On the active site for hydrolysis of aryl amides and choline esters by human cholinesterases

Article abstract of DOI:10.1016/j.bmc.2006.02.021

Cholinesterases, in addition to their well-known esterase action, also show an aryl acylamidase (AAA) activity whereby they catalyze the hydrolysis of amides of certain aromatic amines. The biological function of this catalysis is not known. Furthermore, it is not known whether the esterase catalytic site is involved in the AAA activity of cholinesterases. It has been speculated that the AAA activity, especially that of butyrylcholinesterase (BuChE), may be important in the development of the nervous system and in pathological processes such as formation of neuritic plaques in Alzheimer's disease (AD). The substrate generally used to study the AAA activity of cholinesterases is N-(2-nitrophenyl)acetamide. However, use of this substrate requires high concentrations of enzyme and substrate, and prolonged periods of incubation at elevated temperature. As a consequence, difficulties in performing kinetic analysis of AAA activity associated with cholinesterases have hampered understanding this activity. Because of its potential biological importance, we sought to develop a more efficient and specific substrate for use in studying the AAA activity associated with BuChE, and for exploring the catalytic site for this hydrolysis. Here, we describe the structure-activity relationships for hydrolysis of anilides by cholinesterases. These studies led to a substrate, N-(2-nitrophenyl)trifluoroacetamide, that was hydrolyzed several orders of magnitude faster than N-(2-nitrophenyl)acetamide by cholinesterases. Also, larger N-(2-nitrophenyl)alkylamides were found to be more rapidly hydrolyzed by BuChE than N-(2-nitrophenyl)acetamide and, in addition, were more specific for hydrolysis by BuChE. Thus, N-(2-nitrophenyl)alkylamides with six to eight carbon atoms in the acyl group represent suitable specific substrates to investigate further the function of the AAA activity of BuChE. Based on the substrate structure-activity relationships and kinetic studies, the hydrolysis of anilides and esters of choline appears to utilize the same catalytic site in BuChE.

Full text of DOI:10.1016/j.bmc.2006.02.021

Bioorganic & Medicinal Chemistry 14 (2006) 4586–4599
On the active site for hydrolysis of aryl amides and choline esters
by human cholinesterases
c
c
c
Sultan Darvesh,a,b,c, Robert S. McDonald, Katherine V. Darvesh, Diane Mataija,
Sam Mothana,c Holly Cook,c Karina M. Carneiro,c Nicole Richard,c
Ryan Walshb,c and Earl Martinc
*
aDepartment of Medicine (Neurology), Dalhousie University, Halifax, NS, Canada
bDepartment of Anatomy and Neurobiology, Dalhousie University, Halifax, NS, Canada
cDepartment of Chemistry, Mount Saint Vincent University, Halifax, NS, Canada
Received 18 January 2006; revised 8 February 2006; accepted 9 February 2006
Available online 28 February 2006
Abstract—Cholinesterases, in addition to their well-known esterase action, also show an aryl acylamidase (AAA) activity whereby
they catalyze the hydrolysis of amides of certain aromatic amines. The biological function of this catalysis is not known. Further-
more, it is not known whether the esterase catalytic site is involved in the AAA activity of cholinesterases. It has been speculated that
the AAA activity, especially that of butyrylcholinesterase (BuChE), may be important in the development of the nervous system and
in pathological processes such as formation of neuritic plaques in Alzheimer’s disease (AD). The substrate generally used to study
the AAA activity of cholinesterases is N-(2-nitrophenyl)acetamide. However, use of this substrate requires high concentrations of
enzyme and substrate, and prolonged periods of incubation at elevated temperature. As a consequence, difficulties in performing
kinetic analysis of AAA activity associated with cholinesterases have hampered understanding this activity. Because of its potential
biological importance, we sought to develop a more efficient and specific substrate for use in studying the AAA activity associated
with BuChE, and for exploring the catalytic site for this hydrolysis. Here, we describe the structure–activity relationships for hydro-
lysis of anilides by cholinesterases. These studies led to a substrate, N-(2-nitrophenyl)trifluoroacetamide, that was hydrolyzed several
orders of magnitude faster than N-(2-nitrophenyl)acetamide by cholinesterases. Also, larger N-(2-nitrophenyl)alkylamides were
found to be more rapidly hydrolyzed by BuChE than N-(2-nitrophenyl)acetamide and, in addition, were more specific for hydrolysis
by BuChE. Thus, N-(2-nitrophenyl)alkylamides with six to eight carbon atoms in the acyl group represent suitable specific substrates
to investigate further the function of the AAA activity of BuChE. Based on the substrate structure–activity relationships and kinetic
studies, the hydrolysis of anilides and esters of choline appears to utilize the same catalytic site in BuChE.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
that of BuChE, is involved in this process.4,5 The AAA
activity of BuChE has also been implicated in the
pathology of Alzheimer’s disease (AD). In AD, there
is a significant increase in the levels of BuChE in the
brain and this enzyme activity has been detected in all
the neuropathological hallmarks of this disorder, includ-
ing neuritic plaques and neurofibrillary tangles.6–13
Acetylcholinesterase (AChE, EC 3.1.1.7) and butyryl-
cholinesterase (BuChE, EC 3.1.1.8), in addition to their
well-known esterase action,1,2 also have an aryl acylam-
idase (AAA) activity whereby they catalyze the hydroly-
sis of amides of certain aromatic amines.3 The biological
function of this catalysis is not known. However, fluctu-
ations of this enzyme activity over the course of the
development of the nervous system have led to specula-
tion that the AAA activity of cholinesterases, especially
Enzymes from a variety of animal sources have been
found to exhibit the ability to hydrolyze acid derivatives
of aromatic amines (anilides) such as acetanilide, first
reported by Mirkowski in 1909 (cited in Ref. 14) and
sulfa drugs.15 Detection of this aryl acylamidase activity
(AAA, EC 3.1.5.13) was facilitated by the use of the
chromogenic substrate, N-(2-nitrophenyl)acetamide.16
Using this substrate, it was later shown that AAA activ-
ity could be detected in mammalian brain and that this
Keywords: Aryl acylamidase; Butyrylcholinesterase; N-(2-Nitrophen-
yl)anilides; Alzheimer’s disease.
*
Corresponding author. Tel.: +1 902 473 2490; fax: +1 902 473
7133; e-mail: sultan.darvesh@dal.ca
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.021

S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
4587
hydrolytic activity was associated with AChE,3,17 the en-
zyme known to catalyze the breakdown of the neuro-
transmitter acetylcholine.1,2 Subsequently, BuChE was
also shown to have this ability to hydrolyze anilides.18
tral data were consistent with the structures of the
analogues.
2.1.2. Thiocholine derivatives. All thiocholine derivatives
used in this study have been described before.2,31 Acetyl-
thiocholine (I), propionylthiocholine (II), and butyryl-
thiocholine (III) were purchased commercially.
Octanoylthiocholine (IV) was synthesized according to
a published procedure,31 and the spectral data were con-
sistent with the structure.
Within the context of multiple catalytic activities of cho-
linesterases, it is unclear whether the esterase and AAA
activities utilize the same active site. However, it has
been suggested that the AAA catalytic site overlaps with
the esteratic site in cholinesterases.19,20 Given the possi-
ble biological significance of the AAA activity of
BuChE, further study of this hydrolytic activity was
undertaken.
2.2. General evaluation of anilide substrates
All compounds were first monitored for susceptibility to
hydrolysis by doing repetitive UV–vis scans in the pres-
ence of either BuChE, AChE or AAAPf for up to 7 h.
Since one of the main objectives of this study was to ob-
tain a more efficient anilide substrate for BuChE, such
repetitive scans were able to show which substrates were
more rapidly hydrolyzed than the substrate commonly
used, namely, N-(2-nitrophenyl)acetamide (2). For
example, Figure 1 shows two repetitive scans, in the
presence of BuChE, of N-(2-nitrophenyl)acetamide (2)
(Fig. 1a) and N-(2-nitrophenyl)hexanamide (12)
(Fig. 1b). These scans indicate that compound (12) is
hydrolyzed 50 times more rapidly by BuChE than (2).
The most commonly used substrate for examining the
AAA activity is N-(2-nitrophenyl)acetamide (2) (Table
1).16 The major limitation in the use of this compound
as a cholinesterase substrate is that the reaction is
slow, especially for BuChE, and requires high concen-
trations of enzyme and substrate, relative to what is
required for monitoring esterase activity.21 Further-
more, measurement of enzymatic activity is difficult
with this compound because it absorbs strongly in
the same region of the visible spectrum as the prod-
uct, 2-nitroaniline, the formation of which is used to
monitor the catalytic hydrolysis of the substrate by
cholinesterases.
Of the 22 compounds tested (Table 2), twelve com-
pounds were hydrolyzed by BuChE (2, 7–14, 16, 18,
and 22), eight were hydrolyzed by AChE (2, 8, 9, 11,
13, 14, 16, and 22) and twelve by AAAPf (1–5, 7–12,
and 22). Five compounds (6, 15, 17, 19, and 21) were
not hydrolyzed by any of these enzymes.
The present study was undertaken to assess a series of
analogues of N-(2-nitrophenyl)acetamide with a view
to exploring the requirements for hydrolysis and for
obtaining more efficient and enzyme-specific substrates
for studying aryl acylamidase activity of BuChE.
Through structure–activity explorations involving kinet-
ic studies, calculations of molecular parameters, and
NMR analysis, a number of substrate features are iden-
tified that are essential for, or facilitate hydrolysis by,
aryl acylamidase-type hydrolysis by cholinesterases.
2.2.1. The effect of the 2-nitro group of the aniline ring.
The role of the nitro group in providing a chromophore
for the spectrophotometric detection of the AAA activ-
ity of cholinesterases is well established.3,18 However, its
function in affecting the hydrolysis of the aryl amide
bond is unknown. As summarized in Table 2, no hydro-
lysis by cholinesterases was observed, even after 7 h of
exposure to AChE or BuChE, for compounds such as
acetanilide (1), benzanilide (17), and N-phenyltrifluoro-
acetamide (21), compounds that do not have a nitro
group on the aniline ring.
Here we show that longer acyl chain anilides are more
efficient substrates for the AAA activity of BuChE than
is N-(2-nitrophenyl)acetamide (2). Furthermore, based
on kinetic studies and the comparison of anilide hydro-
lysis with that of analogous long chain thioesters of cho-
line, we suggest that the hydrolysis of acyl anilides and
esters of choline occurs at the same catalytic site.
When the nitro group is present in position 2 on the ani-
line ring, as in compounds (2, 18, and 22), hydrolysis of
these compounds by at least one of the cholinesterases
occurred. This implies a more important function for
the nitro group in influencing the hydrolysis of the
amide bond, beyond simply increasing the sensitivity
for detection of anilide hydrolysis.
2. Results and discussion
A total of 22 compounds (Table 1) were examined as po-
tential substrates for hydrolysis by aryl acylamidase
activity of AChE, BuChE, and the enzyme aryl acylam-
idase from Pseudomonas fluorescens (AAAPf, EC
3.1.1.13) (Table 2).
To determine whether the position of the nitro group in
the aniline ring is important in promoting the hydrolysis
of the amide bond, compounds (3) and (4) with the nitro
group in 3- or 4-position, were compared with the iso-
meric lead compound 2. Changing the position of the ni-
tro group from the 2-position led to loss of hydrolysis by
BuChE and AChE, although AAAPf hydrolysis was en-
hanced (Table 2). These results indicate that a nitro
group in the 2-position of the aniline ring is important
2.1. Synthetic chemistry
2.1.1. Anilide derivatives. Compounds 1, 2, and 4 were
purchased from commercial sources and the rest were
synthesized by standard methods (see Section 4). Physi-
cal data for the synthetic compounds corresponded well
with the published data (where available), and the spec-

4588
S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
Table 1. Structures of anilides tested
Compound
Name
Structure
O
CH
3
1
2
3
4
5
N-Phenylacetamide
NH
O
CH
3
NH
N-(2-Nitrophenyl)acetamide
N-(3-Nitrophenyl)acetamide
N-(4-Nitrophenyl)acetamide
N-(2,4-Dinitrophenyl)acetamide
+
-
N
O
O
+
O
CH
3
NH
O
N
-
O
O
-
-
O
O
CH
3
+
N
NH
O
O
CH
3
+
N
NH
O
+
-
N
O
O
-
O
O
+
N
O
CH
3
6
N-(2,6-Dinitrophenyl)acetamide
NH
+
-
N
O
O
O
CH
3
NH
7
8
N-(3-Methoxy-2-nitrophenyl)acetamide
N-(5-Methoxy-2-nitrophenyl)acetamide
+
-
H C
O
N
O
3
O
O
H C
O
3
CH
3
NH
+
-
N
O
O
O
NH
CH
3
9
10
11
12
N-(2-Nitrophenyl)propanamide
N-(2-Nitrophenyl)butanamide
N-(2-Nitrophenyl)pentanamide
N-(2-Nitrophenyl)hexanamide
+
+
+
+
N
O
O
-
O
O
O
(CH )
2
2
NH
CH
3
N
-
O
O
(CH )
2
3
NH
CH
3
N
-
O
O
(CH )
2
4
NH
O
CH
3
N
-
O

S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
4589
Table 1 (continued)
Compound
Name
Structure
O
(CH )
2
5
NH
O
CH
3
13
14
N-(2-Nitrophenyl)heptanamide
N-(2-Nitrophenyl)octanamide
+
N
-
O
O
(CH )
2 6
NH
O
CH
3
+
N
-
O
O
CH
3
NH
H C
15
16
17
18
19
20
21
22
N-(2-Nitrophenyl)-3,3-dimethylbutanamide
N-(2-Nitrophenyl)phenylacetamide
N-Phenylbenzamide
3
CH
3
+
N
O
O
-
O
NH
O
+
N
-
O
O
NH
O
NH
N-(2-Nitrophenyl)benzamide
+
+
+
N
-
O
O
O
CH
3
N
N-Methyl-N-(2-nitrophenyl)acetamide
N-Methyl-N-(2-nitrophenyl)butanamide
N-Phenyltrifluoroacetamide
CH
3
N
O
O
-
O
N
O
CH
3
CH
3
N
-
O
O
CF
CF
3
3
NH
O
NH
N-(2-Nitrophenyl)trifluoroacetamide
+
-
N
O
O
for the hydrolysis of anilides by cholinesterases. A nitro
group in either the 2- or 4-position has an electron-with-
drawing effect on the benzene ring.32 Therefore, the ac-
tion of the 2-nitro group in promoting anilide
hydrolysis by cholinesterases is thus probably not an
electron-withdrawing one since the 4-nitro analogue
(4), which is electronically similar to compound 2, is
not hydrolyzed (Table 2). Instead, these results suggest
a more direct involvement of the 2-nitro group in pro-
moting cholinesterase-catalyzed anilide hydrolysis. This
direct effect may be through the formation of a six-mem-
bered cyclic species, involving intramolecular hydrogen
bonding, between the oxygen of the nitro group and
the hydrogen of the amide moiety (Fig. 2). Hydrogen
bonding in 2-nitroanilides was suggested earlier, based
on spectrophotometric studies.33–35 We postulated that

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S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
Table 2. Initial rates (DA/h) of hydrolysis of selected acyl anilides by
human butyrylcholinesterase (BuChE), acetylcholinesterase (AChE),
and aryl acylamidase from Pseudomonas fluorescens (AAAPf)
a
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0 Min
O
10 Min
20 Min
30 Min
40 Min
50 Min
60 Min
CH
3
Compound
BuChE
AChE
AAA
NH
1
2
X
X
0.2540
1.0341
1.3835
0.3150
6.2998
9.7167
X
+
-
N
O
0.1101
X
O
(2)
3
X
4
X
X
5
X
X
6
X
X
7
0.0133
0.0746
0.0995
0.9124
2.9244
4.8789
0.5499a
1.4312a
X
X
0.2310
0.0480
0.8070
0.3749
0.3666
0.1491
X
8
0.3819
0.0692
X
9
10
11
12
13
14
15
16
17
18
19
20
21
22
250
300
350
400
450
500
550
0.0226
X
0.0074a
0.0631a
X
Wavelength (nm)
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
b
X
O
0 min
2 min
4 min
6 min
8 min
X
0.1136
X
0.0184
X
X
NH
X
CH
3
+
-
N
O
0.0241
X
X
X
O
X
X
(12)
X
X
X
10 min
X
14.3079
X
0.9445
X
0.2823
All assays carried out at 430 nm except for 1 (280 nm), 3 (360 nm), and
5 (340 nm).
X: No observed hydrolysis of substrate. Assays contained 10 units
(0.16 nm) of BuChE, or 10 units (0.07 nm) AChE or 0.125 units
250
300
350
400
450
500
550
AAAPf
.
a Initial rates of hydrolysis determined at 3.33 · 10ꢀ5 M due to solu-
Wavelength (nm)
bility. All other substrates were 3.33 · 10ꢀ4 M.
Figure 1. Repetitive scans using 3.33 · 10ꢀ4 M of: (a) N-(2-
nitrophenyl)acetamide (2) (total time 60 min) and (b) N-(2-nitrophen-
yl)hexanamide (12) (total time 10 min), as substrate for BuChE.
this intramolecular hydrogen bond could facilitate
hydrolysis of the amide bond.
O
R
To test further the contribution of hydrogen bonding in
facilitating hydrolysis, two analogues without the amide
hydrogen were prepared and examined. Replacement of
the hydrogen on the nitrogen of the amide with a methyl
group, as in N-methyl-N-(2-nitrophenyl)acetamide (19)
and N-methyl-N-(2-nitrophenyl)butanamide (20), com-
pounds that are unable to form the intramolecular
hydrogen bond, led to complete loss of hydrolysis
(Table 2).
N
H-
O-
N+
O
Figure 2. Hydrogen bonding in N-2-nitroanilides.
In an attempt to strengthen the hydrogen bond and
facilitate hydrolysis, compounds with an additional
nitro group in the 4-position, as in N-(2,4-dinitrophe-
nyl)acetamide (5), or 6-position, as in N-(2,6-dinitrophe-
nyl)acetamide (6), were synthesized and examined.
Neither of these compounds was hydrolyzed by cho-
linesterases (Table 2). Another approach was to add
an electron-donating group at the 3-position as in N-
(3-methoxy-2-nitrophenyl)acetamide (7), or 5-position,
as in N-(5-methoxy-2-nitrophenyl)acetamide (8). Once
again, neither of these compounds showed significant
improvement in hydrolysis by cholinesterases over the
lead compound, 2. These observations suggest that the
presence of additional functional groups on the aniline
ring may disrupt the formation of the hydrogen bond
or alter other molecular parameters that may be impor-
tant in the hydrolytic process. In an effort to clarify these
1
questions, H NMR analyses and molecular calculation
studies were undertaken.
2.2.2. NMR analysis. Comparison of the 1H NMR spec-
tra of the three isomeric substrates, N-(2-nitrophen-
yl)acetamide (2), N-(3-nitrophenyl)acetamide (3), and
N-(4-nitrophenyl)acetamide (4), in dilute CDCl3 solu-
tion revealed a unique feature of the presence of a nitro
group in the 2-position. Each spectrum contained a
broad singlet that integrated for one proton and was as-
signed to the N–H proton of the amide moiety. The po-
sition of this signal was first established for the parent
compound N-phenylacetamide (1). The signal for this
proton appeared at d 7.47 for this compound (Table
3). The signal for the amide proton appeared in a com-
parable position for the N-(3-nitrophenyl)acetamide (3)

S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
4591
Table 3. Physical parameters of compounds examined as potential substrates for aryl acylamidase activity of butyrylcholinesterase
˚
H-bond distance (A)
Compound
Name
Chem shift N–H d (ppm)
Atomic charges
Amide-N Amide-H
1
2
N-Phenylacetamide
N-(2-Nitrophenyl)acetamide
7.47
NA
2.63
NA
NA
2.63
2.65
2.67
2.63
2.63
2.63
2.63
2.63
2.63
2.62
2.63
2.63
NA
2.63
NA
NA
NA
2.62
ꢀ0.709
ꢀ0.736
ꢀ0.710
ꢀ0.718
ꢀ0.741
ꢀ0.714
ꢀ0.740
ꢀ0.738
ꢀ0.755
ꢀ0.758
ꢀ0.758
ꢀ0.759
ꢀ0.759
ꢀ0.759
ꢀ0.752
ꢀ0.743
ꢀ0.737
ꢀ0.784
ꢀ0.505
ꢀ0.519
ꢀ0.723
ꢀ0.762
+0.328
+0.385
+0.337
+0.345
+0.390
+0.380
+0.378
+0.385
+0.386
+0.386
+0.386
+0.386
+0.386
+0.386
+0.390
+0.388
+0.333
+0.403
NA
10.32
7.44
7.42
3
N-(3-Nitrophenyl)acetamide
N-(4-Nitrophenyl)acetamide
4
5
N-(2,4-Dinitrophenyl)acetamide
N-(2,6-Dinitrophenyl)acetamide
N-(3-Methoxy-2-nitrophenyl)acetamide
N-(5-Methoxy-2-nitrophenyl)acetamide
N-(2-Nitrophenyl)propanamide
N-(2-Nitrophenyl)butanamide
N-(2-Nitrophenyl)pentanamide
N-(2-Nitrophenyl)hexanamide
N-(2-Nitrophenyl)heptanamide
N-(2-Nitrophenyl)octanamide
N-(2-Nitrophenyl)-3,3-dimethylbutanamide
N-(2-Nitrophenyl)phenylacetamide
N-Phenylbenzamide
10.64
9.48
8.27
6
7
8
10.76
10.37
10.36
10.36
10.36
10.36
10.36
10.31
10.25
7.85
9
10
11
12
13
14
15
16
17
18
19
20
21
22
N-(2-Nitrophenyl)benzamide
11.34
NA
NA
N-Methyl-N-(2-nitrophenyl)acetamide
N-Methyl-N-(2-nitrophenyl)butanamide
N-Phenyltrifluoroacetamide
NA
+0.353
+0.406
8.21
11.38
N-(2-Nitrophenyl)trifluoroacetamide
and N-(4-nitrophenyl)acetamide (4) (d7.44 and
7.42 ppm, respectively). However, in N-2-nitrophenylac-
etamide (2), this signal appeared much further downfield
at d 10.32 ppm (Table 3). This downfield shift of d
2.9 ppm for the N–H proton in compound 2 is interpret-
ed as indicating the presence of a deshielding intramo-
lecular hydrogen bond in this isomer, which cannot be
present in either 3- or 4-nitro isomers or in N-phenylac-
etamide (1). These 1H NMR analyses support the notion
that intramolecular hydrogen bonding (Fig. 2) is an
important feature of 2-nitroanilides that facilitates their
hydrolysis by cholinesterases (Table 2). However, the
observation that N-(2,4-dinitrophenyl)acetamide (5),
N-(2,6-dinitrophenyl)acetamide (6), and N-(2-nitro-
phenyl)-3,3-dimethylbutanamide (15), all show the
downfield N–H shift indicative of hydrogen bonding
(Table 3), but are not readily hydrolyzed by cholinester-
ases (Table 2), suggests that other factors, in addition to
hydrogen bonding, must also be important in determin-
ing whether substrate hydrolysis will occur.
2.2.3. Computational analysis. To explore the possible
contribution of molecular parameters, such as preferred
conformation, atomic distances, and atomic charges, to
the facilitation of anilide hydrolysis, computational
analyses were carried out.
Figure 3. Calculated optimal geometries of anilide derivatives: (a) N-
(2-nitrophenyl)acetamide (2) showing hydrogen bonding with all
relevant atoms in plane and (b) N-(3-methoxy-2-nitrophenyl)acetam-
ide (7) with the nitro oxygen out of plane (52.51ꢁ) and concomitant
weakening of hydrogen bonding.
Geometry optimization of the molecules involved
obtaining the lowest energy conformer, first using
force-field methods (PC Spartan Pro, Wavefunction,
Inc., 18401 Von Karman, Suite 370, Irvine, CA
92612), ultimately led to an optimized geometry at the
higher B3LYP/6-31G(d) level of theory.36–38
lyzed by cholinesterases. As is shown in Figure 3a, the
2-nitro group oxygen atom and the amide N–H of com-
pound 2 are directed toward each other in the proper
orientation to form an intramolecular hydrogen bond.
Figure 3a shows optimized geometry of one of the sub-
strates, N-(2-nitrophenyl)acetamide (2), that is hydro-

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S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
It is significant to note that the most common occur-
rence of intramolecular hydrogen bonding arises when
the H-bonded species can form a six-membered ring,
in which the hydrogen atom is one of the six atoms.32
As can be seen for the representative compound in Fig-
ure 3a, the 2-nitroanilides that are hydrolyzed by cho-
linesterases adopt a conformation such that, with the
hydrogen bond, a planar, six-membered ring is pro-
duced. The preferred conformation of N-(3-methoxy-2-
nitrophenyl)acetamide (7) indicates displacement of the
nitro group oxygen atom out of the plane of the amide
hydrogen (Fig. 3b). This compound is not readily hydro-
lyzed by cholinesterases. In fact, the rate of hydrolysis of
compound (7) by BuChE is much slower than that of the
lead compound (2), and it is not hydrolyzed by AChE.
The resulting weaker hydrogen bonding can be consid-
ered a factor that adversely affects hydrolysis of 7 by
AChE and BuChE (Tables 2 and 3).
yl)benzamide (18). This compound was only hydrolyzed
by BuChE and at a very slow rate (Table 2). Thus, like
the NMR and bond distance results described above, an
electron-rich amide nitrogen appears to be an important
contributing factor for the efficient hydrolysis of anilides
by cholinesterases.
2.2.4. The effect of the acyl group on anilide hydrolysis.
The hydrolysis of esters of choline by cholinesterases has
long been known to be affected by the size of the acyl
function of the molecule.2 For example, AChE and
BuChE readily hydrolyze acetylcholine. However, as
the number of carbon atoms is increased through butyr-
ylcholine, hydrolysis by AChE is diminished consider-
ably, while the rate of hydrolysis by BuChE is
enhanced. In this context, the effect of the size of the acyl
group was examined in a series of 2-nitroanilide deriva-
tives. Increasing the number of carbons from the lead
compound, N-(2-nitrophenyl)acetamide (2), was found
to diminish the hydrolysis of the anilide by AChE, while
the hydrolysis by BuChE was considerably enhanced
(Table 2). For example, compound 2 was hydrolyzed
by both AChE and BuChE, while N-(2-nitrophen-
yl)butanamide (10) was hydrolyzed only by BuChE.
Furthermore, comparison of compounds 9 and 12, in
which the number of acyl carbons is doubled, indicates
a 50-fold increase in the rate of anilide hydrolysis by
BuChE (Table 2). Figure 4 shows a bar diagram summa-
rizing the rates of hydrolysis of straight chain 2-nitroani-
lides as the number of carbons in the acyl group is
increased from two to eight. As has been observed for
the esters of choline, increasing the number of carbons
in the acyl group leads to diminished hydrolysis by
AChE. In the present work, it was observed that with
the N-2-nitroanilides there was indeed loss of hydrolysis
by AChE as the number of acyl carbons was increased
from 3 to 6 (compounds 9–12). However, for the acyl
anilides with seven and eight carbon atoms in the acyl
side chain (compounds 13 and 14), some hydrolysis by
AChE was observed (Fig. 4). Based on the crystal struc-
ture of AChE, 40 it is recognized that the active site is at
For optimum hydrogen bond interactions between an
amide nitrogen and an oxygen atom, the donor (N)
and acceptor (O) atoms should be at a distance from
each other not greater than 2.9A.39 As the results in Ta-
˚
ble 3 indicate, the hydrogen bond distances for the com-
pounds that are hydrolyzed by AChE and BuChE range
˚
between 2.61 and 2.63 A, which are well within the range
for hydrogen bonding to take place. However, there are
exceptions. For example, compounds 5, 6, 15, and 18 are
not hydrolyzed despite optimal hydrogen bond distanc-
es. This suggests that intramolecular hydrogen bonding
alone is not sufficient to effect hydrolysis by
cholinesterases.
Other parameters such as atomic charges on the atoms
in the vicinity of the amide bond were also considered
as potential indicators of the ability of particular sub-
strates to undergo hydrolysis. Calculated atomic charges
for the amide nitrogen and amide hydrogen are summa-
rized in Table 3. These calculated atomic charges, like
the hydrogen bond distance determinations, did not
provide a clear explanation of the patterns for substrate
hydrolysis noted in Table 2. However, if one assumes
that increasing the electron density on the amide nitro-
gen should facilitate proton transfer to this nitrogen,
to create the amino leaving group, then some observa-
tions can be made with respect to this parameter in
the hydrolytic process. For example, N-phenylacetamide
(1), not hydrolyzed by cholinesterases, has an atomic
charge on the amide nitrogen of ꢀ0.709, while that for
the lead compound, N-(2-nitrophenyl)acetamide (2), is
more electron-rich at ꢀ0.736 (Table 3). The 3- and 4-ni-
tro isomers, that is, compounds 3 and 4, which cannot
form an intramolecular hydrogen bond and do not
undergo cholinesterase hydrolysis, also show diminished
amide nitrogen electron density, at ꢀ0.710 and ꢀ0.718,
respectively. On the other hand, compounds that show
improved cholinesterase hydrolysis over that of com-
pound 2, such as compounds 9–14 and 22, all show a
greater negative charge on the amide nitrogen (Table
3). That having an amide nitrogen charge of ꢀ0.740 to
ꢀ0.760 is not sufficient to guarantee efficient hydrolysis
by cholinesterases is evident from the very negative
amide nitrogen (ꢀ0.784) calculated for N-(2-nitrophen-
˚
the bottom of a 20 A deep gorge. Hydrolysis involves a
1.6
1.4
1.2
BuChE
AChE
1
0.8
0.6
0.4
0.2
0
2
3
4
5
6
7
8
Number of carbon atoms in the acyl group
Figure 4. Initial rates of hydrolysis of alkyl acyl anilides with 2–8
carbon atoms in the acyl group (compounds
2 and 9–14,
3.33 · 10ꢀ5 M) by human cholinesterases (10 units).

S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
4593
catalytic triad of serine, histidine, and glutamate resi-
dues, as well as an acyl pocket that accommodates the
acyl group during catalysis. Although the catalytic triad
is common in AChE and BuChE, the acyl pocket in
AChE is small, relative to that in BuChE,41 which ex-
plains, in part, why substrates with larger acyl groups
are not hydrolyzed by AChE, but are hydrolyzed by
BuChE. If the catalytic site for the hydrolysis of esters
of choline and anilides is the same, then the observation
that compounds 13 and 14 are hydrolyzed by AChE,
albeit slightly, suggests that the larger substrates may
be hydrolyzed by an alternate mechanism.31
cent to the carbonyl facilitate the hydrolysis of the
amide bond by cholinesterases. NMR analysis, calculat-
ed charge on the amide nitrogen and atomic distances
for hydrogen bonding (Table 3), all point toward favor-
able hydrolysis of this compound (22) by cholinesteras-
es. Furthermore, replacement of the hydrogen atoms by
larger fluorine atoms may account for the more rapid
hydrolysis of 22 by BuChE than by AChE.
2.2.5. N-2-Nitroanilide kinetic constants. The straight
chain 2-nitroanilide substrates that were effectively
hydrolyzed by cholinesterases (2, 9–14, and 22) were
examined for their BuChE affinities and relative maxi-
mum velocities of hydrolysis under identical conditions.
The Km and Vmax values obtained through Lineweaver–
Burk plots are summarized in Table 4. Although there
were no large differences in the BuChE affinity constants
for most of the anilides, that of N-(2-nitrophenyl)triflu-
oroacetamide (22) was distinctly different, this substrate
showing some 1000-fold lower affinity for BuChE than
the straight-chain alkyl 2-nitroanilides (2 and 9–14).
On the other hand, the rate of hydrolysis of this sub-
strate was found to be much higher. For example,
although the Km values reveal that 22 has a 200-fold
lower affinity for BuChE than 2, it is hydrolyzed about
40,000 times faster than the reference compound 2.
These differences in the kinetic behavior of various 2-
nitroanilides are depicted in Figure 5. These Michae-
lis–Menten plots are based on the observed Km and Vmax
values for BuChE (Table 4) with each substrate. Using
the Michaelis–Menten equation, and the observed Km
and Vmax values, the rate of product formation at each
substrate concentration was calculated. This simulation
shows that, as the number of carbon atoms is increased
in these unsubstituted straight chain acyl anilides (2 and
9–14), the rate of hydrolysis increases proportionately.
The exception is compound 22 in which the presence
of the electron-withdrawing fluorine atoms results in a
dramatic increase in the rate of hydrolysis by BuChE.
The esteratic substrate benzoylcholine has long been
known to be specifically hydrolyzed by BuChE, but at
a slower rate than the analogous alkyl esters.2 To test
whether an anilide derivative behaved similarly, N-(2-
nitrophenyl)benzamide (18) was prepared and tested.
Once again, this was hydrolyzed only by BuChE, and
at a very low rate compared to the alkyl derivatives, 2
and 9–14 (Table 2). In order to examine whether this
was partly a consequence of the highly conjugated rigid
system in 18, compound 16, with a more flexible methy-
lene bridge between the carbonyl and the benzene ring in
the acyl moiety, was examined. This compound showed
enhanced (5-fold) hydrolysis by BuChE and even per-
mitted hydrolysis by AChE (Table 2). To test the effect
of branching in the acyl group, hydrolysis of N-(2-nitro-
phenyl)hexanamide (12) was compared to that of the
highly branched 3,3-dimethylbutanamide derivative
(15), which also has a six carbon acyl group. This com-
pound (15) was not hydrolyzed by BuChE, suggesting
that a highly branched moiety cannot be accommodated
by the binding mechanism of this enzyme.
The effect on hydrolysis of electron-withdrawing groups
in the vicinity of the carbonyl in the acyl moiety was
examined by using N-(2-nitrophenyl)trifluoroacetamide
(22). This compound was very rapidly hydrolyzed by
cholinesterases, especially BuChE (Table 2). The rate
of hydrolysis of compound 22 by AChE was about 4
times faster than that of the analogous lead compound,
2, by the same enzyme. On the other hand, the hydroly-
sis of 22 by BuChE was roughly 100 times faster than
the hydrolysis of 2 by this enzyme. A comparison of
the rates of hydrolyses of N-(2-nitrophenyl)trifluoroace-
tamide (22) by the two cholinesterases (Table 2) showed
that this compound was hydrolyzed about 15 times fast-
er by BuChE than AChE. These results indicate that the
electron-withdrawing fluorine atoms on the carbon adja-
2.2.6. Comparison of anilide and thioester hydrolysis. As
with anilide hydrolysis (Fig. 4), cleavage of thioesters of
choline by AChE drops off rapidly from the acetyl (I) to
the propanoyl (II) derivative and is virtually lost for the
butanoyl (III) substrate (Fig. 6). On the other hand, the
hydrolysis of esters of choline by BuChE is found to in-
crease from the acetyl to the butanoyl ester (Table 5), as
has been observed previously.2 Because N-(2-nitrophen-
yl)octanamide (14) was hydrolyzed by BuChE, even
more rapidly than N-(2-nitrophenyl)butanamide (10)
Table 4. Kinetic parameters of substrates for aryl acylamidase activity of butyrylcholinesterase
Compound
Name
Km (·10ꢀ4 M)
Vmax (·10ꢀ6 M/min)
kcat (minꢀ1
)
2
9
N-(2-Nitrophenyl)acetamide
N-(2-Nitrophenyl)propanamide
N-(2-Nitrophenyl)butanamide
N-(2-Nitrophenyl)pentanamide
N-(2-Nitrophenyl)hexanamide
N-(2-Nitrophenyl)heptanamide
N-(2-Nitrophenyl)octanamide
N-(2-Nitrophenyl)trifluoroacetamide
6.03 0.34
17.8 1.9
0.29 0.03
4.88 0.62
3.77 0.54
18.3 5.0
3
46
10
11
12
13
14
22
3.55 0.74
5.62 1.58
4.85 0.95
3.77 0.70
1.43 0.03
1410 110
35
172
24.5 3.6
31.9 6.3
230
299
34.8 3.8
10,700 3000
326
100,312
The kcat values were calculated by dividing Vmax by the moles of BuChE (0.16 nm) in the assay and multiplying by the reaction volume of 1.5 mL.

4594
3x10-5
S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
lides and choline esters may undergo hydrolysis at the
14
22
same catalytic site via a similar mechanism. However,
the mechanism of hydrolysis for longer acyl derivatives
of both classes of substrates by AChE may differ from
that utilized in the hydrolysis of derivatives with shorter
chain acyl groups.
13
2x10-5
1x10-5
12
11
The broad spectrum of compounds that BuChE is capa-
ble of hydrolyzing may indicate the involvement of this
enzyme in other important biological functions. For
example, BuChE has been suggested to be involved in
the deactivation of the acylated form of the growth hor-
mone secretagogue ghrelin.42 The hormone is a 28 ami-
no acid residue protein which is active when the third
serine residue from the amino terminal is an octanoyl
ester. This active form of the hormone is reported to
be deactivated via deoctanoylation by BuChE.42
10
9
2
2x10-4
8x10-4
4x10-4
6x10-4
1x10-3
0
[S] mM
Figure 5. Simulated plots showing calculated velocities of hydrolysis of
2-nitroanilide substrates based on observed affinity constants (Km) and
maximum velocity (Vmax) values.
2.2.7. Probing the active site of butyrylcholinesterase with
anilides and esters of choline. When hydrolysis of buty-
rylthiocholine by BuChE is examined in the presence
and absence of the comparable, non-chromogenic sub-
strate, butyrylcholine a competition between the sub-
strates is observed.2 Lineweaver–Burk plots of the
hydrolysis of butyrylthiocholine in the presence of
butyrylcholine exhibit an apparent competitive ‘inhibi-
tion’ (‘Ki’ = 1.42 · 10ꢀ6 M). It was reasoned that hydro-
lysis of an anilide substrate in the presence of
butyrylcholine, may give an indication of whether these
two substrates may be competing for the same catalytic
site in BuChE. When the rapidly hydrolyzed N-2-nitro-
phenyltrifluoroacetamide (22) was incubated with
BuChE in the presence of varying concentrations of
butyrylcholine, the time of the appearance of the hydro-
lysis product of 22, namely, 2-nitroaniline, increased
with increasing amounts of butyrylcholine present
(Fig. 7). This suggests that the ester and anilide sub-
strates compete for the some catalytic site and that no
significant hydrolysis of the anilide (22) takes place until
the preferred choline ester hydrolysis has been
completed.
1200
BuChE
1000
AChE
800
600
400
200
0
2
3
4
8
Number of carbon atoms in the acyl group
Figure 6. Initial rates of hydrolysis of thiocholine esters with 2–4 and 8
carbon atoms in the acyl group (compounds I–IV) (3.33 · 10ꢀ5 M) by
human cholinesterases (0.04 units).
(Figs. 4 and 5), the octanoylthiocholine derivative (IV)
was synthesized and its initial rate of hydrolysis was
compared with those of the acetyl (I), propanoyl (II),
and butanoyl (III) choline esters. As observed earlier2
and, as with the 2-nitroanilide derivatives (Table 4),
the rate of hydrolysis by AChE decreased rapidly from
the acetyl- (I) through the butanoyl- (III) derivatives,
whereas the reverse was true for BuChE (Fig. 6). The
octanoyl ester (IV), like its anilide counterpart (14),
was also found to be efficiently hydrolyzed by BuChE.
Furthermore, like the N-(2-nitrophenyl)octanamide
(14), the analogous octanoylthiocholine (IV) was also
slightly hydrolyzed by AChE (Fig. 6). This parallel be-
tween the two classes of substrates suggests that the ani-
3. Conclusions
N-2-Nitroanilides with longer straight chain acyl groups
represent efficient and highly specific substrates for
examining the aryl acylamide activity of BuChE. The
hydrolysis of anilides by cholinesterases has a require-
ment for intramolecular hydrogen bonding between
the amide hydrogen and an adjacent group (e.g., nitro)
on the aniline ring. Hydrolysis is further enhanced by
electron-withdrawing groups (e.g., fluoro) adjacent to
Table 5. Hydrolysis of thiocholine esters by human butyrylcholinesterase
Compound
Name
Km (·10ꢀ5 M)
Vmax (·10ꢀ5 M/minꢀ1
)
kcat (minꢀ1
)
I
Acetylthiocholine
5.07 0.58
4.02 0.55
3.47 1.38
1.99 0.80
1.92 0.11
3.06 0.85
3.77 0.19
3.00 0.21
45,000
72,000
88,000
70,000
II
Propionylthiocholine
Butyrylthiocholine
Octanoylthiocholine
III
IV
The kcat values were calculated by dividing Vmax by the moles of BuChE (0.64 pm) in the assay and multiplying by the reaction volume of 1.5 mL.

S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
4595
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
solution was stirred at room temperature (or at reflux
for the slower reactions) until thin-layer chromatogra-
phy revealed that all aniline had reacted. The solution
was poured slowly into sufficient 5% aqueous sodium
bicarbonate to neutralize all the acid produced. After
vigorous stirring for 1–2 h, the layers were separated
and the organic layer washed with a second portion of
bicarbonate solution, followed by water. The organic
layer was dried (MgSO4) and the solvent removed on a
rotary evaporator. The crude anilide product was puri-
fied by column chromatography and/or recrystallization
from a dichloromethane/petroleum ether mixture.
0
20
40
60
80
100
120
An alternate procedure was found to be superior for the
highly deactivated anilines (2,4- and 2,6-dinitroaniline).
These compounds were acetylated by stirring the aniline
in a 10- to 15-fold excess of pure acetyl chloride at room
temperature. Workup was achieved by pouring the reac-
tion mixture into excess ice-cold 5% aqueous sodium
bicarbonate and stirring the mixture until all gas evolu-
tion had ceased. The precipitated crude anilides were
purified by recrystallization from ethanol–water.
Time (seconds)
Figure 7. Delay in hydrolysis of N-(2-nitrophenyl)trifluoroacetamide
(22) (3.3 · 10ꢀ4 M) caused by increasing concentrations of butyrylch-
oline. No butyrylcholine (s), 0.3 mM butyrylcholine (m), 1.7 mM
butyrylcholine (d), and 3.3 mM butyrylcholine (j).
the carbonyl in the acyl moiety. Comparison of the cho-
linesterase hydrolysis of esters of choline and 2-nitroani-
lides suggests that both classes of substrates generally
utilize the same acyl binding pocket and the same cata-
lytic triad as part of the hydrolytic mechanism.
In each case, the purified anilides were found to be
homogeneous by thin-layer chromatography, had melt-
ing points which agreed well with literature values
1
(where available) and had spectral data (IR, H NMR,
13C NMR, and mass spectra) that were consistent with
the structure.
The data presented here, with more efficient and en-
zyme-specific 2-nitroanilides, provide a basis to aid in
further elucidation of the biochemical nature and possi-
ble biological function of the aryl acylamidase activity
of BuChE.
4.2.2. Thiocholines. Of the four thiocholine derivatives
used in this study, three (I–III) were commercially avail-
able. The fourth, octanoylthiocholine (IV), was synthe-
sized by a previously reported procedure.31
4. Experimental
4.1. Materials
4.3. Analysis of synthesized compounds
Melting points were determined on a Mel-Temp II appa-
ratus 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 or thin films between sodi-
um chloride plates on a Nicolet Avatar 330 FT-IR spec-
trometer. Peak positions were obtained in the ‘find
Recombinant human acetylcholinesterase (AChE, EC
3.1.1.7), aryl acylamidase from P. fluorescens (EC
3.1.1.13), N-phenylacetamide (acetanilide) (1), N-(4-
nitrophenyl)acetamide (4-nitroacetanilide) (4), and all
the reactants used to synthesize the anilide substrates,
except those indicated below, were purchased from Sig-
ma–Aldrich (St. Louis, MO). N-(2-Nitrophenyl)acetam-
ide (2) was purchased from TCI America (Portland,
OR), 3-chloro-2-nitroaniline from Frinton Laboratories
(Vineland, NJ), butyryl chloride from Acros Laborato-
ries (NJ), phenylacetyl chloride from Lancaster Synthe-
sis (Pelham, NH), and benzoyl chloride from Eastman
Kodak (Rochester, NY). Purified human wild-type
butyrylcholinesterase (BuChE, EC 3.1.1.8) was a gift
from Dr. Oksana Lockridge (University of Nebraska
Medical Center).
peaks’ mode and were reproducible within 1–2 cmꢀ1
.
Nuclear magnetic resonance spectra were recorded at
the Atlantic Region Magnetic Resonance Centre at Dal-
housie University on a Bruker Avance spectrometer,
operating at 500 MHz for proton and 125 MHz for car-
bon. Chemical shifts are reported in parts per million
relative to TMS, usually in CDCl3 solution. Mass spec-
tra 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 con-
trolled peak-matching to appropriate PFK reference
ions. Mass measurements were routinely within 3 ppm
of the calculated value.
4.2. Synthesis of substrates
4.2.1. Anilides. All of the anilides were synthesized by one
of the standard procedures.22–30 Briefly, to a well-stirred
solution containing equivalent quantities of the substi-
tuted aniline and triethylamine in dichloromethane was
added slowly 2–4 equiv of the acid chloride or trifluoro-
acetic anhydride also in dichloromethane solution. The

4596
S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
4.4. Analytical data
4.4.6. N-(2-Nitrophenyl)propanamide (9). Bright yellow
needles, mp 62.7–63.6 ꢁC (lit. mp 64 ꢁC).27 IR (Nujol):
3356, 1721, 1606, 1587, 1499, 1315, 1270, 1147, 1070,
4.4.1. N-(3-Nitrophenyl)acetamide (3). Pale yellow crys-
tals, mp 150–151 ꢁC (lit. mp 151–153 ꢁC).23 IR (Nujol):
3262, 3192, 3130, 3097, 1674, 1600, 1551, 1530, 1351,
1326, 1295, 887, 824, 806, 742 cmꢀ1. 1H NMR (CDCl3):
2.24 (s, 3H), 7.44 (br s, 1H), 7.49 (t, J = 8.2 Hz, 1H),
7.94–7.98 (m, 2H), 8.34 (m, 1H). 13C NMR (acetone-
d6): 24.3, 114.3, 118.4, 125.5, 130.7, 141.6, 149.5,
169.6. EI-MS (m/z): 180 (M+), 139, 138 (base), 108, 92,
80, 65. HR-MS: M+ found, 180.0543; calcd for
C8H8N2O3, 180.0535.
1
741 cmꢀ1. H NMR (CDCl3): 1.29 (t, J = 7.6 Hz, 3H),
2.54 (q, J = 7.6 Hz, 2H), 7.17 (m, 1H), 7.64 (m, 1H),
8.20 (dd, J = 8.6 and 1.6 Hz, 1H), 8.80 (dd, J = 8.6
and 1.6 Hz, 1H), 10.37 (br s, 1H). 13C NMR (CDCl3):
9.3, 31.7, 122.1, 123.0, 125.7, 135.0, 136.0, 136.2,
172.8. EI-MS (m/z): 194 (M+), 148, 138 (base), 108, 92,
90, 80, 65, 63, 57. HR-MS (EI): M+ found, 194.0695;
calcd for C9H10N2O3, 194.0691.
4.4.7. N-(2-Nitrophenyl)butanamide (10). Nearly color-
less needles, mp 52–54 ꢁC (lit. mp 49–49.5 ꢁC).29 IR
(Nujol): 3282, 1665, 1590, 1517, 1500, 1273, 1200, 777,
4.4.2. N-(2,4-Dinitrophenyl)acetamide (5). Beige needles,
mp 119–121 ꢁC (lit. mp 119–120 ꢁC).22 IR (Nujol): 3339,
1711, 1600, 1347, 1306, 1264, 1222, 1136, 924, 857, 841,
768, 744 cmꢀ1. 1H NMR (CDCl3): 2.38 (s, 3H), 8.48 (dd,
J = 9.5 and 2.6 Hz, 1H), 9.10 (d, J = 9.5 Hz, 1H), 9.14
(d, J = 2.6 Hz, 1H), 10.64 (br s, 1H). 13C NMR (ace-
tone-d6): 25.2, 122.4, 123.6, 130.2, 137.6, 140.1, 142.7,
170.1. EI-MS (m/z): 225 (M+), 184, 183 (base), 167,
153, 137, 107, 91, 69, 63. HR-MS (EI): M+ found,
225.0386; calcd for C8H7N3O5, 225.0385.
1
740 cmꢀ1. H NMR (CDCl3): 1.04 (t, J = 7.2 Hz, 3H),
1.81 (sextet, J = 7.4 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H),
7.17 (dt, J = 8.6 and 1.4 Hz, 1H), 7.65 (dt, J = 8.6 and
1.4 Hz, 1H), 8.21 (dd, J = 8.6 and 1.4 Hz, 1H), 8.81
(dd, J = 8.6 and 1.4 Hz, 1H), 10.36 (br s, 1H). 13C
NMR (CDCl3): 13.7, 18.8, 40.6, 122.2, 123.1, 125.7,
135.0, 136.0, 136.2, 172.1. EI-MS (m/z): 208 (M+), 162,
138 (base), 121, 108, 91, 90, 79, 71, 62. HR-MS (EI):
M+ found, 208.0835; calcd for C10H12N2O3, 208.0848.
4.4.3. N-(2,6-Dinitrophenyl)acetamide (6). Greenish-yel-
low needles, mp 196–197 ꢁC (lit. mp 193–195 ꢁC).23 IR
(Nujol): 3283, 1677, 1609, 1583, 1535, 1347, 1299,
4.4.8. N-(2-Nitrophenyl)pentanamide (11). Pale yellow
crystals, mp 42.4–43.7 ꢁC. IR (Nujol): 3363, 1717,
1610, 1586, 1500, 1338, 1273, 742 cmꢀ1 1H NMR
.
1
1262, 907, 819, 739, 716 cmꢀ1. H NMR (CDCl3): 2.27
(CDCl3): 0.97 (t, J = 7.4 Hz, 3Hz), 1.40–1.48 (m, 2H),
1.72–1.78 (m, 2H), 2.50 (t, J = 7.6 Hz, 2H), 7.15–7.19
(m, 1H), 7.62–7.66 (m, 1H), 8.21 (dd, J = 8.5 and
1.5H), 8.80 (dd, J = 8.5 and 1.2 Hz, 1H), 10.36 (br s,
1H). 13C NMR (CDCl3): 13.8, 22.3, 27.4, 38.4, 122.2,
123.0, 125.7, 135.1, 136.0, 136.3, 172.3. EI-MS (m/z):
222 (M+), 180, 139, 138 (base), 92, 85, 57, 56. HR-MS
(EI): M+ found, 222.1003; calcd for C11H14N2O3,
222.1004.
(s, 3H), 7.46 (t, J = 8.2 Hz, 1H), 8.29 (d, J = 8.2 Hz,
2H), 9.48 (br s, 1H). 13C NMR (acetone-d6): 24.0,
127.0, 127.5, 130.8, 147.0, 170.0. EI-MS (m/z): 225
(M+), 184, 183 (base), 179, 166, 153, 121, 107, 91, 75,
63. HR-MS (EI): M+ found, 225.0385; calcd for
C8H7N3O5, 225.0385.
4.4.4. N-(3-Methoxy-2-nitrophenyl)acetamide (7). This
compound was prepared from 3-chloro-2-nitroaniline
by the procedure of Nozary et al.28 Reddish-brown
crystals, mp 129–131.5 ꢁC. IR (Nujol): 3255, 1667,
4.4.9. N-(2-Nitrophenyl)hexanamide (12). Pale yellow
crystals, mp 46.4–48.5 ꢁC. IR (Nujol): 3250, 1665,
1590, 1521, 1278, 1098, 972, 855 cmꢀ1 1H NMR
.
(CDCl3): 2.19 (s, 3H), 3.91 (s, 3H), 6.81 (d,
J = 8.4 Hz, 1H), 7.43 (t, J = 8.4 Hz, 1H), 7.86 (br d,
J = 8 Hz, 1H), 8.27 (br s, 1H). 13C NMR (CDCl3):
24.8, 56.7, 108.1, 115.1, 131.9, 132.7 (2 peaks),
152.7, 168.5. EI-MS (m/z): 210 (M+), 174, 172, 168,
164 (base), 121, 110, 108, 107, 95, 80, 77, 67, 65,
64, 63, 53. HR-MS (EI): M+ found, 210.0639; calcd
for C9H10N2O4, 210.0640.
1591, 1499, 1359, 1270, 1252, 1190, 784 cmꢀ1 1H
.
NMR (CDCl3): 0.91–0.95 (m, 3H), 1.36–1.42 (m, 4H),
1.74–1.80 (m, 2H), 2.49 (t, J = 7.5Hz, 2H), 7.15–7.18
(m, 1H), 7.62–7.66 (m, 1H), 8.21 (dd, J = 8.4 and
1.5 Hz, 1H), 8.81 (dd, J = 8.6 and 1.2 Hz, 1H), 10.36
(br s, 1H). 13C NMR (CDCl3): 13.9, 22.4, 25.1, 31.3
38.7, 122.2, 123.1, 125.7, 135.1, 136.0, 136.3, 172.3. EI-
MS (m/z): 236 (M+), 152, 151, 138, 134, 133, 122 (base),
108, 100, 99, 93, 92. HR-MS (EI): M+ found, 236.1159;
calcd for C12H16N2O3, 236.1161.
4.4.5. N-(5-Methoxy-2-nitrophenyl)acetamide (8). This
compound was prepared from 5-chloro-2-nitroaniline
by the procedure of Nozary et al.28 Yellow crystals.
mp 122–125 ꢁC (lit. mp 123–125ꢁ).28 IR (Nujol): 3313,
1696, 1607, 1587, 1326, 1274, 1240, 1218, 1090, 1014,
4.4.10. N-(2-Nitrophenyl)heptanamide (13). Pale yellow
crystals, mp 32.5–33.5 ꢁC. IR (thin film): 3363, 2924,
2854, 1716, 1610, 1586, 1499, 1458, 1377, 1338, 1273,
1
1
865, 760 cmꢀ1. H NMR (CDCl3): 2.30 (s, 3H), 3.91
1147, 742 cmꢀ1. H NMR (CDCl3): 0.88–0.91 (m, 3H),
(s, 3H), 6.55 (dd, J = 9.4 and 2.8 Hz, 1H), 8.19 (d,
J = 9.4 Hz, 1H), 8.42 (d, J = 2.8 Hz, 1H), 10.76 (br s,
1H). 13C NMR (CDCl3): 25.8, 56.1, 104.2, 110.7,
128.0, 129.4, 137.8, 165.7, 169.4 ppm. EI-MS (m/z):
210 (M+), 169, 168 (base), 165, 164, 138, 122, 120, 79,
63. HR-MS (EI): M+ found, 210.0639; calcd for
C9H10N2O4, 210.0640.
1.30–1.43 (m, 6H), 1.73–1.79 (m, 2H), 2.49 (t,
J = 7.5Hz, 2H), 7.15–7.19 (m, 1H), 7.63–7.66 (m, 1H),
8.21 (dd, J = 8.4 and 1.5 Hz, 1H), 8.81 (dd, J = 8.5
and 1.2 Hz, 1H), 10.36 (br s, 1H). 13C NMR (CDCl3):
14.0, 22.4, 25.3, 28.8, 31.5, 38.7, 122.2, 123.0, 125.7,
135.0, 135.9, 136.2, 172.3. EI-MS (m/z): 250 (M+), 180,
151, 139, 138 (base), 122, 113, 90, 85, 65, 55. HR-MS

S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
4597
(EI): M found, 250.1315; calcd for C13H18N2O3,
250.1317.
HR-MS (EI): M+ found, 242.0694; calcd for
C13H10N2O3: 242.0691.
4.4.11. N-(2-Nitrophenyl)octanamide (14). Pale yellow
crystals, mp 40.0–41.8 ꢁC. IR (Nujol): 3287, 1662,
4.4.16. N-Methyl-N-(2-nitrophenyl)acetamide (19). Yel-
low crystals, mp 69.1–70.2 ꢁC (lit. mp 70–71.5 ꢁC).34
IR (Nujol): 1664, 1602, 1524, 1346, 1309, 1142, 1070,
1591, 1536, 1518, 1495, 1362, 778 cmꢀ1 1H NMR
.
1
(CDCl3): 0.87–0.90 (m, 3H), 1.29–1.43 (overlapping m,
8H), 1.73–1.79 (m, 2H), 2.49 (t, J = 7.6 Hz, 2H), 7.15–
7.18 (m, 1H), 7.62–7.66 (m, 1H), 8.21 (dd, J = 8.4 and
1.5 Hz, 1H), 8.81 (dd, J = 8.6 and 1.2 Hz, 1H), 10.36
(br s, 1H). 13C NMR (CDCl3): 14.0, 22.5, 25.3, 28.9,
29.0, 31.6, 38.7, 122.1, 123.0, 125.7, 135.0, 135.9,
136.2, 172.2. EI-MS (m/z): 264 (M+), 218, 180, 151,
139, 138 (base), 127, 122, 92, 90, 57, 55. HR-MS (EI):
M+ found, 264.1479; calcd for C14H20N2O3, 264.1474.
973, 794, 708 cmꢀ1. H NMR (CDCl3): product exists
as a 3.3:1 mixture of two amide conformers. Major com-
ponent (76%): 1.82 (s, 3H), 3.22 (s, 3H), 7.42 (dd, J = 7.9
and 1.3 Hz, 1H), 7.57–7.61 (m, 1H), 7.71–7.75 (m, 1H),
8.02 (dd, J = 8.1 and 1.4 Hz, 1H). Minor component
(24%): 2.25 (s, 3H), 3.45 (s, 3H), 7.34 (dd, J = 8.0 and
1.3 Hz, 1H), 7.43–7.47 (overlapping m, 1H), 7.65–7.69
(m, 1H), 7.97 (dd, J = 8.2 and 1.3 Hz, 1H). 13C NMR:
(a) major conformer: 22.0, 36.7, 125.6, 129.5, 131.0,
134.5, 137.6, 146.8, 169.8 ppm; (b) minor conformer:
22.0, 39.3, 125.1, 128.0, 129.3, 134.2, 137.4, 146.5,
171.2 ppm. EI-MS (m/z): 194 (M+), 153, 152, 148 (base),
135, 106, 105, 104, 79, 78, 77. HR-MS: M+ found,
194.0692; calcd for C9H10N2O3, 194.0691.
4.4.12. N-(2-Nitrophenyl)-3,3-dimethylbutanamide (15).
Bright yellow crystals, mp 64.0–66.2 ꢁC. IR (Nujol):
3360, 1692, 1609, 1586, 1500, 1341, 1272, 1147, 1119,
748 cmꢀ1 1H NMR (CDCl3): 1.12 (s, 9H), 2.35 (s,
.
2H), 7.15–7.19 (m, 1H), 7.62–7.66 (m, 1H), 8.21 (dd,
J = 8.6 and 1.5 Hz, 1H), 8.82 (dd, J = 8.5 and 1.1 Hz,
1H), 10.30 (br s, 1H). 13C NMR (CDCl3): 29.8, 31.4,
52.7, 122.1, 123.1, 125.7, 135.0, 136.0, 136.3, 170.9. EI-
MS (m/z): 236 (M+), 221, 180, 139, 138 (base), 99, 92,
83, 71, 57. HR-MS (EI): M+ found, 236.1157; calcd
For C12H16N2O3, 236.1161.
4.4.17. N-Methyl-N-(2-nitrophenyl)butanamide (20).
Yellow liquid. IR (thin film): 2967, 1710, 1670, 1602,
1530, 1488, 1352, 1293, 1192, 1136, 1081, 1043, 851,
1
785, 760, 707 cmꢀ1. H NMR (CDCl3): product exists
as 3.5:1 mixture of amide conformers. Major compo-
nent (78%): 0.97 (t, J = 7.5 Hz, 3H), 1.65–1.73 (m,
2H), 2.33 (t, J = 7.4 Hz, 2H), 3.23 (s, 3H), 7.39 (dd,
J = 8.7 and 1.2 Hz, 1H), 7.57–7.60 (m, 1H), 7.70–
7.74 (m, 1H), 8.02 (dd, J = 8.3 and 1.3 Hz, 1H). Min-
or component (22%): 1.00 (t, J = 7.5 Hz, 3H), 1.56–
1.62 (m, 2H), 2.47 (t, J = 7.4 Hz, 2H), 3.43 (s, 3H),
7.32–7.34 (m, 1H), 7.43–7.46 (m, 1H), 7.64–7.67 (m,
1H), 7.96–7.98 (m, 1H). 13C NMR: (a) major con-
former: 13.6. 18.2, 18.5, 35.9, 125.7, 129.5, 131.2,
134.1, 134.5, 137.6, 172.74; (b) minor conformer:
13.9, 17.9, 36.9, 125.1, 128.0, 129.4, 137.3, 173.8. EI-
MS (m/z): 222 (M+), 176, 153, 152 (base), 106, 105,
104, 79, 77, 71. HR-MS (EI): M+ found, 222.1004;
calcd for C11H14N2O3, 222.1004.
4.4.13. N-(2-Nitrophenyl)phenylacetamide (16). Bright
yellow crystals, mp 81.2–82.6 ꢁC. IR (Nujol): 3360,
1705, 1608, 1584, 1494, 1339, 1272, 1240, 1162, 1131,
1073, 1033, 853, 786, 743 cmꢀ1 1H NMR (CDCl3):
.
3.81 (s, 2H), 7.11–7.14 (m, 1H), 7.35–7.44 (overlapping
multiplets, 5H), 7.58–7.62 (m, 1H), 8.13 (dd, J = 8.4
and 1.5 Hz, 1H), 8.78 (dd, J = 8.6 and 1.2 Hz, 1H),
10.25 (br s, 1H). 13C NMR (CDCl3): 45.8, 122.0,
123.3, 125.7, 128.0, 129.3, 129.7, 133.3, 134.7, 135.9,
136.3, 170.3. EI-MS (m/z): 256 (M+, base), 210, 119,
118, 92, 91, 90, 89, 65. HR-MS (EI): M+ found,
256.0839; calcd for C14H12 N2O3, 256.0848.
4.4.14. N-Phenylbenzamide (17). Colorless crystals, mp
162–163 ꢁC (lit. mp 163ꢁ).26 IR (Nujol): 3342, 1655,
4.4.18. N-Phenyltrifluoroacetamide (21). Colorless nee-
dles, mp 86.5–88.9 ꢁC (lit. mp 85–86 ꢁC).30 IR (Nujol):
3318, 1704, 1603, 1551, 1308, 1287, 1244, 1152, 1078,
1
1599, 1578, 1529, 1321, 1260, 749, 716, 690 cmꢀ1. H
1
NMR (CDCl3): 7.15 (t, J = 7.4 Hz, 1H), 7.37 (t,
J = 8.0 Hz, 2H), 7.48 (t, J = 7.4 Hz, 2H), 7.55 (m, 1H),
7.86 (br s, 1H), 7.87 (d, J = 8 Hz, 2H). 13C NMR
(CDCl3): 120.4, 124.8, 127.2, 129.0, 129.3, 132.0,
135.2, 138.1, 165.9. EI-MS (m/z): 197 (M+), 106, 105
(base), 77, 76, 65. HR-MS (EI): M+ found, 197.0845;
calcd for C13H11NO, 197.0841.
921, 896, 755, 731, 690 cmꢀ1. H NMR (CDCl3): 7.23
(t, J = 7.7 Hz, 1H), 7.36 (m, 2H), 7.55 (d, J = 7.8 Hz,
2H), 8.21 (br s, 1H). 13C NMR (proton-decoupled;
CDCl3): 115.7 (q, J1CF ¼ 288 Hz), 120.7, 126.4, 129.3,
135.0, 155.0 (q, J2CF ¼ 37 Hz). EI-MS (m/z): 189 (M+)
(base), 121, 120, 119, 94, 92, 91, 77, 69, 65, 52. HR-
MS (EI): M+ found, 189.0405; calcd for C8H6NOF3,
189.0401.
4.4.15. N-(2-Nitrophenyl)benzamide (18). Bright yellow
needles, mp 89.5–91.5 ꢁC (lit. mp 90–92 ꢁC).22,24 IR
(Nujol): 3361, 1683, 1606, 1589, 1502, 1341, 1278, 742,
4.4.19. N-(2-Nitrophenyl)trifluoroacetamide (22). Pale
yellow needles, mp 88–89 ꢁC (lit. mp 89–90 ꢁC).25 IR
(Nujol): 3298, 1728, 1596, 1519, 1275, 1171, 1158,
1
706 cmꢀ1. H NMR (CDCl3): 7.22 (d, J = 7.6 Hz and
1.3 Hz, 1H), 7.54 (t, J = 7.5 Hz, 2H), 7.61 (m, 1H),
7.71 (dt, J = 7.8 Hz and 1.3 Hz, 1H), 8.00 (dd,
J = 7.1 Hz and 1.3 Hz, 1H), 8.27 (dd, J = 8.4 Hz and
1.5 Hz, 1H), 9.01 (dd, J = 8.5 Hz and 1.3 Hz, 1H),
11.34 (br s, 1H). 13C NMR (CDCl3): 122.1, 123.3,
125.9, 127.4, 129.1, 132.6, 134.1, 135.4, 136.2, 136.5,
165.7. EI-MS (m/z): 242 (M+), 196, 106, 105 (base), 77.
1139, 740 cmꢀ1 1H NMR (CDCl3): 7.38 (dt, J = 8.5
.
and 1.4 Hz, 1H), 7.77 (dt, J = 8.5 and 1.4 Hz, 1H),
8.32 (dd, J = 8.5 and 1.4 Hz, 1H), 8.74 (dd, J = 8.5
and 1.4 Hz, 1H), 11.38 (br s, 1H). 13C NMR (proton-de-
coupled; CDCl3): 115.4 (q, J1CF ¼ 288 Hz), 122.2, 125.6,
126.2, 132.1, 136.4, 137.0, 155.4 (q, J2CF ¼ 38 Hz). EI-
MS (m/z): 234 (M+), 188 (base peak), 168, 165, 148,

4598
S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
121, 120, 102, 91, 90, 89, 77, 69, 64, 62. HR-MS (EI): M+
found, 234.0268; calcd for C8H5N2O3F3, 234.0252.
ged from 8 · 10ꢀ6 to 3.5 · 10ꢀ4 M in the reaction mix-
ture. The rate of formation of the aniline product was
determined using a Milton Roy 1200 UV–vis spectro-
photometer or an Ultrospec 2100 pro UV–vis spectro-
photometer with Swift II applications software. The
molar extinction coefficient (e) for 2-nitro aniline used
to convert the change in absorbance at k = 430 nm to
moles of product was 3954 Mꢀ1 cmꢀ1. Kinetic constants
(Km and Vmax) were determined using the modified-di-
rect linear plot method.43 The kcat values were calculated
by dividing Vmax by the moles of BuChE (0.16 nm) in
the assay and multiplying by the reaction volume of
1.5 mL.
4.4.20. Octanoylthiocholine (IV). Colorless powder, mp
129.6–133.8 ꢁC. IR (Nujol): 1694, 1417, 1124, 1048,
1
1017, 968, 932, 909 cmꢀ1. H NMR (D2O): 0.84–0.87
(m, 3H), 1.2–1.3 (overlapping m, 8H), 1.55–1.61 (m,
2H), 2.64 (t, J = 7.3 Hz, 2H), 3.14 (s, 9H), 3.24–3.27
(m, 2H), 3.40–3.44 (m, 2H). 13C NMR (D2O): 13.8,
21.2, 21.9, 24.8, 28.0, 28.2, 30.9, 43.1, 52.1, 63.6, 197.8.
ESI-MS (m/z): 246.1 (M+), 188, 187, 127, 109.
4.5. Enzyme assays
4.5.1. Aryl acylamidase activity. The aryl acylamidase
activity of purified human recombinant AChE and puri-
fied human plasma BuChE, as well as aryl acylamidase
from P. fluorescens (AAAPf) was studied using a modi-
fied Fujimoto spectrophotometric method.3 Briefly, in
a quartz cuvette, with a 1 cm path-length, were placed
1.35 mL of 0.06 M Tris–HCl buffer (pH 8.0), 0.05 mL
of 50% aqueous acetonitrile or 0.05 mL of inhibitor in
this solvent or H2O, and 0.05 mL of up to 10 mM sub-
strate in 50% acetonitrile. The reagents were mixed
and zeroed at the appropriate wavelength, 0.05 mL of
enzyme (10 units, defined below), of AChE or BuChE
or 0.125 unit of AAAPf were added (final volume
1.50 mL), mixed by inversion, and the absorbance mea-
sured every minute for an appropriate time period, most
often 10 min, using a Milton Roy 1200 UV–vis spectro-
photometer at 23 ꢁC.
4.5.2. Esterase activity. The esterase activity of AChE
and BuChE was determined by a modification44 of the
method described by Ellman et al.45 Briefly, 1.35 mL
of buffered 5,50-dithio-bis(2-nitrobenzoic acid) solution
(pH 8.0), 0.05 mL of AChE (0.03 units) or BuChE
(0.04 units) in 0.1% aqueous gelatin, and 0.05 mL of
50% aqueous acetonitrile were mixed in a quartz cuvette
of 1 cm path-length. The mixture was zeroed at 412 nm,
and the reaction was initiated by the addition of a thi-
ocholine in an aqueous solution to give a final substrate
concentration of 2.0 · 10ꢀ4 M or less. The reactions
were performed at 23 ꢁC. The rate of change of absor-
bance (DA/min), reflecting the rate of hydrolysis of the
thiocholine, was recorded every 5 s for 1 min, using a
Milton-Roy 1201 UV–vis spectrophotometer. The mo-
lar extinction coefficient (e) for the Ellman product used
to convert the change in absorbance at k = 412 nm to
moles of product was 14,150 Mꢀ1 cmꢀ1. These experi-
ments were generally done at least in triplicate and the
values averaged. Modified-direct linear plots43 were gen-
erated by using a fixed amount of cholinesterase and
varying amounts of substrate (2.0 · 10ꢀ5 to
2.0 · 10ꢀ4 M).
Under the conditions employed, 0.1 unit of BuChE was
defined as that amount of enzyme that led to a change of
absorbance of 1.00 in one minute in the presence of
1.60 · 10ꢀ4 M butyrylthiocholine. This represents a rate
of butyrylthiocholine hydrolysis of 2.1 · 10ꢀ7 M/min.
Under the conditions employed, 0.1 unit of AChE was
defined as above, but using 1.60 · 10ꢀ4 M acetylthioch-
oline as the substrate. For AAAPf, 1 unit is defined as
that amount of enzyme that will convert 1.0 · 10ꢀ6 M/
min of N-acetyl-p-aminophenol to p-aminophenol at
pH 9.0 and 37 ꢁC, as defined by the supplier.
4.6. Calculations of molecular parameters
Geometry optimization of the anilide substrates was car-
ried out in a three-step process. First, the lowest energy
conformer was obtained by the molecular mechanics
(MM) method using the MMFF94 force-field (PC Spar-
tan Pro, Wavefunction, Inc., 18401 Von Karman, Suite
370, Irvine, CA, 92612). Second, the geometry was opti-
mized at the Hartree–Fock (HF) level using the STO-3G
basis set starting from the preferred MM conformer. In
step three, the HF/STO-3G optimized geometry became
the starting point for an optimization at a higher level of
theory using the B3LYP functional with 6-31G(d) basis
set.36–38 The latter two steps were carried out using the
Gaussian 98 suite of programs.46 HF and B3LYP geom-
etry optimizations were followed by a frequency analysis
to ascertain that the optimized species were the true min-
ima on their potential surfaces. In some cases, the opti-
mization was on the basis of negligible forces. The
rationale for carrying out higher-level quantum chemi-
cal calculations was 2-fold. First, it was noted that
B3LYP/6-31G(d) geometries more closely resembled
X-ray crystallographic data than did their counterparts
obtained at MM level of theory.47 Second, any study
of the relationship between electronic properties and
Repetitive scans for detecting AAA activity of cholines-
terases and AAAPf were performed using a reaction mix-
ture containing 1.4 mL of 0.06 M Tris buffer, pH 8, and
0.05 mL of enzyme as above. The reactions were com-
menced with the addition of 0.05 mL of anilide com-
pound (10 mM or 1 mM, depending on solubility)
dissolved in 50% aqueous acetonitrile. Changes in absor-
bance were monitored over a spectral range of 200–
600 nm using an Ultrospec 2100 UV–vis spectropho-
tometer with Swift II applications software. The time
interval for these scans varied depending on the rate of
hydrolysis.
Determination of kinetic constants for hydrolysis of ani-
lide substrates by BuChE was done using reaction mix-
ture containing 1.4 mL of 0.06 M Tris buffer, pH 8, and
0.05 mL of enzyme. The reactions were commenced with
the addition of 0.05 mL of substrate dissolved in 50%
aqueous acetonitrile. Final substrate concentrations ran-

S. Darvesh et al. / Bioorg. Med. Chem. 14 (2006) 4586–4599
4599
24. Wrasidio, W.; Levine, H. H. J. Polym. Sci., Part A:
Polym. Chem. 1964, 2, 4795.
25. Rutherford, K. G.; Ing, S. Y.-S.; Thibert, R. J. Can. J.
Chem. 1965, 43, 541.
26. Tani, J.; Oine, T.; Inoue, T. Synthesis 1975, 11, 714.
27. Chiron, R.; Graff, Y. Spectrochim. Acta 1976, 32A, 1303.
28. Nozary, H.; Piguet, C.; Rivera, J.-P.; Tissot, P.; Bernar-
activity would be impossible at the force-field level since
electrons are not explicitly included in force-field calcu-
lations. Atomic charges were calculated via Mulliken
population analysis. Calculated properties included low-
est energy conformations, extent of linearity between
donor and acceptor atoms, extent of planarity of the
anilide derivatives, and atomic charges.
dinelli, G.; Vulliermet, N.; Weber, J.; Bunzli, J.-C. G.
¨
Inorg. Chem. 2000, 39, 5286.
29. Fonseca, T.; Gigante, B.; Gilchrist, T. L. Tetrahedron
2001, 57, 1793.
Acknowledgments
´
30. Salazar, J.; Lopez, S. E.; Rebollo, O. J. J. Fluorine Chem.
2003, 124, 111.
31. Yamada, M.; Marui, Y.; Hayashi, C.; Miki, Y.; Takem-
ura, S. Clin. Chem. 2001, 47, 1962.
32. Smith, M. B, March, J. March’s Advanced Organic
Chemistry Reactions, Mechanisms and Structures, 5th ed.;
John Wiley & Sons: New York, Chichester, Weiheim,
Brisbane, Singapore, Toronto, 2001; p 98.
33. Lutskii, A. E.; Konel’skaya, V. N.; Kondratenko, B. P.
Zh. Obshch. Khim. 1960, 30, 3782.
This work was supported by Canadian Institutes of
Health Research, Dalhousie University Internal Medi-
cine Research Fund, Queen Elizabeth II Health Science
Centre Research Fund, Heart and Stroke Foundation of
NS, the Natural Sciences and Engineering Research
Council of Canada, and the Committee on Research
and Publications of Mount Saint Vincent University.
We would like to thank Jennifer Uhlman, Lesek
Webber, and Heather Smith for excellent technical help.
34. Bartels-Keith, J. R.; Cieciuch, R. F. W. Can. J. Chem.
1968, 46, 2593.
35. Skulski, L. J. Org. Chem. 1963, 28, 3565.
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