Carbamates with differential mechanism of inhibition toward acetylcholinesterase and butyrylcholinesterase

Article abstract of DOI:10.1021/jm8002075

Most carbamates are pseudoirreversible inhibitors of cholinesterases. Phenothiazine carbamates exhibit this inhibition of acetylcholinesterase but produce reversible inhibition of butyrylcholinesterase, suggesting that they do not form a covalent bond with the catalytic serine. This atypical inhibition is attributable to π-π interaction of the phenothiazine moiety with F329 and Y332 in butyrylcholinesterase. These residues are in a helical segment, referred to here as the E-helix because it contains E325 of the catalytic triad. The involvement of the E-helix in phenothiazine carbamate reversible inhibition of butyrylcholinesterase is confirmed using mutants of this enzyme at A328, F329, or Y332 that show typical pseudoirreversible inhibition. Thus, in addition to various domains of the butyrylcholinesterase active site gorge, such as the peripheral anionic site and the π-cationic site of the Ω-loop, the E-helix represents a domain that could be exploited for development of specific inhibitors to treat dementias.

Full text of DOI:10.1021/jm8002075

4200
J. Med. Chem. 2008, 51, 4200–4212
Carbamates with Differential Mechanism of Inhibition Toward Acetylcholinesterase and
Butyrylcholinesterase
Sultan Darvesh,*,†,‡,§ Katherine V. Darvesh,§ Robert S. McDonald,§ Diane Mataija,§ Ryan Walsh,‡ Sam Mothana,§
Oksana Lockridge,| and Earl Martin§
Department of Medicine (Neurology), Dalhousie UniVersity, Halifax, NoVa Scotia, Canada, Department of Anatomy and Neurobiology,
Dalhousie UniVersity, Halifax, NoVa Scotia, Canada, Department of Chemistry, Mount Saint Vincent UniVersity, Halifax, NoVa Scotia, Canada,
Eppley Institute, UniVersity of Nebraska Medical Center, Omaha, Nebraska
ReceiVed February 27, 2008
Most carbamates are pseudoirreversible inhibitors of cholinesterases. Phenothiazine carbamates exhibit this
inhibition of acetylcholinesterase but produce reversible inhibition of butyrylcholinesterase, suggesting that
they do not form a covalent bond with the catalytic serine. This atypical inhibition is attributable to π-π
interaction of the phenothiazine moiety with F329 and Y332 in butyrylcholinesterase. These residues are in
a helical segment, referred to here as the E-helix because it contains E325 of the catalytic triad. The
involvement of the E-helix in phenothiazine carbamate reversible inhibition of butyrylcholinesterase is
confirmed using mutants of this enzyme at A328, F329, or Y332 that show typical pseudoirreversible
inhibition. Thus, in addition to various domains of the butyrylcholinesterase active site gorge, such as the
peripheral anionic site and the π-cationic site of the Ω-loop, the E-helix represents a domain that could be
exploited for development of specific inhibitors to treat dementias.
Introduction
beenusedtotreatotherdisordersofcholinergicneurotransmission.8–12
Furthermore, selection of unique carbamate molecular properties
has proved valuable in targeting specific disorders. Thus, a
carbamate such as pyridostigmine is used to treat motor
weakness in myasthenia gravis because this molecule, because
of its polar nature, does not readily cross the blood-brain barrier
and predominantly targets cholinesterases in the periphery.8
Hydrophobic carbamates, such as rivastigmine9 and
phenserine,12 readily cross the blood-brain barrier to inhibit
cholinesterases in the central nervous system, leading to
improved cognition in dementias.
Derivatives of phenothiazine are known to inhibit cholinest-
erases, especially BuChE,13–15 and one BuChE-specific phe-
nothiazine inhibitor, ethopropazine, has been shown to improve
cognitive function.16 To explore further structure-activity
relationships for inhibition of cholinesterases by phenothiazines,
a number of phenothiazine carbamate derivatives were synthe-
sized and evaluated for their inhibitory properties toward human
cholinesterases. Although carbamates are generally known to
inhibit AChE and BuChE by a common mechanism involving
covalent bond formation at the active site serine (Figure 1),
phenothiazine carbamates show reversible inhibition of BuChE
while exhibiting the more common pseudoirreversible inhibition
of AChE. It has been suggested that phenothiazine derivatives
inhibit BuChE by binding to F329 and Y332 (Figure 1) near
the mouth of the active site gorge.15,17 Such an interaction with
the corresponding F338 and Y341 in AChE is not possible
because of steric interference by Y33717 (Figure 1). We therefore
hypothesized that phenothiazine carbamates exhibited reversible
inhibition of BuChE because of the interaction between F329
and Y332 of BuChE with the benzene rings of the phenothiazine
tricycle. This would preclude the carbamate from reaching, and
carbamylating, S198 of the BuChE catalytic triad. To test this
hypothesis, a number of BuChE mutants were examined that
had alterations in the helical segment from E325 of the catalytic
triad to Y332 at the mouth of the active site gorge. For ease of
reference, this helical portion of the active site gorge will be
referred to as the E-helix because it contains a component of
Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcho-
linesterase (BuChE, EC 3.1.1.8) are serine hydrolases that
catalyze the hydrolysis of acetylcholine, thus regulating cho-
linergic neurotransmission.1–3 In disorders such as Alzheimer’s
disease (AD),a where there is diminished cholinergic activity,
treatment with cholinesterase inhibitors has been shown to be
beneficial.4 A number of different types of cholinesterase
inhibitors are known and have been found to affect these
enzymes in a variety of ways. Compounds like donepezil and
galantamine produce reversible inhibition where the inhibitor
interferes with substrate hydrolysis by transiently interacting
with the enzyme, without forming a covalent bond.5–8 On the
other hand, organophosphates (such as diisopropylfluorophos-
phate) inhibit the same enzymes irreversibly by forming a strong
covalent bond with the serine of the catalytic triad.1,8 Carbamates
tend to fall between these extremes in that they carbamylate
the catalytic serine, but the inhibition is “pseudoirreversible”
or “slowly reversible” because the acylated intermediate is
slowly hydrolyzed to reactivate the enzyme.8
Cholinesterase inhibitors, including carbamates, have gained
much attention since they have been successfully used in the
treatment of a number of diseases involving cholinergic
dysfunction. In fact, a carbamate, the naturally occurring
physostigmine, was the first clinically employed cholinesterase
inhibitor, used by Laqueur over a century ago to treat glaucoma.8
Since then, many carbamates have been synthesized and have
* To whom correspondence should be addressed. Phone: 902-473-2490.
Fax: 902-473-7133. E-mail: sultan.darvesh@dal.ca. Address: Room 1308,
Camp Hill Veterans’ Memorial Building 1, 5955 Veterans’ Memorial Lane,
Halifax, Nova Scotia, Canada, B3H 2E1.
†
Department of Medicine (Neurology), Dalhousie University.
Department of Anatomy and Neurobiology, Dalhousie University.
Department of Chemistry, Mount Saint Vincent University.
Eppley Institute, University of Nebraska Medical Center.
‡
§
|
a Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholinest-
erase; AD, Alzheimer’s disease; DTNB, 5,5′-dithiobis (2-nitrobenzoic acid);
MMFF, Merck molecular force field; SMILES, simplified molecular input
line entry system.
10.1021/jm8002075 CCC: $40.75
2008 American Chemical Society
Published on Web 06/21/2008

Carbamates with Mechanism of Inhibition for AChE and BuChE
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4201
Figure 1. Active site regions of human acetylcholinesterase and human butyrylcholinesterase. The figure was generated using crystal structure of
acetylcholinesterase5 (PDB ID: 1EVE) and butyrylcholinesterase33 (PDB ID: 1POI) obtained from the protein databank34 and using PyMol.35
the enzyme catalytic triad, E325 in BuChE and E334 in AChE
(Figure 1). Changes in amino acid residues in the E-helix in
BuChE are observed to alter the reversible inhibition by
phenothiazine carbamates, seen for wild-type enzyme, to
pseudoirreversible inhibition of the mutant enzyme species.
Certain domains of the active site gorge, such as the peripheral
anionic site and the π-cationic site, that are part of the
Ω-loop,18,19 have been shown to be involved in reversible
inhibitor binding.5,6 Evidence presented here suggests that an
additional domain, the E-helix region containing F329 and Y332,
represents a domain that could be exploited as a target for
development of specific cholinesterase inhibitors to treat de-
mentias such as AD.
Figure 2. General reaction scheme. Scheme for synthesis of N-(10)-
substituted phenothiazine carbamate derivatives. The R groups are
shown in Tables 1 and 2.
Results and Discussion
A total of 29 carbamate derivatives of phenothiazine were
synthesized according to the reaction scheme depicted in Figure
2. Of these, 10 compounds (1,20 2,21 4,21 6,21 8,20,22 9,20 10,22
11,20 13,23 and 1424) have been synthesized previously. The
structures of all 29 compounds are shown in Tables 1 and 2.
All the derivatives were N-(10)-carbamates with variously
substituted alkyl-, aminoalkyl-, and aryl-side groups. Each
carbamate derivative was examined for its ability to inhibit
human AChE and BuChE.
Synthetic Chemistry. The alkyl and aryl phenothiazine
carbamates were prepared by refluxing, in dichloromethane,
phenothiazine-10-carbonyl chloride with an excess of the alcohol
or phenol and an equivalent amount of triethylamine to
neutralize the generated HCl. In the preparation of the alkyl-
amino carbamates, triethylamine was not used. Because the alkyl
and aryl carbamates could not be cleanly separated from
remaining phenothiazine-10-carbonyl chloride by thin layer
chromatography or column chromatography, any unreacted
carbonyl chloride was reacted with propylamine to form the
corresponding propyl urea derivative of phenothiazine, which
could be readily separated from the desired phenothiazine
carbamates. After workup, carbamate purification was achieved
by silica gel column chromatography and crystallization. The
alkylamino phenothiazines were isolated as hydrochloride salts.
All purified compounds were homogeneous by thin layer
chromatography. HPLC analysis and 1H NMR revealed all
synthesized compounds to be more than 98% pure. All
1
compounds were fully characterized by IR, H, and 13C NMR
spectroscopy, as well as low- and high-resolution (accurate
mass) mass spectrometry. Physical data were consistent with
the structure (Tables 1 and 2) of each molecule synthesized.
Enzyme Kinetic Studies. All assays were carried out using
a modification25 of the Ellman method26 as outlined in detail
in the Experimental Section. Each phenothiazine carbamate was
tested for its ability to effect time-dependent deactivation of
AChE and BuChE. This was done by determining the extent of
inhibition of AChE or BuChE at time zero (enzyme added to
the assay last to initiate the reaction) and preincubation of the

4202 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Table 1. Alkyl and Aminoalkyl Phenothiazine Carbamatesa
DarVesh et al.
a ka values for inhibition by rivastigmine: AChE ) 1.13 × 103 M-1 min-1; BuChE ) 129 × 103 M-1 min-1. Ki values for inhibition by donepezil: AChE
) 0.024 × 10-6 M; BuChE ) 2.21 × 10-6 M. Ki values for inhibition by galantamine: AChE ) 0.52 × 10-6 M; BuChE ) 2.09 × 10-6 M.7
enzyme with the inhibitor at regular time intervals for up to 30
min. None of the derivatives showed pseudoirreversible inhibi-
tion of purified human plasma wild-type BuChE, as evidenced
by the absence of additional loss of enzyme activity after
preincubation of enzyme with inhibitor. Conversely, all the
phenothiazine carbamates described here showed time-dependent
deactivation of purified recombinant human AChE (Tables 1
and 2) because there was an increase in the extent of inhibition
of this enzyme with increase in time of preincubation of the
enzyme and inhibitor.
The second-order rate constants (ka values) for AChE
deactivation were obtained by comparing enzyme activity at
time zero (enzyme added to the assay mixture last) to the activity
obtained after preincubation of enzyme with a carbamate for
periods of up to 30 min. The ka value for each carbamate was
determined from this data by methods described earlier.7,27,28
An example of such a determination is shown in Figure 3, where
ka is obtained as the slope of the straight line (see Experimental
Section for details).
Human wild-type BuChE, which showed no pseudoirrevers-
ible inhibition by phenothiazine carbamates, was tested for
immediate reversible inhibition by comparing assay in the
absence of the inhibitor, to a reaction mixture containing various
amounts of inhibitor. This gave an enzyme activity-inhibitor
concentration profile from which could be selected inhibitor
concentrations suitable for kinetic studies. Inhibition constants
for BuChE (Ki values, Tables 1 and 2) were obtained from
Lineweaver-Burk double reciprocal plots, an example of which
is shown in Figure 4 (see Experimental Section for details).
Molecular Computational Studies. Computational methods
were used to examine molecular characteristics that could
influence cholinesterase inhibition by phenothiazine carbamates.
One of these parameters was total molecular volume, derived
from molecular mechanics calculations of the minimum energy
conformation for each derivative. Molecular mechanics calcula-
tions were carried out using Spartan ’0629 in which the Merck
molecular force field (MMFF) was employed, with the keyword
“equilibrium conformer”, so that the most stable conformer
would be selected.
Also calculated by molecular mechanics for each derivative
was the “butterfly angle” for the phenothiazine tricyclic ring
system. The butterfly angle refers to the angle between the two
planes defined by the benzene rings of the phenothiazine
tricycle.14 For all compounds, this was consistently 146 ( 0.1°.
The butterfly conformation of the phenothiazine tricycle provides
an appropriate shape for π-π stacking of the benzene rings of
this moiety with F329 and Y332 (Figure 1) within the BuChE
active site gorge as suggested earlier.14,15,17
For each derivative, log P value was calculated in order to
predict the ability of the derivative to cross the blood-brain
barrier.15,30 Experimentally, the log P value of a particular
molecule is determined by partitioning the molecule between
polar water and nonpolar n-octanol as competing solvents.
However, when solubility and detection limits do not permit
experimental determination, as with phenothiazines, log P values
can be calculated via programs such as the ALOGPS v. 2.0,
which is available on line.30 This method compares the structure
of the molecule being considered to a large database of log P
values for known molecules. The larger the value of log P, the
greater the probability that the molecule will be able to cross
the blood-brain barrier. Although very high log P values can
be associated with decreased aqueous solubility and potentially
limited absorption, all of the phenothiazine derivatives examined
here had calculated log P values of 3 to less than 7 (Tables 1
and 2). These values are comparable to those calculated earlier
for drugs currently used to treat AD.15
Structure-Activity Relationships. Most known carbamates
deactivate cholinesterases by time-dependent covalent bond

Carbamates with Mechanism of Inhibition for AChE and BuChE
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4203
Table 2. Aryl Phenothiazine Carbamates
a No inhibition detected up to [I] ) 7.33 × 10-5 M, the solubility limit. b No inhibition detected up to [I] ) 2.36 × 10-5 M, the solubility limit.
formation (carbamylation) at the active-site serine1 that prevents
substrate hydrolysis (Figure 5A,B,C). The resulting carbamy-
lated serine residue subsequently undergoes hydrolysis to
regenerate the active enzyme (Figure 5D,E,F), leading to the
designation of such compounds as pseudoirreversible inhibitors.
Typically, when such a compound inhibits cholinesterases, the
mechanism of inhibition is the same for both AChE and BuChE
because they both have the same catalytic triad composed of
the nucleophilic serine, histidine, and glutamate (Figure 1).
However, phenothiazine carbamates did not exhibit the same
type of inhibition for the two cholinesterases. All did show the
expected pseudoirreversible carbamylation of AChE (Tables 1
and 2) but, in stark contrast, BuChE inhibition was found to be
the reversible type, indicating the absence of covalent bond
formation with this enzyme (Tables 1 and 2).
Inhibition of Acetylcholinesterase by Phenothiazine
Carbamates. The carbamate-induced deactivation of AChE was
evaluated by measuring the second-order rate constant for loss
of enzyme activity (ka value).27,28 Most alkyl phenothiazine
carbamates (Table 1) exhibited relatively small ka values, ranging
from 135 to 492 M-1min-1 and indicating low potency as AChE
inhibitors. The single exception was the isopropyl derivative
(4), which had a ka value of 1800 M-1 min-1, comparable to
that of rivastigmine (1130 M-1 min-1).25
these carbamates poor inhibitors of AChE. However, it is not
unexpected that alkyl carbamates should be less potent pseudoir-
reversible inhibitors of AChE than rivastigmine because the
latter carbamate generates a resonance-stabilized phenoxide
anion as a leaving group in the enzyme carbamylation step
(Figure 5C). In contrast, the alkyl carbamates must produce a
highly basic alkoxide ion in the same step. This type of
unfavorable leaving group should also be true for the isopropyl
carbamate (4), which makes its relatively high inhibitor potency
difficult to explain unless the compact nature of the isopropyl
substituent facilitates interaction of the carbamate with the aryl-
rich gorge of AChE. Such affinity could promote passage of
the inhibitor down the gorge to S203 of the catalytic triad. This
notion is supported by the observation that other compact alkyl
groups (as in 1 and 7) also show more potent AChE inhibition
when compared to most of the alkyl carbamates in Table 1.
Several alkylamino phenothiazine carbamates, specifically 8,
9, 10, and 13 (Table 1), were, like most of the alkyl carbamates,
weak inhibitors of AChE. The unstable alkoxide leaving group
from carbamylation by these derivatives may again be a
dominant factor in the low inhibitor potency. However, within
this group of compounds there were also notable exceptions.
Compared to rivastigmine, the 2-pyrrolidinylethyl carbamate
(11) was a 40-fold better inhibitor, while 2-piperidinylethyl
derivative (12) was roughly 3 times better at inactivating AChE.
It is possible that the earlier arguments for delivery of the more
potent compact alkyl carbamates (1, 4, and 7) are also operating
The molecular volumes of the alkyl phenothiazine derivatives
(Table 1) were comparable to the estimated active site gorge
volume for AChE (302 Å3)17 so that size alone would not make

4204 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
DarVesh et al.
Figure 3. Kinetic plot for inhibition of human acetylcholinesterase by 1-naphthyl phenothiazine carbamate (28). The ka value was calculated by
plotting the extent of inhibition {ln (eo/et)/[I]} against time, where e0 is the enzymatic activity at time zero (without preincubation of enzyme and
inhibitor), et is the enzymatic activity at time t min of preincubation, and [I] ) 5.18 × 10-7 M 28. The slope of this plot gave the second-order rate
constant (ka value M-1 min-1).
Figure 4. Lineweaver-Burk plot, where the reciprocal of the velocity of the reaction V (M min-1) is plotted against the reciprocal of the substrate
concentration s (M), for reversible inhibition of wild-type human butyrylcholinesterase in the absence and presence of 1-naphthyl phenothiazine
carbamate (28): (2) no inhibitor; (b) 4.32 × 10-8 M 28; (9) 8.64 × 10-8 M 28. Replot of the slopes of the lines versus inhibitor concentration
[I] (M) gave the inhibitor constant Ki (M) as the x-intercept (inset).
here with 11 and 12. In addition, in view of the superior AChE
inhibition by 11, it is possible that the protonated heterocyclic
nitrogen atom of the pyrrolidinyl group may be so positioned
as to permit favorable π-cationic interaction with W86 of the
enzyme (Figure 1), thereby stabilizing the inhibitor for reaction
in the active site gorge, as occurs with acetylcholine at this site.
Furthermore, in 11, the presence of a cationic nitrogen may help
stabilize the oxyanion leaving group through intramolecular
general acid catalysis.
Most aryl phenothiazine carbamates (Table 2) were signifi-
cantly better at inactivating AChE than rivastigimine. All of
these carbamates, including rivastigmine, produce a resonance-
stabilized phenoxide anion leaving group in the carbamylation
step (Figure 5C). To determine whether electronic effects on
the stability of the phenoxide leaving group could be contribut-
ing to enhanced potency of phenothiazine carbamates, variously
substituted phenyl carbamates were examined (Table 2). There
was no clear pattern to suggest predominant electronic effects

Carbamates with Mechanism of Inhibition for AChE and BuChE
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4205
Figure 5. General mechanism of pseudoirreversible cholinesterase inhibition by carbamates. Enzyme deactivation is initiated by nucleophilic
attack of the catalytic triad serine oxygen on the carbonyl group of the carbamate.
because identical substituents in the ortho-, meta- or para-
positions did not lead to consistent predictable pattern in
inhibitor potencies (Table 2). The one possible exception was
with the strongly electron-releasing methoxy substituent in
compounds 18, 19, and 20. This series exhibited a weakening
effect on ka when this group was in the ortho- (18) or para-
(20) position where electron donation could destabilize the
phenoxide leaving group. This is reflected in the low ka values
of 18 and 20 when compared to the meta-derivative (19), where
the methoxy group tends to be electron-withdrawing, or to the
unsubstituted phenyl carbamate (14).
being roughly 60-fold less potent than the para-counterpart (26).
Steric effects may also make 2-naphthyl (29) some 150-fold
less potent than the corresponding 1-naphthyl derivative (28)
that presents a much different conformation because of its point
of attachment to the phenothiazine, as was seen with comparable
amide derivatives.15
Of all the synthesized phenothiazine carbamates, the best
inhibitor of AChE was 3-(N,N-dimethylamino)phenyl (24) (ka
) 19.2 × 106 M-1 min-1). The potency of this derivative is
not readily explained on electronic or steric grounds. It is
possible that, for this compound (24), the side group nitrogen
or phenyl ring may enhance the binding of the compound to
AChE, possibly with W86. Such interaction could greatly
Steric hindrance within the AChE gorge produced by the
bulky 2-t-butyl derivative (25) helps explain this carbamate

4206 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
DarVesh et al.
improve the affinity of the enzyme for this molecule and
facilitate rapid carbamylation of S203 (Figure 1).
One of the most striking features of the aryl phenothiazine
carbamates is the very high inhibitor potency for AChE seen
for derivatives such as 24, 27, and 28 in spite of the large total
molecular volume of these compounds (Table 2) relative to the
AChE gorge volume (302 Å3).17 This implies that only the
portion of the molecule containing the carbamate moiety need
be delivered to S203 for covalent bond formation to occur.
Inhibition of Wild-Type Butyrylcholinesterase by
Phenothiazine Carbamates. All the phenothiazine carbamate
derivatives examined inhibited BuChE, except 26 and 27, the
latter compounds being subject to solubility limitations. Of the
27 derivatives that inhibited BuChE, 19 were comparable to,
or better than, donepezil or galantamine in their ability to inhibit
this enzyme (Tables 1 and 2). In all cases where BuChE
inhibition by phenothiazine carbamates occurred, the type of
inhibition was reversible, that is, no time-dependent deactivation
of the enzyme was observed. The absence of the usual
pseudoirreversible effect, generally characteristic of carbamates,
can be attributed to favorable π-π interaction between the
phenothiazine tricycle and aromatic amino acid residues F329
and Y332 above the catalytic triad in the BuChE active site
Figure 6. Simulated binding of 3-(N,N-dimethyl aminophenyl) phe-
nothiazine carbamate (24) to wild-type butyrylcholinesterase through
F329 and Y332 on the E-helical segment and utilizing the “butterfly”
conformation of the benzene rings of the phenothiazine tricycle to effect
π-π stacking. The figure was generated using HyperChem program,32
and the crystal structure coordinates of BuChE33 (PDB ID: 1POI) were
downloaded from the protein databank.34
with phenothiazine binding,17 the comparable A328 in BuChE
is small and does not interfere with the π-π overlap of the
aromatic rings of enzyme and inhibitor (Figure 6). The reversible
inhibition of BuChE by phenothiazine carbamates appears,
therefore, to involve a common transient interaction of the
phenothiazine moiety with F329 and Y332, and the potency of
inhibition can often be attributed to the size of the substituent
projecting into the gorge. In addition to blocking substrate, the
binding of these phenothiazines to F329 and Y332, these
residues being part of the helical polypeptide segment containing
E325 of the catalytic triad (E-helix, Figure 1), could result in
conformational changes in the triad that, of themselves, could
have a negative impact on substrate hydrolysis.
Inhibition of Butyrylcholinesterase Mutants. The selective
inhibition of human BuChE by phenothiazine derivatives15,17
has been ascribed, at least in part, to the interaction between
the two aromatic rings of the phenothiazine moiety of these
compounds with two aromatic amino acid residues of the
enzyme, F329 and Y332 (Figure 6), in the helical segment of
the enzyme that contains E325 (E-helix, Figure 1). In fact, this
π-π interaction near the top of the gorge appears to be so
influential in the present study of phenothiazine carbamates that
it consistently prevents the typical pseudoirreversible reaction
between these carbamates and BuChE (Tables 1 and 2). The
same π-π interaction is prevented in AChE because of
interference from Y337 that replaces A328 of BuChE (Figure
1), fostering the typical carbamylation of this enzyme. It was
therefore hypothesized that altering critical residues in the
BuChE active site gorge could alter the type of inhibition
produced in BuChE by phenothiazine carbamates. That is,
appropriate BuChE mutations at positions 328, 329, and 332
on the E-helix should have a profound effect on the type of
observed BuChE inhibition, reversible becoming pseudoirre-
versible. To test this hypothesis, the carbamate derivative
3-(N,N-dimethylamino phenyl) carbamate (24), was compared
for inhibition of wild-type human BuChE and several site-
directed recombinant BuChE mutants, affected in the 328-332
region31 of the enzyme. This carbamate (24) was chosen
because, of all the phenothiazine carbamates, it was the most
potent pseudoirreversible inhibitor of AChE and a rather poor
reversible inhibitor of BuChE (Ki ) 1.91 × 10-6 M). Wild-
type BuChE showed no time-dependent deactivation but dis-
played typical reversible inhibition with 24 (see Table 2), where
full inhibition is instantaneous and is not altered by preincu-
bation of inhibitor and enzyme. On the other hand, recombinant
BuChE mutants tested (Figure 7) were all found to become
15,17
gorge.
All seven alkyl phenothiazine carbamates were poor inhibitors
compared with either donepezil or galantamine (Table 1). This
may be because these alkyl side groups are small and, despite
interaction of the phenothiazine moiety with the enzyme, these
side groups may have limited ability to block substrate access
to the catalytic triad. In contrast, the six aminoalkyl phenothi-
azine carbamates were comparable or better inhibitors of BuChE
relative to donepezil and galantamine (Table 1). This may reflect
additional interaction of the protonated nitrogen atom in these
side groups with the enzyme. For example, the nitrogen cation
of the derivative could interact with the W82 via π-cationic
association to provide an additional enzyme-inhibitor ligand.
Of the 14 aromatic phenothiazine carbamates that inhibited
BuChE, 12 were comparable to, or superior to, donepezil or
galantamine as BuChE inhibitors (Table 2). In general, substitu-
tion in the aromatic side group did not have a significant effect
on the extent of inhibition. This suggests that electronic effects
in the side group do not significantly influence the ability of
the phenothiazine moiety to bind to the enzyme. However, the
planar nature of the aryl side groups in these derivatives may
constitute a major factor in blocking substrate access to the
catalytic triad, once the phenothiazine tricycle is bound to the
enzyme. For example, the 1-naphthyl derivative (28), with a
fused bicyclic ring system and larger volume, is roughly a 16-
fold more potent inhibitor of BuChE than the phenyl carbamate
(14).
The putative π-π interaction between the phenothiazine
moiety and the two aromatic residues, F329 and Y332,17 in
BuChE must be highly favored because there is little indication
that phenothiazine carbamates reach the bottom of the BuChE
active site gorge to react with S198. One factor that may favor
the BuChE-phenothiazine π-π interaction is the “butterfly”
shape of the phenothiazine tricycle that facilitates π-π stacking
of the aromatic rings of the tricycle with F329 and Y332 in the
gorge.15 For all of the carbamates reported here, the “butterfly
angle” was found to be virtually the same (146.0 ( 0.1°),
permitting optimum interaction of the two aromatic rings of
phenothiazine with the side chain rings of F329 and Y332 in
the BuChE gorge (Figure 6). Unlike the prominent 4-hydrox-
yphenyl side chain of Y337 in AChE (Figure 1), that interferes

Carbamates with Mechanism of Inhibition for AChE and BuChE
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4207
Table 3. Time-Dependent Deactivation of Wild-Type and Mutant
Butyrylcholinesterase by 4-Biphenyl Phenothiazine Carbamate (27) (2.1
× 10-6 M)
a
%
residual enzymatic activity
butyrylcholinesterase-type
0 min
5 min
10 min
15 min
wild-type native
D70G
A539T
Y332A
A328Y
A328F
A328W
F329S
100
100
100
100
100
100
100
100
100
100
99.5
99
95
85
88
74
51
46
100
90
94
87
67
75
57
15
26
100
76
93
85
63
59
35
11
12
F329A
a Obtained from the calculation [∆A/min at time t min/∆A/min at time
zero] × 100.
Figure 7. Pseudo-irreversible inhibition of recombinant butyrylcho-
linesterase mutants by 3-(N,N-dimethyl aminophenyl) phenothiazine
carbamate (24) indicated by diminished residual enzyme activity over
time. The low concentration of 24 used (4.0 × 10-8 M) produced no
instantaneous inhibition of wild-type serum or wild-type recombinant
butyrylcholinesterase but showed time-dependent inhibition of mutant
butyrylcholinesterases indicating that this carbamate was able to react
with S198 of mutant BuChEs but not of the wild-type BuChE.
to assume that the altered binding of the inhibitor to the BuChE
mutant enzyme species exposes S198 to carbamylation.
Conclusions
Carbamate derivatives of phenothiazine are unique in that
they generally exhibit only reversible inhibition of BuChE, while
producing the common pseudoirreversible inhibition of AChE.
The atypical BuChE-phenothiazine carbamate binding is at-
tributable to dominant π-π interaction between residues F329
and Y332 in BuChE with the phenothiazine aromatic tricycle.
This is confirmed with mutations of F329 and Y332 that result
in time-dependent deactivation of BuChE.
Interference with similar noncovalent binding in AChE, due
toY337 projecting into the gorge (Figure 1), permits delivery
of the phenothiazine carbamate moiety to the bottom of the
gorge for reaction with S203. AChE gorge volume is not as
restrictive here as it is in reversible inhibition because only the
carbamate moiety needs to be delivered to the catalytic triad
serine. This is confirmed with BuChE mutants in which A328
is converted to amino acid residues with aryl side chains
comparable to Y337 of AChE. These mutations, replacing A328
in BuChE with Y328, F328, or W328, also result in conversion
of phenothiazine carbamate inhibition from reversible to pseudoir-
reversible.
increasingly deactivated over time, the phenomenon typically
observed for carbamylation of the catalytic serine residue. In
contrast, neither wild-type native BuChE nor wild-type recom-
binant BuChE showed time-dependent deactivation by 24
(Figure 7). All six BuChE mutant types depicted in Figure 7
involve residues in the E-helix at positions 328, 329, and 332
(see Figure 1). It was considered that converting F329 or Y332
to alkyl residues should greatly alter the π-π interaction that
is the putative means for reversible inhibition between wild-
type BuChE and phenothiazines in general (Figure 6). Likewise,
replacing A328 in BuChE with an aromatic amino acid residue
should make BuChE more like AChE, which has Y337 in that
position (see Figure 1). This should also make the enzyme
generally resistant to reversible inhibition by phenothiazine
derivatives by disrupting the inhibitor-enzyme π-π stacking
due to new aryl residues at position 328. Figure 7 clearly
indicates that such BuChE mutations at 328, 329, or 332 alter
the type of inhibition produced by a phenothiazine carbamate,
as indicated by increased loss of enzyme activity over time. Of
the mutants tested, the most effective change was with F329
converted to the small alkyl side chains of alanine or serine.
This inhibition was still some 10-fold less potent than that of
the same carbamate (24) on AChE (Table 2) but indicated a
profound change over the effect of 24 on wild-type native
BuChE and wild-type recombinant BuChE, both of which
showed no inhibition by 24 at a concentration (4.0 × 10-8 M)
that caused time-dependent deactivation of all the mutant
enzymes (Figure 7). Replacing A328 with an aryl side chain
(A328W, A328F, and A328Y), making BuChE more like AChE
in the E-helical segment of the enzyme, had the next most
significant effect on BuChE (Figure 7), while the mutant Y332A
was the BuChE mutant slowest to be deactivated by 24.
Furthermore, the effect of 4-biphenyl phenothiazine carbamate
(26) on the same BuChE mutants, as well as two additional
mutant enzyme species D70G (the atypical variant) and A539T
(the K-variant), also gave predictable results, as summarized
in Table 3. That is, a mutation well away from the active site
(A539T) showed little tendency to pseudoirreversible inhibition
by this phenothiazine carbamate. On the other hand, altering
D70, which, like Y332, is considered a component of the BuChE
peripheral anionic site, showed similar limited time-dependent
deactivation to that observed for Y332A. It seems reasonable
Phenothiazine carbamates with resonance-stabilized aromatic
oxyanion leaving groups tend to be the most powerful pseudoir-
reversible AChE inhibitors.
The lack of covalent bond formation between phenothiazine
carbamates and wild-type BuChE implies that such molecules,
unlike other carbamates, will not be subject to the typical
scavenging by serum BuChE. This, combined with log P values
favorable for crossing the blood-brain barrier, suggests these
hydrophobic phenothiazine carbamates could be developed for
treatment of dementia, following appropriate analysis of their
effects on other neurotransmitter systems.
In addition to various domains of the active site gorge known
to be involved in reversible inhibitor binding, such as peripheral
anionic site and the π-cationic site, which are part of the Ω-loop,
the E-helix represents a domain that could be exploited as a
target for development of specific inhibitors of cholinesterases
for treatment of dementias.
Experimental Section
Materials. Purified human plasma butyrylcholinesterase (BuChE,
EC 3.1.1.8), purified recombinant wild-type, and mutant BuChEs
(Y332A, F329A, A328Y, A328W, A328F, D70G, and A539T) were
from Dr. Oksana Lockridge (University of Nebraska Medical
Center) (Masson et al., 1997).31 Purified recombinant human
acetylcholinesterase (AChE, EC 3.1.1.7), acetylthiocholine, bu-

4208 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
DarVesh et al.
tyrylthiocholine, and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
were purchased from Sigma (St. Louis, MO). Phenothiazine-10-
carbonyl chloride was purchased from Acros Chemicals. The
alcohols and phenols were purchased from either Fischer Scientific
or Aldrich Chemical Co. Inc. (Milwaukee, Wi).
A modified procedure was required for the preparation of the
t-butyl carbamate derivative. In this case, to a solution of phe-
nothiazine-10-carbonyl chloride (10 mmol) in THF (30 mL) was
added dropwise a 1.0 M solution of potassium t-butoxide in THF
(10 mL; 10 mmol) and the resulting solution stirred for 2 days at
room temperature. After treatment with propylamine, water (50 mL)
was added and the THF removed under reduced pressure. CH2Cl2
(50 mL) was added, and the resulting solution was washed and the
product isolated and purified as outlined above.
Organic Synthesis. Synthesis of Aryl-Substituted
Carbamates. Typically, a mixture of phenothiazine-10-carbonyl
chloride (5.0 mmol), phenol or substituted phenol (20-25 mmol)
and triethylamine (5.0 mmol) in CH2Cl2 (50 mL) was heated at
reflux until analysis by thin layer chromatography revealed no
unreacted phenothiazine-10-carbonyl chloride remained or no
further change was observed. Reflux periods were typically in the
range of 2-10 days. One complication for some of the carbamates
was that the Rf values of the products are very similar to that of
phenothiazine-10-carbonyl chloride, although sometimes the result-
ing spots took on a different color after short irradiation periods.
As a result, to aid in product purification, propylamine (5.0 mmol)
was added to the final reaction mixture to consume any unreacted
starting material, which may have been present. The propyl urea
thus formed has a much lower Rf value than the carbamates and
was readily removed during column chromatography.
For the aryl carbamates, the cooled reaction mixture was poured
into a separatory funnel and washed with 5% aqueous sodium
hydroxide (2 × 50 mL) to remove unreacted phenolic reactant,
followed by water (2 × 50 mL). The organic layer was dried
(MgSO4), filtered, and the solvent removed under reduced pressure.
The crude product was then further purified by column chro-
matography using silica gel (60-230 mesh) and CH2Cl2 as eluent.
Fractions containing only the desired product were combined, the
solvent removed, and the resulting solid recrystallized using 2:1
petroleum ether:CH2Cl2. Activated charcoal was used for the solids
that were highly colored. Isolated product yields varied from 31 to
75%; no attempt was made to optimize these yields.
The procedure above did not work well for the 2-t-butylphenyl
carbamate preparation, presumably due to severe steric effects. In
this case, a solution of phenothiazine-10-carbonyl chloride (10
mmol) and 2-t-butylphenoxide [15 mmol; prepared from the phenol
(20 mmol) and potassium t-butoxide (15 mmol)] in tetrahydrofuran
(40 mL) was stirred for 21 h at room temperature. After treatment
with propylamine (10 mmol) and the addition of water (50 mL),
the THF was removed under reduced pressure, CH2Cl2 (100 mL)
added, and the resulting solution washed with 5% NaOH (2 × 100
mL) followed by water (100 mL). Further isolation and purification
followed the procedure above.
Synthesis of Alkylamino Carbamates. The synthetic procedure
used for this series was very similar to that outlined above with
the elimination of the triethylamine. The products did not move
from the baseline using CH2Cl2 as eluent for TLC analysis, but
this did not compromise monitoring of reaction progress. The
solvent system used for purification by column chromatography
was 10-20% (by volume) CH3OH/CH2Cl2. Derivatives with the
pyrrolidinyl, piperidinyl, and morpholino ring systems were isolated
and recrystallized as the free bases. The three acyclic systems
produced oils in the free base form; therefore, these were converted
to the hydrochloride salts and recrystallized from water. Isolated
product yields ranged from 21 to 53%.
Analysis of Synthesized Compounds. Melting points were
determined on a Mel-Temp II apparatus and are uncorrected. Thin
layer chromatography was carried out using silica gel sheets with
fluorescent indicator (0.20 mm thickness; Macherey-Nagel) and
dichloromethane or a dichloromethane/ethyl acetate mixture as
developing solvent. Plates were visualized using a short wavelength
UV lamp. Infrared spectra were recorded as Nujol mulls between
sodium chloride plates on a Nicolet model 205 or a Nicolet Avatar
330 FT-IR spectrometer. Peak positions were reproducible within
1-2 cm-1. Nuclear magnetic resonance spectra were recorded at
the Atlantic Region Magnetic Resonance Centre, Dalhousie Uni-
versity, on a Bruker AC-250F operating at 250.1 MHz for proton
and 62.9 MHz for carbon or a Bruker AVANCE 500 operating at
500.13 MHz for proton and 125.76 MHz for carbon-13. Chemical
shifts are reported in ppm relative to TMS, in CDCl3 solution. Mass
spectra were recorded at Dalhousie University on a CEC 21-110B
spectrometer using electron ionization at 70 V and an appropriate
source temperature with samples being introduced by means of a
heatable port probe. Accurate mass measurements were also made
on this machine operated at a mass resolution of 8000 by computer
controlled peak matching to appropriate PFK reference ions. Mass
measurements were routinely within 3 ppm of the calculated value.
The purity of the synthesized compounds was also determined using
either an Agilent Technologies 1200 series HPLC system with silica
gel column and dichloromethane as the mobile phase or a Waters
HPLC system using C18 reverse phase column with methanol as
the mobile phase.
Analytical Data. Methyl Carbamate (1). Colorless crystals, mp
116-118 °C (lit. mp 92-93 °C;20 118-120 °C21). IR (Nujol):
1
1712, 1330, 1307, 1288, 1263, 1228, 1052, 760 cm -1. H NMR
(CDCl3): 3.79 (s, 3H), 7.13-7.20 (m, 2H), 7.25-7.32 (m, 2H),
7.35 (dd, J ) 7.6 and 1.5 Hz, 2H), 7.54 (dd, J ) 7.9 and 1.2 Hz,
2H). 13C NMR (CDCl3): 53.54, 126.46, 126.90, 127.02, 127.58,
132.24, 138.34, 154.27. EI-MS (m/z): 257 (M+; base), 200, 199,
198, 154. HR-MS (EI): M+ found, 257.0525; calcd for C14H11NO2S,
257.0510.
Ethyl Carbamate (2). Off-white crystals, mp 102-103.5 °C (lit.
mp 116-117 °C21). IR (Nujol): 1707, 1322, 1259, 1223, 1044,
-1
1029, 946, 764 cm
.
1H NMR (CDCl3): 1.30 (t, J ) 7.5 Hz,
3H), 4.29 (q, J ) 7.5 Hz, 2H), 7.15-7.21 (m, 2H), 7.26-7.33 (m,
2H), 7.38 (broad d, J ∼ 7.5 Hz, 2H), 7.57 (broad d, J ∼ 7.5 Hz,
2H). 13C NMR (CDCl3): 14.40, 62.68, 126.34, 126.80, 127.07,
127.55, 132.18, 138.44, 153.70. EI-MS (m/z): 271 (M+), 200, 199,
198 (base), 197, 167, 166, 154, 140, 127. HR-MS (EI): M+ found,
271.0678; calcd for C15H13NO2S, 271.0667.
Propyl Carbamate (3). Colorless crystals, mp 106.5-107.5 °C.
IR: (Nujol): 1708, 1322, 1305, 1288, 1257, 1220, 1066, 909, 755
cm -1. 1H NMR (CDCl3): 0.90 (t, J ) 7.4 Hz, 3H), 1.62-1.69 (m,
2H), 4.16 (t, J ) 6.7 Hz, 2H), 7.14-7.17 (m, 2H), 7.25-7.28 (m,
2H), 7.35 (dd, J ) 7.7 and 1.3 Hz, 2H), 7.54 (dd, J ) 8.0 and 0.8
Hz, 2H). 13C NMR (CDCl3): 10.38, 22.00, 68.13, 126.23, 126.65,
126.97, 127.41, 132.09, 138.33, 153.73. EI-MS (m/z): 285 (M+),
199, 198 (base), 171, 167, 166, 154, 140, 127. HRMS (EI): M+
found, 285.0828; calcd for C16H15NO2S, 285.0823.
Isopropyl Carbamate (4). Colorless crystals, mp 137.5-138.5
°C (lit. mp 135 -137 °C21). IR (Nujol): 1712, 1319, 1304, 1259,
1228, 1181, 1095, 1038, 767, 752 cm -1. 1H NMR (CDCl3): 1.26
(d, J ) 6.3 Hz, 6H), 5.05 (septet, J ) 6.3 Hz, 1H), 7.13-7.16 (m,
2H), 7.24-7.28 (m, 2H), 7.34 (broad d, J ∼ 7.7 Hz, 2H), 7.53
(broad d, J ∼ 8.1 Hz, 2H). 13C NMR (CDCl3): 21.91, 70.62, 126.21,
126.69, 127.10, 127.52, 132.16, 138.56, 153.25. EI-MS (m/z): 285
Synthesis of Alkyl and Cycloalkyl Carbamates. Reaction of
phenothiazine-10-carbonyl chloride with simple alcohols is much
slower than that with phenols. For this reason, the alcohol (50 mL)
was used as solvent and the reaction of the phenothiazine-10-
carbonyl chloride (5 mmol) with triethylamine (5 mmol) was carried
out at reflux. Reaction periods were in the range of 7-14 days
and, again, because the Rf values of the carbamates were so similar
to that of phenothiazine-10-carbonyl chloride, any possible unre-
acted phenothiazine-10-carbonyl chloride was removed by treatment
with propylamine (5 mmol).
Workup involved removal of the excess alcohol under reduced
pressure, followed by partitioning of the product between CH2Cl2
and water. The organic solution was washed and treated and the
product purified as outlined above. Isolated product yields were in
the range 16-76%.

Carbamates with Mechanism of Inhibition for AChE and BuChE
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4209
(M+), 199, 198 (base), 170, 167, 166, 153, 140, 126. HR-MS (El):
M+ found, 285.0827; calcd for C16H15NO2S, 285.0823.
243, 199, 198, 98, 97 (base), 84. HR-MS (EI): M+ found, 340.1268;
calcd for C19H20N2O2S, 340.1245.
Butyl Carbamate (5). Colorless crystals, mp 51-53 °C. IR
2-(1-Piperidinyl)ethyl Carbamate (12). Colorless crystals, mp
92.2-93.5 °C. IR (Nujol): 1719, 1313, 1287, 1260, 1048, 1036,
984, 756 cm -1. 1H NMR (CDCl3): 1.37-1.43 (m, 2H), 1.51-1.58
(m, 4H), 2.35-2.42 (m, 4H), 2.59 (t, J ) 5.9 Hz, 2H), 4.32 (t, J )
5.9 Hz, 2H), 7.14-7.18 (m, 2H), 7.24-7.29 (m, 2H), 7.34 (dd, J
) 7.8 and 1.1 Hz, 2H), 7.59 (broad d, J ∼ 8.1 Hz, 2H). 13C NMR
(CDCl3): 24.24, 26.15, 54.74, 57.25, 64.26, 126.33, 126.75, 127.17,
127.47, 132.19, 138.44, 153.62. EI-MS (m/z): 354 (M+), 199, 198,
167, 154, 113, 112 (base), 111, 98. HR-MS (EI): M+ found,
354.1414; calcd for C20H22N2O2S, 354.1402.
2-(4-Morpholino) Ethyl Carbamate (13). Pale-yellow crystals,
mp 99.5-102 °C (lit. mp of hydrochloride 213-214 °C23), 218
°C (Szabo et al., 198028)). IR (Nujol): 1724, 1324, 1305, 1257,
1224, 1152, 1116, 1056, 758 cm -1. 1H NMR (CDCl3): 2.41-2.45
(m, 4H), 2.61 (t, J ) 5.7 Hz, 2H), 3.63-3.68 (m, 4H), 4.33 (t, J )
5.7 Hz, 2H), 7.15-7.19 (m, 2H), 7.25-7.29 (m, 2H), 7.36 (dd, J
) 7.7 and 1.3 Hz, 2H), 7.57 (dd, J ) 8.1 and 1.0 Hz, 2H). 13C
NMR (CDCl3): 53.72, 56.95, 63.75, 67.03, 126.40, 126.73, 127.09,
127.49, 132.22, 138.31, 153.55. EI-MS (m/z): 356 (M+), 243, 199,
198, 114 (base), 113, 100. HR-MS (EI): M+ found, 356.1193; calcd
for C19H20N2O3S, 356.1194.
(Nujol): 1728, 1322, 1287, 1259, 1220, 1091, 1064, 1033, 728 cm
1
-1. H NMR (CDCl3): 0.90 (t, J ) 7.4 Hz, 3H), 1.30-1.38 (m,
2H), 1.59-1.65 (m, 2H), 4.20 (t, J ) 6.7 Hz, 2H), 7.14-7.17 (m,
2H), 7.25-7.29 (m, 2H), 7.34 (dd, J ) 7.8 and 1.2 Hz, 2H), 7.53
(dd, J ) 8.1 and 0.8 Hz, 2H). 13C NMR (CDCl3): 13.65, 19.13,
30.70, 66.47, 126.28, 126.70, 127.03, 127.47, 132.16, 138.43,
153.78. EI-MS (m/z): 299 (M+; base), 200, 199, 198, 197, 171,
167, 166, 154. HR-MS (EI): M+ found, 299.0984; calcd for
C17H17NO2S, 299.0980.
t-Butyl Carbamate (6). Colorless crystals, mp 116-117 °C (lit.
mp 108-110 °C21). IR (Nujol): 1709, 1334, 1256, 1232, 1157,
-1
1021, 951, 846, 764 cm
.
1H NMR (CDCl3): 1.48 (s, 9H),
7.12-7.15 (m, 2H), 7.23-7.27 (m, 2H), 7.33 (dd, J ) 7.8 and 1.4
Hz, 2H), 7.52 (dd, J ) 8.1 and 1.1 Hz, 2H). 13C NMR (CDCl3):
28.15, 82.05, 126.03, 126.52, 127.19, 127.41, 132.13, 138.72,
152.45. EI-MS (m/z): 299 (M+), 244, 243, 200, 199 (base), 198,
167, 166, 154, 140, 128, 127, 57. HR-MS (EI): M+ found,
299.0972; calcd for C17H17NO2S, 299.0980.
Cyclopentyl Carbamate (7). Colorless crystals, mp 95-97 °C.
IR (Nujol): 1708, 1364, 1329, 1288, 1259, 1225, 1168, 1040, 971,
-1
763 cm
.
1H NMR (CDCl3): 1.5-2.0 (overlapping m, 8H),
Phenyl Carbamate (14). Colorless crystals, mp 165-166 °C
(lit. mp 166 °C24). IR (Nujol): 1725, 1589, 1331, 1287, 1267, 1214,
1197, 1126, 1023, 764, 749 cm -1. 1H NMR (CDCl3): 7.16-7.43
(overlapping m, 11H), 7.68 (dd, J ) 8.0 and 1.1 Hz, 2H). 13C NMR
(CDCl3): 121.43, 125.72, 126.65, 126.91, 126.95, 127.62, 129.31,
132.27, 137.99, 149.92, 150.90. EI-MS (m/z): 319 (M+, base), 199,
198, 166, 154, 77. HR-MS (EI): M+ found, 319.0661; calcd for
C19H13O2 NS, 319.0667.
5.20-5.27 (m, 1H), 7.11-7.17 (m, 2H), 7.22-7.29 (m, 2H), 7.34
(dd, J ) 7.7 and 1.5 Hz, 2H), 7.51 (dd, J ) 8.0 and 1.1 Hz, 2H).
13C NMR (CDCl3): 23.49, 32.59, 79.58, 126.11, 126.53, 126.89,
127.36, 131.95, 138.37, 153.30. EI-MS (m/z): 311 (M+), 199, 198,
154, 127, 69 (base), 67. HR-MS (EI): M+ found, 311.0974; calcd
for C18H17NO2S, 311.0980.
2-(N,N-Dimethylamino)ethyl Carbamate HCl (8). Colorless
solid, mp 207-208 °C (lit. mp 212-213 °C20); 213-214 °C22).
2-Methylphenyl Carbamate (15). Pale-yellow crystals, mp
1
IR (Nujol): 1710, 1331, 1219, 1099, 1055, 1033, 760 cm -1. H
199-200 °C. IR (Nujol): 1732, 1329, 1228, 1173, 1110, 1012, 944,
NMR (DMSO-d6): 2.67 (s, 6H), 3.3-3.4 (overlaps with DMSO
signal), 4.51 (t, J ∼ 6 Hz, 2H), 7.26-7.30 (m, 2H), 7.36-7.40 (m,
2H), 7.49 (dd, J ∼ 7.7 and 2.0 Hz, 2H), 7.74 (broad d, J ∼ 8 Hz,
2H), 11.1 (very broad s, 1H). 13C NMR (DMSO-d6): 42.44, 54.93,
60.94, 126.83, 127.21, 127.31, 127.50, 131.31, 137.67, 152.29. EI-
MS (m/z): 314 (M+), 243, 200, 199, 198, 172, 167, 166, 154, 127,
116, 72, 58 (base). HR-MS (EI): M+ found, 314.1091; calcd for
C17H18N2O2S, 314.1089.
-1
766, 753 cm
.
1H NMR (CDCl3): 2.21 (s, 3H), 7.11-7.26
(overlapping m, 6H), 7.31-7.36 (m, 2H), 7.44 (dd, J ) 7.6 and
1.5 Hz, 2H), 7.70 (dd, J ) 7.9 and 1.1 Hz, 2H). 13C NMR (CDCl3):
16.52, 122.08, 126.16, 126.95, 127.07, 127.20, 127.36, 127.86,
130.47, 131.30, 132.71, 138.42, 149.76, 152.22. EI-MS(m/z): 333
(M+), 201, 199 (base), 171, 167, 154, 127, 91, 77, 65. HR-MS
(EI): M+ found, 333.0827; calcd for C20H15O2NS, 333.0823.
3-Methylphenyl Carbamate (16). Colorless crystals, mp
146-147.5 °C. IR (Nujol): 1722, 1615, 1588, 1333, 1240, 1215,
1142, 1031, 1020, 1000, 770, 762, 748 cm -1. 1H NMR (CDCl3):
2.32 (s, 3H), 6.96-7.03 (overlapping m, 3H), 7.16-7.24, (overlap-
ping m, 3H), 7.27-7.31 (m, 2H), 7.39 (dd, J ) 7.8 and 1.5 Hz,
2H), 7.66 (dd, J ) 8.0 and 1.2 Hz, 2H). 13C NMR (CDCl3): 21.53,
118.64, 122.31, 126.79, 126.91, 127.22 (2 peaks), 127.88, 129.32,
132.55, 138.35, 139.80, 151.15, 152.52. EI-MS (m/z): 333 (M+),
201, 199 (base), 171, 167, 154, 127, 92, 77, 65. HR-MS (EI): M+
found, 333.0820; calcd for C20H15O2NS, 333.0823.
2-(N,N-Diethylamino)ethyl Carbamate HCl (9). Colorless
crystals, mp 161-163 °C (lit. mp 163-164 °C20). IR (Nujol): 1728,
1712, 1330, 1263, 1219, 1099, 1033, 759 cm -1. 1H NMR (D2O):
0.90 (t, J ) 7.2 Hz, 6H), 2.79 (q, J ) 7.2 Hz, 4H), 3.11 (broad s,
2H), 4.31 (broad t, J ≈ 4,5 Hz, 2H), 6.92-6.95 (m, 2H), 7.02 (broad
d, J ∼ 7.3 Hz, 2H), 7.10-7.14 (m, 2H), 7.35 (broad d, J ∼ 8.1 Hz,
2H). In DMSO-d6 solution, the N-H signal occurs at 11.13 (broad
s, 1H). 13C NMR (D2O): 8.36, 48.49, 50.00, 61.46, 127.23, 127.34,
127.66, 127.69, 131.91, 137.26, 153.70. EI-MS (m/z): 342 (M+),
338, 337, 270, 199, 198, 163, 136, 135, 100, 99, 86 (base). HR-
MS (EI): M+ found, 342.1405; calcd for C19H22O2 N2S, 342.1402.
3-(N,N-Diethylamino)propyl Carbamate HCl (10). Colorless
crystals, mp 188-189 °C (lit. mp 195-197 °C22). IR (Nujol): 2577,
4-Methylphenyl Carbamate (17). Colorless needles, mp 168-169
°C. IR (Nujol): 1735, 1722, 1508, 1333, 1260, 1221, 1191, 1013,
793, 759 cm -1. 1H NMR (CDCl3): 2.34 (s, 3H), 7.09 (d, J ) 8.4
Hz, 2H), 7.17 (d, J ) 8.3 Hz, 2H), 7.20-7.23 (m, 2H), 7.30-7.34
(m, 2H), 7.42 (dd, J ) 7.8 and 1.0 Hz, 2H), 7.69 (broad d, J ∼ 8.0
Hz, 2H). 13C NMR (CDCl3): 20.77, 121.07, 126.57, 126.90 (2
peaks), 127.57, 129.77, 132.24, 135.30, 138.07, 148.71, 152.29.
EI-MS (m/z): 333 (M+; base), 199, 198, 166, 154, 91. HR-MS (EI):
M+ found, 333.0813; calcd for C20H15O2NS, 333.0823.
2-Methoxyphenyl Carbamate (18). Colorless crystals, mp
156.5-158 °C. IR (Nujol): 1737, 1607, 1586, 1501, 1481, 1330,
1310, 1261, 1209, 1195, 767, 754 cm -1. 1H NMR (CDCl3): 3.92
(s, 3H), 6.90-6.93 (m, 1H), 6.98 (dd, J ) 8.3 and 1.2 Hz, 2H),
7.10 (dd, J ) 7.9 and 1.6 Hz, 1H), 7.16-7.20 (overlapping m,
3H), 7.27-7.31 (m, 2H), 7.39 (dd, J ) 7.8 and 1.2 Hz, 2H), 7.77
(dd, J ) 8.1 and 0.9 Hz, 2H). 13C NMR (CDCl3): 56.00, 112.41,
120.72, 123.02, 126.47, 126.75, 126.88, 126.99, 127.49, 132.11,
138.30, 140.17, 151.32, 151.92. EI-MS (m/z): 349 (M+), 198, 171,
166, 154, 140, 127, 95, 92, 79, 77 (base). HR-MS (EI): M+ found,
349.0766; calcd for C20H15NO3S, 349.0772.
2490, 1718, 1318, 1286, 1259, 1214, 1045, 1034, 789, 751 cm -1
.
1H NMR (D2O): 0.97 (t, J ) 7.2 Hz, 6H), 1.68-1.75 (m, 2H),
2.71 (t, J ) 8.1 Hz, 2H), 2.83 (t, J ) 7.2 Hz, 4H), 4.03-4.09 (m,
2H), 6.87-6.91 (m, 2H), 6.96 (broad d, J ∼ 7.7 Hz, 2H), 7.09-7.13
(m, 2H), 7.36 (broad d, J ∼ 8.0 Hz, 2H). 13C NMR (D2O): 8.22,
22.43, 47.42, 47.86, 63.78, 126.97, 127.11, 127.41 (2 peaks),
131.60, 137.49, 153.90. EI-MS (m/z): 356 (M+ for freebase; base
peak), 341, 284, 199, 198, 114, 86. HR-MS (EI): M+ found,
356.1552; calcd for C20H24N2O2S, 356.1558.
2-(1-Pyrrolidinyl)ethyl carbamate (11). Colorless crystals, mp
61-62 °C (lit. mp of hydrochloride 215-217 °C20). IR (Nujol):
1728, 1307, 1261, 1221, 1160, 1053, 755 cm -1. 1H NMR (CDCl3):
1.68-1.77 (m, 4H), 2.46-2.49 (m, 4H), 2.73 (t, J ) 6.1 Hz, 2H),
4.33 (t, J ) 6.1 Hz, 2H), 7.14-7.18 (m, 2H), 7.25-7.29 (m, 2H),
7.35 (dd, J ) 7.8 and 1.2 Hz, 2H), 7.57 (dd, J ) 8.1 and 1.0 Hz,
2H). 13C NMR (CDCl3): 23.60, 54.22, 54.50, 65.72, 126.36, 126.78,
127.17, 127.48, 132.24, 138.41, 153.64. EI-MS (m/z): 340 (M+),

4210 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
DarVesh et al.
3-Methoxyphenyl Carbamate (19). Colorless crystals, mp
107-109 °C. IR (Nujol): 1725, 1611, 1333, 1304, 1290, 1254, 1218,
1143, 1050, 755 cm -1. 1H NMR (CDCl3): 3.76 (s, 3H), 6.74-6.80
(overlapping m, 3H), 7.17-7.21 (m, 2H), 7.22-7.26 (m, 1H),
7.28-7.32 (m, 2H), 7.40 (dd, J ) 7.8 and 1.2 Hz, 2H), 7.66 (dd,
J ) 8.1 and 0.8 Hz, 2H). 13C NMR (CDCl3): 55.38, 107.34, 111.80,
113.52, 126.63, 126.88, 126.93, 127.60, 129.64, 132.25, 137.97,
151.84, 152.96, 160.39. EI-MS (m/z): 349 (M+; base), 260, 200,
199, 198, 154, 95. HR-MS (EI): M+ found, 349.0777; calcd for
C20H15NO3S, 349.0772.
7.34-7.41 (overlapping m, 4H), 7.67 (broad d, J ∼ 8.0 Hz, 2H).
13C NMR (CDCl3): 31.37, 34.42, 120.71, 126.20, 126.57, 126.89,
126.92, 127.57, 132.23, 138.10, 148.57, 152.27. EI-MS (m/z): 375
(M+; base), 200, 199, 198, 166, 154. HR-MS (EI): M+ found,
375.1297, calcd for C23H21NO2S, 375.1293.
4-Biphenyl Carbamate (27). Colorless crystals, 226-227 °C.
IR (Nujol): 1731, 1333, 1262, 1219, 1168, 1019, 1006, 760 cm
1
-1. H NMR (CDCl3): 7.19-7.33 (m, 2H), 7.24-7.28 (m, 2H),
7.29-7.34 (overlapping m, 3H), 7.39-7.43 (overlapping m, 4H),
7.53-7.58 (overlapping m, 4H), 7.69 (dd, J ) 8.1 and 0.9 Hz,
2H). 13C NMR (CDCl3): 121.69, 126.70, 126.93, 126.98, 127.08,
127.31, 127.65, 128.05, 128.76, 132.31, 138.02, 138.88, 140.31,
150.36, 152.13. EI-MS (m/z): 395 (M+; base), 200, 199, 198, 170,
166, 154, 152. HR-MS (EI): M+ found, 395.0988, calcd for
C25H17NO2S, 395.0980.
4-Methoxyphenyl Carbamate (20). Colorless needles, mp
127-128 °C. IR (Nujol): 1717, 1596, 1509, 1332, 1304, 1256, 1213,
-1
1179, 1038, 1011, 804, 759 cm
.
1H NMR (CDCl3): 3.75 (s,
3H), 6.84-6.87 (m, 2H), 7.07-7.11 (m, 2H), 7.16-7.20 (m, 2H),
7.27-7.31 (m, 2H), 7.39 (dd, J ) 7.9 and 1.1 Hz, 2H), 7.66 (dd,
J ) 8.1 and 0.9 Hz, 2H). 13C NMR (CDCl3): 55.52, 114.29, 122.21,
126.57, 126.89 (2 peaks), 127.56, 132.21, 138.04, 144.42, 152.46,
157.16. EI-MS(m/z): 349 (M+), 199, 198 (base), 171, 166, 154,
127, 123, 107, 95, 92, 77. HR-MS (EI): M+ found, 349.0773; calcd
for C20H15NO3S, 349.0772.
2-Chlorophenyl Carbamate (21). Colorless crystals, mp 222-224
°C. IR (Nujol): 1737, 1334, 1261, 1221, 1059, 1031, 1007, 945,
757 cm -1. 1H NMR (DMSO-d6): 7.30-7.34 (overlapping m, 3H),
7.38-7.44 (overlapping m, 3H), 7.48 (dd, J ) 8.1 and 1.5 Hz,
1H), 7.56-7.58 (overlapping m, 3H), 7.77 (broad d, J ∼ 7.9 Hz,
2H). 13C NMR (DMSO-d6): 124.35, 126.08, 127.04, 127.29, 127.53,
127.82 (2 peaks), 128.54, 130.15, 131.62, 137.42, 146.59, 150.46.
EI-MS (m/z): 353 (M+), 226, 199, 198, 154. HR-MS (EI): M+
found, 353.0279; calcd for C19H12NO2SCl, 353.0277.
3-Chlorophenyl Carbamate (22). Colorless crystals, mp 132-133
°C. IR (Nujol): 1727, 1586, 1334, 1260, 1216, 1134, 1090, 1014,
949, 874, 770, 760, 752 cm -1. 1H NMR (CDCl3): 7.08-7.10 (m,
1H), 7.17-7.26 (overlapping m, 5H), 7.29-7.33 (m, 2H), 7.41 (dd,
J ) 7.8 and 1.2 Hz, 2H), 7.64 (broad d, J ∼ 8 Hz, 2H). 13C NMR
(CDCl3): 119.80, 122.06, 125.96, 126.80 (2 peaks), 126.99, 127.66,
129.99, 132.31, 134.53, 137.75, 151.31, 151.62. EI-MS (m/z): 353
(M+; base), 199, 198, 171, 154, 140, 127, 111. HR-MS (EI): M+
found, 353.0282; calcd for C19H12NO2SCl, 353.0277.
4-Chlorophenyl Carbamate (23). Colorless crystals, mp 179-180
°C. IR (Nujol): 1737, 1330, 1307, 1220, 1132, 1087, 1009, 850,
806, 764, 756 cm -1. 1H NMR (DMSO-d6): 7.29-7.34 (overlapping
m, 4H), 7.39-7.42 (m, 2H), 7.45-7.48 (m, 2H), 7.54 (dd, J ) 7.8
and 1.3 Hz, 2H), 7.79 (broad d, J ∼ 7.9 Hz, 2H). 13C NMR
(DMSO-d6): 123.75, 127.06, 127.08, 127.41, 127.70, 129.37,
130.08, 131.40, 137.50, 149.41, 151.09. EI-MS (m/z): 353 (M+),
198, 171, 166, 154, 140, 128, 127, 113, 111 (base), 99, 75. HR-
MS (EI): M+ found, 353.0262; calcd for C19H12NO2SCl, 353.0277.
3-(N,N-Dimethylamino) Phenyl Carbamate (24). Colorless
crystals, mp 181-184 °C. IR (Nujol): 1718, 1622, 1510, 1327,
1260, 1213, 1139, 1018, 999, 763, 750 cm -1. 1H NMR (CDCl3):
2.92 (s, 6H), 6.46-6.59 (overlapping m, 3H), 7.18-7.21 (overlap-
ping m, 3H), 7.29-7.32 (m, 2H), 7.40 (dd, J ) 7.8 and 1.2 Hz,
2H), 7.68 (dd, J ) 8.0 and 0.9 Hz, 2H). 13C NMR (CDCl3): 40.49,
105.55, 109.02, 109.93, 126.53, 126.92, 126.97, 127.57, 129.52,
132.22, 138.17, 151.67, 152.02, 152.26. EI-MS (m/z): 362 (M+),
298, 263, 261, 200, 199, 198 (base), 197, 171, 166, 154, 140, 137,
127, 120, 86, 84, 69. HR-MS (EI): M+ found, 362.1093; calcd for
C21H18N2O2S, 362.1089.
1-Naphthyl Carbamate (28). Colorless crystals, mp 132-133
°C. IR (Nujol): 1720, 1598, 1510, 1322, 1254, 1226, 1208, 1150,
1091, 1043, 1014, 788, 765 cm -1. 1H NMR (CDCl3): 7.18-7.22
(m, 2H), 7.29-7.33 (m, 2H), 7.40-7.48 (overlapping m, 6H),
7.67-7.70 (m, 1H), 7.76 (dd, J ) 8.1 and 0.9 Hz, 2H), 7.78-7.82
(m, 2H). 13C NMR (CDCl3): 117.85, 121.15, 125.31, 125.91,
126.34, 126.49, 126.81, 126.94, 127.05, 127.12, 127.69, 127.91,
132.53, 134.60, 138.17, 146.87, 152.25. EI-MS (m/z): 369 (M+),
198, 171, 166, 154, 127, 115 (base), 101, 89, 77. HR-MS (EI):
M+ found, 369.0831; calcd for C23H15NO2S, 369.0823.
2-Naphthyl Carbamate (29). Colorless crystals, mp 205.5-206.5
°C. IR (Nujol): 1720, 1333, 1262, 1216, 1151, 1136, 1013, 804,
751 cm -1. 1H NMR (DMSO-d6): 7.31-7.35 (m, 2H), 7.42-7.59
(overlapping m, 7H), 7.82-7.87 (overlapping m, 3H), 7.91 (broad
d, J ∼ 7.9 Hz, 1H), 7.96 (broad d, J ∼ 7.8 Hz, 1H), 7.99 (broad d,
J ∼ 8.9 Hz, 1H). 13C NMR (DMSO-d6): 118.48, 121.44, 125.88,
126.75, 127.08, 127.13, 127.44 (2 peaks), 127.70 (2 peaks), 129.31,
130.99, 131.47, 133.26, 137.66, 148.28, 151.52. EI-MS (m/z): 369
(M+), 198, 154, 127, 115 (base), 101, 89, 77. HR-MS (EI): M+
found, 369.0810; calcd for C23H15NO2S, 369.0823.
Enzyme Kinetic Studies. The esterase activity of AChE and
BuChE was determined by a modification25 of the Ellman method26
using a Milton-Roy 1201 UV-visible spectrophotometer (Milton-
Roy, Ivyland, PA), set at λ ) 412 nm. Briefly, 1.35 mL of buffered
DTNB solution (pH 8.0), 0.05 mL of enzyme (0.04 units of either
human recombinant AChE, purified human serum BuChE, or
recombinant BuChE in 0.1% aqueous gelatin containing 0.01%
sodium azide and 0.05 mL of 50% aqueous acetonitrile or one of
the phenothiazine carbamates dissolved in this solvent at 0.5-5
mM, depending on solubility, in a stoppered cuvette of 1 cm path
length). After mixing and bringing the absorbance to zero, substrate
(acetylthiocholine for AChE and butyrylthiocholine for BuChE) at
0.03 to 0.16 mM, as appropriate, was added to initiate reaction.
Assays were carried out at 23 °C over a 1 min period, taking
readings every 5 s, after initial 5 s delay. The ∆A/min at 412 nm
was converted to velocity as described earlier.25
To test for enzyme deactivation over time, loss of enzyme activity
was monitored by adding 0.05 mL of 4.8 mM aqueous thiocholine
substrate solution in the cuvette after incubation of the enzyme with
inhibitor and buffered DTNB for periods of up to 30 min. A zero-
time sample was also obtained by adding enzyme last to initiate
reaction and the second-order rate constants for enzyme deactivation
(ka values) were determined.7,27,28 The ka value was calculated by
plotting ln (e0/et)/[I] against time, where e0 is the enzymatic activity
at time zero (without preincubation of enzyme and inhibitor), et is
the enzymatic activity at time t min of preincubation, and [I] is the
molar concentration of inhibitor. The slope of this plot gave the
second-order rate constant (ka value; see Figure 3 as an example).
For study of reversible inhibition of BuChE, an initial experiment
at the highest inhibitor concentration, with subsequent 1:10 serial
dilutions of each compound were carried out to obtain an enzyme
activity-inhibitor concentration profile. Experiments were generally
done at least in triplicate and the values averaged.
2-t-Butylphenyl Carbamate (25). Colorless crystals, 134-135
°C. IR (Nujol): 1728, 1327, 1306, 1285, 1261, 1238, 1211, 1186,
1010, 766, 756 cm -1. 1H NMR (CDCl3): 1.16 (s, 9H), 7.18-7.22
(m, 1H), 7.24-7.40 (overlapping, m, 7H), 7.48 (broad d, J ∼ 7.8
Hz, 2H), 7.74 (broad d, J ∼ 7.9 Hz, 2H). 13C NMR (CDCl3): 29.95,
34.19, 123.87, 125.58, 126.76, 126.80, 126.98, 127.03, 127.35,
127.54, 132.71, 138.09, 141.33, 149.39, 152.34. EI-MS (m/z): 375
(M+), 200, 199, 198 (base), 91. HR-MS (EI): M+ found, 375.1300;
calcd for C23H21NO2S, 375.1293.
4-t-Butylphenyl Carbamate (26). Colorless crystals, 139.5-140.3
°C. IR (Nujol): 1725, 1512, 1332, 1266, 1215, 1173, 1012, 866,
Lineweaver-Burk plots were generated for reversible inhibitors
by using a fixed amount of BuChE (0.04 unit) and varying amounts
of substrate (0.03-0.16 mM, final concentration) in the presence
-1
859, 807, 764, 756 cm
.
1H NMR (CDCl3): 1.29 (s, 9H),
7.09-7.12 (m, 2H), 7.17-7.21 (m, 2H), 7.27-7.31 (m, 2H),

Carbamates with Mechanism of Inhibition for AChE and BuChE
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4211
(8) Taylor, P. Anticholinesterase agents. In Goodman & Gilman’s The
Pharmacological Basis of Therapeutics 11th ed.; Brunton, L. L., Lazo,
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or absence of the inhibitor. The replot of the slopes of the above
double reciprocal plots against inhibitor concentration gave the
inhibitor constant (Ki) as the intercept on the x-axis (see Figure 4
as example).
Calculation of Molecular Parameters. Molecular mechanics
calculations were carried out using the MMFF force field in Spartan
’0629 in which the Merck molecular force field (MMFF) was
employed, with the keyword “equilibrium conformer”, so that the
most stable conformer would be selected.
The structure for the graphical abstract was generated using
HyperChem program.32 Briefly, the crystal structure coordinates
of BuChE (PDB ID: 1POI) were downloaded from the protein data
bank.33,34 All amino acids were then deleted from the structure
except for the residues contained in the active site. Compound 24,
which can be seen in the active site of this figure was optimized
separately at the molecular mechanics level using the HyperChem
program.32
Butterfly angles were determined using Spartan ’06.29 Briefly,
two planes were created using three atoms in each of the aromatic
rings of the phenothiazine moiety. Once the planes were created,
the angle between these planes was calculated, which gave rise to
the butterfly angle.
Log P calculations were determined using the AlogPS program,
which is available online.30 This method compares the input
structure of the compound to a large collection of organic
compounds in a database. The compound was built using a template
provided by the program. A simplified molecular input line entry
system (SMILES) notation was then generated for the compound.
The database assessed the compound based on the SMILES notation
and a theoretical Log P value was determined that statistically best
described the molecule compared to the other molecules in the
database based on a variety of parameters.
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trypsin. Cell. Mol. Neurobiol. 2001, 21, 285–296.
Acknowledgment. Canadian Institutes of Health Research,
Vascular Health and Dementia Initiative (DOV-78344) (through
partnership of Canadian Institutes of Health Research, Heart &
Stroke Foundation of Canada, the Alzheimer Society of Canada,
and Pfizer Canada Inc.), Natural Sciences and Engineering
Research Council of Canada, Capital District Health Authority
Research Fund, Nova Scotia Health Research Foundation, Brain
Tumour Foundation of Canada, the Committee on Research and
Publications of Mount Saint Vincent University. We would like
to thank Jennifer Uhlman for excellent technical support. We
also thank Dr. Ian Pottie (Mount Saint Vincent University,
Halifax) for the use of his Agilent Technologies 1200 series
HPLC system purchased through funds from Canada Foundation
for Innovation.
Supporting Information Available: Tables of purity and HPLC
chromatograms of all the synthesized compounds.This material is
available free of charge via the Internet at http://pubs.acs.org.
(26) Ellman, G. L.; Courtney, K. D.; Andres, V. J.; Featherstone, R. M. A
new and rapid colorimetric determination of acetylcholinesterase
activity. Biochem. Pharmacol. 1961, 7, 88–95.
(27) Webb, J. L. Enzyme and metabolic inhibitors, Vol I.: New York:
Academic Press, 1963, 535-603..
(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 103-119..
(29) Spartan ’06; Wavefunction, Inc.: Irvine, CA, 2006.
(30) Tetko, I. V.; Tanchuk, V. Y.; Villa, A. E. Prediction of n-octanol/
water partition coefficients from PHYSPROP database using artificial
neural networks and E-state indices. J. Chem. Inf. Comput. Sci. 2001,
41, 1407–1421.
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