces of sodium
(about 1 mg) were added into a solution of mixture of 6a and 7a
(24.4 mg, 0.127 mmol) in 5 mL of anhydrous ether at room
temperature. This mixture was then stirred for 2 min, and ethyl
isocyanate (30.1 µL, 0.381 mmol) was next added to the reaction
mixture in one portion. An hour into the reaction at room
temperature, 1.4 mL of water was added and the ether layer was
separated. After drying over sodium sulfate and filtering, the filtrate
was evaporated to remove solvent. The residue was chromato-
graphed on silica gel plate (EtOAc/hexane ) 1/3) to afford products
(+)-(3aR)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-b]benzo-
furan-5-yl N-(o-Tolyl)carbamate (9b) and (-)-(5R)-5-Methyl-
4,5-dihydro-2,5-methano-1,3-benzodioxepin-7-yl N-(o-Tolyl)-
carbamate (12b). Under a nitrogen atmosphere, two small pieces
of sodium (about 1 mg) were added into a solution of mixture of
6b and 7b (18 mg, 0.09 mmol) in 5 mL of anhydrous ether at room
temperature. The mixture was stirred for 2 min, and then o-tolyl
isocyanate (18.3 µL, 0.145 mmol) was added in one portion. The
reaction was continued for a further 35 min at room temperature,
4 mL of water then was added and the ether layer was separated.
After drying over sodium sulfate and filtering, the filtrate was
evaporated to remove solvent. The residue was chromatographed
on silica gel plate (EtOAc/hexane ) 1/3) to afford products 9b
and 12b. Product 9b (11.0 mg, 74.9%), [R]D26 +104.2° (c ) 0.095,
26
8a and 11a. Product 8a (11.6 mg, 69.7%): [R]D -92.2° (c )
0.09, CHCl3); ee ) 100%; 1H NMR (CDCl3) δ 6.87-6.60 (m, 3H,
Ar-H), 5.77 (s, 1H, C8a-H), 4.89 (br s, 1H, NH), 4.06-3.56 (m,
2H, C2-H), 3.23 (m, 2H, C5-CH2NHCOO), 2.13-1.91 (m, 2H,
C3-H), 1.47 (s, 3H, C3a-CH3), and 1.14 (t, 3H, C5-CH3CH2-
NHCOO) ppm; CI-MS (CH4) m/z 264 (MH+), 193, 175, and 72.
Anal. (C14H17NO4) N, H; C, calcd 63.87, found 63.14. Product 11a
(14.1 mg, 84.6%): [R]D25 -10.0° (c ) 0.12, CHCl3), ee ) 100%;
1H NMR (CDCl3) δ 6.82-6.67 (m, 3H, Ar-H), 5.69 (d, J ) 1.80
Hz, 1H, C2-H), 4.88 (br s, 1H, C7-NHCOO), 4.17, 3.70 (AB,
Jgem ) 7.20 Hz, 2H, C4-H), 3.22 (m, 2H, C-7CH2NHCOO), 2.19-
1.19 (m, 2H, C10-H), 1.41 (s, 3H, C5-CH3), and 1.16 (t, 3H,
C7-CH3CH2NHCOO) ppm; CI-MS (CH4) m/z 264 (MH+), 246,
220, 193, 175, 149, and 72. Anal. (C14H17NO4) C, H; N, calcd 5.32,
found 4.91.
1
CHCl3); ee ) 100%; H NMR (CDCl3) δ 7.84 (br s, 1H, NH),
7.32-6.70 (m, 7H, Ar-H), 5.89 (s, 1H, C8a-H), 4.18-3.68 (m,
2H, C2-H), 2.39-2.05 (m, 2H, C3-H), 2.31 (s, 3H, Ar-CH3)
and 1.56 (s, 3H, C3a-CH3) ppm; CI-MS (CH4) m/z 326 (MH+).
Anal. (C19H19NO4) C, H; N, calcd 4.31, found 3.78. Product 12b
(+)-(3aR)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-b]benzo-
furan-5-yl N-Ethylcarbamate (8b) and (+)-(5R)-5-Methyl-4,5-
dihydro-2,5-methano-1,3-benzodioxepin-7-yl N-Ethylcarbamate
(11b). Under a nitrogen atmosphere, two small pieces of sodium
(about 1 mg) were added into a solution of mixture of 6b and 7b
(26 mg, 0.135 mmol) in 5 mL of anhydrous ether at room
temperature. The mixture was stirred for 2 min, and thereafter, ethyl
isocyanate (32.8 µL, 0.408 mmol) was added to the reaction mixture
in one portion. The reaction was continued for an hour at room
temperature, 4.0 mL of water was then added, and the ether layer
was separated. After drying over sodium sulfate and filtering, the
filtrate was evaporated to remove solvent. Thereafter, the residue
was subjected to chromatography on a silica gel plate (EtOAc/
hexane ) 1/3) to afford products 8b and 11b. Product 8b (14.8
27
(9.5 mg, 64.4%), [R]D -29.0° (c ) 0.10, CHCl3); ee ) 100%;
1H NMR (CDCl3) δ 7.87 (br s, 1H, C7-NHCOO), 7.33-6.70 (m,
7H, Ar-H), 5.79 (d, J ) 1.80 Hz, 1H, C2-H), 4.22, 3.79 (AB,
Jgem ) 6.84 Hz, 2H, C4-H), 2.37-2.01 (m, 2H, C10-H), 2.31 (s,
3H, Ar-CH3) and 1.51 (s, 3H, C5-CH3) ppm; CI-MS (CH4) m/z
326 (MH+). Anal. (C19H19NO4) N; Calcd C 70.14, N 5.89; Found
C 69.49, N 5.46.
(-)-(3aS)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-b]benzo-
furan-5-yl N-(p-Isopropylphenyl)carbamate (10a) and (+)-(5S)-
5-Methyl-4,5-dihydro-2,5-methano-1,3-benzodioxepin-7-yl N-(p-
Isopropylphenyl)carbamate (13a). Under a nitrogen atmosphere,
two small pieces of sodium (about 1 mg) were added into a solution
of mixture of 6a and 7a (16 mg, 0.083 mmol) in 5 mL of anhydrous
ether at room temperature. The mixture was stirred for 2 min, and
p-isopropylphenyl isocyanate (14 µL, 0.086 mmol) was added to
the mixture of 6a and 7a in one portion. The reaction was continued
for an additional 45 min at room temperature; thereafter, 4 mL of
water was added and the ether layer separated. After drying over
sodium sulfate and filtering, the filtrate was evaporated to remove
solvent. The residue was chromatographed on silica gel plate
(EtOAc/hexane ) 1/3) to afford products 10a and 13a. Product
10a (8 mg, 54.4%): [R]D26 -91.4° (c ) 0.35, CHCl3); ee ) 100%;
1H NMR (CDCl3) δ 7.31-6.71 (m, 8H, Ar-H, HNCOO), 5.80 (s,
26
1
mg, 83.1%): [R]D +92.5° (c ) 0.08, CHCl3); ee ) 100%; H
NMR (CDCl3) δ 6.87-6.60 (m, 3H, Ar-H), 5.77 (s, 1H, C8a-
H), 4.89 (br s, 1H, NH), 4.06-3.56 (m, 2H, C2-H), 3.23 (m, 2H,
C5-CH2NHCOO), 2.13-1.91 (m, 2H, C3-H), 1.47 (s, 3H, C3a-
CH3), and 1.14 (t, 3H, C5-CH3CH2NHCOO) ppm; CI-MS (CH4)
m/z 264 (MH+), 193, 175, and 72. Anal. (C14H17NO4) C, H; N,
26
calcd.5.32, found 4.78. Product 11b (10.4 mg, 58.4%): [R]D
+10.0° (c ) 0.05, CHCl3); ee ) 100%. 1H NMR (CDCl3) δ 6.82-
6.67 (m, 3H, Ar-H), 5.69 (d, J ) 1.80 Hz, 1H, C2-H), 4.88 (br
s, 1H, C7-NHCOO), 4.17, 3.70 (AB, Jgem)7.20 Hz, 2H, C4-H),
3.22 (m, 2H, C-7CH2NHCOO), 2.19-1.19 (m, 2H, C10-H), 1.41

Inhibition of Acetyl- and Butyrylcholinesterase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2183
1H, C8a-H), 4.09-3.59 (m, 2H, C2-H), 2.82 (septet, J ) 5.24
Hz, 1H, CHMe2), 2.19-1.96 (m, 2H, C3-H), 1.49 (s, 3H, C3a-
CH3), and 1.18 (d, J ) 5.24 Hz, 6H, CH3CCH3) ppm; CI-MS (CH4)
m/z 354 (MH+). Anal. (C21H23NO4) H; alcd C 71.37, N 3.94; Found
C 70.86, N 3.30. Product 13a (9 mg, 61.2%): [R]D26 +50.0° (c )
0.2, CHCl3); ee ) 100%; 1H NMR (CDCl3) δ 7.30-6.72 (m, 8H,
Ar-H, HNCOO), 5.71 (d, J ) 1.80 Hz, 1H, C2-H), 4.17, 3.70
(AB, Jgem ) 5.40 Hz, 2H, C4-H), 2.81 (septet, J ) 5.22 Hz, 1H,
CHMe2), 2.19-1.94 (m, 2H, C10-H), 1.48 (s, 3H, C5-CH3), and
1.19 (d, J ) 5.22 Hz, 6H, CH3CCH3) ppm; CI-MS (CH4) m/z 354
(MH+). Anal. (C21H23NO4) C, H; N, calcd 3.96, found 4.56.
X-ray Crystallography. A clear colorless crystal of dimensions
0.30 × 0.23 × 0.12 mm2 was mounted on glass fiber using a small
amount of Epoxy. Data were collected on a Bruker three-circle
platform diffractometer equipped with a SMART 6000 CCD
detector. The crystals were irradiated by use of a rotating anode
Cu KR source (λ ) 1.541 78) with incident beam Go¨bel mirrors.
Data collection was performed and the unit cell was initially refined
with SMART (version 5.625).53a Data reduction was performed with
SAINT (version 6.36A)53b and XPREP (version 6.12).53c Corrections
were applied for Lorentz, polarization, and absorption effects by
use of SADABS (version 2.03).53d The structure was solved and
refined with the aid of the programs in the SHELXTL-plus (version
6.10) system of programs.53e The full-matrix least-squares refine-
ment on F2 included atomic coordinates and anisotropic thermal
parameters for all non-H atoms. The H atoms were included by
use of a riding model. The absolute configuration of C3 was
established by making reference to unchanging chiral centers (C19,
C21, and C24) in the synthetic procedure, with a resulting Flack
parameter of -0.3(2).53f The X-ray data of compound 3 also can
be found at the Cambridge Crystallographic Data Center (CCDC),
http://www.ccdc.cam.ac.uk/. The reference number of this crystal
is 285734.
Quantitation of Anticholinesterase Activity. The action of
enantiomers 8-13 and compounds 17b, 18b, 22b, and 23b to inhibit
the ability of freshly prepared human AChE and BChE to
enzymatically degrade their respective specific substrates, acetyl-
(â-methyl)thiocholine and s-butyrylthiocholine (0.5 mmol/L) (Sigma
Chemical Co., St. Louis, MO), was quantified.13-17,19-21 Samples
of AChE and BChE were derived from freshly collected human
whole red blood cells and plasma, respectively. 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 concentration range that spanned
from 0.3 nM to 30 µM. Tween 80/EtOH was diluted to in excess
of 1:5000. No inhibitory action on either AChE or BChE was
detected in separate experiments where the ChEI activity of the
known compound 15a was quantified in excess and without Tween
80/EtOH.
(+)-(3aR)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-b]benzo-
furan-5-yl N-(p-Isopropylphenyl)carbamate (10b) and (-)-(5R)-
5-Methyl-4,5-dihydro-2,5-methano-1,3-benzodioxepin-7-yl N-(p-
Isopropylphenyl)carbamate (13b). Under a nitrogen atmosphere,
two small pieces of sodium (about 1 mg) were added into a solution
of mixture of 6b and 7b (18 mg, 0.094 mmol) in 5 mL of anhydrous
ether at room temperature. The mixture was stirred for 2 min, and
then p-isopropylphenyl isocyanate (14.6 µL, 0.096 mmol) was
added in one portion. The reaction was continued for a further 45
min at room temperature, and then 4 mL of water was added and
the ether layer separated. After drying over sodium sulfate and
filtering, the filtrate was evaporated to remove solvent. The residue
was chromatographed on a silica gel plate (EtOAc/hexane ) 1/3)
to afford products 10b and 13b. Product 10b (13.7 mg, 82.1%):
27
1
[R]D +91.4° (c ) 0.14, CHCl3); ee ) 100%; H NMR (CDCl3)
δ 7.31-6.71 (m, 8H, Ar-H, HNCOO), 5.80 (s, 1H, C8a-H),
4.09-3.59 (m, 2H, C2-H), 2.82 (septet, J ) 5.24 Hz, 1H, CHMe2),
2.19-1.96 (m, 2H, C3-H), 1.49 (s, 3H, C3a-CH3), and 1.18 (d,
J ) 5.24 Hz, 6H, CH3CCH3) ppm; CI-MS (CH4) m/z 354 (MH+).
Anal. (C21H23NO4‚1/8H2O) C, H, N. Product 13b (10.1 mg,
27
1
60.4%): [R]D -48.0° (c ) 0.1, CHCl3); ee ) 100%; H NMR
(CDCl3) δ 7.30-6.72 (m, 8H, Ar-H, HNCOO), 5.72 (d, J ) 1.80
Hz, 1H, C2-H), 4.17, 3.70 (AB, Jgem ) 5.40 Hz, 2H, C4-H), 2.81
(septet, J ) 5.22 Hz, 1H, CHMe2), 2.19-1.94 (m, 2H, C10-H),
1.48 (s, 3H, C5-CH3), and 1.19 (d, J ) 5.22 Hz, 6H,CH3CCH3)
ppm; CI-MS (CH4) m/z 354 (MH+). Anal. (C21H23NO4) H, N; C,
calcd 71.37, found 70.75.
For the preparation of 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). Plasma was
carefully checked to ensure an absence of hemolysis. For AChE
preparation, 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 were diluted with an additional
19 volumes of buffer to a final dilution of 1:200.
(+)-(3aR)-1,3a,8-Trimethyl-1,2,3,3a,8,8a-hexahydropyrrolo-
[2,3-b]indol-5-ol (14b). Compound 14b was synthesized, according
to the procedure for its enantiomer, from N-methylphenetidine.14
(+)-(3aR)-1,3a,8-Trimethyl-1,2,3,3a,8,8a-hexahydropyrrolo-
[2,3-b]indol-5-yl N-(2′-Methylphenyl)carbamate (17b). Com-
pound 17b was made, according to the procedure for its antipode,
26
from o-tolyl isocyanate and (+)-eseroline:21 [R]D +68.2° (c )
1
0.2, CHCl3); H NMR and MS were the same as reported for its
Analysis of anticholinesterase activity was undertaken by utilizing
a 25 µL sample of each enzyme preparation, 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 at room
temperature) and then were incubated with their respective sub-
strates and with 5,5′-dithiobis(2-nitrobenzoic acid) (25 min, 37 °C).
The substrate/enzyme interaction was immediately halted by the
addition of excess enzyme inhibitor (physostigmine, 1 × 10-5 M)
and production of a yellow thionitrobenzoate anion was then
measured by spectrophotometry 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 (15a)], and the associated alteration in
absorbance was subtracted from that observed through the con-
centration range of each test compound. Each agent was analyzed
on four separate occasions and assayed alongside physostigmine
(15a), as a control and external standard whose activity we have
previously reported.13-17,19-21 The mean enzyme activity at each
concentration of test compound was then expressed as a percent of
the activity in the absence of compound. This was transformed into
a logit format (where logit ) ln (% activity/100 minus % activity))
and then was plotted as a function of its log concentration. Inhibitory
activity was calculated as an IC50, defined as the concentration of
compound (nanomolar) required to inhibit 50% of enzymatic
activity, which was determined from a correlation between log
enantiomer.21 Anal. (C21H25N3O2) H; Calcd C 71.70, N 11.95; found
C 70.08, N 11.45.
(+)-(3aR)-1,3a,8-Trimethyl-1,2,3,3a,8,8a-hexahydropyrrolo-
[2,3-b]indol-5-yl N-(4′-Isopropylphenyl)carbamate (18b). Com-
pound 18b was synthesized, according to the procedure for its
antipode, from p-isopropylphenyl isocyanate and (+)-eseroline:21
[R]D26 +67.5° (c ) 0.2, CHCl3); 1H NMR and MS were the same
as reported for its enantiomer.21 Anal. (C23H29N3O2) H, N; C, calcd
72.79, found 71.70.
(+)-(3aR)-3a,8-Dimethyl-2,3,3a,8a-tetrahydrofuro[2,3-b]indol-
5-ol (19b). Compound 19b was made according to the procedure
for synthesis of its enantiomer.14,17
(+)-(3aR)-3a,8-Dimethyl-2,3,3a,8a-tetrahydrofuro[2,3-b]indol-
5-yl N-(2′-Methylphenyl)carbamate (22b). Compound 22b was
synthesized, according to the procedure for its antipode, from o-tolyl
26
isocyanate and (+)-eseroline:21 [R]D +27.5° (c ) 0.2, CHCl3);
1H NMR and MS were the same as reported for its enantiomer.20
Anal. (C20H22N2O3) C, H; N, calcd 8.28, found 7.84.
(+)-(3aR)-3a,8-Dimethyl-2,3,3a,8a-tetrahydrofuro[2,3-b]indol-
5-yl N-(4′-Isopropylphenyl)carbamate (23b). Compound 23b was
made, according to the procedure for its antipode, from p-
26
isopropylphenyl isocyanate and (+)-eseroline:16 [R]D +32.0° (c
) 0.2, CHCl3); 1H NMR and MS were the same as reported for its
enantiomer.17 Anal. (C22H26N2O3) C, H; N, calcd 7.64, found 7.13.

2184 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Luo et al.
(10) Greig, N. H.; Pei, X. F.; Soncrant, T. 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.
(11) Greig, N. H.; Sambamurti. K.; Yu, Q. S.; Brossi, A.; Bruinsma, G.;
Lahiri, D. K. An overview of phenserine tartrate, a novel acetyl-
cholinesterase inhibitor for the treatment of Alzheimer’s disease. Curr.
Alzheimer Res. 2005, 2, 281-291
(12) Greig, N. H.; Sambamurti, K.; Yu, Q. S.; Perry, T. A.; Holloway,
H. W.; Haberman, F,; Brossi, A,; Ingram, D. K.; Lahiri, D. K.
Butyrylcholinesterase: its selective inhibition and relevance to
Alzheimer’s disease. In Butyrylcholinesterase: Its Function and
Inhibition; Giacobini, E., Ed.; Martin Dunitz Ltd.: London, 2003;
pp 69-90.
(13) Pei, X. F.; Greig, N. H.; Bi, S.; Broosi, A.; Toome, V. Inhibition of
Human Acetylcholinesterase by Unnatural (+)-(3aR)-N1-Nor-
physostigmine and Arylcarbamate Analogues. Med. Chem. Res. 1995,
5, 265-270.
(14) Yu, Q. S.; Luo, W.; Holloway, H. W.; Utsuki, T.; Perry, T. A.; Lahiri,
D. K.; Greig, N. H.; Brossi, A. Racemic N1-Norphenserine and Its
Enantiomers: Unpredicted Inhibition of Human Acetyl- and Butylyl-
cholinesterase and â-Amyloid Precursor Protein in Vitro. Heterocycles
2003, 619, 529-539.
(15) Yu, Q. S.; Liu, C.; Brzostowska, M.; Chrisey, L.; Brossi, A.; Greig,
N. H. Physovenines: Efficient Synthesis of (-)- and (+)-Physovenine
and Synthesis of Carbamate Analogues of (-)-Physovenine. Anti-
cholinesterase Activity and Analgesic Properties of Optically Active
Physovenines. HelV. Chim. Acta 1991, 74, 761-766.
(16) Brzostowska, M.; He, X. S.; Greig, N. H.; Rapoport, S.; Brossi, A.
Phenylcarbamates of (-)-Eceroline and (-)-Physovenol: Selective
Inhibition of Acetyl and, or Butyrylcholinesterases by Phenylcar-
bamates. Med. Chem. Res. 1992, 2, 238-246.
(17) Luo, W.; Yu, Q. S.; Zhan, M.; Parrish, D.; Deschamps, J. R.;
Kulkarrni, S. S.; Holloway, H. W.; Alley, G.M.; Lahiri, D. K.; Brossi,
A.; Greig, N. H. Novel Acetylcholinesterase based on the Molecular
Skeletons of Furobenzofuran and Methanobenzodioxepine. J. Med.
Chem. 2005, 48, 986-994.
(18) Luo, W.; Yu, Q. S.; Holloway, H. W.; Parrish D. A.; Greig, N. G.;
Brossi, A. Syntheses of Tetrahydrofurobenzofurans and Dihydro-
methanobenzodioxepines from 5-Hydroxy-3-methyl-3H-benzofuran-
2-one. Rearrangement and Ring Expansion under Reductive Condi-
tions on Treatment with Hydrides. J. Org. Chem. 2005, 70, 6171-
6176.
(19) Yu, Q. S.; Holloway, H. W.; Utsuki, T.; Brossi, A.; Greig, N. H.
Phenserine-based synthesis of novel selective inhibitors of butyryl-
cholinesterase for Alzheimer’s disease. J. Med. Chem., 1999, 42,
1855-1861.
(20) Yu, Q. S.; Zhu, X.; Holloway, H. W.; Whittaker, N. F.; Utsuki, T,;
Brossi, A.; Greig, N. H. Anticholinesterase activity of compounds
related to geneserine tautomerssN-oxides and 1,2-oxazines. J. Med.
Chem. 2002, 45, 3684-3691.
(21) Yu, Q. S.; Holloway, H. W.; Flippen-Anderson, F.; Hoffman, B.;
Brossi, A.; Greig, N. H. Methyl analogues of the experimental
Alzheimer drug, phenserine: synthesis and structure/activity relation-
ships for acetyl- and butyrylcholinesterase inhibitory action. J. Med.
Chem. 2001, 44, 4062-4071.
(22) Kamal, M. A.; Greig, N. H.; Alhomida, A. S.; Al-Jafari, A. A. Human
acetylcholinesterase inhibition kinetics of the experimental Alzheimer
therapeutic, tolserine. Biochem. Pharmacol. 2000, 60, 561-570.
(23) Al-Jafari, A. A.; Kamal, M. A.; Alhomida, A. S.; Greig, N. H. Brain
acetylcholinesterase inhibition kinetics of two experimental Alz-
heimer’s disease drugs, phenserine and tolserine, in the rat. J.
Biochem. Mol. Biol. Biophys. 2000, 4, 323-335.
(24) Kamal, M. A.; Al-Jafari, A. A.; Greig, N. H. A new, simple and
economical approach to analyze the inhibition kinetics of the enzyme,
acetylcholinesterase, by using the experimental Alzheimer drug,
tolserine. Emir. Med. J. 2002, 20, 333-337.
(25) Marshall, G. R.; Barry, C. D. Functional Representation of Molecular
Volume for Computer-Aided Drug Design. Abstracts from the
American Crystallographic Association, Honolulu, HI, 1979.
(26) Sufrin, J. R.; Dunn, D. A.; Marshall, G. R. Steric Mapping of the
L-Methionine Binding Site of ATP: L-Methionine S-Adenosyltrans-
ferase. Mol. Pharmacol. 1981, 19. 307-313.
(27) Brufani, M.; Filocamo, L. Rational design of cholinesterase inhibitors.
In Cholinesterases and Cholinesterase Inhibitors; Giacobini, E., Ed.;
Martin Dunitz Ltd.: London, 2000; pp 27-46.
(28) Reiner, E.; Radic, Z. Mechanism of action of cholinesterase inhibitors.
In Cholinesterases and Cholinesterase Inhibitors; Giacobini, E., Ed.;
Martin Dunitz Ltd.: London, 2000; pp 27103-119.
concentration and logit activity. Only results obtained from cor-
relation coefficients of r2 g -0.98 were considered acceptable.
Studies that did not obtain this threshold were repeated.
Computer-Aided Molecular Modeling. Computer-aided mo-
lecular modeling was undertaken by using Sybyl version 7.0 (Tripos
Inc., St. Louis, MO). The numbering of the amino acid residues is
based on that for TcAChE. Molecular volume calculations were
performed by using multiple volume comparison routine. The
atomic coordinates of the transition state of TcAChE were obtained
from the protein data bank (PDB entry 1AMN)38 and were used
for transition-state studies. In undertaking this, (i) molecules of
water were removed, (ii) the small molecule m-(N,N,N-trimethyl-
ammonio)-2,2,2-trifluoroacetophenone (TMTFA) was extracted, and
(iii) the carbonyl carbon of TMTFA was retained in the tetrahedral
conformation. (iv) The carbonyl carbon of the inhibitor, (-)-(3aS)-
N-(2′-methylphenyl)carbamoyl phenol of furobenzofuran (9a), was
modified into the tetrahedral conformation and then superimposed
with that of TMTFA, keeping both phenyl groups on the same side
and, as much as possible, in a superimposed conformation.
Thereafter, compound 9a was merged into the active domain of
TcAChE. The covalent bond between carbonyl carbon of compound
9a and γ-O of Ser200 was created. To maintain the hydrogen bonds
associated with acylation, the distances between the NH of Gly118
,
Gly119, Ala201 and oxygen of the ligand carbonyl, γ-O of Ser200
and a single H, the hydrogen and N of His440, the NH of His440
and oxygen of Glu327 were constrained. All the residues outside a
radius of 10 Å from the compound 9a were aggregated. The energy
of the enzyme-inhibitor complex was minimized with the conjugate
gradient algorithm.
Acknowledgment. We are grateful to the Medicinal Chem-
istry Section, National Institute on Drug Abuse, NIH, for use
of chemical characterization and molecular modeling equipment.
The research was supported, in part, by the Intramural Research
Program of the National Institute on Aging, NIH. W.L. and
Q.-S.Y. are supported via the MedStar Research Institute,
Baltimore, MD, and S.S.K. is supported through a National
Institutes of Health Visiting Fellowship. D.L. is funded via NIH
grants (AG18379 and AG 18884) and the Alzheimer’s Associa-
tion.
Supporting Information Available: X-ray crystallographic data
for compound 3 in CIF format; chiral HPLC spectra of compounds
1, 4, and 5; and elemental analysis data. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Drachman, D. A.; Leavitt, J. Human memory and the cholinergic
system. A relationship to aging? Arch. Neurol. 1974, 30, 113-21.
(2) Small, D. H. Do acetylcholinesterase inhibitors boost synaptic scaling
in Alzheimer’s disease? Trends Neurosci. 2004, 27, 245-9.
(3) Lahiri, D. K.; Rogers, J. T.; Sambamurti, K.; Greig, N. H. Rationale
for the development of cholinesterase inhibitors as anti-Alzheimer
agents. Curr. Pharm. Des. 2004, 10, 3111-9.
(4) Cummings, J. L. Cholinesterase inhibitors: A new class of psycho-
tropic compounds. Am. J. Psychiatry 2000, 157, 4-15.
(5) Courtney, C.; Farrell, D.; Gray, R.; Hills, R.; Lynch, L.; Sellwood,
E.; Edwards, S.; Hardyman, W.; Raftery, J.; Crome, P.; Lendon, C.;
Shaw, H.; Bentham, P. AD2000 Collaborative Group. Long-term
donepezil treatment in 565 patients with Alzheimer’s disease
(AD2000): randomised double-blind trial. Lancet 2004, 363 (9427),
2105-15.
(6) Lopez, O. L.; Becker, J. T.; Saxton, J.; Sweet, R. A.; Klunk, W.;
DeKosky, S. T. Alteration of a clinically meaningful outcome in the
natural history of Alzheimer’s disease by cholinesterase inhibition.
J. Am. Geriatr. Soc. 2005, 53, 83-7.
(7) Conworth, E. In The Alkaloids; Manskel, R. H. F., Ed.; Academic:
New York, 1965; Vol. 8, pp 27-46.
(8) (a) Robinson, B. In The Alkaloids; Manskel, R. H. F., Ed.;
Academic: New York, 1967; Vol. 10, pp 383-401. (b) Robinson,
B. In The Alkaloids; Manskel, R. H. F., Ed.; Academic: New York,
1971; Vol. 13, pp 213-226.
(9) Brossi, A. Bioactive Alkaloids. 4. Results of Recent Investigations
with Colchicine and Physostigmine. J. Med. Chem. 1990, 33, 2311-
2319.
(29) Greenblatt, H. M.; Dvir, H.; Silman, I.; Sussman, J. L. Acetylcho-
linesterase: a multifaceted target for structure-based drug design of
anticholinesterase agents for the treatment of Alzheimer’s disease.
J. Mol. Neurosci. 2003, 20, 369-83.

Inhibition of Acetyl- and Butyrylcholinesterase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2185
(30) Krasiski, A.; Radi, Z.; Manetsch, R.; Raushel, J.; Taylor, P.; Sharpless,
K. B.; Kolb H. C. In Situ Selection of Lead Compounds by Click
Chemistry: Target-Guided Optimization of Acetylcholinesterase
Inhibitors J. Am. Chem. Soc. 2005, 127, 6686-6692.
(43) Porschke, D.; Creminon, C.; Cousin, X.; Bon. C.; Sussman, J. L.;
Silman, I. Electrooptical measurements demonstrate a large permanent
dipole moment associated with acetylcholinesterase. Biophys J. 1996,
4, 1603-8.
(44) Felder, C. E.; Botti, S. A.; Lifson, S.; Silman, I,; Sussman, J. L.
External and internal electrostatic potentials of cholinesterase models.
J. Mol. Graphics Modell. 1997, 15, 318-27 and 335-7.
(45) Schowen, R. L. In Transition States of Biochemical Processes;
Gandour, R. D., Schowen, R. L., Eds.; Plenum: New York, 1978;
pp 555-578.
(46) Harel, M.; Quinn, D. Q.; Nair, H. K.; Silman, I.; Sussman, J. L. The
X-ray Structure of a Transition State Analogue Complex Reveals
the Molecular Origins of the Catalytic Power and Substrate Specificity
of Acetylcholinesterase. J. Am. Chem. Soc. 1996, 118, 2340-
2346.
(47) Perola, E.; Cellai, L.; Lamba, D.; Filocamo, L.; Brufani, M. Long
Chain Analogues of Physostigmine as Potential Drugs for Alz-
heimer’s Disease: New Insights Into the Mechanism of Action in
the Inhibition of Acetylcholinesterase. Biochim. Biophys. Acta 1997,
1343, 41.
(48) Bartolucci, C.; Perola, E.; Cellai, L.; Brufani, M.; Lamba, D. “Back
Door” Opening Implied by the Crystal Structure of Carbamoylated
Acetylcholinesterase. Biochemistry 1999, 38, 5714.
(49) Koellner, G.; Steiner, T.; Millard, C. B.; Silman, I.; Sussuman, J. L.
A Neutral Molecule in Cation-binding Site: Specific Binding of a
PEG-SH to Acetylcholinesterase from Torpedo californica. J. Mol.
Biol. 2002, 320, 721-725.
(50) Yu, Q. S.; Greig, N. H.; Holloway, H. W.; Brossi, A. 4′-Hydroxy-
phenylcarbamates of (3aS)-eseroline and (3aS)-N(1)-noreseroline:
potential metabolites of the Alzheimer’s anticholinesterase drug
phenserine. Heterocycles 1999, 50, 95-102.
(51) Francis, P. T.; Nordberg, A.; Arnold, S. E. A preclinical view of
cholinesterase inhibitors in neuroprotection: do they provide more
than symptomatic benefits in Alzheimer’s disease? Trends Pharma-
col. Sci. 2005, 26, 104-11.
(31) Mun˜oz-Ruiz, P.; Rubio, L.; Garc´ıa-Palomero, E.; Dorronsoro, I.;
Monte-Milla´n, M.; Valenzuela, R.; Usa´n, P.; Austra, C.; Bartolini,
M.; Andrisano, V.; Bidon-Chanal, A.; Orozco, M.; Luque, F. J.;
Medina, M.; Mart´ınez, A. Design, synthesis and biological evaluation
of dual binding site acetylcholinesterase inhibitors: new disease
modifying agents for Alzheimer’s disease. J. Med. Chem. 2005, 48,
7223-7233.
(32) Otoguro, K.; Kuno, F.; Omura, S. Arisugacins, selective acetylcho-
linesterase inhibitors of microbial origin. Pharmacol. Ther. 1997,
76, 45-54.
(33) Soreq, H.; Seidman, S. Acetylcholinesterasesnew roles for an old
actor. Nat. ReV. Neurosci. 2001, 2, 294-302.
(34) Massoulie, J. Molecular forms and anchoring of acetylcholinesterase.
In Cholinesterases and Cholinesterase Inhibitors; Giacobini, E., Ed.;
Martin Dunitz Ltd.: London, 2000; pp 81-102.
(35) Soreq, H.; Gnatt, A.; Loewenstein, Y.; Neville, L. F. Excavations
into the active-site gorge of cholinesterases. Trends Biochem. Sci.
1992, 17, 353-8.
(36) Dvir, H.; Wong, D. M.; Harel, M.; Barril, X.; Orozco, M.; Luque,
F. J.; Munoz-Torrero, D.; Camps, P.; Rosenberry, T. L.; Silman, I.;
Sussman, J. L. 3D structure of Torpedo californica acetylcholinest-
erase complexed with huprine X at 2.1 A resolution: kinetic and
molecular dynamic correlates. Biochemistry 2002, 41, 2970-81.
(37) Silman, I.; Millard, C. B.; Ordentlich, A.; Greenblatt, H. M.; Harel,
M.; Barak, D.; Shafferman, A.; Sussman, J. L. A preliminary
comparison of structural models for catalytic intermediates of
acetylcholinesterase. Chem.-Biol. Interact. 1999, 119-120, 43-52.
(38) Silman, I.; Harel, M.; Axelsen, P.; Raves, M.; Sussman, J. L. Three-
dimensional structures of acetylcholinesterase and of its complexes
with anticholinesterase agents. Biochem. Soc. Trans. 1994, 22, 745-
9.
(39) Nachon, F.; Asojo, O, A.; Borgstahl, G. E. O.; Masson, P. and
Lockridge, O. Role of waterin aging of human butylcholinesterase
inhibited by echothiophate: the crystal structure suggest two alterna-
tive mechanisms of aging. Biochemistry 2005, 44, 1154. (PDB
codes: 1XLW, 1XLV, 1XLU).
(40) Nicolet, Y.; Lockridg, O.; Masson, P.; Fontecilla-Camps, J. C.;
Nachon, F. Crystal Structure of Human Butyrylcholinesterase and
of its Complexes with Substrate and Products. J. Biol. Chem. 2003,
278, 41141 (PDB codes 1PO1, 1POM, 1POP, and 1POQ).
(41) Nachon, F.; Nicolet, Y.; Viguie´, N.; Masson, P.; Fontecilla-Camps,
J. C.; Lockridg, O. Engineering of a monomeric and low-glycosylated
form of human butyrylcholinrsterase: expression, purification,
characterization and crystallization. Eur. J. Biochem. 2002, 269, 630.
(42) Inestrosa, N. C.; Sagal, J. P.; Colombres, M. Acetylcholinesterase
interaction with Alzheimer amyloid beta. Subcell. Biochem. 2005,
38, 299-317.
(52) Hashimoto, M.; Kazui, H.; Matsumoto, K.; Nakano, Y.; Yasuda, M.;
Mori, E. Does donepezil treatment slow the progression of hippo-
campal atrophy in patients with Alzheimer’s disease? Am. J.
Psychiatry 2005, 162, 676-82.
(53) (a) SMART version 5.625; Bruker AXS Inc.: Madison, WI, 2001.
(b) SAINT version 6.36A; Bruker AXS Inc.: Madison, WI, 2002.
(c) XPREP version 6.12; Bruker AXS Inc.: Madison, WI, 2001. (d)
SADABS version 2.03; Bruker AXS Inc.: Madison, WI, 2000. (e)
SHELXTL version 6.10; Bruker AXS Inc.: Madison, WI, 2000. (f)
Flack, H. D. Acta Crystallogr. 1983, A39, 876-881.
JM050578P