New efficient imidazolium aldoxime reactivators for nerve agent-inhibited acetylcholinesterase

Article abstract of DOI:10.1016/j.bmcl.2014.10.055

Herein, we described a new class of uncharged non-pyridinium reactivators for nerve agent-inhibited acetylcholinesterase (AChE). Based on a dual site binding strategy, we conjugated the imidazolium aldoxime to different peripheral site ligands (PSLs) of A

Full text of DOI:10.1016/j.bmcl.2014.10.055

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5743–5748
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New efficient imidazolium aldoxime reactivators for nerve
agent-inhibited acetylcholinesterase
Zhao Wei a, Yan-qin Liu b, Xin-bo Zhou a, Yuan Luo b, Chun-qian Huang b, Yong-an Wang b,
,
⇑
Zhi-bing Zheng a, , Song Li a
⇑
a Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
b Department of Military Toxicology and Biochemical Pharmacology, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2014
Revised 26 September 2014
Accepted 17 October 2014
Available online 22 October 2014
Herein, we described a new class of uncharged non-pyridinium reactivators for nerve agent-inhibited
acetylcholinesterase (AChE). Based on a dual site binding strategy, we conjugated the imidazolium aldox-
ime to different peripheral site ligands (PSLs) of AChE through alkyl chains. Compared with the known
quaternary pyridinium reactivators, two of the resulting conjugates (7g and 7h) were highlighted to be
the first efficient non-pyridinium oxime conjugates exhibiting similar or superior ability to reactivate
sarin-, VX- and tabun-inhibited AChE. Moreover, they were more broad-spectrum reactivators.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Human acetylcholinesterase
Reactivator
Nerve agent
Imidazolium aldoxime
Peripheral site ligand
Organophosphate (OP) nerve agents (e.g., sarin, VX, tabun, and
soman) are highly toxic compounds and pose potential threats to
the military and the public (such as the terrorist attacks in Tokyo
subway in 1995 and the recent Syria civil war).1 Acetylcholinester-
ase (AChE) is a hydrolase which terminates cholinergic neurotrans-
mission by hydrolyzing the neurotransmitter acetylcholine (ACh).2
The acute toxic effect of OPs stems from irreversible inhibition of
AChE by forming a covalent P–O bond in the active site (A-site)
of the enzyme, and the resulting accumulation of unhydrolyzed
ACh in synapse would lead to cholinergic crisis, respiratory dis-
tress, convulsive seizures and ultimately death.3
HON
NOH
NOH
HON
NH2
N
X
N
N
Obidoxime (X=O)
TMB-4 (X=CH2)
2-PAM
O
HON
N
O
N
N
N
NOH
HI-6
MMB-4
Figure 1. Current available pyridinium oximes in the treatment of OP poisoning.
Current available drugs for OP-poisoning include an AChE reac-
tivator, such as one of the standard pyridinium oximes (e.g., prali-
doxime or 2-PAM, trimedoxime or TMB-4, obidoxime, HI-6, and
MMB-4 Fig. 1),4,5 a muscarinic receptor antagonist (e.g., atropine)
and an anticonvulsant (i.e., diazepam).6,7 It is generally believed
that the highly nucleophilic oximes could break the P–O bond and
restore the enzyme’s activity.8 Whereas one drawback for these
quaternary reactivators is that they provide little or no protection
against neurological effects of OP exposure in the central nervous
system (CNS), because the permanent charges seriously limit their
blood–brain barrier (BBB) penetration,9 while the brain is a major
target of nerve agents.10 Another weakness is that there is no
universal broad-spectrum oxime suitable for the antidotal treat-
ment of various OP-poisoning, especially in the cases of tabun
and soman. For instance, HI-6, the best available reactivators to
date, is inefficient in reactivating tabun-inhibited hAChE.4,5 So it
remains a challenge to develop effective antidotes for OP exposure.
Over the past several decades, a number of strategies have been
developed to overcome the problems mentioned above.11–13 It was
proven that nonionic reactivators (e.g., monoisonitrosoacetone or
MINA, amidine-oximes, Fig. 2) would facilitate the BBB penetration
as a result of increased lipophilicity, and they showed obvious supe-
riority to charged 2-PAM as antidotes for CNS poisoning.14–17 Nev-
ertheless, the absence of charge will cause decreasing reactivation
potency because the uncharged reactivators don’t properly bind
⇑
Corresponding authors. Tel.: +86 01066931642.
E-mail address: zzbcaptain@aliyun.com (Z.-b. Zheng).
http://dx.doi.org/10.1016/j.bmcl.2014.10.055
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

5744
Z. Wei et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5743–5748
O
R4
R2
N
R3
O
NOH
N
N
Ph
n
N
O
N
N
NOH
R1
NOH
NOH
O
n
Ph
O
Amidine-oxime (n=1, 2)
MINA
1 (n=1,2,3)
O
HO
HON
N
R=
R=
R=
N
R
O
N
N
n
H
Ph
6 (n=2)
7 (n=3)
2 (n=2)
3 (n=3)
4 (n=2)
5 (n=3)
Figure 2. Chemical structures of some reported nonionic oximes.
to the aromatic moiety of the A-site,18 which buries at the bottom of
a deep active gorge into the AChE.19 However, there is a peripheral
site (P-site) at the entrance of the active gorge, serving as binding
site for distinctive substrates.20,21 Accordingly, a dual site binding
strategy was proposed, a peripheral site ligand (PSL) was conju-
gated to the oxime group to contribute extra affinity for AChE at
the P-site.22,23 Based on this strategy, a series of promising reactiva-
tors were synthesized (conjugates 1–7, Fig. 2), and among these
compounds, conjugate 3 outperformed all others. But all these con-
jugates are pyridyl aldoximes and they are difficult to prepare due
to a long synthetic route, moreover, their reactivation potencies
against sarin-inhibited hAChE are still unknown.24–27 Hence we
sought to develop alternatives of the pyridyl aldoximes.
From the beginning, a kind of imidazolium aldoximes drew our
attention, whose chemical structures are similar to the amidine oxi-
mes mentioned above.16,17 Early in 1990s, quaternary imidazolium
aldoximes had been intensively investigated as reactivators,28–31
and recently, a series of nonquaternary imidazolium aldoximes
(Fig. 3) were reported as antidotes for OP poisoning.32–35 Neverthe-
less, the reactivation abilities of these oximes are still not satisfying
and they need further modification for more efficient reactivators.
Therefore, enlightened by the dual site binding strategy, the PSL
(phenyl-tetrahydroisoquinoline, PIQ) from conjugates 3 was con-
nected to the imidazolium oxime through flexible alkyl chains,
aiming at developing more efficient tertiary reactivators (Fig. 3).
The designed conjugates were expected to possess enhanced BBB
penetration due to dramatic improvement of calculated lipophilic-
ity (a higher value of S + logP indicates higher lipophilicity; values
are listed in Table 1). In this study, we prepared eight new tertiary
imidazolium reactivators (Fig. 3), the in vitro screening essays
demonstrated that conjugates 7g and 7h were efficient and rela-
tively broad-spectrum reactivators.
approaches, the starting materials tetrahydroisoquinoline (TIQ)
1a and PIQ 1b were firstly synthesized. In path A, it was started
with N-alkylation of 1a and 1b with two different halide 2a and
2b to give the intermediates 3a, 3d, 3e and 3h. They were treated
with TsCl in pyridine to produce the tosylates 4a, 4d, 4e and 4h.
Then N-alkylation of imidazole-2-carboxaldehyde 5a with the
tosylates provided compounds 6a, 6d, 6e and 6h. Finally, treatment
of the latters with hydroxylammonium chloride afforded the target
conjugates 7a, 7d, 7e and 7h, whose methylene length is either 3 or
6 units. Unfortunately, efforts to obtain conjugates with methylene
length of 4 or 5 units using method A failed, because unexpected
compounds were produced in the N-alkylation of 5a. Thus, we
turned to path B and synthesized the desired compounds success-
fully. The starting materials 2b and 2c were converted to tosylates
3b and 3c in a similar way as in path A. In comparison with halide
moiety in 3b and 3c, the tosylate moiety was much easier to
undergo an N-alkylation reaction with 5a to obtain the intermedi-
ates 4b and 4c, and then they were readily converted to oximes 6b
and 6c by treating with hydroxylammonium chloride. At last,
N-alkylation was conducted again between 6b, 6c and 1a, 1b to
give the desired compounds 7b, 7c, 7f and 7g. In addition, com-
pound 3 (Fig. 3), one of the most promising uncharged reactivators
in the literature, was prepared in a similar way as described by
Mercey et al.26 In contrast to 7a–7h, the synthetic route of pyri-
dine-aldoxime 3 was long as ten steps from the starting material
1b. Furthermore, the synthesis of 3 involved a Sonogashira cou-
pling reaction and two protection and deprotection reactions,
which were complex, costly and poor yield, while the synthesis
of conjugates 7a–7h was simple, economical and high-yield.
The in vitro experiments were conducted with fresh human
whole blood serving as enzyme source. The enzyme activity was
measured using a similar method of Ellman et al.36 and the exper-
imental detail was described in the section of supporting informa-
tion. Two oxime concentrations (1 mM and 0.1 mM) were selected
Two different approaches were utilized for the synthesis of con-
jugates 7a–7h. They are outlined in Schemes 1 and 2. In both
R6
R4
N
N
N
n
NOH
NOH
N
NOH
N
N
R5
Quarternary
imidazolium aldoxime
Nonquaternary
imidazolium aldoxime
Amidine-oxime
A-site
ligand
NOH
HO
HON
O
O
O
P-site
ligand
N
N
N
N
N
O
n
Ph
R
3
n=1, 2, 3, 4; R=H, Ph
Target compounds
Figure 3. Chemical structures of the designed target compounds based on a dual site binding strategy.

Z. Wei et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5743–5748
5745
Table 1
Reactivation of nerve agent-inhibited hAChE by 3, 5b, 7a–7h and the reference reactivators (1 mM and 0.1 mM)
Oxime
Reactivation rate (%)
VX
S + logP
Sarin
Tabun
1 mM
0.1 mM
1 mM
0.1 mM
1 mM
0.1 mM
Obidoxime
35
84
49
18
68
À2
8
14
34
2
7
7
25
45
3
64
À2
0
2
6
0
4
1
0
1
0
6
2
0
0
0
0
0
1
84
83
71
80
84
31
68
89
89
13
0
2
3
4
4
4
1
2
1
3
54
59
23
24
2
0
3
4
84
10
21
91
68
2
1
7
17
1
5
5
0
0
5
7
1
0
2
2
1
2
2
1
0
24
4
5
58
54
À1
1
5
32
0
1
0
1
5
5
1
0
4
1
0
2
À4.33
À3.51
À3.65
À3.46
5.06
2.34
2.6
2.99
3.37
3.73
4.06
4.45
4.84
0.12
HI-6
MMB-4
TMB-4
3
7a
7b
7c
7d
7e
7f
1
3
4
1
1
1
2
0
2
1
3
7
2
68 16
À1
28
46
54
3
0
0
4
5
0
5
5
0
0
27
69
94
5
47 14
6
3
7g
7h
5b
21
50 7
3
92
80
8
3
2
0
67
84
7
47
53
10
33 13
44
3
3
0
1
Experiments were performed in triplicate at 37 °C in phosphate buffer (0.10 M, pH = 7.4); AChE inhibition percentages ranging 85–98% were firstly achieved before the
reactivation assays. Data shows the average and standard deviation. The values of S + logP (calculated by ADMET Predictor 5.5) were also given.
R
R
R
NaI, K2CO3,DMF
O
O
O
O
O
O
N
N , 0°C,
2
OH
2h
N ,100°C
NH
,16h
N
2
OTs
n
n
TsCl, pyridine
HO
Cl
3a n=1, R=H, 95%
3d n=4, R=H, 60%
3e n=1, R=Ph, 71%
3h n=4, R=Ph, 89%
n
4a n=1, R=H
4d n=4, R=H
4e n=1, R=Ph
4h n=4, R=Ph
1a R=H
1b R=Ph
2a n=1
2d n=4
NH
NaI,K2CO3, CH3CN
N2,reflux,6h
O
N
5a
HON
O
N
O
O
O
HONH2HCl, NaOAc
C2H5OH, RT, 2h
N
N
n
N
N
N
O
n
R
R
7a n=1, R=H, 13%
7d n=4, R=H, 81%
7e n=1, R=Ph, 66%
7h n=4, R=Ph, 77%
6a n=1, R=H, 58% from 3a
6d n=4, R=H, 24% from 3d
6e n=1, R=Ph, 51% from 3e
6h n=4, R=Ph, 45% from 3h
Scheme 1. Preparation of imidazolium aldoxime conjugates 7a, 7d, 7e, 7h, path A.
K2CO3, NaI, CH3CN
O
S
N2, reflux, 5h
NH
TsCl, pyridine
HO
Cl
n
Cl
N
N
O
Cl
O
n
N2, 0°C, 2h
n
O
5a
O
4b n=2, 56%
4c n=3, 80%
2b n=2
2c n=3
3b n=2, 79%
3c n=3, 88%
N
HONH2HCl, NaOAc
C2H5OH, RT, 2h
HON
O
O
NaI, K2CO3 , CH3CN
N2, reflux, 25h
N
N
N
N
n
Cl
N
R
n
NOH
1a, 1b
7b n=2, R=H, 28%
7c n=3, R=H, 45%
7f n=2, R=Ph, 24%
6b n=2, 83%
6c n=3, 64%
7g n=3, R=Ph, 13%
Scheme 2. Preparation of imidazolium aldoxime conjugates 7b, 7c, 7d, 7e, path B.
in the biological essays, because the current available oximes
generally show obvious reactivation potency only in relatively high
concentrations, especially in the cases of tabun (for examples,
obidoxime exhibited high reactivation rate for tabun-inhibited
hAChE at 1 mM concentration, but the reactivation rate greatly
decreased at 0.1 mM concentration, data shown in Table 1).

5746
Z. Wei et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5743–5748
inhibition of hAChE
inhibition of hAChE
not washed
oxime concentration(1 mM)
oxime concentration(0.1 mM)
120
110
100
90
80
70
60
50
40
30
20
10
0
110
100
90
80
70
60
50
40
30
20
10
0
washed once
Figure 4. Inhibition of hAChE by conjugates 3, 7a–7h and the reference oximes at different concentrations (1 mM and 0.1 mM, left figure) and under different treatments
(1 mM, right figure). Data depicted shows the average and standard deviation (calculated as a fraction of positive control samples without oximes) for AChE activity (n = 3
incubations per oxime) after inhibition. Unless otherwise indicated, the standard deviation bars were smaller than the data marker.
Considering the fact that the inhibition ability would help to pro-
vide an insight into the oximes binding affinity for hAChE, and a
strong or irreversible inhibitor of AChE would lead to toxicity,37,38
we firstly evaluated the AChE inhibition potency of these new
reactivators (7a–7h and 3).
to the poisoned control (85–98% AChE inhibition) and the results
are shown in Table 1. In the case of sarin, the most promising
uncharged reactivators were 7h, 7g and 3. Their reactivation rates
equaled and even exceeded those of MMB-4 and HI-6, while 7d
and 7f showed relatively low reactivation potency. For VX-inhib-
ited hAChE, it is inspiring that most of the novel oximes were
promising reactivators; some of them even exhibited higher reac-
tivation abilities than obidoxime and HI-6 (such as 7c, 7d, 7g, 7h
and 3). In terms of tabun-inhibited hAChE, to our best knowledge,
oxime 3, 7g and 7h were the most promising nonquaternary reac-
tivators reported until now, while HI-6 and oximes 7a–7f only
slightly reactivated tabun-inhibited hAChE. For conjugate 3, our
results are in line with what Mercey et al. had reported, but they
did not evaluate the reactivation ability against sarin-inhibited
hAChE.24,25 In contrast to those quaternary oximes, 7g, 7h and 3
emerged as more broad-spectrum for sarin-, VX- and tuban-
inhibited hAChE, while obidoxime and TMB-4 were less efficient
for sarin; HI-6 and MMB-4 were much less efficient for tabun
poisoning. We also determined the reactivation potency of
imidazolium-2-aldoxime (5b), the results demonstrated that most
conjugates showed improved reactivation potency, especially in
the cases of 7d, 7g and 7h, which justified the concept of dual-site
biding strategy.
In addition, in order to get a complete comprehension of the
reactivation of conjugates 7g and 7h, we measured their reactiva-
tion rate constant Kr, dissociation constant KD and second order
reactivation rate constant Kr2 for sarin-, VX- and tabun-inhibited
hAChE,39 the results were presented in Table 2. As a result of
improved Kr or KD, their reactivation efficiencies, or Kr2 (Kr/KD),
equaled and even exceeded those of obidoxime, HI-6 and TMB-4
in similar conditions, which were in line with the conclusions we
got above. For details, conjugate 7h was more efficient than HI-6
and 3 for sarin poisoning due to a better affinity (lower KD). For
Four quaternary reactivators (obidoxime, HI-6, MMB-4 and
TMB-4, Fig. 1) were used as reference compounds in the inhibition
experiments, the results are depicted in Figure 4. It is apparent that
these new oximes, including 3, were moderate or weak inhibitors
of hAChE, while the reference reactivators just showed slight
inhibitory abilities (Fig. 4). At 1 mM oxime concentration, enzyme
inhibition percentages by 3, 7g, 7h were 51%, 36% and 47% respec-
tively, while the inhibition percentages by all other oximes were
below 40%. In contrast, at 0.1 mM oxime concentration, the
enzyme activity was almost completely released (>85%). We fur-
ther determined the IC50 of 3, 7g and 7h (data show in Table 2)
and confirm that they were weak inhibitors of AChE. In order to
determine whether the inhibition was reversible, the erythrocyte
(AChE bound to it covalently) was washed once with PBS (0.1 M,
pH = 7.4). For most tested oximes, the activities of the hAChE were
greatly restored (>85%) after wash (Fig. 4). For examples, at 1 mM
oxime concentration, hAChE activities increased from 53% to 68%
for 7h, 49% to 73% for 3 and 64% to 95% for 7g, respectively. On
the contrary, it was found that there was no change in the activity
of the nerve agent-inhibited hAChE in an extra experiment. There-
fore, we concluded that the synthesized oximes inhibited hAChE in
a moderate and reversible way, this could not only provide suitable
AChE affinity for reactivation of OP poisoning, but also avoid
toxicology caused by strong inhibition of AChE. These results
encouraged us to proceed to the reactivation experiments.
Sarin, VX and tabun were used to determine the reactivation
potency of oximes 7a–7h,
3 and the reference compounds
mentioned above. The reactivation rates were calculated relatively
Table 2
Reactivation rate constant (Kr), dissociation constant (KD), second order reactivation rate constant (Kr2) and IC50 of obidoxime, HI-6, TMB-4, 3, 7g and 7h
Oxime
Kr/minÀ1
VX
KD/
l
M
Kr2/mMÀ1minÀ1
IC50 (mM)
Sarin
Tabun
Sarin
VX
Tabun
Sarin
VX
Tabun
Obidoxime
0.011 0.002
0.048 0.003
n.d.
0.063 0.022
0.040 0.008
0.033 0.004
0.22 0.02
0.25 0.01
n.d.
0.22 0.02
0.14 0.02
0.21 0.01
0.028 0.002
n.d.a
0.056 0.004
0.027 0.003
0.009 0.001
0.012 0.001
90 39
93 19
n.d.
88 77
143 78
41 16
32
27
n.d.
22
24
25
9
3
66 16
n.d.
79 20
18 10
26 15
0.12
0.52
n.d.
0.72
0.28
0.81
6.95
9.30
n.d.
10.03
5.75
8.43
0.43
n.d.
0.70
1.54
0.36
0.86
2.21
1.36
2.57
0.89
0.93
0.87
HI-6
TMB-4
3
7g
7h
7
8
4
14
8
a
not determined.

Z. Wei et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5743–5748
5747
Figure 5. Docked conformation of molecules 3 (left) and 7h (right) in the active site gorge of sarinnonaged-mAChE (pdb code: 2WHP). The docked conformations of the
reactivators are depicted as stick model in yellow and the key amino acid residues as stick model in grey.
VX-hAChE, 7h was almost as efficient as HI-6, obidoxime and 3 due
to similar Kr and KD. For tabun poisoning, despite a lower reactiva-
tion rate Kr, conjugate 7h was more efficient than obidoxime and
TMB-4 for greatly improved affinity for tabun-hAChE (lower KD),
while conjugate 3 was the best reactivator for tabun due to rela-
tively high Kr and low KD.
In this study, TIQ, an analogue of PIQ, was also chosen as the
PSL, which had been proven to be effective P-site binders of reac-
tivators for OP poisoning,40 but replacement of PIQ by TIQ
decreased the reactivation efficacy. The failure of TIQ may be due
to its low affinity for P-site of AChE, while PIQ has been confirmed
as a moderate P-site binder.41 However, a PSL possessing strong
affinity towards the P-site should be avoided, because it would
lead to toxicity.37,38 Therefore, a proper PSL may play a critical role
for an efficient reactivators.
greatly enhanced reactivation potency and two conjugates (7g
and 7h) emerged as first efficient and more broad-spectrum non-
pyridinium reactivators than those quaternary reactivators for
sarin-, VX- and tuban-inhibited hAChE. For conjugates 3, we deter-
mined its reactivation potency against sarin-inhibited hAChE for
the first time, confirming its relatively wide scope efficiency. Con-
sidering the simple synthesis route in comparison to conjugates 3,
these novel imidazolium aldoximes show economical superiority
and hold promise for further design of more efficient uncharged
reactivators. Finally, it should be noted that PIQ was used as a race-
mic mixture in this study, the separation of the enantiomers is
ongoing and it will be discussed in a future paper.
Acknowledgments
Furthermore, in order to determine the influence of alkyl length
between the A-site ligand and PSL on the reactivation potency, alkyl
chains ranging from 3 to 6 methylene units were investigated. It is
clear that, along with the increasing linker length (from compounds
7a to 7d and 7e to 7h), both inhibition and reactivation potency
increased in most cases. The calculated KD for 7g and 7h indicated
that an increased linker length may provide better affinity for poi-
soned hAChE. Thus, we hypothesized that a relatively longer linker
may provide more flexibility for the reactivators to have a better
conformation to bind to AChE, and result in higher reactivation abil-
ity. To valid our hypothesis, preliminary molecular docking studies
of the most promising reactivators 3 and 7h into the active gorge of
AChE were conducted and compared. The results demonstrated
that the two conjugates interacted with AChE in a similar dual site
binding mode (Fig. 5). The PSLs (PIQ) of both conjugates interact
with the P-site in an optimized position (key amino acid residues
include Tyr72, Tyr124, Trp286 and Tyr341and Asp74),20,21 which
The financial support from the Major Program of Ministry of
Science and Technology of China (No: 2013ZX09J109-02C) is grate-
fully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.10.055.
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