Conjugates of salicylaldoximes and peripheral site ligands: Novel efficient nonquaternary reactivators for nerve agent-inhibited acetylcholinesterase

Article abstract of DOI:10.1016/j.bmc.2017.06.041

A new family of nonquaternary reactivators for nerve agent-inhibited human acetylcholinesterase (hAChE) were designed, synthesized and tested in this paper. It was found that salicylaldoximes were able to quickly cleave the P–S bond of organophosphate and avoid the reinhibition phenomenon in the reactivation process, but they lacked reactivating ability due to poor affinity for AChE. Based on a dual site binding strategy, different peripheral site ligands of AChE were introduced to achieve extra affinity. The in vitro reactivation experiments demonstrated that some of the yielding conjugates exhibited similar or even superior ability to reactivate sarin-, VX- or tabun-inhibited hAChE in comparison with the mono- and bis-pyridinium aldoximes currently used. Moreover, due to greatly improved lipophilicity, these nonquaternary conjugates hold promise for the development of efficient centrally activating reactivators.

Full text of DOI:10.1016/j.bmc.2017.06.041

Accepted Manuscript
Conjugates of salicylaldoximes and peripheral site ligands: novel efficient non-
quaternary reactivators for nerve agent-inhibited acetylcholinesterase
Zhao Wei, Yan-qin Liu, Sheng-zheng Wang, Lin Yao, Hui-fang Nie, Yong-an
Wang, Xue-Ying Liu, Zhi-bing Zheng, Song Li
PII:
S0968-0896(17)30981-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.041
BMC 13824
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
12 May 2017
13 June 2017
25 June 2017
Please cite this article as: Wei, Z., Liu, Y-q., Wang, S-z., Yao, L., Nie, H-f., Wang, Y-a., Liu, X-Y., Zheng, Z-b.,
Li, S., Conjugates of salicylaldoximes and peripheral site ligands: novel efficient nonquaternary reactivators for
nerve agent-inhibited acetylcholinesterase, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
1
0.1016/j.bmc.2017.06.041
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Graphical Abstract
Based on a dual site binding strategy, the salicylaldoximes were
conjugated to different peripheral site ligands through alkyl
chains for construction of novel nonquaternary reactivators.
Compared with the known quaternary pyridinium reactivators,
some of the resulting compounds emerged as efficient and
relatively broad-spectrum reactivators for nerve agent-inhibited
human acetylcholinesterase.
Conjugates of salicylaldoximes and
peripheral site ligands: novel efficient
nonquaternary reactivators for nerve
agent-inhibited acetylcholinesterase
a,*
b
a
Zhao Wei , Yan-qin Liu , Sheng-zheng Wang ,
a
a
b
Lin Yao , Hui-fang Nie , Yong-an Wang ,
a,*
c,*
c
Xue-Ying Liu , Zhi-bing Zheng , Song Li
a
Department of Medicinal Chemistry, School of Pharmacy, Fourth Military Medical University, Xi’an 300071,
China
b
Department of Military Toxicology and Biochemical Pharmacology, Institute of Pharmacology and
Toxicology, Academy of Military Medical Sciences, Beijing 100850, China
c
Department of Medicinal Chemistry, Institute of Pharmacology and Toxicology, Academy of Military Medical
Sciences, Beijing 100850, China

Bioorganic & Medicinal Chemistry
Conjugates of salicylaldoximes and peripheral site ligands: novel efficient
nonquaternary reactivators for nerve agent-inhibited acetylcholinesterase
a,*
b
a
a
a
b
Zhao Wei , Yan-qin Liu , Sheng-zheng Wang , Lin Yao , Hui-fang Nie , Yong-an Wang , Xue-Ying
a,*
c,*
c
Liu , Zhi-bing Zheng , Song Li
a
Department of Medicinal Chemistry, School of Pharmacy, Fourth Military Medical University, Xi’an 300071, China
Department of Military Toxicology and Biochemical Pharmacology, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing
00850, China
b
1
c
Department of Medicinal Chemistry, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A
new family of nonquaternary reactivators for nerve agent-inhibited human
acetylcholinesterase (hAChE) were designed, synthesized and tested in this paper. It was found
that salicylaldoximes were able to quickly cleave the P-S bond of organophosphate and avoid
the reinhibition phenomenon in the reactivation process, but they lacked reactivating ability due
to poor affinity for AChE. Based on a dual site binding strategy, different peripheral site ligands
of AChE were introduced to achieve extra affinity. The in vitro reactivation experiments
demonstrated that some of the yielding conjugates exhibited similar or even superior ability to
reactivate sarin-, VX- or tabun-inhibited hAChE in comparison with the mono- and
bis-pyridinium aldoximes currently used. Moreover, due to greatly improved lipophilicity, these
nonquaternary conjugates hold promise for the development of efficient centrally activating
reactivators.
Available online
Keywords:
Nerve agent,
Human acetylcholinesterase,
Nonquaternary reactivator,
Salicylaldoxime conjugates,
Peripheral site ligand
2009 Elsevier Ltd. All rights reserved.
1
. Introduction
organophosphorus pesticides have been proved of limited
success, resulting in a serious public health issue with about 3
Organophosphate (OP) nerve agents (such as sarin, tabun,
000 000 acute intoxications and over 200 000 fatalities annually
soman and VX, Fig. 1) and pesticides (such as such as parathion,
chlorpyrifos) are highly toxic compounds. The acute toxicity of
OP stems from the phosphorylation of the active site serine
residue in acetylcholinesterase (AChE), which terminates
worldwide, mostly in developing countries [6]. Therefore, the
development of more effective antidotes against OP poisoning
remains a challenging issue.
cholinergic
neurotransmission
by
hydrolyzing
the
Currently approved antidotal therapies for the treatment of OP
poisoning in humans combine intramuscular injections of an
AChE reactivator of the pyridinium aldoxime family (e.g.,
pralidoxime (2-PAM), trimedoxime (TMB-4), obidoxime, HI-6,
Fig. 2) [7, 8], a muscarinic receptor antagonist (e.g., atropine),
and an anticonvulsant (e.g., diazepam) [9, 10]. Pyridinium
aldoxime therapy is directed toward nucleophilic reactivation of
AChE covalently inhibited by OPs to restore the enzyme’s
activity [11].
neurotransmitter acetylcholine (ACh) [1], the resulting
irreversible inhibition of AChE causes accumulation of ACh at
both peripheral and central cholinergic synapses, leading to
cholinergic crisis, respiratory distress, convulsive seizures and
ultimately death [2].
Figure 1. Chemical structures of some nerve agents
OPs represent potential threats to both military personnel and
civilians [3]. To date, the nerve agents had been used in terrorist
attacks (e.g., subway attack in Tokyo in 1995) [4], in murder
Figure 2. Current available pyridinium oximes in the treatment of
OP poisoning
(e.g., Kim Jong-nam’s Killing in Kuala Lumpur in 2017) and in
the armed conflicts (e.g., Gulf Wars) [5] as weapons of mass
destruction. In addition, due to their relatively simple chemical
synthesis, efforts to control the proliferation of the
During the past several decades, researchers have studied
various monopyridinium oximes and especially more efficient

bispyridinium oximes, but all known reactivators have several
drawbacks: (1) there is no universal broad-spectrum oxime
suitable for the antidotal treatment of various OP-poisoning. For
instance, HI-6, the best available reactivators to date, rapidly
reactivates sarin-, soman- and VX-inhibited AChE, but it’s
inefficient for tabun [7, 8]. (2) Phosphylated oximes (compound c,
Fig. 4, left) formed during reactivation may act as a nerve agent
and reinhibit AChE, which would reduce the antidotal efficacy of
these pyridinium reactivators [12, 13]. (3) Most of all, due to
their permanent positive, these quaternary reactivators are unable
to cross the blood-brain barrier (BBB) and show poor
reactivation efficiency in the central nervous system (CNS) [14],
while the brain is a major target of nerve agents [15, 16]. Thereby,
reactivators of broad-spectrum efficiency and especially of CNS
antidotal efficiency are needed.
(Fig. 3) can penetrate the BBB and rapidly undergo oxidation in
the brain to produce functionally active 2-PAM [17]. But the
synthesis of prodrug is rather difficult and pro-2-PAM is unstable
to autoxidation [18].
A recent spur of nonquaternary AChE reactivators has drawn
our attention [19-22]. Due to increased lipophilicity, some
nonquaternary reactivators (e.g. monoisonitrosoacetone (MINA),
Fig. 3) [23, 24] readily penetrate the BBB and display superior in
vivo reactivation activity to charged 2-PAM as antidotes for CNS
poisoning. However, the absence of charge reduced the affinity to
AChE and resulted in decreasing reactivation potency [25].
Kalisiak et al had designed and synthesized a class of
amidine-oximes (Fig. 3), the basic amidine residue was expected
to undergo protonation under physiological conditions to form a
positively charged center similar to pyridinium oximes,
increasing the binding affinity for OP-inhibited AChE. The in
vitro reactivating ability for OP-inhibited AChE of
amidine-oximes was improved in contrast to MINA, but still
lower than 2-PAM [26, 27].
A number of strategies have been developed to address the
problems mentioned above. Based on the use of prodrug, the
charged pyridine ring of 2-PAM is replaced with a significantly
less charged dihydropyridyl moiety, the resulting pro-2-PAM
Figure 3. Chemical structures of some reported nonionic oximes
The bispyridinium oximes (e.g., HI-6 and obidoxime) exhibit
superior reactivation properties to monopyridinium oximes due
to the fact that the second cation interacts with the peripheral
anionic site (P-site) of AChE and increases binding affinity [28].
Accordingly, De Koning and Mercey et al proposed to attach
nonquaternary pyridine aldoxime to a ligand able to bind with the
P-site, which lies at the entrance of the active gorge of AChE [29,
heteroaryl keto-oximes (Fig. 3, compounds 8), which showed
obvious reactivation potency for cyclosarin-inhibited hAChE
[34]. Similarly, we used the dual site binding strategy to design
and synthesize a series of imidazolium aldoxime conjugates,
some of them (Fig. 3, compounds 7h) exhibited similar or
superior ability to reactivate sarin-, VX- and tabun-inhibited
AChE in comparison to HI-6 and obidoxime [35]. Their synthesis
is relatively easy and economical, but imidazolium aldoxime may
encounter the problem of reinhibition (Fig. 4, left). Furthermore,
it is noticeable that Katz et al had reported that some Mannich
phenols (Fig. 3, compounds 9 and 10) were efficient reactivators
for organophosphate inhibited AChE [36]. Coincidentally, we
also designed and synthesized a series of Mannich phenols which
showed obvious reactivating ability for nerve agent inhibited
hAChE, and the corresponding research will be reported soon.
30]. It was found some of the resulting conjugates (Fig. 3,
compounds 2-7) exhibited dramatically enhanced reactivation
potency as a result of greatly improved affinity for AChE [30-33].
These dual site binding nonquaternary reactivators hold promise
for the development of more efficient centrally activating
reactivators for nerve agent poisoning. However, the synthesis of
these pyridine aldoxime conjugates is difficult and their
reactivation potency against sarin-inhibited AChE is still
unknown. In addition, McHardy et al reported a series of
Figure 4. The mechanism of reactivation, reinhibition and intra-molecular cyclization of the phosphylated oximes

Recently, it was found that salicylaldoxime was able to cleave
the P-O bond of OP, and then yielded corresponding isoxasole i
and non-toxic phosphonic acids f through an intra-molecular
attack of the phenol onto the nitrogen atom (Fig. 4, right) [37, 38].
The rapid formation of this isoxasole could thus avoid
reinhibition by the active phosphylated oximes and allow the
discovery of more efficient reactivators. Furthermore,
enlightened by the structure of donepezil, a highly selective
AChE inhibitor for Alzheimer's disease treatment, the piperidine
ring was introduced as a linker between aldoximes and peripheral
site ligands (PSLs) (Fig. 5), it was expected to interact with the
AChE active gorge and contribute extra affinity for the enzyme
[39], and it had been used by McHardy et al to design and
synthesize novel reactivators (compound 8) [34]. As
a
comparison, methylene was also used as linkage of aldoximes
and PSLs. As a preliminary attempt, the moderate AChE P-site
binder phenyltetrahydroisoquinoline (PIQ) and its analogue
tetrahydroisoquinoline (TIQ) were chosen as PSLs (Fig. 5),
which can avoid toxicology produced by heavy AChE inhibition
[40].
Figure 5. Construction of the novel dual site binding conjugates for reactivation of inhibited AChE
Consequently, a novel series of salicylaldoxime conjugates
Fig. 6) based on the dual site binding strategy were constructed.
In this paper, we prepared 28 new tertiary salicylaldoxime
reactivators (Fig. 6), the in vitro screening essays demonstrated
that conjugates 3c, 4c and 5c were efficient and relatively broad
spectrum reactivators. They have advantageous properties
including (1) improved chemical stability (compared to
pro-2-PAM) and synthesis feasibility (compared to pyridine
aldoxime conjugates 2-7); (2) greatly improved lipophilicity
(compared to quaternary oximes); (3) and relatively increased
in vitro reactivation efficacy of nerve agents-inhibited AChE
(compared to quaternary oximes).
(
It was supposed that salicylaldoxime would lie at the bottom of
AChE active gorge to reactivate phosphoryl serine, while the
PSLs interacted with aromatic group in the P-site and
compensate the salicylaldoxime’s affinity for AChE (Fig. 5). The
designed conjugates were expected to possess enhanced BBB
penetration due to dramatic improvement of calculated
lipophilicity (higher value of S+logP indicates higher
lipophilicity, values are listed in table 1).
Figure 6. Novel designed and synthesized compounds in this paper
double triplet, t: triplet, td triple doublet, tm, triple multiplet, q:
quartet, quint: quintuplet, m: multiplet, br: broad).
Low-resolution mass spectra were obtained using an API 3000
LC/MS with an ESI source or an Agilent 620B TOF LC/MS with
an ESI source. Thin-layer chromatography (TLC) was carried out
on alumina sheets precoated with silica gel 10-40 um (pH
2
. Materials and methods
2.1. Chemicals
All reagents and solvents were used as received from
1
13
commercial sources. H NMR and C NMR spectra were
recorded at 400 MHz and 100 MHz on a JNM-ECA-400
instrument in in CDCl or DMSO-d , respectively. Proton and
3 6
carbon chemical shifts are expressed in parts per million (ppm)
relative to internal tetramethylsilane (TMS) and coupling
constants (J) are expressed in Hertz (Hz). The splitting pattern
abbreviations are as follows: multiplicity (s: singlet, d: doublet,
dd: double doublet, dm: double multiplet, ds: double single, dt:
6
(
2
.2-6.8) and the chromatography were performed on silica gel
200-300 mesh), compounds were visualized under UV light at
54 nm, R values were given for guidance. 2-PAM was
f
purchased from Sigma-Aldrich. HI-6 and obidoxime were
synthesized ourselves [41-43]. The synthetic procedures are
described in detail in the supporting information.

2
.2. General in vitro hAChE screening information
enzyme was gotten and different nerve agents were diluted more
than 7000-fold, indicating that most of the nerve agent was
removed and inhibition of hAChE had been terminated. Then the
inhibited enzyme (20 U/L, 80 μL) was incubated with oximes (40
μL, 0.3 mM) at 37 °C for 30 min (final concentration of oximes
was 0.1 mM). At different time intervals, 15 μL of the incubation
mixture was taken and enzyme activity was measured by using
same method described above.
Human acetylcholinesterase (hAChE), bovine serum albumin
(BSA), acetylthiocholine (ATCh), 5, 5’-dithiodis-2-nitrobenzoic
acid (DTNB) and 2-PAM were purchased from Sigma-Aldrich.
Sarin, VX, tabun and soman were from Anti chemical command
and Engineering Institute of the Chinese people's Liberation
Army. (Caution! Nerve agents used in our research are highly
toxic and must be handled with extreme care by well-trained
personnel. Use of these materials has been approved by Anti
chemical command and Engineering Institute of the Chinese
people's Liberation Army. After reactivation studies, biochemical
samples were neutralized by stirring with 2 M NaOH for 12 h,
the remaining solutions were brought back to pH ∼7 and
disposed in chemical waste.) Centrifugation was conducted at 4
Blank samples were run in parallel and consisted of: (a) A
positive control (P): uninhibited enzyme (80 μL) was used
instead of the inhibited enzyme; (b) a negative control (N): PBS
(40 μL, 0.1 M, pH 7.4, 0.1% BSA) was used instead of oximes.
Reactivation was calculated using the formula: %Reactivation
100*(S-N)/(P-N) [29, 45].
°
C in a 3-18K instrument from Sigma, the absorption was
2.5. Determination of reactivation kinetics
measured on a Bio Rad Microplate Reader Model 550 (Parts).
r
In order to determine the reactivation rate constant (K ),
dissociation constant (K ) and second order reactivation rate
D
constant (Kr2) of the selected reactivators, the reactivation rate at
different time intervals and at different concentrations were
measured in the same way as described above. The observed
first-order rate constant Kobs for each oxime concentration, the
The in vitro experiments were conducted with hAChE serving
as enzyme source. At the beginning of each experiments, the
stock solution of hAChE (dissolved in 20 mM HEPES, pH 8.0,
contain 0.1% TRITON X-100) were diluted by PBS (0.1 M,
pH=7.4, 0.1% BSA) and stored at 0-4°C. 10 mM oxime
concentration solutions were prepared in water contain 2.5%
acetic acid and 10% PEG-400 and they were further diluted by
PBS (0.1 M, pH=7.4) to the required concentrations. It was found
that there was no effect of CH3COOH and PEG-400 on hAChE
by a control experiment. All the biological evaluation experiment
were conducted triplicate in 96-well plate, the enzyme activity
was measured by the time-dependent hydrolysis of ATCh in
which the product thiocholine was detected by reaction with the
Ellman’s reagent DTNB and absorbance at 415 nm [44].
dissociation constant K
EP-OX) and the reactivation rate constant K
D
of inhibited enzyme-oxime complex
were calculated by
(
r
non-linear fitting using the standard oxime concentration
dependent reactivation equation derived from the following
scheme [46, 47]:
2.3. hAChE inhibition experiments
Initially 10 μL of hAChE (20 U/L in 0.1 M PBS, pH 7.4, 0.1%
BSA) was incubated with 5 μL of each oxime (final
concentrations: 0.2, 2, 100, 500 and 1000 µM) for 30 min at
25 °C. A blank experiment (positive control) was run in parallel
in which oxime was replaced by PBS. For each sample above in
the 96-well plate, 30 µl of ATCh (3.0 mM in 0.1 M PBS, pH 8.0,
In this scheme, EP is the phosphylated enzyme, [EP-OX] is
the reversible Michaelis-type complex between EP and the oxime
[OX], E is the active enzyme and P-OX the phosphylated oxime.
K is equal to the ratio (K +K )/K , and it typically approximates
0.1% BSA) along with 150 µl DTNB (0.75 mM in 0.1 M PBS,
pH 7.0) and 10 µl HCl (0.1 M) was added in each well. Reaction
product was monitored every 5 minutes, up to 20 minutes by
testing the absorption value at 415 nm (0<abs<2.5). Enzyme
activity was calculated by using the formula: %Activity=
D
-1
r
1
the dissociation constant of the [EP-OX] complex, where from it
follows that: K = K /K .
r2
r
D
Experimental details to determine the concentration
dependence of the apparent reactivation rate Kobs for the
reactivation of VX-, sarin- and tabun-inhibited hAChE are
described in the supporting information.
100*S/P. (S=absorption value of test substance; P = absorption
value of positive control (100% activity)). IC50 was determined
by non-linear fitting using the standard IC50 equation: %Activity
=
100*IC50/(IC50+[Ox]).
2.6. ADMET Predictions
2.4. hAChE reactivation experiments
All reagents and solvents were used as received from
A stock solution of hAChE (20 U/mL, from sigma) was
commercial sources. Initially structures of the compounds were
saved in the ‘sdf’ format using ChemBio3D Ultra software
(Cambridge Soft Corporation, 2013). The ‘sdf’ file of molecular
structures of these compounds was uploaded into the ADMET
predictor software, pH value was set at 7.4, pKa was limited
between 1.0 to 14.0 and the ‘activate-one-out in all associative
models’ option was selected. The program was executed to
diluted 50-fold with PBS (0.1 M, pH 7.4, 0.1% BSA). Different
nerve agents was diluted to proper concentrations (VX,
-6
-5
-6
-8
3*10 M; sarin, 2.75* 10 M; tabun, 1*10 M; soman, 5*10 M)
with PBS (0.1 M, pH 7.4, 0.1% BSA)， which had been
determined by a pre-experiment. The diluted hAChE (175 μL)
was incubated with different nerve agents (325 μL) at 37 °C for
30 min (at 4 °C for soman to delay rapid aging) and an excess of
calculate various parameters such as S+logP, S+S
S+logBBB, S+V and so on. The predicted data could be saved
as output file in ‘xls’ format.
w
, S+Peff,
nerve agents was removed from inhibited enzyme immediately
by filtration through a 10 kDa MWCO filter with a modified PES
membrane (Amicon Ultra-0.5, Millipore Corporation, Billerica,
MA) at 4 °C, followed by two washes prior to the final
resuspension in PBS/BLG (pH 7.4). The remaining solution in
the filter (50 μL) was diluted to 3.5 mL with PBS (0.1 M, pH 7.4,
d
3. Results and discussion
3.1. Synthesis
0.1% BSA). Under these conditions, 95% or greater inhibition of

A representative synthesis route highlighted in Scheme 1 was
used to prepare the conjugate templates 3a and 3b as shown in
The synthetic route outlined in Scheme 2 was used to prepare
the conjugates 1c, 1d to 7c, 7d and the details for oximes 3c and
3d were presented. Firstly, piperidinemethanol 4 were protected
by Boc to produce the intermediate 5, it was converted to tosylate
6 by treating with tosyl chloride (TsCl) in pyridine. Then
N-alkylation between the tosylate 6 and TIQ 2 or PIQ 3 provided
the intermediates 7 or 8, and a subsequent deprotection of Boc
was followed to give the tertiary amines 9 or 10. Finally,
condensation between 9, 10 and 3f was conducted and the
resulting compounds 3i and 3j were treated with
hydroxylammonium chloride to furnish the desired oximes 3c
and 3d.
Fig. 6. It was started with
a ring close reaction of
dimethoxyphenylethylamine 1 to give TIQ 2 and PIQ 3. At the
same time, salicylaldehyde derivatives such as 3e underwent a
chloromethylation reaction with aldehyde and concentrated
hydrochloric to provide the chloromethyl derivatives 3f in
excellent yields. Then condensation of 2, 3 and 3f afforded the
intermediates 3g and 3h. Finally, they were readily converted to
oximes 3a and 3b by treating with hydroxylammonium chloride.
The rest conjugates from 1a, 1b to 7a, 7b were obtained in a
similar way to 3a and 3b.
Scheme 1. Preparation of conjugates 3a and 3b. Conditions and reagents: a1, R=H) HCOOH(98%), 50 °C, 75%; a2, R = Ph) benzaldehyde,
TFA, reflux, 76%; b) con. HCl, 70 °C, 87%; c) DCM, NEt , r.t. 78%; d) HONH HCl, Na CO , EtOH/DCM, 79%.
3
2
2
3
Scheme 2. Preparation of conjugates 3c and 3d. Conditions and reagents: a) Boc
2
O, DCM, r.t. 76%; b) DMAP, pyridine, 0 °C, 87%；c) NaI,
HCl, NaOAc, EtOH/DCM, 91%.
K
2
CO , CH CN, reflux, 69%; d) TFA, DCM, r.t. 91%; e) DCM, NEt , r.t. 82%; f) HONH
3
3
3
2
3
.2. ADMET Predictions
HI-6 as a result of their greatly tremendous S+logBBB values,
especially for compounds (such as 6b, 1c-6c and 4d) with higher
predicted S+LogBBB values.
Lipophilicity and BBB penetration potency of all the
compounds were evaluated through in silico predictions by using
ADMET Predictor software version 7.0 (Pharmogo Co.,
Limited.), which was recommended as an accurate, quick and
useful tool to predict physicochemical and biological properties
of drug-like chemicals [48, 49]. S+logP is predicted
octanol-water partition coefficient and S+logBBB is predicted
log(Cbrain/Cblood), where Cbrain stands for the concentration of
compound in the brain and Cblood stands for the concentration of
compound in the blood. A higher S+LogP value indicates higher
lipophilicity and a higher S+logBBB value indicates higher BBB
penetration ability. The calculated results are listed in table 1.
3.3. Inhibition evaluation
A proper affinity to AChE is essential for a good reactivator,
while strong inhibition of AChE should avoided because it would
result in heavy toxicity [29, 45, 52]. Thus it is necessary to
evaluate the AChE inhibition abilities of these oximes firstly.
Quaternary oximes 2-PAM, HI-6 and obidoxime were used as
reference compounds. IC50 was determined by using multiple
concentrations of the oximes, and the results are displayed in
table 1. It can be found that these new synthesized oximes were
moderate or weak inhibitors of hAChE with IC50 greater than 100
μM, which might allow a proper affinity to hAChE for
reactivation of the poisoned enzyme. The results and the previous
studies in this field [26, 27, 45] confirmed us that an oxime
concentration of 100 μM is suitable for the in vitro reactivation
experiments.
Sakurada et al reported that the concentration of 2-PAM in the
corpus striatum is about 10% of the plasma level in rat in vivo
[50]. Falb and Erdmann et al reported that central nervous system
concentrations of obidoxime amounting to 3-5% of plasma levels
in both rats and mice in vivo [51]. It can be found that the
predicted S+logBBB values of 2-PAM (-0.69) and obidoxime
3.4. in vitro reactivation evaluation
(-1.26) (table 1) are almost in accordance with these experimental
results. Accordingly, these new synthesized compounds were
expected to penetrate BBB much more easily than 2-PAM and

During the course of the in vitro reactivation experiment, four
most common nerve agents (VX, sarin, tabun and soman) were
used for the inhibition of hAChE, and reactivation studies of
these new synthesized oximes were evaluated together with three
reference quaternary oximes (i.e., 2-PAM, HI-6 and obidoxime).
Firstly, the diluted hAChE was incubated with sufficient amounts
of the nerve agent to get a 95% or greater inhibition of the
enzyme and the excess of nerve agents were removed by
filtration immediately. Then the inhibited AChE was incubated
with different oximes (0.1 mM) for reactivation and the enzyme
activity was measured. The percentage of reactivated enzyme
The reactivation study shows that: (1) For VX-inhibited
hAChE, depart from 1a, 3a and 7b, methylene linked conjugates
1a-7a and 1b-7b were poor reactivators, while piperidine linked
conjugates 1c-7c and 1d-7d exhibited superior reactivation
ability, some of them even exhibited higher reactivation potency
as compared to obidoxime and HI-6 (such as 1c, 4c, 6c, 3d, 6d
and 7d). It was supposed that a methylene linker was not long
and flexible enough for the reactivators to have an optimal
orientation in regard to the reactivation reaction. Furthermore,
compounds whose linker in the ortho position to the phenol
function seemed to be better reactivators than compounds whose
linker in the para position (such as 1a vs 2a, 1c vs 2c and 1d vs
2d), it was also noteworthy that the phenol para linked
conjugates were heavier inhibitor of hAChE than the
corresponding ortho linked conjugates.
(%Reactivation) was calculated as the ratio of the recovered
enzyme activity and activity in the control and the results are
listed in table 1.
Table 1. a. Reactivation of sarin-, VX-, tabun- and soman-inhibited hAChE by new synthesized and the reference oximes (0.1 mM). b. IC50
,
calculated S+LogP and S+LogBBB of the reference and new synthesized oximes.
Reactivation (%)
Oximes
IC50
S+LogP
S+LogBBB
VX
sarin
tabun
soman
2
-PAM
HI-6
68.4±5.9
76.8±1.9
86.0±2.8
45.0±3.4
18.9±1.9
69.2±1.4
2.0±0.2
6.1±1.1
3.3±0.4
15.3±4.7
0.4±0.3
2.9±0.8
31.1±3.5
52.9±6.1
34.2±3.7
3.4±0.2
5.6±1.2
10.0±1.6
0.9±0.2
1.2±0.1
1.2±0.1
0.2±0.2
1.0±0.1
0.3±0.4
1.3±0.1
0.9±0.0
1.2±0.2
1.2±0.2
1.0±0.1
27.8±2.5
14.8±5.4
48.4±12.5
51.5±7.4
51.4±9.5
34.2±7.0
33.4±7.4
4.5±0.3
7.0±0.1
19.9±0.3
22.9±12.0
15.6±10.3
4.0±0.7
7.8±0.9
13.4±0.3
2.2±0.1
36.7±1.7
3.2±0.4
7.7±1.2
6.7±0.3
2.5±0.6
1.2±0.5
1.3±0.1
3.6±1.2
0.6±0.2
2.8±1.3
1.0±0.7
0.1±0.1
1.6±0.3
0.3±0.3
0.6±0.1
16.7±0.1
0.3±0.4
38.6±1.9
26.6±0.5
25.0±1.0
20.1±1.4
19.7±1.3
3.1±0.3
1.8±0.1
17.5±1.3
9.5±0.8
8.9±1.3
6.2±1.7
13.1±1.1
7.8±2.2
36.1±2.6
9.0±0.5
1.6±0.3
1.1±0.2
1.9±0.8
1.6±0.9
3.6±1.2
3.3±0.5
2.0±0.2
2.1±0.6
2.5±0.4
1.8±1.1
2.4±0.8
2.6±0.8
1.4±0.6
1.4±0.3
8.0±3.8
4.7±0.1
6.7±0.8
7.1±0.2
6.5±0.8
6.3±0.8
7.2±0.3
1.1±0.2
1.4±0.3
1.5±0.1
1.1±0.2
4.5±0.4
1.1±0.6
2.1±0.9
995.7±107
667.8±60.8
2169±234
1265±227
559.3±85.2
-2.81
-5.03
-4.61
3.50
3.24
3.89
4.17
4.17
3.73
3.32
4.69
4.54
5.07
5.22
5.24
4.87
4.50
4.20
3.97
4.64
4.85
4.86
4.48
4.10
5.65
5.31
6.03
6.14
6.09
5.57
5.50
-0.69
-1.39
-1.26
-0.44
-0.42
-0.49
-0.24
-0.37
-0.18
-0.51
-0.32
-0.38
-0.39
-0.14
-0.27
-0.06
-0.45
-0.05
-0.06
-0.09
0.16
0.03
0.17
-0.17
-0.19
-0.10
-0.25
-0.03
-0.17
-0.21
-0.33
obidoxime
1a
2a
3a
4a
5a
6a
7a
1b
2b
3b
4b
5b
6b
7b
1058±109
1567±173
956.7±79.2
692.5±107
1382±67.0
907.8±168
625.6±119
1124±158
1207±144
895.8±123
754.7±107
911.3±87.9
0.1±0.0
1.2±0.2
1.1±0.1
29.2±4.6
38.6±1.3
88.5±0.9
55.3±0.5
76.7±0.9
84.8±0.9
81.2±5.4
83.1±3.2
80.9±1.7
77.5±3.2
33.8±2.3
82.9±1.9
61.4±2.9
72.8±5.1
79.2±0.3
82.6±7.0
1
2
3
4
5
6
7
c
c
c
c
c
c
c
802.4±63.0
393.3±26.0
417.5±42.6
636.7±33.6
568.0±46.3
658.1±55.6
847.9±78.7
740.4±67.1
233.7±13.5
524.7±77.7
940.8±173
977.4±158
684.5±104
543.4±81.6
1
2
3
4
5
6
7
d
d
d
d
d
d
d
Experiments were performed in duplicate at 37 °C in phosphate buffer (0.10 M, pH 7.4), data shows the average and standard deviation, the
values of S+logP and S+logBBB were calculated by ADMET Predictor 7
(2) In the case of sarin, a similar trend was observed,
(3c, 4c and 5c) were even more potent reactivators for sarin
piperidine linked conjugates and whose linker in the ortho
position to the phenol function outperformed the other oximes
tested, such as 1c, 3c-7c and 4d. It was also noticeable that
conjugates containing a TIQ as PSL were more potent
reactivators than those containing PIQ. Additionally, methyl or
halogen substitution in the para position of salicylaldoxime
created more efficient reactivators such as 3c-6c, some of them
poisoning in comparison with 2-PAM and obidoxime.
(3) Tabun-inhibited hAChE is an OP complex known to be
reluctant to reactivation due to its weak electrophilicity and to the
steric hindrance imposed on the phosphorus atom in the
tabun-hAChE adduct. All of the new synthesized and reference
oximes displayed relatively lower reactivating ability for
tabun-inhibited hAChE than that for VX and sarin, but same
reactivation trend was concluded. Conjugates with TIQ and

linked to the ortho position to the phenol function of
salicylaldoxime though piperidine, such as 3c-7c, were still
efficiency reactivators superior to 2-PAM, whereas not good
enough than obidoxime.
reactivating ability towards VX-, sarin- and tabun-inhibited
hAChE. However, conjugation of the salicylaldoximes and PSLs
through a flexible linker would dramatically improve the
reactivation potency, such as reactivators 3c-5c. Thereby, we
believed that a proper PSL was essential for improvement of
reactivating ability of these salicylaldoximes. In general, based
on the reactivation results, TIQ and piperidine were considered as
proper PSL and linker, and piperidine should link to the ortho
position to the phenol function of salicylaldoxime. Moreover,
methyl and halogen substitution in the para position of
salicylaldoxime would achieve improvement of reactivating
ability.
(4) It was a pity that no satisfying results were obtained for
soman-inhibited hAChE, which is notoriously difficult to
reactivate, except that HI-6 showed obvious reactivation ability.
To further confirm the efficiency of the dual site binding
strategy, PSL1 and oxime2-oxime4 (Fig. 5) were also tested in
the reactivation experiment together with 3c-5c, the results (Fig.
7
) showed that PSL1 and oxime2-oxime4 exhibited slight or no
Reactivation
100
VX
Sarin
Tabun
80
60
40
20
0
1
2
3
4
L
3c
4c
5c
S
me
me
i
me
P
i
i
Ox
Ox
Ox
oximes
Figure 7. Reactivation of PSL1, 3c-5c, and oxime2-oxime4 for VX-, sarin- and tabun-inhibited hAChE
3.5. Determination of reactivation kinetics
It can be found that all of the tested conjugates were efficient
reactivators for VX-inhibited hAChE, especially the halogen
substitution 4c-7c, even exceeded that of HI-6, and while the
methyl substituted 3c just remained a little less efficient than
HI-6. In terms of sarin, conjugates 3c-5c exhibited higher
reactivation ability respectively. Compared with halogen
In order to get a deeper comprehension of the reactivation
mechanism of selected conjugates 3c-7c, we measured their
reactivation rate constant K , dissociation constant K and second
r
D
order reactivation rate constant Kr2 for sarin-, VX- and
tabun-inhibited hAChE. Reactivation kinetics were compared to
those of known reactivators, including HI-6 and obidoxime.
OP-inhibited hAChE was incubated at 37 °C with at least 5
concentrations of oxime in phosphate buffer (0.1 M, pH 7.4,
substituted 4c and 5c, despite the reactivation rate constant K
c is quite lower, its second order reactivation rate constant Kr2
was greatly improved due to increased affinity towards the
enzyme (lower dissociation constant K ). Although none of these
r
of
3
D
0
.1%BSA). At time intervals ranging from 5 to 180 min
oximes do not surpassed the reactivating efficiency of HI-6 for
sarin poisoning, they represented an interesting new starting new
starting point for further improvement. Quite remarkably,
conjugates 3c-5c displayed even higher reactivation efficiency
for the obstinate tabun-inhibited hAChE. Despite the reactivation
depending on the reactivation rate, the reactivation rate at
different concentrations of different oximes were measured. The
corresponding reactivation kinetics were calculated by non-linear
fit using the standard oxime concentration dependent reactivation
equation and the details were described in the experimental
section. The results were presented in table 2.
r
rate constant (K ) was lower compared to obidoxime, compound
Table 2. Reactivation rate constant (K
r D
), dissociation constant (K ), second order reactivation rate constant (Kr2) of obidoxime, HI-6, and
3
c-7c.
-
3
-1
-1
-1
oxime
K
r
/10 min
D
K /μM
K
r2/mM min
VX
48±0.6
sarin
344±156
Tabun
n.d.
VX
15±0.5
sarin
234±142
Tabun
n.d.
VX
3.09
sarin
1.47
Tabun
n.d.
HI-6
Obidoxime
30.0±1.4
5.6±0.6
3.4±0.2
2.9±0.2
5.2±0.2
4.9±0.1
91±9.2
7.6±4.0
8.9±2.9
7.2±2.7
53±4.3
51±3.0
0.325
0.739
0.382
0.412
0.098
0.097
n.d.
49±2.8
n.d.
19±1.5
n.d.
23±2.7
n.d.
38±9
n.d.
2.10
n.d.
0.502
3
4
5
6
7
c
c
c
c
c
-
-
-
-
127±1.7
107±2.1
41±4.4
91±5.7
-
-
-
-
262±55
204±66
268±44
512±42
7.19±0.68
5.20±0.50
6.18±0.48
3.10±0.19
0.485
0.521
0.153
0.177
Experiments were performed in duplicate at 37 °C in phosphate buffer (0.10 M, pH 7.4), data shows the nonlinear fitting results and standard
deviation.

trifluoracetic acid; DMAP, 4-dimethylaminopyridine; DCM,
dichloromethane; PE, petroleum ester; EA, ethylamine; TLC,
thin-layer chromatography; TMS, tetramethylsilane; ppm, parts
per million; r.t., room temperature; n.d., not determined; ATCh,
acetylthiocholine; DTNB, 5, 5’-dithiodis-2-nitrobenzoic acid;
BSA, albumin from bovine serum; BLG, β-lactoglobulin; PBS,
phosphate buffered saline.
3
c was 2-fold more efficient than obidoxime as a result of its
greatly increased affinity. However, compounds 6c and 7c
exhibited poor reactivation of tabun-inhibited hAChE due to
decreased affinity towards the enzyme. Interestingly, it can be
D
found that the dissociation constants (K ), indicating the binding
affinity for the inhibited hAChE, were always in consistent with
IC50 of these reactivators, but they were not proportional to each
other. Thus we proposed that a relatively moderate inhibition of
reactivators for hAChE might achieve improvement in
reactivating efficiency as a result of increasing affinity for the
target enzyme
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4. Conclusion
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(
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Corresponding Author
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Abbreviations
1
-methyl-1,6-dihydropyridine-2-carbaldoxime, a pro-drug of
OP, organophosphate; hAChE, human acetylcholinesterase; ACh,
N-methylpyridinium-2- carbaldoxime chloride. J. Med. Chem. 1976, 19,
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acetylcholine;
2-PAM,
pralidoxime;
MINA,
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monoisonitrosoacetone; BBB, blood-brain barrier; CNS, central
nervous system; P-site, peripheral anionic site; PSL, peripheral
site
ligand;
TIQ,
tetrahydroisoquinoline;
PIQ,
phenyltetrahydroisoquinoline; TsCl, tosyl chloride; TFA,

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7. Louise-Leriche, L.; Paunescu, E.; Saint-André, G.; Baati, R.; Romieu,

Highlights:
1
2
3
4
.
.
.
.
Novel
nonquaternary
reactivators
for
nerve
agent-inhibited AChE were constructed.
Some exhibited superior in vitro reactivation efficiency
in comparison to 2-PAM.
Introduction of peripheral site ligand dramatically
enhanced reactivation ability.
These conjugates may develop as efficient centrally
activating reactivators.