Bisquaternary pyridinium oximes: Comparison of in vitro reactivation potency of compounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

Article abstract of DOI:10.1016/j.bmc.2009.11.052

Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two different classes of compounds with varying linker chains was compared and observed that the structure of the connecting chain is an important factor for the activity of the reactivators. At a higher concentration (10^-3 M), compounds bearing aliphatic linker showed better reactivation than compounds with heterocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration (10^-3 M), whereas their ability to reactivate was increased at lower concentrations (10^-4 M and 10^-5 M). Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reactivation of EeAChE, respectively, at 10^-5 M. These compounds showed 14%, 6% and 15% reactivation of rHuAChE at 10^-4 M. Amongst newly synthesized analogs with heterocyclic linkers (26-35 and 45-46), compound 31, bearing furan linker chain, was found to be the most effective reactivator with a k_r 0.042 min^-1, which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a k_r 0.0041 min^-1 that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE.

Full text of DOI:10.1016/j.bmc.2009.11.052

Bioorganic & Medicinal Chemistry 18 (2010) 787–794
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bisquaternary pyridinium oximes: Comparison of in vitro reactivation
potency of compounds bearing aliphatic linkers and heteroaromatic linkers for
paraoxon-inhibited electric eel and recombinant human acetylcholinesterase
Sandip B. Bharate a, Lilu Guo b, Tony E. Reeves c, Douglas M. Cerasoli c, Charles M. Thompson a,b,
*
a NIH COBRE Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
b ATERIS Technologies LLC, 901 N Orange Street, Missoula, MT 59802, USA
c USAMRICD, Research Division, Physiology and Immunology Branch, 3100 Ricketts Point Road, APG, MD 21010, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication
and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the
present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited
by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with
some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated
for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase
(EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most
of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two
different classes of compounds with varying linker chains was compared and observed that the structure
of the connecting chain is an important factor for the activity of the reactivators. At a higher concentra-
tion (10ꢀ3 M), compounds bearing aliphatic linker showed better reactivation than compounds with het-
erocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration
(10ꢀ3 M), whereas their ability to reactivate was increased at lower concentrations (10ꢀ4 M and 10ꢀ5 M).
Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reac-
tivation of EeAChE, respectively, at 10ꢀ5 M. These compounds showed 14%, 6% and 15% reactivation of
rHuAChE at 10ꢀ4 M. Amongst newly synthesized analogs with heterocyclic linkers (26–35 and 45–46),
compound 31, bearing furan linker chain, was found to be the most effective reactivator with a kr
0.042 minꢀ1, which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed
a kr 0.0041 minꢀ1 that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE.
Received 4 October 2009
Revised 18 November 2009
Accepted 21 November 2009
Available online 27 November 2009
Keywords:
Organophosphorus
Acetylcholinesterase
Bisquaternary oximes
Reactivation
Paraoxon
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
biochemical studies. Oximes reactivate phosphorylated cholines-
terases by cleaving the phosphoester bond between the active site
Exposure to organophosphorus compounds (OPs) such as sarin,
paraoxon, soman, and VX causes acute intoxication inclusive of
convulsions and paralysis of the respiratory muscles. The toxic ac-
tion of organophosphorus compounds is generally attributed to the
inhibition of acetylcholinesterase (AChE; EC 3.1.1.7) thus blocking
its physiologic action—hydrolyzing the neurotransmitter acetyl-
choline at central and peripheral synapses. Acetylcholine accumu-
lation results in an over-stimulation of cholinergic receptors and,
depending on the type and dose of the OP can lead to respiratory
arrest and death.1,2 Reactivation of AChE is the primary therapeutic
approach to reverse the ill effects of OP poisoning and oximes are
the universal choice for this therapeutic intervention. Oximes are
also important tools in vitro serving in kinetic, mechanistic and
serine and the phosphorus atom. By virtue of their high affinity for
AChE and their inherent nucleophilicity, oxime-based reactivation
is relatively efficient forming a dephosphorylated, restored enzyme
and a transient phosphorylated oxime.3
The search for oxime-based reactivators dates back to the early
1950s and led to the discovery of 2-pyridine aldoxime methiodide
(2-PAM, 1) as an effective reactivator used to treat human OP poison-
ings.4,5 The next compound of interest was trimedoxime (TMB-4, 2)
which was synthesized and tested by Poziomek et al.6 and indepen-
dently by Hobbiger et al.7 This bispyridinium derivative was superior
to 2-PAM, particularly in tabun poisoning. Obidoxime (3) was synthe-
sized by Luttringhaus and Hagedorn in the early 1960s8 and studied in
pesticide-poisoned patients by Erdmann and von Clarmann.9 In the
following decades, Hagedorn et al. synthesized numerous oximes in
order to increase the efficacy against soman, resulting in the develop-
ment of HI-6 (4).10–12 HI-6 is considered to be superior to 2-PAM, obi-
* Corresponding author. Tel.: +1 406 243 4643; fax: +1 406 243 4227.
E-mail address: charles.thompson@umontana.edu (C.M. Thompson).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.052

788
S. B. Bharate et al. / Bioorg. Med. Chem. 18 (2010) 787–794
doxime and TMB-4 in reactivating AChE after nerve agent poisoning.
Apart from these well known oxime reactivators, several bisquaterna-
ry pyridinium oximes bearing varying linker chains with p-electron
say.32 Substrate and reporter reagents (AtChI at 1 mM and DTNB at
0.3 mM) were added in 0.1 M phosphate buffer solution pH 7.2.
The OP paraoxon was used to inhibit both AChEs, and pralidoxime
(1), trimedoxime (2) and obidoxime (3) were used as the reference
oxime reactivators. Reactivation results are summarized in Table 1.
Among compounds bearing aliphatic linkers, 25, 41 and 44 at
10ꢀ5 M promoted reactivation of EeAChE (68%, 56% and 52%,
respectively) at levels equal to or greater than that of pralidoxime
(1), but less than that of trimedoxime (2). These compounds, along
with newly synthesized compound 42, showed 58%, 80%, 71% and
74% reactivation of EeAChE, respectively at 10ꢀ4 M. Compound 42
also reactivated rHuAChE to 20% at 10ꢀ4 M. For rHuAChE, com-
pounds 41 and 44 were the most active with 64% and 73% reacti-
vation at 10ꢀ3 M, respectively. Compounds with heterocyclic
linker chains (thiophene, furan and isoxazole) displayed a reverse
concentration-activity relationship. These compounds (26–35 and
45), with the exception of 46, showed 6 7% reactivation of EeAChE
and no reactivation (63%) of rHuAChE at the highest concentration
(10ꢀ3 M). Lower concentrations of oxime (10ꢀ4–10ꢀ5 M) 26–35
and 45–46 were better reactivators of both enzymes suggesting
that these compounds may inhibit AChE at higher concentrations
and explain, in part, the inability to reactivate inhibited AChE at
10ꢀ3 M.18,33 To confirm this change in mechanism, compounds 2,
3, 26, 28, 31 and 46 were tested as inhibitors against rHuAChE
and EeAChE. The results indicated that the compounds (except obi-
doxime, 3) gave 23–87% inhibition of both AChE’s at 10ꢀ3 M while
<3% inhibition was observed at 10ꢀ5 M (Table 2). Pyridinium oxi-
mes bearing heterocyclic linkers 26, 28, 31 and 46 were better
inhibitors than those oximes bearing aliphatic linkers 2 and 3.
These results explain, in part, the inability of pyridinium oximes
with heterocyclic linkers to reactivate paraoxon-inhibited AChE
at 10ꢀ3 M. The lower reactivation potency of bisquaternary oximes
with heterocyclic linkers compared with oximes bearing aliphatic
linkers might also be due to the restricted flexibility of the hetero-
cycles in the AChE active site cavity. Since the concentration
10ꢀ3 M is not attainable with in vivo experiments,34 10ꢀ5 M is
more appropriate and attainable for clinical use.1
functionalities are reported in literature viz. (E)-but-2-ene linker 5
(55% reactivation of tabun-inhibited AChE),13,14 CH2CH2OCH2CH2
linker 6 (17% reactivation of cyclosarin-inhibited AChE),15 CH2O
(CH2)2OCH2 linker 7 (63% reactivation of DFP-inhibited AChE)16,17
and xylene linker 8 (44% reactivation of paraoxon-inhibited AChE).18
Structures of pyridinium oximes 1-8 are shown in Figure 1. Overall,
reactivators differ mainly in the number of quaternary pyridinium
rings (1 or 2), length and structure of connecting chain and/or in the
number and position of functional oxime groups.
From a toxicological standpoint, the reactivating efficacy of oxi-
mes has been mainly investigated in rodents.19 However, owing to
its ready availability, an extensive number of in vitro investigations
have been conducted over the years with electric eel AChE
(EeAChE). As a result, it is generally necessary to conduct the
screening of new oximes with EeAChE for comparison purposes.
Nevertheless, the structural and functional differences between
human, animal and electric eel AChE may result in a different affin-
ity and reactivity of oximes. Several studies indicate substantial
species-dependent marked differences in the ability of oximes to
reactivate OP-inhibited AChE.20–22 Overall, measurements of spe-
cies differences can be crucial for the assessment of oxime efficacy
in humans. Experiments were undertaken to determine percentage
of reactivation and reactivation rate constant of several newly syn-
thesized as well as some known bisquaternary oximes for recom-
binant human and electric eel AChE inhibited by paraoxon.
Paraoxon has been responsible for more cases of poisoning than
any other OP insecticide,23 so it is vitally important to determine
the potency of new oximes for this OPI. Recently, we reported the
reactivation potency of a series of newly synthesized monoquaterna-
ry pyridinium oximes against paraoxon-inhibited electric eel AChE
(EeAChE) and recombinant human AChE (rHuAChE).24 In the present
paper, a series of bisquaternary pyridinium oximes bearing heterocy-
clic linkers along with several known bisquaternary oximes bearing
aliphatic 3–4 atom chain linkers were synthesized and evaluated for
their reactivation potency against paraoxon-inhibited electric eel
AChE (EeAChE) and recombinant human AChE (rHuAChE). A compar-
ison of the reactivation profile of two different classes of oxime reac-
tivators against rHuACHE and EeAChE is also discussed.
At 10ꢀ5 M, compounds 26, 31 and 46 were the best reactivators
of both enzymes (59%, 52% and 49% reactivation of EeAChE, and
57%, 77% and 7% reactivation of rHuAChE). The efficacy of each
reactivator varied with concentration, and four trends were identi-
fied from the data (Table 1) (Fig. 2). First, percent reactivation cor-
related with increasing concentration of oximes 20, 22, and 40.
Second, we observed that an increasing reactivation correlated
with a decreasing concentration of oximes 2, 21, 23, 25, 26–28,
31, and 35.
2. Results and discussion
2.1. Synthesis of bisquaternary pyridinium oximes
Bisquaternary reactivators were prepared as shown in Schemes
1 and 2. A series of known bisquaternary pyridinium oximes bear-
ing 3–4 atom aliphatic linkers (2, 3, 19–25, 39–41, 44)25–31 along
with two new structural analogs (42 and 43) were synthesized.
Furthermore, a series of new bisquaternary pyridinium oximes
bearing the heterocyclic linkers viz. thiophene (26–28, 45–46), fur-
an (29–32) and isoxazole (33–35) were synthesized. Heterocyclic
Third, oximes 3, 19, 24, 29, 30, 39, 41–46 showed the best per-
cent reactivation at midrange concentrations 10ꢀ4 M. This might
be explained by a combination of inefficient reactivation at low
concentrations coupled with inhibitory activity at high concentra-
tions.33 Last, 2- and 4-oximes were better reactivators than 3-oxi-
mes. Control compound 32, which does not possess any oxime
functionality, did not reactivate either enzyme.
linkers were used to alter the lipophilicity and include a
characteristic capable of interacting with the AChE active site res-
idues via or cation- interactions. Additionally, various posi-
tions of the oxime functional groups on the pyridinium ring were
used to optimize any angular dependence between cation and
nucleophile to afford the effective reactivation of AChE. All com-
pounds were characterized by 1H NMR, 13C NMR, ESI-MS and IR
data and have purity of >95% from NMR spectra.
p-electron
All synthesized pyridinium oximes were also tested for their
cytotoxic effects towards mammalian kidney fibroblasts (Vero
cells). None of the compounds showed cytotoxicity up to the con-
p
–p
p
centration of 4.67 l
g/ml (unpublished results).35
2.3. Determination of reactivation rate constants (kr)
Owing to their greater activity (Table 1) amongst newly synthe-
sized compounds (26–35, 42, 45–46), compounds 26, 31, 42 and 46
were selected for analysis of the oxime-mediated reactivation rate
constants (kr; characterizes the dissociation of the enzyme and the
phosphorylated-oxime). The kr was calculated using the equation
from the linear portion of activity curves (0–15 min).36
2.2. In vitro reactivation screening
All compounds were assayed for their capacity to reactivate
rHuAChE and EeAChE using a 96-well format modified Ellman as-

S. B. Bharate et al. / Bioorg. Med. Chem. 18 (2010) 787–794
789
2.X
2.Br
HO N
R2
O
R1
N
N
A
N
N
N
N
OH
NH2
I
Trimedoxime (2):A = CH2, R1 = R2 = 4-CHNOH, X = Br
Obidoxime (3): A = O, R1= R2 = 4-CHNOH, X = Cl or OMs
HI-6 (4): A = O, R1 = 2-CHNOH, R2 = 4-CONH2, X = Cl or OMs
5
1
OH
N
HO
N
OH
N
N
O
O
N
N
O
N
N
N
N OH
2.Br
HO N
2.Cl
2.Cl
N
HO
6
7
8
Figure 1. Examples of mono- and bisquaternary pyridinium oximes.
R
N
N
A
N
+
a
R1
X
A
X
R2
2. X-
19. R1= R2 = 2-CHNOH, A = O, X = OSO2CH3
20. R1= R2 = 3-CHNOH, A = O, X = OSO2CH3
3. R1= R2 = 4-CHNOH, A = O, X = OSO2CH3
21. R1= R2 = 2-CHNOH, A = CH2, X = Br
22. R1= R2 = 3-CHNOH, A = CH2, X = Br
2. R1= R2 = 4-CHNOH, A = CH2, X = Br
23. R1= R2 = 2-CHNOH, A = CH2CH2, X = Br
24. R1= R2 = 3-CHNOH, A = CH2CH2, X = Br
25. R1= R2 = 4-CHNOH, A = CH2CH2, X = Br
13. A = O; X = OSO2CH3
14. A = CH2; X = Br
15. A = CH2CH2; X = Br
16. A = Thiophen-2,5-yl; X = Cl
17. A = Furan-2,5-yl; X = Br
18. A = Isoxazol-3,5-yl; X = Br
9. R = 2-CHNOH
10. R = 3-CHNOH
11. R = 4-CHNOH
12. R = 4-CONH2
26. R1= R2 = 2-CHNOH, A = Thiophen-2,5-yl; X = Cl
27. R1= R2 = 3-CHNOH, A = Thiophen-2,5-yl; X = Cl
28. R1= R2 = 4-CHNOH, A = Thiophen-2,5-yl; X = Cl
29. R1= R2 = 2-CHNOH, A = Furan-2,5-yl; X = Br
30. R1= R2 = 3-CHNOH, A = Furan-2,5-yl; X = Br
31. R1= R2 = 4-CHNOH, A = Furan-2,5-yl; X = Br
32. R1= R2 = 4-CONH2, A = Furan-2,5-yl; X = Br
33. R1= R2 = 2-CHNOH, A = Isoxazol-3,5-yl; X = Br
34. R1= R2 = 3-CHNOH, A = Isoxazol-3,5-yl; X = Br
35. R1= R2 = 4-CHNOH, A = Isoxazol-3,5-yl; X = Br
Scheme 1. Reagents and conditions: (a) DMF, 80 °C, 2–24 h, 24–89%.
O
O
O
NH2
NH2
2. X-
NH2
N
A
N
9-11
14-16
a
X
A
N
X-
R
b
39. R = 2-CHNOH; A = CH2; X = Br
40. R = 3-CHNOH; A = CH2; X = Br
41. R = 4-CHNOH; A = CH2; X = Br
N
36. A = CH2; X = Br
37. A = CH2CH2; X = Br
12
38. A = Thiophen-2,5-yl; X = Cl
42. R = 2-CHNOH; A = CH2CH2; X = Br
43. R = 3-CHNOH; A = CH2CH2; X = Br
44. R = 4-CHNOH; A = CH2CH2; X = Br
45. R = 3-CHNOH; A = Thiophen-2,5-yl; X = Cl
46. R = 4-CHNOH; A = Thiophen-2,5-yl; X = Cl
Scheme 2. Reagents and conditions: (a) MeCN, 70–80 °C, 2–3 h, 60–75%; (b) DMF, 80 °C, 2–24 h, 55–75%.
In agreement with the preliminary data (Table 1), these oximes
(26, 31, 42 and 46) showed 10–26-fold greater kr values for reacti-
vation of paraoxon-inhibited EeAChE than rHuAChE (Table 3).
Compounds 26, 31, 42 and 46 showed kr values (0.034, 0.042,
0.064 and 0.041 minꢀ1
, respectively) better than obidoxime
(0.033 minꢀ1) for EeAChE. In case of rHuAChE, 26, 31 and 42
showed kr values (0.0035, 0.0041 and 0.0045 minꢀ1, respectively)
comparable to that of pralidoxime (0.0041 minꢀ1). None of

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S. B. Bharate et al. / Bioorg. Med. Chem. 18 (2010) 787–794
Table 1
Reactivation potency of bisquaternary pyridinium oximes for paraoxon-inhibited acetylcholinesterase
AChE
% Reactivation = [(Ar ꢀ Ai)/(Ao ꢀ Ai)] ꢁ 100
EeAChE (%R SEM)
10ꢀ4 Ma
rHuAChE (%R SEM)
10ꢀ4 Ma
Reactivator
10ꢀ3 Ma
10ꢀ5 Ma 10ꢀ3 Ma
10ꢀ5 Ma
Pralidoxime (1)
Trimedoxime (2)
Obidoxime (3)
67
43
78
6
49
0
56
0
21
28
2
0
3
3
7
6
0
0
0
1
2
2
67
60
80
21
19
5
50
6
45
58
42
2
24
10
14
38
1
3
3
4
1
0
0
1
0
63
74
57
12
3
3
4
3
1
42
61
75
0
1
1
1
0
1
0
0
0
0
3
0
0
0
0
1
0
1
19
64
12
20
73
0
2
3
2
22
38
36
3
4
1
10
1
11
26
14
2
5
1
5
15
0
1
0
7
4
2
1
1
7
1
18
10
3
2
1
2
3
3
14
5
4
11
1
1
7
0
1
1
4
1
2
10
4
2
8
2
7
1
1
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
39
40
41
42
43
44
45
46
1
0
0
0
1
0
1
0
0
0
3
2
1
2
0
2
0
1
0
7
15
33
22
68
59
14
47
2
8
52
0
0
1
31
3
4
56
25
9
52
3
2
2
0
4
5
2
3
1
0
1
2
1
2
2
3
1
1
2
0
0
0
1
1
1
1
0
1
1
0
1
2
2
0
0
1
2
1
1
0
0
0
3
2
1
1
0
0
0
0
1
2
1
2
1
1
3
0
0
0
0
1
1
0
1
0
4
1
29
18
28
80
74
40
71
13
52
2
1
2
3
3
2
1
1
3
16
38
77
59
38
61
5
2
3
4
4
2
2
1
1
1
2
1
1
1
12
35
20
13
40
2
2
3
2
1
1
1
0
1
0
1
2
4
1
0
1
17
1
49
1
0
6
a
Reactivator concentration.
Table 2
Inhibition of AChE by pyridinium oximes
AChE
% Inhibition SEM
EeAChE
10ꢀ4 Ma
rHuAChE
10ꢀ4 Ma
Oxime/paraoxon
10ꢀ3 Ma
10ꢀ5 Ma
10ꢀ3 Ma
10ꢀ5 Ma
Trimedoxime (2)
Obidoxime (3)
26
28
31
46
Paraoxon
29
0
2
0
0
0
28
16
5
0
0
0
3
0
0
23
16
42
85
78
82
100
4
4
1
1
0
1
13
5
0
31
16
15
4
3
0
0
0
0
0
0
100
62
87
74
55
100
1
1
1
1
1
2
2
1
2
4
4
100
100
100
a
Oxime/paraoxon concentration.
Table 3
Reactivation rate constant (kr) values for 26, 31, 42 and 46
b
Reactivatora
kr (minꢀ1
)
EeAChE
rHuAChE
Pralidoxime (1)
Trimedoxime (2)
Obidoxime (3)
26
31
42
46
0.1766 0.0134
0.1070 0.0059
0.0330 0.0037
0.0347 0.0022
0.0423 0.0051
0.0642 0.0024
0.0417 0.0004
0.0041 0.0003
0.0182 0.0018
0.0083 0.0013
0.0035 0.0002
0.0041 0.0004
0.0045 0.0001
0.0016 0.0001
a
10ꢀ5 M oxime concentration used.
kr—First order rate constant for reactivation.
b
Figure 2. Comparison of reactivation potency at three different concentrations
(0.001–0.00001 M) for selected compounds with aliphatic linker (20, 22, 24 and 42)
and heterocyclic linkers (30, 26 and 31) at three different concentrations (0.001–
0.00001 M) for EeAChE. Three different trends [(i) 20 and 22; (ii) 24, 42 and 30; (iii)
26 and 31] of % reactivation with increasing concentration of oxime are also shown.
Bars are represented as % reactivation SEM.
compounds have kr values greater than that of trimedoxime with
either enzyme. The trend of kr values was 1 > 2 > 42 > 31 > 46 >
26 > 3 for EeAChE and 2 > 3 > 42 > 31 > 26 > 1 > 46 for rHuAChE
(Table 3).

S. B. Bharate et al. / Bioorg. Med. Chem. 18 (2010) 787–794
791
2.4. Reactivation activity of pyridinium oximes bearing
aliphatic linkers versus heterocyclic linkers
The comparison of reactivation potency of selected compounds
for EeAChE and rHuAChE at 10ꢀ5 M is depicted in Figure 4. For rHu-
AChE, among heterocyclic linked compounds, the greatest reacti-
vation was obtained with compounds 26, 31 (14% and 15% at
10ꢀ4 M) and 35 (19% at 10ꢀ3 M). Compounds 41 and 44, which
are both aliphatic chain linked compounds, displayed 64% and
73% reactivation of rHuAChE when present at 10ꢀ3 M. Compounds
26 and 31 showed 59% and 52% reactivation of EeAChE at 10ꢀ5 M,
while aliphatic chain linked compounds such as 25 (68%, 10ꢀ5 M),
41 (80%, 10ꢀ4 M), 44 (71%, 10ꢀ4 M) were even more effective. Thus,
in general, most compounds showed better reactivation of EeAChE
than of rHuAChE, with the exception of compound 39 (which
showed only 16% reactivation of EeAChE and 68% reactivation for
rHuAChE at 10ꢀ3 M).
Paraoxon-inhibited EeAChE undergoes greater than 57% reacti-
vation with obidoxime (3) from 10ꢀ3 M to 10ꢀ5 M (Table 1). Con-
versely, paraoxon-inhibited rHuAChE is barely reactivatable by
obidoxime (3) at 10ꢀ5 M (10%). This suggests that over time rHu-
AChE will eventually become unable to reactivate at all following
inhibition by paraoxon. The diethylphosphoryl hAChE adduct is
Linker structure influences the reactivation potential of bis-
pyridinium oximes. At 10ꢀ3 M, compounds bearing aliphatic link-
ers showed better reactivation potential than compounds with
heterocyclic linkers (Fig. 3). None of the compounds bearing het-
erocyclic linkers showed >17% reactivation of EeAChE (46: 17%)
at 10ꢀ3 M, while those with aliphatic chains (22, 40–42 and 44)
showed >50% reactivation of EeAChE at this concentration. At a
lower concentration (10ꢀ5 M), compounds bearing both heterocy-
clic linkers (26, 31) and aliphatic linkers (25, 41, 44) showed
>50% reactivation of EeAChE. Compounds 26, 31, 42 and 46 were
found to be faster reactivators compared with obidoxime 3 for
EeAChE. Amongst compounds with heterocyclic linkers, com-
pounds 31, a 4,40-bisoxime with a furan linker was found to be a
fast reactivator (kr 0.0423 and 0.0041 minꢀ1 for EeAChE and rHu-
AChE, respectively) for both enzymes. This rate is better than obi-
doxime 3 (kr 0.033 minꢀ1) for EeAChE and equal to pralidoxime (1)
for rHuAChE (0.0041 minꢀ1). Compound 31 is a structural analog of
obidoxime (3) differing in the linker portion by the presence of fur-
an ring in lieu of the central dimethyl ether unit. The comparison of
reactivation potency of four 4,40-bisoximes differing from each
other by linker chain is depicted in Figure 3.
known to undergo aging slowly with
a rate constant of
ka = 0.022 hꢀ1 37
,
therefore, the low reactivatibility of diethyl-rHu-
AChE adducts may be due to one or more non-reactivation pro-
cesses including denaturation that occur to a greater extent with
rHuAChE. The reason for this 10–26-fold greater rate of reactiva-
tion observed for EeAChE is not obvious from the amino acid se-
quences of rHuAChE38 and EeAChE39 that are 89% homologous.
In general, it was noted that 2,20 and 4,40-bisoximes were more
efficient reactivators of AChE than 3,30-bisoximes. When bisquater-
nary pyridinium bisoximes bearing two different types of linkers
were compared, the following differences were observed: (i)
among 2,20-bisoximes, compounds containing a heterocyclic linker
(e.g., 26) showed better reactivation activity (59%) than aliphatic
chain linked compounds for EeAChE (e.g., 21 and 23: 7% and
33%) at 10ꢀ5 M; (ii) among 4,40-bisoximes, aliphatic chain linked
compounds (2 and 25: 74% and 68% reactivation) were better than
heterocycle linked compounds (28 and 31: 47% and 52% reactiva-
tion) for EeAChE at 10ꢀ5 M. The bisquaternary pyridinium mono-
oximes 44 (aliphatic linker with 4-oxime) and 46 (thiophene linker
with 4-oxime) had similar reactivation potencies (52% and 49%
reactivation for EeAChE and 8% and 7% for rHuAChE, respectively)
at 10ꢀ5 M. For rHuAChE, compounds with aliphatic linkers had
greater reactivation activity than compounds with heterocyclic
linkers. Compounds 41 and 44, bearing aliphatic linkers, resulted
in 64% and 73% reactivation of rHuAChE at 10ꢀ3 M, while com-
pound 35 (with an isoxazole linker) had 19% reactivation at
10ꢀ3 M.
3. Experimental
3.1. General
Chemicals were purchased from Sigma–Aldrich and were used
without further purification. Electric eel AChE (EeAChE) and hu-
man recombinant AChE (rHuAChE) were purchased from Sigma.
Paraoxon was synthesized in our laboratory by reaction of p-nitro-
phenol with diethyl chlorophosphate using a method reported by
Ghanem et al.40 Para-nitrophenol and diethyl chlorophosphate
were purchased from Aldrich. 1H NMR spectra were recorded on
a Varian 400 MHz NMR spectrometer with TMS as the internal
standard. Mass spectra were obtained on a Waters LCT Premier
time-of-flight mass spectrometer and IR spectra on Nexus 670 FTIR
spectrometer. Reactivation assay readings were measured using
VERSAmax microplate reader. Melting points are uncorrected.
2.5. Reactivation of EeAChE versus rHuAChE
3.2. Synthesis of bisquaternary pyridinium bis-oximes 2, 3, 19–
31, 33–35 and bis-amide 32
Results of the reactivation studies indicate that most of the
compounds are better reactivators of EeAChE than of rHuAChE.
A mixture of pyridine aldoxime 9–11 or isonicotinamide (12)
(1 mmol) and halo-methyl compounds 13–18 (0.6 mmol) in DMF
Figure 3. Comparison of reactivation potency of 4,40-bisoximes differing by a linker
chain (aliphatic—2, 3, 25; heterocyclic—28, 32, 35) for EeAChE at 10ꢀ3 M and
10ꢀ5 M. Bars are represented as % reactivation SEM.
Figure 4. Comparison of reactivation potency for selected compounds at 10ꢀ5 M for
EeAChE and rHuAChE. Bars are represented as % reactivation SEM.

792
S. B. Bharate et al. / Bioorg. Med. Chem. 18 (2010) 787–794
was refluxed for 2–24 h. The reaction mixture was allowed to cool
to room temperature and acetone was added to precipitate the
product. The precipitate was filtered and the residue was washed
with acetone (3 ꢁ 20 mL). The solid residue was dried under vac-
uum to obtain the desired bisquaternary pyridinium compounds
2, 3 and 19–35 in 24–89% yield. Compounds 2, 3, and 19–25 were
characterized by comparison of their spectral data with literature
values.25,28,29,31
J = 6.4 Hz, 4H), 6.70 (s, 2H), 5.64 (s, 4H); 13C NMR (D2O,
100 MHz): 149.47, 147.87, 146.20, 144.54, 124.98, 114.50,
56.44; IR (Neat): mmax 3073, 3018, 2963, 2963, 2867, 2734, 1504,
1300 cmꢀ1; ESI-MS: m/z 416.97 [M]+ (calcd for [C18H18N4O3Br]+
417.06); 337.22 [M]+ (calcd for [C18H17N4O3]2+ 337.14)
d
3.2.7. 4,40-Bis(carbamoyl)-1,10-(2,5-furandiyldimethylene)-
bispyridinium dibromide (32)
Light brown solid; yield: 70%; mp 222–224 °C; 1H NMR (D2O,
400 MHz): d 8.92 (d, J = 6.8 Hz, 4H), 8.21 (J = 6.8 Hz, 4H), 6.76 (s,
2H), 5.76 (s, 4H); 13C NMR (D2O, 100 MHz): d 163.07, 149.59,
147.59, 145.60, 126.70, 115.15, 57.02; IR (Neat): mmax 3354, 3120,
3041, 2908, 2829, 1663, 1644, 1615, 1567, 1452, 1403 cmꢀ1; ESI-
MS: m/z 416.98 [M]+ (calcd for [C18H18N4O3Br]+ 417.06).
3.2.1. 2,20-Bis(hydroxyiminomethyl)-1,10-(2,5-
thiophenediyldimethylene)-bispyridinium dichloride (26)
Dark green solid; yield: 59%; mp 185–187 °C; 1H NMR (D2O,
400 MHz):
d 8.75 (d, J = 6.0 Hz, 2H), 8.55 (s, 2H), 8.39 (t,
J = 8.0 Hz, 2H), 8.20 (d, J = 8.4 Hz, 2H), 7.84 (t, J = 8.0 Hz, 2H), 7.01
(s, 2H), 5.98 (s, 4H); 13C NMR (D2O, 100 MHz): d 147.2, 146.52,
145.57, 142.25, 136.93, 130.40, 128.37, 127.88, 56.66; IR (KBr):
mmax 3295, 3070, 2951, 2816, 2721, 1508, 1145, 1023 cmꢀ1; ESI-
MS: m/z 353.17 [M]+ (calcd for [C18H17N4O2S]2+ 353.11).
3.2.8. 2,20-Bis(hydroxyiminomethyl)-1,10-(3,5-
isoxazoldiyldimethylene)-bispyridinium dibromide (33)
Brown viscous oil; yield: 24%; 1H NMR (CD3OD, 400 MHz): d
9.14 (d, J = 6.4 Hz, 1H), 9.02 (d, J = 6.3 Hz, 1H), 8.76 (s, 1H), 8.61
(m, 3H), 8.52 (t, J = 4.0 Hz, 2H), 8.10 (m, 2H), 6.94 (s, 1H), 6.34 (s,
2H), 6.24 (s, 2H); 13C NMR (CD3OD, 100 MHz): d 165.56, 158.87,
148.98, 148.78, 148.61, 146.67, 146.54, 146.44, 140.78, 140.69,
127.84, 127.59, 127.11, 126.46, 104.71, 53.82, 52.65; IR (Neat):
3.2.2. 3,30-Bis(hydroxyiminomethyl)-1,10-(2,5-
thiophenediyldimethylene)-bispyridinium dichloride (27)
Cream colored solid; yield: 68%; mp 245–247 °C; 1H NMR (D2O,
400 MHz): d 8.97 (s, 2H), 8.75 (m, 2H), 8.57 (m, 2H), 8.15 (s, 2H),
7.91 (m, 2H), 7.25 (s, 2H), 5.83 (s, 4H); 13C NMR (100 MHz, D2O):
d 144.75, 144.03, 143.12, 142.15, 136.93, 134.01, 131.98, 128.63,
mmax 3322, 3032, 2900, 2726, 1646, 1604, 1557, 1412, 1203 cmꢀ1
ESI-MS: m/z 338.04 [M]+ (calcd for [C17H16N5O3]2+ 338.12).
;
58.92; IR (Neat): mmax 3098, 3037, 2974, 2870, 2762, 1511 cmꢀ1
;
ESI-MS: m/z 388.17 (calcd for [C18H17N4O2SCl]+ 388.08); 353.12
3.2.9. 3,30-Bis(hydroxyiminomethyl)-1,10-(3,5-
isoxazoldiyldimethylene)-bispyridinium dibromide (34)
[M]+ (calcd for [C18H17N4O2S]2+ 353.11).
Brown viscous oil; yield: 24%; 1H NMR (CD3OD, 400 MHz): d 9.38
(d, J = 9.6 Hz, 2H), 9.11 (m, 2H), 8.84 (d, J = 8.4 Hz, 2H), 8.30 (d,
J = 3.6 Hz, 2H), 8.17 (m, 2H), 7.13 (s, 1H), 6.22 (s, 2H), 6.13 (s, 2H);
13C NMR (CD3OD, 100 MHz): d 165.36, 158.84, 145.03, 144.72,
143.31, 142.96, 142.81, 142.40, 135.29, 128.73, 128.50, 106.18,
56.04, 54.88; IR (Neat): mmax 3324, 3100, 2900, 2728, 1653, 1605,
1527, 1300 cmꢀ1; ESI-MS:m/z 417.79 [M]+ (calcd for [C17H17BrN5O3]+
418.05); 338.06 [M]+ (calcd for [C17H16N5O3]2+ 338.12).
3.2.3. 4,40-Bis(hydroxyiminomethyl)-1,10-(2,5-
thiophenediyldimethylene)-bispyridinium dichloride (28)
Light brown solid; yield: 65%; mp 244–246 °C; 1H NMR (D2O,
400 MHz):
J = 6.4 Hz, 4H), 7.23 (s, 2H), 5.82 (s, 4H); 13C NMR (D2O,
100 MHz): 149.46, 146.29, 144.25, 137.08, 131.80, 125.11,
d 8.71 (d, J = 6.8 Hz, 4H), 8.18 (s, 2H), 8.02 (d,
d
58.22; IR (Neat): mmax 3345, 3117, 3040, 2824, 2721, 1689, 1660,
1640, 1606, 1567, 1445, 1297, 1150 cmꢀ1; ESI-MS: m/z 388.19
(calcd for [C18H17N4O2SCl]+ 388.08); 353.11 [M]+ (calcd for
[C18H17N4O2S]2+ 353.11).
3.2.10. 4,40-Bis(hydroxyiminomethyl)-1,10-(3,5-
isoxazoldiyldimethylene)-bispyridinium dibromide (35)
Dark brown sticky mass; yield: 26%; 1H NMR (CD3OD,
400 MHz): d 9.07 (d, J = 6.4 Hz, 2H), 9.03 (d, J = 6.4 Hz, 2H), 8.66
(s, 2H), 8.37 (d, J = 5.2 Hz, 2H), 8.27 (d, J = 6.0 Hz, 2H), 7.07 (s,
1H), 6.13 (s, 2H), 6.04 (s, 2H); 13C NMR (CD3OD, 100 MHz): d
165.63, 158.97, 151.28, 151.11, 145.73, 145.65, 144.48, 124.89,
124.63, 122.40, 105.82, 55.22, 54.11; IR (Neat): mmax 3372, 3100,
2958, 2739, 1653, 1640, 1537, 1303 cmꢀ1; ESI-MS: m/z 338.04
[M]+ (calcd for [C17H16N5O3]2+ 338.12).
3.2.4. 2,20-Bis(hydroxyiminomethyl)-1,10-(2,5-
furandiyldimethylene)-bispyridinium dibromide (29)
Black solid; yield: 28%; mp 73–75 °C; 1H NMR (D2O, 400 MHz):
d 8.71 (d, J = 6.4 Hz, 2H), 8.52 (s, 2H), 8.38 (t, J = 8.0 Hz, 2H), 8.19 (d,
J = 6.8 Hz, 2H), 7.87 (dd, J = 1.6, 6.4 Hz, 2H), 6.60 (s, 2H), 5.78 (s,
4H); 13C NMR (D2O, 100 MHz,): d 150.92, 147.40, 146.48, 145.91,
141.93, 128.28, 127.17, 113.35, 54.50; IR (Neat): mmax 3421, 3107,
3031, 2967, 2829, 1660, 1638, 1601, 1458, 1419, 1389, 1288,
1151 cmꢀ1; ESI-MS: m/z 337.21 [M]+ (calcd for [C18H18N4O3]2+
337.13).
3.3. Synthesis of bisquaternary pyridinium mono-oximes 39–
46
3.2.5. 3,30-Bis(hydroxyiminomethyl)-1,10-(2,5-
A mixture of isonicotinamide (12, 1 mmol) and halo-methyl
compound 14–16 (3 mmol) in acetonitrile was stirred at 70–
80 °C for 2–3 h. The reaction mixture was cooled to room temper-
ature and the precipitated product was filtered, washed with ace-
tone (3 ꢁ 20 mL), and then dried under vacuum to produce
monoquaternary compounds 36–38 in 60–75% yield. Compounds
36–38 (1 mmol) were further treated with pyridine aldoximes 9–
11 (1.5 mmol) in DMF at 80 °C for 2–24 h. The reaction mixture
was cooled to room temperature and acetone was added to precip-
itate the product. The precipitate was filtered and the residue was
washed with acetone (3 ꢁ 20 mL). Finally, the solid residue was
dried under vacuum to obtain the desired bisquaternary pyridini-
um compounds 39–46 in 60–80% yield. Compounds 39–41 and
44 were characterized by comparison of their spectral data with
literature values.26,27,30
furandiyldimethylene)-bispyridinium dibromide (30)
Light brown solid; yield: 89%; mp 234–236 °C; yield: 54%; 1H
NMR (D2O, 400 MHz): d 8.91 (s, 2H), 8.72 (d, J = 6.0 Hz, 2H), 8.55
(d, J = 8.4 Hz, 2H), 8.13 (s, 2H), 7.91 (t, J = 6.0 Hz, 2H), 6.72 (s,
2H), 5.70 (s, 4H); 13C NMR (D2O, 100 MHz): d 147.70, 144.65,
144.35, 143.22, 142.28, 133.89, 128.60, 114.77, 57.11; IR (Neat):
mmax 3073, 3018, 2964, 2867, 2773, 2733, 1505, 1300 cmꢀ1; ESI-
MS: m/z 416.97 [M]+ (calcd for [C18H18BrN4O3]+ 417.06); 337.23
[M]+ (calcd for [C18H17N4O3]2+ 337.13).
3.2.6. 4,40-Bis(hydroxyiminomethyl)-1,10-(2,5-furandiyl
dimethylene)-bispyridinium dibromide (31)
Light brown solid; yield: 61%; mp 163–166 °C; 1H NMR (D2O,
400 MHz):
d 8.67 (d, J = 6.4 Hz, 4H), 8.16 (s, 2H), 8.01 (d,

S. B. Bharate et al. / Bioorg. Med. Chem. 18 (2010) 787–794
793
3.3.1. 2-(Hydroxyiminomethyl)-40-(carbamoyl)-1,10-(1,4-
butylene)-bispyridinium dibromide (42)
The % reactivation for each reactivator is reported as an average
with n P 3:
Cream colored solid; yield: 55%; mp 196–198 °C; 1H NMR
(CD3OD, 400 MHz): d 9.19 (d, J = 6.0 Hz, 2H), 9.00 (d, J = 6.4 Hz,
1H), 8.73 (d, J = 6.8 Hz, 2H), 8.52–8.43 (m, 2H), 8.02 (t, J = 8.0 Hz,
1H), 7.85 (d, J = 6.0 Hz, 1H), 4.84–4.75 (m, 4H), 2.24–2.18 (m,
2H), 2.11–2.06 (m, 2H); 13C NMR (CD3OD, 100 MHz): d 166.82,
149.11. 145.80, 145.42, 145.01, 143.02, 134.03, 128.41, 126.84,
58.22, 55.21, 32.12; IR (Neat): mmax 3354, 3120, 3041, 2908, 2829,
1663, 1644, 1615, 1567, 1452, 1403 cmꢀ1; ESI-MS: m/z 379.06
[M]+ (calcd for [C16H19N4O2Br]+ 379.08); 299.13 [M]+ (calcd for
[C16H19N4O2]2+ 299.15).
% reactivation ¼ ½ðAr ꢀ AiÞ=ðAo ꢀ AiÞꢂ ꢁ 100
A control experiment (without inhibitor) was performed to
determine the AChE inhibition ability of oximes at different con-
centrations (1–0.01 mM). Paraoxon was used as reference inhibi-
tor. The % AChE inhibition for each oxime is reported as an
average with n P 3.
3.5. Determination of the reactivation rate constant (kr)
Reactivation rate constants (kr) were determined for com-
pounds 1, 2, 3, 26, 31, 42 and 46 using a similar experimental pro-
tocol to that indicated for the in vitro reactivation screening except
using different reactivation time period (0–15 min). A 10ꢀ5 M
oxime concentration was used in this experiment. By plotting
ln(reactivation % ꢀ age) versus reactivation time (t), kr is presented
as the negative slope of the plot according to the equation given
below.36 Each experiment was repeated with n P 3. Plots with R2
>0.9
3.3.2. 3-(Hydroxyiminomethyl)-40-(carbamoyl)-1,10-(1,4-
butylene)-bispyridinium dibromide (43)
White solid; yield: 75%; mp 238–240 °C; 1H NMR (CD3OD,
400 MHz):
d 9.24 (s, 1H), 9.22 (d, J = 6.0 Hz, 2H), 9.01 (d,
J = 6.4 Hz, 1H), 8.78 (d, J = 6.5 Hz, 1H), 8.64 (d, J = 6.8 Hz, 2H),
8.30 (s, 1H), 8.12 (t, J = 8.0 Hz, 1H), 4.00 (m, 4H), 2.22 (m, 4H);
13C NMR (D2O, 100 MHz): d 166.83, 148.81, 145.70, 144.91,
144.42, 142.78, 142.52, 133.92, 128.63, 126.73, 61.27, 27.45,
27.37; IR (Neat): mmax 3361, 3114, 3054, 2900, 2823, 1655, 1634,
1610, 1563, 1444, 1200 cmꢀ1; ESI-MS: m/z 378.99 [M]+ (calcd for
[C16H19N4O2Br]+ 379.08); 299.10 [M]+ (calcd for [C16H19N4O2]2+
299.15).
ꢀ
ꢁ
Ao ꢀ At
2:3 log 100 ꢃ
¼ kr ꢃ t
Ao ꢀ A0
o
were chosen for kr calculations.
3.3.3. 3-(Hydroxyiminomethyl)-40-(carbamoyl)-1,10-(2,5-
thiophenediyldimethylene)-bispyridinium dichloride (45)
Cream colored solid; yield: 60%; mp 220–222 °C; 1H NMR
(CD3OD, 400 MHz,): d 9.30 (s, 1H), 9.22 (d, J = 6.0 Hz, 2H), 9.01 (d,
J = 6.8 Hz, 1H), 8.78 (d, J = 6.0 Hz, 1H), 8.42 (d, J = 6.0 Hz, 2H),
9.26 (s, 1H), 8.11 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 2.0 Hz, 2H), 6.18
(s, 2H), 6.10 (s, 2H); 13C NMR (CD3OD, 100 MHz): d 149.88,
145.54, 143.80, 142.43, 142.10, 137.70, 137.60, 131.93, 131.87,
131.69, 128.56, 126.59, 58.61, 58.45; IR (Neat): mmax 3400, 3071,
2951, 2810, 2721, 1574, 1508, 1145, 1022 cmꢀ1; ESI-MS: m/z
353.11 [M]+ (calcd for [C18H17N4O2S]2+ 353.11).
4. Conclusion
The results demonstrate key differences in the reactivation pro-
file of paraoxon-inhibited AChE depending on the type of oxime,
concentration of oxime and species. Oximes bearing aliphatic lin-
ker were better reactivators at higher concentration (10ꢀ3 M) com-
pared with oximes bearing heterocyclic linkers while at a lower
concentration (10ꢀ5 M), both class of oximes exhibited comparable
reactivation potency. Another important finding from this study is
that most of the oximes are better reactivators of EeAChE than of
rHuAChE which may be due to one or more non-reactivation pro-
cesses including denaturation or aging that occur to a greater ex-
tent with rHuAChE. Amongst newly synthesized oximes,
thiophene-containing analogs 26 and 46 and furan containing ana-
log 31 were the better reactivators with 59%, 52% and 49% reacti-
vation of EeAChE at 10ꢀ5 M and 14%, 15% and 6% reactivation of
rHuAChE at 10ꢀ4 M. Compounds 26, 31, 42 also showed a higher
kr value than obidoxime (3) for paraoxon-inhibited EeAChE and
equal to pralidoxime (1) for paraoxon-inhibited rHuAChE.
Although the newly synthesized compounds showed slower reac-
tivation rate, interesting structure–activity dependence was found.
Introduction of a highly electron rich heterocycle in linker resulted
in oximes that were most effective at lower concentrations. Among
these, compounds bearing a thiophene and furan linker exhibited
better reactivation compared to compounds with the isoxazole
ring.
3.3.4. 4-(Hydroxyiminomethyl)-40-(carbamoyl)-1,10-(2,5-
thiophenediyl dimethylene)-bispyridinium dichloride (46)
Light yellow solid; yield: 64%; mp 214–216 °C; 1H NMR (D2O,
400 MHz): d 8.92 (d, J = 5.2 Hz, 2H), 8.71 (d, J = 6.8 Hz, 2H), 8.20
(d, J = 6.0 Hz, 2H), 8.18 (s, 1H), 8.03 (d, J = 6.8 Hz, 2H), 7.27 (m,
2H), 5.94 (s, 2H), 5.83 (s, 2H); 13C NMR (D2O, 100 MHz): d
146.26, 145.27, 144.25, 137.41, 136.38, 132.42, 132.27, 131.83,
126.70, 125.09, 58.84, 58.19; IR (Neat): mmax 3070, 2949, 2909,
2816, 2727, 1508, 1145, 1022 cmꢀ1; ESI-MS: m/z 388.90 [M]+
(calcd for [C18H17N4O2SCl]+ 389.08); 353.01 [M]+ (calcd for
[C18H17N4O2S]2+ 353.11).
3.4. In vitro reactivation screening
An AChE stock solution (0.2 mg/mL in 0.1 M sodium phosphate
buffer, pH 7.2) was treated with ethanol (1% v/v) and paraoxon (1%
Acknowledgments
v/v of 100 lM in ethanol; final concentration of paraoxon is 1 lM)
as control and experiment vessels. After 20–50 min, 90% AChE
This research was supported by NIH UO1-ES016102 (C.M.T.)
and SBIR grants to ATERIS Technologies LLC (R43 ES016392 and
U44 NS058229). Support from the Core Laboratory for Neuromo-
lecular Production (NIH P30-NS055022) and the Center for Struc-
tural and Functional Neuroscience (NIH P20-RR015583) is
appreciated.
Opinions, interpretations, conclusions, and recommendations are
those of the authors and are not necessarily endorsed by the U.S. Army
or the Department of Defense.
inhibition was achieved and halted with a 32-fold dilution (PBS).
A 16
to 20
l
l
L aliquot from control and experiment vessels was diluted
L with PBS, and incubated for 30 min at 25 °C as activity
control and inhibition control. The initial activity (A0) or inhibition
(Ai) was analyzed by adding DTNB (final concentration of 0.3 mM,
200
l
L total volume) and ATChI (final concentration of 1 mM,
L total volume). Oxime reactivator (4 L, 1–0.01 mM final
L aliquot and after 30 min incubation,
the reactivated activity was determined by Ellman assay as Ar.
200
l
l
conc) was added to a 16
l

794
S. B. Bharate et al. / Bioorg. Med. Chem. 18 (2010) 787–794
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