Synthesis and in vitro kinetic evaluation of N-thiazolylacetamido monoquaternary pyridinium oximes as reactivators of sarin, O-ethylsarin and VX inhibited human acetylcholinesterase (hAChE)

Article abstract of DOI:10.1016/j.bmc.2015.05.027

Presently available medications for treatment of organiphosphorus poisoning are not sufficiently effective due to various pharmacological and toxicological reasons. In this regard, herein we report the synthesis of a series of N-thiazolylacetamide monoquaternary pyridinium oximes and its analogs (1a-1b to 6a-6b) with diversely substituted thiazole ring and evaluation of their in vitro reactivation efficacies against nerve agent (sarin, O-ethylsarin and VX) inhibited human erythrocyte acetylcholinesterase (hAChE). Reactivation kinetics was performed to determine dissociation constant (K_D), reactivity rate constant (k_r) and the second order rate constant (k_r2) for all the compounds and compared their efficacies with commercial antidotes viz. 2-PAM and obidoxime. All the newly synthesized oximes were evaluated for their physicochemical parameters (pKa) and correlated with their respective reactivation efficacies to assess the capability of the oxime reactivator. Three of these novel compounds showed promising reactivation efficacies toward OP inhibited hAChE. Molecular docking studies were performed in order to correlate the reactivation efficacies with their interactions in the active site of the AChE.

Full text of DOI:10.1016/j.bmc.2015.05.027

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro kinetic evaluation of N-thiazolylacetamido
monoquaternary pyridinium oximes as reactivators of sarin,
O-ethylsarin and VX inhibited human acetylcholinesterase (hAChE)
a
Aditya Kapil Valiveti a, Uma M. Bhalerao a, Jyotiranjan Acharya a, , Hitendra N. Karade ,
⇑
Badri Narayan Acharya a, G. Raviraju a, Anand K. Halve b, Mahabir Parshad Kaushik a
a Process Technology Development Division, Defence Research & Development Establishment, Jhansi Road, Gwalior 474 002, India
b School of Studies in Chemistry, Jiwaji University, Gwalior, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Presently available medications for treatment of organiphosphorus poisoning are not sufficiently
effective due to various pharmacological and toxicological reasons. In this regard, herein we report the
synthesis of a series of N-thiazolylacetamide monoquaternary pyridinium oximes and its analogs
(1a–1b to 6a–6b) with diversely substituted thiazole ring and evaluation of their in vitro reactivation effi-
cacies against nerve agent (sarin, O-ethylsarin and VX) inhibited human erythrocyte acetylcholinesterase
(hAChE). Reactivation kinetics was performed to determine dissociation constant (KD), reactivity rate
constant (kr) and the second order rate constant (kr2) for all the compounds and compared their efficacies
with commercial antidotes viz. 2-PAM and obidoxime. All the newly synthesized oximes were evaluated
for their physicochemical parameters (pKa) and correlated with their respective reactivation efficacies to
assess the capability of the oxime reactivator. Three of these novel compounds showed promising
reactivation efficacies toward OP inhibited hAChE. Molecular docking studies were performed in order
to correlate the reactivation efficacies with their interactions in the active site of the AChE.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 29 January 2015
Revised 13 May 2015
Accepted 15 May 2015
Available online xxxx
Keywords:
Acetylcholinesterase
Thiazolylacetamides
Nerve agents
Reactivation
Kinetics
1. Introduction
From the mechanism point of view the toxic OP compounds
irreversibly bind to the serine hydroxyl group at the active site of
Organophosphorus compounds (OPs) have highly been indus-
trialized owing to their wider applicability in the field of veterinary
and human medicine, in the agriculture as pesticides and also have
been misused for military purpose. The major revolution in the
research of OPs began at the dawn of World War II that led to
the accidental discoveries of various deadliest chemicals called
nerve agents viz. sarin, soman, Tabun and VX1 (Fig. 1). Owing to
their acute toxicity toward mammalian systems these nerve agents
have been used as chemical weapons of mass destruction in wars2
as well as in the terrorist activities worldwide.3 In addition, self-
poisoning with OP pesticides is another important and current
issue of global concern. Annually 300,000 deaths (either acciden-
tally or intentionally) have been recorded due to the acute OP pes-
ticide intoxications.4 All these consequences strictly demand an
effective medical treatment regimen against the acute OP-
poisoning.
enzyme AChE (AChE; E.C. 3.1.1.7). This results in elevated levels
of ACh (acetylcholine; a neurotransmitter) at the pre- and post-
synaptic junctions of the nerve ends. The unwanted accretion of
ACh levels in peripheral and central nervous system leads to a
variety of cholinergic manifestation that includes depression of
respiratory center followed by peripheral neuromuscular blockade
causing respiratory paralysis and death.5 The behavior of
phosphylated (represents, phosphonylation and phosphorylation)
AChE is highly dependent on the structural variations and toxicity
of the nerve agent used.6 After inhibition, the OP–AChE adduct
undergoes several biochemical pathways7 viz., hydrolysis (with
an effective nucleophilic attack at the P–O bond), spontaneous
reactivation and aging process (in case of soman poisoning).8
However, the recovery of original AChE activity to its normal func-
tioning is highly essential to avoid the cholinergic break down and
death.9
In general, the immediate therapeutic intervention for acute OP
nerve agent poisoninginvolves administration of an anti-muscarinic
agent (atropine),10 anti-convulsant (Diazepam)11 and enzyme reac-
tivator oximes (Fig. 2; 2-PAM, Obidoxime and HI-6).12,13
⇑
Corresponding author. Tel.: +91 751 2390205, +91 751 2340245; fax: +91 751
2341148.
E-mail addresses: jracharya01@gmail.com, j.acharya@drde.drdo.in (J. Acharya).
http://dx.doi.org/10.1016/j.bmc.2015.05.027
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Valiveti, A. K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.027

2
A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
the present study, we have synthesized various thiazolylac-
etamides sandwiched between substituted phenyl group and the
quaternary pyridinium moieties. These aromatic moieties in the
reactivator were ensembled in order to facilitate various non-cova-
lent interactions with the aromatic residues lined up along the rim
of the gorge in the peripheral anionic site of AChE. Additionally the
quaternary nitrogen in the pyridinium ring enables the cation-
p
interactions with the indole and imidazole based amino acid (Trp
& His) residues present at the base of the AChE gorge.40,41 The
hydrogen bond donor and acceptor capability of the acetamido
linkages between the thiazole and pyridinium rings may facilitate
significant electrostatic interactions (hydrogen bonding, salt bridge
and hydrophobicity) inside the narrow confines of the gorge which
may aid in achieving proper orientations of the reactivator toward
the phosphylated complex of AChE. Further, in vitro reactivation
efficacies of the newly prepared oximes were tested against human
erythrocyte AChE (hAChE) inhibited by three structurally different
OPs viz. sarin, O-ethylsarin and VX.
Figure 1. Structure of the organophosphorus nerve agents.
3. Results
There are several factors which limit the scope of oxime therapy
in the context of acute OP poisoning. Being quaternary ammonium
salts, majority of these oximes are unable to cross the blood brain
barrier.14,15 Further, oxime treatments are ineffective toward the
aged enzyme. Moreover, theintrinsic toxicitiesof these oximes exert
deleterious side effects.16,17 All these drawbacks strictly underscore
the need of a versatile antidotal treatment in the event of acute OP
nerve agent and pesticide poisoning.18 Over past few decades,
numerous structurally divergent oxime reactivators were synthe-
sized and evaluated for their efficacies against acute OP poison-
ing.19–27 As newly synthesized compounds cannot be evaluated on
humans for ethical reasons hence studies on animals and human
enzymes are the mainstay for understanding the pharmacological
and toxicological interactions between the antidotes and the OP–en-
zyme complexes.28–30 In the development of versatile and effective
medical countermeasures against acute OP poisoning, extensive
variations in the structural features of oxime reactivators are needed
for further refinement to shape-up a broad spectrum antidote.31,32
3.1. Reactivation of sarin inhibited hAChE
It wasobservedthatthecompoundscontainingoximegroupat4-
position (1a–6a) of the pyridinium ring have shown better reactiva-
tion efficacy as compared to the compounds containing oxime group
at 3-position (1b–6b) in case of sarin inhibition (Table 1). Variations
in the KD values of the oximeshave beennoticed and corroboratedby
their respective kr2 values (Table 1). Among all the 4-pyridinium
compounds, oximes 6a, 5a and 3a have shown considerable reacti-
vation efficacies than other oximes as evident by their lower KD
(42.0 lM, 46.9 lM, 67.5 lM) and higher second order rate constant
values kr2 (1.70 mMꢀ1 minꢀ1, 1.60 mMꢀ1 minꢀ1, 1.04 mMꢀ1 minꢀ1).
However, in case of 3-pyridinium oximes (1b–6b), higher KD values
have been observed which revealed their poor affinity and lower
reactivity toward the OP–AChE adduct and hence resulted in lower
kr2 values ranging from 0.14 to 0.68 mMꢀ1 minꢀ1. Compound 3b
was the least efficacious and was unable to show any reactivation
efficacy among all the oximes studied. Further, none of the synthe-
sized oximes have been found to be effective than the standard oxi-
2. Synthesis and chemistry of N-thiazolylacetamido pyridinium
oximes
mes 2-PAM (kr2
;
2.95 mMꢀ1 minꢀ1
)
and obidoxime (kr2;
6.38 mMꢀ1 minꢀ1). The overall reactivation efficacy order for all
the oximes against sarin inhibited AChE was obidoxime > 2-
PAM > 6a > 5a > 3a > 1a > 2a > 5b > 1b > 4a > 6b > 2b > 4b > 3b.
In continuation to our work on the development of antidotes
against OP poisoning,19,21 herein we report the synthesis and
in vitro evaluation of a series of mono-pyridinium oximes having
substituted thiazolylacetamides as side chain linkers. Thiazoles
are aromatic and have most therapeutic access as drug candidates
for various diseases.31–36 These heterocyclic entities are capable of
binding with specific enzyme targets and act as reversible inhibi-
tors in the treatment of various neurological disorders.37–39 In
3.2. Reactivation of O-ethylsarin inhibited hAChE
In case of O-ethylsarin inhibited hAChE, except 4a, majority of the
oximes (having oxime at 4-position, 1a–6a) have shown an
appreciable reactivity toward the OP–AChE complex. It was
observed that the oximes 6a, 1a and 5a have higher reactivation
efficacy as evidenced by their lower KD (6a; 14.7
l
M, 1a; 29.9
lM
and 5a; 34.9
l
M) and higher kr2 (6a; 4.31 mMꢀ1 minꢀ1, 1a;
2.66 mMꢀ1 minꢀ1and 5a; 2.05 mMꢀ1 minꢀ1) values. In termsofboth
affinity and ability of the reactivator, oxime 6a revealed better
reactivation efficacy as compared to the standard oximes 2-PAM
(kr2; 2.19 mMꢀ1 minꢀ1) and obidoxime (kr2; 3.9 mMꢀ1 minꢀ1).
Further, oximes 3a (50.6 lM) and 2a (37.8 lM) have shown slightly
lower affinities toward the phosphylated AChE in comparison to the
oximes 6a, 1a and 5a. However, these oximes have recorded
lower reactivation efficacies as indicated by their lower kr2 values
3a: 1.77 mMꢀ1 minꢀ1 and 2a: 1.25 mMꢀ1 minꢀ1
,
respectively
(Table 1). Among the 4-positioned oximes, oxime 4a was found to
be least effective reactivator with higher KD (90.6
affinity) and lower kr2 (0.66 mMꢀ1 minꢀ1) values.
lM; i.e., lower
Figure 2. Structure of the pyridinium oxime reactivators.
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A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Table 1
Reactivation constants for oxime-induced reactivation of OP-inhibited hAChE
Oxime
KD
l
M ( SE)
kr minꢀ1 ( SE)
O-Ethylsarin
kr2 (mMꢀ1 minꢀ1
O-Ethylsarin
)
Sarin
O-Ethylsarin
VX
Sarin
VX
Sarin
VX
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
2-PAM
Obid.
63.0 (11.5)
115.0 (40.6)
86.9 (10.4)
682.7 (27.7)
67.5 (25.2)
nd
83.7 (25.4)
641.0 (429)
46.9 (1.3)
109.2 (22.7)
42.0 (4.4)
75.5 (24.8)
28.8 (7.6)
19.5 (4.6)
29.9 (7.3)
87.6 (18.9)
37.8 (9.7)
762.6 (129.9)
50.6 (14.1)
nd
90.6 (25.6)
nd
34.9 (2.9)
104.5 (22.3)
14.67 (3.0)
nd
15.4 (2.2)
98.3 (20.9)
21.4 (5.1)
350.6 (111.7)
57.2 (15.5)
145.6 (54.8)
73.6 (17.8)
113.8 (14.9)
12.9 (2.9)
97.7 (25.2)
17.0 (4.4)
58.4 (12.6)
17.77 (69.7)
14.4 (3.0)
0.059 (0.003)
0.076 (0.007)
0.082 (0.002)
0.129 (0.003)
0.070 (0.006)
nd
0.053 (0.004)
0.092 (0.029)
0.075 (0.0005)
0.074 (0.004)
0.072 (0.002)
0.046 (0.004)
0.085 (0.005)
0.125 (0.005)
0.080 (0.004)
0.079 (0.004)
0.047 (0.003)
0.129 (0.011)
0.089 (0.005)
nd
0.059 (0.004)
nd
0.072 (0.001)
0.079 (0.004)
0.063 (0.002)
nd
0.060 (0.002)
0.095 (0.005)
0.057 (0.003)
0.094 (0.011)
0.067 (0.004)
0.056 (0.005)
0.055 (0.003)
0.097 (0.003)
0.067 (0.002)
0.086 (0.006)
0.072(0.003)
0.054 (0.003)
0.070 (0.003)
0.129 (0.005)
0.95
0.66
0.95
0.19
1.04
nd
0.63
0.14
1.60
0.68
1.70
0.60
2.95
6.38
2.66
0.89
1.25
0.17
1.77
nd
0.66
nd
2.05
0.76
4.31
nd
3.92
0.96
2.70
0.27
1.17
0.38
0.75
0.85
5.19
0.88
4.23
0.92
3.92
9.03
34.7 (7.1)
20.2 (5.8)
0.076 (0.003)
0.079 (0.004)
2.19
3.9
nd: not determined.
Decrements in the reactivation efficacies were observed in case
of 3-pyridinium oxime derivatives 1b–6b. Majority of the oximes
(3b, 4b and 6b) failed to reactivate the OP inhibited hAChE in the
60 min course of the reactivation due to their poor affinity toward
the tetrahedral complex of OP and hAChE. Again, oximes 1b, 5b and
2b showed poor reactivation efficacies as represented by their
were of paramount importance to understand the mechanism of
oxime and inhibitor interactions within the AChE active site.42 In
this investigation, three structurally divergent OP inhibitors have
been used to study variations in the reactivation efficacy of the
newly synthesized oximes. The reactivation kinetics data were
generated by performing experiments under identical experimen-
tal protocols as reported in the literature 6.
lower second order rate constant kr2 values (0.89 mMꢀ1 minꢀ1
,
0.76 mMꢀ1 minꢀ1, 0.17 mMꢀ1 minꢀ1). Among these, 2b was the
The findings of the present study demonstrated that the posi-
tion of the oxime group in the pyridinium ring was a significant
factor in assessing the affinity, ability and the specific reactivity
of the oxime reactivator. Marked differences were noticed with
respect to their kinetic parameters among the tested oximes. The
improved reactivation efficacies in terms of affinity toward the
phosphylated AChE complex was observed in case of VX and O-
ethylsarin inhibited hAChE compared to that of sarin inhibition.
Majority of the compounds containing oxime at 4-position (1a–
6a) have shown greater reactivation efficacy as compared to the
compounds containing oxime at 3-position (1b–6b) of the pyri-
dinium ring. This finding inferred that the position of the oxime
moiety in the pyridinium ring was a critical factor in deciding
the ability of the oxime as reactivator. The overall reactivating
potencies of all the tested oximes against the three OP nerve agents
followed the order VX > O-ethylsarin > sarin. The percentage reac-
tivation efficacy for all the tested oximes (1a–6a and 1b–6b) in
comparison to 2-PAM and obidoxime at 60 minute against the
nerve agents (sarin, O-ethylsarin and VX) has been illustrated in
Figure 4.
It was found that the standard obidoxime has shown better
reactivation efficacy toward all the three nerve agents inhibited
hAChE. Being bis-pyridinium in nature, the presence of a second
quaternary center in the obidoxime molecule might be responsible
for their higher binding affinity toward OP–AChE complex in the
active site of the enzyme gorge. This has resulted in higher rate
constant and reactivity of obidoxime toward OP inhibited AChE.
In contrast, in the present investigation all the studied oximes
were of mono-quaternary in nature. These oximes were chosen
with an aim of enhancing the hydrophobicity of the reactivator
and also to observe the presence of non-covalent interactions (if
least effective reactivator with the highest KD (762.6
3.3. Reactivation of VX inhibited hAChE
lM) value.
The ability of all the tested oximes to reactivate the VX inhib-
ited hAChE has been presented in Table 1. It was worth noticing
that in case of VX inhibited hAChE, reactivation efficacy was
significantly higher compared to other OP agents (sarin and
O-ethylsarin) used in this study. Among the 4-positioned oximes
(1a–6a), oximes 5a and 6a have shown higher reactivation effica-
cies as revealed by their lower KD values: 12.89
lM and 17.0 lM
and higher kr2 values: 5.19 mMꢀ1 minꢀ1 and 4.23 mMꢀ1 minꢀ1
,
respectively than the standard oxime 2-PAM. In addition, oximes
1a (kr2; 3.92 mMꢀ1 minꢀ1) and 2a (kr2; 2.70 mMꢀ1 minꢀ1) have
also shown comparable reactivation efficacies to that of 2-PAM.
Oxime 4a with highest dissociation constant KD (73.6 lM) and
lowest kr2 values (0.75 mMꢀ1 minꢀ1) was found to be least effec-
tive in case of VX inhibited hAChE. Further, 3-pyridinium oximes
(1b–6b) demonstrated lower reactivation efficacies as evident
from their higher KD and lower kr2 values. Among these, oximes
1b, 6b, 5b and 4b have shown much less kr2 (0.96 mMꢀ1 minꢀ1
;
0.92 mMꢀ1 minꢀ1; 0.88 mMꢀ1 minꢀ1; 0.85 mMꢀ1 minꢀ1, respec-
tively) values revealing their insignificant efficacies toward VX
inhibited hAChE. Oxime 2b was found to be the least effective
reactivator among all the synthesized oximes with highest KD
(350.6 l
M) and least kr2 (0.27 mMꢀ1 minꢀ1) values.
The relative time and concentration dependent reactivation
profile of oxime 5a and the plot of kobs against oxime concentration
for all the three OP inhibitors were depicted in Figure 3.
any) viz. cation–p, p–p and hydrogen boding interactions with
4. Discussion
the aromatic amino acid residues present inside the gorge of AChE.
The variations observed in the reactivation abilities of the oxi-
mes used in this study might be due to the discrepancy in the occu-
pied molecular volumes of the tested OP inhibitors in the active
site of AChE. The difference in the molecular volumes could be
due to the presence of O-alkyl substituent in the OP inhibitors. In
case of VX and O-ethylsarin, after the departure of their respective
Determination of kinetic parameters and in vitro experiments
has extensively been used in the assessment of the reactivation
efficacies of newly synthesized oximes. These kinetic parameters
have also been used to establish the correlation between the struc-
ture of the inhibitor and reactivation ability of new oxime reactiva-
tors.6,28,42–44 Therefore determination of the kinetic parameters
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A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 3. Relative time and concentration dependent reactivation profile of oxime 5a against (A) sarin, (B) O-ethylsarin and (C) VX inhibited human AChE (A–C).
Corresponding plot of kobs against oxime concentration [5a] (D–F). Data are means of SE, n = 2.
leaving groups the formed OP–AChE adducts were structurally
same as compared to the sarin-AChE conjugate (Scheme 1).
Hence, the active site of the AChE gorge was mainly occupied by
the bulky isopropyl group of the sarin followed by the O-ethyl
group of the VX and O-ethylsarin.44 Therefore the active site of
AChE inhibited by VX and O-ethylsarin was least occupied and
hence there might be greater access of the incoming nucleophile
(oxime) toward the P-atom. This could be the plausible explana-
tion that majority of the oximes have shown greater affinity (KD)
and ability (kr2) to reactivate the VX followed by O-ethylsarin
inhibited hAChE (oxime 5a, 6a and 1a; Table 1). However, no sim-
ilar trends have been observed in the reactivation profiles of the
tested oximes in case of VX and O-ethylsarin inhibition. This could
be due to unknown pharmacological or toxicological implications
which remain to be explored.
In the past, several reactivators linked with heterocyclic side
chains to the pyridinium ring were reported in order to augment
various interactions of the oxime moiety with
p-stacking clouds
of the amino acid residues present inside the 20 Å deep gorge of
AChE. Docking studies and calculation of binding energies
performed on these type oximes revealed the importance of the
various non-covalent interactions viz. hydrogen bonding, cation–
p
and
p–p interactions that influenced the reactivation efficacy
of the reactivators.20,23–25 In this study, heteroaromatic thiazoles
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A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 4. Percentage reactivation efficacies (%) of the oximes (1a–6a and 1b–6b) in comparison to 2-PAM and obidoxime against OP-inhibited hAChE. Oxime concentration:
1 mM, pH: 7.4 and temperature: 37 °C.
Scheme 1. Comparative inhibition process of AChE by (a) VX (b) O-ethylsarin and (c) sarin.
Table 2
possessing extended conjugation with aromatic rings were incor-
porated in to the pyridinium oximes in order to observe the effect
of aromatic clouds on the orientation and reactivation of the reac-
tivator. Oximes 5a and 6a showed highest reactivation efficacy in
case of VX and O-ethylsarin inhibited AChE as indicated by their
lowest KD value and highest second order rate constant kr2 than
any other tested oximes (Table 1). Therefore, oximes with ester
substituted thiazole linker might have imparted a better orienta-
tion toward the phosphylated AChE complex in the active site of
the gorge. This could be the plausible reason that among all the
synthesized oximes, the ester substituted thiazole linkers (5a and
6a) have shown superior reactivation efficacies and improved
affinities in case of O-ethylsarin and VX inhibited AChE.
Therefore oximes 5a and 6a were further subjected for docking
studies with nerve agent modified human AChE in order to under-
stand their superior reactivation efficacy. The average binding
energies for 5a and 6a were presented in Table 2. Both the oximes
have interacted in a similar manner with the active site of the
inhibited enzyme through hydrogen bonding (H-bonding). In
Figure 5, it can be seen that, the flexible amide linker attached to
the thiazole moiety facilitates the orientations of the ligand in
the active site of the AChE mainly through H-bonding (green dot-
ted lines). The H-bonding interactions are primarily observed
between various amino acid residues of AChE and the amide linker
as well as the carbonyl oxygen of the ester tail linked to the
thiazole moiety.
Binding energy values of oxime 5a and 6a bound to the sarin, VX/O-ethylsarin
modified hAChE
Oxime
Binding energy (Kcal/mol)
Sarin inhibited AChE VX/Ethyl sarin inhibited AChE
5a
6a
ꢀ9.76
ꢀ11.74
ꢀ11.31
ꢀ10.69
In case of sarin modified AChE, it was noticed that the oxime 5a
has two H-bonding interactions with TYR 337 residue. Another H-
bonding was observed between the nitrogen atom of the oxime
group of 5a and PHE 295 (Fig. 5A). 6a has one H-bonding between
TYR 337 and the nitrogen atom of the thiazole ring. In addition
another interaction was observed between HIS 447 and the nitro-
gen atom of oxime functionality (Fig. 5B). Even having one H-bond
less, 6a has shown better binding energy than 5a. The greater size
of 5a responsible for higher conformation energy resulted due to
steric hindrance with nearby amino acid residues of AChE than
6a. This might be the reason for the greater reactivation efficacy
of 6a in case of sarin inhibited hAChE. This was further manifested
by better rate constant of 6a against sarin inhibited hAChE. In case
of VX/O-ethylsarin modified AChE, 5a has H-bondings between the
carbonyl group of the amide linkage and the NH dipole of the
amide group with amino acid residues TYR 337 and TYR 124,
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A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 5. Top-scored docking poses of compounds 5a and 6a. (A) Compound 5a bound to sarin modified hAChE; (B) compound 6a bound to sarin modified hAChE; (C)
compound 5a bound to VX/O-ethylsarin modified hAChE; (D) compound 6a bound to VX/O-ethylsarin modified hAChE.
respectively. Further, an additional interaction was observed
between TRP 86 and the carboxylic oxygen of the ester group pre-
sent in the tail of the reactivator (Fig. 5C). Oxime 6a has H-bonding
with the amino acid residues TYR 72 and TYR 341 respectively
(Fig. 5D). Compounds 5a and 6a have shown comparably similar
reactivation efficacies in case of VX and O-ethylsarin modified
hAChE. This was indicated by the close binding energies obtained
for both the oximes (Table 2).
Compounds 5a and 6a have also shown similar binding energy
and reactivation efficacy in case of VX/O-ethylsarin modified
hAChE but different binding energy and reactivation efficacy
against sarin inhibited hAChE. This may be explained on the basis
of their molecular sizes. It is worth mentioned here that VX/O-
ethyl sarin adduct of SER 203 are smaller in size by one methyl
group compared to sarin modified AChE. On the other hand, the
oxime 5a is one methylene group larger than 6a. Therefore the
effect of size of 5a is marginalized in case of VX/O-ethyl sarin but
realized in sarin modified hAChE. This has been apparent by the
reactivation kinetics of both 5a and 6a. Overall, oxime 6a has
shown better binding energies with sarin and VX/O-ethylsarin
modified AChE. This was further reflected by its higher reactivation
efficacies against the three tested inhibitors.
Among the 4-positioned oximes, 5a, 6a and 1a have compara-
tively lower inhibition potencies as reflected by their higher IC50
values. Oximes 2a and 3a (0.99 and 0.97 mM) with least IC50 values
revealed their dominant inhibition character toward the enzyme
AChE and this might be responsible for their lower reactivation
efficacies than the other tested oximes. In upshot, oximes 5a, 6a
and 1a with lower inhibitory activity have demonstrated higher
reactivation efficacies against the three OP inhibited AChE
(Fig. 6). However, oxime 2a with higher inhibitory activity was able
to reactivate up to 60% of VX, 50% of O-ethylsarin and 39% of sarin
inhibited human AChE. The overall inhibitory potencies of the
tested oximes followed the order: 3a > 2a > 4a > 5a > 1a > 6a.
The dephosphorylation of phosphorylated enzyme is dependent
on various factors, such as physicochemical properties, steric and
electronic factors, lipophilic–hydrophilic balance and acid dissoci-
ation constant (pKa) of the reactivator.9,17 The reactivation of the
inhibited AChE is brought about by the attack of
a strong
Table 3
IC50 values for the oximes 1a–6a
Reactivator
IC50 (mM)
Inhibition potency of reactivators is an essential criterion while
assessing the efficiency of a drug molecule. Ideally an efficient drug
should not affect the native activity of the enzyme. The IC50 values
were determined for the 4-positioned oximes (1a–6a) (Table 3) in
order to correlate the inhibition potencies of the reactivators with
their reactivation efficiencies.
1a
2a
3a
4a
5a
6a
2.36
0.99
0.97
1.66
2.27
2.49
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A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
(2-nitrobenzoic acid) (DTNB), potassium dihydrogenphosphate,
dipotassium hydrogenphosphate, trizma-base and trizma-hy-
drochloride, solvents (isopropanol and n-hexane of spectroscopic
grade) were purchased from Sigma–Aldrich, USA and used without
further purification. Glycine was obtained from E. Merck (India) and
used without further purification. Chloroacetyl chloride, anhydrous
sodium sulfate, anhydrous sodium bicarbonate and anhydrous
potassium carbonate were purchased from Qualigens, India.
Solvents (ethanol, dichloromethane, acetonitrile, ethyl acetate, ace-
tone, and methanol) were purchased from SD Fine Chemicals
(India) were dried and distilled before use. VX, sarin and O-ethyl-
sarin were prepared in this laboratory with >98% purity (GC and
31P NMR). 2-PAM was prepared according to the method of
Wilson and Ginsburg.45 Obidoxime was synthesized using reported
methods.46 All the synthesized compounds as well as standards
were characterized by IR, 1H NMR, 13C NMR spectral data and ele-
mental analysis. The progress of reaction and purity of the com-
pounds were checked by thin layer chromatography (TLC) using
commercially available pre-coated silica on aluminum sheets pur-
chased from E. Merck, India. The stock solutions of OP inhibitors
were prepared in isopropanol and stored at ꢀ20 °C. Further dilu-
tions of OP inhibitors were prepared in deionized water. Oxime
stock solutions and their further dilutions were prepared freshly
in triple distilled water. All the working solutions were kept on
ice until the completion of the experiment.
Figure 6. IC50 of the oxime 6a.
nucleophile in order to break the covalent bond formed between the
OP and OH group of the serine residue in the catalytic triad of AChE.
For this, ionization of oxime into oximate anion is an essential process
under the physiological conditions in the reactivation of OP inhibited
AChE. Therefore, the pKa values should be taken into consideration
while searching for new oxime reactivators.19,56 The determined
pKa values of all the compounds are given in the Table 4.
It has been observed that the 4-pyridinium oximes have pKa in
the range of 8–8.5, whereas those of the 3-pyridinium oximes were
found in the range of 8.5–8.9. The higher pKa values of the 3-pyri-
dinium oximes (2b, 3b, 4b and 5b) represented the poorer ten-
dency of the oxime dissociation into the oximate anion. This may
be the probable reason that majority of the 3-pyridinium oximes
recorded lower reactivation efficacies and lower second order rate
constants (kr2) against sarin, O-ethylsarin and VX inhibited AChE.
All the 4-pyridinium oximes with lower pKa values exhibited
higher reactivation efficacies. The oximes 5a (pKa: 8.41) and 6a
(pKa: 8.31) showed higher reactivation efficacies which were
reflected in their higher second order rate constants against VX
and O-ethylsarin inhibited AChE.
Human blood samples were collected from the volunteers of
our laboratory as per norms and the hemoglobin free erythrocyte
ghost AChE were prepared using the reported methods.6,47 The
activity of the human AChE was adjusted to that of the whole blood
activity and stored at ꢀ80 °C until use. Prior to start the inhibition
and reactivation experiments, the aliquots of the erythrocyte
ghosts were homogenized on ice-bath with the help of an ultra-
sonic homogenizer (Model 3000, Biologics. Inc., Manassas, USA).
5.2. Synthesis of monoquaternary pyridinium oximes
A three step simple protocol depicted in Scheme 2 was used to
synthesize all the monoquaternary pyridinium oximes (1a–6a and
1b–6b). The following synthetic procedure described for the com-
pound 2a was the representative of the whole series.
5. Experimental
5.1. Materials and methods
5.2.1. Synthesis of 4-phenylthiazol-2-amine
Thiourea, substituted phenacylbromides, 2-aminothiazole,
ethyl 4-chloroacetoacetate, ethyl bromopyruvate, 2-, 3- and 4-
pyridinealdoxime, acetylthiocholine iodide (ATChI), 5,50-dithiobis-
Phenacylbromide (5.0 g, 25 mM) and thiourea (1.9 g, 25 mM)
were taken in a 50 mL round bottom flask equipped with a con-
denser, calcium chloride guard tube and magnetic stirrer. The mix-
ture was dissolved in dry ethanol (30 mL) and stirred at room
temperature for overnight. Excess solvent was distilled off and
the concentrated reaction mixture was slowly poured into a beaker
containing ice-cooled saturated sodium bicarbonate solution. The
yellow colored precipitate obtained was filtered off, washed thor-
oughly with chilled water and dried under vacuum (4.2 g, yield:
95%). TLC (ethyl acetate/hexane (1:3), Rf = 0.14). The product
obtained was used in the next step without further purification.
Table 4
Physicochemical data and general chemical structure of the synthesized oximes
H N
O
S
O
R
X
N
N
N
H
Oxime Oxime position
X
R
Yield (%) Mp (°C) pKa
5.2.2. Synthesis of 2-chloro-N-(4-phenylthiazol-2-yl)acetamide
A suspension of 4-phenylthiazol-2-amine (2 g, 11.4 mM) and
anhydrous potassium carbonate (1.89 g, 13.6 mM) in dichloro-
methane (20 mL) was stirred for 30 min at room temperature.
The reaction mixture was then cooled on an ice bath followed by
addition of ice-cooled solution of chloroacetyl chloride (1.5 g,
13.6 mM) in dichloromethane (25 mL) in drop wise manner over
a period of 30 min. The reaction mixture was then stirred at room
temperature overnight followed by reflux for additional 30 min.
The excess solvent was distilled off and the residue obtained was
neutralized with aqueous sodium bicarbonate solution (5% w/v).
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
4
3
4
3
4
3
4
3
4
3
4
3
Cl –H
Cl –H
9
17
21
83
9
6
46
26
216–218 8.39 0.03
238–240 8.47 0.04
235–237 8.27 0.03
209–211 8.85 0.05
220–222 8.38 0.05
198–200 8.77 0.04
218–220 8.54 0.05
201–203 8.92 0.03
235–237 8.42 0.04
216–218 8.54 0.04
243–245 8.32 0.04
212–214 8.17 0.04
Cl –Ph-4-H
–Ph-4-H
I
Cl –Ph-4-CH3
Cl –Ph-4-CH3
I
I
–Ph-4-OCH3
–Ph-4-OCH3
Cl –CH2COOC2H5 14
Cl –CH2COOC2H5
Cl –COOC2H5
Cl –COOC2H5
8
15
19
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8
A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Scheme 2. Synthetic route for the preparation of monoquaternary pyridinium oximes (1a–6a and 1b–6b).
The solid product formed was filtered off, washed thoroughly with
chilled water and dried under vacuum (2.3 g, yield: 80%). TLC
(ethyl acetate/hexane (1:3), Rf = 0.31). This product was sufficiently
pure and used in the next step without further purification.
5.3. Spectroscopic data for the synthesized oximes
5.3.1. 4-((Hydroxyimino)methyl)-1-(2-oxo-2-(thiazol-2-
ylamino)ethyl)pyridinium chloride (1a)
Creamish powder; IR (KBr) mmax 3053, 3046, 2957, 1687, 1640,
1592, 1461, 1322 cmꢀ1
; d
1H NMR (DMSO-d6, 400 MHz):
5.2.3. Synthesis of 4-((hydroxyiminomethyl)-1-(2-oxo-2-(4-phe-
nylthiazol-2-ylamino)ethyl)pyridinium chloride (2a)
(ppm) = 5.756 (s, 2H, –NCH2), 7.296 (d, 1H, J = 3.6 Hz, @CH–S),
7.524 (d, 1H, J = 3.6 Hz, @CH–N), 8.299 (d, 1H, J = 6.8 Hz, –Py),
8.456 (s, 1H, –CH@N), 9.02 (d, 2H, J = 6.8 Hz, –Py), 12.992 (s, 1H,
–OH); 13C NMR (DMSO-d6, 100 MHz): d (ppm) = 61.2, 114.1,
123.4, 129.2, 144.9, 146.6, 149.2, 157.1, 168.2; Anal. Calcd for
4-Pyridinealdoxime (0.32 g, 2.6 mM), dissolved in dry acetoni-
trile (25 mL) was 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-phenylthiazol-2-
yl)acetamide (0.8 g, 3.2 mM) 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 tem-
perature followed by reflux for another 3 h. The solid product
obtained was filtered off, washed with dry hot acetonitrile
(3 ꢁ 15 mL) followed by dry hot acetone. The crude product was
dried (0.88 g, yield: 75%) and recrystallized from methanol-ace-
tone mixture. TLC (acetone/methanol (2:1), Rf = 0.10), mp:
235–237 °C.
Note: The iodo variants (2b, 4a and 4b) of compound B in
Scheme 2 were prepared by exchanging the chloro group with
iodide using sodium iodide, by refluxing in dry acetone.
Except 1a and 1b, all other compounds were prepared by
following the same protocol as mentioned above while 1a
and 1b were prepared directly from the second step of the
above protocol. Melting points were determined with open
capillary in conventional method and were uncorrected
(Table 4).
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 disks on a Bruker TENSOR-27 FTIR spec-
trophotometer. 1H NMR (DMSO-d6) spectra of the synthesized
oximes were recorded on Bruker Avance 400 spectrometer at
400 MHz using tetramethylsilane (TMS) as internal standard
and expressed in the d (ppm) values. 13C NMR (DMSO-d6) chem-
ical shifts values were obtained using the same instrument at
100 MHz.
C11H11ClN4O2S: C, 44.22; H, 3.71; N, 18.75; S, 10.73. Found: C,
44.64; H, 4.06; N, 18.34; S, 10.31.
5.3.2. 3-((Hydroxyimino)methyl)-1-(2-oxo-2-(thiazol-2-ylamino)
ethyl)pyridinium chloride (1b)
Off-white powder; IR (KBr) mmax 3191, 3070, 3015, 1683, 1629,
; d
1582, 1469, 1326 cmꢀ1 1H NMR (DMSO-d6, 400 MHz):
(ppm) = 5.851 (s, 2H, –NCH2), 7.296 (d, 1H, J = 3.6 Hz, @CH–S),
7.52 (d, 1H, J = 3.2 Hz, @CH–N), 8.245 (dd, 1H, J = 6.0 Hz & 2.0 Hz,
–Py), 8.376 (s, 1H, –CH@N), 8.808 (d, 1H, J = 8.4 Hz, –Py), 9.079
(d, 1H, J = 6.0 Hz, –Py), 9.349 (s, 1H, –Py), 12.363 (s, 1H, –NH),
12.979 (s, 1H, –OH); 13C NMR (DMSO-d6, 100 MHz):
d
(ppm) = 61.9, 114.1, 127.6, 133.0, 142.6, 143.0, 143.8, 145.9,
159.1, 168.3; Anal. Calcd for C11H11ClN4O2S: C, 44.22; H, 3.71; N,
18.75; S, 10.73. Found: C, 43.94; H, 3.67; N, 18.46; S, 10.40.
5.3.3. 4-((Hydroxyimino)methyl)-1-(2-oxo-2-(4-phenylthiazol-
2-ylamino)ethyl)pyridinium chloride (2a)
Pale yellow powder; IR (KBr)
mmax 3117, 3022, 2944, 1694, 1642,
1610, 1469, 1350 cmꢀ1 1H NMR (DMSO-d6, 400 MHz):
;
d
(ppm) = 5.818 (s, 2H, –NCH2), 7.346 (t, 2H, J = 7.6 Hz, –Ph), 7.45
(t, 2H, J = 7.6 Hz, –Ph), 7.717 (s, 1H, @CH–S), 7.916 (d, 2H,
J = 7.6 Hz, –Ph), 8.316 (d, 1H, J = 6.4 Hz, –Py), 8.466 (s, 1H,
–CH@N), 9.06 (d, 2H, J = 6.4 Hz, –Py), 13.017 (s, 1H, –NH), 13.108
(s, 1H, –OH); 13C NMR (DMSO-d6, 100 MHz): d (ppm) = 61.1,
108.7, 123.5, 125.7, 127.9, 128.7, 133.9, 144.9, 146.7, 149.1,
149.3, 157.1, 164.3; Anal. Calcd for C17H15ClN4O2S: C, 54.47; H,
4.03; N, 14.95; S, 8.55. Found: C, 54.09; H, 4.26; N, 14.89; S, 8.21.
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A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
5.3.4. 3-((Hydroxyimino)methyl)-1-(2-oxo-2-(4-phenylthiazol-
2-ylamino)ethyl)pyridinium chloride (2b)
5.3.9. 1-(2-(4-(2-Ethoxy-2-oxoethyl)thiazol-2-ylamino)-2-
oxoethyl)-4-((hydroxyimino)methyl)pyridinium chloride (5a)
Creamish powder; IR (KBr) mmax 3108, 3076, 2966, 1688,
White powder; IR (KBr) mmax 3115, 3085, 2981, 1739, 1694,
1634, 1599, 1501, 1344 cmꢀ1
;
1H NMR (DMSO-d6, 400 MHz): d
1644, 1556, 1469, 1298 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d
;
(ppm) = 5.810 (s, 2H, –NCH2), 7.350 (t, 2H, J = 7.2 Hz, –Ph),
7.455 (t, 2H, J = 7.6 Hz, –Ph), 7.722 (s, 1H, @CH–S), 7.915 (d,
2H, J = 7.6 Hz, –Ph), 8.267 (dd, 1H, J = 6.4 Hz & 1.6 Hz, –Py),
8.393 (s, 1H, –CH@N), 8.82 (d, 1H, J = 8.0 Hz, –Py), 9.032 (d,
1H, J = 6.0 Hz, –Py), 9.322 (s, 1H, –Py), 12.317 (s, 1H, –NH),
(ppm) = 1.186 (t, 3H, J = 7.2 Hz, –CH3), 3.716 (s, 2H, –CH2), 4.087
(q, 2H, J = 7.2 Hz, –CH2), 5.737 (s, 2H, –NCH2), 7.071 (s, 1H, @CH–
S), 8.30 (d, 2H, J = 6.8 Hz, –Py), 8.455 (s, 1H, –CH@N), 9.011 (d,
2H, J = 6.8 Hz, –Py), 12.951 (s, 1H, –NH), 12.988 (s, 1H, –OH); 13C
NMR (DMSO-d6, 100 MHz): d (ppm) = 14.0, 36.5, 60.2, 61.0, 111.1,
123.4, 144.9, 146.6, 149.2, 156.2, 159.2, 168.7, 169.8; Anal. Calcd
for C15H17ClN4O4S: C, 46.81; H, 4.45; N, 14.56; S, 8.33. Found: C,
46.46; H, 4.39; N, 14.56; S, 8.12.
13.110 (s, 1H, –OH); 13C NMR (DMSO-d6, 100 MHz):
d
(ppm) = 61.8, 108.7, 125.6, 127.6, 127.9, 128.7, 132.9, 133.9,
142.9, 143.2, 143.8, 145.9, 163.8; Anal. Calcd for
C17H15ClN4O2S: C, 54.47; H, 4.03; N, 14.95; S, 8.55. Found: C,
54.87; H, 4.03; N, 14.91; S, 8.83.
5.3.10. 1-(2-(4-(2-Ethoxy-2-oxoethyl)thiazol-2-ylamino)-2-
oxoethyl)-3-((hydroxyimino)methyl)pyridinium chloride (5b)
Off-white powder; IR (KBr) mmax 3124, 3031, 2994, 1731, 1696,
5.3.5. 4-((Hydroxyimino)methyl)-1-(2-oxo-2-(4-p-tolylthiazol-
2-ylamino)ethyl)pyridinium chloride (3a)
1635, 1558, 1471, 1341 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d
;
Tan powder; IR (KBr)
mmax 3108, 3029, 2914, 1694, 1644, 1567,
(ppm) = 1.186 (m, 3H, –CH3), 3.717 (s, 2H, –CH2), 4.088 (q, 2H,
J = 7.2 Hz, –CH2), 5.792 (s, 2H, –NCH2), 7.072 (s, 1H, @CH–S),
8.242 (dd, 1H, J = 6.0 Hz & 2.0 Hz, –Py), 8.374 (s, 1H, –CH@N),
8.80 (d, 1H, J = 8.0 Hz, –Py), 9.036 (d, 1H, J = 6.0 Hz, –Py), 9.319
(s, 1H, –Py), 12.320 (s, 1H, –NH), 12.944 (s, 1H, –OH); 13C NMR
(DMSO-d6, 100 MHz): d (ppm) = 14.0, 40.1, 60.2, 61.7, 111.1,
127.6, 133.0, 142.7, 143.0, 143.8, 145.9, 152.9, 158.7, 162.3,
169.8; Anal. Calcd for C15H17ClN4O4S: C, 46.81; H, 4.45; N, 14.56;
S, 8.33. Found: C, 46.09; H, 4.38; N, 14.45; S, 8.09.
1467, 1322 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d (ppm) = 2.333
;
(s, 3H, –CH3), 5.789 (s, 2H, –NCH2), 7.254 (d, 2H, J = 8.0 Hz, –Ph),
7.627 (s, 1H, @CH–S), 7.799 (d, 2H, J = 8.0 Hz, –Ph), 8.311 (d, 1H,
J = 6.4 Hz, –Py), 8.463 (s, 1H, –CH@N), 9.041 (d, 2H, J = 6.4 Hz,
–Py), 12.995 (s, 1H, –OH); 13C NMR (DMSO-d6, 100 MHz): d
(ppm) = 20.7, 107.8, 123.4, 125.6, 129.2, 130.3, 136.8, 137.2,
145.0, 146.6, 149.2, 150.2, 164.1, 169.2; Anal. Calcd for
C18H17ClN4O2S: C, 55.59; H, 4.41; N, 14.41; S, 8.25. Found: C,
55.91; H, 4.41; N, 14.74; S, 8.58.
5.3.6. 3-((Hydroxyimino)methyl)-1-(2-oxo-2-(4-p-tolylthiazol-
2-ylamino)ethyl)pyridinium chloride (3b)
5.3.11. 1-(2-(4-(Ethoxycarbonyl)thiazol-2-ylamino)-2-oxoethyl)-
4-((hydroxyimino)methyl)pyridinium chloride (6a)
Tan powder; IR (KBr) mmax 3137, 3046, 2951, 1700, 1634, 1556,
White powder; IR (KBr) mmax 3119, 3042, 2983, 1726, 1698,
1428, 1300 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d (ppm) = 2.336
;
1642, 1610, 1463, 1290 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d
;
(s, 3H, –CH3), 5.820 (s, 2H, –NCH2), 7.257 (d, 2H, J = 8.0 Hz, –Ph),
7.632 (s, 1H, @CH–S), 7.80 (d, 2H, J = 8.0 Hz, –Ph), 8.254 (m, 1H,
–Py), 8.385 (s, 1H, –CH@N), 8.811 (d, 1H, J = 8.0 Hz, –Py),
9.043 (d, 1H, J = 6.0 Hz, –Py), 9.328 (s, 1H, –Py), 12.297 (s, 1H,
–NH), 13.019 (s, 1H, –OH); Anal. Calcd for C18H17ClN4O2S: C,
55.59; H, 4.41; N, 14.41; S, 8.25. Found: C, 55.25; H, 4.43; N,
14.46; S, 8.66.
(ppm) = 1.301 (m, 3H, –CH3), 4.287 (q, 2H, J = 7.2 Hz, –CH2),
5.750 (s, 2H, –NCH2), 8.129 (s, 1H, @CH–S), 8.303 (d, 2H,
J = 6.8 Hz, –Py), 8.454 (s, 1H, –CH@N), 8.999 (d, 2H, J = 6.8 Hz,
–Py), 12.968 (s, 1H, –NH), 13.306 (s, 1H, –OH); 13C NMR (DMSO-
d6, 100 MHz): d (ppm) = 14.1, 24.8, 60.6, 61.0, 123.3, 123.4, 141.1,
145.0, 146.6, 149.3, 157.3, 160.7, 164.7; Anal. Calcd for
C14H15ClN4O4S: C, 45.35; H, 4.08; N, 15.11; S, 8.65. Found: C,
45.17; H, 4.03; N, 15.16; S, 8.52.
5.3.7. 4-((Hydroxyimino)methyl)-1-(2-(4-(4-methoxyphenyl)
thiazol-2-ylamino)-2-oxoethyl)pyridinium chloride (4a)
Yellow powder; IR (KBr) mmax 3117, 3026, 2957, 1705, 1642,
5.3.12. 1-(2-(4-(Ethoxycarbonyl)thiazol-2-ylamino)-2-
oxoethyl)-3-((hydroxyimino)methyl)pyridinium chloride (6b)
Creamish powder; IR (KBr) mmax 3139, 3089, 2985, 1727, 1696,
1605, 1488, 1324 cmꢀ1
; d
1H NMR (DMSO-d6, 400 MHz):
(ppm) = 3.798 (s, 3H, –OCH3), 5.722 (s, 2H, –NCH2), 7.009 (d, 2H,
J = 8.8 Hz, –Ph), 7.541 (s, 1H, @CH–S), 7.84 (d, 2H, J = 8.8 Hz, –Ph),
8.31 (d, 1H, J = 6.4 Hz, –Py), 8.462 (s, 1H, –CH@N), 8.99 (d, 2H,
J = 6.4 Hz, –Py), 12.924 (s, 1H, –OH); 13C NMR (DMSO-d6,
100 MHz): d (ppm) = 55.7, 61.1, 103.1, 107.2, 114.6, 124.0, 127.5,
140.1, 145.6, 147.1, 149.7, 157.5, 159.6, 164.5; Anal. Calcd for
1638, 1556, 1465, 1285 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d
;
(ppm) = 1.302 (t, 3H, J = 7.2 Hz, –CH3), 4.287 (q, 2H, J = 7.2 Hz,
–CH2), 5.795 (s, 2H, –NCH2), 8.127 (s, 1H, @CH–S), 8.248 (dd, 1H,
J = 6.4 Hz & 1.6 Hz, –Py), 8.376 (s, 1H, –CH@N), 8.803 (d, 1H,
J = 8.0 Hz, –Py), 9.017 (d, 1H, J = 6.4 Hz, –Py), 9.307 (s, 1H, –Py),
12.293 (s, 1H, –NH), 12.850 (s, 1H, –OH); Anal. Calcd for
C18H17ClN4O3S: C, 53.40; H, 4.23; N, 13.84; S, 7.92. Found: C,
C14H15ClN4O4S: C, 45.35; H, 4.08; N, 15.11; S, 8.65. Found: C,
55.37; H, 4.11; N, 13.84; S, 7.49.
45.55; H, 4.29; N, 15.46; S, 8.31.
5.3.8. 3-((Hydroxyimino)methyl)-1-(2-(4-(4-methoxyphenyl)
thiazol-2-ylamino)-2-oxoethyl)pyridinium chloride (4b)
Pale yellow powder; IR (KBr) mmax 3191, 3074, 2988, 1700,
5.4. AChE assay
1631, 1607, 1488, 1318 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d
;
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 proto-
col.48–50 Quartz cuvettes of 3 mL were used to measure the activity
of the assay mixture. ATChI (0.48 mM, in distilled water) as sub-
strate and DTNB as the colorimetric indicator (0.3 mM in 0.1 M
phosphate buffer, pH = 7.4) were used to assay the activity. 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.
(ppm) = 3.798 (s, 3H, –OCH3), 5.784 (s, 2H, –NCH2), 7.009 (d,
2H, J = 8.8 Hz, –Ph),7.544 (s, 1H, @CH–S), 7.841 (d, 2H,
J = 8.8 Hz, –Ph), 8.386 (s, 1H, –CH@N), 9.014 (d, 1H, J = 6.4 Hz,
–Py), 9.304 (s, 1H, –Py), 12.265 (s, 1H, –NH), 12.951 (s, 1H,
–OH); 13C NMR (DMSO-d6, 100 MHz):
d (ppm) = 55.1, 61.8,
106.6, 114.1, 126.7, 127.0, 127.6, 132.9, 142.9, 143.2, 143.8,
145.9, 159.1, 163.8, 167.2; Anal. Calcd for C18H17ClN4O3S: C,
53.40; H, 4.23; N, 13.84; S, 7.92. Found: C, 53.05; H, 4.55; N,
13.91; S, 7.56.
Please cite this article in press as: Valiveti, A. K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.027

10
A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5.5. AChE inhibition by OP
The determined kinetic parameters and rate constants of all the
oximes were presented in the Table 1.
The initial stock solutions of OP inhibitors (sarin, O-ethylsarin
and VX) were prepared in isopropanol and stored in refrigerator.
Subsequent dilutions of OP inhibitors were freshly prepared in
deionized water prior to start the experiment. The enzyme
hAChE was taken in phosphate buffer (pH 7.4, 0.1 M) was inhibited
by appropriate OP concentrations at 37 °C for 10–15 min to achieve
95–98% inhibition of the control activity. Excess OP nerve agent
was removed by extraction of the inhibition cocktail with six fold
volume of n-hexane.51,52
5.7. Data analysis
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).
5.8. Docking simulations
Simulation for receptor—ligand docking was carried out on Auto
Dock 4.0 (Sanner, 1999)53 using a reported method with slight
modification (Musilek et al., 2011).24 Briefly, structure of human
AChE (PDB–ID 1B41) was obtained from the protein data bank
and used in docking studies. After eliminating the inhibitor
‘Fasciculin-2’ and water molecules, SER 203 was modified with
sarin and VX reaction products, respectively. O-Ethylsarin reaction
product was same as VX reaction product. Oximes were drawn,
energy minimized and prepared as ligands. Pre-calculated electro-
static grid map for each atom type present in ligands were gener-
5.6. Oxime assisted reactivation kinetics of OP inhibited hAChE
The OP inhibited hAChE was subjected to the reactivation by the
addition of the synthesized oximes at final concentrations ranging
from 10 to 1000
100, 500 and 1000
constants at different time points (1, 3, 5, 7, 10, 15, 20 25, 30, 40,
50, 60 min). An aliquot of 50 L from the reactivation cocktail (con-
taining inhibition cocktail and oxime reactivator) was transferred
to the cuvette containing 3.0 mL of phosphate buffer and 100
of DTNB. Shortly afterward the AChE activity was assayed by add-
ing 20 L of ATChI at 37 °C (final volume 3.17 mL). The oxime
lM. Five different oxime concentrations (10, 50,
lM) were used for the determination of rate
l
lL
ated. All maps were created with 0.375 Å spacing and the c-oxygen
atom of SER 203 was selected as grid center. The dimension of grid
box was set at 60 Å ꢁ 60 Å ꢁ 60 Å, the accessible space for ligand
around grid center. Docking calculations were carried out using
Lamarckian genetic algorithm (LGA). A population of 150 random
individuals, a maximum of 2,500,000 energy evaluations, a maxi-
mum of 27,000 generations, maximum 1 top individual surviving,
0.02 rate of gene mutation and 0.8 rate of crossover were used as
parameters for LGA. At the end of docking, the resulting positions
(10 runs) were clustered according to root mean square criterion
of 0.5 Å.
l
induced reactivation of the OP-inhibited AChE was monitored at
different time intervals over a period of 60 min. Spontaneous reac-
tivation of inhibited AChE was assayed using the same protocol
(the reaction mixture containing enzyme and OP but no oxime).
Under these conditions spontaneous reactivation was found to be
insignificant. All the values were corrected for their oxime induced
hydrolysis of ATChI.
The oxime assisted reactivation of the OP-inhibited AChE pro-
ceeds according to the Scheme 3.
Where [EP] phosphylated AChE; [OX] the reactivator;
[EPOX] reversible Michaelis-type phosphyl–AChE–oxime complex;
[E] reactivated enzyme and [POX] phosphylated oxime.
[EP]*[OX]/[EPOX] estimates the dissociation constant (KD) and
inversely proportional to the affinity of the oxime to [EP]. kr
represents the rate constant for the displacement of the phosphyl
residue from [EPOX] by the oxime and indicates the reactivation
potency of the oxime reactivator.
5.9. Determination of acid dissociation constant (pKa)
5.9.1. Reagents
Oxime stock solutions (5 ꢁ 10ꢀ3 M) were freshly prepared in
triple distilled water. Buffer solutions were prepared from the
methods reported in literature (Gomori, 1955).54 The analytical
wavelengths for the protonated and deprotonated species were
obtained by using 0.1 M hydrochloric acid and 0.1 M sodium
hydroxide solutions.
In case of complete reactivation and with [OX] ꢂ [EP]o a pseudo
first-order rate equation can be derived for the reactivation process
as represented in Eq. 1
The acid dissociation constants (pKa) of the oximes were deter-
mined using the method of Albert and Sergeant (1971).55,56 The
method was based on direct determination of the ratio of molecu-
lar species (protonated) to dissociated (deprotonated) species in a
series of non-absorbing buffer solutions. For this purpose, spectra
of molecular species were obtained first in buffer solution of par-
ticular pH in which compounds of interest would be present
kr½OXꢃ
kobs
¼
ð1Þ
KD þ ½OXꢃ
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 analysis6 using Eq. 2
entirely in either form. Oxime stock solutions (15–35 lL,
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 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,
were of protonated (Dm) and deprotonated (Di) molecules (Fig. 7).
Eleven different pH values, ranging from 6.85 to 9.76 were selected
to determine the pKa of oximes. For this, appropriate buffers con-
sisting of phosphate (pH: 6.85–7.76), tris (pH: 8.17–8.56) and gly-
cine-NaOH (pH: 9.03–9.76) were used to determine the
obsꢁt
vt
¼
vo ꢁ ð1 ꢀ eꢀk
Þ
ð2Þ
kr and KD were obtained by the non-linear fit of the relationship
between kobs versus [OX]. Both the rate constants kr and KD follow
Michaelis–Menten type kinetics and the second order reactivation
rate constant kr2 was obtained from the ratio of kr and KD.
dissociation constants of oximes. 15–35 lL of aqueous solutions
of oximes were diluted to 3 mL in each buffer and optical densities
were determined at analytical wavelengths using buffer blank at
25 1.0 °C. A set of eleven values of pKa were obtained using the
following Eq. 3:
Scheme 3. Oxime assisted reactivation of OP-inhibited AChE.
Please cite this article in press as: Valiveti, A. K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.027

A. K. Valiveti et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
Germany, for his valuable suggestions during optimization of the
experimental protocol. Authors highly appreciate the help of the
Microbiology, Biotechnology and Biochemistry Divisions, of DRDE,
Gwalior for their help and technical support in the isolation of
human AChE. Authors are thankful to Mr. Ajay Pratap for spectral
characterization of the synthesized compounds. Authors are
thankful to Prof. K. K. Ghosh, PRSU, Raipur for his kind help in the cal-
culation of kinetic rate constants. Aditya Kapil Valiveti is thankful to
Defence Research & Development Organization (DRDO), New Delhi
for financial support.
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