Synthesis and in vitro kinetic study of novel mono-pyridinium oximes as reactivators of organophosphorus (OP) inhibited human acetylcholinesterase (hAChE)

Article abstract of DOI:10.1016/j.cbi.2015.06.007

A series of mono pyridinium oximes linked with arenylacetamides as side chains were synthesized and their in vitro reactivation potential was evaluated against human acetylcholinesterase (hAChE) inhibited by organophosphorus inhibitors (OP) such as sarin, VX and tabun. The reactivation data of the synthesized compounds were compared with those obtained with standard reactivators such as 2-PAM and obidoxime. The dissociation constant (K_D) and specific reactivity (k_r) of the oximes were also determined by performing reactivation kinetics against OP inhibited hAChE. Among the synthesized compounds, oximes 1-(2-(4-cyanophenylamino)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium chloride (12a) and 4-((hydroxyimino)methyl)-1-(2-(4-methoxyphenylamino)-2-oxoethyl)pyridinium chloride (2a) were found most potent reactivators for hAChE inhibited by sarin. In case of VX inhibited hAChE majority of the oximes have shown good reactivation efficacies. Among these oximes 1-(2-(benzylamino)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium chloride (18a), 4-((hydroxyimino)methyl)-1-(2-(4-(methoxycarbonyl)phenylamino)-2-oxoethyl)pyridinium-chloride (14a) and 12a were found to surpass the reactivation potential of 2-PAM and obidoxime. However, the synthesized oximes showed marginal reactivation efficacies in case of tabun inhibited hAChE. The pKa value of the oximes were determined and correlated with their observed reactivation potential.

Full text of DOI:10.1016/j.cbi.2015.06.007

Chemico-Biological Interactions 237 (2015) 125–132
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Synthesis and in vitro kinetic study of novel mono-pyridinium
oximes as reactivators of organophosphorus (OP) inhibited
human acetylcholinesterase (hAChE)
a
a
a,
⇑
a
a
Aditya Kapil Valiveti , Uma M. Bhalerao , Jyotiranjan Acharya , Hitendra N. Karade , Raviraju Gundapu ,
b
a
Anand K. Halve , Mahabir Parshad Kaushik
a
Process Technology Development Division, Defence Research & Development Establishment, Jhansi road, Gwalior 474 002, India
School of Studies in Chemistry, Jiwaji University, Gwalior, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of mono pyridinium oximes linked with arenylacetamides as side chains were synthesized and
their in vitro reactivation potential was evaluated against human acetylcholinesterase (hAChE) inhibited
by organophosphorus inhibitors (OP) such as sarin, VX and tabun. The reactivation data of the synthe-
sized compounds were compared with those obtained with standard reactivators such as 2-PAM and
Received 12 March 2015
Received in revised form 26 May 2015
Accepted 3 June 2015
Available online 10 June 2015
D r
obidoxime. The dissociation constant (K ) and specific reactivity (k ) of the oximes were also determined
by performing reactivation kinetics against OP inhibited hAChE. Among the synthesized compounds,
oximes 1-(2-(4-cyanophenylamino)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium chloride (12a)
and 4-((hydroxyimino)methyl)-1-(2-(4-methoxyphenylamino)-2-oxoethyl)pyridinium chloride (2a)
were found most potent reactivators for hAChE inhibited by sarin. In case of VX inhibited hAChE majority
of the oximes have shown good reactivation efficacies. Among these oximes 1-(2-(benzylamino)-2-oxoe
thyl)-4-((hydroxyimino)methyl)pyridinium chloride (18a), 4-((hydroxyimino)methyl)-1-(2-(4-(methoxy
carbonyl)phenylamino)-2-oxoethyl)pyridinium-chloride (14a) and 12a were found to surpass the
reactivation potential of 2-PAM and obidoxime. However, the synthesized oximes showed marginal
reactivation efficacies in case of tabun inhibited hAChE. The pKa value of the oximes were determined
and correlated with their observed reactivation potential.
Keywords:
Acetylcholinesterase
Nerve agents
Tabun
Reactivation
Inhibition
Oxime
Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1
. Introduction
Poisoning by organophosphorus (OP) nerve agents viz. VX, sarin
developing countries around the globe [4]. Despite serious and
continued efforts to prevent synthesis, storage and use of these
compounds by the Chemical Weapons Convention (CWC) [5],
repeated use of chemical warfare agents during military conflicts
and terrorist attacks indicate that they constitute a persistent
threat for the civilization [6].
The OP compounds (Fig. 1A) exert their toxicity by inhibiting
the activity of the enzyme acetylcholinesterase (AChE), an enzyme
responsible for hydrolysis of neurotransmitter acetylcholine (ACh).
This lead to the accumulation of endogenous ACh thereby trigger-
ing a variety of clinical manifestations in the autonomic nervous
system (both central and peripheral nervous system) and finally
leading to death due to respiratory failure [7].
Current medical protection against the toxicity of OP poisoning
consists of a regimen of anti-cholinergic drugs, such as atropine to
counteract the accumulation of acetylcholine, an anti convulsant
e.g. diazepam to reduce the CNS related symptoms and an oxime
reactivator to reactivate OP-inhibited AChE [8]. Quaternary pyri-
dinium oximes such as 2-pyridinealdoxime methochloride
and tabun (Fig. 1A) and pesticides pose serious life-threatening
situations to the mankind [1]. The ready availability of the raw
materials, ease in their preparation and extreme toxicities of these
toxicants made them as chemical weapons of mass destruction [2].
The use of nerve agents on the civilian population was witnessed
by several instances in the history viz. Iran–Iraq war (1980–
1
988), sarin attack in Tokyo subway (1995) and recent sarin gas
attack at Damascus in Syrian civil war (August 2013) [3]. All these
incidents produced severe casualties all over the world. In addition,
approximately 300,000 deaths have been recorded annually
because of intentional (suicidal) and unintentional (occupational)
means of poisoning by OP pesticides and insecticides in the
⇑
Corresponding author. Tel.: +91 751 2390205, +91 751 2340245; fax: +91 751
341148.
E-mail addresses: jracharya@rediffmail.com, j.acharya@drde.drdo.in (J. Acharya).
2
http://dx.doi.org/10.1016/j.cbi.2015.06.007
009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
0

1
26
A.K. Valiveti et al. / Chemico-Biological Interactions 237 (2015) 125–132
Fig. 1. (A) Structures of organophosphorus nerve agents; (B) structures of oxime reactivators.
(
2-PAM), trimedoxime (TMB-4), obidoxime and HI-6 (Fig. 1B) are
currently used as reactivators in the treatment of OP poisoning
9]. Though, these oximes have proven their efficacies against OP
(dichloromethane, acetonitrile, acetone, and methanol) were pur-
chased from S.D. Fine Chemicals (India) and dried and distilled
before use. Sarin, tabun and VX were prepared in house with
[
3
1
nerve agent poisoning, however there are certain limitations which
constraint their scope in shaping them as universal antidote.
2
>98% purity (GC and P NMR). 2-PAM was prepared according to
the method of Wilson and Ginsburg [20]. Obidoxime was synthe-
sized using reported methods [21]. The synthesized compounds
-PAM is being used as an effective drug in many countries against
1
13
OP poisoning, however its efficacy is limited to sarin and VX inhib-
ited AChE and has marginal efficacy in case of other nerve agents.
In view of the above, research is being continued for a far more
effective reactivator that can be used as an antidote against a broad
spectrum of nerve agents [10,11].
Recently several studies on bis- and mono-quaternary oximes
connected with various bridging chains (prop-1,3-diyl, xylene link-
ers, aliphatic and heteroaromatic linkers) and side chain (benzyl,
heterocyclic and functionalized aliphatic moieties) were reported
for their efficacies against OP inhibited AChE [12–17]. Few of these
reactivators have shown promising reactivation efficacies against
specific OP inhibited AChE. Therefore further modifications in the
structural features of AChE reactivator are highly essential in order
to establish broad spectrum antidote in OP poisoning.
as well as standards were characterized by their IR, H NMR,
C
NMR spectral and elemental analysis data (Supplementary infor-
mation). The progress of reaction and purity of the compounds
were checked by thin layer chromatography (TLC) using commer-
cially available pre-coated silica on aluminum sheets purchased
from E. Merck, India.
2.2. Synthesis of oximes
A two step simple (Scheme 1) protocol was used to synthesize
the mono-pyridinium oximes (1a–22a).
2.2.1. Step 1: synthesis of 2-chloro-N-(4-nitrophenyl)acetamide
A suspension of 4-nitroaniline (7 g, 0.051 mol) and anhydrous
Bis-pyridinium oximes and their analogs have been widely
studied against OP poisoning, however 2-PAM has been used
worldwide [18]. Moreover, various studies have proved that the
diffusion rate of monoquaternary oximes into the brain is higher
in comparison to their bis-analogs [19]. Therefore, in continuation
to our work on antidotes against nerve agents, herein we report the
synthesis and in vitro evaluation of a series of mono-pyridinium
oximes (connected to aromatic and aliphatic acetamide side
chains) as reactivators of three different OP nerve agents (sarin,
VX and tabun) inhibited hAChE.
potassium carbonate (7 g, 0.051 mol) in dichloromethane (30 mL)
was stirred for 30 min at room temperature. The reaction mixture
was cooled on an ice bath. To this was added an ice-cooled solution
of chloroacetylchloride (5.7 g, 0.051 mol) in dichloromethane
(
20 mL) drop wise over a period of 30 min. The reaction mixture
was stirred overnight at room temperature followed by reflux for
additional 30 min. Excess solvent was removed and the residue
was neutralized with aqueous sodium bicarbonate solution (5%
w/v). The product obtained was filtered off and washed thoroughly
with cold water. The crude product obtained was dried under vac-
f
uum (10 g, yield: 92%). TLC (chloroform, R = 0.5). The product was
2
. Materials and methods
sufficiently pure and used in the next step directly.
2.1. Materials
2.2.2. Step 2: synthesis of 4-((hydroxyimino)methyl)-1-(2-(4-nitro-
phenylamino)-2-oxoethyl)pyridinium chloride
Substituted aromatic amines, benzylamine, cyclohexylamine,
4-Pyridinealdoxime (1.0 g, 0.0082 mol) dissolved in dry ace-
tonitrile (30 mL) taken in a two neck round bottom flask equipped
with a magnetic stirrer, condenser and calcium chloride guard tube
was stirred at room temperature. 2-chloro-N-(4-nitrophenyl)
acetamide (1.76 g, 0.0082 mol) dissolved in dry acetonitrile
(20 mL) was added to the reaction mixture slowly over a period
of 10 min. The reaction mixture was then stirred for 3 h at room
temperature followed by reflux for another 3 h. The product
obtained was filtered off, washed with dry hot acetonitrile
(2 ꢀ 15 mL) followed by dry hot acetone. Finally, the crude product
was dried (0.52 g, yield 19%) and recrystallized from
c
-aminobutyric acid, 6-aminocaproic acid, propargylamine, 2-, 3-
0
and 4-pyridinealdoxime, acetylthiocholineiodide (ATChI), 5,5 -dit
hiobis-(2-nitrobenzoic acid) (DTNB), isopropanol (spectroscopic
grade), potassium dihydrogenphosphate, dipotassium hydrogen
phosphate, trizma-base and trizma-HCl were purchased from
Sigma–Aldrich, USA and used without further purification.
Glycine was obtained from E. Merck (India) and used without
further purification. Chloroacetylchloride, anhydrous sodium
sulfate, anhydrous sodium bicarbonate and anhydrous potassium
carbonate were purchased from Qualigens, India. Solvents

A.K. Valiveti et al. / Chemico-Biological Interactions 237 (2015) 125–132
127
Scheme 1. Synthetic route for the preparation of mono-pyridinium oximes (1a–23a).
mixture. ATChI (0.48 mM, in distilled water) as substrate and
DTNB as the colorimetric indicator (0.3 mM in 0.1 M phosphate
buffer, pH = 7.4) were used to assay the activity. Oxime stock solu-
tions and their dilutions were prepared freshly in triple distilled
water. All the working solutions were kept on an ice bath until
completion of the experiment. All experiments were performed
at 37 °C in phosphate buffer at pH 7.4. All concentrations in the
above assay mixture refer to the final concentrations.
The stock solutions of OP inhibitors (sarin, VX and tabun) were
prepared in isopropanol once in a week and were stored at ꢁ20 °C.
The subsequent dilutions of OP inhibitors were freshly prepared
every day in deionized water prior to start of the experiment.
The enzyme hAChE taken in phosphate buffer (pH 7.4, 0.1 M)
was inhibited by appropriate OP concentrations at 37 °C around
Table 1
Physicochemical data and chemical structure of the synthesized oximes.
Oxime Oxime position
X
–R
Cl Ph-4-CH
Cl Ph-4-OCH
Yield (%) MP (°C)
pKa
1a
2a
3a
4a
5a
6a
7a
8a
4
4
4
4
4
4
4
4
2
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3
40
43
19
57
50
58
32
38
15
5
19
51
16
36
14
28
41
35
50
76
14
35
22
11
231–233 8.33
216–218 8.31
242–244 8.22
258–260 8.49
260–262 8.21
228–230 8.32
207–209 8.13
192–194 8.29
202–204 8.08
244–246 8.34
219–221 8.23
235–237 8.28
239–241 8.26
250–252 8.19
222–224 8.15
224–226 7.87
219–221 8.25
241–243 8.37
145–147 8.27
188–190 8.43
3
Cl Ph-4-NO
Cl Ph-3-NO
Cl Ph-2-NO
Cl Ph-4-Cl
Cl Ph-3-Cl
Cl Ph-4-H
2
2
2
8
b
I
Ph-4-H
9
a
Cl Ph-4-COOH
Cl Ph-3-COOH
Cl Ph-4-F
Cl Ph-4-CN
Cl Ph-3-CN
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
1
0–15 min to achieve 95–98% inhibition of the control activity.
Cl Ph-4-COOCH
3
The residual inhibitor was removed by the concomitant extraction
with six times excess volume of n-hexane [27–29].
The OP-inhibited hAChE was reactivated by the addition of
oxime reactivator at a final concentration ranging from 10 to
Cl Ph-4-COOC
Cl Ph-4-COCH
Cl Ph-4-CF
Cl CH
Cl
Cl CH
Cl (CH
Cl (CH
Cl CH
2 5
H
3
3
2
-Ph-4-H
C
6
H
11
1000
volume of cocktail: 600
oxime reactivator) at different time intervals (1, 3, 5, 7, 10, 15,
0, 25, 30, 40, 50 and 60 min) was transferred to the cuvette con-
taining 3.0 mL of phosphate buffer and 100 L of DTNB. The AChE
activity was assayed by adding 20 L of ATChI at 37 °C (final vol-
l
M. An aliquot of 50
lL from the reactivation cocktail (total
2
-CONH
2
–
–
lL, containing inhibition cocktail and
2
)
)
4
-COOH
-COOH
194–196 8.42
180–182 8.39
2
5
2
2
C„H
–
8.11
l
l
ume 3.17 mL). The oxime induced reactivation of the
OP-inhibited AChE was monitored at different time intervals over
a period of 60 min. Spontaneous reactivation of inhibited AChE
was assayed using the same protocol (the reaction mixture con-
taining enzyme and OP but no oxime). Under these conditions
spontaneous reactivation was found to be insignificant. All the val-
ues were corrected for their oxime induced hydrolysis of ATChI.
methanol-acetone mixture. TLC (acetone/methanol (2:1), R
m.p: 235–237 °C.
f
= 0.10),
The physicochemical parameters of the synthesized oximes
(1a–23a) were determined and depicted in Table 1. Elemental
analyses were conducted on an ELEMENTAR, vario MICRO cube,
Universal micro analyzer and the values were within ±0.4% of the
calculated values. Infra-red (IR) spectra were obtained from KBr
1
discs on a Bruker TENSOR-27 FTIR spectrophotometer. H NMR
2.4. Reactivation kinetics
(
6
DMSO-d ) spectra for the synthesized oximes were recorded on
Bruker Avance 400 spectrometer at 400 MHz using tetramethylsi-
The oxime assisted reactivation of the OP-inhibited AChE pro-
ceeds according to the Scheme 2.
lane (TMS) as internal standard and expressed in the d (ppm) val-
1
3
ues. C NMR (DMSO-d
6
) was recorded using same instrument at
Where [EP] is the phosphylated enzyme, [OX] is the reactivator,
1
00 MHz (Supplementary information).
[
EPOX] is the Michaelis-type complex between phosphylated-
AChE and oxime reactivator, [E] is the reactivated enzyme, [POX]
is the phosphylated oxime, K is the dissociation constant which
is inversely proportional to the affinity of reactivator towards the
phosphylated enzyme [EP], k is the rate constant for the displace-
2.3. Enzyme assay, inhibition and reactivation
D
Human blood samples were collected from the volunteers as
r
per norms. The hemoglobin free erythrocyte ghost AChE was pre-
pared using the reported method [22] and stored at ꢁ80 °C until
use. AChE activities were measured at 412 nm by using
UV–Visible spectrophotometer (Cary 100, Agilent Technologies,
USA) assisted with PCB 1500 water peltier system with a modified
Ellman protocol [23,24]. Prior to start the experiments, aliquots of
the erythrocyte ghosts were homogenized on an ice-bath with the
help of an ultrasonic homogenizer (Model 3000, Biologics Inc.,
Manassas, USA), three times for 5 s with 30 s intervals to achieve
homogenous matrix for the kinetic experiments [25,26]. Quartz
cuvettes of 3 mL were used to measure the activity of the assay
ment of phosphyl residue from [EPOX] by oxime and it expresses
the reactivation efficiency of the reactivator.
Scheme 2. Oxime assisted reactivation of OP-inhibited AChE.

1
28
A.K. Valiveti et al. / Chemico-Biological Interactions 237 (2015) 125–132
In case of complete reactivation and with [OX] ꢂ [EP]
o
a pseudo
(15–35 lL) of oximes were diluted to 3 mL in each buffer and opti-
first-order rate equation can be derived for the reactivation process
as represented in Eq. (1)
cal densities were determined at analytical wave lengths using
buffer blank at 25 ± 1.0 °C. A set of ten values of pKa were obtained
using the following Eq. (3):
k
r
½OXꢃ
k
obs ¼ K
ð1Þ
þ ½OXꢃ
pKa ¼ pH þ log½ðD
where, D and D corresponds to the optical density of protonated
i
ꢁ DÞ=ðD ꢁ D
m
Þꢃ
ð3Þ
D
where kobs is the observed first-order rate constant of the reactiva-
tion at any given oxime concentration and it was calculated by
non-linear regression analysis [24,25] using Eq. (2)
m
i
and deprotonated forms of the oxime, and D is the optical density
in the buffer. The average value of the ten measurements was con-
sidered the pKa of the compound with respect to oximino function-
ality. UV–Visible spectrophotometer assisted with PCB 1500 water
peltier system was used for spectrophotometric analysis. The tem-
perature of the peltier system was adjusted at 25 ± 1 °C. The pH of
the buffer solutions were determined by using Eutech 1500
Cyberscan pH meter. The pH meter was calibrated at 25 °C with
standard buffer solutions pH 7.00 and 9.21.
k
obs
m
t
¼
m
0
ꢀ ð1 ꢁ eꢁ ꢀtÞ
ð2Þ
k
r
and K
between kobs versus [OX]. Both the rate constants k
Michaelis–Menten type kinetics and the second order reactivation
rate constant kr2 was obtained from the ratio of k and K
D
were obtained by the non-linear fit of the relationship
r
and K follow
D
r
D
.
The determined kinetic parameters and rate constants of the
oximes were presented in the Table 2.
2
.6.1. Reagents
ꢁ
3
Oxime stock solutions (5 ꢀ 10 M) were freshly prepared in
2
.5. Data analysis
triple distilled water. Buffer solutions were prepared as per
reported protocol [31]. The analytical wavelengths for the proto-
nated and deprotonated species were obtained by using 0.1 M
hydrochloric acid and 0.1 M sodium hydroxide solutions
respectively.
The kinetic rate constants were determined by processing the
experimental data with non-linear regression analysis using curve
fitting programs provided by Prism™ Vers. 6.0 (Graph Pad soft-
ware, San Diego, USA).
2.6. Determination of acid dissociation constant (pKa)
3. Results and discussion
The acid dissociation constants (pKa) of the oximes were deter-
All the newly synthesized oximes were subjected to evaluation
of their reactivation efficacies against three structurally different
nerve agents (sarin, VX and tabun) inhibited hAChE. Depending
on the structural features of nerve agent and the oxime reactiva-
tors used in this study, significant differences were observed in
mined using the method of Albert and Sergeant (1971) [30]. The
method is based on direct determination of the ratio of molecular
species (protonated) to dissociated (deprotonated) species in a ser-
ies of non-absorbing buffer solutions. For this purpose, the spectra
of molecular species were obtained first in buffer solution of par-
ticular pH in which compounds of interest would be present
the reactivation efficacies and kinetic rate constants (k
r D
, K and
kr2) of the oximes. The nerve agents used in this study have suffi-
entirely in either form. Oxime stock solutions (15–35
l
L,
cient aging time and hence the oxime induced reactivation was
carried out for the reactivated enzyme but not for the aged one.
The affinity of an oxime toward the OP-AChE adduct has been
ꢁ3
5
0
ꢀ 10 M) were diluted to 3 mL in a cuvette containing either
.1 M hydrochloric acid or 0.1 M sodium hydroxide solution and
the absorption spectra of oxime in acid or alkali were recorded
over the wavelength range of 200–600 nm with a reference blank
solution at 25 ± 1.0 °C. The spectra thus obtained in acid or alkali
interpreted by their respective K
ations in the K values were observed among the tested oximes.
Majority of the oximes showed higher affinity (lower K ) toward
sarin and VX inhibited AChE. On the other hand, lower affinities
(higher K ) were observed in case of the reactivation of tabun
inhibited hAChE which were further characterized by their lower
r2 values. The reactivation efficacy of an oxime reactivator was
assessed by calculating the reactivity rate constant k . The respec-
tive k values for the oximes were presented in Table 2. It was
D
(Section 2.4) values. Large vari-
D
D
were of protonated (D
m i
) and deprotonated (D ) molecules
(Fig. 2). Ten different pH values, ranging from 5.94 to 10.03 were
D
selected to determine the pKa of oximes. For this, appropriate buf-
fers consisting of phosphate (pH: 5.94–7.60), tris (pH: 8.00–8.60)
and glycine–NaOH (pH: 9.06–10.03) were used. Aqueous solutions
k
r
r
observed that the efficacy of the oximes to reactivate sarin and
VX inhibited hAChE was significantly higher than that of tabun
r D
inhibited hAChE. Both the rate constants k (reactivity) and K
(
affinity) follow Michaelis–Menten type kinetics. The quantifica-
tion of specific reactivity of an oxime was carried out by calculating
its second order reactivation rate constant (kr2) which depend on
its k and K values (Table 2).
r D
3.1. Reactivation of sarin inhibited hAChE
Moderate results were obtained in case of sarin inhibited
hAChE. Few of the tested oximes have shown better reactivation
efficacies than those of the standard oximes as evidenced by their
higher second order rate constants. Among the studied oximes,
1
2a, 2a, 22a, 9a and 8b have shown higher affinities (K
D
)
4
.51 M; 6.62 M; 8.83 M; 9.24 M; 13.27 M respectively for
l
l
l
l
l
the phosphylated complex of sarin inhibited AChE. This was fur-
ther reflected by their higher second order rate constants
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
Fig. 2. Spectrophotometric determination of pKa of the oxime 12a.
(kr2) 10.41 mM min ; 9.92 mM min ; 6.90 mM min ;

A.K. Valiveti et al. / Chemico-Biological Interactions 237 (2015) 125–132
129
Fig. 3. Reactivation profile of oxime 18a against sarin (a), VX inhibited hAChE (c); plot of kobs vs. [18a] (mM) for sarin (b), VX inhibited hAChE (d).
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
6
.67 mM min ; 6.73 mM min respectively. The time depen-
dent reactivation profile and kobs for the oxime 18a was illustrated
in Fig. 3a and b. The lower K values of the oximes represent their
greater affinity and better reactivity toward sarin-hAChE complex
&
k
r2
:
8.61 mM min
)
and 12a (K
7.41 mM min ) have shown significantly higher reactivation
efficacies than that of the standard 2-PAM (K : 26.59 M & kr2
2.73 mM min ) and comparable reactivity with obidoxime
D r2
: 10.18 lM & k :
ꢁ1
ꢁ1
D
D
l
:
ꢁ1
ꢁ1
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
in the active site of the gorge. Oximes 18a (6.14 mM min ),
D
(K : 14.4 lM & kr2: 9.03 mM min ). Oximes 2a, 6a, 8a, 8b,
ꢁ
ꢁ1
1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
1
(
7a
4.39 mM min ) and 11a (4.28 mM min ) have also shown
higher reactivity than that of the standard 2-PAM
(4.70 mM min ),
23a
(4.59 mM
min ),
13a
15a, 16a, 19a and 23a have shown higher reactivation efficacies
than that of the standard 2-PAM as represented by their higher
second order rate constant values (Table 2). However oxime 1a,
9a, 11a, 17a and 22a have shown nominal reactivation efficacies
against VX inhibited hAChE. These oximes also recorded signifi-
cantly lower reactivation efficacies than that of obidoxime.
Among all the synthesized compounds, oximes 21a and 5a dis-
played larger K (111.6 and 87.5 lM) and lower second order rate
D
constant (kr2) (0.52 mM min and 0.58 mM min ) inferring
their least affinity and reactivity towards the VX-hAChE complex.
ꢁ
1
ꢁ1
ꢁ
1
ꢁ1
1
(
4.07 mM min ) and comparable reactivity with that of obidox-
ꢁ
ꢁ1
ime (6.39 mM min ). Further, oximes 1a, 5a, 6a, 14a, 15a and
9a showed comparable reactivity to those of 2-PAM and obidox-
ime (Table 2). Oximes 4a (72.57 M) and 3a (41.41 M) have dis-
played larger K values demonstrating their least reactivity toward
the phosphylated complex of sarin-inhibited hAChE and thereby
resulted in lower second order rate constants
1
l
l
D
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
r2
k ;
ꢁ1
ꢁ1
ꢁ1
ꢁ1
1
.06 mM min and 1.57 mM min respectively.
3.3. Reactivation of tabun inhibited hAChE
3.2. Reactivation of VX inhibited hAChE
Tabun inhibited AChE was least reactivated by any of the newly
The reactivation efficacy of the synthesized oximes (1a–23a) to
reactivate VX inhibited hAChE was presented in Table 2. It was
observed that the K values for the oximes against VX inhibition
were largely varied between 8 and 111 M. Majority of the oximes
have shown greater reactivation efficacy than that of the standard
oxime 2-PAM. Among the oximes, 18a (Fig. 3c and d) has shown
synthesized oximes. In the present study majority of the tested
oximes failed to reactivate tabun inhibited AChE even at higher
concentrations of the reactivator. The observed higher dissociation
D
l
constants (K
toward the highly resistant tabun inhibited hAChE. Among all the
tested oximes, 8a has recorded lower K (62.76 M) and higher
r2 (0.91 mM min ) thereby explaining its better affinity and
D
) of these oximes demonstrated their least affinity
D
l
ꢁ1
ꢁ1
greater affinity (K
D
: 8.83
lM) toward phosphylated hAChE. This
k
was further reflected by its higher second order rate constant
reactivity than other oximes. Oximes 12a, 14a and 1a have
ꢁ1
ꢁ1
(kr2: 9.70 mM min ) which indicate its higher reactivation effi-
recorded nominal reactivity as revealed by their respective lower
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ1
cacy among all the tested oximes. Again, oxime 14a (K : 9.64
D
lM
k
r2 (0.75 mM min
;
0.69 mM min
;
0.72 mM min )

1
30
A.K. Valiveti et al. / Chemico-Biological Interactions 237 (2015) 125–132
Fig. 4. Reactivation profile of oxime 14a against Tabun-inhibited hAChE (a); plot of kobs vs. [14a] (mM) for Tabun-inhibited hAChE (b).
Fig. 5. Percentage reactivation efficacies of the oximes (1a–23a) in comparison to 2-PAM and obidoxime against sarin, VX and Tabun-inhibited hAChE. Oxime concentration:
mM, pH: 7.4, temperature: 37 °C, time of reactivation: 60 min.
1
values. Majority of these oximes (including 2-PAM) have failed to
reactivate tabun inhibited AChE thereby explaining the resistivity
of the tabun-AChE complex toward oxime assisted reactivation.
However, only obidoxime has shown moderate reactivity
persistent reactivation efficacy of the oximes while the percentage
reactivation efficacies (Fig. 5) reflect only the percentage of AChE
reactivation at a particular concentration after certain time. In
Fig. 5, 2-PAM and obidoxime (at 1 mM concentration) have shown
higher percentage reactivation than 12a (1 mM) at 60 min. But the
kinetic data (Table 2) of 12a has been found to be better than those
of 2-PAM and obidoxime. Therefore the oxime 12a has been iden-
tified as a superior reactivator than 2-PAM and obidoxime. Further,
at higher oxime concentration (1 mM), oxime 18a (92%), 11a (71%)
and 17a (66%) have shown higher reactivation efficacy of sarin
inhibited hAChE. However their affinities toward the phosphylated
complex have shown to be lower as indicated by their lower sec-
ond order rate constants. In case of tabun inhibited hAChE, all
the oximes have recorded lower reactivation efficacies even at
higher oxime concentration. However, oximes 5a, 8a, 8b and 18b
have recorded nominal reactivation efficacies against tabun as
compared to other nerve agents (VX and sarin) at 1 mM oxime
concentration.
ꢁ1
ꢁ1
D
(K : 50.73 lM and kr2: 0.99 mM min ) as compared to all other
oximes. The time dependent reactivation profile and plot of kobs vs.
reactivator concentration [14a] was depicted in Fig. 4.
It is worth noticing (Table 2) that the tested oximes have shown
several fold greater efficacies toward VX and sarin inhibited hAChE
than that of tabun inhibited hAChE. The overall order of the reacti-
vation efficacies of the oximes toward nerve agents were found to
be VX > sarin > tabun.
The percentage reactivation efficacy of the oximes (1a–23a) at
1
mM oxime concentration in comparison to 2-PAM and obidox-
ime at 60 min against all the three nerve agents (sarin, VX and
tabun) was illustrated in Fig. 5. Shortlisting a better reactivator
solely on the basis of their percentage reactivation is difficult.
Assessment of efficacy of an oxime has generally been made by
evaluating their kinetic data (Table 2). Majority of the tested oxi-
mes have exhibited higher reactivation efficacies at concentration
Several studies were made to highlight the importance of the
interactions present in the gorge of OP inhibited AChE and con-
1
mM against VX inhibited hAChE in comparison to sarin and
Tabun (Fig. 5). Among the tested oximes 12a (97%), 14a (85%),
8a (81%) and 16a (80%) have shown higher reactivation efficacies
cluded that the reactivators capable of p–p and cation-p
interactions were highly significant in order to achieve a proper
orientation of the reactivator toward the OP-hAChE complex
[13–16]. In order to enhance the hydrophobicity of the reactivator,
various aryl and aliphatic side chains with a variety of substituents
1
indicating their appreciable reactivity of the VX inhibited hAChE
complex. The second order rate constant kr2 signify the overall

A.K. Valiveti et al. / Chemico-Biological Interactions 237 (2015) 125–132
131
Table 2
Reactivation constants for oxime-induced reactivation of sarin, VX and tabun- inhibited hAChE.
min 1 (±SE)
ꢁ
k
r2 (mM min
ꢁ1
ꢁ1
)
Oxime
K
D
lM (±SE)
k
r
Sarin
VX
Tabun
Sarin
VX
Tabun
Sarin
VX
Tabun
1
2
3
4
5
6
7
8
a
a
a
a
a
a
a
a
21.02 (0.0043)
6.62 (0.0009)
41.41 (0.0126)
72.57 (0.0081)
24.92 (0.0032)
17.00 (0.0031)
28.86 (0.0029)
32.16 (0.0088)
13.27 (0.0013)
9.24 (0.0013)
20.36 (0.0025)
14.95 (0.0036)
4.51 (0.0008)
15.43 (0.0019)
36.45 (0.0027)
11.80 (0.0016)
27.44 (0.0051)
14.79 (0.0014)
7.721 (0.0013)
22.13 (0.0042)
20.57 (0.0043)
24.27 (0.0027)
38.46 (0.0039)
87.46 (0.0156)
11.31 (0.0022)
29.60 (0.0046)
12.06 (0.0015)
19.95 (0.0046)
92.46 (0.0253)
139.2 (0.0351)
191.1 (0.1279)
98.88 (0.0193)
0.061 (0.0023)
0.066 (0.0012)
0.065 (0.0045)
0.077 (0.0022)
0.058 (0.0015)
0.049 (0.0017)
0.062 (0.0013)
0.064 (0.0037)
0.089 (0.0015)
0.062 (0.0014)
0.026 (0.0006)
0.064 (0.0027)
0.047 (0.0009)
0.068 (0.0015)
0.094 (0.0015)
0.034 (0.0007)
0.037 (0.0014)
0.069 (0.0011)
0.047 (0.0012)
0.072 (0.0026)
0.085 (0.0033)
0.051 (0.0011)
0.063 (0.0015)
0.051 (0.0025)
0.053 (0.0017)
0.034 (0.0011)
0.072 (0.0015)
0.097 (0.0042)
0.662 (0.005)
0.081 (0.006)
0.077 (0.0164)
0.033 (0.0017)
2.89
9.92
1.57
1.06
2.33
2.91
2.13
2.00
6.73
6.67
1.26
4.28
10.41
4.39
2.58
2.84
1.33
4.70
6.14
3.25
4.12
2.07
1.63
0.58
4.65
1.15
6.00
4.87
3.81
ns
3.79
7.41
2.01
8.61
5.99
6.23
3.68
9.70
5.08
1.20
0.52
3.56
5.77
2.73
9.03
0.72
0.59
0.41
0.34
94.79 (0.0115)
0.040 (0.0013)
0.42
*
*
*
127.3 (0.0336)
62.76 (0.0245)
0.058 (0.0044)
0.057 (0.0056)
0.45
0.91
8
b
77.80 (0.023)
0.056 (0.0045)
0.72
*
*
*
9
a
17.53 (0.0044)
0.067 (0.0030)
*
*
*
*
*
*
*
*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
0a
1a
2a
3a
4a
5a
6a
7a
8a
9a
0a
1a
2a
3a
PAM
16.13 (0.0039)
10.18 (0.0017)
45.14 (0.0075)
9.639 (0.0017)
14.48 (0.0032)
10.63 (0.0022)
16.21 (0.0032)
8.83 (0.0009)
15.87 (0.0016)
41.14 (0.0133)
111.6 (0.030)
15.97 (0.0034)
15.24 (0.0037)
26.59 (0.0087)
14.4 (0.003)
0.061 (0.0026)
0.075 (0.0019)
0.091 (0.0035)
0.083 (0.0023)
0.087 (0.0033)
0.066 (0.0021)
0.059 (0.0021)
0.086 (0.0013)
0.081 (0.0014)
0.049 (0.0036)
0.058 (0.0044)
0.057 (0.0022)
0.088 (0.0036)
0.072 (0.0048)
0.129 (0.0047)
74.28 (0.0167)
0.055 (0.0032)
0.75
*
*
*
60.85 (0.0132)
39.40 (0.0143)
0.042 (0.0023)
0.023 (0.0018)
0.69
0.59
103.3 (0.0192)
0.045 (0.0023)
0.43
*
*
*
71.96 (0.0143)
0.02 (0.001)
0.28
*
*
*
24.94 (0.0061)
0.079 (0.0038)
3.15
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
31.53 (0.0021)
8.831 (0.0013)
17.63 (0.0035)
20.46 (0.0036)
19.5 (0.0046)
0.045 (0.0006)
0.061 (0.0014)
0.081 (0.0029)
0.083 (0.0028)
0.125 (0.0055)
1.41
6.90
4.59
4.07
6.39
Obid.
50.73 (0.0235)
0.051 (0.0056)
0.99
*
Insgnificant reactivation; data are means of ±SE (n = 2).
connected with the amide linkages to the quaternary N- atom of
the pyridinium ring were chosen. In addition, the electron donor
and acceptor capability of the acetamido moieties present in the
reactivator might facilitate the conventional mode of interactions
the P–O bond of the phosphylated complex. In order to assess
the reactivation efficacies of new reactivators as antidotes against
OP poisoning, determination of physico-chemical parameters such
as acid dissociation constants (pKa) are important to understand
the nucleophilicity of the oxime reactivator. Therefore, pKa of the
oximes (1a–23a) were determined spectrophotometrically
(Table 1 and Fig. 2) and were found in the range of 7.81–8.41.
The lower range of these values indicated ease in their ionization.
This was further reflected in their reactivation efficacies against
sarin and VX Inhibited AChE. Oxime 4a has recorded the highest
pKa (8.49) among all the tested oximes and therefore characterized
by its lower second order rate constant against all the three nerve
agents inhibited hAChE. Overall the lower pKa values exhibited by
the synthesized oximes might be the one of the reasons for their
significantly higher reactivation efficacies observed in this study.
(
H-bonding, salt bridge and hydrophobic interactions) between
the gorge residues and the reactivator. This might promote the
suitable orientation of the reactivator toward the active site of
the AChE in the OP-hAChE complex.
The greater reactivation efficacies of majority of the oximes in
case of VX inhibited hAChE might be due to the greater molecular
access of the reactivator toward the VX-inhibited AChE than the
other two nerve agents (sarin and tabun). After inhibition of the
2 2 3 7 2
enzyme active site, the bulky residue of VX i.e. –S(CH ) N(C H )
is departed from the active site thereby leaving more free space
for the approach of the incoming nucleophile (oxime) [32]. This
might be the plausible reason that majority of the tested oximes
have exhibited significant efficacies in the reactivation of VX inhib-
ited hAChE as indicated by their higher second order reactivation
constants (Table 2).
Previously mono-pyridinium oximes having oxime group at the
position 4-have been synthesized and evaluated for their reactiva-
tion efficacy against tabun inhibited hAChE [33]. Some of these
reactivators were found to be most efficacious against tabun inhib-
ited hAChE. However, in the present study majority of the
4. Conclusions
In the present investigation, a series of monoquaternary oximes
were synthesized and evaluated for their reactivation efficacies
against three different nerve agents (sarin, VX and tabun) inhibited
hAChE. Significant reactivation efficacies against VX and sarin
inhibited hAChE were observed by all the tested oximes. Oxime
12a and 2a have shown higher reactivation efficacies against sarin
inhibited hAChE. Further, oximes 18a, 14a and 12a have shown
superior reactivation efficacies among all the tested oximes against
VX inhibited hAChE. Based upon this study, the oximes 2a, 8b, 12a,
14a, 18a and 22a may provide a useful therapeutic potential for the
reactivation of AChE inhibited by sarin and VX. The detailed study
of antidotal efficacy including in vivo reactivation against sarin and
other nerve agents is under progress and will be reported in due
course.
4
-substituted pyridinium oximes were unresponsive toward reac-
tivation of tabun inhibited hAChE.
In the present investigation few of the synthesized oximes con-
taining aliphatic moieties attached to the pyridinium ring (21a,
2
2a, 19a, 20a, and 23a) have displayed an appreciable reactivity
toward sarin and VX inhibited hAChE complex as compared to
-PAM and obidoxime. However, none of these oximes have shown
2
sufficient reactivation against tabun inhibited hAChE, thus repre-
senting their poor reactivity and affinity toward tabun-hAChE
complex.
Conflict of Interest
During the reactivation of OP inhibited hAChE, ionization of
the oxime into oximate anion is essential to initiate cleavage of
The authors declare that there are no conflicts of interest.

1
32
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