New series of monoquaternary pyridinium oximes: Synthesis and reactivation potency for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

Article abstract of DOI:10.1016/j.bmcl.2009.07.035

The preparation of a series of monoquaternary pyridinium oximes bearing either a heterocyclic side chain or a functionalized aliphatic side chain and the corresponding in vitro evaluation for reactivation of paraoxon-inhibited electric eel acetylcholinest

Full text of DOI:10.1016/j.bmcl.2009.07.035

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5101–5104
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New series of monoquaternary pyridinium oximes: Synthesis and
reactivation potency for paraoxon-inhibited electric eel and recombinant
human acetylcholinesterase
Sandip B. Bharate a, Lilu Guo b, Tony E. Reeves c, Douglas M. Cerasoli c, Charles M. Thompson a,b,
*
a NIH COBRE Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
b ATERIS Technologies LLC, 901 N Orange Street, Missoula, MT 59802, USA
c USAMRICD, Research Division, Physiology and Immunology Branch, 3100 Ricketts Point Road, APG, MD 21010, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation of a series of monoquaternary pyridinium oximes bearing either a heterocyclic side chain
or a functionalized aliphatic side chain and the corresponding in vitro evaluation for reactivation of
paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinester-
ase (rHuAChE) are reported. Several newly synthesized compounds efficiently reactivated inhibited
EeAChE, but were poor reactivators of inhibited rHuAChE. Compounds bearing a thiophene ring in the
side chain (20, 23, 26 and 29) showed better reactivation (24–37% for EeAChE and 5–9% for rHuAChE)
compared to compounds with furan and isoxazole heterocycles (0–8% for EeAChE and 2–3% for rHuAChE)
Received 3 June 2009
Revised 21 June 2009
Accepted 2 July 2009
Available online 10 July 2009
Keywords:
Organophosphorus
Acetylcholinesterase
Reactivators
Monoquaternary pyridinium oximes
Pralidoxime
at 10ꢀ5 M. The N-pyridyl-CH2COOH analog 8 reactivated EeAChE (36%) and rHuAChE (15%) at 10ꢀ4
with a kr value better than 2-pyridine aldoxime methiodide (2-PAM) for rHuAChE.
M
Ó 2009 Elsevier Ltd. All rights reserved.
2-PAM
Reactivators of acetylcholinesterase (AChE; EC 3.1.1.7) are
important therapeutic agents for the treatment of toxicity resulting
from organophosphorus inhibitors (OPI) such as sarin, paraoxon,
tabun, chlorpyrifos, cyclosarin, VX, and DFP. Some of these OPIs
have been used as warfare agents (such as sarin, soman, tabun,
VX) and are some of the most toxic man made compounds known.1
OPIs inhibit AChE via formation of a covalent bond at a serine hy-
droxyl group within the enzyme’s active site. Once inhibited, AChE
is not able to hydrolyze acetylcholine and accumulates in the syn-
aptic cleft leading to cholinergic over-stimulation, compromised
respiration and sometimes death.1 AChE inhibited by OPI can be
reactivated by 2-pyridine aldoxime methiodide (2-PAM, pralidox-
ime; 1)2 that nucleophilically attacks the phosphoryl moiety dis-
placing it from the active site serine (Fig. 1). In addition to 2-
PAM,3–6 the bis-oximes trimedoxime (TMB-4, 2),7,8 obidoxime
(3)9,10 and HI-6 (4)11,12 show efficacy as reactivators in vivo and
are widely used for the treatment of OPI poisoning.13,14 Recently,
several new pyridinium oxime reactivators were reported includ-
ing bisquaternary pyridinium oximes connected with different lin-
ker chains viz. alkylene linker,15–18 but-2-ene linker,19–22 aromatic
ring containing linkers23–25 and oxygenated linker chain.26–28
Monoquaternary pyridinium oximes include 2-PAM analogs with
varying side chain29,30 and 4-PAM analogs with modified oximes.31
Amongst these, but-2-ene linked bisquaternary 4,40-bisoxime22
and 2-PAM analogs with a propyl side chain29 showed reactivation
of paraoxon-inhibited AChE comparable to clinically known
reactivators.
Although bis-quaternary oximes are considered more effective
AChE reactivators, many countries use 2-PAM as the therapeutic
agent.32,33 The efficacy of the oxime used is also dependent on
the AChE type. Certain AChE types undergo rapid reactivation
O
Z
X
Y
P
X
Y
O
P
Inhibition
AChE
AChE
Ser OH
Ser O
Reactivation
Acetylcholinesterase
Inhibited AChE
RCH=NOH
Oximes 1-4
2X
A
R2
N
R1
N
Me
OH
N
N
I
Trimedoxime (2): A = CH2, R1 = R2 = 4-CHNOH, X = Br
Obidoxime (3): A = O, R1= R2 = 4-CHNOH, X = Cl
HI-6 (4): A = O, R1 = 2-CHNOH, R2 = 4-CONH2, X = Cl
2-PAM or
Pralidoxime (1)
* Corresponding author. Tel.: +1 406 243 4643.
Figure 1. Equation representing inhibition and reactivation of AChE by OPI and
E-mail address: charles.thompson@umontana.edu (C.M. Thompson).
oximes. Selected examples of oximes are shown.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.035

5102
S. B. Bharate et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5101–5104
whereas others show little to no appreciable activity after oxime
treatment. This and other limitations highlight that there has been
no single broad-spectrum oxime reactivator identified to date.1,34
Therefore, the development and selection of new, more effective
AChE oxime reactivators is very important. The focus of this work
was to determine the effect of different heterocyclic and aliphatic
side chains (capable of hydrogen bonding) on the ability of mono-
quaternary pyridinium oximes to reactivate two forms of AChE
inhibited by paraoxon.
Monoquaternary pyridinium oximes 5–35 were prepared in
40–92% yield via reaction of 2-, 3- or 4-pyridine aldoximes with
different substituted alkyl halides in acetonitrile or acetone at
60–80 °C for 1–24 h.35 Monoquaternary pyridinium oximes 5–16
were designed to contain an aliphatic side chain with a group capa-
ble of hydrogen bonding viz. carbamoyl, carboxyl and hydroxyl.
Furthermore, highly electron rich heterocycles, viz. thiophene, fur-
an and isoxazole were used as side chains 17–35 to explore inter-
vate EeAChE and rHuAChE using a modified Ellman assay.37,38 2-
PAM and paraoxon were chosen as a reference reactivator and as
representative OPI.
Reactivation results are summarized in Table 1. Oximes with
aliphatic side chains mostly showed concentration-dependent
reactivation of AChE. Compounds containing carboxamide or car-
boxylic acid groups paired with the 2 or 4-oxime position (5, 7
and 8) gave 40–45% reactivation of EeAChE while compounds with
hydroxyethyl side chain 11 and 13 showed 50% and 70% reactiva-
tion of EeAChE at 10ꢀ3 M comparable to 2-PAM 1. Compound 8
also showed promise as
a reactivator of rHuAChE (15%) at
10ꢀ3 M. Compounds with aromatic side chains (thiophene, furan
and isoxazole) displayed a reverse concentration–activity relation-
ship. These compounds, with the exception of 19 and 35, showed
68% reactivation of EeAChE and no reactivation of rHuAChE at
the highest concentration (10ꢀ3 M). Lower concentrations of oxime
(10ꢀ4–10ꢀ5 M) 17–29 were better reactivators of both enzymes
suggesting that these compounds may inhibit AChE at higher con-
centrations and explain, in part, the inability to reactivate inhibited
AChE at 10ꢀ3 M.19,23
actions with cation-containing (cation–p bonding) or aromatic
residues ( bonding) at or near the AChE active site. The oxime
p–p
group was positioned at 2, 3 and 4 on the pyridinium ring to deter-
mine the best position for reactivation of AChE using the N-deriv-
atized groups. All compounds were characterized by 1H NMR, 13C
NMR, ESI-MS and IR prior to assay.35 Compounds 10, 11 and 13
have been reported as reactivators but not against EeAChE or rHu-
AChE.7,36 All compounds were assayed for their capacity to reacti-
Each reactivator varied with respect to the optimal concentra-
tion used, and four trends were identified from the data (Table
1). First, a correlation was found between percent reactivation
and increasing concentration of oxime (5, 7, 8, 11–16). A second
trend was found that inversely correlates increasing reactivation
Table 1
Synthesis and in vitro reactivation potency of monoquaternary pyridinium oximes 5–35 for paraoxon-inhibited EeAChE and rHuAChE
CHNOH
CHNOH
acetone or CH3CN, 60-80 ºC, 1-24 h*
+
R
X
N
40-92 %
N
X
R
Entry
R
Oxime position
X
Reaction
time (h)
% Yield
% Reactivation = [(Ar ꢀ Ai)/(Ao ꢀ Ai)] ꢁ 100
EeAChE (% R SEM) rHuAChE (% R SEM)
10ꢀ3 Ma 10ꢀ4 Ma 10ꢀ5 Ma 10ꢀ3 Ma 10ꢀ4 Ma 10ꢀ5 Ma
1
5
6
7
8
H
2
2
3
4
2
3
4
2
3
4
2
3
4
2
3
4
2
3
4
2
3
4
2
3
4
2
3
4
3
2
3
4
I
I
I
I
I
I
I
—
24
2
2
24
2
2
24
2
2
24
2
2
12
1
1
24
2
2
12
1
1
24
2
—
67
41
1
44
45
1
4
50
8
70
10
5
15
8
1
19
1
1
3
1
2
3
0
0
4
1
2
1
0
1
1
1
1
0
67
9
0
6
36
0
0
37
0
3
0
63
1
0
0
34
0
0
13
0
3
0
0
0
18
0
3
34
1
12
37
8
10
24
8
3
0
42
2
0
0
15
0
0
2
0
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
22
2
1
1
15
1
1
5
1
3
1
1
2
7
3
3
9
4
4
7
5
5
7
2
2
2
2
3
3
7
3
2
2
0
0
0
2
0
0
0
0
0
0
0
0
1
0
1
2
0
1
0
1
1
1
0
0
0
0
0
0
1
1
1
7
1
2
1
5
1
2
2
1
2
1
1
1
3
2
2
9
5
5
8
6
4
8
5
2
5
2
2
2
3
2
2
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
1
1
0
1
0
0
0
0
0
0
CONH2
CONH2
CONH2
COOH
COOH
COOH
CH2OH
CH2OH
CH2OH
CH2COOH
CH2COOH
CH2COOH
4-Methoxybenzen-1-yl
4-Methoxybenzen-1-yl
4-Methoxybenzen-1-yl
Thiophen-2-yl
Thiophen-2-yl
Thiophen-2-yl
5-Bromothiophen-2-yl
5-Bromothiophen-2-yl
5-Bromothiophen-2-yl
Benzthiophen-3-yl
Benzthiophen-3-yl
Benzthiophen-3-yl
Thiophen-3-yl
Furan-2-yl
62
76
84
61
71
78
58
60
68
48
56
68
70
84
92
58
65
78
74
84
90
67
79
85
79
62
60
46
40
64
67
1
3
2
1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Br
Br
Br
Br
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
3
2
1
0
1
2
1
2
0
0
0
19
0
11
24
3
12
29
7
14
28
5
0
8
0
14
0
25
0
24
2
2
1
2
1
2
2
0
1
2
1
1
2
0
0
2
1
1
3
1
1
2
0
3
0
4
3
0
1
1
0
0
0
0
0
8
0
7
0
20
2
24
1
1
1
24
2
2
1
28
0
1
0
8
0
6
1
2
3
2
Furan-2-yl
Furan-3-yl
3-Methyl-isoxazol-5-yl
3-Methyl-isoxazol-5-yl
3-Methyl-isoxazol-5-yl
1
1
2
1
1
0
*
Compounds 5–16: acetone, 60–70 °C, 2–24 h; 17–35: MeCN, 70–80 °C, 2–24 h.
Reactivator concentration.
a

S. B. Bharate et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5101–5104
5103
Table 2
(R43 ES016392 U44 NS058229). Support from the Core Laboratory
for Neuromolecular Production (NIH P30-NS055022) and the Cen-
ter for Structural and Functional Neuroscience (NIH P20-
RR015583) is appreciated.
Reactivation rate constant (kr) for 8, 13, 20, 23 and 26
Entry
kr (minꢀ1
)
EeAChE
rHuAChE
Opinions, interpretations, conclusions, and recommendations are
those of the authors and are not necessarily endorsed by the U.S. Army
or the Department of Defense.
2-PAM (1)
8
13
20
23
26
0.1766 0.0134
0.0406 0.0021
0.0108 0.0002
0.0339 0.0026
0.0189 0.0004
0.0138 0.0008
0.0041 0.0003
0.0048 0.0004
0.0010 0.0002
0.0016 0.0001
0.0012 0.0001
0.0010 0.0001
References and notes
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compounds 17, 21, 25, 26, 31, 33, 35 showed the best percent reac-
tivation at mid-range concentrations of oxime.19 Last, analogs
bearing the oxime in the 2-position were better reactivators than
those with oximes in the 3- or 4-position. Of the compounds with
heteroaromatic side chains, thiophene analogs 20, 23, 26 and 29
with the 2-oxime group reactivated paraoxon-inhibited EeAChE
34%, 37%, 24% and 28% at 10ꢀ5 M, respectively. These thiophene
analogs also reactivated rHuAChE but only ꢂ5% at 10ꢀ5 M. Oximes
with a thiophen-2-yl side chain (e.g., 20 and 23) were slightly bet-
ter reactivators than the corresponding thiophen-3-yl structures
(26 and 29). The furan analogs 30–32 were poor reactivators but
the 3-methylisoxazole analogs 33 and 35 showed 24–25% reactiva-
tion of EeAChE at 10ꢀ4 M. The 4-methoxybenzyl analogs 17–19
were poor reactivators of paraoxon-inhibited EeAChE and rHuAChE
although the 2- and 4-positional oximes 17 and 19 reactivated
AChE up to 19% of its activity.
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Owing to their greater activity, compounds 8, 13, 20, 23 and 26
were selected for analysis of the oxime-mediated reactivation rate
constants (kr; characterizes the dissociation of the enzyme and the
phosphorylated-oxime).39 The kr was calculated using the equation
from the linear portion of activity curves (0–15 min).40
As expected from the preliminary data (Table 1), these oximes
showed 8–21-fold greater kr values for reactivation of paraoxon-
inhibited EeAChE than rHuAChE. Compound 8 showed the highest
kr values among the analogs tested against for both EeAChE and
rHuAChE, and 8 was superior to 2-PAM for rHuAChE reactivation.
Also interesting was the trend of kr values 8 > 20 > 23 > 26 P 13
that was the same for both EeAChE and rHuAChE (Table 2).
Paraoxon-inhibited EeAChE undergoes greater than 60% reacti-
vation with 2-PAM from 10ꢀ3 M to 10ꢀ5 M (Table 1). Conversely, 2-
PAM reactivates paraoxon-inhibited rHuAChE to a far lesser extent
at 10ꢀ3 M (42%) and is barely reactivatable at 10ꢀ5 M (7%). This
suggests that rHuAChE will eventually become unable to reactivate
at all following inhibition by paraoxon. This may be due to one or
more non-reactivation processes including denaturation or aging.
The amino acid sequences of rHuAChE41 and EeAChE42 are 89%
homologous and do not reveal any obvious differences to account
for the 8–21-fold greater rate of reactivation observed for EeAChE.
However, species-specific differences in reactivation rates follow-
ing inhibition by a variety of OP agents are known43,44 and any data
comparisons should consider these differences.
24. Acharya, J.; Gupta, A. K.; Dubey, D. K.; Raza, S. K. Eur. J. Med. Chem. 2009, 44, 1335.
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35. Typical procedure for synthesis of monoquaternary pyridinium salts 5–35:
Hydroxyiminomethylpyridine (1 mmol) and the alkylating agent (1.5 mmol)
in solvent (20 mL) were stirred at 60–80 °C for 1–24 h. The mixture was cooled
to rt, the precipitate collected, washed with acetone (3 ꢁ 20 mL), dried under
vacuum, and characterized by 1H NMR, 13C NMR, ESI-MS and IR. Representative
compounds as follows: 1-Carboxymethyl-2-hydroxyimino methylpyridinium
iodide (8): light green solid; yield: 61%; mp 138–140 °C; 1H NMR (400 MHz,
CD3OD): d 8.80 (d, J = 5.2 Hz, 1H), 8.65 (s, 1H), 8.58–8.42 (m, 2H), 8.01 (t,
J = 8.0 Hz, 1H), 4.41 (s, 2H); 13C NMR (100 MHz, CD3OD): d 169.11, 147.01,
In conclusion, a new series of monoquaternary pyridinium oxi-
mes with heteroaromatic side chains have been reported of which
thiophene-containing analogs 20, 23 and 26 were the better reac-
tivators. Compound 8, bearing an acetic acid side chain, was found
to be the most effective reactivator of the 31 novel compounds
tested with a higher kr value than pralidoxime (1) for paraoxon-
inhibited rHuAChE.
146.53, 145.36, 141.25, 127.52, 125.99, 46.46; IR (Neat): mmax 3059, 3098, 3048,
2965, 2880, 2743, 1692, 1628, 1508, 1480, 1288, 1160, 1018 cmꢀ1; ESI-MS: m/z
181.06 [M]+ (calcd for [C8H9N2O3]+ 181.06). 1-(2-Hydroxy)-ethyl-2-
hydroxyimino methyl pyridinium bromide (11): brown solid; yield: 58%; mp
184–186 °C; 1H NMR (400 MHz, D2O): d 8.64 (d, J = 6.4 Hz, 1H), 8.57 (s, 1H),
8.37 (t, J = 8.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.87 (t, J = 6.8 Hz, 1H), 4.71 (t,
J = 4.4 Hz, 2H), 3.89 (t, J = 4.4 Hz, 2H); 13C NMR (100 MHz, D2O): d 147.20,
146.38, 145.85, 142.49, 127.78, 127.03, 60.33, 60.07; IR (Neat): mmax 3377,
3072, 2991, 2865, 2734, 2620, 1627, 1590, 1504, 1430, 1313, 1152, 1072,
1000 cmꢀ1 ESI-MS: m/z 167.08 [M]+ (calcd for [C8H11N2O2]+ 167.08). 1-
;
Acknowledgments
(Thiophen-2-yl)-methyl-2-hydroxyiminomethyl-pyridinium chloride (20): off
white solid; yield: 58%; mp 150–152 °C; 1H NMR (400 MHz, D2O): d 8.73 (d,
J = 6.4 Hz, 1H), 8.63 (s, 1H), 8.38 (t, J = 8.0 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.86
(t, J = 6.4 Hz, 1H), 7.40 (d, J = 5.2 Hz, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.94 (t,
The research in this study was supported by NIH UO1-
ES016102 (CMT) and SBIR grants to ATERIS Technologies LLC

5104
S. B. Bharate et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5101–5104
J = 4.0 Hz, 1H), 5.98 (s, 2H); 13C NMR (100 MHz, D2O): d 146.15, 145.23, 142.28,
139.01, 133.52, 130.28, 129.20, 128.25, 127.94, 127.50, 56.61; IR (Neat): mmax
38. Ellman, G. L.; Courtney, K. D.; Andres, V. J.; Feather-Stone, R. M. Biochem.
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3069, 3013, 2945, 2831, 2735, 1711, 1628, 1577, 1514, 1474, 1321, 1253,
39. Determination of reactivation rate constant (kr) for selected compounds: Similar
experiments as in vitro reactivation screening were accomplished with
different reactivation time (0–15 min). By plotting ln(reactivation%–age)
versus reactivation time (t), kr is presented as the negative slope of the plot.
ln(reactivation%–age) = ꢀkrꢃt. Each experiment was repeated with n P 3. Plots
with R2 > 0.9 were chosen for kr calculations.
1020 cmꢀ1; ESI-MS: m/z 219.21 [M]+ (calcd for [C11H11N2OS]+ 219.06).
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37. In vitro reactivation screening: An AChE stock solution (0.2 mg/mL in PBS7.2)
was treated with ethanol (0.1% v/v) and paraoxon (100
as control and experiment vessels. After 20–50 min, 90% AChE inhibition was
achieved and halted with a 32-fold dilution (PBS). A 16 L aliquot from control
and experiment vessels was diluted to 20 L with PBS, and incubated for
lM in ethanol, 0.1% v/v)
l
40. Thompson, C. M.; Ryu, S.; Berkman, C. E. J. Am. Chem. Soc. 1992, 114,
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l
30 min as activity control and inhibition control. The initial activity (A0) or
inhibition (Ai) was analyzed by adding DTNB (final concentration of 0.3 mM,
41. Kryger, G.; Harel, M.; Giles, K.; Toker, L.; Velan, B.; Lazar, A.; Kronman, C.;
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200
volume). Oxime reactivator (4
16 aliquot and after 30 min incubation, the reactivated activity was
l
L total volume) and ATChI (final concentration of 1 mM, 200
lL total
l
L, 1–0.01 mM final concn) was added to a
lL
determined by Ellman assay as Ar. The % reactivation for each reactivator
was determined with n P 3: % reactivation = [(Ar ꢀ Ai)/(A0 ꢀ Ai)] ꢁ 100.
43. Kuca, K.; Cabal, J.; Kassa, J. J. Enzyme Inhib. Med. Chem. 2005, 20, 227.
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