In vitro reactivation of organophosphorus (OP)-inhibited electric eel acetylcholinesterase by novel monoquaternary pyridinium oximes

Article abstract of DOI:10.1007/s00044-012-0128-5

The synthesis of a series of monoquaternary pyridinium oximes bearing either a long-chain alkyloxymethyl or benzyloxymethyl side chain and the corresponding in vitro evaluation for the reactivation of electric eel acetylcholinesterase-inhibited organophosphorus inhibitors viz. sarin, DFP, and VX is reported. The data were compared with that of 2-PAM and obidoxime. Compounds bearing a benzyloxymethyl and 4-methylbenzyloxymethyl side chain showed better reactivation compared to obidoxime. However, none of the newly synthesized oximes bearing an aliphatic side chain could surpass the reactivation potential of 2-PAM. The pKa of the new oximes were determined and correlated with the observed reactivation potential.

Full text of DOI:10.1007/s00044-012-0128-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1277–1286
DOI 10.1007/s00044-012-0128-5
ORIGINAL RESEARCH
In vitro reactivation of organophosphorus (OP)-inhibited electric
eel acetylcholinesterase by novel monoquaternary pyridinium
oximes
•
Jyotiranjan Acharya Hemlata Rana
•
•
V. Aditya Kapil M. P. Kaushik
Received: 3 November 2011 / Accepted: 23 May 2012 / Published online: 10 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract The synthesis of a series of monoquaternary
pyridinium oximes bearing either a long-chain alkyloxym-
ethyl or benzyloxymethyl side chain and the corresponding
in vitro evaluation for the reactivation of electric eel acet-
ylcholinesterase-inhibited organophosphorus inhibitors viz.
sarin, DFP, and VX is reported. The data were comparedwith
that of 2-PAM and obidoxime. Compounds bearing a ben-
zyloxymethyl and 4-methylbenzyloxymethyl side chain
showed better reactivation compared to obidoxime. How-
ever, none of the newly synthesized oximes bearing an ali-
phatic side chain could surpass the reactivation potential of
2-PAM. The pKa of the new oximes were determined and
correlated with the observed reactivation potential.
neurotransmitter acetylcholine (ACh) (Tougu, 2001).
Hydrolysis of ACh is efficiently brought about by the
serine hydroxyl moiety present at the bottom of the narrow
˚
20 A active site gorge of the enzyme AChE (Sussman
et al., 1991). AChE is the primary target of organophos-
phorus compounds (OPs) that irreversibly inhibit the
enzyme (MacPhee-Quigly et al., 1985). Some of these OPs
have been developed in the past as chemical warfare nerve
agents viz. sarin, soman, tabun, VX etc. Further, OP pes-
ticides such as paraoxon, malathion, chlorpyriphos etc., are
also used in the agriculture (Eyer, 2003). These OPs con-
tinue to be threat to the society due to their possible use
during military conflicts or in terrorist acts (MacIlwain,
1993; Nagao et al., 1997). The broad distribution of OPs
causes several millions intoxication each year, of which
some are fatal (Kwong, 2002; Gunnel and Eddleston,
2003).
Keywords Pyridinium oximes ꢀ Reactivators ꢀ
2-PAM ꢀ Acetylcholinesterase ꢀ Organophosphorus
inhibitors
OPs inhibit AChE by forming a stable alkyl phosphonate,
phosphate or phosphoramidate conjugates that are cova-
lently attached to the serine hydroxy group present in the
active site (Bajgar, 2004). The function of the AChE can be
restored by removal of the OP moiety from OP–AChE
conjugate, a process known as reactivation. Strong nucleo-
philes such as oximes are reactivators of OP-inhibited AChE
(Volans, 1996). The strength of the nucleophile, the orien-
tation of the nucleophile with respect to the phosphate
conjugated to the active center serine, and dealkylation of
the OP–AChE conjugate (aging) are well known factors that
affect the process of reactivation (Wong et al., 2000). Based
on these factors, various quaternary pyridinium oximes viz.
2-PAM, obidoxime, HI-6, and TMB-4 (Fig. 1) were subse-
quently developed and used as therapeutic agents in the
treatment of OP intoxication.
Abbreviations
AChE
Acetylcholinesterase
2-PAM 2-(hydroxyiminomethyl)-1-methylpyridinium
chloride
Introduction
Acetylcholinesterase (AChE) terminates cholinergic neu-
rotransmission by catalyzing the hydrolysis of the
J. Acharya (&) ꢀ H. Rana ꢀ V. Aditya Kapil ꢀ M. P. Kaushik
Process Technology Development Division,
Defence Research & Development Establishment,
Jhansi Road, Gwalior 474002, India
However, none of the currently available oximes is
able to reactivate inhibited AChE due to diversity in the
e-mail: jracharya@rediffmail.com
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Med Chem Res (2013) 22:1277–1286
present in the active site gorge of the AChE (Tougu, 2001).
The synthesized oximes were evaluated for their in vitro
reactivation potential against electric eel AChE inhibited by
sarin, VX, and diisopropylfluoro phosphate (DFP) (Fig. 2).
Materials and methods
Chemicals
Chloromethyl ethers were prepared according to the known
synthetic method (Farren et al., 1925). Sarin, DFP, and VX
were prepared in this laboratory with [98 % purity (GC
and 31P NMR). 2-PAM was prepared according to the
method of Ginsburg and Wilson (1957). The synthesis of
pyridinium oximes involved alkylations of 3-, and 4-pyri-
dine aldoxime with chloromethoxy alkane, chlorometh-
oxymethyl benzene, and 1-chloromethoxymethyl-4-methyl
benzene (Scheme 1). The method afforded compounds
3a–3r with good yields within 3–6 h (Table 1). 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 and
characterized by their elemental analysis and spectral data.
Fig. 1 Commonly used reactivators
F
O
O
F
P
P
O
O
O
DFP
Sarin
O
S
P
N
O
VX
Fig. 2 Structure of OP inhibitors used in this study
Chemistry
structure of OP compounds (Bajgar, 1996). Therefore, an
effective therapy by a single oxime to all the known nerve
agents is still lacking.
General procedure for the synthesis of monoquaternary
pyridinium oximes (3a–3r)
The numerous new oximes have been synthesized and
tested in the last decade (Musilek et al., 2007; Bharate et al.,
2009; Jeong et al., 2009; Worek et al., 2007). Recently
several bis-pyridinium oximes linked by variable lengths of
alkylene and bis-methoxy alkane chain were reported as
AChE reactivators (Pang et al., 2003; Acharya et al., 2009a,
b; Musilek et al., 2006). 2-PAM and 4-PAM analogs of
quaternary monopyridinium oximes with varying side chain
are also reported (Odzak et al., 2007; Bharate et al., 2009).
Again, docking studies with these types of compounds with
OP-inhibited AChE led to the assumption that the linker
between pyridinium and aromatic ring should be longer to
achieve better reactivation (Odzak et al., 2007).
Hydroxyiminomethyl pyridine (1.0 g, 8.2 mmol) in 50 mL
dry acetonitrile was taken in a two-neck round bottom flask
equipped with a calcium chloride guard tube, magnetic
stirrer, and dropping funnel. To this was added chloro-
methoxy alkane, (10.0 mmol) slowly over 30 min with
stirring at room temperature. It was then further stirred for
3–6 h at room temperature and monitored by TLC. The
solid obtained was filtered off, washed repeatedly with hot
dry acetone and recrystallized with dry methanol–acetone
mixture to give pure product.
Dodecyloxymethyl-4-(hydroxyiminomethyl)-pyridinium
bromide (3a)
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
(Epstein et al., 1978; Katrolia et al., 1994). Therefore, in
continuation of our work on antidotes against nerve agents
(Acharya, et al., 2008, 2009a, b, 2010), herein we report the
synthesis of a new series of monoquaternary pyridinium
oximes bearing either a long-chain alkyloxymethyl or a ben-
zyloxymethyl side chain to enhance the lipid solubility and
stability, which is found to be low with the currently available
antidotes (Gray, 1984) as well as to allow the hydrophobic
interactions with some of the aromatic residues that are
FTIR (KBr cm-1): 3423 (O–H str), 3011, 2920, 2853 (C–H
str), 1646, 1467 (C–C aryl), 1301, 1159 (C–N str), 1111,
CH=NOH
CH=NOH
CH3CN, r.t.
O
Cl
Cl
O
R
stirring, 3-6h
N
N
R
1
2
3
Scheme 1 Synthesis of quaternary monopyridinium oximes
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Med Chem Res (2013) 22:1277–1286
1279
Table 1 Physical data of monopyridinium oximes
Oxime
–CH=NOH
R
Time (h)
Yielda (%)
MPb (°C)
pKa
3a
4
4
4
4
4
4
4
4
4
3
3
3
3
3
3
3
3
3
C12H25
3
3
3
4
4
5
5
5
6
3
3
4
5
5
5
6
5
6
76
70
77
78
68
62
68
65
57
78
70
66
60
69
51
62
63
66
156–60
206–10
190–92
186–90
196–198
198–200
208–10
138–40
140–42
172–74
174–76
170–74
158–60
160–62
152–54
180–82
144–46
144–48
8.23 0.04
8.20 0.03
8.17 0.03
8.15 0.03
8.21 0.05
8.22 0.04
8.20 0.05
8.25 0.03
8.23 0.03
9.17 0.04
9.23 0.05
9.25 0.05
9.41 0.04
9.25 0.05
9.24 0.05
9.27 0.05
9.25 0.04
9.14 0.03
7.83 0.04
7.85 0.04
3b
C
C
11H23
10H21
3c
3d
C9H19
3e
C8H17
3f
C7H15
3g
C6H13
3h
–CH2C6H5
–CH2C6H4-4-CH3
3i
3j
C
C
C
12H25
11H23
10H21
3k
3l
3m
C9H19
3n
C8H17
3o
C7H15
3p
C6H13
3q
–CH2C6H5
–CH2C6H4-4-CH3
3r
2-PAM
Obidoxime
a
Isolated yields
b
Uncorrected melting points
1018 (C–O–C str), 778 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.85 (t, J = 8 Hz, 3H, –CH3),
1.21 (m, 18H, –CH2–), 1.59 (m, 2H, –CH2–), 3.56
(t, J = 6 Hz, 2H, OCH2), 5.89 (s, 2H, NCH2), 8.29 (d,
J = 8 Hz, 2H, –Py), 8.47 (s, 1H, CH=N), 9.08 (d,
J = 8 Hz, 2H, –Py), 12.98 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 13.92, 22.14, 25.24, 28.66, 28.73,
28.98, 29.03, 29.06, 31.33, 70.28, 88.23, 124.06, 143.68,
145.26, 150.04; Anal. Calcd for C19H33ClN2O2: C, 63.95;
H, 9.25; N, 7.85. Found: C, 63.54; H, 9.37; N, 8.18;
*ESI–MS: m/z 321.35 [M]? [C19H33N2O2]?.
N, 8.17. Found: C, 63.47; H, 9.24; N, 7.91; *ESI–MS:
?.
m/z 307.25 [M]? [C18H31N2O2]
Decyloxymethyl-4-(hydroxyiminomethyl)-pyridinium
bromide (3c)
FTIR (KBr cm-1): 3429 (O–H str), 3009, 2921, 2853 (C–H
str),1645, 1466 (C–C aryl), 1301, 1155 (C–N str), 1110,
1016 (C–O–C str), 778 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.84 (t, J = 6 Hz, 3H, –CH3),
1.23 (m, 14H, –CH2–), 1.52 (m, 2H, –CH2–), 3.56
(t, J = 6 Hz, 2H, OCH2), 5.90 (s, 2H, NCH2), 8.29 (d,
J = 8 Hz, 2H, –Py), 8.47 (s, 1H, CH=N), 9.09 (d,
J = 8 Hz, 2H, –Py), 12.98 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 13.94, 22.06, 25.17, 28.60, 28.64,
28.91, 31.25, 70.15, 88.09, 123.96, 143.68, 145.14, 149.97;
Anal. Calcd for C17H29ClN2O2: C, 62.10; H, 8.82; N, 8.52.
Undecyloxymethyl-4-(hydroxyiminomethyl)-pyridinium
bromide (3b)
FTIR (KBr cm-1): 3416 (O–H str), 3010, 2922, 2852 (C–H
str), 1644, 1463 (C–C aryl), 1302, 1169 (C–N str), 1111
(C–O–C str), 1007, 779 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.85 (t, J = 8 Hz, 3H, –CH3),
1.22 (m, 16H, –CH2–), 1.61 (m, 2H, –CH2–), 3.57
(t, J = 6 Hz, 2H, OCH2), 5.91 (s, 2H, NCH2), 8.29 (d,
J = 8 Hz, 2H, –Py), 8.47 (s, 1H, CH=N), 9.10 (d,
J = 8 Hz, 2H, –Py), 13.00 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 13.94, 22.07, 25.18, 28.61, 28.67,
28.92, 28.96, 31.26, 70.17, 88.11, 123.97, 143.67, 145.16,
149.97; Anal. Calcd for C18H31ClN2O2: C, 63.06; H, 9.05;
*
Found: C, 62.33; H, 8.36; N, 8.23; ESI–MS: m/z 293.28
[M]? [C18H31N2O2]?.
Nonyloxymethyl-4-(hydroxyiminomethyl)-pyridinium
bromide (3d)
FTIR (KBr cm-1): 3410 (O–H str), 3016, 2924, 2853 (C–H
str), 1647, 1463 (C–C aryl), 1403, 1292 (C–N str), 1154,
1103 (C–O–C str), 999, 785 cm-1 –(CH2)n–; 1H NMR
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Med Chem Res (2013) 22:1277–1286
(400 MHz, DMSO-d6) d 0.84 (t, J = 6 Hz, 3H, –CH3),
1.22 (m, 12H, –CH2–), 1.50 (m, 2H, –CH2–), 3.56
(t, J = 6 Hz, 2H, OCH2), 5.90 (s, 2H, NCH2), 8.30 (d,
J = 8 Hz, 2H, –Py), 8.47 (s, 1H, CH=N), 9.11 (d,
J = 8 Hz, 2H, –Py), 13.00 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 13.98, 22.11, 25.22, 25.66, 28.44,
28.81, 29.34, 31.29, 70.75, 88.81, 124.06, 143.68, 145.26,
150.04; Anal. Calcd for C16H27ClN2O2: C, 61.04; H, 8.58;
N, 8.90. Found: C, 60.65; H, 8.97; N, 8.56; *ESI–MS:
m/z 279.21 [M]? [C16H27N2O2]?.
J = 8 Hz, 2H, OCH2), 5.91 (s, 2H, NCH2), 8.29 (d,
J = 8 Hz, 2H, –Py), 8.47 (s, 1H, CH=N), 9.10 (d,
J = 8 Hz, 2H, –Py), 13.00 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 13.94, 22.12, 25.33, 25.69, 28.51,
29.21, 31.29, 70.21, 88.95, 123.48, 142.61, 145.66, 148.42;
Anal. Calcd for C13H21ClN2O2: C, 57.24; H, 7.70; N,
10.27. Found: C, 57.57; H, 7.88; N, 9.80; *ESI–MS:
m/z 237.19 [M]? [C13H21N2O2]?.
Benzyloxymethyl-4-(hydroxyiminomethyl)-pyridinium
bromide (3h)
Octyloxymethyl-4-(hydroxyiminomethyl)-pyridinium
bromide (3e)
FTIR (KBr cm-1): 3436 (O–H str), 3012, 2939, 2822 (C–H
str), 1637, 1596, 1459 (C–C aryl), 1293 (C–N str), 1159,
1108 (C–O–C str), 1006, 753 cm-1; H NMR (400 MHz,
1
FTIR (KBr cm-1): 3434 (O–H str), 3010, 2925, 2855(C–H
str), 1644, 1463(C–C aryl), 1302(C–N str), 1161,
1108(C–O–C str), 1015, 779 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.84 (t, J = 6 Hz, 3H, –CH3),
1.21 (m, 10H, –CH2–), 1.52 (m, 2H, –CH2–), 3.57
(t, J = 6 Hz, 2H, OCH2), 5.91 (s, 2H, NCH2), 8.30 (d,
J = 4 Hz, 2H, –Py), 8.47 (s, 1H, CH=N), 9.10 (d,
J = 4 Hz, 2H, –Py), 13.01 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 14.38, 22.50, 25.65, 29.03, 29.07,
31.64, 70.62, 88.55, 124.42, 144.16, 145.59, 150.45; Anal.
Calcd for C15H25ClN2O2: C, 59.90; H, 8.31; N, 9.31.
DMSO-d6) d 4.72 (s, 2H, OCH2), 6.04 (s, 2H, NCH2), 7.33
(m, 5H, –Ph), 8.26 (d, J = 8 Hz, 2H, –Py), 8.46 (s, 1H,
CH=N), 9.14 (d, J = 8 Hz, 2H, –Py), 13.05 (s, 1H, OH);
13C NMR (100 MHz, DMSO-d6) d 72.04, 87.83, 122.22,
123.89, 128.07, 128.43, 136.15, 143.87, 145.10, 149.99;
Anal. Calcd for C14H15ClN2O2: C, 60.32; H, 5.38; N,
10.05. Found: C, 60.44; H, 5.67; N, 9.80; *ESI–MS:
m/z 243.25 [M]? [C14H15N2O2]?.
(4-Methyl-benzyloxy)-4-(hydroxyiminomethyl)-pyridinium
bromide (3i)
*
Found: C, 59.78; H, 8.66; N, 8.88; ESI–MS: m/z 265.22
[M]? [C15H25N2O2].
FTIR (KBr cm-1): 3427 (O–H str), 3018, 2947, 2832 (C–H
str), 1642, 1607, 1458 (C–C aryl), 1304 (C–N str), 1154,
Heptyloxymethyl-4-(hydroxyiminomethyl)-pyridinium
bromide (3f)
1
1099 (C–O–C str), 1011, 798 cm-1; H NMR (400 MHz,
DMSO-d6) d 2.28 (s, 3H, –CH3) 4.67 (s, 2H, OCH2), 6.02
(s, 2H, NCH2), 7.18 (m, 4H, –Ph), 8.26 (d, J = 8 Hz, 2H,
–Py), 8.47 (s, 1H, CH=N), 9.12 (d, J = 8 Hz, 2H, –Py),
13.04 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) d
21.21, 72.41, 88.21, 124.37, 128.07, 129.04, 129.44,
133.52, 144.26, 145.60, 150.43; Anal. Calcd for
C15H17ClN2O2: C, 61.53; H, 5.81; N, 9.57. Found: C,
61.18; H, 6.28; N, 9.61; *ESI–MS: m/z 257.23 [M]?
[C15H17N2O2]?.
FTIR (KBr cm-1): 3425 (O–H str), 3029, 2928, 2862(C–H
str), 1641, 1458 (C–C aryl), 1301(C–N str), 1157,
1111(C–O–C str), 998, 796 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.84 (t, J = 8 Hz, 3H, –CH3),
1.22 (m, 8H, –CH2–), 1.52 (m, 2H, –CH2–), 3.58
(t, J = 6 Hz, 2H, OCH2), 5.93 (s, 2H, NCH2), 8.30 (d,
J = 8 Hz, 2H, –Py), 8.48 (s, 1H, CH=N), 9.13 (d,
J = 4 Hz, 2H, –Py), 13.05 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 13.90, 22.03, 25.46, 25.67, 28.43,
29.22, 31.25, 70.40, 88.81, 123.27, 142.72, 145.59, 148.67;
Anal. Calcd for C14H23ClN2O2: C, 58.63; H, 8.02; N, 9.77.
Dodecyloxymethyl-3-(hydroxyiminomethyl)-pyridinium
bromide (3j)
*
Found: C, 58.48; H, 8.22; N, 9.54; ESI–MS: m/z 251.23
[M]? [C14H23N2O2]?.
FTIR (KBr cm-1): 3420 (O–H str), 3015, 2936, 2838 (C–H
str), 1651, 1460(C–C aryl), 1410, 1299 (C–N str), 1152,
1106 (C–O–C str), 1009, 789 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.85 (t, J = 6 Hz, 3H, –CH3),
1.23 (m, 18H, –CH2–), 1.52 (m, 2H, –CH2–), 3.58 (t,
J = 6 Hz, 2H, OCH2), 5.96 (s, 2H, NCH2), 8.22 (m, 1H,
–Py), 8.41 (s, 1H, CH=N), 8.81 (d, J = 8 Hz, 1H, –Py),
9.10 (d, J = 4 Hz, 1H, –Py), 9.30 (s, 1H, –Py), 12.32 (s,
1H, OH); 13C NMR (100 MHz, DMSO-d6) d 13.92, 22.05,
25.16, 28.58, 28.66, 28.91, 28.98, 31.25, 70.40, 88.81,
Hexyloxymethyl-4-(hydroxyiminomethyl)-pyridinium
bromide (3g)
FTIR (KBr cm-1): 3414 (O–H str), 3010, 2924, 2855(C–H
str), 1645, 1463(C–C aryl), 1302(C–N str), 1158, 1108,
1015 (C–O–C str),933, 778 cm-1 –(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.83 (t, J = 6 Hz, 3H, –CH3),
1.24 (m, 6H, –CH2–), 1.52 (m, 2H, –CH2–), 3.58 (t,
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Med Chem Res (2013) 22:1277–1286
1281
128.11, 133.50, 140.81, 142.89, 143.29, 144.05; Anal.
Calcd for C19H33ClN2O2: C, 63.95; H, 9.25; N, 7.85.
25.17, 25.71, 28.65, 29.22, 31.27, 70.41, 88.84, 125.94,
138.22, 142.91, 143.07, 143.33, 144.45; Anal. Calcd for
C16H27ClN2O2: C, 61.04; H, 8.58; N, 8.90. Found: C,
61.39; H, 8.68; N, 8.76; *ESI–MS: m/z 279.23 [M]?
[C16H27N2O2]?.
*
Found: C, 63.60; H, 9.38; N, 8.14; ESI–MS: m/z 321.31
[M]? [C19H33N2O2]?.
Undecyloxymethyl-3-(hydroxyiminomethyl)-pyridinium
bromide (3k)
Octyloxymethyl-3-(hydroxyiminomethyl)-pyridinium
bromide (3n)
FTIR (KBr cm-1): 3434 (O–H str), 3017, 2934, 2856(C–H
str), 1644, 1456 (C–C aryl), 1290 (C–N str), 1159, 1108
(C–O–C str), 1002, 796 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.84 (t, J = 6 Hz, 3H, –CH3),
1.23 (m, 16H, –CH2–), 1.52 (m, 2H, –CH2–), 3.59 (t,
J = 8 Hz, 2H, OCH2), 5.96 (s, 2H, NCH2), 8.22 (m, 1H,
–Py), 8.41 (s, 1H, CH=N), 8.81 (d, J = 8 Hz, 1H, –Py),
9.11 (d, J = 8 Hz, 1H, –Py), 9.31 (s, 1H, –Py), 12.34 (s,
1H, OH); 13C NMR (100 MHz, DMSO-d6) d 13.94, 22.18,
25.29, 28.44, 28.71, 28.89, 28.92, 31.34, 70.51, 88.82,
126.36, 133.51, 140.49, 142.82, 143.48, 144.11; Anal.
Calcd for C18H31ClN2O2: C, 63.06; H, 9.05; N, 8.17.
FTIR (KBr cm-1): 3427 (O–H str), 3009, 2941, 2838 (C–H
str), 1645, 1609, 1463(C–C aryl), 1296(C–N str), 1159,
1098 (C–O–C str), 1008, 787 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.84 (t, J = 8 Hz, 3H, –CH3),
1.23 (m, 10H, –CH2–), 1.51 (m, 2H, –CH2–), 3.59 (t,
J = 6 Hz, 2H, OCH2), 5.97 (s, 2H, NCH2), 8.22 (m, 1H,
–Py), 8.41 (s, 1H, CH=N), 8.81 (d, J = 8 Hz, 1H, –Py),
9.12 (d, J = 8 Hz, 1H, –Py), 9.32 (s, 1H, –Py), 12.35 (s,
1H, OH); 13C NMR (100 MHz, DMSO-d6) d 13.90, 22.05,
25.50, 28.71, 29.20, 31.21, 66.92, 87.84, 126.81, 137.25,
142.40, 142.86, 143.34, 143.87; Anal. Calcd for
C15H25ClN2O2: C, 59.90; H, 8.31; N, 9.31. Found: C,
59.68; H, 8.24; N, 9.59; *ESI–MS: m/z 265.19 [M]?
[C15H25N2O2]?.
*
Found; C, 63.31; H, 8.81; N, 8.29; ESI–MS: m/z 307.29
[M]? [C18H31N2O2]?.
Decyloxymethyl-3-(hydroxyiminomethyl)-pyridinium
bromide (3l)
Heptyloxymethyl-3-(hydroxyiminomethyl)-pyridinium
bromide (3o)
FTIR (KBr cm-1): 3443 (O–H str), 3012, 2929, 2858(C–H
str), 1649, 1461(C–C aryl), 1302(C–N str), 1153,
1111(C–O–C str), 1019, 777 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.85 (t, J = 6 Hz, 3H, –CH3),
1.23 (m, 14H, –CH2–), 1.52 (m, 2H, –CH2–), 3.59 (t,
J = 8 Hz, 2H, OCH2), 5.97 (s, 2H, NCH2), 8.22 (m, 1H,
–Py), 8.41 (s, 1H, CH=N), 8.81 (d, J = 8 Hz, 1H, –Py),
9.11–9.12 (d, J = 4 Hz, 1H, –Py), 9.32 (s, 1H, –Py), 12.35
(s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) d 13.92,
22.06, 25.17, 25.69, 28.61, 29.01, 29.21, 31.25, 70.37,
88.75, 126.49, 139.40, 142.36, 142.83, 143.59, 144.03;
Anal. Calcd for C17H29ClN2O2: C, 62.10; H, 8.82; N, 8.52.
FTIR (KBr cm-1): 3435 (O–H str), 3011, 2928, 2856 (C–H
str), 1639, 1454 (C–C aryl), 1308, (C–N str), 1165, 1094
(C–O–C str), 998, 779 cm-1–(CH2)n; 1H NMR (400 MHz,
DMSO-d6) d 0.83 (t, J = 6 Hz, 3H, –CH3), 1.22 (m, 8H,
–CH2–), 1.52 (m, 2H, –CH2–), 3.58 (t, J = 6 Hz, 2H,
OCH2), 5.98 (s, 2H, NCH2), 8.21 (m, 1H, –Py), 8.41 (s, 1H,
CH=N), 8.81 (d, J = 8 Hz, 1H, –Py), 9.09 (d, J = 8 Hz,
1H, –Py), 9.33 (s, 1H, –Py), 12.39 (s, 1H, OH; 13C NMR
(100 MHz, DMSO-d6) d 13.90, 22.03, 25.46, 25.67, 28.43,
29.22, 31.25, 66.94, 94.49, 126.72, 139.62, 141.23, 142.42,
143.12, 143.96; Anal. Calcd for C14H23ClN2O2: C, 58.63;
H, 8.02; N, 9.77. Found: C, 58.71; H, 8.50; N, 9.48;
*ESI–MS: m/z 251.27 [M]? [C14H23N2O2]?.
*
Found: C, 62.36; H, 8.65; N, 8.22; ESI–MS: m/z 293.27
[M]? [C18H31N2O2]?.
Nonyloxymethyl-3-(hydroxyiminomethyl)-pyridinium
bromide (3m)
Hexyloxymethyl-3-(hydroxyiminomethyl)-pyridinium
bromide (3p)
FTIR (KBr cm-1): 3439 (O–H str), 3015, 2936, 2830 (C–H
str), 1631, 1594, 1461(C–C aryl), 1297 (C–N str), 1155,
1106 (C–O–C str), 998, 768 cm-1–(CH2)n–; 1H NMR
(400 MHz, DMSO-d6) d 0.84 (t, J = 6 Hz, 3H, –CH3),
1.23 (m, 12H, –CH2–), 1.52 (m, 2H, –CH2–), 3.59 (t,
J = 8 Hz, 2H, OCH2), 5.95 (s, 2H, NCH2), 8.22 (m, 1H,
–Py), 8.41 (s, 1H, CH=N), 8.81 (d, J = 8 Hz, 1H, –Py),
9.08 (d, J = 4 Hz, 1H, –Py), 9.29 (s, 1H, –Py), 12.31 (s,
1H, OH); 13C NMR (100 MHz, DMSO-d6) d 13.94, 22.08,
FTIR (KBr cm-1): 3424 (O–H str), 3010, 2924, 2856(C–H
str), 1645, 1459(C–C aryl), 1296(C–N str), 1158, 1110,
1015 (C–O–C str), 786 cm-1–(CH2)n; 1H NMR (400 MHz,
DMSO-d6) d 0.83 (t, J = 8 Hz, 3H, –CH3), 1.23 (m, 6H,
–CH2–), 1.51 (m, 2H, –CH2–), 3.60 (t, J = 6 Hz, 2H,
OCH2), 5.98 (s, 2H, NCH2), 8.22 (m, 1H, –Py), 8.41 (s, 1H,
CH=N), 8.82 (d, J = 8 Hz, 1H, –Py), 9.13 (d, J = 4 Hz,
1H, –Py), 9.33 (s, 1H, –Py), 12.37 (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6) d 13.92, 22.12, 25.38, 25.61, 28.48,
123

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Med Chem Res (2013) 22:1277–1286
29.52, 31.34, 67.22, 94.29, 126.77, 139.58, 141.14, 142.47,
143.22, 143.89; Anal. Calcd for C13H21ClN2O2: C, 57.24;
H, 7.70; N, 10.27. Found: C, 57.33; H, 7.92; N, 10.02;
*ESI–MS: m/z 237.25 [M]? [C13H21N2O2]?.
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 consisting of
phosphate (6.04–7.90), tris (8.55–9.15), and glycine–NaOH
(9.47–10.37), were used to determine the dissociation con-
stants of oximes. Aqueous solutions of oximes (30–50 lL)
were diluted to 3 mL in each buffer and optical densities
were determined at analytical wave lengths using buffer
blank at 25 1 °C. A set of 11 values of pKa were obtained
using Eq. 1
Benzyloxymethyl-3-(hydroxyiminomethyl)-pyridinium
bromide (3q)
FTIR (KBr cm-1): 3430 (O–H str), 3016, 2935, 2820(C–H
str), 1635, 1598, 1463(C–C aryl), 1293(C–N str), 1160,
1
1108 (C–O–C str), 1007, 765 cm-1; H NMR (400 MHz,
DMSO-d6) d 4.76 (s, 2H, OCH2), 6.09 (s, 2H, NCH2), 7.32
(m, 5H, –Ph), 8.19 (m, 1H, –Py), 8.39 (s, 1H, CH=N), 8.78
(d, J = 8 Hz, 1H, –Py), 9.13 (d, J = 4 Hz, 1H, –Py), 9.33
(s, 1H, –Py), 12.34 (s, 1H, OH); 13C NMR (100 MHz,
DMSO-d6) d 72.32, 88.61, 126.60, 127.70, 128.27, 128.48,
133.05, 133.40, 138.05, 142.86, 143.37, 144.01; Anal.
Calcd for C14H15ClN2O2: C, 60.32; H, 5.38; N, 10.05.
pKa ¼ pH þ log½ðDi ꢁ DÞ=ðD ꢁ DmÞꢂ
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.
ð1Þ
The average value of the 11 measurements was con-
sidered as pKa of the compound with respect to oximino
functionality. Thermospectronic Unicam 300 UV–Visible
double beam spectrophotometer 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 1.0 °C). The
pH values of buffers were determined using a Mettler-
Toledo SevenEasy pH meter equipped with Inlab@ Expert
Pro glass electrode with an accuracy of 0.01 U. The pH
meter was calibrated at 25 °C using the two point cali-
bration method with commercially available Mettler-
Toledo standard buffer solutions pH 7.00 and 9.21.
*
Found: C, 59.88; H, 5.47; N, 10.18; ESI–MS: m/z 243.21
[M]? [C14H15N2O2]?.
(4-Methyl-benzyloxy)-3-(hydroxyiminomethyl)-pyridinium
bromide (3r)
FTIR (KBr cm-1): 3427 (O–H str), 3012, 2947, 2832 (C–H
str), 1642, 1607, 1458 (C–C aryl), 1304 (C–N str), 1154,
1
1090 (C–O–C str), 1011, 787 cm-1; H NMR (400 MHz,
DMSO-d6) d 2.22 (s, 3H, –CH3) 4.59 (s, 2H, OCH2), 6.06
(s, 2H, NCH2), 7.17 (m, 4H, –Ph), 8.18 (m, 1H, –Py), 8.38
(s, 1H, CH=N), 8.78 (d, J = 8 Hz, 1H, –Py), 9.11 (d,
J = 4 Hz, 1H, –Py), 9.29 (s, 1H, –Py), 12.34 (s, 1H, OH);
13C NMR (100 MHz, DMSO-d6) d 20.72, 72.24, 88.47,
126.48, 127.58, 128.70, 128.94, 133.05, 133.40, 137.57,
142.70, 143.07, 143.11; Anal. Calcd for C15H17ClN2O2: C,
61.53; H, 5.81; N, 9.57. Found: C, 61.66; H, 5.94; N, 9.71;
*ESI–MS: m/z 257.25 [M]? [C15H17N2O2]?.
Reagents
Freshly prepared standard solutions of oximes (5 9 10-3 M)
in distilled water were used as stock solutions. Buffer solu-
tions of appropriate pH were prepared according to the
reported method (Gomori, 1955). Solutions of oximes in
0.1 M hydrochloric acid and 0.1 M sodium hydroxide were
used for determining the analytical wavelength of undisso-
ciated and dissociated forms.
Determination of acid dissociation constant (pKa)
The acid dissociation constants (pKa) of all the oximes were
determined using the method of Albert and Sergeant (1971).
The method is based on direct determination of the ratio of
molecular species (protonated) to dissociated (deproto-
nated) 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 wholly in either
form. Oxime stock solutions (30–50 lL, 5 9 10-3 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 mea-
sured over the wave-length range of 200–600 nm with a
reference to blank solution at 25 1 °C. The spectra, thus
In vitro reactivation studies
In vitro reactivation of OP (sarin, DFP, and VX)-inhibited
AChE using test oximes were carried out in triplicate in
phosphate buffer (0.1 M, pH 8.0 at 37 °C) using the
method of Ellman et al. (1961). Values depicted in figures
are average of triplicate runs with maximum relative
standard deviation of 2.5 %. AChE stock solution (stock
A) was prepared in phosphate buffer (pH 7.6, 0.1 M)
(332 U/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 9
10-2 M; DFP, 1.08 9 10-2 M; and VX, 1.33 9 10-2 M)
123

Med Chem Res (2013) 22:1277–1286
1283
Fig. 3 Efficacy of tested
60
50
40
30
20
10
0
oximes in reactivation of OP-
inhibited AChE in comparison
with 2-PAM and obidoxime.
Source of enzyme: electric eel,
inhibitor agent sarin, DFP, and
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 SD 2.5 %
Sarin
DFP VX
Oxime
a
70
60
50
40
30
20
10
0
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 sub-
strate stock (acetylthiocholine iodide, 75 mM) was pre-
pared in distilled water. The incubation mixture was
prepared by the addition of 50 lL of OP (sarin, 1.4 9
10-6 M; DFP, 1.08 9 10-4 M, and VX, 3.32 9 10-7 M)
to a mixture of 50 lL enzyme (stock B) in 350 lL phos-
phate buffer pH 8.0 (0.1 M). The mixture was allowed to
stand for 15 min at ambient temperature to give 96–98 %
inhibition of enzyme activity. It was then followed by
addition of 50 lL of oximes test solution (10-2 M) to start
reactivation. The final volume of the reactivation cocktail
was 500 lL. The final concentration of OP inhibitor and
oxime was diluted tenfold in the reactivation cocktail.
After 10 min of reactivation the enzyme activity was
assayed by Ellman’s method (Fig. 3). An aliquot of reac-
tivation cocktail (20 lL) was transferred to a cuvette
containing 50 lL DTNB in phosphate buffer (pH 8.0,
0.1 M). The enzyme activity was then assayed by addition
of 50 lL of substrate to the cuvette against a blank con-
taining 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 and
1.25 mM, respectively. The reactivation of inhibited
enzyme was then studied at an interval of 10 min and
followed up to 1 h (Fig. 4). Percentage reactivation was
calculated using the following equation (Acharya et al.,
2008, 2009a, b, 2010)
3e
3h
3i
3q
3r
2-PAM
Obidoxime
10'
20'
30'
40'
50'
60'
Time (min)
b
60
50
40
30
20
10
0
3h
3i
3q
3r
2-PAM
Obidoxime
10'
20'
30'
40'
50'
60'
Time (min)
c
70
60
50
40
30
20
10
0
3d
3e
3h
3i
3m
3q
3r
2-PAM
Obidoxime
% Reactivation ¼ ðEr ꢁ Ei=Eo ꢁ EiÞ ꢃ 100:
10'
20'
30'
40'
50'
60'
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
Time (min)
Fig. 4 Time-dependent reactivation profile a sarin, b DFP, and c VX
of synthesized monopyridinium oximes
123

1284
Med Chem Res (2013) 22:1277–1286
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
not oxime. Under these conditions spontaneous reactivation
was found to be insignificant. All the values are corrected for
their oxime-induced hydrolysis.
reactivation by 2-PAM and obidoxime at the same oxime
concentration and same interval of time. In case of sarin-
inhibited AChE, none of these oximes could surpass the
reactivation potential of 2-PAM. However, the oxime 3h
showed only 26 % reactivation of sarin-inhibited AChE
which was at par with that of obidoxime.
The greater activity of some of the oximes warranted to
study their time-dependent reactivation profile (Fig. 4).
This is worth noticing that, in case of sarin- and DFP-
inhibited AChE (Fig. 4a, b), the reactivation potential of
most of the synthesized oximes either decreased gradually
or remained constant over 30–40 min. However, the reac-
tivation of VX-inhibited AChE (Fig. 4c) by the synthesized
oximes increased gradually with time and reached maxi-
mum in 40 min. The oxime 3h was able to reactivate 59 %
the VX-inhibited AChE after 40 min as compared to 63
and 54 % reactivation observed by 2-PAM and obidoxime,
respectively.
Results and discussion
The oximes studied in this investigation represent a homol-
ogous series that differ only in the length of the alkyl group in
the alkoxymethyl and methyl group in the benzyloxymethyl
side chain. With an increasing number of carbon atoms, the
lipophilicity of the compound increases and the dissociation
ability of the oxime group increases as well (Patocka and
Bielavsky 1972). The in vitro reactivation data of sarin, DFP,
and VX-inhibited electric eel AChE by monoquaternary
pyridinium oximes bearing alkoxymethyl side chain at
10-3 M oxime concentration is presented in Fig. 3. Most of
the oximes with aliphatic side chains showed either poor or
insignificant reactivation. Oximes containing benzyloxym-
ethyl side chain have showed increased reactivation for
DFP- and VX-inhibited AChE. It is evident from these data
that best reactivation was observed with the oxime 1-benzyl-
oxymethyl-4-(hydroxyiminomethyl)-pyridinium chloride
(3h) and 4-(hydroxyimino-methyl)-1-(4-methyl-benzyl-
oxymethyl)-pyridinium chloride (3i) in case of DFP- and
VX-inhibited AChE. The oximes, 3h and 3i respectively
reactivated 52 and 42 % of DFP-inhibited AChE in com-
parison to respectively 46 and 35 % reactivation by 2-PAM
and obidoxime at a concentration of 10-3 M after 10 min.
Similarly, both these oximes (3h and 3i) were also able
to reactivate 54 and 41 % of the VX-inhibited AChE,
respectively in comparison to 52 and 45 % respective
The reactivation of OP-inhibited AChE is also depen-
dent on factors such as physicochemical properties of the
reactivator including steric and electronic factors, lipo-
philic–hydrophilic balance, pKa etc. (Aldridge and Reiner,
1972). Since the oximate anion is involved in the nucleo-
philic attack during the reactivation of OP-inhibited AChE,
and its concentration is pH dependent, therefore the pKa
values should be taken into consideration while searching
for new oxime reactivators (Aldridge and Reiner, 1972,
Sinko et al., 2006). In the series of pyridinium oximes, the
optimum pKa value for the reactivator should be in the
range of 7.6–8.0 (Hegedorn et al., 1972; Eto, 1974). In this
regard, the pKa values of the oximes were determined
spectrophotometrically in the pH range of 6.04–10.37
(Fig. 5). Two characteristic maxima were obtained in UV
in the region 200–600 nm. These maxima correspond to
the absorption of different oxime ionized forms as a result
Fig. 5 Spectrophotometric
determination of pKa of oxime
3a
pH:
10.37
10.01
Isobestic point
9.47
9.15
8.82
8.55
7.90
7.56
7.16
6.74
6.04
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Med Chem Res (2013) 22:1277–1286
1285
of p ? p* transitions within the aromatic system (Odzak
et al., 2007). The absorption around 280 and 345 nm were
due to absorption of non-dissociated and dissociated form
of 4-pyridinium oximes, respectively (Fig. 5). However,
for 3-pyridinium oximes, the characteristic maxima were at
around 250 and 290 nm for non-dissociated and dissociated
form of the oxime group, respectively. The sharp isobestic
point at 305–310 nm referred to the acid–base equilibrium
for 4-pyridinium oximes. The pKa of bis-pyridinium oxi-
mes were found to be in the range of 8.15–9.41 (Table 1).
The reactivation of OP-inhibited AChE by quaternary
pyridinium oximes depends on several factors such as
structure of the inhibitor, structure of the reactivator,
position of the oxime group on the pyridinium ring and in
case of bis-pyridinium compounds, the nature of the con-
necting chain between the pyridinium rings (Kuca et al.,
2006). From the present study it is evident that the oximes
3h and 3i were found to be most active in reactivating
DFP-inhibited AChE. The common structural feature of
these oximes found most active in reactivating DFP-
inhibited AChE is placement of oximino moiety on the
position 4 of the pyridinium ring. This results correlates
with many other studies (Kassa et al., 2007). However, in
case of VX-inhibited AChE, no such preference for the
positioning of the oxime on the pyridinium 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 thereby leaving more free space for
oxime access to the phosphorylated site with least steric
hindrance compared to DFP or sarin (Maxwell et al.,
2008). This is manifested in the reactivation data that most
of the oximes in this study have shown significant reacti-
vation toward VX-inhibited AChE. It is further reported
that aromatic side chains in the pyridinium rings are sta-
bilized with the p–p interactions with the aryl residues
present near the peripheral anionic site of the active site
gorge of the AChE (Odzak et al., 2007). Thus, an analogy
of bis-pyridinium oximes with monopyridinium oximes 3h
and 3i can be drawn, where benzyl or 4-methyl benzyl
moiety resides at the peripheral anionic site. This locali-
zation is facilitated by p–p interactions leading to direct the
pyridinium oxime function at phosphorylated site. Absence
of such an interaction in aliphatic monooximes could be the
reason of poor or no reactivation of inhibited AChE.
It is worth noticing that the pKa of bis-pyridinium oxi-
mes having oximino function at position 4 on the pyridi-
nium ring were found to be in the lower the range
(8.15–8.25) as compared to the oximes where oximino
function is attached at position 3 on the pyridinium ring
(9.14–9.41). This finding is in close agreement to our
earlier reports (Acharya et al., 2010). The high reactivation
potential of oximes 3h and 3i for OP-inhibited AChE may
be due to their low value of pKa which make the oximes to
dissociate sufficiently to be active at the pH used for the
study.
Conclusion
In conclusion, we have synthesized a new series of quater-
nary monopyridinium oximes and evaluated their in vitro
reactivation efficacy against OP-inhibited AChE. The oxi-
mes with alkoxymethyl side chain were found to be poor
reactivators of AChE. However, oximes with benzyloxym-
ethyl and 4-methyl-benzyloxymethyl side chain have shown
significant reactivation potential against OP-inhibited
AChE. Based upon this study, pyridinium oximes with
substituted aryloxymethyl side chain may be synthesized to
study the effect of substitution in the aromatic ring on the
reactivation potential of AChE inhibited by various OP
inhibitors.
Acknowledgments Authors thank Dr. R. Vijayraghavan, Director,
Defence Research and Development Establishment, Gwalior for his
keen interest in this study. The authors are also thankful to Dr. D. K.
Dubey and Dr. B. K. Bhattacharya for their valuable suggestions.
VAK thanks DRDO, New Delhi for financial support.
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