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)

Article abstract of DOI:10.1016/j.bmc.2014.03.023

A series of bis-quaternary pyridinium derivatives 3a-3i of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide (2) have been synthesized. The synthesized pyridinium compounds have an amide group in conjugation to the oxime moiety. These compounds were evaluated in vitro for their reactivation efficacy against organophosphorus (OP) nerve agents (NAs) (sarin and VX) inhibited human erythrocyte ghost acetylcholinesterase (hAChE) and compared with the reactivation efficacy of 2-PAM and obidoxime. The pK_a values of the synthesized compounds were found closer to the pK_a values of 2- and 4-pyridinium oxime reactivators such as 2-PAM and obidoxime. Some of the compounds have shown better reactivation efficacy than 2-PAM, and obidoxime against sarin and VX inhibited AChE.

Full text of DOI:10.1016/j.bmc.2014.03.023

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
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)
⇑
Hitendra N. Karade, Aditya Kapil Valiveti, Jyotiranjan Acharya , Mahabir Parshad Kaushik
Process Technology Development Division, Defence Research & Development Establishment, Jhansi Road, Gwalior 474 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bis-quaternary pyridinium derivatives 3a–3i of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide
(2) have been synthesized. The synthesized pyridinium compounds have an amide group in conjugation
to the oxime moiety. These compounds were evaluated in vitro for their reactivation efficacy against
organophosphorus (OP) nerve agents (NAs) (sarin and VX) inhibited human erythrocyte ghost acetylcho-
linesterase (hAChE) and compared with the reactivation efficacy of 2-PAM and obidoxime. The pKa values
of the synthesized compounds were found closer to the pKa values of 2- and 4-pyridinium oxime reacti-
vators such as 2-PAM and obidoxime. Some of the compounds have shown better reactivation efficacy
than 2-PAM, and obidoxime against sarin and VX inhibited AChE.
Received 16 September 2013
Revised 13 March 2014
Accepted 14 March 2014
Available online xxxx
Keywords:
Acetylcholinesterase
2-(Hydroxyimino)-N-(pyridin-3-
yl)acetamide
Ó 2014 Elsevier Ltd. All rights reserved.
Organophosphorus
Nerve agents
Reactivators
1. Introduction
by forming the covalent bond between serine residue of the active
site of AChE and phosphorus atom of the OP inhibitor.3 The inhibi-
The use of the organophosphorus (OP) compounds as pesticides
(parathion, malathion, chlorpyriphos) have been restricted in
many countries but still are in use in the developing countries ow-
ing to their effectiveness and low cost.1 Unfortunately, some of
these OP compounds were evolved as chemical warfare (CW)
agents in the form of nerve agents (NA) such as sarin, soman, tabun
and VX (Fig. 1).2 With growing threats of recent terrorist activities
worldwide, one should not deny the possibility of the repeated use
of NAs on a mass scale in the future. Further, the decontamination
and/or destruction of large stockpile of NAs envisage a serious
threat to human health. In this regard, the development of effective
medical treatment regimen of OP poisoning has attracted the
attention of many researchers.
tion of AChE leads to the overstimulation of the cholinergic recep-
tor in the synapse which finally results in the breakdown of
neuromuscular function. The inhibited AChE can be reactivated
by the dephosphorylation of the serine residue by a nucleophile
such as pyridinium oxime. At present, the standard treatment for
OP poisoning involves the use of a muscarinic receptor antagonist
such as atropine, an oxime as reactivator of inhibited AChE, and an
anticonvulsant agent such as diazepam.4 The commonly used reac-
tivators are quaternary mono- or bis-pyridinium compounds car-
rying the hydroxyiminomethyl (oxime) group as nucleophilic
agent. Oximes viz. 2-hydroxyiminomethyl-1-methylpyridinium
chloride (2-PAM), 1,3-bis-(4-hydroxyiminomethylpyridino-1-yl)
propane dibromide (TMB-4), 1-(2-hydroxyimino-methylpyridino-
1-yl)-3-(4-carbamoylpyridino-1-yl)-2-oxapropane dichloride (HI-6)
and 1,3-bis-(4-hydroxyiminomethylpyridino-1-yl)-2-oxapropane
dichloride (obidoxime) are commercially available oxime reactiva-
tors used in the treatment of OP poisoning (Fig. 2).5 The binding
interaction of the OP compounds with the enzyme AChE differs
owing to their diverse structural features which results in the var-
iation of the reactivation efficacy of 2-PAM and other oximes.6 Each
type of OP needs the AChE reactivator having specific structure and
Acetylcholinesterase (AChE) is the key enzyme responsible for
the hydrolysis of the neurotransmitter, acetylcholine (ACh). Intox-
ication by the OP compounds inhibit the enzyme AChE irreversibly
Abbreviations: AChE, acetylcholinesterase; 2-PAM, 2-(hydroxyiminomethyl)-1-
methylpyridinium chloride; OP, organophosphorus; NA, nerve agent.
⇑
Corresponding author. Tel.: +91 751 2340245; fax: +91 751 2341148.
E-mail address: jracharya01@gmail.com (J. Acharya).
http://dx.doi.org/10.1016/j.bmc.2014.03.023
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Karade, H. N.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.03.023

2
H. N. Karade et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 1. Structure of the organophosphorus nerve agents.
Figure 2. Structure of the pyridinium oxime reactivators.
hence a broad spectrum reactivator is lacking till date. Therefore,
the development of universal and effective oxime reactivators of
AChE in the treatment of OP poisoning is very important. To over-
come these difficulties, a large number of oxime reactivators were
synthesized and screened for the search of an effective and univer-
sal AChE reactivator during last several decades.7
Previously, we have reported the effect of various types of link-
ers on the reactivation efficacy of the bis-quaternary pyridinium
oximes.8 The intermediate 2-(hydroxyimino)-N-(pyridin-3-yl)acet-
amide (2) was prepared from 3-aminopyridine (1), chloral hydrate
and hydroxylamine hydrochloride in water. The coupling of oxime
2 with various terminal dibromo aliphatic and xylene linkers
yielded the desired bis-quaternary products (3a–3i) (Scheme 1).
The attempted synthesis of corresponding 2-(hydroxyimino)-N-
2. Result and discussion
(pyridin-2-yl)acetamide
and
2-(hydroxyimino)-N-(pyridin-4-
yl)acetamide using 2-aminopyridine and 4-aminopyridine,
respectively, resulted only trace amount of conversions. This may
be due to the less nucleophilic –NH2 group in conjugation to the
ring nitrogen in 2- and 4-aminopyridine.
2.1. Chemical structures of the studied oximes
In continuation to our study on antidotes against OP intoxica-
tion,8 herein we report the synthesis of bis-quaternary derivatives
of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide (3a–3i). The
amide group (–NHCO–) has both the features of a hydrogen bond
donor (–NH–) and hydrogen bond acceptor (>C@O), which may re-
sult in better binding interactions with the enzyme AChE. This kind
of binding interaction may be responsible for the reduced KD (dis-
sociation constant) value of some of the oximes (3a–3i) in compar-
ison to 2-PAM and obidoxime (Fig. 3).
2.2. In vitro reactivation kinetics of the synthesized oximes
against NA-inhibited hAChE
The reactivation kinetic parameters (rate constants and dissoci-
ation constant) depend on the source of enzyme, experimental buf-
fer pH, temperature and the experimental protocols. The
synthesized compounds (3a–3i) were assayed for their in vitro
Figure 3. Conjugation of oxime group with amidine, carbonyl and amide group.
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H. N. Karade et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Scheme 1. Synthesis of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide (2) and corresponding mono and bis-quaternary pyridinium compounds (3a–3i).
Figure 4. Time and concentration dependent reactivation profile of oxime 3d against sarin (a) and VX (c) inhibited hAChE; plot of kobs versus [3d] (mM) for sarin (b) and VX
(d) inhibited hAChE.
reactivation potency at five different concentrations (0.1–1 mM)
using erythrocyte ghost hAChE inhibited by nerve agents: sarin
and VX (Fig. 4). Reactivation study was monitored till one hour
at different time intervals and kinetic data were generated (Fig. 5
and Table 1).
The OP inhibitors were chosen on the basis of their structural
divergence feature. The in vitro reactivation data were compared
with those of 2-PAM and obidoxime. The affinity of the reactiva-
tor towards the OP-inhibited enzyme is expressed in terms of
The efficiency of the oxime for the de-phosphorylation of the
phosphoryl residue from the OP-inhibited AChE is expressed in
terms of reactivity constant (kr minꢀ1). The rate constant kr for
the oximes 3a–3i towards the sarin and VX inhibited AChE were
found in the range of 0.045–0.0.11 and 0.048–0.23 minꢀ1, respec-
tively, (Table 1). The second order rate constant kr2 gives the spe-
cific reactivity of the oxime and it depends on KD and kr. In
comparison to the kr2 of 2-PAM (2.23 mMꢀ1 minꢀ1) and obidoxime
(8.03 mMꢀ1 minꢀ1), the kr2 of the oximes 3a–3i ranges between 1.1
and 10.85 mMꢀ1 minꢀ1, for the sarin inhibited AChE. Similarly, in
case of VX-inhibited AChE the kr2 of the obidoxime (8.31 mMꢀ1
dissociation constant (KD
mes 3a–3i (Table 1) against sarin inhibited AChE varies largely
between 20 and 57 M, except the oxime 3f (84.29 M). As
compared to 2-PAM (KD 25.72 M) and obidoxime (KD
21.48 M), both the oximes 3g (KD 20.82 M) and 3i (KD
8.55 M) showed better affinity towards sarin inhibited AChE.
lM). The values of the KD for the oxi-
l
l
minꢀ1) is more than all oximes 3a–3i (1.95–5.04 mMꢀ1 minꢀ1
)
l
(Table 1).
l
l
In addition to the kinetic parameters, the inhibition potential of
the oximes (IC50) towards the enzyme is also the important factor.
Therefore the inhibition potential (IC50) values of the oximes 3a–3i
were also determined and found in the range of 0.93–2.53 mM. The
synthesized oximes (3a–3i) can act as good reactivator if they have
l
In case of VX–AChE conjugate, oximes 3d, 3e, 3i showed better
affinity towards phosphorylated enzyme as compared to 2-PAM
and obidoxime.
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H. N. Karade et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 5. Reactivation efficacy (%) of the oximes 3a–3i in comparison to 2-PAM and obidoxime at 60 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).
Table 1
In vitro reactivation kinetic constants and IC50 values for the oximes against NA-inhibited hAChE
Oxime
KD
lM ( SE)
kr minꢀ1 ( SE)
kr2 (mMꢀ1 minꢀ1
)
IC50 (mM)
Sarin
VX
Sarin
VX
Sarin
VX
3a
3b
3c
3d
3e
3f
3g
3h
42.86 (4.6)
48.66 (8.2)
88.86 (12.7)
86.12 (3.3)
19.71 (2.1)
16.86 (2.0)
65.82 (5.9)
22.57 (3.0)
54.93 (9.7)
17.46 (1.5)
31.39 (3.9)
21.86 (1.6)
0.05 (0.0007)
0.06 (0.0018)
0.08 (0.0002)
0.05 (0.0007)
0.05 (0.0005)
0.12 (0.0036)
0.05 (0.0003)
0.10 (0.0012)
0.09 (0.0027)
0.06 (0.0004)
0.17 (0.0019)
0.20 (0.0048)
0.23 (0.0065)
0.23 (0.0017)
0.07 (0.0006)
0.07 (0.0005)
0.13 (0.0019)
0.05 (0.0008)
0.12 (0.0032)
0.09 (0.0005)
0.11 (0.0014)
0.18 (0.0011)
1.25
1.10
1.46
1.50
2.16
1.42
2.24
2.16
10.85
2.23
8.03
4.17
2.62
2.63
3.76
3.97
1.95
3.47
2.17
5.04
3.59
8.31
2.53
1.07
1.06
1.79
1.43
0.93
1.7
1.67
2.1
1.19
0.87
57.96 (10.5)
52.32 (9.7)
30.23 (4.7)
24.39 (2.9)
84.29 (13.2)
20.82 (1.8)
48.38 (4.0)
8.55 (2.0)
3i
2-PAM
Obid.
25.72 (2.1)
21.48 (3.0)
Table 2
Physical and elemental analysis data of the compounds 3a–3i
Entry
Oxime
Yield (%)
MP (°C)
pKa
C
H
N
C
H
N
Calculated (%)
Found (%)
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
3g
3h
3i
65
60
63
65
62
58
62
65
67
200–202
238–240
240–242
216–218
241–243
201–203
244–246
256–258
243–245
8.14 0.03
8.17 0.04
8.16 0.03
8.14 0.05
8.25 0.05
8.22 0.04
8.10 0.05
8.23 0.03
7.95 0.04
37.09
38.37
39.58
40.73
41.83
42.87
44.46
44.46
44.46
3.50
3.79
4.06
4.32
4.56
4.80
3.73
3.73
3.73
16.22
15.79
15.39
15.00
14.63
14.29
14.14
14.14
14.14
36.99
38.33
39.51
40.76
41.78
42.81
44.39
44.39
44.41
3.55
3.81
4.14
4.41
4.61
4.91
3.81
3.77
3.66
16.18
15.82
15.46
14.91
14.61
14.21
14.16
14.02
14.05
10
considerable nucleophilicity which can be determined on the basis
of their acid dissociation constant (pKa). The pKa of the oximes
3a–3i were found in the range of 7.95–8.2 (Table 2). Now, the com-
parison of the kinetic data, pKa and IC50 values can lead to the
meaningful conclusion for the oximes 3a–3i as reactivators against
OP-inhibited AChE.
in the OP–AChE conjugate during the reactivation process. It is well
known that the substituents having negative inductive/resonance
effect in conjugation to the oxime group enhances its ionization
tendency into oximate anion (better nucleophile) which in turn in-
creases the reactivation efficacy of the oxime reactivator.13 The
oxime group of the monoisonitrosoacetone (MINA) is in conjuga-
tion with carbonyl group and therefore was found to show good
reactivation efficacy against OP inhibited AChE.14 Recently ami-
dine–oximes15a and amide conjugated oximes15b,c were reported
as reactivators against OP-inhibited AChE. The electron withdraw-
ing functional groups such as carbonyl, amidine and amide in con-
jugation to the oxime group can assist the dissociation of oxime
group into more active oximate anion thereby increasing their
reactivation efficacy (Fig. 3).
3. Discussion
The reactivation of the OP-inhibited AChE depends on several
factors including structure of the reactivator,9 structure of the
inhibitor (OP), source of the enzyme,10 on post-inhibitory reactions
such as spontaneous dealkylation (aging)11 and spontaneous
dephosphorylation (spontaneous reactivation) of the OP–AChE
complex.12 The oxime moiety present in the pyridinium reactiva-
tors acts as strong nucleophile and effectively breaks the OAP bond
The structures of OP inhibitors, VX and sarin are different;
therefore variation in the reactivation of OP-inhibited AChE by
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H. N. Karade et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
the reactivators was expected. The steric influence that differs in
the structures of VX and sarin might contribute to the variation to-
wards in vitro oxime reactivation which can be explained on the
basis of molecular volumes of these OP agents. Inhibition of AChE
by OP NAs leads to the cleavage of the leaving group to form
alkylmethylphosphonate–AChE conjugates. The differences in the
molecular volume of sarin and VX arise mainly because of the O-al-
kyl substituents. Therefore the 20 Å deep active site gorge of AChE
will be largely occupied by sarin followed by VX inhibited AChE.16
dissociation of the oxime group into the more nucleophilic oximate
anion can be attributed from its pKa value. All the synthesized com-
pounds (3a–3i) have 3-substituted pyridinium ring and their pKa
value were found in the range of 7.95–8.25 (Table 2 and Fig. 6).
The pKa values of the compounds (3a–3i) were found closer to
those of 2- and 4-pyridinium oxime reactivators (pKa 7.8–8.5).
Generally it is observed that the 3-pyridinium oxime reactivators
have pKa P9.0 which reflect their lesser tendencies to dissociate
into oximate anion.19 This might have resulted in the lower reacti-
vation efficacy of the 3-pyridinium oxime reactivators as compared
to their 2- and 4-isomers. However, in the synthesized compounds
(3a–3i), the oxime group is not directly attached to 3-position of
the pyridinium ring but is attached to the side chain at 3-position.
Hence the resonance effect of the pyridinium ring may not affect
directly to the nucleophilicity of 3-substituted side chain oxime
group which in fact is reflected in their lower pKa values (pKa
7.95–8.25). In addition, the conjugation of the amide group with
oxime group may assist the dissociation of the compounds 3a–3i
which might be responsible for their lower pKa value as compared
to 3-pyridinium oximes. The pKa of the oximes may affect the val-
ues of kinetic rate constant (kr and kr2). The oxime 3i has the least
pKa value (7.95) among the oximes 3a–3i, which might be one of
the reasons responsible for the higher second order rate constant
(kr2) against sarin and VX inhibited AChE.
The IC50 values of the oximes 3a–3i (Fig. 7) showed that 3a, 3d,
3e, 3g, 3h and 3i were less inhibitors as compared to 2-PAM and
obidoxime (Table 1). Among the synthesized oximes (3a–3i), the
oxime 3f has least IC50 value (0.93 mM) followed by oxime 3b
(1.07 mM) and 3c (1.06 mM). Therefore these three oximes (3b,
3c and 3f) inhibit the enzyme AChE relatively more as compared
to other oximes, which might be one of the plausible reasons
responsible for their less reactivation efficacy. Overall, the oximes
3a, 3d, 3e, 3g, and 3i were found to show relatively low inhibition
and better reactivation efficacy against VX-inhibited AChE while
oxime 3i showed better reactivation against sarin-inhibited AChE.
The compound 3g–3i have the aromatic xylene linker (
which can interact well with the AChE active site residues via
or cation– interactions. This may be the major reason that
p-electrons)
p–p
p
amongst the synthesized compounds, the compound 3i showed
far better reactivation efficacy than 2-PAM and obidoxime against
sarin and VX inhibition. As compared to ortho and meta-xylene
linkers (compound 3g and 3h, respectively, Scheme 1), the para-
xylene linker (3i) disposed the two oxime groups far apart from
each other. This kind of orientation of the oxime 3i may allow
one of the two pyridinium rings to reside on the rim, and other
pyridinium ring penetrates into gorge of the enzyme where phos-
phorylation of active site of enzyme takes place.8b,18b This kind of
the interaction of aromatic xylene linker might be responsible for
the lower value of the rate constant KD for the oximes 3g and 3i
than other synthesized oximes, toward sarin and VX-inhibited
AChE. Similarly the AChE reactivators and/or inhibitor having con-
formationaly flexible longer aliphatic chain (six to seven carbon
chain) might orient well within the range of peripheral to catalytic
site of enzyme AChE.17 This might be the plausible reason that the
oximes 3d and 3e showed better affinity (less KD value) toward sar-
in and VX inhibited AChE, in comparison to 2-PAM and obidoxime.
The reactivation efficacy of the pyridinium oxime reactivators
depend on certain structural and physicochemical parameters such
as position of the oxime group in the pyridinium ring, steric fac-
tors, length and nature of the connecting chain, and most impor-
tantly, its acid dissociation constant (pKa) value.18 The extent of
Figure 6. Spectrophotometric determination of pKa of the oxime 3d.
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H. N. Karade et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Ellman assay.23 ATCh (0.45 mM) as substrate and DTNB (0.3 mM)
as chromogen were used in 0.1 M phosphate buffer (pH 7.4). Cuv-
ette contained total 3.0 mL volume of assay mixture.
5.3. Enzyme inhibition by NA
A
freshly prepared stock solution of OP inhibitor (sarin,
1.4 ꢁ 10ꢀ2 M and VX, 1.33 ꢁ 10ꢀ2 M) was in isopropanol and
stored under refrigeration. It was then diluted appropriately with
triple distilled water just before use. Around 300 lL of enzyme sus-
pension (0.1 M phosphate buffer, pH 7.4) was incubated by 5–
10
l
L of NA (sarin, 1.4 ꢁ 10ꢀ6 M and VX, 3.32 ꢁ 10ꢀ7 M) at room
temperature for 10–15 min. AChE activity was measured to deter-
mine the extent of inhibition (95–98%). Excess of NA was removed
by the extraction of NA inhibited enzyme mixture with tenfold vol-
ume of n-hexane. The mixture of NA inhibited enzyme was stored
at ꢀ60 °C and consumed on the same day.
Figure 7. IC50 of the oxime 3d.
4. Conclusions
5.4. Reactivation kinetics of NA-inhibited hAChE by oxime
We have synthesized the series of bis-quaternary pyridinium
acetamide oxime reactivators (3a–3i) from 2-(hydroxyimino)-N-
(pyridin-3-yl)acetamide (2). These are the pyridinium reactivators
having amide group in conjugation to the oxime functionality. The
synthesized compounds were evaluated in vitro for their reactiva-
tion efficacy against sarin and VX inhibited hAChE. Based upon this
study, 3a, 3d, 3e, 3g, and 3i may provide a useful therapeutic po-
tential for the reactivation of AChE inhibited by OP NAs. The de-
tailed study of antidotal efficacy including in vivo reactivation
against sarin and VX will be reported in due course of time.
The NA inhibited hAChE suspension (20 lL) was transferred to
the cuvette containing phosphate buffer (0.1 M, pH 7.4), DTNB
and an oxime (0.1–1 mM). AChE activity was measured after differ-
ent time intervals at 37 °C by adding ATCh. Spontaneous reactiva-
tion of the inhibited AChE was assayed using the same protocol,
the reaction mixture contained enzyme and OP but no oxime. Un-
der these conditions spontaneous reactivation was found to be
insignificant. All the values were corrected for their oxime induced
hydrolysis. The IC50 values of the oximes were determined by using
the same protocol, for which oxime induced inhibition of enzyme
was studied at different concentration.
5. Experimental sections
5.1. Materials
The process of oxime induced reactivation of the NA-inhibited
AChE can be represented by Scheme 2.
[EP] is the phosphylated enzyme, [OX] is the reactivator, [EPOX]
is the Michaelis-type complex between phosphylated-AChE and
oxime reactivator, [E] is the reactivated enzyme, [POX] is the phos-
phylated oxime, KD is the dissociation constant and is inversely
proportional to the affinity of reactivator towards the phosphylat-
ed enzyme [EP], kr is the rate constant for the displacement of
phosphoryl residue from [EPOX] by oxime and it expresses the
reactivation efficiency of the reactivator.
Acetylthiocholineiodide (ATCh), 5,50-dithiobis-(2-nitrobenzoic
acid) (DTNB), 3-aminopyridine, chloral hydrate, hydroxylamine
hydrochloride, aliphatic linkers and
a,
a0-dibromoxylene, potas-
sium dihydrogenphosphate, dipotassium-hydrogen phosphate, tri-
zma-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. Anhy-
drous sodium sulfate was purchased from Qualigens, India. Sol-
vents (DMF, acetone, and methanol) were purchased from S.D.
Fine Chemicals (India) and dried before use. VX and sarin were pre-
pared in this laboratory with >98% purity (GC and 31P NMR).
2-PAM was prepared according to the method of Ginsburg and
Wilson.20 Obidoxime was synthesized using reported methods.21
The bis-quaternary pyridinium salts were synthesized, character-
ized (I.R., 1H and 13C NMR spectral data) and their purity was
checked by commercially available pre-coated cellulose on alumina
sheets TLC plates (E. Merck) with 1-butanol/acetic acid/water
(3:1:1) as solvent system (Table 2). Hemoglobin free human eryth-
rocyte ghost enzyme acetylcholinesterase (hAChE) was prepared
using the reported protocol.22
For the reactivation process where complete reactivation occurs
with [OX]>>[EP]0 then pseudo first order rate equation24 can be
derived;
kr½OXꢂ
Kobs ¼
ð1Þ
KD þ ½OXꢂ
where kobs is the observed first-order rate constant of the reactiva-
tion at any given oxime concentration.
The value of kobs is not proportional to the oxime concentration
but underlies a saturation kinetics.25 The rate constants kr and KD
follow Michaelis–Menten kinetics.26 The second order reactivation
rate constant kr2 is the ratio of kr and KD;
kr
KD
kr2
¼
ð2Þ
5.2. Enzyme assays
All the kinetic rate constants were calculated by processing the
experimental data with nonlinear regression analysis using curve-
fitting programs provided by Prism™ Vers. 6.0 (GraphPad software,
San Diego, USA).
The activity of hAChE was measured spectrophotometrically
(Carry 100 UV–Visible spectrophotometer) at 37 °C, using modified
5.5. Determination of acid dissociation constant (pKa)
The acid dissociation constants (pKa) of all the compounds (3a–
3i) were determined spectrophotometrically using the method of
Scheme 2. Oxime induced reactivation of OP-inhibited AChE.
Please cite this article in press as: Karade, H. N.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.03.023

H. N. Karade et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Albert and Sergeant.27 The method is based on the direct determi-
5.6.2. Synthesis of 1,10-(pentane-1,5-diyl)bis(3-(2-
nation of the ratio of molecular species (protonated) to the disso-
ciated (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 particular pH in which com-
pounds of the interest would be present wholly in either form.
(hydroxyimino)acetamido)pyridinium) dibromide (3d)
In a typical experimental procedure, 2-(hydroxyimino)-N-(pyri-
din-3-yl)acetamide (1.66 g, 10.1 mM) and 1,5-dibromo pentane
(1.14 g, 5 mM) were dissolved in 15 mL dry DMF in a 50 mL round
bottom flask fitted with a water condenser and calcium chloride
guard tube. It was then stirred at 80 °C for 6 h and monitored by
TLC. The reaction mixture was then brought to room temperature,
cooled in an ice bath and the brown colored solid appeared was
then filtered off and washed repeatedly by hot dry methanol
followed by hot dry acetone to give the desired product 1,10-
(pentane-1,5-diyl)bis(3-(2-(hydroxyimino)acetamido)pyridinium)
dibromide (3d). Yield: 3.65 g (65%); mp: 216–218 °C. All other
products were synthesized using the same protocol.
30–50
l
L of oxime stock solutions (5 ꢁ 10ꢀ3 M) were diluted to
3 mL in a cuvette containing either 0.1 M hydrochloric acid or
0.1 M sodium hydroxide solution and the absorption spectra of
the oxime in acid or alkali were measured over the wavelength
range of 200–600 nm with a reference to blank solution at
25 1 °C. The spectra, thus obtained in acid or alkali, were of pro-
tonated (Dm) and deprotonated (Di) molecules. Eleven different pH
values, ranging from 5.97 to 10.48 were selected to determine the
pKa of oximes. For this, appropriate buffers consisting of phosphate
(pH 5.97–8.05), tris (pH 8.44–9.12) and glycine–NaOH (pH 9.52–
10.48), were used to determine the dissociation constants of oxi-
5.6.3. Characterization of the synthesized compounds 3a–3i
Purity of the synthesized pyridinium compounds were checked
by thin-layer chromatography (TLC, cellulose, Merck) with 1-buta-
nol/acetic acid/water (3:1:1) as mobile phase. Melting points were
determined with open capillary tube on a Gallenkamp (variable
heater) melting point apparatus and were uncorrected. The struc-
ture of the synthesized compounds was confirmed by their ele-
mental analysis and spectral data. Elemental analyses were
conducted on an ELEMENTAR, vario MICRO cube, Universal micro
analyzer and were within 0.4% of the calculated values (Table 2).
Infra-red (I.R.) spectra was obtained as KBr discs on a Bruker TEN-
SOR-27 FTIR spectrometer. 1H NMR (DMSO-d6) spectra were re-
corded on Bruker Avance 400 spectrometer at 400 MHz using
tetramethylsilane as internal standard and expressed in the d
(ppm) values. The –OH and –NH protons appeared as a singlet in
the range of d 12.00 and 11.00, respectively, and were exchange-
able with D2O. 13C NMR (DMSO-d6) chemical shifts values were ob-
tained using the same instrument at 100 MHz (Supplementary
data).
mes. 30–50 lL of aqueous solutions of oximes was diluted to
3 mL in each buffer and optical densities were determined at ana-
lytical wave lengths using buffer blank at 25 1 °C. A set of 11 val-
ues of pKa were obtained using Eq. 3;
pKa ¼ pH þ log½ðDi ꢀ DÞ=ðD ꢀ DmÞꢂ
ð3Þ
where, Dm, and Di, correspond to the optical density of protonated
and deprotonated forms of the compounds (3a–3i), and D is the
optical density in the buffer. The average value of the eleven mea-
surements was considered as the pKa of the compound with respect
to oximino functionality. Thermospectronic Unicam 300 UV–Visible
double beam spectrophotometer with quartz cells of 10 mm were
used for spectrometric analysis. The quartz cells were attached to
a thermostatic water bath (Julabo) for maintaining the constant
temperature (25 1.0 °C). The pH values of the buffers were deter-
mined using a Mettler-Toledo Seven Easy pH meter equipped with
Inlab@ Expert Pro glass electrode with an accuracy of 0.01 units.
The pH meter was calibrated at 25 °C using the two point calibra-
tion method with commercially available Mettler-Toledo standard
buffer solutions pH 7.00 and 9.21.
Reagents: Freshly prepared standard solutions of oximes
(5 ꢁ 10ꢀ3 M) in distilled water were used as stock solutions. Buffer
solutions of appropriate pH were prepared according to the re-
ported method.28 Solutions of the oximes in 0.1 M hydrochloric
acid and 0.1 M sodium hydroxide were used for determining the
analytical wavelength of the undissociated and dissociated forms
respectively.
Spectroscopic characterization of the synthesized compounds:
Compound 2: Tan colored power. IR (KBr) mmax (cmꢀ1) 3499,
3397, 3258, 3064, 1826, 1689, 1563, 1482, 993, 799, 645; 1H
NMR (400 MHz, DMSO-d6) d 7.34 (m, 1H, Ar-H), 7.65 (s, 1H,
–CH@NOH), 8.08 (1H, Ar-H), 8.28 (m, 1H, Ar-H), 8.83 (d, J = 2.4 Hz,
1H, Ar-H), 10.38 (s, –NH), 12.28 (s, –OH); 13C NMR (100 MHz,
DMSO-d6)
160.85; ESI-MS: m/z 166.0837 [M+] (calcd for C7H7N3O2 165.15).
Compound 3a: Brown powder. IR (KBr)
d
123.63, 127.06, 135.12, 141.51, 143.72, 144.65,
m
max (cmꢀ1) 3403, 3077,
2970, 1710, 1589, 1564, 992, 825, 676; 1H NMR (400 MHz, DMSO-
d6) d 5.27 (s, 4H, –CH2), 7.72 (s, 2H, Ar-H), 8.13 (m, 2H, Ar-H), 8.60
(d, J = 8.4 Hz, 2H, Ar-H), 8.75 (d, J = 6.4 Hz, 2H, Ar-H), 9.48 (s, 1H,
Ar-H), 9.58 (s, 1H, –CH@NOH), 11.18 (s, 1H, –NH), 11.23 (s, 1H,
–NH), 12.57 (s, 1H, –OH), 12.58 (s, 1H, –OH); 13C NMR (100 MHz,
5.6. Synthetic procedure
5.6.1. Synthesis of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide
(2)
In
a 50 mL round bottom flask, 3-aminopyridine (2.82 g,
DMSO-d6)
d 60.12, 128.03, 128.33, 135.69, 135.88, 136.06,
30 mM) was dissolved in a mixture of 10 mL water and 2.5 mL
conc. hydrochloric acid at room temperature. In another 250 mL
round bottom flask, chloral hydrate (4.98 g, 30.12 mM) was dis-
solved in 70 mL of water and sodium sulfate (50 g, 352.01 mM)
was added to it. The reaction mixture was stirred at room temper-
ature. Previously prepared solution of 3-aminopyridine was added
to the reaction mixture of chloral hydrate followed by addition of
hydroxylamine hydrochloride (6.6 g, 95 mM). The reaction mixture
was then refluxed on an oil bath and stirred for 10 min. It was then
brought to the room temperature and neutralized by adding 28–
30% aqueous ammonia solution till the product, 2-(hydroxyimi-
no)-N-(pyridin-3-yl)acetamide was precipitated out. Tan colored
solid product was filtered off, washed repeatedly by cold water
and dried under vacuum (3.21 g; yield: 65%; mp: 186–188 °C). Pur-
ity of the product was checked by pre-coated silica on alumina
sheets TLC plates (Merck), (ethyl acetate/hexane 8:2, Rf = 0.31).
136.63, 137.65, 138.55, 138.77, 140.26, 142.84, 142.94, 161.20,
161.49; ESI-MS: m/z 192.3547 (calcd for C9H10N3O+2 192.19).
Compound 3b: Light brown powder. IR (KBr) mmax (cmꢀ1) 3425,
3076, 2982, 1701, 1611, 1563, 986, 823, 695; 1H NMR (400 MHz,
DMSO-d6) d 2.6 (m, 2H, –CH2–), 4.74 (m, 4H, –CH2–), 7.73 (s, 2H,
Ar-H), 8.13 (m, 2H, Ar-H), 8.61 (d, J = 9.2 Hz, 2H, Ar-H), 8.82 (d,
J = 5.6 Hz, 2H, Ar-H), 9.52 (s, 2H, –CH@NOH), 11.21 (s, 2H, –NH),
12.57 (s, 2H, –OH); 13C NMR (100 MHz, DMSO-d6) d 31.82, 58.14,
128.21, 135.06, 135.54, 138.60, 139.76, 142.95, 161.38; ESI-MS:
m/z 372.4724 (calcd for C17H20N6O24+ 372.38).
Compound 3c: Light brown powder. IR (KBr) mmax (cmꢀ1) 3455,
3086, 2976, 1701, 1614, 1565, 993, 830, 713; 1H NMR (400 MHz,
DMSO-d6) d 1.96 (m, 4H, –CH2–), 4.69 (m, 4H, –CH2–), 7.72 (s,
2H, Ar-H), 8.11 (m, 2H, Ar-H), 8.61 (d, J = 9.6 Hz, 2H, Ar-H), 8.83
(d, J = 6.0 Hz, 2H, Ar-H), 9.49 (s, 2H, –CH@NOH), 11.22 (s, 2H,
–NH), 12.58 (s, 2H, –OH); 13C NMR (100 MHz, DMSO-d6) d 27.11,
Please cite this article in press as: Karade, H. N.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.03.023

8
H. N. Karade et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
60.44, 128.15, 134.96, 135.44, 138.52, 139.67, 142.99, 161.43;
The authors are also thankful to Dr. D.K. Dubey and Dr. B.K. Bhat-
tacharya for their valuable suggestions.
ESI-MS: m/z 386.5322 (calcd for C18H22N6O24+ 386.40).
Compound 3d: Brown powder. IR (KBr) m
max (cmꢀ1) 3445, 3063,
2977, 1704, 1632, 1561, 1000, 829, 683; 1H NMR (400 MHz, DMSO-
d6) d 1.13 (m, 2H, –CH2–), 1.93 (m, 4H, –CH2–), 4.62 (m, 4H, –CH2–),
7.74 (s, 2H, Ar-H), 8.11 (m, 2H, Ar-H), 8.61 (d, J = 8.4 Hz, 2H, Ar-H),
8.85 (d, J = 6.0 Hz, 2H, Ar-H), 9.48 (s, 2H, –CH@NOH), 11.22 (s, 2H,
–NH), 12.58 (s, 2H, –OH); 13C NMR (100 MHz, DMSO-d6) d 21.72,
29.83, 60.82, 128.06, 134.84, 135.46, 138.43, 139.63, 142.94,
161.37; ESI-MS: m/z 400.6017 (calcd for C19H24N6O24+ 400.43).
Compound 3e: Light brown powder. IR (KBr) mmax (cmꢀ1) 3392,
3067, 2866, 1707, 1639, 1564, 995, 826, 682; 1H NMR (400 MHz,
DMSO-d6) d 1.33 (m, 4H, –CH2–), 1.90 (m, 4H, –CH2–), 4.62 (m,
4H, –CH2–), 7.74 (s, 2H, –CH@NO–), 8.10 (m, 2H, Ar-H), 8.62 (d,
J = 8.4 Hz, 2H, Ar-H), 8.85 (d, J = 6.0 Hz, 2H, Ar-H), 9.47 (s, 2H,
–CH@NOH), 11.21 (s, 2H, –NH), 12.57 (s, 2H, –OH); 13C NMR
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.023.
References and notes
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24.57, 30.24, 61.05, 128.04, 134.75,
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135.42, 138.39, 139.60, 142.91, 161.34; ESI-MS: m/z 414.2576
(calcd for C20H26N6O24+ 414.46).
Compound 3f: Tan powder. IR (KBr) mmax (cmꢀ1) 3436, 3059,
2978, 1706, 1657, 1557, 989, 809, 679; 1H NMR (400 MHz,
DMSO-d6) d 1.28 (m, 6H, –CH2–), 1.88 (m, 4H, –CH2–), 4.61 (m,
4H, –CH2–), 7.72 (s, 2H, Ar-H), 8.10 (m, 2H, Ar-H), 8.62 (d,
J = 9.6 Hz, 2H, Ar-H), 8.84 (d, J = 6.0 Hz, 2H, Ar-H), 9.46 (s, 2H,
–CH@NOH), 11.20 (s, 2H, –NH), 12.57 (s, 2H, –OH); 13C NMR
(100 MHz, DMSO-d6) d 25.44, 28.08, 30.89, 31.18, 61.65, 128.52,
135.27, 135.89, 138.90, 140.03, 143.44, 161.87; ESI-MS: m/z
428.3424 (calcd for C21H28N6O24+ 428.48).
Compound 3g: Off white powder. IR (KBr) mmax (cmꢀ1) 3140,
3065, 2975, 1702, 1590, 1559, 990, 809; 1H NMR (400 MHz,
DMSO-d6) d 6.15 (s, 4H, benzylic –CH2), 7.36 (m, 2H, Ar-H), 7.57
(m, 2H, Ar-H), 7.69 (s, 2H, Ar-H), 8.13 (m, 2H, Ar-H), 8.70 (d,
J = 9.2 Hz, 2H, Ar-H), 8.83 (d, J = 6.0 Hz, 2H, Ar-H), 9.37 (s, 2H,
–CH@NOH), 11.25 (s, 2H, –NH), 12.55 (s, 2H, –OH); 13C NMR
(100 MHz, DMSO-d6) d 60.69, 128.45, 130.43, 130.52, 132.42,
135.09, 135.24, 138.86, 139.85, 142.94, 161.46; ESI-MS: m/z
433.3020 (calcd for C22H22N6O24+ 434.45).
´
Amitai, G.; Green, C.; Radic, B.; Fokin, V. V.; Sharpless, K. B.; Taylor, P. J. Biol.
Compound 3h: Light brown powder. IR (KBr) mmax (cmꢀ1) 3428,
3083, 2973, 1701, 1588, 1556, 1502, 995, 807, 736, 673; 1H NMR
(400 MHz, DMSO-d6) d 5.94 (s, 4H, benzylic –CH2), 7.57 (m, 3H,
Ar-H), 7.69 (m, 3H, Ar-H), 8.14 (m, 2H, Ar-H), 8.66 (m, 2H, Ar-H),
8.97 (d, J = 6.0 Hz, 2H, Ar-H), 9.53 (s, 2H, Ar-H), 11.25 (s, 2H,
–NH), 12.56 (s, 2H, –OH); 13C NMR (100 MHz, DMSO-d6) d 63.25,
128.42, 129.27, 129.83, 130.15, 134.90, 135.23, 135.30, 138.74,
139.80, 142.96, 161.45; ESI-MS: m/z 434.3869 (calcd for
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C
22H22N6O24+ 434.45).
Compound 3i: Light brown powder. IR (KBr) mmax (cmꢀ1) 3424,
3115, 2972, 1704, 1589, 1561, 998, 812, 730, 673; 1H NMR
(400 MHz, DMSO-d6) d 5.91 (s, 4H, benzylic –CH2), 7.60 (m, 4H,
Ar-H), 7.68 (s, 2H, Ar-H), 8.12 (m, 2H, Ar-H), 8.63 (d, J = 8.8 Hz,
2H, Ar-H), 8.95 (d, J = 6.0 Hz, 2H, Ar-H), 9.55 (s, 2H, –CH@NOH),
11.18 (s, 2H, –NH), 12.56 (s, 2H, –OH); 13C NMR (100 MHz,
DMSO-d6)
139.78, 142.97, 161.45; ESI-MS: m/z 433.3914 (calcd for
22H22N6O24+ 434.45).
d 63.08, 128.45, 129.62, 135.14, 135.28, 138.76,
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A
Acknowledgments
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1955; Vol. I, p 138.
Authors thank to the Director, Defence Research and Develop-
ment Establishment, Gwalior for his keen interest in this work.
Please cite this article in press as: Karade, H. N.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.03.023