Synthesis and in-vitro reactivation screening of imidazolium aldoximes as reactivators of sarin and VX-inhibited human acetylcholinesterase (hAChE)

Article abstract of DOI:10.1016/j.cbi.2016.04.034

Post-treatment of organophosphate (OP) poisoning involves the application of oxime reactivator as an antidote. Structurally different oximes are widely studied to examine their kinetic and mechanistic behavior against OP-inhibited cholinesterase enzyme. A series of structurally related 1,3-disubstituted-2-[(hydroxyiminomethyl)alkyl]imidazolium halides (5a–5e, 9a–9c) were synthesized and further evaluated for their in-vitro reactivation ability to reactivate sarin- and VX-inhibited human acetylcholinesterase (hAChE). The observed results were compared with the reactivation efficacy of standard reactivators; 2-PAM, obidoxime and HI-6. Amongst the synthesized oximes, 5a, 9a and 9b were found to be most potent reactivators against sarin-inhibited hAChE while in case of VX only 9a exhibited comparable reactivity with 2-PAM. Incorporation of pyridinium ring to the imidazole ring resulted in substantial increase in the reactivation strength of prepared reactivator. Physicochemical properties of synthesized reactivators have also been evaluated.

Full text of DOI:10.1016/j.cbi.2016.04.034

Accepted Manuscript
Synthesis and in-vitro reactivation screening of imidazolium aldoximes as reactivators
of sarin and VX-inhibited human acetylcholinesterase (hAChE)
Rahul Sharma, Bhanushree Gupta, Arvind Kumar Sahu, Jyotiranjan Acharya,
Manmohan L. Satnami, Kallol K. Ghosh
PII:
S0009-2797(16)30157-0
10.1016/j.cbi.2016.04.034
CBI 7673
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 17 November 2015
Revised Date: 10 March 2016
Accepted Date: 24 April 2016
Please cite this article as: R. Sharma, B. Gupta, A.K. Sahu, J. Acharya, M.L. Satnami, K.K. Ghosh,
Synthesis and in-vitro reactivation screening of imidazolium aldoximes as reactivators of sarin and VX-
inhibited human acetylcholinesterase (hAChE), Chemico-Biological Interactions (2016), doi: 10.1016/
j.cbi.2016.04.034.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and in-vitro reactivation screening of imidazolium aldoximes as
reactivators of sarin and VX-inhibited human acetylcholinesterase (hAChE)
Rahul Sharmaa Bhanushree Guptaa, Arvind Kumar Sahua, Jyotiranjan Acharyab,
,
Manmohan L. Satnamia, Kallol K. Ghosha*
aSchool of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur (C.G.) 492010,
India
bProcess Technology Development Division, Defence Research & Development Establishment, Jhansi
Road, Gwalior (M.P.) 474002, India
Abstract
Post-treatment of organophosphate (OP) poisoning involves the application of oxime reactivator
as an antidote. Structurally different oximes are widely studied to examine their kinetic and
mechanistic behavior against OP-inhibited cholinesterase enzyme. A series of structurally related
1,3-disubstituted-2-[(hydroxyiminomethyl)alkyl]imidazolium halides (5a-5e, 9a-9c) were
synthesized and further evaluated for their in-vitro reactivation ability to reactivate sarin- and
VX- inhibited human acetylcholinesterase (hAChE). The observed results were compared with
the reactivation efficacy of standard reactivators; 2-PAM and obidoxime. Amongst the
synthesized oximes, 5a, 9a and 9b were found to be most potent reactivators against sarin-
inhibited hAChE while in case of VX only 9a exhibited comparable reactivity with 2-PAM.
Incorporation of pyridinium ring to the imidazole ring resulted in substantial increase in the
reactivation strength of prepared reactivator. Physicochemical properties of synthesized
reactivators have also been evaluated.
Keywords:- Organophosphates, Acetylcholinesterase, Oximes, Antidote, Reactivators,
Reactivation kinetics.
*Author for correspondence:
Tel: +91-771-2263146 (O), 2262249 (R),
Fax: +91-771-2262583
Email: kallolkghosh@yahoo.com
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1. Introduction
The widespread, frequent use and misuse of highly perilous organophosphate (OP) compounds
(Fig. 1) as pesticides (paraoxon, parathion, diazinon etc.) and nerve agents (sarin, soman, VX
etc.) underlines the requirement of effective clinical therapy [1] in military conflicts (e.g. Gulf
war, Syrian attack) and terrorist attacks (e.g. Subway attack in Tokyo) [2]. Nerve agents are
rather toxic compounds and could be worn as weapons of mass destruction and thus represent a
potential threat to humans worldwide. The toxic symptomatology of OP is reflected by the
phosphylation of active serine residue of acetylcholinesterase (AChE). Consequently, its
physiological function is discontinued and reflected in permanent stimulation of cholinergic
receptors leading to failure of respiratory system and subsequent death from suffocation [3]. This
indeed, requires the development of efficient, easily affordable and simply administered
countermeasures for instant defense of common population from toxic OP exposures. Currently
approved antidotal therapies against OP-intoxication entails co-administration of AChE
“reactivator” such as 2-PAM, HI-6, obidoxime, trimedoxime, or similar agents (Fig. 2) with
anticholinergic (atropine) and anticonvulsant (benzodiazepine) drugs. Reactivator restores the
activity of enzyme while atropine antagonizes the effect of accumulated acetylcholine and
benzodiazepine acts as an anticonvulsant [4].
Fig.1. Structures of Chemical Warfare Agents
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The interaction of AChE with inhibitors and reactivators is fully dependent on their structural
features thus results into different inhibitory and reactivation potencies [5]. The available
conventional oximes are not broad spectrum for all classes of OP-poisoning and this
ineffectiveness encourages the synthesis of novel and efficient oximes with several structural
modifications. In search of a universal antidote, several research groups have been engaged in
synthesizing a number of oximes reactivators against all kind of OPs [5-9].
Fig. 2. Structures of Standard oximes
In order to characterize efficient reactivators, synthesized oximes are firstly evaluated for their
physicochemical properties. Generally acid dissociation constant (pKa), lipophilicity (logP),
polar surface area (PSA), hydrogen bond donor and acceptor counts (HBD and HBA) are
included as essential physicochemical properties or molecular descriptors [10].
Determination of pKa of an oxime is essential to diagnose the effective pH at which the
oximate moiety can attack the OP-AChE adduct to reactivate the enzyme [11]. Lipophilicity is
first of the primary descriptors known for BBB penetration [12]. It describes the involvement of
oxime molecule in lipid phase with respect to hydrophilic phase and their ability to penetrate the
lipid bilayer membrane to reach the target site [13]. PSA defines the sum of polar atoms
(nitrogen and oxygen) to which hydrogen is present in the oxime reactivator. It is a significant
parameter to predict drug transport properties [14]. Hydrogen bond donor (articulated as the sum
of OHs and NHs) and acceptor (represents the sum of Ns and Os) counts [15] also play crucial
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role in prediction of minimal distribution of a molecule through BBB. Voicu et al. [14] well
documented the function of several molecular descriptors as an assortment tool for a series of
pyridinium aldoximes derivatives. They further [14] described their pharmacokinetic and
pharmacodynamic importance and associated therapeutic consequences for oximes. However,
the real potency of a reactivator is quantified by applying reactivation kinetics to inhibited-AChE
along with the primary information facilitated by their physicochemical properties.
Tertiary and quaternary imidazolium aldoximes were broadly studied as mono-oxime
reactivators of inhibited-AChE [16-17] and also as bis-oximes in combination with pyridinium
and quinuclidinium oximes [18-19]. Some of these oxime reactivators were evaluated for both
in-vitro and in-vivo investigations and observed as promising candidates for reactivation of
tabun- and soman-inhibited AChE. Recently, Sit et al. [20] have synthesized a series of tertiary
and quaternary imidazole-based aldoximes and studied their reactivation strength against
different OP-inhibited BChE.
Therefore, in the sequence of development of antidotes against OP intoxication [21-24],
herein, we report the synthesis of non-quaternary imidazole aldoxime, ethylimidazolium oximes
and also bis-oximes in combination of N-alkylated 4-pyridinium aldoxime. Since efficacy of
imidazolium oximes are generally reported with tabun and soman [18] hence we examined their
physicochemical properties/molecular descriptors along with reactivation kinetics against sarin
and VX-inhibited human AChE (hAChE).
2. Chemistry
The synthesis of non-quaternary imidazole-2-aldoxime derivatives (4a-4e) was achieved by N-
alkylation of imidazole-2-carboxaldehyde (1) with the requisite alkyl iodide (2a-2e) by varying
alkylated side chain at N1 position. This was then treated with hydroxylamine hydrochloride as
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shown in Scheme 1. To increase the nucleophilicity and solubility of resulted compounds (4a-
4e), process of quaternization has been undertaken. Further their quaternary salts (5a-5e) were
prepared by reaction with iodoethane.
I-
N
N
N
N
b
c
a
R
I
N
H
N
R
N
R
N
R
N OH
N OH
O
O
2a-2e
1
2a, R= CH3
2b, R= C2H5
3a-3e
5a-5e
4a-4e
2c
, R= C3H7
2d, R= C4H9
2e
, R= C5H11
Reagents: a) K2CO3, DMF, 80°C; b) NH 2OH.HCl, Na2CO3, EtOH, 70°C; c) C 2H5I, ACN, 75°C
Scheme 1. Synthesis of non-quaternary and quaternary imidazole aldoxime
In addition to 10 non-quaternary and quaternary mono-imidazolium compounds, three bis-
quaternary imidazolium compounds were also synthesized. Compounds (9a-9c) were
synthesized by treatment of 4-PAM derivative of N-alkylated monopyridinium oxime (8a-8c)
with 2-[(hydroxyimino) methyl]-l-methylimidazol (4a) (Scheme 2). Compounds (9a-9c)
incorporate both the 4-[(hydroxyimino)- methylpyridinium moiety and the 2-[(hydroxyimino)-
methyl] imidazolium moiety, thus combining the important antidotal properties of both types of
reactivators. Elemental analysis of prepared compounds is presented in Table 1.
OH
N
OH
N
OH
N
N
N
N OH
Acetoen, reflux
4-6h
4a
Br
Br
n
N
Br-
Br
8a-8c
N
N
DMF, 75°C
n
n
7a-7c
6
2Br-
OH
N
7a, n=propane
7b,
N
N
n=butane
7c, n=pentane
9a-9c
Scheme 2. Synthesis of bis-quaternary imidazole aldoxime
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The prototype structure of a mono-imidazolium compound (5a) was established on its
1
spectroscopic data. In the H NMR spectrum, compound 5a, exhibited the OH proton of oxime
as singlet at δ 12.99, olefinic proton as singlet at δ 8.54, aromatic proton in range of δ 7.89-7.83,
*N-CH2 proton as quartet at δ 4.37 with J value 7.2 Hz. The N-CH3 proton was observed as
singlet at δ 3.91 and CH3 as triplet at δ 1.38 in the aliphatic region with J value 7.2 Hz. Similarly
the structure of bis-quaternary imidzolium oxime (9a) was also established using NMR
1
techniques. The H NMR spectrum of compound 9a exhibited different peaks for OH group of
oxime functionality at δ 12.96 and at δ 12.83. The aromatic protons of the pyridinium ring was
observed at δ 9.11 and δ 8.26 with J value 6.4 Hz, olefinic proton as singlet at δ 8.58. Further the
aromatic imidazolium proton was obtained at singlet; δ 8.26 and δ 7.94. *N-CH2 proton of
pyridinium ring was found as triplet at δ 4.71 and *N-CH2 proton of imidazolium ring as triplet
at δ 4.48. CH3 proton was observed as singlet at δ 3.94 and CH2 proton of the linker in aliphatic
region of NMR spectrum at δ 2.50. All the synthesized compounds have shown their appropriate
shifting value in 13C NMR spectrum also.
Table 1. Elemental analysis and acid dissociation constants (pKa) of synthesized compounds 5a–
5e and 9a-9c.
R1
X-
N
OH
N
N
R2
Oximes
5a
R1
R2
X- Yield (%)
pKa
C2H5
C2H5
C2H5
C2H5
C2H5
Methyl
Ethyl
I-
I-
I-
I-
I-
84
74
79
78
78
7.65±0.11
7.79±0.09
7.92±0.13
7.96±0.12
8.10±0.07
5b
5c
Propyl
Butyl
5d
5e
Pentyl
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9a
9b
Methyl Br-
80
75
8.52±0.10
8.69±0.13
N
HO
N
Methyl Br-
N
HO
N
9c
Methyl Br-
73
8.92±0.12
N
HO
N
3.0 Materials and Methods
3.1 Materials
Acetylthiocholineiodide (ATChI), 5, 5’-dithiobis-(2-nitrobenzoic acid) (DTNB), 4-
pyridinealdoxime, hydroxylamine hydrochloride, imidazole-2-carboxaldehyde, alkyl iodide,
sodium carbonate and aliphatic linkers were purchased from Sigma-Aldrich, USA and used
without
further
purification.
Potassium
dihydrogenphosphate
and
dipotassium
hydrogenphosphate were obtained from E. Merck (India) and used without further purification.
Solvents (DMF, acetonitrile, acetone, methanol) were purchased from SD Fine Chemicals (India)
and dried before use. Nerve agents sarin and VX were prepared in Process Technology
Development Division, Defence Research & Development Establishment, Jhansi Road, Gwalior
31
(M.P.) 474002, India with >98% purity (GC and P NMR). 2-PAM was prepared according to
the method of Ginsburg and Wilson [25]. Obidoxime was synthesized using reported methods
1
13
[26]. The oximes were synthesized and characterized by their H NMR & C NMR spectral
data. Purity of the synthesized oximes was checked by TLC (cellulose, DSO, Fluka) with 1-
butanol:acetic acid:water (3:1:1) as solvent system.
3.2 Determination of acid dissociation constant (pKa)
The pKa of all the oximes were determined by using the method of Albert and Sergeant [27]. The
method is based on direct determination of the ratio of molecular species (protonated) to
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dissociated (deprotonated) species in a series of non-absorbing buffer solutions. For this purpose,
the spectra of molecular species were obtained first in buffer solution of a particular pH in which
compound of interest would be present wholly in either form. An aliquot (1-3ml) of oxime stock
solutions (5 X 10-4 M) were diluted to 25ml buffer solution (combination of 70 mM di-sodium
hydrogen phosphate and 70 mM potassium dihydrogen phosphate), Different pH values ranging
from 6.35 to 10.32 were maintained using different composition of phosphate buffer, further pH
was adjusted to the desired value by the addition of dilute sodium hydroxide solution (100 mM).
After each pH adjustment, the solution was transferred into the cuvette and the absorption
spectra were recorded over the wave-length range of 200–500 nm with a reference to blank
solution at 25ºC ± 1˚C.
AbsΨ − AbsHOx
AbsOx − AbsΨ
(1)
pKa = pHexp - log
Where, AbsΨ represents the absorbance of partially ionized oxime species at particular pH;
AbsHOx is the absorbance of unionized form of oxime; and AbsOx is the absorbance of the
completely ionized form of oxime. The average value of the 11 measurements was considered as
the pKa of the compound with respect to oximino functionality. Thermofisher scientific evolution
3000 UV-Visible double beam spectrophotometer with quartz cells of 10 mm was used for
spectrometric analysis. The quartz cells were attached to a thermostatic peltier for maintaining
the constant temperature (25 ±1.0ºC). pH values of buffers were determined using a Eutech (pH
700), pH meter equipped with Inlab@ Expert Pro glass electrode with an accuracy of ±0.01 units.
The pH meter was calibrated at 25ºC using the two point calibration method with commercially
available standard buffer solutions pH 7.00 and 9.21.
3.3 Preparation of human erythrocyte ghost
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The source of AChE was native intact human erythrocytes, prepared according to the method of
Worek et al. [28]. Volunteers (n = 2) were male, aged 27-28 years, healthy, non-smoking, non-
alcoholic and weighed ≈70 kg. Blood (10 mL each) was withdrawn into tubes containing EDTA,
centrifuged (3000g, 10 min) and plasma was removed. Erythrocytes were washed three times
with two volumes of phosphate buffer (0.1 M, pH 7.4). Packed erythrocytes were diluted in 20
volumes of hypotonic phosphate buffer (6.6 mM, pH 7.4) to facilitate hemolysis followed by
centrifugation at 40,000g for 30 min at 4ºC. The supernatant was removed and pellets re-
suspended in hypotonic phosphate buffer. After two additional washing cycles the pellets were
combined and re-suspended in 3 mL phosphate buffer (0.1 M, pH 7.4). The erythrocyte ghosts
were concentrated by centrifugation at 100,000g for 30 min at 4ºC. Aliquots of erythrocyte
ghosts were stored at -80ºC until use. Prior to use, ghosts were homogenized on ice on
homogenizer (Sonics vibra cell, USA); three times for 5s with 9s interval to achieve a
homogenous matrix for the enzyme assay.
3.4 AChE Assay: Inhibition and Reactivation
The activities of AChE were monitored spectrophotometrically by Perkin Elmer Lambda 35 UV-
Visible double beam spectrophotometer at wavelength 412 nm with a modified Ellman assay
[29] using quartz cuvettes and 0.48 mM ATChI as substrate and 0.32 mM DTNB as chromogen
in 0.1 M phosphate buffer (pH 7.4). All experiments were performed at 37ºC temperature and pH
7.4. All mentioned concentrations refer to final concentrations. Oxime stock solutions and their
dilutions were done freshly in triple distilled water. The stock solutions of OP inhibitors (sarin
and VX) were prepared in isopropanol once in a week and were stored at -20ºC. The subsequent
dilutions of these inhibitors were freshly prepared every day in deionized water just before to
start the experiment. The hAChE was incubated with appropriate OP concentrations; sarin (0.5 x
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10-7 M) and VX (0.5x 10-7 M) at 37ºC for 10–15 mins to achieve 95–98% inhibition of the
control activity. The residual inhibitor was removed by the concomitant extraction with six times
excess volume of n-hexane.
The reactivation of OP-inhibited hAChE was achieved by the addition of oxime
reactivator at a final concentration ranging from 0.1 mM-1.0 mM. An aliquot of 50
reactivation cocktail (total volume of cocktail; 750 l, containing inhibition cocktail and oxime
reactivator) were withdrawn at specified time intervals and diluted in to cuvettes containing 3000
l phosphate buffer and 100 l of DTNB to monitor change in the enzyme activity after addition
of 20 l ATChI. The oxime induced reactivation of the OP-inhibited AChE was monitored at
ꢀl of
ꢀ
ꢀ
ꢀ
ꢀ
different time intervals over a period of 60-90 mins. Spontaneous reactivation of inhibited AChE
was assayed using the same protocol (the reaction mixture containing enzyme and OP but no
oxime). In these conditions spontaneous reactivation was found to be insignificant. All the values
were corrected for their oxime induced hydrolysis of ATChI.
3.5 Reactivation Kinetic Studies
The activity of enzyme in the control remained constant during the experiment. The reactivation
percentage of inhibited enzyme was calculated as the ratio of the recovered enzyme activity and
activity in the control. The apparent reactivation rate constant kobs for each oxime concentration,
the dissociation constant KD of inhibited enzyme-oxime complex [EPOX] and the maximal
reactivation rate constant kr, were calculated by non-linear fit using the standard oxime
concentration-dependent reactivation Eq. 2 derived from the following Scheme 3 [30].
Calculation of kinetic constants was performed by non-linear regression analysis using curve
fitting programs using PrismTM Vers. 6.0 (Graph Pad Software, San Diego, USA).
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kr
KD
[E] + [POX]
[EP] + [OX]
[EPOX]
kr2
Scheme 3. Schematic representation of reactivation of OP-inhibited AChE by oxime
kr x [OX]
KD + [OX]
kobs
=
(2)
4.0 Results
The synthesized oximes may act as efficient reactivator if they have considerable nucleophilcity.
The nucleophilicity of reactivators can be assessed by determining their pKa values. Therefore
acid dissociation constants (pKa) of all the oximes (5a-5e, and 9a-9c) were determined (Table 1)
using the method of Albert and Sergeant [27]; Eq 1. pKa is based on direct estimation of the ratio
of molecular species (protonated) to ionised (deprotonated) species in a series of non-absorbing
phosphate buffer solutions. A prototype spectrum of oxime 9a is shown in Fig.3.
On the basis of pharmacokinetic principles, it is concluded that the blood brain barrier (BBB)
is relatively impermeable to hydrophilic molecules like quaternary oximes due to their charged
structures and hydrophilic oxime groups. Thus, make them inefficient to penetrate BBB and cure
CNS-poisoning. The molecular descriptors serve as a screening tool for prediction of
permeability of oximes (or lead compound) through biological membranes and are key
parameters for efficient drug design [10]. Hence a variety of descriptors have been evaluated
(Table 2) for synthesized oximes such as molecular weight (MW), polar surface area (PSA),
hydrogen bond donor (HBD), hydrogen bond acceptor (HBA), and lipophilicity (logP) using
MarvinSketch version 6.2.2, 2014 (http://www.chemaxon.com). For favourable penetration
through BBB, all oximes should have [14]; MW <500 Da; PSA≈
60-70Å2, HBD: <5, HBA: <10,
log P: >2 to <5. Most of the tested reactivators fulfilled the mentioned requirements. Only
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charged oximes (5a-9c) showed high negative logP values and may have insignificant ability for
BBB permeability. It was observed that imidazole oximes in combination with pyridinium ring
exceed in term of permissible PSA value and have a range of 77.87-80.70 Å2.
Fig. 3. Spectrophotometric determination of pKa of oxime 9a
.
Table 2. Calculated values of other molecular descriptors of tested oximes (at pH 7.4)
Oximes
MW (Da) PSA (Å2)
HBD
HBA
Log P
5a
5b
5c
5d
5e
9a
9b
9c
281.09
295.02
309.15
323.17
337.20
449.14
463.17
477.19
41.40
41.40
41.40
41.40
41.40
80.70
80.70
77.87
1
1
1
1
1
2
2
2
2
2
2
2
2
4
4
4
-3.21
-2.85
-2.33
-1.88
-1.44
-7.14
-6.63
-6.18
All the synthesized oximes were subjected to kinetic analysis to evaluate their reactivation
potential. Experimentally observed kinetic parameters for studied oximes (Table 3) against
hAChE inhibited by sarin and VX revealed that the different affinity and reactivity are dependent
12

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on the structures of both OP and oximes used. Synthesized oximes (5a-5e and 9a-9c) were
assayed for their in-vitro reactivation efficiency (Figs 4-5) at five different concentrations
ranging from 0.1mM to 1.0mM using erythrocyte ghost hAChE inhibited by tested nerve agents
over a period of 60-90 minutes (Table 3 and Fig. 6). The affinity of oximes toward phosphylated
AChE was reflected by their KD values. For both the nerve agents, all the studied molecules
exhibited KD values within the range of (21.86
comparatively higher KD value i.e. 106.00 M and only oxime 9a showed comparable value with
2-PAM. For VX–AChE conjugate, synthesized oximes possessed slightly higher KD values
(71.68 M to 144.40 M) and displayed lower affinity toward OP-AChE complex as compared
ꢀM- 144.40 ꢀM). In case of sarin 5c displayed
ꢀ
ꢀ
ꢀ
to standard 2-PAM and obidoxime.
The ability of the oxime for the de-phosphorylation of the phosphoryl residue from the
OP-AChE adduct is articulated in terms of reactivity rate constant (kr, min-1). The rate constant kr
for the oximes 5a-5e and 9a-9c against the reactivation of sarin- and VX-inhibited AChE were
found to be in the range of 0.040–0.156 min-1 and 0.076–0.169 min-1 respectively (Table 3). Bis-
oxime 9a (0.156 min-1) showed comparable reactivity rate constant with obidoxime (0.170 min-1)
and was having much higher kr value than 2-PAM (0.06 min-1). In case of VX-inhibited AChE,
again oxime 9a (0.169 min-1) showed higher reactivity rate constant than standard 2-PAM (0.110
min-1) and displayed comparable values with obidoxime (0.18 min-1).
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(a)
(b)
Fig. 4. Time and concentration-dependent reactivation of sarin-inhibited hAChE by 9a. (a) Data were
analyzed by non-linear regression analysis to determine kobs (b) Plot of kobs vs [9a] enabled the
calculation of KD and kr.
(a)
(b)
Fig. 5. Time and concentration-dependent reactivation of VX-inhibited hAChE by 9a. (a) Data were
analyzed by non-linear regression analysis to determine kobs (b) Plot of kobs vs [9a] enabled the
calculation of KD and kr.
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The second order rate constant kr2 gave the specific reactivity of the reactivator and is dependent
on the value of KD and kr. In case of sarin- and VX-inhibited AChE the observed kr2 values of
synthesized oximes 5a-5e and 9a-9c ranged between 0.56 to 3.59 mM-1min-1 and 0.73 to 2.36
mM-1min-1. Amongst the synthesized oximes, compound 9a (kr2; 3.59 mM-1min-1) surpassed the
second order rate constant value of 2-PAM (2.23 mM-1min-1), while 9b (2.05 mM-1min-1) showed
quite good reactivity against sarin inhibited erythrocyte ghost AChE. For VX-inhibited AChE, no
tested oxime could cross the ability of 2-PAM and obidoxime but 9a (2.36 mM-1min-1) displayed
comparable kr2 value with standard 2-PAM (Table 3). Non-quaternary imidazole aldoximes (4a-
4e) didn’t show any remarkable reactivation efficiency against any of the OP inhibited hAChE.
Fig. 6. Comparative percentage reactivation profile of synthesized oximes 5a–5e and 9a-9c
and standard oximes at 90 min against sarin and VX inhibited hAChE (1.0 mM
concentration of oximes, 0.1 M phosphate buffer, pH 7.4 and temperature 37 ˚C).
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Table 3 Reactivation constants for oxime-induced reactivation of OP-inhibited AChE
Oximes
KD (ꢀM)
kr (min-1)
kr2 (mM-1min-1)
%R
IC50
(mM)
Sarin
VX
Sarin
VX
Sarin VX
Sarin
VX
5a
58.14±4.4
74.14±4.4 0.077±0.0009 0.098±0.0011 1.72±0.51 1.32±0.42 55.15±0.21 45.15±0.21 1.39
70.21±3.6
83.22±18.6 0.040±0.0004 0.098±0.0037 0.56±0.10 1.18±0.20 33.90±0.14 35.25±0.35 1.97
5b
106.00±20.4 73.61±10.2 0.075±0.0041 0.076±0.0032 0.70±0.17 1.03±0.16 20.50±0.71 37.70±0.42 1.90
88.16±6.9 78.45±11.0 0.054±0.0010 0.085±0.0025 0.61±0.14 1.08±0.12 27.20±0.28 41.10±0.14 2.08
55.68±11.4 144.40±12.1 0.060±0.0020 0.105±0.0019 1.07±0.16 0.73±0.14 21.55±0.78 31.60±0.57 1.87
5c
5d
5e
9a
61.85±0.21 52.20±0.28
43.42±4.9
67.11±4.6
75.27±2.4
25.72±2.1
21.48±3.0
71.68±8.5 0.156±0.0022 0.169±0.0033 3.59±0.48 2.36±0.60
1.38
89.21±13.8 0.138±0.0013 0.110±0.0045 2.05±0.49 1.23±0.53 50.20±0.28 43.40±0.57 1.34
84.81±7.7 0.081±0.0007 0.099±0.0030 1.07±0.45 1.18±0.74 50.55±0.78 45.80±0.28 1.17
9b
9c
2-PAMa
Obidoximea
31.39±3.9
21.86±1.6
0.06±0.0004 0.110±0.0014 2.23±0.41 3.59±1.34 54.95±0.07 58.50±0.71 0.87
0.17±0.0019 0.180±0.0011 8.03±1.20 8.31±1.54 80.50±0.71 60.60±0.85 1.19
The pseudo first order rate constant kobs was determined by non-linear regression analysis and these data were used for the calculation
of the reactivity rate constant kr, the dissociation constant KD and the hybrid reactivation rate constant kr2 (from the ratio of kr/KD).
Data are given as means ±SD (n = 2). Maximum reactivation gives the highest AChE activity after 90 mins incubation of inhibited
AChE by the respective oxime. a= Ref.31.
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In addition to the kinetic parameters, the inhibition potencies (IC50 values) of synthesized oximes
(5a-5e and 9a-9c) have also been determined and found in the range of 1.17 to 2.08 mM (Table
3). The observed IC50 values indicated that prepared oximes are less toxic in comparison to 2-
PAM and obidoxime, except oxime 9c which has least IC50 value (1.17mM).
5.0 Discussion
The reactivation of OP-inhibited AChE is affected by several factors such as the strength of
nucleophile (oxime), the orientation of nucleophile with respect to phosphate group conjugated
to the active centre serine and dealkylation of the OP–AChE conjugate (aging) [23]. Due to
structural diversity of OP-based pesticides and nerve agents; none of the pyridinium based oxime
reactivators (Fig. 2) is efficient to regenerate inhibited AChE [23]. Therefore, an effective and
quick remedy for all kind of OP-poisoning is still lacking. Hence we tried to develop alternatives
of pyridyl oximes with lower molecular weight and of smaller size. From the last few years,
imidazolium aldoximes which have structural similarity to amidine oximes have drawn attention
of researchers across the globe. In early 1990’s, imidazolium aldoximes were studied extensively
as cholinesterase reactivators [32-33] and recently a series of non-quaternary imidazolium
aldoxime were reported against OP poisonings [20, 34]. In continuation of search of
cholinesterase reactivators, we selected imidazole ring system. The selection of imidazole ring as
compared to pyridinium ring was based on the synthetic limberness, sound isosteric similarity
with pyridine, ease of quaternization [35] (for Coulombic attraction to anionic sites of AChE),
and the standard requirement for oximes with pKa near 8.0 [16]. Nevertheless, non-quaternary
oxime resulted in decreased reactivation efficacy because they don’t bind properly to the
aromatic group of the anionic site, which is located at the bottom of the deep gorge of the AChE
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[6]. This is the reason we have quternized the imidazole aldoxime and evaluated further. Apart
from this we have also studied the effect combination of pyridinium and imidazolium ring on the
reactivation of AChE inhibited by tested OP agents. Previously published uncharged oximes
reactivators by Sit et al. [36] revealed that simple N-alkyl substituted imidazole aldoximes were
good reactivators of OP-hBChE conjugates. The group tried to identify the optimal uncharged
reactivators of OP-hAChE, unfortunately the synthesized imidazole aldoxime didn’t surface as
optimal candidates. However, the reactivation results of present investigation didn’t follow any
regular trend in case of (5a-5e), but the insertion of pyridinium ring (9a-9c) thoroughly enhanced
reactivation ability as well as affinity of synthesized oximes towards OP-AChE conjugates.
5.1 Structure activity relationship (SAR)
The evaluation of physicochemical properties along with reactivation kinetics may facilitate
suitable structure-activity-relationships for the synthesized oximes. An increase in carbon chain
length for quaternary imidazolium oximes (5a-5e) didn’t display any notable change in acid
dissociation constant (pKa) value (Table 1), while insertion of pyridinium ring connected to
various, alkyl chains considerably affected the pKa (Table 1). All the synthesized charged
aldoximes (5a-5e and 9a-9c) exhibited negative logP values (Table 2). The reactivation strength
of synthesized oximes significantly improved with the incorporation of pyridinium oxime (9a-
9c) on the other end of the alkyl chain of imidazolium ring. Presence of pyridinium ring is an
important factor for cation-π interactions with aromatic amino acid residues of the peripheral
anionic site (PAS) of AChE. A plausible explanation of the enhanced reactivation percentage of
9a-9c is the presence of additional π -π interactions and thereby results in strong binding with
AChE active site. The order of reactivity of compound 9a was 1.5 folds higher as compare to 2-
PAM against sarin-inhibited hAChE. The recognized optimal chain length for the moderate
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reactivity of examined reactivator was 3-4 carbon atoms. The findings are in well agreement
with the earlier reported trend [5, 7, 16]. Compounds 9a-9c showed moderate to significant reac-
tivation potency against tested nerve agents.
Reactivation potency of investigated oximes was also influenced by structure of nerve
agents. Out of the synthesized oximes, compounds 9a-9c showed comparable reactivation ability
for VX-inhibited hAChE. This may be accounted on the basis of least occupied gorge of VX-
inhibited AChE due to the removal of bulkier leaving group: N, N-diisopropyl-S-ethyl, hence
facilitated more space for oximes to attack phosphorylated active site with minimum steric
hindrance as compared to sarin [11].
6.0 Conclusion
We have synthesized a series of mono- and bis-quaternary imidazolium aldoximes (5a-5e and
9a-9c) from imidazol-2-carboxaldehyde. All the synthesized oximes were assayed for their in-
vitro reactivation efficacy against sarin- and VX- inhibited-hAChE. Effect of presence and
absence of pyridinium ring along with imidazole ring was also studied. Apart from reactivation
kinetic studies, physicochemical studies were also performed. Based upon the study it was found
that all the oximes have pKa with in the range of 7.65-8.92 and compound 9a showed best
interaction with AChE sites and resulted into good reactivation potential against both sarin- and
VX- inhibited hAChE. Overall assessment led to the conclusion that compounds 5a, 9a-9c may
provide useful therapeutic potential for the reactivation of hAChE inhibited by toxic
organophosphorus nerve agents. The detail study of antidotal efficacy of synthesized compounds
along with their in-vivo reactivation potential will be reported in due course of time.
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7.0 Conflict of interest statement
Authors declare that there are no conflicts of interest.
8.0 Acknowledgment
The financial assistance from DRDO project (New Delhi) (No.ERIP/ER/1003906/M/01/1393) is
gratefully acknowledged. BG is grateful to CSIR, New Delhi for providing senior research
fellowship. Technical support from NMR Division, Defence Research and Development
Establishment is highly acknowledged for providing spectral data. Authors thank Prof. (Dr.) M.
P. Kaushik, Former Director, Defence Research and Development Establishment, Gwalior for
his keen interest in this work. We are grateful to the reviewers for their valuable suggestions and
comments that helped us to improve the quality of paper.
References
1.
J. Bajar, Organophosphates/nerve agent poisoning: mechanism of action, diagnosis,
prophylaxis, and treatment, Adv. Clin. Chem. 38 (2004) 38 151−216.
M. Nagao, T. Takatori, Y. Nakajima, H. Iwase, K. Iwadate, Definitive evidence for the
acute sarin poisoning diagnosis in the Tokyo subway, Toxicol. Appl. Pharmacol. 144
(1997) 198−203.
2.
3.
P. Eyer, F. Worek, Oximes. In: T.C. Marrs, R.L. Maynard, F.R. Sidell, Eds., Chemical
Warfare Agents: Toxicology and Treatment. John Wiley & Sons, Chichester, 2007 pp.
305–329.
4.
5.
H. Thiermann, F. Worek, K. Kehe, Limitations and challenges in treatment of acute
chemical warfare agent poisoning, Chem. Biol. Interact. 206 (2013) 435−443.
G. Mercey, T. Verdelet, J. Renou, M. Kliachyna, R. Baati, F. Nachon, Reactivators of
acetylcholinesterase inhibited by organophosphorus nerve agents, Acc. Chem. Res. 45
(2012) 756–766.
6.
K. Kuca, D. Jun, K. Musilek, Structural requirements of Acetylcholinesterase reactivators,
Min. Rev. Med. Chem. 6 (2006) 109–120.
20

ACCEPTED MANUSCRIPT
7.
8.
J. Kassa, K. Kuca, J. Karasova, K. Musilek, Structural requirements of
Acetylcholinesterase reactivators. Min. Rev. Med. Chem. 8 (2008) 1134-1143.
G.E. Garcia, A.J. Campbell, J. Olson, D. Moorad-Doctor, Morthole, V.I., Novel oximes as
blood–brain barrier penetrating cholinesterase reactivators, Chem. Biol. Interact. 187
(2010) 199–206.
9.
J. Kalisiak, E.C. Ralph, J. Zhang, J.R. Cashman, Amidine-oximes: reactivators for
organophosphate exposure, J. Med. Chem. 54 (2011) 3319–3330.
10. B. Gupta, N. Singh, R. Sharma, B. Foretić, K. Musilek, K. Kuca, J. Acharya, M.L.
Satnami, K.K. Ghosh, Assessment of antidotal efficacy of cholinesterase reactivators
against paraoxon: in vitro reactivation kinetics and physicochemical properties, Bioorg.
Med. Chem. Lett. 24 (2014) 4743-4748.
11. J. Acharya, H. Rana, M.P. Kaushik, Synthesis and in vitro evaluation of xylene linked
carbamoyl bis-pyridinium monooximes as reactivators of organophosphorus (OP) inhibited
electric eel acetylcholinesterase (AChE), Eur. J. Med. Chem. 46 (2011) 3926-3933.
12. H. Pajouhesh, G.R. Lenz, Medicinal Chemical Properties of Successful Central Nervous
System Drugs, NeuroRx 2 (2005) 541-553.
13. F. Lombardo, M.Y. Shalaeva, K.A. Tupper, F. Gao, M.H. Abraham, ElogPoct:ꢁ A Tool for
Lipophilicity Determination in Drug Discovery, J. Med. Chem. 43 (2000) 2922-2928.
14. V.A. Voicu, J. Bajgar, A. Medvedovici, F.S. Radulescu, D.S. Miron, Pharmacokinetics and
pharmacodynamics of some oximes and associated therapeutic consequences: a critical
review, J. Appl. Toxicol. 30 (2010) 719-729.
15. F.S. Radulescu, V.A. Voicu, D.S. Miron, C. Mircioiu, C. Draghici, The Role of Molecular
Descriptors as Screening Tools for the Pyridinealdoxime Methiodide Derivatives
Biopharmaceutical Profile, Rev. Chim. (Bucharest) 62 (2011) 601-605
16. C.D. Bedford, R.N, III Harris, R.A. Howd, A. Miller, H.W. III, Nolen, R.A. Kenley,
Structure-activity relationships for reactivators of organophosphorus-inhibited
acetylcholinesterase: Quaternary salts of 2-[(hydroxyimino)methyl]imidazole, J. Med.
Chem. 27 (1984) 1431−1438.
17. G.A. Koolpe, S.M. Lovejoy, D.A. Goff, K.Y. Lin, D.S. Leung, C.D. Bedford, H.A.
Musallam,
I.
Koplovitz,
R.N.
Harris,
Quaternary
salts
of
2-
21

ACCEPTED MANUSCRIPT
[(Hydroxyimino)methyl]imidazole. 5. Structureactivity relationships for side-chain nitro-,
sulfone-, amino-, and aminosulfonyl-substituted analogues for therapy against
anticholinesterase intoxication, J. Med. Chem. 34 (1991) 1368−1376.
18. I. Primožić, R. Odžak, S. Tomić, V. Simeon-Rudolf, E. Reiner, Pyridinium, imidazolium,
and quinuclidinium oximes: Synthesis, interaction with native and phosphorylated
cholinesterases, and antidotes against organophosphorus compounds, J. Med. Chem. Biol.
Radiol. Def. 2 (2004) 1-30.
19. E. Reiner, V. Simeon, Pyridinium, imidazolium and quinuclidinium compounds: Toxicity
and antidotal effects against the nerve agents tabun and soman, Arh. Hig. Rada Toksikol.
57 (2006) 171−179.
20. R.K. Sit, V.V. Fokin, G. Amitai, K.B. Sharpless, P. Taylor, Z. Radic, Imidazole Aldoximes
Effective in Assisting Butyrylcholinesterase Catalysis of Organophosphate Detoxification,
J. Med. Chem. 57 (2014) 1378−1389.
21. B. Gupta, R. Sharma, N. Singh, K. Kuca, J.R. Acharya, In vitro reactivation kinetics of
paraoxon- and DFP-inhibited electric eel AChE using mono- and bis-pyridinium oximes.
Arch. Toxicol. 88 (2014) 381-390.
22. R. Sharma, B. Gupta, J. Acharya, M.P. Kaushik, K. K. Ghosh, Interactions between
xylene-linked carbamoyl bis-pyridinium mono-oximes and organophosphates inhibited-
AChE: A kinetic study. Toxicology 316 (2014) 1– 8.
23. R. Sharma, B. Gupta, N. Singh, J. Acharya, K. Musilek, K. Kuca, K.K. Ghosh,
Development and structural modifications of cholinesterase reactivators against chemical
warfare agents in last decade: a review. Mini-Rev Med Chem 15 (2015) 58-72.
24. J. Acharya, D.K. Dubey. A.K. Srivastava, S.K. Raza, In vitro reactivation of sarin-inhibited
human acetylcholinesterase (AChE) by bis-pyridinium oximes connected by xylene
linkers, Toxicol. in Vit. 25 (2011) 251-256.
25. S. Ginsburg, I.B. Wilson, Oximes of the Pyridine Series J. Am. Chem. Soc. 79 (1957) 481-
485.
26. A.Luttringhaus, I. Hegedorn, Quaternary hydroxyimminomethylpyridinium salts, [Quärtare
Hydroxyimminomethylpyridinium Salze, in German]. Arzneimittelforschung 14 (1964) 1-
5.
22

ACCEPTED MANUSCRIPT
27. A.Albert, E.P. Sergeant, In The Determinations of Ionization Constants, A Laboratory
Manual, Chapman and Hall, London 1971.
28. F. Worek, G. Reiter, P. Eyer, L. Szinicz, Reactivation kinetics of acetylcholinesterase from
different species inhibited by highly toxic organophosphates, Arch. Toxicol. 76 (2002)
523–529.
29. F. Worek, U. Mast, D. Kiderlen, C. Diepold, P. Eyer, Improved determination of
acetylcholinesterase activity in human whole blood, Clin. Chim. Acta. 288 (1999) 73–90.
30. W.N. Aldridge, E. Reiner, Enzyme inhibitors as substrates – interactions of esterases with
esters of organophosphorus and carbamic acids, North-Holland Publishing Company,
Amsterdam, London, 1972.
31. H.N. Karade, A.K. Valiveti, J. Acharya, M.P. Kaushik, Synthesis and in vitro evaluation of
bis-quaternary 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide derivatives as reactivators
against sarin and VX inhibited human acetylcholinesterase (hAChE), Bioorg. Med. Chem.,
22 (2014) 2684-2691.
32. M. Mesic, A. Deljac, V. Deljac, Z. Binenfeld, Reactivators of acetylcholinesterase
inhibited by organophosphorus compounds. Imidazole derivatives. II Acta. Pharm. Jugosl.
41 (1991) 203-210.
33. M. Mesic, A. Deljac, V. Deljac, Z. Binenfeld, M. Maksimovic, V. Kilibarda, Reactivators
of acetylcholinesterase inhibited by organophosphorus compounds. Imidazole derivatives.
III. Acta Pharm. Jugosl. 42 (1992) 169-172.
34. Z. Kovarik, N. Macek, R. K. Sit, Z. Radic, V.V. Fokin, K. B. Sharpless, P. Taylor,
Centrally acting oximes in reactivation of tabun-phosphoramidated AChE. Chem. Biol.
Interact. 203 (2013) 77-80.
35. L.W. Deady,Rates of N-methylation of azoles with methyl iodide and dimethyl sulphate,
Aust. J. Chem. 26 (1973) 1949-1953.
36. R.K. Sit, Z. Radić, V. Gerardi, L. Zhang, E. Garcia, M. Katalinić, G. Amitai, Z. Kovarik,
V.V Fokin, K.B. Sharpless, P. Taylor, New structural scaffolds for centrally acting oxime
reactivators of phosphylated cholinesterases, J. Biol. Chem. 286 (2011) 19422−19430.
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Highlights
• A series of 1,3-disubstituted imidazolium aldoximes were synthesized.
• Analysis of physico-chemical properties of prepared oximes were also studied.
• Synthesized oximes were evaluated against sarin and VX inhibited human AChE.
• Insertion of pyridinium ring to imidazolium oxime resulted in better reactivity.