Synthesis and in vitro evaluation of xylene linked carbamoyl bis-pyridinium monooximes as reactivators of organophosphorus (OP) inhibited electric eel acetylcholinesterase (AChE)

Article abstract of DOI:10.1016/j.ejmech.2011.05.064

A series of carbamoyl bis-pyridinium monooximes linked with xylene linker were synthesized and their in-vitro reactivation potential was evaluated against acetylcholinesterase (AChE) inhibited by organophosphorus inhibitors (OP) such as sarin, DFP and VX

Full text of DOI:10.1016/j.ejmech.2011.05.064

European Journal of Medicinal Chemistry 46 (2011) 3926e3933
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro evaluation of xylene linked carbamoyl bis-pyridinium
monooximes as reactivators of organophosphorus (OP) inhibited electric eel
acetylcholinesterase (AChE)
*
Jyotiranjan Acharya , Hemlata Rana, M.P. Kaushik
Process Technology Development Division, Defence Research & Development Establishment, Jhansi Road, Gwalior 474002, 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 carbamoyl bis-pyridinium monooximes linked with xylene linker were synthesized and their
in-vitro reactivation potential was evaluated against acetylcholinesterase (AChE) inhibited by organo-
phosphorus inhibitors (OP) such as sarin, DFP and VX and the data were compared with reactivation
obtained with 2-PAM and obidoxime. Amongst the synthesized compounds, 3-carbamoyl-
20hydroxyiminomethyl-1-10-(1,4-phenylenedimethyl)-bispyridinium dibromide (5e) 3-carbamoyl-
20hydroxyiminomethy l-1-10-(1,3-phenylenedimethyl)-bispyridinium dibromide (5k) and 4-carbamoyl-
20hydroxyiminomethyl-1-10-(1,3-phenylenedimethyl)-bispyridinium dibromide (5l) were found to be
the most potent reactivators for electric eel AChE inhibited by sarin and DFP. However, in case of VX
inhibited AChE, none of the synthesized oximes could surpass the reactivation potential of 2-PAM and
obidoxime. The pKa values of all the oximes were determined and correlated with their observed
reactivation potential.
Received 1 February 2011
Received in revised form
18 April 2011
Accepted 26 May 2011
Available online 12 June 2011
Keywords:
Bis-pyridinium oximes
Reactivators
2-PAM
Acetylcholinesterase
Xylene linkers
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
phosphorylated AChE. Quaternary pyridinium oximes such as 2-
pyridine aldoxime methochloride (2-PAM), trimedoxime (TMB-4),
Quaternary mono- and bis-pyridinium oximes are important
therapeuticagents used asreactivatorsof acetylcholinesterase (AChE)
in the treatment of poisoning resulting from the exposure of organ-
ophosphorus inhibitors (OP) [1]. Some of these OPs have been
synthesized and used as warfare agents (nerve agents e.g., sarin,
soman, tabun and VX) in the past (Fig. 1) [2,3]. They have also been
used as pesticides (e.g., parathion, malathion, chlorpyriphos) result-
ing in severe occupational hazards [4]. The use of nerve agents during
war and acts of terrorism has emphasized the development of an
effective medical treatment regimen of OP poisoning. The intoxica-
tion with nerve agents leads to inhibition of acetylcholinesterase by
phosphorylation of theiractive site serine residue [5]. The subsequent
accumulation of the neurotransmitter acetylcholine and over-
stimulation of cholinergic receptors results in a generalized cholin-
ergic crisis including breakdown of neuromuscular function [6].
The reactivation of OP-inhibited AChE can be achieved effec-
tively by the nucleophillic attack of a strong nucleophile on the P-
atom which breaks the phosphorus-AChE bond in the
obidoxime and HI-6 (Fig. 2) are currently used as reactivators in the
treatment of OP poisoning [7]. However, these OP nerve agents
behave differently towards reactivation due to their broad structure
variability and most often their deleterious side effects become
difficult to counteract [8]. Furthermore an effective therapy by
a single oxime to all the known nerve agents is still lacking.
2. Chemistry
Several new oximes have been synthesized and tested in the last
decades [9e11]. The reactivation of OP-inhibited AChE depends on
several factors including structure of the reactivator [12], structure
of the inhibitor (OP), source of the enzyme AChE [13] as well as on
post-inhibitory reactions such as spontaneous dealkylation (aging)
[14] and spontaneous dephosphorylation (spontaneous reac-
tivation) of the OP-AChE complex [15].
In the past, reactive oxime moiety has been incorporated into
the varieties of micelle forming molecules, resulting in significant
enhancement of hydrolysis of OP compounds [16,17]. Recently, N-
benzyl-4-(hydroxyiminomethyl) pyridinium bromide and its anal-
ogous were reported to be more active in the reactivation of tabun-
inhibited human erythrocyte AChE. Again the docking studies with
these types of compounds led to the assumption that the linker
Abbreviations: AChE, Acetylcholinesterase; 2-PAM, 2-(hydroxyiminomethyl)-1-
methylpyridinium chloride; DFP, diisopropyl flurophosphate.
* Corresponding author. Tel.: þ91 751 2340245; fax: þ91 751 2341148.
E-mail address: jracharya@rediffmail.com (J. Acharya).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.064

J. Acharya et al. / European Journal of Medicinal Chemistry 46 (2011) 3926e3933
3927
3. Results and discussion
F
O
O
F
P
O
P
O
O
H3C
The in-vitro reactivation data of OP (sarin, DFP and VX) inhibited
electric eel AChE by carbamoyl bis-pyridinium monooximes
bearing xylene bridge at two distinct concentrations (10ꢀ3 M &
10ꢀ4 M) is presented in Fig. 3. It is evident from these data that best
reactivation was observed with 3-carbamoyl-20hydroxyimi
nomethyl-1-10-(1,4-phenylenedimethyl)-bispyridinium dibromide
(5e) 3-carbamoyl-20hydroxyiminomethyl-1-10-(1,3-phenylenedim
ethyl)-bispyridinium dibromide (5k) and 4-carbamoyl-20hydro
xyiminomethyl-1-10-(1,3-phenylenedimethyl)-bispyridinium dibro
mide (5l) in case of sarin (Fig. 3a) and DFP (Fig. 3b) inhibited electric
eel AChE. The oximes, 5e, 5k and 5l respectively reactivated 47%,
48% and 45% of sarin inhibited AChE in comparison to respectively
42% and 21% reactivation by 2-PAM and obidoxime at the concen-
tration 10ꢀ3 M after 10 min. However at a lower oxime concen-
tration (10ꢀ4 M), the reactivation potential of oxime 5l only was
found to be greater than that of obidoxime and 2-PAM in reac-
tivating sarin inhibited AChE. In case of DFP inhibited AChE, the
oximes 5e, 5k and 5l were able to reactivate 54%, 54% and 48%
respectively in comparison to 51% and 43% reactivation by 2-PAM
and obidoxime at the concentration 10ꢀ3 M after 10 min. It may
also be noted that at lower oxime concentration, oxime 5k was
found to be more active than 2-PAM and obidoxime in reactivating
DFP inhibited AChE. Furthermore, the reactivation potential of
other two oximes i.e., 5e and 5l was at par with 2-PAM and more
than that of obidoxime. It is worth noticing that in case of VX
inhibited AChE (Fig. 3c), synthesized oximes such as 5b, 5c, 5d, 5j,
5k and 5l have shown significant reactivation potential. However,
none of these could surpass the reactivation potential of 2-PAM.
The best reactivation was observed with 2-PAM (52%) followed by
5b (43%) at 10ꢀ3 M after 10 min. The oximes 5d and 5j reactivated
40% of the VX inhibited AChE which was at par with that of obi-
doxime at the same concentration. However at lower concentra-
tion, oxime 5d showed highest reactivation ability followed by 2-
PAM, obidoxime and 5j.
The time dependent reactivation profile of some selected
oximes indicated that for most of the oximes, reactivation increased
with time and reached maximum in 30e40 min. The oxime 5k was
able to reactivate 83% followed by 5e (75%) and 5l (73%) of the sarin
inhibited AChE after 40 min as compared to 63% and 34% reac-
tivation observed by 2-PAM and obidoxime respectively. However
in case of DFP inhibited AChE, oxime 5k was able to reactivate 81%
followed by 56% reactivation each by 2-PAM and 5e, obidoxime
(54%) and 5l (41%). It may also be noted that the reactivation
potential of oxime 5l in reactivating DFP inhibited AChE decreased
gradually with time. Maximum reactivation potential of 2-PAM and
obidoxime in reactivating VX inhibited AChE was 63% and 54%
respectively after 40 min as compared to 45% reactivation by oxime
5j in 30 min. The OP inhibitors used in this study have aging half-
life of more than an hour [26]. Thus no appreciable amount of
aged enzyme was present during the course of reactivation studies.
Therefore, the observed time-dependent reactivation of inhibited
AChE is of non-aged enzyme only and obtained plateau of reac-
tivation vs time indicates maximum possible reactivation achiev-
able with lowest time.
Sarin
DFP
O
S
P
O
N
VX
Fig. 1. Structure of OP inhibitors used in this study.
between pyridinium and benzyl ring should be long enough to
achieve desired reactivation [18]. Xylene linked bis-pyridinium
oximes have gained considerable interest in the recent past
owing to their improved reactivation potential for OP inhibited
AChE [19]. Further, structure-activity relationship (SAR) and dock-
ing studies with xylene linked pyridinium oximes revealed
cation- interactions within the enzyme active-sites [20]. Previ-
p-p and
p
ously, we have also reported symmetrical bis-pyridinim oximes
linked by a xylene, methoxyalkane and methoxymethyl benzene as
AChE reactivators [21e23]. Some of these oximes have shown
promising reactivation potential against OP inhibited AChE. Reac-
tivation potential of such oximes attracted our attention to further
explore xylene linked pyridinium oximes. In this context we
focused our attention on carbamoyl bis-pyridinium monooximes
having a xylene linker between two pyridinium rings with an aim
to enhance the lipid solubility and stability, which is found to be
low with the currently available antidotes [24] as well as to allow
the hydrophobic interactions with some of the aromatic residues
that remain in the active site gorge of the enzyme acetylcholines-
terase [25]. Therefore, in continuation of our work on antidotes
against nerve agents, herein we report the synthesis and in-vitro
evaluation of a series of carbamoyl bis-pyridinium monooximes
linked by xylene bridge as reactivators of electric eel AChE inhibited
by sarin, VX and a model OP inhibitor diisopropyl flurophosphate
(DFP).
By adopting a simple and mild two-step synthetic protocol, we
have synthesized a series of carbamoyl bis-pyridinium mono-
oximes. The intermediate mono quaternary pyridinium mono-
oximes (3ae3i) were prepared according to the reported method
[19], by the reaction of isomeric pyridine aldoxime (1ae1c) with
a,
a0 dibromoxylene (2ae2c) (Scheme 1). The subsequent conden-
sation of intermediate 3 with 3- and 4-carbamoylpyridine (4a and
4b) gave desired products (5ae5r) with good yields within 3e6 h
(Table 1).
NOH
NOH
NOH
2Br
C
H
N
CH3
Cl
O
N
N
TMB-4
2-PAM
The pKa values should be taken into consideration while
searching for new oxime reactivators [27] essentially due to the fact
that deprotonated oxime group is involved in the nucleophilic
attack during reactivation and its concentration is pH dependant.
Therefore the pKa values of the oximes were determined spectro-
photometrically in the pH range of 6.04e10.37 (Fig. 4). In the series
of pyridinium oximes, the optimum pKa value for the reactivator
should be in the range of 7.6e8.0 [28,29]. Two characteristic
maxima were obtained in UV in the region 200e600 nm. These
NOH
CH
NOH
HC
H2N
2Cl
O
2Cl
N
N
N
O
N
CH
HON
Obidoxime
HI-6
Fig. 2. Commonly used reactivators.

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J. Acharya et al. / European Journal of Medicinal Chemistry 46 (2011) 3926e3933
Br
CHNOH
CHNOH
CHNOH
CONH2
CONH2
2Br
DMF, 70-90 o
stirring, 3-6h
C
Acetone, r.t.,
stirring, 3-8h
N
Br
N
N
N
N
Br
Br
2a-2c
1a-1c
3a-3i
5a-5r
4a-4b
2a = 1,4-phenylene
2b = 1,3-phenylene
2c = 1,2-phenylene
4a = 3-CONH2
4b = 4-CONH2
Scheme 1. Synthesis of carbamoyl bis-pyridinium monooximes.
maxima correspond to the absorption of different oxime ionized
forms as a result of
* transitions within the aromatic system
plausible explanation for observed activity of 5e, 5k and 5l over
other oximes. This is in close agreement with reported study by
Musilek et al. [30], where oxime group in position 2 or 3 have
shown increased reactivation towards tabun inhibited AChE, as
compared to similar compounds with oxime in position 4.
However in case of VX inhibited AChE, no such preference for the
position of the oxime on the pyridinium ring with respect to
reactivation was observed. This may be attributed to the fact that,
in case of VX inhibited AChE, the active site gorge is least occupied
(due to the cleavage of bulkier N, N-diisopropyl-S-ethyl leaving
group) thereby leaving more free space for oxime access to the
phosphorylated site with least steric hindrance compared to DFP
or sarin [31]. This is manifested in the reactivation data that most
of the oximes in this study have shown significant reactivation
towards VX inhibited AChE irrespective of the position of the
oxime moiety on the pyridinium ring, the nature of the linker and
position of the carbamoyl moiety.
The dephosphorylation of phosphorylated enzyme is also
dependent on factors such as physicochemical properties of the
reactivator including steric and electronic factors, lipophilic-
hydrophilic balance and pKa [21]. The pKa of bis-pyridinium
oximes were found to be in the range of 7.69e8.79 (Table 1). It is
worth noticing that the pKa of bis-pyridinium oximes having oxi-
mino function at position 2 or 4 on the pyridinium ring were found
to be in the lower range (7.69e8.15) as compared to the oximes
where oximino function is attached at position 3 on the pyridinium
ring (8.59e8.79). This difference in the pKa values may be attrib-
uted to the resonating structures in which the isomer with 2 and 4
p
/ p
[18]. The first absorption around 280 nm was a result of absorption
of non-dissociated oxime group and the maximum above 340 nm
was a result of the absorption of dissociated oxime group (Fig. 4).
The sharp isobestic point at 305e308 nm referred to the acidebase
equilibrium.
From the present study it is evident that the oximes 5e, 5k and
5l were found to be most active in reactivating both sarin and DFP
inhibited AChE. The common structural feature of the oximes
found most active in reactivating DFP and sarin inhibited AChE is
that the oximino moiety is on the position 2 of the pyridinium ring
with placement of the amide function at position 3- or 4-. The best
activity of 5e, 5k and 5l may be attributed to the optimal length of
the reactivator imparted by position of amide and oxime func-
tionality. This optimal length positions amide at the periphery of
the 20 Å gorge of AChE [25] and extends oxime function to the
active site via xylene linker through the gorge. There are two
factors which determine the distance between oxime and amide.
First is the positioning of the amide function in the pyridinium
ring, and second is the linkage of xylene (1, 4- or 1, 3-). Close
examination of 5e, 5k and 5l reveals that the xylene linkage of 1,
4- or 1, 3- and position of amide at 3- (if the xylene linkage is 1, 4-
or 1, 3-) or 4- (if the xylene linkage is 1,3-) provides appropriate
length between amide and oxime functions. While in case of other
oximes connected by 1, 2- or 1, 4- xylene linkers with corre-
sponding placement of amide function at position 4- in the pyr-
idinium ring, alters the required optimal length. This could be the
Table 1
Physical and elemental analysis data of carbamoyl bis-pyridinium oximes.
Oxime
eCH ¼ NOH
Xylene
eCONH2
Temp. (ꢁC)
Time (h)
Yielda (%)
M.Pb(ꢁC)
pKa
C
H
N
C
H
N
Calculated (%)
Found (%)
4.15
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
4
4
3
3
2
2
4
4
3
3
2
2
4
4
3
3
2
2
4
2
1,4-
1,4-
1,4-
1,4-
1,4-
1,4-
1,3-
1,3-
1,3-
1,3-
1,3-
1,3-
1,2-
1,2-
1,2-
1,2-
1,2-
1,2-
e
3-
4-
3-
4-
3-
4-
3-
4-
3-
4-
3-
4-
3-
4-
3-
4-
3-
4-
e
75
70
80
70
80
80
75
75
75
70
80
80
75
75
80
80
90
90
e
3
3
4
4
5
5
6
6
8
7
8
8
5
4
6
6
6
7
e
e
82
75
71
65
56
58
72
78
77
80
62
66
67
62
55
48
46
48
e
272e74
278e80
248e50
270e72
234e36
238e40
224e26
198e200
140e44
230e32
248e50
299e201
230e34
240e42
230e32
190e92
222e24
188e90
e
8.15 ꢂ 0.05
8.09 ꢂ 0.04
8.79 ꢂ 0.05
8.77 ꢂ 0.05
7.72 ꢂ 0.03
7.76 ꢂ 0.03
7.95 ꢂ 0.03
8.01 ꢂ 0.04
8.76 ꢂ 0.05
8.75 ꢂ 0.05
7.75 ꢂ 0.03
7.69 ꢂ 0.03
7.85 ꢂ 0.03
7.94 ꢂ 0.03
8.69 ꢂ 0.05
8.59 ꢂ 0.05
7.79 ꢂ 0.04
7.77 ꢂ 0.03
7.83 ꢂ 0.04
7.85 ꢂ 0.04
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
e
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
e
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
e
46.95
47.17
47.61
47.54
46.87
46.97
47.45
46.91
47.48
46.89
46.97
47.32
47.41
46.91
47.27
47.52
46.92
47.11
e
11.24
11.41
10.71
11.04
11.37
10.84
11.24
11.27
10.95
11.32
11.23
11.35
10.88
11.34
11.18
10.81
11.12
11.25
e
4.16
3.78
4.21
4.09
3.95
4.14
3.77
4.11
3.76
4.10
3.67
4.02
4.25
3.75
4.11
3.98
4.05
e
5q
5r
Obidoxime
2-PAM
e
e
e
e
e
e
e
e
e
e
e
a
Isolated yield.
The melting points are uncorrected.
b

J. Acharya et al. / European Journal of Medicinal Chemistry 46 (2011) 3926e3933
3929
0.001M 0.0001M
a
60
50
40
30
20
10
0
Oxime
0.001M 0.0001M
b
60
50
40
30
20
10
0
Oxime
0.001M 0.0001M
c
60
50
40
30
20
10
0
Oxime
Fig. 3. Efficacy of tested oximes in reactivation of OP-inhibited AChE in comparison with 2-PAM and obidoxime. Source of enzyme: electric eel, inhibitor agent (a) sarin, (b) DFP and
(c) VX; time of inhibition 15 min; time of reactivation 10 min; pH 8.0 & Temperature 37 ꢁC. The values are average of three runs with a maximum S.D. of ꢂ2.5%.
position passes through a resonating structure where the positive
charge of the pyridinium ring resides on the carbon at 2 and 4
position. This makes the oxime function to realize more electron
withdrawing effect as compared to 3-isomer which is manifested in
lower pKa of 2, 4 isomers [32]. The high reactivation potency of
oximes 5e, 5k and 5l for AChE inhibited by sarin or DFP at pH 8.0
may be due to their low value of pKa (7.69e7.75) which is closer to
the physiological pH and make the oximes to dissociate sufficiently
to be active at the pH under study.
and 5l may provide a useful therapeutic potential for the reac-
tivation of AChE inhibited by sarin and DFP. The detailed study of
antidotal efficacy including in-vivo reactivation against sarin and
DFP and determination of kinetic rate constant with human AChE is
under progress and will be reported in due course of time.
4. Experimental section
4.1. Chemistry
In conclusion, we have synthesized series of carbamoyl bis-
pyridinium monooximes and evaluated their in-vitro reactivation
efficacy against OP inhibited AChE. Based upon this study, 5e, 5k
Electric eel AChE (EC.3.1.1.7), 5, 50-dithiobis-(2-nitrobenzoic acid)
(DTNB), acetylthiocholineiodide, 2-, 3-,
& 4-pyridinealdoxime,

3930
J. Acharya et al. / European Journal of Medicinal Chemistry 46 (2011) 3926e3933
Fig. 4. Spectrophotometric determination of pKa of oxime 5f.
nicotinamide, isonicotinamide,
a
,
a0 dibromoxylene, Potassium
4.0 mmol) and nicotinamide (0.61 g, 5.0 mmol) in 20 mL dry DMF
dihydrogenphosphate, dipotassium-hydrogen phosphate trizma-
base and trizma-HCl were purchased from SigmaeAldrich, USA
and used without further purification. Glycine were obtained from
E. Merck (India) and used without further purification. Solvents
(DMF, acetonitrile, acetone, methanol) were purchased from S.D.
Fine Chemicals (India) and dried before use. VX, sarin and DFP was
prepared in this laboratory with >98% purity (GC and 31P NMR). 2-
PAM was prepared according to the method of Ginsburg and Wilson
[33] The bis-pyridinium oximes and obidoxime were synthesized,
characterized by their I.R., 1H NMR & 13C NMR spectral data (Table 2)
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.
was stirred at 70e75 ꢁC for 3 h. The solids appeared were filtered off
and washed repeatedly with hot dry acetone. Re-crystallization
from methanol-acetonitrile or methanol-acetone mixture gave
the pure compound (5a). Yield: 1.66 g (82%). All other compounds
were synthesized as per the reaction conditions shown in Table 1.
4.2.3. Spectral characterization of Carbamoyl bis-pyridinium
monooximes
Purity of the synthesized compounds were checked by thin-
layer chromatography (TLC, cellulose, DS-O, Fluka) with 1-
butanol, acetic acid, water (3:1:1) as mobile phase. Melting points
were determined using melting point apparatus IA9200 Electro-
thermal, U.K., and are uncorrected. The structure of the compounds
was confirmed by their elemental analysis and spectral data.
Elemental analyses was conducted on an ELEMENTAR, vario MICRO
cube, Universal micro analyzer and were within ꢂ0.4% of the
calculated values (Table 1). Infra-red (I.R.) spectra was obtained as
KBr discs on a Bruker TENSOR-27 FTIR spectrophotometer (Table 2).
IR absorption in the range of 3393e3342 cmꢀ1 was due to oximino
OH group. The other characteristic absorptions were at
3025e3035 cmꢀ1 CeH str (aryl) and at 2988e2965 cmꢀ1 for CeH
str (aliphatic). The absorptions in the range of 1678e1709 cmꢀ1 is
due to amide carbonyl group. The characteristic absorptions for
oximes were quite distinct and appeared at 1642-1600 cmꢀ1 and
1010-990 cmꢀ1 1H NMR (DMSO-d6) spectra were recorded on
Bruker Avance 400 spectrometer at 400 MHz using tetramethylsi-
4.2. Synthetic procedure
4.2.1. Synthesis of mono-pyridinium oxime intermediate
In
pyridinealdoxime (2.44 g, 20.00 mmol) in 50 mL dry acetone was
added slowly to a stirred solution of
a0 dibromo-p-xylene (13.2 g,
a typical experimental procedure a solution of 4-
a,
50.00 mmol) in 100 mL dry acetone at room temperature. The
mixture was stirred at r.t. for 4 h. The solids appeared were filtered
off and washed repeatedly with hot dry acetone to give the inter-
mediate 1-(4-bromomethyl benzyl)-4-(hydroxyimino methyl)-
pyridinium bromide. The compound thus obtained was sufficiently
pure for use in the next step. m.p. 194e96 ꢁC. Yield: 5.65 g (73%). All
other intermediate pyridinium monooximes were prepared in
a similar manner.
lane as internal standard and expressed in the
OH protons appeared as a singlet in the range of
were exchangeable with D2O. Signals at 5.85e6.19 were due to
NeCH2 protons. The aryl protons appeared in the range of
d
(ppm) values. The
d
12.24e13.10 and
d
4.2.2. Synthesis of Carbamoyl bis-pyridinium monooximes
3-carbamoyl-40-hydroxyiminomethyl-1,10-(1,4-
phenylenedimethyl)-bispyridinium dibromide (5a). In a typical
experimental procedure, a mixture of 1-(4-bromomethyl benzyl)-
4-(hydroxyimino methyl)-bispyridinium bromide (3a) (1.54 g,
d
7.23e8.27. Pyridine and N]CH protons appeared in the range of
d
8.17e9.92. 13C NMR (DMSO-d6) chemical shifts values were
obtained using the same instrument at 100 MHz (Table 2). The
methylene carbon attached to pyridinium nitrogen (NeCH2)

J. Acharya et al. / European Journal of Medicinal Chemistry 46 (2011) 3926e3933
3931
Table 2
Spectral data of carbamoyl bis-pyridinium oximes.
Oxime
I.R. (KBr) gmax(cmꢀ1
)
1H NMR(400 MHz)
, ppm, DMSO-d6)
13C NMR (100 MHz)
d, ppm, DMSO-d6)
d
5a
3279, 3143, 3028, 2946,
1703, 1646, 1505, 1434,
1396, 1278, 1262, 1004,
775, 667
5.87 (s, 2H, NCH2), 5.95 (s, 2H, NCH2), 7.61e7.69(m, 4H, -Ph),
8.20 (s, 1H, NH2), 8.24e8.25 (d, 2H, -Py), 8.27e8.31 (t, 1H, -Py),
8.41 (s, 1H, CH]N), 8.64 (s,1H, NH2), 8.97e8.99 (d,1H),
9.17e9.18 (d, 2H, -Py), 9.34e9.35 (d, 1H, -Py), 9.69 (s, 1H, -Py),
12.87 (s, 1H, OH)
62.12, 62.95, 124.41, 128.29, 128.41, 129.71,
129.97, 134.15, 135.03, 135.45, 144.09, 145.33,
146.11, 148.89
5b
5c
3361, 3169, 3118, 3038,
2997, 1669, 1623, 1570,
1454, 1430, 1399, 1279,
1129, 995, 777, 754
3270, 3148, 3025, 2955,
1696, 1638, 1514, 1441,
1403, 1282, 1275, 1011,
768, 671
5.85 (s, 2H, NCH2), 5.93 (s, 2H, NCH2), 7.60e7.63 (m, 4H, -Ph)
8.24e8.25 (d, 2H, -Py), 8.29 (s, 1H, NH2), 8.40 (s, 1H, CH]N),
8.44e8.46 (d,2H), 8.67 (s,1H, NH2), 9.15e9.16 (d, 2H, -Py),
9.38e9.39 (d, 2H, -Py), 12.87 (s, 1H, OH)
5.95 (s, 2H, NCH2), 5.98 (s, 2H, NCH2), 7.66e7.71 (m, 4H, -Ph),
8.17e8.22 (t, 1H, -Py), 8.28e8.32 (m, 2H, -Py), 8.36 (s, 1H,
CH]N),
8.66 (s, 1H, NH2), 8.73e8.75 (d, 1H, -Py), 8.99e9.01 (d, 1H, -Py),
9.23e9.24 (d, 1H, -Py), 9.38e9.39 (d, 1H, -Py), 9.47 (s, 1H, eNH2),
9.72 (s, 1H, -Py), 12.27 (s, 1H, OH)
61.18, 62.82, 123.11, 126.22, 128.98, 129.61,
134.77, 135.85, 144.37, 145.42, 148.07, 148.93
64.31, 64.55, 128.63, 129.96, 130.19, 130.51,
133.95, 134.22, 134.33, 142.99, 144.46,
144.73, 144.85
5d
5e
3355, 3144, 3108, 3037,
2988, 1657, 1631, 1560,
1459, 1404, 1260, 1123, 631
5.93 (s, 2H, NCH2), 5.95 (s, 2H, NCH2), 7.66 (s, 4H, -Ph), 8.17e8.21
(t, 1H, -Py), 8.30 (s, 1H, NH2), 8.35 (s, 1H, CH]N), 8.45e8.47
(d, 1H), 8.70 (s, 1H, NH2), 8.73e8.75 (d, 1H, -Py), 9.19e9.21
(d, 2H, -Py), 9.41 (s, 1H, -Py), 9.43e9.45 (d, 2H, -Py), 12.28
(s, 1H, OH)
5.95 (s, 2H, NCH2), 6.12 (s, 2H, NCH2), 7.65 (s, 4H, -Ph),
8.20e8.22 (m, 2H, -Py), 8.25 (s, 1H, NH2), 8.44e8.46
(d, 1H, -Py), 8.67e8.71 (m, 3H, -Py), 8.98e9.01 (d, 1H, -Py),
9.23e9.24 (d, 1H, -Py), 9.39e9.41 (d, 1H, -Py), 9.65 (s,1H, NH2),
13.10 (s, 1H, OH).
62.51, 63.32, 125.11, 128.28, 129.49, 133.25,
134.89, 141.66, 142. 69, 143.55, 145.35,
146.10, 148.88
3350, 3291,3148, 3025,
2937, 1696, 1646, 1583,
1508, 1442, 1393, 1188, 672
60.32, 62.14, 125.63, 128.52, 129.11, 129.96,
130.36, 130.89, 134.02, 134.57, 142.01,
143.40, 144.08, 146.11, 146.53, 147.38,
147.74, 148.58
5f
3357, 3150,3115, 3031, 2994,
1678, 1622, 1568, 1455, 1399,
1264, 1131, 621
5.92 (s, 2H, NCH2), 6.10 (s, 2H, NCH2), 7.67 (s, 4H, -Ph), 7.98
(s, 1H, NH2), 8.18e8.21 (t, 1H, -Py), 8.34 (s, 1H, NH2), 8.38
(s, 1H, CH]N), 8.43e8.47 (m, 2H, -Py), 8.63e8.69 (m, 3H),
9.20e9.21 (d, 1H, -Py), 9.33e9.35 (m, 1H, -Py), 13.07 (s, 1H, OH).
5.86 (s, 2H, NCH2), 5.95 (s, 2H, NCH2), 7.52e7.59 (m, 3H, -Ph),
7.76 (s, 1H, -Ph), 8.21 (s, 1H, NH2), 8.25e8.30 (m, 3H, -Py), 8.44
(s, 1H, CH]N), 8.64 (s, 1H, NH2), 8.99e9.01 (d, 1H, -Py),
9.14e9.16 (d, 2H, -Py), 9.31e9.32 (d, 1H, -Py), 9.64 (s,1H, -Py),
12.89 (s, 1H, OH)
59.82, 61.21, 126.43, 127.51, 128.17,
129.36, 130.09, 133.91, 135.27, 135.84,
141.08, 145.50, 146.18, 146.72, 147. 53,
148.10
62.05, 62.76, 123.84, 127.63, 129.14,
129.75, 130.41, 130.93, 134.16, 134.88,
135.79, 144.14, 145. 21, 145.79, 146.20,
147.01, 148.94
5g
3266, 3135, 3034, 2943, 1697,
1649, 1500, 1441, 1390, 1282,
1266, 1001, 776, 661
5h
5i
3246, 3124, 3049, 2955, 1705,
1641, 1507, 1435, 1388, 1298,
1279, 998, 772, 669
5.86 (s, 2H, NCH2), 5.94 (s, 2H, NCH2), 7.52e7.59 (m, 3H, -Ph),
7.75 (s, 1H, -Ph), 8.26e8.27 (d, 2H, -Py), 8.31 (s, 1H, NH2), 8.44
(s, 1H, CH]N), 8.47e8.48 (d, 2H, -Py), 8.71 (s,1H, NH2),
9.15e9.17 (d, 2H, -Py), 9.38e9.40 (d, 2H, -Py), 12.89
(s, 1H, OH)
5.97 (s, 2H, NCH2), 6.00 (s, 2H, NCH2), 7.50e7.54 (m, 2H, -Ph),
7.71 (s, 1H, -Ph), 8.19 (s, 1H, -Ph), 8.22e8.24 (t, 1H, -Py),
8.28e8.32 (m, 2H, -Py), 8.39 (s, 1H, CH]N), 8.66 (s, 1H, NH2),
8.76e8.78 (d, 1H, -Py), 9.01e9.03 (d, 1H, -Py), 9.24e9.25
(d, 1H, -Py), 9.40e9.41 (d, 1H, -Py), 9.49 (s, 1H, eNH2), 9.73
(s, 1H, -Py), 12.24 (s, 1H, OH)
62.25, 62.77, 124.35, 126.21, 128.95,
129.88, 130.69, 135.70, 136.42, 144.91,
146.65, 147.90, 148.33
3231, 3130, 3029, 2948, 1699,
1652, 1505, 1448, 1397, 1287,
1268, 1006, 770, 667
62.91, 63.17, 127.89, 128.55, 129.17,
129.54, 130.61, 134.58, 135.00, 135.88,
142.65, 143.25, 143.96, 144.46, 144.98,
146.27, 147.11
5j
3228, 3137, 3065, 2943, 1709,
1654, 1496, 1450, 1392, 1280,
1263, 1005, 772, 669
5.92 (s, 2H, NCH2), 5.94 (s, 2H, NCH2), 7.51e7.59 (m, 3H, -Ph),
7.76 (s, 1H, -Ph), 8.18e8.21 (m, 1H, -Py), 8.28 (s, 1H, NH2), 8.38
(s, 1H, CH]N), 8.46e8.48 (d, 2H, -Py), 8.69 s,1H, NH2),
8.75e8.77 (d, 1H, -Py), 9.14e9.16 (d, 1H, -Py), 9.37e9.41
(m, 3H, -Py), 12.26 (s, 1H, OH)
62.75, 63.01, 125.56, 128.88, 129.31,
129.62, 130.20, 133.48, 134.85, 142.08,
142.82, 143. 59, 144.61, 146.04, 148.92
5k
3223, 3110, 3071, 3005, 2940,
1682, 1633, 1573, 1513, 1458,
1257, 1182, 1135, 1006, 771, 676
5.92 (s, 2H, NCH2), 6.10 (s, 2H, NCH2), 7.29e7.31 (d, 1H, -Ph),
7.48e7.52 (m, 2H, -Ph), 7.57e7.59 (d, 1H, -Ph), 8.19e8.22
(m, 2H, -Py), 8.27 (s,1H, NH2), 8.41e8.43 (d, 1H, -Py),
8.61e8.66 (m, 3H), 8.98e9.01 (d, 1H, -Py), 9.20e9.21
(d, 1H, -Py), 9.26e9.28 (d, 1H, -Py), 9.65 (s,1H, NH2), 13.04
(s, 1H, OH).
62.11, 62.93, 125.81, 127.55, 127.90,
129.17, 129.45, 129.73, 130.04, 134.28,
135.16, 141.11, 144.02, 144.86, 145.44,
146.37, 146.91, 147.48
5l
3298, 3137, 3039, 2969, 1685,
1658, 1573, 1515, 1459, 1385,
1311, 1170, 998, 780, 727
5.91 (s, 2H, NCH2), 6.09 (s, 2H, NCH2), 7.27e7.28 (d, 1H, -Ph),
7.48e7.57 (m, 3H, -Ph), 7.94 (s, 1H, NH2), 8.19e8.23 (t, 1H, -Py),
8.30 (s, 1H, NH2), 8.42e8.47 (m, 3H), 8.63e8.69 (m, 3H),
9.20e9.21 (d, 1H, -Py), 9.33e9.35 (m, 1H, -Py), 13.07
(s, 1H, OH).
61.91, 63.08, 125.82, 127.45, 127.66,
128.71, 129.55, 130.50, 135.17, 142.03,
145.46, 145.83, 146.19, 146.72, 147.87,
148.26
5m
5n
3404, 3379, 3141, 3104, 3058,
2981, 1691, 1618, 1565, 1458,
1393, 1275, 1129, 875, 729
6.09 (s, 2H, NCH2), 6.17 (s, 2H, NCH2), 7.25e7.26 (d, 2H, -Ph),
7.51e7.53 (d, 2H, -Ph), 8.23 (s, 1H, NH2), 8.28e8.29 (d, 1H, -Py),
8.32e8.35 (m, 2H, -Py), 8.48 (s, 1H, NH2), 8.65 (s, 1H, CH]N),
9.04e9.05 (m, 3H, -Py), 9.18e9.20 (d, 1H, -Py), 9.53 (s, 1H, -Py),
12.93 (s, 1H, OH).
6.08 (s, 2H, NCH2), 6.15 (s, 2H, NCH2), 7.23e7.27 (m, 2H, -Ph),
7.51e7.53 (m, 2H, -Ph), 7.94 (s, 1H, NH2), 8.28e8.31 (m, 2H, -Py),
8.47 (s, 1H, CH]N), 8.48e8.50 (d, 2H, -Py), 8.73 (s, 1H, NH2),
9.02e9.03 (d, 2H, -Py), 9.24e9.26 (d, 2H, -Py), 12.92 (s, 1H, OH)
59.34, 59.79, 125.08, 128.43, 129.31,
129.78, 132.17, 132.76, 134.25, 144.01,
145. 19, 145.34, 145.70, 146.50, 148.85
3398, 3366, 3154, 3109, 3034,
2983, 1690, 1634, 1571, 1455,
1411, 1286, 1137, 874, 724
59.75, 60.27, 124.421, 126.29, 129.12,
129.65, 130.12, 132.50, 132.87, 145.08,
145.20, 145.64, 146.13, 148.77, 149.14
(continued on next page)

3932
J. Acharya et al. / European Journal of Medicinal Chemistry 46 (2011) 3926e3933
Table 2 (continued)
Oxime
I.R. (KBr) gmax(cmꢀ1
)
1H NMR(400 MHz)
, ppm, DMSO-d6)
13C NMR (100 MHz)
d, ppm, DMSO-d6)
d
5o
3436, 3391, 3134, 3110, 3041,
2987, 1674, 1632, 1576, 1459,
1409, 1271, 1140, 861, 720
6.15 (s, 2H, NCH2), 6.18 (s, 2H, NCH2), 7.25e7.32 (m, 2H, -Ph),
8.20e8.24 (m, 2H, -Ph), 8.32e8.35 (m, 2H, -Py), 8.39 (s, 1H, NH2),
8.64(s, 1H, CH]N), 8.79e8.81 (d, 1H, -Py), 9.04e9.06
(d, 2H, -Py), 9.18e9.20 (d, 2H, -Py), 9.26 (s, 1H, NH2), 9.53
(s, 1H, -Py), 12.27 (s, 1H, OH)
61.59, 61.79, 128.87, 128.95, 129.01, 130.77,
131.65, 131.83, 134.17, 134.28, 142.54, 143.46,
144.51, 144.62, 145.05, 145.79, 146.49, 146.80
5p
5q
5r
3430, 3356, 3161, 3109, 3051,
2978, 1682, 1634, 1576, 1459,
1398, 1274, 1128, 874, 729, 609
6.14 (s, 2H, NCH2), 6.15 (s, 2H, NCH2), 7.25e7.29 (m, 2H, -Ph),
7.95 (s, 1H, -Ph), 8.21e8.23 (m, 1H, -Ph), 8.32 (s, 1H, NH2),
8.39 (s, 1H, CH]N), 8.64 (s, 1H, NH2),8.79e8.81 (d, 2H),
9.04e9.06 (d, 2H), 9.18e9.20 (d, 2H), 9.26 (s, 1H, -Py), 9.53
(s, 1H, -Py),12.27 (s, 1H, OH)
6.17 (s, 2H, NCH2), 6.19 (s, 2H, NCH2), 7.26e7.27 (m, 2H, -Ph),
7.51e7.53 (m, 2H, -Ph), 8.22e8.24 (m, 2H, -Py), 8.29
(s, 1H, NH2), 8.41e8.43 (d, 1H, -Py), 8.62e8.67 (m, 3H, -Py),
9.01e9.03 (d, 1H, -Py), 9.26e9.27 (d, 1H, -Py), 9.41e9.44
(d, 1H, -Py), 9.61 (s, 1H, NH2), 13.01 (s, 1H, OH).
6.12 (s, 2H, NCH2), 6.14 (s, 2H, NCH2), 7.23e7.24 (m, 2H, -Ph),
7.55e7.57 (m, 2H, -Ph), 8.18e8.21 (t, 1H, -Py), 8.33 (s, 1H, NH2),
8.39 (s, 1H, CH]N), 8.43e8.47 (m, 2H, -Py), 8.63e8.67
(m, 3H), 9.23e9.24 (d, 1H, -Py), 9.31e9.33 (m, 2H, -Py),
13.08 (s, 1H, OH).
59.46, 59.98, 125.77, 128.38, 129.20, 130.55,
132.23, 132.35, 134.02, 141.91, 142.34, 143.12,
144.55, 146.01, 148.24
3384, 3166, 3072, 3032, 2937,
1709, 1671, 1644, 1583, 1440,
1400, 1191, 1140, 735, 663
58.09, 59.85, 126.42, 126.88, 128.24, 128.75,
129.18, 129.46, 129.94, 131.10, 132.31, 133.62,
141.30, 143.96, 145.15, 146.05, 146.34, 146.88,
148.08
3428, 3375, 3145, 3113, 3042,
2993, 1672, 1629, 1565, 1451,
1403, 1279, 1132, 870, 721, 604
58.14, 59.78, 125.59, 125.91, 128.04, 129.49,
129.75, 130.22, 131.30, 132.48, 142.09, 146.33,
147.81, 148.36
appeared in the regions of
ring absorbed in the range of
idinum rings appeared in the range of
d
57.8e62.9. Aromatic carbons of phenyl
124.5e138.5 and carbons on pyr-
129.2e148.9. The oximino
144.50e150.8. The detailed spectral
% Reactivation ¼ (ErꢀEi/EoꢀEi) ꢃ 100
d
d
Where Eo is the control enzyme activity at 0 min (without
inhibitor and oxime), Ei is the inhibited enzyme activity (without
oxime) determined in the similar manner described above and Er is
the activity of reactivated enzyme after incubation with the oxime
test compounds. Spontaneous reactivation of inhibited AChE was
assayed using the same protocol, the reaction mixture contained
enzyme and OP but no oxime. Under these conditions spontaneous
reactivation was found to be insignificant. All the values are cor-
rected for their oxime induced hydrolysis.
Caution! Sarin, VX and DFP are highly toxic compounds and
should be handled by trained personals only with proper protec-
tion using efficient fume hood. Great caution should be exercised
especially while preparing their dilute solutions and residues
containing these agents after each experiment must be properly
destroyed by using 20% alkali solution.
carbon (CH]N) appeared at
d
data is presented in Table 2.
4.3. In-vitro reactivation studies
The in-vitro reactivation of OP (sarin, DFP and VX)-inhibited
AChE using test oximes were carried out in triplicate in phos-
phate buffer (0.1 M, pH 8.0 at 37 ꢁC) using the method of Ellman
et al. [34]. Values depicted in figures are average of triplicate runs
with maximum relative standard deviation of ꢂ2.5%. AChE stock
solution (stock A) was prepared in phosphate buffer (pH 7.6, 0.1 M)
(332 units/0.5 mL). An aliquot of stock A was then diluted 50 times
with phosphate buffer to give stock B. A freshly prepared stock
solution of OP inhibitor (sarin, 1.4 ꢃ 10ꢀ2 M; DFP 1.08 ꢃ 10ꢀ2 M and
VX, 1.33 ꢃ 10ꢀ2 M) was in isopropanol and stored under refriger-
ation. It was then diluted appropriately with triple distilled water
just before use. All oxime stock solutions were prepared in triple
distilled water. DTNB stock solution (10 mM) was prepared in
phosphate buffer (pH 7.6, 0.1 M). The substrate stock (acetylth-
iocholine iodide, 75 mM) was prepared in distilled water. The
4.4. Determination of acid dissociation constant (pKa)
The acid dissociation constants (pKa) of all the oximes were
determined using the method of Albert and Sergeant [35]. The
method is based on direct determination of the ratio of molecular
species (protonated) to 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 particular pH in which compounds of interest would be present
incubation mixture was prepared by the addition of 50 mL of OP
(sarin, 1.4 ꢃ 10ꢀ6 M; DFP 1.08 ꢃ 10ꢀ4 M and VX, 3.32 ꢃ 10ꢀ7 M) to
a mixture of 50 mL enzyme (stock B) in 350 mL phosphate buffer pH
wholly in either form. 30e50
mL of oxime stock solutions
8.0 (0.1 M). The mixture was allowed to stand for 15 min at ambient
(5 ꢃ 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 measured over
the wavelength range of 200e600 nm with a reference to blank
solution at 25 ꢂ 1 ꢁC. The spectra, thus obtained in acid or alkali,
were of protonated (Dm) and deprotonated (Di) molecules. Eleven
different pH values, ranging from 6.04 to 10.37 were selected to
determine the pKa of oximes. For this, appropriate buffers con-
sisting of phosphate (6.04e7.90), tris (8.55e9.15) and glycine-NaOH
(9.47e10.37), were used to determine the dissociation constants of
temperature to give 96e98% inhibition of enzyme activity. It was
then followed by addition of 50
10ꢀ3 M) to start reactivation. The final volume of the reactivation
cocktail was 500 L. The final concentration of OP inhibitor and
m
L of oximes test solution (10ꢀ2 M &
m
oxime was diluted 10 fold in the reactivation cocktail. After 10 min
of reactivation the enzyme activity was assayed by Ellman’s
method (Fig. 3). 20
a cuvette containing 50
0.1 M). The enzyme activity was then assayed by addition of 50
m
L of reactivation cocktail was transferred to
L DTNB in phosphate buffer (pH 8.0,
L of
m
m
substrate to the cuvette against a blank containing reactivation
cocktail without substrate. The final volume of the assay mixture
was adjusted to 3 mL and final concentration of DTNB and substrate
was 0.16 mM and 1.25 mM respectively. The reactivation of
inhibited enzyme was then studied at an interval of 10 min and
followed up to 60 min. Percentage reactivation was calculated using
the following equation [16e18];
oximes. 30e50 mL of aqueous solutions of oximes were diluted to
3 mL in each buffer and optical densities were determined at
analytical wave lengths using buffer blank at 25 ꢂ 1 ꢁC. A set of 11
values of pKa were obtained using Eq.(1)
pKa ¼ pH þ log½ðDi ꢀ DÞ=ðD ꢀ DmÞꢄ
(1)

J. Acharya et al. / European Journal of Medicinal Chemistry 46 (2011) 3926e3933
[8] J. Bajger, Act. Med. 39 (1996) 101e105.
3933
where, Dm, and Di, correspond to the optical density of protonated
and deprotonated forms of the oxime, and D is the optical density in
the buffer.
[9] C.S. Rao, V. Venkateswahlu, G. Achaiah, Bioorg. Med. Chem. Lett. 16 (2006)
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(2009) 1214e1217.
The average value of the 11 measurements was considered the
pKa of the compound with respect to oximino functionality. Ther-
mospectronic Unicam 300 UVeVisible double beam spectropho-
tometer with quartz cells of 10 mm was used for spectrometric
analysis. The quartz cells were attached to a thermostatic water
bath (Julabo) for maintaining the constant temperature
(25 ꢂ 1.0 ꢁC). pH values of buffers were determined using a Mettler-
Toledo SevenEasy pH meter equipped with Inlab@ Expert Pro glass
electrode with an accuracy of ꢂ0.01 units. The pH meter was cali-
brated at 25 ꢁC using the two point calibration method with
commercially available Mettler-Toledo standard buffer solutions pH
7.00 and 9.21.
Reagents - Freshly prepared standard solutions of oximes
(5 ꢃ 10ꢀ3 M) in distilled water were used as stock solutions. Buffer
solutions of appropriate pH were prepared according to the
reported method [36]. Solutions of oximes in 0.1 M hydrochloric
acid and 0.1 M sodium hydroxide were used for determining the
analytical wavelength of undissociated and dissociated forms
respectively.
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Acknowledgement
Authors thank Dr. R. Vijayraghavan, Director, Defence Research
and Development Establishment, Gwalior for his keen interest in
this work. The authors are also thankful to Dr. D. K. Dubey and Dr. B.
K. Bhattacharya for their valuable suggestions.
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