In-vitro regeneration of sarin inhibited electric eel acetylcholinesterase by bis-pyridinium oximes bearing xylene linker

Article abstract of DOI:10.1016/j.ejmech.2008.02.020

A series of bis-pyridinium oximes connected by xylene linker were synthesized and their in-vitro reactivation potential was evaluated against acetylcholinesterase (AChE) inhibited by nerve agent, sarin. Among the synthesized compounds, α,α′xylene-bis-[3,3

Full text of DOI:10.1016/j.ejmech.2008.02.020

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European Journal of Medicinal Chemistry 44 (2009) 1326e1330
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Short communication
In-vitro regeneration of sarin inhibited electric eel acetylcholinesterase
by bis-pyridinium oximes bearing xylene linker
*
Jyotiranjan Acharya , Arvind Kumar Gupta, Avik Mazumder, Devendra Kumar Dubey
Process Technology Development Division, Defence Research & Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh 474002, India
Received 17 August 2007; received in revised form 17 December 2007; accepted 8 February 2008
Available online 5 March 2008
Abstract
A series of bis-pyridinium oximes connected by xylene linker were synthesized and their in-vitro reactivation potential was evaluated against
acetylcholinesterase (AChE) inhibited by nerve agent, sarin. Among the synthesized compounds, a,a0xylene-bis-[3,30-(hydroxyiminomethyl)
pyridinium] dibromide (3b) was found to be most potent reactivator for AChE inhibited by sarin. The oxime 3b exhibits 34% regeneration
of inhibited AChE, in comparison to 20 and 15% regeneration by 2-PAM and obidoxime, respectively, at a concentration of 10ꢀ4 M within
10 min.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Bis-pyridinium oximes; Reactivators; 2-PAM; Organophosphorus nerve agents; Sarin; Acetylcholinesterase; Xylene linkers
1. Introduction
nerve agents leads to inhibition of acetylcholinesterase by
phosphorylation of their active site serine residue [4]. The sub-
sequent accumulation of the neurotransmitter acetylcholine
and over-stimulation of cholinergic receptors results in a gener-
alized cholinergic crisis including breakdown of neuromuscu-
lar function.
Some of the most deadly compounds ever known to man
were developed in the past for use as chemical warfare agents
[1]. Phosphate esters and related Phosphorous (V) compounds
viz. isopropyl methylphosphonofluoridate (sarin), pinacolyl
methylphosphonofluoridate (soman), O-ethyl,N,N-dimethyl-
phosphoramidocyanidate (tabun) and O-ethyl-S-(2-diisopropy-
laminoethyl) methylphosphonothioate (VX) are classified as
nerve agents. In spite of world wide efforts to prevent synthe-
sis, storage and use of these compounds, the repeated use
of chemical warfare agents during military conflicts [2] and
terrorist attacks [3] displays that they constitute a persistent
threat for the population.
The standard treatment of nerve agent poisoning includes
a muscarine antagonist, e.g., atropine, and a reactivator of
OP-inhibited AChE [5]. The presently available reactivators
are oximes such as pralidoxime (2-PAM) and obidoxime
(Fig. 1). These compounds are well tried as antidotes against
OP poisoning but are considered to be rather ineffective
against certain nerve agents [6,7]. Numerous new oximes
have been synthesized and tested in the last decades [8e10].
The efficacy of oximes depends on post-inhibitory reactions
such as spontaneous dealkylation (aging) [11] and spontane-
ous dephosphorylation (spontaneous reactivation) [12] of the
OP-AChE complex. However, these organophosphorous nerve
agents behave differently towards reactivation due to their
broad structure variability and most often their deleterious
side effects become difficult to counteract [13]. Furthermore,
an effective therapy by a single oxime to all the known nerve
agents is still lacking.
Hence, efforts are on to develop an effective medical
treatment regimen of OP poisoning. The intoxication with
Abbreviations: AChE,; acetylcholinesterase; 2-PAM,; 2-(hydroxyimino-
methyl)-1-methylpyridinium chloride.
* Corresponding author. Tel.: þ91 751 2340245/1148; fax: þ91 751
2341148.
E-mail address: jracharya@rediffmail.com (J. Acharya).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.020

J. Acharya et al. / European Journal of Medicinal Chemistry 44 (2009) 1326e1330
1327
NOH
on a Bruker TENSOR-27 FTIR spectrophotometer. IR absorp-
tion in the range of 3393e3342 cmꢀ1 was due to oximino OH
group. The other characteristic absorptions were at 3025e
3035 cmꢀ1 for CeH str (aryl) and at 2988e2965 cmꢀ1 for
CeH str (aliphatic). The characteristic absorptions for oximes
were quite distinct and appeared at 1642e1600 cmꢀ1 and
NOH
HC
HC
NOH
C
H
N
Cl
N
O
N
2Cl
CH3
1
1010e990 cmꢀ1. H NMR (DMSO-d6) spectra was recorded
2-PAM
Obidoxime
on Bruker Avance 400 spectrometer at 400 MHz using tetra-
methylsilane as internal standard and expressed in the
d (ppm) values. The OH protons appeared as a singlet in the
range of d 12.41e13.01 and were exchangeable with D2O.
Signals at d 5.9e6.0 were due to NeCH2 protons. Pyridine
and N]CH protons appear in the range of d 8.2e9.1. 13C
NMR (DMSO-d6) chemical shifts values were obtained using
the same instrument at 100 MHz (Table 2). The methylene car-
bon attached to pyridinium nitrogen (NeCH2) appeared in the
regions of d 57.8e62.9. Aromatic carbons of phenyl ring ab-
sorbed in the range of d124.5e138.5 and carbons on pyridi-
num rings appeared in the range of 129.2e148.9 d. The
oximino carbon (CH]N) appeared at d 144.50e150.8. The
detailed spectral data are presented in Table 2.
Fig. 1. Oximes used as reactivatores of inhibited AChE.
2. Chemistry
Recently, several bis-pyridinium aldoximes linked by a vari-
able length alkylene chain were reported as AChE reactivators
[14,15]. Some of such oximes have shown promising reactiva-
tion profile against OP-inhibited AChE. Reactivation potential
of such oximes attracted our attention to further explore the
synthesis of bis-pyridinium oximes with different kind of
linkers. In the past, reactive oxime moiety has been incorpo-
rated into the varieties of micelle forming molecules, resulting
in significant enhancement of hydrolysis of OP compounds
[16,17]. In this context, we focused our attention on sym-
metrical bis-pyridinium oximes analogous of TMB-4 and
obidoxime class of compounds having a xylene linker between
two pyridinium rings with an aim to enhance the lipid solubil-
ity and stability, which is found to be low with the currently
available antidotes [18] as well as to allow the hydrophobic
interactions with some of the aromatic residues that are
present in the active site gorge of the enzyme acetylcholines-
terase [19].
Herein, we report the synthesis and in-vitro reactivation
studies of a series of pyridinium oximes linked with a xylene
bridge. By adopting a simple and mild synthetic method [20],
we have synthesized a series of bis-pyridinium oximes. The
reaction involved alkylations of isomeric pyridinealdoxime
(1) with a,a0 dibromoxylene (2) (Scheme 1). The method af-
forded the desired products (3aeh) with good yields within
3e6 h (Table 1). Purity of the synthesized compounds was
checked by thin-layer chromatography (TLC, cellulose,
DSO, Fluka) with 1-butanol:acetic acid:water (3:1:1) as mo-
bile phase. Melting points were determined using IA9200
Electrothermal, U.K., melting point apparatus and are uncor-
rected. Elemental analysis was conducted on a NOD 1106
Carlo Erba Instrumentazione analyser and is within ꢁ0.4%
of the calculated values (Table 1).
3. Results and discussion
The in-vitro reactivation of sarin inhibited AChE by bis-
pyridinium oximes bearing xylene bridge at three different
concentrations (10ꢀ4 M, 5 ꢂ 10ꢀ5 M and 10ꢀ5 M) is depicted
in Fig. 2. It is evident from these data that best reactivation
was observed with N,N0-p-xylene-bis[(3,30-hydroxyimino-
methyl)-pyridinium] dibromide (3b) and N,N0-m-xylene-
bis[(3,30-hydroxyiminomethyl)-pyridinium] dibromide (3e).
The oximes, 3b and 3e, respectively, reactivated 35 and 25%
of sarin inhibited eel AChE in comparison to, respectively,
21 and 15% reactivation by 2-PAM and obidoxime at the con-
centration 10ꢀ4 M after 10 min. It may also be noted that the
reactivation potential of two other oximes of the series i.e.,
N,N0-p-xylene-bis[(2,20-hydroxyiminomethyl)-pyridinium] di-
bromide (3c) and N,N0-o-xylene-bis[(3-hydroxyiminomethyl)-
pyridinium] dibromide (3g) was found to be at par with that of
2-PAM. At the lower concentration of 10ꢀ5 M (probably at-
tainable concentration in vivo), the oximes 3b and 3e, respec-
tively, exhibited reactivation potency of 20 and 10% in
comparison to 7% reactivation exhibited by 2-PAM within
10 min. It is generally known that increasing of reactivation
potency to 5e10% is sufficient for survival [9]. This suggests
that these oximes may be applicable for in vivo experiments in
animal models.
The structure of the compounds was confirmed by their
spectral data. Infra-red (IR) spectra was obtained as KBr discs
HC
NOH
CH
HC
HON
NOH
Br
DMF, stirring
r.t-90°C, 3-6h
N
N
N
2Br
Br
1
2
3a-h
Scheme 1. Synthesis of bis-pyridinium compounds.

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J. Acharya et al. / European Journal of Medicinal Chemistry 44 (2009) 1326e1330
Table 1
Physical data of compounds
Oxime
eCH]NOH
Xylene
Reaction temperature (ꢃC)
Time (h)
Yielda (%)
M.Pb (ꢃC)
Calculated (%)
Foundc (%)
C
H
N
C
H
N
3a
3b
3c
3d
3e
3f
3g
3h
a
4
3
2
4
3
4
3
2
para
para
para
meta
meta
ortho
ortho
ortho
Ambient
Ambient
Ambient
Ambient
50
3
4
4
3
5
4
6
6
82
80
70
81
81
78
72
55
268e270
258e260
205e207
240e242
234e236
238e240
208e10
47.24
47.24
47.24
47.24
47.24
47.24
47.24
47.24
3.93
3.93
3.93
3.93
3.93
3.93
3.93
3.93
11.02
11.02
11.02
11.02
11.02
11.02
11.02
11.02
47.52
46.91
46.95
47.61
47.32
47.48
46.87
47.11
4.11
4.25
4.15
3.78
3.67
4.11
4.09
4.05
10.81
11.34
11.24
10.71
11.35
10.95
11.37
11.25
70
70
90
202e204
Isolated yield.
The melting points are uncorrected.
b
c
Elemental analyses (C, H, N) were within ꢁ0.4% of the calculated values.
All the conventional oximes reported to date differ from
nerve agents is under progress and will be reported in due
course of time.
each other by the number of pyridinium rings (mono-pyridi-
nium or bis-pyridinium), the position of the oxime group on
the pyridinium ring, and, in the case of bis-pyridinium
oximes, by the chemical structure and length of the bridge
between the pyridinium rings [16,17]. That is why the
compounds differing in these features were synthesized and
evaluated. It is worth noting that these oximes showed better
reactivation of inhibited enzyme even in comparison to estab-
lished antidote 2-PAM. On changing the position of oxime
functionality from 3 to 4 i.e., the 4-isomers of bis-pyridinium
oximes (3a, 3d, & 3f) decreased the reactivation potential.
Similarly by changing the oxime group from position 3 to
2 (3c & 3h), reactivation efficiency was found to be at par
with 2-PAM. Further, from the study of the effect of different
isomers of xylene bridge (Fig. 2), it was observed that the
oximes connected by p-xylene linkers showed the highest re-
activation followed by the oximes having m- and o-xylene
linkers (3b, 3e, & 3g). It has been found that the best in-vitro
reactivation of sarin inhibited AChE of these bis-pyridinium
oximes is observed with the placement of oxime functionality
at position 3 in the pyridinium ring with a p-xylene linker.
Although the synthesized compounds did not differ in the
number of bonds between two pyridinium rings yet the
disposition of alkyl residues at 1,4 ( para-) and 1,3 (meta-)
positions of the xylene linkage does make the two oxime
functions to remain at two different locations, which is
responsible for the difference in the reactivation profile of
two compounds. Thus the 1,4-isomer (3b) does make the
oxime functions to fall farther apart than that of 1,3-isomer
(3e). The maximum reactivation activity of 3b is well in
4. Experimental section
4.1. Chemistry
Electric eel AChE (EC.3.1.1.7), 5,50-dithiobis-(2-nitroben-
zoic acid) (DTNB), acetylthiocholineiodide, 2-, 3-, & 4-pyri-
dinealdoxime and a,a0 dibromoxylenes were purchased from
Sigma-Aldrich, USA and used without further purification.
Potassium dihydrogenphosphate and dipotassium hydrogen-
phosphate were obtained from E. Merck (India) and used with-
out further purification. Solvents (DMF, acetonitrile, acetone,
methanol) were purchased from SD Fine Chemicals (India)
and dried before use. Nerve agent sarin was prepared in this
laboratory with >98% purity (GC and 31P NMR). 2-PAM
was prepared according to the reported method [23]. The
bis-pyridinium oximes were synthesized and characterized
1
by their IR, H NMR & 13C NMR spectral data (Table 2)
Purity of the synthesized oximes was checked by TLC (cellu-
lose, DSO, Fluka) with 1-butanol:acetic acid:water (3:1:1) as
solvent system.
4.2. General synthetic procedure
In a typical experimental procedure, a mixture of 4-pyridi-
nealdoxime (1.34 g, 11.00 mmol) and a,a0 dibromo-p-xylene
(1.32 g, 5.00 mmol) in 25 mL dry DMF was stirred at room
temperature for 3 h. The solids appeared were filtered off
and washed repeatedly with hot dry acetone. Re-crystallization
from methanoleacetonitrile or methanoleacetone mixture
gave the pure compound (3a). Yield: 2.10 g (82%). All other
compounds were synthesized as the per reaction conditions
shown in Table 1.
˚
agreement with 20 A active site gorge of AChE [21,22],
where one of the two pryridinium rings might reside at the
rim, and other pyridinium ring penetrates into gorge of the
enzyme where phosphorylation of active site of enzyme takes
place.
In conclusion, we have synthesized series of bis-pyridinium
oximes and evaluated their in-vitro reactivation efficacy
against sarin inhibited AChE. Based upon this study, 3b may
provide a useful therapeutic potential for the reactivation of
AChE inhibited by sarin. The detailed study of antidotal effi-
cacy including in vivo reactivation against sarin and other
4.3. In-vitro reactivation studies
The in-vitro reactivation of sarin inhibited AChE using test
oximes were carried out in triplicate in phosphate buffer (pH
8.0 at 37 ꢃC, 0.1 M) using the method of Ellman et al. [24].

J. Acharya et al. / European Journal of Medicinal Chemistry 44 (2009) 1326e1330
1329
Table 2
Spectral data of compounds
Oxime
IR (KBr) nmax(cmꢀ1
)
1H NMR (400 MHz) d/ppm, (DMSO-d6)
13C NMR (100 MHz) d/ppm, (DMSO-d6)
3a
3393, 3031, 2966, 1602,
1435, 1295,1152, 1004, 760
3368, 3029, 2988, 1625,
1430, 1290, 1153, 990, 761
5.87 (s, 4H, NCH2), 7.63(s, 4H, ePh) 8.25e8.27 (d, 4H, ePy),
8.43 (s, 2H, CH]N) 9.17e9.18 (d, 4H, ePy), 12.26 (s, 2H, OH)
5.95 (s, 4H, NCH2), 7.67 (s, 4H, ePh), 8.18e8.21
(t, 2H, ePy), 8.36 (s, 2H, N]CH),
62.12, 124.50, 127.18, 128.81, 129.73,
135.46, 145.20, 148.92, 150.80.
3b
3c
62.84, 128.63, 129.71, 129.77, 133.89,
135.25, 142.14, 142.51, 143.32, 144.56.
8.74e8.76 (d, 2H, ePy), 9.21e9.23 (d, 2H, ePy), 9.46
(s, 2H, ePy), 12.26 (s, 2H, OH)
3378, 3028, 2967, 1656,
1431, 1312, 1008, 764
6.07 (s, 4H, NCH2), 7.29 (s, 4H, -Ph), 8.14e8.17
(t, 2H,- Py), 8.43e8.45 (d, 2H, -Py),
60.15, 126.10, 127.91, 128.10, 134.69,
136.54, 141.39, 146.05, 146.60, 147.32.
8.58e8.62 (t, 2H, ePy) 8.66 (s, 2H, N ¼ CH), 9.14e9.16
(d, 2H, ePy), 12.78(s, 2H, OH)
5.92 (s, 4H, NCH2), 7.94 (s, 3H, ePh), 7.70
3d
3e
3385, 3025, 2966, 1635,
1426, 1292, 1005, 756
62.31, 124.48, 129.35, 129.62, 130.16,
135.23, 145.16, 148.88
(s, 1H, ePh), 8.26e8.28 (d, 4H, ey),
8.45 (s, 2H, N]CH) 9.14e9.15 (d, 4H, ePy) 12.88 (s, 2H, OH)
d 5.92 (s, 4H, NCH2), 7.57 (s, 3H, ePh), 7.3 (s, 1H, -Ph),
8.18e8.21 (t, 2H, ePy), 8.37 (s, 2H, N]CH),
8.75e8.77 (d, 2H, ePy), 9.14e9.16 (d, 2H, ePy),
9.40 (s, 2H, ePy), 12.27(s, 2H, OH)
3342, 3032, 2971, 1629,
1435, 1290, 992, 767
62.96, 128.63, 129.61, 129.83, 130.15, 133.84,
135.02, 142.06, 142.68, 143.32, 144.67.
3f
3390, 3035, 2968, 1631,
1425, 1308, 1012, 760
6.09 (s, 4H, NCH2), 7.24e7.26 (t, 2H, ePh),
60.19, 124.93, 130.12, 130.60,
133.30, 145.63, 145.87, 149.54
7.50e7.53 (t, 2H, ePh), 8.28e8.30 (d, 4H, ePy),
8.48 (s, 2H, N]CH), 9.03e9.05 (d, 4H, ePy), 12.92 (s, 2H, OH)
6.17 (s, 4H, NCH2), 7.32e7.34 (t, 2H, ePh),
3g
3388, 3030, 2967, 1615,
1442, 1298, 992, 768
60.52, 128.65, 129.94, 130.27, 132.65,
133.89, 142.32, 142.79, 143.34, 144.86.
7.56e7.58 (t, 2H, ePh), 8.24e8.28 (t, 2H, ePy),
8.43 (s, 2H, N]CH), 8.83e8.85 (d, 2H, ePy), 9.07e9.08
(d, 2H, ePy), 9.29 (s, 2H, ePy), 12.32 (s, 2H, OH)
6.26 (s, 4H, NCH2), 6.56e6.58 (t, 2H, ePh), 7.35e7.37
(t, 2H, ePh), 8.23e8.27 (t, 2H, ePy), 8.60e8.62
(d, 2H, ePy), 8.65 (s, 2H, N]CH), 8.71e8.74 (t, 2H, ePy),
8.93e8.95 (d, 2H, ePy), 12.35 (s, 2H, OH)
3h
3377, 3031, 2965, 1635,
1440, 1305, 998, 762
57.81, 125.52, 125.85, 128.44, 129.29,
131.52, 141.31, 146.10, 146.41, 148.15.
Values depicted in figures are average of triplicate runs with
a maximum relative standard deviation of ꢁ2%. AChE stock
solution (stock A) was prepared in phosphate buffer (pH 7.6,
0.1 M) (360 units/0.5 mL). An aliquot of stock A was then di-
luted 50 times with phosphate buffer to give stock B. A freshly
prepared stock solution of sarin (1.4 ꢂ 10ꢀ2 M) was in
isopropanol and stored under refrigeration. 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 (acetylthiocholine
iodide, 75 mM) was prepared in distilled water. The incuba-
tion mixture was prepared by the addition of 50 mL of sarin
(1.4 ꢂ 10ꢀ6 M) to a mixture of 50 mL enzyme (stock B) in
350 mL phosphate buffer (pH 8.0, 0.1 M). The mixture was al-
lowed to stand for 15 min at ambient temperature to give
96 ꢁ 1% inhibition of enzyme activity. It was then followed
by addition of 50 mL of oximes test solution (10ꢀ3 M,
5 ꢂ 10ꢀ4 M & 10ꢀ4 M) to start reactivation. The final volume
of the reactivation cocktail was 500 mL. Thus, reactivation
cocktail indeed composed of 0.08 M phosphate buffer at
a pH between 7.6 and 8.0. The final concentration of sarin
was 1.4 ꢂ 10ꢀ7 M and oxime was diluted 10 fold in the
reactivation cocktail. After 10 min of reactivation, the
enzyme activity was assayed by Ellman’s method (Fig. 2).
Twenty micro litres of reactivation cocktail was transferred
to a cuvette containing 50 mL DTNB in phosphate buffer
(pH 8.0, 0.1 M). The enzyme activity was then assayed by
addition of 50 mL of 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
40
0.0001M
35
0.00005 M
0.00001M
30
25
20
15
10
5
0
Oxime
Fig. 2. Efficacy of tested oximes in reactivation of sarin inhibited AChE
in comparison with 2-PAM. Source of enzyme: electric eel, inhibitor
agent sarin, time of inhibition 15 min; time of reactivation 10 min; pH
8.0 & temperature 37 ꢃC. The values are average of three runs with a maxi-
mum S.D. of ꢁ2%.

1330
J. Acharya et al. / European Journal of Medicinal Chemistry 44 (2009) 1326e1330
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to 1 h. Percentage reactivation was calculated using the fol-
lowing equation:
% Reaction ¼ ðErꢀEi=E0ꢀEiÞ ꢂ 100
[7] F. Worek, H. Thiermann, L. Szinicz, P. Eyer, Biochem. Pharmacol. 68
(2004) 2237e2248.
Where E0 is the control enzyme activity at 0 min (without in-
hibitor 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 sarin but no oxime.
Under these conditions spontaneous reactivation was found
to be insignificant. All the values are corrected for their oxime
induced hydrolysis.
[8] C. Bismuth, R.H. Inns, T.C. Marrs, Efficacy, toxicity and clinical use of
oximes in anticholinesterase poisoning, in: B. Ballantyne, T.C. Marrs
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[12] W.N. Aldridge, E. Reiner, Enzyme Inhibitors as Substrates e Interactions
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Acknowledgement
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Authors thank Dr. R. Vijayraghavan, Director, and Prof. M.
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work. The authors are thankful to Dr. B. K. Bhattacharya for
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