Isosorbide-2-carbamate esters: Potent and selective butyrylcholinesterase inhibitors

Article abstract of DOI:10.1021/jm800564y

In this study, we report the SAR and characterization of two groups of isosorbide-based cholinesterase inhibitors. The first was based directly on the clinically used nitrate isosorbide mononitrate (ISMN) retention of the 5-nitrate group and introduction of a series of 2-carbamate functionalities. The compounds proved to be potent and selective inhibitors of human plasma butyrylcholinesterase (huBuChE). In the second group, the nitrate ester was removed and replaced with a variety of alkyl and aryl esters. These generally exhibited nanomolar potency with high selectivity for BuChE over acetylcholinesterase (AChE). The most potent and selective compound was isosorbide-2-benzyl carbamate-5-benzoate with an IC₅₀ of 4.3 nM for BuChE and > 50000 fold selectivity over human erythrocyte AChE. Inhibition with these compounds is time-dependent, competitive, and slowly reversible, indicating active site carbamylation.

Full text of DOI:10.1021/jm800564y

6400
J. Med. Chem. 2008, 51, 6400–6409
Isosorbide-2-carbamate Esters: Potent and Selective Butyrylcholinesterase Inhibitors
Ciaran G. Carolan,† Gerald P. Dillon,† Joanne M. Gaynor,‡ Sean Reidy,‡ Sheila A. Ryder,† Denise Khan,† Juan F. Marquez,†
and John F. Gilmer*,†
School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland
ReceiVed May 14, 2008
In this study, we report the SAR and characterization of two groups of isosorbide-based cholinesterase
inhibitors. The first was based directly on the clinically used nitrate isosorbide mononitrate (ISMN) retention
of the 5-nitrate group and introduction of a series of 2-carbamate functionalities. The compounds proved to
be potent and selective inhibitors of human plasma butyrylcholinesterase (huBuChE). In the second group,
the nitrate ester was removed and replaced with a variety of alkyl and aryl esters. These generally exhibited
nanomolar potency with high selectivity for BuChE over acetylcholinesterase (AChE). The most potent and
selective compound was isosorbide-2-benzyl carbamate-5-benzoate with an IC50 of 4.3 nM for BuChE and
>50000 fold selectivity over human erythrocyte AChE. Inhibition with these compounds is time-dependent,
competitive, and slowly reversible, indicating active site carbamylation.
Introduction
physiological role has been assigned to BuChE, nor has it a
known endogenous substrate; for the present, it takes its name
from butyrylcholine, a synthetic substance it hydrolyses exceed-
ingly rapidly (BuChE was historically referred to as pseudo-
cholinesterase, perhaps disparagingly). BuChE is found ubiq-
uitously throughout the human body but is at high concentrations
in the lung, liver, and serum. This distribution pattern points to
a role in primary metabolism, possibly in protecting the
organism from agents that might inhibit the pivotal functions
of AChE. Indeed, BuChE has a prominent role in the metabolism
of ester drugs and in the activation of ester prodrugs. There are
also suggestions that it may be involved in lipoprotein regula-
tion.11 Evidence for the involvement of BuChE in AD and
support for its role as a drug target may be summarized as
follows: (i) the nullizygous (AChE -/-) mouse while having
a clear phenotype appears to have normal CNS function,
indicating at least a compensatory role for BuChE in the CNS;12
(ii) while synaptic AChE levels decrease to 10-15% of normal
during AD progression, BuChE activity is maintained or even
increased;13 (iii) post mortem tissue analysis on AD patients
shows a high level of BuChE in the hallmark lesions of AD14
(although it might well have a protective role here); (iv) in rats,
the selective BuChE inhibitor cymserine causes elevation of
acetylcholine and augments long-term potentiation and learn-
ing.15 A notable feature of that study was the absence of
peripheral side effects that are associated with AChE inhibitors.
There are two types of cholinesterase enzymes found in
vertebrates: acetylcholinesterase (EC 3.1.1.7; AChEa) and
butyrylcholinesterase (EC 3.1.1.8; BuChE). These are probably
the most widely studied enzymes due to their extraordinary
efficiency and the classical relationship between AChE and the
neurotransmitter acetylcholine. AChE has been a drug target
for the treatment of Alzheimer’s disease (AD) since the
emergence of the cholinergic hypothesis of AD over 25 years
ago. This followed recognition that the cognitive impairments
in AD correlated with cholinergic deficits such as reduced
synaptic acetylcholine synthesis and choline acetyltransferase
(ChAT) activity.1,2 These observations were made just as the
role of the cholinergic system in memory processing and
learning began to be more widely appreciated.3 The introduction
of the cholinesterase inhibitors (CHIs), first tacrine then done-
pezil, rivastigmine, and galantamine, has made an important
contribution to the management and well-being of early stage
AD patients, although significant scientific and clinical questions
about these remain.
In the past few years, CHI medicinal chemistry has diversified
from pure AChE inhibitor design into a number of related
strategies: (i) dual binding site inhibitors that bind at the active
site and at the AChE peripheral site, which catalyzes the
aggregation of ꢀ-amyloid in vitro4,5 (dual binding site occupancy
has also led to the compounds of femtomolar potency6); (ii)
hybrid compounds that affect both the metabolism of ACh and
stimulate its receptor targets;7 (iii) hybrids that contain CHI
functionality and a second noncholinergic modality, e.g., MAO
inhibitory activity, melatonin surrogates, or nitric oxide releasing
capability.8-10 A fourth recent strategy (iv) has been to develop
selective inhibitors for BuChE. In contrast to AChE, no
We reported previously that isosorbide esters tend to undergo
very rapid hydrolysis in the presence of mammalian esterases.16-18
The aspirin ester of isosorbide mononitrate (ISMNA) is one of
the few compounds reported that can act as an aspirin prodrug
(at least in rabbit plasma) and it is a potent inhibitor of platelet
aggregation.16 This is because, in the case of ISMNA, hydrolysis
at the aspirin ester group proceeds faster than at the neighboring
acetyl group. We hypothesized that it might be possible to
generate potent and selective inhibitors for the cholinesterases
by replacing the aspirin group in ISMNA with a carbamate
functionality while retaining the isosorbide-5-nitrate group as
illustrated in Figure 1b. The 5-nitrate appeared to us to be an
important recognition feature for esterase complementarity. In
this paper, we report on the pattern of enzyme inhibition by
isosorbide-2-carbamate-5-nitrates and then isosorbide2-carbam-
ate-5-alkyl and -aryl esters. Some of the compounds in the latter
* To whom correspondence should be addressed. Phone: +353-1-896
2795. Fax: +353-1-896 2793. E-mail: gilmerjf@tcd.ie.
†
School of Pharmacy and Pharmaceutical Sciences, Trinity College.
School of Science, Athlone Institute of Technology, Westmeath, Ireland.
‡
a Abbreviations: AChE, acetylcholinesterase; ATCI, acetylthiocholine
iodide; BTCI, butyrylthiocholine iodide; BuChE, butyrylcholinesterase;
DCC, dicyclohexylcarbodiimide; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid);
huBuCHE, human butyrylcholinesterase; ISMN, isosorbide-5-mononitrate;
ISMNA, isosorbide mononitrate aspirinate; MAO, monoamino oxidase;
SAR, structure activity relationship, TBAF, tetrabutylammonium fluoride;
TBDMS, tertbutyldimethylsilyl; TMS, tetramethylsilane.
10.1021/jm800564y CCC: $40.75
2008 American Chemical Society
Published on Web 09/26/2008

Isosorbide-2-carbamate Esters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6401
IC50 values determined at a single time point are a composite
value, reflecting the initial equilibrium as well as the rates of
the carbamylation and decarbamylation steps and they are a
generally used global measure of carbamate potency: E/I
incubation times of 15-30 min are typical in the literature.21-23
Inhibitory potency was determined in this study following
incubation of the enzyme and test compound for 30 min. Several
of the more potent compounds were also tested at 60 min with
no significant shift in potency. Inhibition was initially evaluated
at 100 µM with IC50 values estimated only for compounds
showing significant inhibition (Table 1). The 5-nitrate com-
pounds (series 1) generally exhibited low micromolar potency
toward BuChE with no significant inhibition of AChE at 100
µM. Potency increased in the series from methyl, ethyl, propyl,
to butyl carbamate; the cyclohexyl carbamate had similar
potency to the ethyl carbamate. The phenyl carbamates 1 g-k
were moderate inhibitors of both cholinesterases with slight
preference in some cases for AChE. The most potent compound
in the nitrate series was the 2-benzylcarbamate 1f with an IC50
of 50 nM and >2000 fold selectivity for BuChE. When the
benzyl group was maintained but the nitrate removed (leaving
the isosorbide-5-position unsubstituted, 3), there was a 74-fold
drop in potency as well as selectivity (3.7 µM) although not
because of any significant increase in potency toward AChE.
Clearly, the 5-subsituent has a marked effect on inhibitory
potency toward BuChE. It was therefore decided to explore
2-benzylcarbamates variously substituted at the 5-position with
aryl and alkyl esters. The acetyl, propionyl, valeryl, and
cyclopropyl ester compounds (2a-e) were moderately potent
but selective inhibitors of BuChE. The cyclopentyl ester was
the most potent of the alkyl ester compounds with an IC50 of
5.8 nM for BuChE and 975-fold selectivity over AChE. The
5-benzoate ester (2i) was the most potent compound in this study
and the most selective BuChE inhibitor reported with an IC50
ratio of >63000 over electric eel AChE (4.3 nM/272 µM). When
purified human erythrocyte AChE was used instead, the IC50
dropped a little to 221 µM and the selectivity ratio to 51000.
The nicotinate esters 2j-k were prepared as potentially more
water soluble analogues, but they were around 20-fold less
potent and selective than the corresponding benzoate. The
influence of the ionisability of the pyridine ring on BuChE
affinity or turnover was evaluated by measuring the IC50 at pH
values in the range 6-8, the typical Ellman pH. The potency
was found to be pH invariant in this range. A number of larger
esters were prepared in order to test the hypothesis that in the
case of BuChE, the isosorbide-5-ester group is accommodated
by a large pocket or possibly back up the main active site
channel. It was found that the benzoate ester could be replaced
by 1- or 2-naphthoyl or biphenyl esters (2l-n) with little loss
in potency. The cinnamate (2o) and coumarin (2q) esters were,
however, significantly less potent and the heptyloxybenzoate
ester (2p) quite inactive (IC50 2.8 µM). Having used the 2-benzyl
carbamate to probe for the optimal 5-ester, we now maintained
the 5-benzoate group but changed the 2-carbamate group. The
5-benzoate-2-methylcarbamate (2r), -2-(3,4-dichlorophenylcar-
bamate) (2s), and 2-butylcarbamate (2t) compounds were
significantly more potent than their nitrate analogues (1a, 1j,
and 1d) as expected, but clearly in this panel of possible acyl
groups, a benzyl carbamate is optimal in the 2-position and a
benzoate ester at the 5-position.
Figure 1. (1a) Carbamate inhibitors of the cholinesterases. (1b) Design
strategy behind isosorbide-based carbamates.
group turned out to be among the most selective BuChE
inhibitors reported. We also report preliminary kinetic and
modeling data that provides an explanation for how these
anomalous compounds work.
Chemistry
Isosorbide-2-carbamate-5-nitrates (series 1, Figure 1b) were
obtained by direct carbamylation of ISMN with the appropriate
isocyanate in DCM in the presence of a triethylamine. The
2-carbamate-5-ester compounds of series 2 were obtained by
removal of the nitrate under reductive conditions (H2, Pd/C)
and esterification with a series of commercially available acid
chlorides. Where an acid chloride for a desired ester was not
available, the acid itself was used instead with DCC/DMAP
coupling (Scheme 1). In a few cases, acylation of the 2-car-
bamate nitrogen was found to compete with 5-esterification, and
it proved difficult to get useful amounts of the target ester. In
these cases, the order of carbamylation and esterification was
reversed. ISMN was TBDMS-protected, the 5-nitrate removed,
and then the 5-position esterified (Scheme 1). The 2-TBDMS
group was then removed (TBAF) and the carbamate introduced
as described already.
Enzyme InhibitionsSAR. Candidate inhibitors were evalu-
ated using Ellman’s spectrophotometric determination of cho-
linesterase activity with either human plasma or purified BuChE
from human plasma.19 For AChE activity, electric eel enzyme
was used because this has quite similar active site structure to
the human enzyme. Initially, the assumption was made that the
inhibitors would act through carbamylation of the enzymes
because their design was based on supersubstrates for huBuChE
(Figure 1b). Carbamate inhibition can be considered a two-step
process with an initial rapidly established reversible interaction
being followed by a slower covalent reaction between enzyme
and inhibitor, forming an enzyme carbamate adduct.20 The
carbamate adduct hydrolyses slowly, liberating the free enzyme.
The slow recovery of enzyme activity provides the fundamental
kinetic distinction between carbamate inhibitors and ester
substrates. Each stage of the carbamate inhibition and hydrolysis
process can be influenced by structural features of the inhibitor.20
Enzyme InhibitionsMode. The relationship between inhibi-
tory potency and assay substrate concentration was surveyed
initially in a classical way in order to determine the mode of
inhibition. Substrate hydrolysis velocity was proportional to

6402 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Carolan et al.
Scheme 1. Showing the Synthesis Routes to Candidate Inhibitorsa
a (i) Isocyanate, pyridine or Et3N/DMAP, DCM, 40 °C, 2 h; (ii) Pd/C, H2, EtOAc, 12 h, RT; (iii) R′(CO)Cl, DMAP, Et3N, DCM, RT, 8 h or R′C(O)OH,
DCC, DMAP, DCM, RT, 8 h; (iv) TBDMSCl, imidazole, DMAP, DCM, 2 h, RT; (v) TBAF, THF, RT, 15 min.
Table 1. Carbamate Test Compounds along with Inhibition Data and Selectivity for BuChE and AChEa
BuChE
IC50 (µM)c
AChE
IC50 (µM)c
b
b
compd
series 1, Rd
%
%
selectivityd
1a -methyl
82
73
93
92
99
96
10
37
11
28
11
5.1 (2.2-11.8)
3.9 (1.6-9.7)
67
1
1
17.2 (6-31)
1b -ethyl
1c -propyl
1d -butyl
1e-cyclohexyl
1f -benzyl
1g -(3-trifluormethylphenyl)
1h -phenyl
1i -(2-chlorophenyl)
1j -(3,4-dichlorophenyl)
1k -(3-chlorophenyl)
series 2, RdBn, R′d
2a-acetyl
2b-propionyl
2c-valeryl
0.639 (0.66-1.2)
0.894 (0.65-1.23)
2.5 (1.7-3.4)
1520 (1300-1700)
1440 (1010-3300)
2383
1610
>40
>2000
32
30
35
12
23
24
78
24
0.051 (0.4-0.7)
9.9 (7.9-12.3)
97
97
95
97
99
85
9.8
25
99
99
97
99
100
99
4.1 (3-5)
12
5
4
55
88
6
>25
>100
>142
∼300
9383
0.98 (0.8-1.21)
0.7 (0.52-0.94)
0.334 (0.25-0.44)
0.006 (0.001-0.0012)
0.359 (0.15-0.88)
2d-cyclopropyl
2e-cyclopentyl
2f-triflate
56.3 (29-79)
>278
2g-mesylate
3
2h-tosylate
66
21
33
24
86
80
89
82
21
81
2i-benzoate
0.0043 (0.001-0.007)
0.057 (0.049-0.068)
0.088 (0.074-0.106)
0.028 (0.025-0.032)
0.032 (0.022-0.048)
0.012 (0.01-0.015)
0.137 (0.103-0.183)
2.8 (2.2-3.2)
272.5 (210-354)
63,255
1754
2j-nicotinate
2k-isonicotinate
2l -(1-naphthoyl)
2m -(2-naphthoyl)
2n-biphenyl
37.6 (21-69)
43.4 (26-71)
51.6 (28-75)
55.9 (52-60)
1342
1367
4187
∼729
35
2o-cinnamate
96
80
100
2p-(4-hepyloxy-)benzoate
2q-coumarin ester
R′, benzoate, R, carbamate
2r-2-methycarbamate-
2s -3,4-dichlorphenyl
2t-2-butyl
0.073 (0.06-0.08)
60.4 (41-81)
821
93
38
99
95
0.669 (0.541-0.768)
54
70
31
15
∼150
0.072 (0.031-0.163)
3.7 (3.3-4.1)
0.028 (0.011-0.041)
0.733 (0.555-0.953)
255 (180-362)
1389
>27
3.7
3-2-benzylcarbamate-5-OH
eserine
iso-OMPA
0.103 (0.61-0.17)
2.5 (0.9-3.7)
0.063 (0.05-0.09)
3.41
2.5e-4
BW284c51
a All experiments performed in triplicate. b Inhibition % was determined at 100 µM inhibitor concentration. c IC50 values are expressed with the 95%
confidence interval. d Selectivity was estimated by dividing the IC50 for AChE by the IC50 for BuChE. In cases where there was negligible inhibition of
AChE at 100 µM and the IC50 was not determined, the selectivity is stated to be greater than the value obtained using 100 µM instead of the IC50.
substrate concentration with increasing Vmax values. Hydrolysis
velocity was inversely proportional to inhibitor concentration
consistent with a competitive mode of inhibition. However KM
values shifted only slightly. Double reciprocal plots yielded
increasing slopes and intercepts, indicating mixed inhibition.
Secondary replots of Lineweaver-Burk and Dixon data further
indicated a significant amount of noncompetitive inhibition. This
pattern can be observed when a reversible model is applied to
pseudoirreversible inhibitors, and the assay is to a large extent
measuring residual enzyme activity after carbamylation has
already occurred. Therefore, the time dependence of inhibition
was evaluated by sampling enzyme activity at successive time
points immediately after introducing the inhibitor and then
substrate. The experiment was performed repeatedly at increas-

Isosorbide-2-carbamate Esters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6403
Figure 2. Percentage inhibition of huBuChE by 2i as a function of
time at a BTCI concentration of 0.5 nM and inhibitor concentrations
of 1.25 mM (+), 333 nM (2), 250 nM (0), 125 nM (b), 83 nM (×),
62.5 nM (O), 50 nM (9).
Figure 3. Recovery of purified huBuChE enzyme activity following
treatment with compound 2i and 2t and dilution.
ing inhibitor but fixed substrate concentrations. The disappear-
ance of enzyme activity for compounds 1f, 2i, and 2t was time-
dependent at low concentration but apparently immediate at high
concentration. At intermediate-low concentration, the disap-
pearance was first order and proportional to inhibitor concentra-
tion as shown in Figure 2 for 2i. The IC50 values for 2i
determined at 60 min (3.6 nM (1.3-9.7)) was similar to the 30
min value. Further experiments were performed at fixed inhibitor
concentration but increasing substrate concentration in the range
0.5-10 mM. The disappearance of enzyme activity was again
time-dependent and the extent of inhibition at maximal inhibition
was inversely proportional to substrate concentration, indicating
competitive inhibition. When the carbamylation experiment was
repeated for 2i in the presence of edrophonium, a reversible
active site directed inhibitor, there was a marked reduction in
the rate and extent of inhibition.24 Another relevant kinetic
feature of carbamate inhibition is the slow regeneration of
enzyme activity as the enzyme-carbamate adduct undergoes
turnover. huBuChE solutions were treated with compound 1f,
2i, or 2t at a concentration giving 100% for two hours.
Compound 2t was selected because it gives rise to an
enzyme-butyl carbamate adduct, whereas 1f and 2i generate
enzyme-benzyl carbamates, albeit with different potencies. In
the case of inhibitors 2i and 2t, the recovery of enzyme activity
was measured following dilution to prevent reinhibition. Activity
recovered slowly over 24 h but with similar rates (Figure 3).
The 1f-E solution was transferred to a dialysis bag and immersed
in a buffered solution, which was then periodically changed as
dialysis proceeded. In this case, there was significant residual
inhibition up to 32 h.
In summary, the evidence for competitive cholinesterase
carbamylation by the isosorbide-based carbamates is: (i) inhibi-
tion is time-dependent at low inhibitor concentration but
independent of it at high concentration; (ii) the extent and rate
of enzyme inhibition is substrate concentration dependent; (iii)
inhibition is attenuated in the presence of the active-site
dependent reversible inhibitor edrophonium; (iv) inhibition
kinetics are first order at low concentration; (v) there is slow
recovery of enzyme activity.
Modeling, Stability, and Structural Studies. Carbamate
inhibitors of the cholinesterases are generally related to phys-
ostigmine, isolated from the calabar bean. They usually consist
of an alkyl or aryl carbamate of a phenol with a distal amino
group that is positively charged at physiological pH (Figure 1a).
The compounds described here depart markedly from this
Figure 4. Conformation for compound 2i produced using a stochastic
search procedure followed by energy minimization using the MMFF94s
force field in MOE. The 5-ester group is endo to the V-shaped ring,
whereas the 2-carbamate is exo.
pattern in being neutral (nonamino) and because they are derived
from the secondary alcohol and pseudosugar, isosorbide. Isos-
orbide is V-shaped because of its cis ring junction with an angle
of approximately 120 ° between two planar rigid rings. (Figure
4). The 2-R-OH group lies exo to the ring and the 5-ꢀ group
endo. Endo substituents are directed into the V, exo groups
outside it. Although this makes the 2-position more sterically
accessible, in isosorbide, the 5-OH is activated because of an
intramolecular H-bond to one of the ring oxygen atoms. In the
2-carbamate-5-esters, the 5-ester is directed orthogonal to the
plane of isosorbide rings, whereas the 2-exo group points away
from the system (Figure 4). In isosorbide diesters, the 2-group
tends to undergo very rapid hydrolysis in human plasma. (This
observation was critical to the carbamate design reported here.)
The 5-endo ester is much less prone to hydrolysis (at least by
butyrylcholinesterase) and in human plasma, isosorbide 2,5-
diesters undergo hydrolysis almost exclusively at the 2-position,
liberating the corresponding 5-monoester. For example, the half-
life for isosorbide-2,5-dibenzoate at Vmax concentration in 50%
human plasma is 19 s with the exclusive liberation of isosorbide-
5-benzoate.18 The 5-benzoate product eventually decays with a
half-life of 41 min. The dibenzoates do not undergo processing
by AChE, indeed, substituted dibenzoates are moderately potent
AChE inhibitors.25
To confirm the stability of the 5-ester under assay conditions,
compound 2i was incubated in 50% human plasma solution in

6404 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Carolan et al.
residues are Ser-198, His-438, and Glu-325. Other residues of
note in BuChE are Trp-82, the principal residue of the cation-π
site where the cationic head of choline substrates bind; the
peripheral site residues, Asp-70 and Tyr-332, which bind ligands
initially and help to orientate them appropriately to slide down
the gorge and interact with the active site, and Glu-116, Glu-
117, and Ala-199, which together form the oxyanion hole that
stabilizes the negative charge on the carbonyl oxygen that
develops subsequent to the nucleophilic attack by O-γ of the
catalytic serine. The principal difference between the enzymes
is the size of the acyl pocket. In AChE, the pocket is occluded
by the aromatic groups of residues Phe-288 and Phe-290
(Torpedo californica AChE numbering is used throughout the
text when referring to residues of AChE). In the larger BuChE
channel, the acyl pocket is lined with the aliphatic residues Leu-
286 and Val-288.
Kinetic studies indicated that inhibition by the isosorbide-
based carbamates proceeds through serine carbamylation. This
involves a tetrahedral intermediate that collapses to the car-
bamylated drug-enzyme adduct. While the transition state
during a reaction process is associated with a first-order saddle
point on the potential energy surface, this tetrahedral intermedi-
ate is an energy minimum on the overall surface and can thus
be modeled using classical force field-based methods as
described by Gao et al.33
Figure 5. Compound 1f docked within the active site gorge of
huBuChE with Connolly interaction surface in yellow and a Gaussian
contact surface as a red mesh.
the range 10-100 µM. No hydrolysis occurred over several
hours as measured by HPLC, and there was no apparent
formation of either benzoic acid or isosorbide-5-benzoate, the
carbamylation product. Enzyme-substrate interactions with the
isosorbide-2-carbamate are preferred over enzyme-5-ester and
the carbamate interaction is inhibitory, abolishing esterase
activity. The failure to detect the carbamylation byproduct in
this experiment is probably due to the negligible depletion of
inhibitor by the tiny amount of enzyme present (BuChE is
present in human plasma at about 5 mg/L with a mass of 86
kD per subunit).26 Although isosorbide-5-esters are products of
the pseudosubstrate carbamate interaction, they do not ac-
cumulate sufficiently in the inhibitory period to have influenced
the inhibitory data. The isosorbide-5-benzoate ester product of
compound 2i is in any case a weak reversible inhibitor of BuChE
(30% at 100 µM). The modeling and kinetic data for series 2
suggest that the 5-ester group promotes carbamylation by
binding in the active site in orientations that are productive for
enzyme carbamylation but not for 5-ester hydrolysis. BuChE
is the most significant esterase in human plasma (at least in
terms of activity). We can anticipate problems with the
compounds in in vivo models because of nonspecific 5-ester
hydrolysis by carboxylesterases present in other tissues that do
not possess the same substrate or inhibitor/substrate as BuChE.27
However, these observations are not relevant to interpretation
of the BuChE-ligand interaction data presented here.
A modeling approach was used to further explore the nature
of 5-ester interaction, the role of the 5-ester group in determining
potency, and the basis for cholinesterase selectivity. Modeling
of cholinesterase enzymes and their inhibitors has been aided
greatly by the elucidation of the structures of enzyme-ligand
complexes for both BuChE and its homologous enzyme,
AChE.28-30 The active sites of both enzymes are at the base of
a gorge of some 20 Å depth in both cases.31,32 On the basis of
crystallographic studies, mutagenesis work, and radiolabeling
studies, a number of subsites have been identified in the catalytic
gorges that are typically involved in binding ligands, both
substrates and inhibitors. While the residues that comprise these
sites vary between BuChE and AChE, the overall structures are
basically similar, and comparisons between the two uncover
the origins of binding specificity and the basis of ligand
selectivity for one enzyme over the other.
Isosorbide inhibitors of BuChE were modeled in the active
site of the enzyme as this tetrahedral intermediate, allowing us
to draw structural conclusions regarding the basis of relative
inhibitor activity. The approach has the virtue of limiting the
potential containment volume of the ligand and has been used
to model cholinesterase inhibitors previously.34 It is structurally
very similar to the reversible Michaelis complex formed upon
initial binding and will indicate the likelihood of initial binding
in a manner that is appropriate for carbamylation.
Modeling of the isosorbide-based inhibitors was based on the
2.0 Å resolution crystal structure of huBuChE. (PDB entry
1p0i).32 When BuChE was crystallized and its structure
established, it was noted that the catalytic Ser-198 was bound
to an unidentified moiety that was ultimately modeled as
butyrate. Apparently, the presence of this butyrate fragment was
essential for crystallization. The butyrate was bound to the serine
residue in a tetrahedral configuration, with one oxygen atom
orientated toward the oxyanion hole. The second oxygen atom
points toward Trp-82, while the butyryl chain points toward
the acyl pocket. The butyrate fragment could thus be used as a
template for the tetrahedral intermediate involved in carbamy-
lation. The assignment of directionality for the other functional
groups was based on the positioning of similar groups in crystal
structures of other enzyme-ligand complexes. The bulky
isosorbide ester fragment could be orientated toward Trp-82,
while the carbamate side chain could be directed toward the
acyl pocket; similar placement of side chains could be observed
in the crystal structures of rivastigmine and trimethylammonium
trifluoroacetophenone with AChE (PDB codes 1gqr and 1amn,
respectively).29,35 The inhibitors were built in situ within the
enzyme active site using the butyrate fragment as a template
and placing each group at the appropriate binding subsite. A
tethered minimization procedure gave rise to the models, two
of which are presented in Figures 5 and 6.
The general activity of the inhibitor series could be explained
by the fact that the isosorbide moiety, when the attached
carbamate is bound by the catalytic serine, closely lines the base
of the gorge (Figure 5) while orientating the two groups at the
2- and 5-position toward appropriate pockets. The relative
These enzymes are serine hydrolases, and a classic catalytic
triad is formed with histidine and glutamate. In huBuChE, these

Isosorbide-2-carbamate Esters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6405
Table 2. Predicted Binding Affinity for a Selected Range of Inhibitors
to BuChE Relative to Compound 2i
compd
affinity (kcal ·mol-1
)
2n
2t
2j
2r
1a
1h
1f
3
-0.656
-4.39
-0.166
-10.997
-16.212
-10.693
-8.340
-14.562
predicted by quantitative analysis, which suggested a 7
kcal·mol-1 difference in binding energy between the two
compounds in favor of compound 1f. However, the incorpora-
tion of a variety of ester derivatives served to significantly
increase potency. Models suggested that compound 2i should
bind more avidly to BuChE than 1f by a factor of 8 kcal ·mol-1
.
This is principally due to the increased interaction with Trp82,
the principal residue of the cation-π site. Longer chain esters
could also be accommodated due to the general orientation of
the 5-group pointing upward toward the gorge entrance. The
biphenyl ester, for example, can interact not only with Trp82
but also Trp430, compensating for its steric bulk. Its predicted
binding energy was then 1 kcal·mol-1 less than compound 2i.
The predicted binding energies of a number of the com-
pounds, relative to the most potent inhibitor in the group,
compound 2i, are shown in Table 2.
Figure 6. Compound 1f covalently bound to huBuChE. Residues Ser-
200, Trp-84, Phe-288, and Phe-290 of TcAChE are also shown, having
been overlaid from the AChE structure from PDB code 1EA5. AChE
residues are shown in orange; BuChE residues are green and the ligand
yellow.
potencies of the different compounds in Table 1 could be
explained by the extent to which the functional groups,
permitting interactions with various residues of the active site
gorge.
In series 1, the carbamate group was altered while a nitrate
group was maintained at the 5-position of isosorbide. The
carbamate function was orientated toward the acyl pocket, and
it was the differential interaction of the various groups with
the residues at this position that gave rise to the variable
potencies of these compounds. Smaller carbamate compounds
tended to be poorer inhibitors of BuChE, largely because they
failed to fully occupy the large pocket formed by residues
Leu286 and Val288. Thus the methyl and ethyl carbamates were
relatively poor inhibitors, but potency increased for propyl and
butyl carbamates as the longer chains could fit better within
the gorge. The phenyl carbamates were also poor inhibitors of
BuChE in general because they similarly could not fill the
pocket. The extra methylene group in the benzyl carbamate
functional group allowed extension of the side chain into the
acyl pocket, however, and the formation of σ-π interactions
between the aromatic ring of the ligand and the aliphatic side
chains of the surrounding residues. These findings were borne
out within the models: the interaction energy between the benzyl
carbamate compounds and BuChE was predicted as being almost
8 kcal ·mol-1 more favorable than the methyl carbamate and 3
kcal·mol-1 more favorable than the phenyl carbamate. The acyl
pocket of AChE is larger and therefore less able to cope with
large carbamate side chains. Phenyl carbamates tend to interact
well with the aromatic side chains of Phe288 and Phe290, a
fact that has been documented previously for a number of
compounds such as phenserine. Extended side chains at this
site tend to favor BuChE selectivity, hence the BuChE selectivity
of benzyl carbamate compounds. This effect is demonstrated
in Figure 6.
Because the models described below are inherently biased
toward the starting pose prior to minimization, a covalent
docking procedure was also carried out in order to examine
whether the placement of the molecules in the active site was
optimal. This was achieved using the Autodock 3 program.36
In this case, the ligand could dock within the gorge in any
orientation without bias toward starting structure. Compound
2i was docked within the active site gorge of huBuChE. When
the results were clustered, the largest of only four molecular
clusters contained molecules docked in the described orientation.
It was satisfying that the docked poses were generally similar
to that produced using the minimization protocol despite the
fact that the protein was rigid in this protocol. The simple and
fast minimization procedure could apparently produce reliable
models of the inhibitors that were replicated by the more
rigorous docking approach (Figure 7).
Conclusion
Isosorbide-2-carbamate-5-nitrate and -benzoate esters are
highly potent and selective inhibitors of huBuChE. They are
structurally distinct from established carbamates, being un-
charged and derived from a secondary alcohol. Their neutrality
is likely to be important to their potential utility. Central and
peripheral BuChE have similar structures at the subunit level
so that inhibitors of plasma BuChE ought to act as inhibitors
of central BuChE, provided they cross the blood-brain barrier.
The compounds act as competitive pseudosubstrates with time-
dependent but reversible inhibition. Molecular models indicate
that they bind with the carbamate functionality pointed into
the acyl pocket and the 5-ester group directed back up the
cholinesterase channel. The 5-position shows some tolerance
to substitution, which might allow for tuning of pharmaceutical
characteristics or the introduction of new pharmacological
modalities. Compounds 2i and 2t are being evaluated in
clinically relevant models of learning and memory in aged mice.
Modification of the group at the 5-position (series 2) gave
rise to compounds of variable potency. In the models that were
prepared, the fragments at the 5-position pointed upward toward
the gorge entrance, aligned with Trp-82 at the so-called cation-π
site and toward the peripheral site residues at the lip of the gorge.
The 5-nitrate group is relatively small and relatively few
interactions were evident between this group and residues of
the catalytic gorge. Its presence was still important; compound
1f is substantially more potent than compound 3, and this was
Experimental Section
Chemistry. Infrared (IR) spectra were obtained using a Perkin-
Elmer 205 FT Infrared Paragon 1000 spectrometer. Band positions

6406 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Carolan et al.
Isosorbide-2-benzylcarbamate (3). 2-(Benzylaminocarbony-
loxy-)-5-O-nitro-1,4:3,6-dianhydro-D-glucitol 1f (mol wt 324.29
g/mol, 46.25 mmol, 15 g) was dissolved in ethyl acetate:methanol
1:1 (100 mL) in a 250 mL round-bottom flask. A spatula tip-full
of 10% palladium on activated carbon was added to the solution.
Air was expelled from the flask, and the mixture was kept under
an atmosphere of hydrogen gas and stirred for 24 h. The solvent
was evaporated under vacuum, and the crude product was dissolved
in DCM and filtered through silica. The filtrate was collected and
evaporated under vacuum to yield 12.60 g as a white crystalline
product (97.5%). mp 76.0 °C. 1H NMR δ (CDCl3): 2.67 (d, 1H, J
) 7.03 Hz), 3.57 (dd, 1H, J ) 5.53, 8.54 Hz), 3.90 (dd, 1H, J )
6.03, 9.54 Hz), 4.00 (dd, 1H, J ) 3.51, 10.54 Hz), 4.10 (d, 1H, J
) 10.45), 4.31 (m, 1H), 4.38 (d, 2H, J ) 6.03 Hz), 4.51 (d, 1H, J
) 4.01 Hz), 4.61 (t, 1H, J ) 4.77 Hz), 5.17 (m, 1H), 5.22 (d, 1H,
J ) 3.01 Hz), 7.23-7.40 (m, 5H). 13C NMR ppm (CDCl3): 44.70
(CH2), 71.8, 73.04, 73.38, 78.39, 81.46, 85.25, 127.13, 127.23,
128.30, 137.6, 154.69. Anal. (C14H17NO5) C, H, N.
Isosorbide-2-benzylcarbamate-5-cyclopropanoate (2d). A white
crystalline product (265.2 mg, 85.2%): mp 96 °C. 1H NMR δ
(CDCl3): 0.87-0.98 (m, 2H), 1.04 (m, 2H), 1.70 (m, 1H), 3.80
(dd, 1H, J ) 5.52, 9.54 Hz), 3.95 (dd, 1H, J ) 6. 03, 9.54 Hz),
3.98-4.08 (m, 2H), 4.37 (d, 2H, J ) 6.02 Hz), 4.52 (d, 1H, J )
4.51 Hz), 4.79 (t, 1H, J ) 4.77 Hz), 5.14 (q, 1H, J ) 5.52, 11.54
Hz), 5.17-5.27 (m, 2H), 7.22-7.40 (m, 5H). 13C NMR ppm
(CDCl3): 44.64, 69.74, 73.25, 73.48, 78.09, 80.27, 85.52, 127.10,
127.17, 128.3, 137.65, 154.81, 173.93. Anal. (C18H21NO6) C, H,
N.
Isosorbide-2-benzylcarbamate-5-cyclopentanoate (2e). A white
crystalline compound (81.9%): mp 94 °C. 1H NMR δ (CDCl3):
1.53-2.00 (m, 8H), 2.83 (m, 1H), 3.73 (dd, 1H, J ) 5.02 and 10.03
Hz), 3.89-4.06 (m, 3H), 4.39 (d, 2H, J ) 6.02 Hz), 4.50 (d, 1H,
J ) 4.51 Hz), 4.81 (t, 1H, J ) 5.02 Hz), 5.08-5.18 (m, 2H), 5.20
(d, 1H, J ) 3.01 Hz), 7.22-7.40 (m, 5H). 13C NMR ppm (CDCl3):
25.35, 25.37, 29.43, 29.73, 43.04, 44.65, 70.12, 73.05, 73.21, 78.06,
80.29, 85.62, 127.10, 127.19, 128.28, 137.61, 154.79, 175.78. Anal.
(C20H25NO6) C, H, N.
Isosorbide-2-(benzylcarbamate)-5-benzoate (2i). Compound 3
(0.6027 g, 2 mmol) was dissolved in dry pyridine (5 mL), and the
solution was cooled to -5 °C using a salt/water bath. Benzoyl
chloride (mol wt 140.57 g/mol, d ) 1.211 g/mL, 5 mmol, 0.56
mL) was added. The mixture was allowed to come to room
temperature, and stirring was continued for 22 h. The mixture was
added to ice-water (approx 200 mL), and the precipitated solid
was filtered. The resultant solid was taken up in chloroform (15
mL) washed first with water (10 mL) then with saturated brine
solution (10 mL), dried with anhydrous sodium sulfate (10 g),
filtered into a round-bottomed flask, and evaporated under reduced
pressure. The resultant semisolid was recrystallized from hot ethanol
to yield 0.74 g of a white crystalline product (96.1%): mp 76.0 °C,
[R]D +13.45 °C. 1H NMR δ (DMSO): 3.84-3.85 (s, 2H),
3.85-3.97 (m, 2H), 4.18-4.20 (d, 2H, J ) 6.4 Hz), 4.45-4.46 (d,
1H, J ) 5.2 Hz), 4.88-4.91 (t, 1H, J ) 5.6 Hz), 5.03 (s, 1H),
5.36-5.38 (t, 1H), 7.23-7.33 (m 5H), 7.53-7.57 (m, 2H),
7.66-7.70 (m, 1H), 7.97-8.01 (m, 3H). 13C NMR ppm (DMSO):
44.10, 70.89, 73.08, 74.79, 77.86, 81.04, 86.29, 127.16-127.37,
128.63, 129.19, 129.56-129.65, 133.86, 139.89, 155.8, 165.30.
Anal. (C21H21NO6) C, H, N.
Isosorbide-2-benzylcarbamate-5-nicotinate (2j). To a solution
of 2-O-(t-(butyl)-dimethylsilyl-)-1,4:3,6-dianhydro-D-glucitol 4 (mol
wt 260.40 g/mol, 1.1521 mmol, 300 mg) in dry THF (50 mL) was
added 1.25 mol equiv of anhydrous nicotinoyl chloride hydrochlo-
ride (mol wt 178.05 g/mol, 1.4400 mmol, 256.4 mg). The mixture
was kept under an atmosphere of nitrogen and stirred at room
temperature for 15 min. To the mixture was added four mol equiv
of triethylamine, and stirring was continued for a further 24 h. The
solvent was evaporated under vacuum to give a white residue, which
was dissolved in a saturated aqueous solution of Na2CO3 (25 mL)
and extracted with DCM (3 × 25 mL). The collected organic
portions were dried over anhydrous sodium sulfate (5 g) and
evaporated to dryness to give a clear oil. The oil was diluted with
Figure 7. Compound 2i modeled as the tetrahedral acylation inter-
mediate in the BuChE active site using Autodock 3.
are given in cm-1. Solid samples were obtained by KBr discs, and
oils were analyzed as neat films on NaCl plates. 1H and 13C spectra
were recorded at 27 °C on a Brucker DPX 400 MHz FT NMR
spectrometer (400.13 MHz 1H, 100.61 MHz 13C) or a Brucker
AV600 (600.13 MHz 1H, 150.6 MHz 13C) in either CDCl3 or
(CD3)2CO with TMS as internal standard. In CDCl3, 1H spectra
were assigned relative to the TMS peak at 0.0 ppm, and 13C spectra
were assigned relative to the middle CDCl3 triplet at 77.00 ppm.
1
In (CD3)2CO, H spectra were assigned relative to the (CD3)2CO
peak at 2.05 ppm, and 13C spectra were assigned relative to the
(CD3)2CO at 29.5 ppm. Coupling constants were reported in hertz
(Hz). HRMS was performed using a Micromass mass spectropho-
tometer with electrospray ionization at the School of Chemistry,
Trinity College Dublin. Elemental analyses were performed at the
Microanalytical Laboratory, Department of Chemistry, University
College Dublin. Flash chromatography was performed on Merck
Kieselgel (particle size: 60 mm).
Isosorbide-2-(butylcarbamate)-5-mononitrate (1d). ISMN
(0.7852 g, 4 mmol) was dissolved in dry pyridine (5 mL). Butyl
isocyanate (mol wt 99.13 g/mol, d ) 0.880 g/mL, 8 mmol, 0.79304
g, 0.9 mL) was added and the mixture heated at 100 °C for 1 h.
The mixture was cooled and methanol (10 mL) added to remove
excess isocyanate. The mixture was then heated for further 10 min
at 100 °C, cooled to room temperature, poured into ice-water, and
stirred. The precipitate was collected and crystallized from hot
methanol to yield 0.69 g of a white crystalline product (59.3%):
mp 101.7 °C. 1H NMR δ (CDCl3): 0.91-0.95 (t, 3H, J ) 7.8 Hz.),
1.32-1.1.39 (m, 2H), 1.45-1.51 (q, 2H, J ) 7.8 Hz), 3.16-3.21
(q, 2H, J ) 7 Hz), 3.88-3.3.92 (m, 1H), 3.96-4.06 (m, 3H),
4.49-4.50 (d, 1H, J ) 5.3 Hz,), 4.79 (s, 1H), 4.95-4.97 (t, 1H, J
) 5.3 Hz), 5.17-5.18 (d, 1H, J ) 2.6 Hz), 5.3-5.35 (m, 1H). 13
C
NMR ppm (CDCl3): 13.2, 19.39, 31.4, 40.3, 68.7, 73.3, 77.1, 80.9,
80.9, 86.34, 154.6. Anal. (C11H18N2O7) C, H, N.
Isosorbide-2-(benzylcarbamate)-5-mononitrate (1f). A white
crystalline product (47.3%): mp 93.4 °C, [R]D +99.01 °C. 1H NMR
δ (CDCl3): 3.88-3.91 (m, 1H), 3.96-4.07 (m, 3H), 4.36-4.38 (d,
2H, J ) 6 Hz), 4.50-4.51 (d, 1H, J ) 4.8 Hz), 4.94-4.96 (t, 1H,
J ) 5.2 Hz), 5.20-5.21 (d, 1H, J ) 2.8 Hz), 5.23 (s, 1H),
5.33-5.36 (m, 1H), 7.28-7.38 (m, 5H). 13C NMR ppm (CDCl3):
69.6, 74.1, 77.2, 78.23, 81.76, 81.85, 87.14, 127.95, 128.07, 129.2,
138.42, 155.54. Anal. (C14H16N2O7) C, H, N.
Isosorbide-2-phenylcarbamate-5-mononitrate (1h). A white
crystalline product (22%): mp 156.6 °C, [R]D +81.0 °. 1H NMR δ
(CDCl3): 3.9 (m, 1H), 4.0 (m, 2H), 4.1 (d, 1H), 4.5 (d, 1H, J ) 4.9
Hz), 5.0 (t, 1H, J ) 4.9 Hz), 5.3 (d, 1H, J ) 3 Hz), 5.4 (m, 1H),
6.7 (s, 1H), 7.1 (t, 1H), 7.3 (m, 4H). 13C NMR ppm (CDCl3): 69.31,
73.59, 78.02, 81.29, 81.53, 86.66, 118.72, 123.93, 129.17, 137.24,
152.05. Anal. (C13H14N2O7) C, H, N.

Isosorbide-2-carbamate Esters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6407
THF (10 mL) and 1.1 mol equiv of TBAF (mol wt 261.47 g/mol,
0.7524 mmol, 196.7 mg, 0.7524 mL of a 1 M solution) was added,
and the mixture was stirred at room temperature for 15 min. The
solution was evaporated to dryness under vacuum giving a brown
oil. DCM (10 mL) was added to the reaction vessel, and the solution
was stirred at room temperature. To the mixture was added
triethylamine (mol wt 101 g/mol, d 0.726 g/mL, 0.7515 mmol, 75.9
mg, 0.1047 mL), benzyl isocyanate (mol wt 133.15 g/mol, d 1.078
g/mL, 0.7515 mmol, 100.2 mg, 0.0929 mL), and DMAP (mol wt
122.17 g/mol, 0.2456 mmol, 30 mg). The mixture was heated to
105 °C for two hours. The mixture was cooled upon completion of
the reaction, and methanol (10 mL) was added to remove excess
isocyanate. The mixture was heated for a further 15 min at 105 °C
and cooled to room temperature. All organic solvent was removed
under vacuum giving a clear oil to which was added DCM (20
mL) and washed with 1 M HCl (20 mL), 5% NaHCO3 (20 mL),
and saturated brine solution (20 mL) and dried over anhydrous
sodium sulfate (2 g). The solution was filtered into a round-
bottomed flask, which was evaporated under vacuum to give a white
crystalline compound. Purification by column chromatography using
hexane and ethyl acetate (3:1 and 1:1) as eluent afforded 2j as a
white crystalline compound (187.7 mg, 42.3%): mp 136 °C, mol.
1H NMR δ (CDCl3): 3.93-4.07 (m, 4H), 4.37 (d, 2H, J ) 6.03
Hz), 4.55 (d, 1H, J ) 4.52 Hz, IsH-3), 4.94 (t, 1H, J ) 5.02 Hz),
5.22 (d, 1H, J ) 2.01 Hz), 5.39 (m, 2H), 7.20-7.37 (m, 5H),
7.38-7.48 (s, 1H), 8.32 (d, 1H, J ) 7.53 Hz) 8.80 (s, 1H), 9.35 (s,
1H). 13C NMR ppm (CDCl3): 44.64, 70.17, 73.18, 74.50, 77.85,
80.44, 85.82, 123.12, 126.78, 127.0, 127.15, 128.25, 136.74, 137.65,
150.47, 153.23, 154.81, 165.36. Anal. (C20H20N2O6) C, H, N.
2-(Benzylaminocarbonyloxy-)-5-O-(1-naphthoyl)-1,4:3,6-dian-
hydro-D-glucitol (2l). A white crystalline product (86.8%): mp 126
°C. 1H NMR δ (CDCl3): 4.01-4.16 (m, 4H), 4.38 (d, 2H, J )
6.03 Hz), 4.61 (d, 1H, J ) 4.52 Hz), 5.03 (t, 1H, J ) 5.02 Hz),
5.11 (s, 1H), 5.26 (s, 1H), 5.51 (q, 1H, J ) 5.53, 11.05 Hz),
7.23-7.40 (m, 5H), 7.50-7.60 (m, 2H), 7.65 (t, 1H, J ) 7.28 Hz),
7.92 (d, 1H, J ) 8.03 Hz), 8.07 (d, 1H, J ) 8.03 Hz), 8.25 (d, 1H,
J ) 7.72 Hz), 8.96 (d, 1H, J ) 8.53 Hz). 13C NMR ppm (CDCl3):
44.69, 70.23, 73.2, 74.11, 78.14, 80.56, 85.80, 124.06, 125.31,
125.85, 125.90, 127.11, 127.21, 127.47, 128.11, 128.30, 130.01,
130.95, 133.30, 133.36, 137.54, 154.77, 166.33. Anal. (C25H23NO6)
C, H, N.
of enzyme inhibition, 25 µL of an inhibitor solution was added to
the test solution instead of the acetonitrile:water (1:1) solution.
Inhibitors were added 30 min before determination of remaining
enzyme activity. IC50 values were calculated using xlfit 4.0 software
and GraphPad Prism 4.02 software. For carbamylation studies with
compounds 2i and 2t, initial testing involved measuring cholinest-
erase activity at various [I] and [S] while incubating the enzyme
and inhibitor over a 1 h period. Readings were obtained at 0, 5,
10, 20, 30, 40, 50, and 60 min with [E] ) 1.2 × 10-3 mg/mL
purifed huBuChE, [DTNB] ) 0.3 mM, [S] and [I] varied. The
relevant amounts of buffer, inhibitor, and enzyme were mixed in a
cuvette and allowed to incubate for the various time intervals. Once
incubation was complete, the DTNB and substrate were added and
the reaction was measured at 412 nm for 5 min. After completion
of the above, the data was transformed so as to plot ln E/ET versus
time. The carbamylation experiments were repeated using com-
pound 2t at 1 mM (giving 100% inhibition) in the presence of the
edrophonium (5 mM). The reversibility of carbamate inhibitors was
determined by using dialysis or by dilution of the enzyme carbamate
adduct and monitoring for recovery of activity. The dialysis
experiments were performed using Visking dialysis tubing (Medicell
International Ltd. supplied from Lennox Laboratory Supplies Ltd.,
Dublin) with an exclusion limit of 12000-14000 Da. The enzyme
was mixed with inhibitor at a concentration giving 100% inhibition.
Each experiment was performed in triplicate. Tubing was sealed
at both ends using dialysis clips (Lennox Laboratory Supplies Ltd.,
Dublin). The samples were dialysed against buffer (1 L) at 37 °C,
with samples being removed and assayed at intervals. The buffer
was renewed at 4-6 h intervals. A control containing no inhibitor
was treated and sampled in parallel with the samples. In the dilution
experiments (compounds 2i and 2t), enzyme was incubated with
sufficient inhibitor for at least 2 h to effect at least 90% inhibition.
The mixture was then diluted 1000-fold in order to minimize the
possibility of reinhibition by excess inhibitor. This dilution was
made using phosphate buffer pH 8.0. The diluted sample was kept
at 37 °C for the duration of the analysis, with aliquots being taken
at defined time intervals and checked for BChE activity as described
previously.
Stability Studies. Pooled plasma solutions (4 mL, 50%) were
prepared by centrifugation of citrated human venous blood and
dilution of the resultant supernatant with pH 7.4 phosphate buffer
(2 mL). The pooled plasma sample gave normal adult BuChE
activity. A 100 µL aliquot of compound 2i in acetonitrile was added
to plasma solution and then incubated (37 °C). Aliquots (250 µL)
of the incubated mixture were withdrawn at appropriate intervals
and transferred to 1.5 mL Eppendorf tubes containing 500 µL of
2% ZnSO4·7H20 in MeCN-H2O (1:1) solution and 250 µL of
acetonitrile. Samples were then vortexed for 30 s and centrifuged
for 3 min at 10000 rpm. A 20 µL aliquot of the clear supernatant
was analyzed by HPLC. A gradient method was used with a Waters
ODS2 4.6 mm × 250 mm column and mobile phase consisting of
phosphate buffer (pH 2.5):acetonitrile, 50:50, grading to 20:80 over
20 min. External standards of isorobide-5-benzoate, benzoic acid,
and compound 2i were used to confirm specificity. The eluent was
monitored at 230 nm and peak identity and homogeneity confirmed
by PDA analysis. The experiment was repeated five times in the
range 1-100 µM.
Isosorbide-2-(methylcarbamate)-5-benzoate (2r). Colorless
1
gum (0.52 g, 65%). H NMR δ (CDCl3): 8.02 (2H, d, J ) 8.03
Hz), 7.55 (1H, t, J ) 7.53 Hz), 7.42 (2H, t, J ) 8.03 Hz), 5.36
(1H, d, J ) 5.52 Hz), 5.15 (1H, s), 4.91 (1H, t, J ) 5.02 Hz), 4.51
(1H, d, J ) 5.02 Hz), 3.91 (4H, m,), 2.75 (3H, d, J ) 4.52 Hz).
13C NMR δ (CDCl3): 165.45, 155.22, 132.85, 129.24, 128.96,
128.01, 86.29, 80.96, 80.49, 77.21, 73.13, 68.70, 27.00. HRMS:
found, (M - Na)+ ) 330.0946; required, (M - Na)+ ) 330.0954.
Isosorbide-2-(butylcarbamate)-5-benzoate (2t). A white crys-
1
talline product; (10.2%): mp 81.7 °C, mol. H NMR δ (DMSO):
0.8-0.9 (t, 3H), 1.2-1.3 (q, 2H), 1.33-1.38 (q, J ) 2.4 Hz),
2.96-2.99 (q, 2H, J ) 6.4 Hz), 3.82-3.83 (s, 2H), 3.89-3.97 (m,
2H), 4.41-4.43 (d, 1H, J ) 5.2 Hz), 4.86-4.89 (t, 1H, J ) 5.2
Hz), 4.99 (s, 1H), 5.36-5.39 (q, 1H, J ) 4 Hz), 7.35-7.38 (t, 1H,
J ) 5.6 Hz), 7.53-7.57 (m, 2H), 7.66 (m, 1H), 7.9-8 (d, 2H).
13C NMR ppm (DMSO): 13.97, 19.74, 31.75, 40.0, 70.87, 73.08,
74.79, 77.55, 81.02, 86.31, 129.18, 129.55, 129.6, 133.84, 155.53,
165.28. Anal. (C18H23O6N) C, H, N.
Docking Studies. The atomic coordinates of the crystal structure
of BuChE with bound butyrate (PDB code 1p0i) were used. All
molecules of water were removed, along with other heteroatoms,
to leave only the bound butyrate and the enzyme itself. Residues
1-3, 378-379, and 455, all of which were missing in the original
crystal structure, were added using Swiss PDB Viewer v3.7. The
added residues were briefly minimized within MOE (Chemical
Computing Group, Montreal, Canada) using a sequence of SD, CG,
and Truncated Newton algorithms; the AMBER99 force field and
atom charges were used to model the protein throughout. Each
inhibitor was rehybridized to sp3 at the carbonyl carbon. The
fragments distal to the carbamate were energy minimized using
the MM94s force field.37,38 The carbonyl oxygen was assigned a
formal charge of -1. Inhibitor molecules were manually superim-
Enzyme Studies. Cholinesterase activity was measured using
the Ellman spectrophotometric method. HuBuChE activity was
measured in replicate samples using a 96-well plate reader. The
total volume of test solution in each well was 250 µL. This consisted
of 25 µL of plasma solution, 150 µL of phosphate buffer pH 8.0,
25 µL of DTNB solution (0.5 mM), and 25 µL of acetonitrile:
distilled water (1:1). The 96-well plate was incubated for 30 min
before 25 µL of BTCI solution (0.5 mM) was added, and the
reaction was measured at 405 nm over 5 min using an Anthos bt2
plate reader. For the determination of AChE activity, 25 µL of
electric eel AChE solution and 25 µL of ATCI solution were used
instead of the plasma solution and BTCI solution. For determination

6408 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Carolan et al.
posed onto the butyrate fragment in the appropriate orientation, as
carried out by Luo et al. in AChE.34 The butyrate fragment was
deleted and the inhibitor molecule covalently joined to O-ꢁ of the
active site serine, forming the desired intermediate. To prepare the
complex for minimization, partial atomic charges for the nonstand-
ard ligand atoms were calculated using MOPAC 7 (AM1-BCC
method)39 interfaced to MOE in a simplified system with the
oxyanion hole residues removed. Because the total charge calculated
for the inhibitor atoms was not an integer due to charge transfer
between the inhibitor and Ser-198 during the reaction, the charge
was altered on O-ꢁ of the serine to give an overall charge of -1
on the inhibitor and Ser-198 atoms. AMBER 99 charges were
calculated for the remaining protein atoms. The system was
optimized for hydrogen bonding and protonation states for each
residue in the protein were assigned using PDB2PQR40 and
Propka;41 it was always ensured that Glu-325 and His-438 were
appropriately protonated, such that the important hydrogen-bonding
network of the catalytic triad was maintained. Subsequently, the
whole system was energy minimized in MOE using sequential SD
(100 steps) and TN (1000 steps or to an rms gradient of <0.01
kcal/(mol·A)) algorithms. The distances between the negatively
charged carbonyl oxygen and the backbone nitrogens of the
oxyanion hole residues were constrained, along with the distances
between the hydrogen-bonding atoms of the catalytic triad. General-
ized Born solvation was used with an interior dielectric constant
of 20 and an exterior dielectric of 80. A nonbonded cutoff was
applied, with a smoothing function applied between 12 and 15 Å.
A second iteration of charge calculation and energy minimization
was performed in an identical manner to arrive at the final models
of the protein-carbamate adducts shown.
Supporting Information Available: Characterization data for
compounds 1a, 1b, 1c, 1e, 1g, 1i, 1j, 1k, 21, 2b, 2c, 2f, 2g, 2h, 2k,
2m, 2n, 2o, 2p, 2q, 2s, 4; elemental analysis data, purity data by
HPLC. This material is available free of charge via the Internet at
http://pubs.acs.org.
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calculated using MOE. The interaction energies reported are the
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Acknowledgment. This work was supported by Science
Foundation Ireland (05/RFP/CHE0046), Enterprise Ireland
(BRGS2002), and IRCSET (Ciaran Carolan). We are grateful
to Schwarz-Pharma, Shannon, Co. Clare for a gift of ISMN.

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