Design, synthesis and biological evaluation of novel tetrahydroacridine pyridine- Aldoxime and -Amidoxime hybrids as efficient uncharged reactivators of nerve agent-Inhibited human acetylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2014.03.044

A series of new uncharged functional acetylcholinesterase (AChE) reactivators including heterodimers of tetrahydroacridine with 3-hydroxy-2-pyridine aldoximes and amidoximes has been synthesized. These novel molecules display in vitro reactivation potencies towards VX-, tabun- and paraoxon-inhibited human AChE that are superior to those of the mono- and bis-pyridinium aldoximes currently used against nerve agent and pesticide poisoning. Furthermore, these uncharged compounds exhibit a broader reactivity spectrum compared to currently approved remediation drugs.

Full text of DOI:10.1016/j.ejmech.2014.03.044

European Journal of Medicinal Chemistry 78 (2014) 455e467
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of novel
tetrahydroacridine pyridine- aldoxime and -amidoxime hybrids as
efficient uncharged reactivators of nerve agent-inhibited human
acetylcholinesterase
Maria Kliachyna a, Gianluca Santoni b, Valentin Nussbaum a, Julien Renou c,
Benoit Sanson b, Jacques-Philippe Colletier b, Mélanie Arboléas d, Mélanie Loiodice d,
,
c
c
,
b
,
d,
a,
*
Martin Weik b , Ludovic Jean , Pierre-Yves Renard , Florian Nachon
, Rachid Baati
*
*
*
a Université de Strasbourg, Faculté de Pharmacie, CNRS/ UMR 7199 BP 24, 74 route du rhin 67401 Illkirch, France
b Commissariat à l’Energie Atomique, Institut de Biologie Structurale, F-38054 Grenoble; CNRS, UMR5075, F-38027 Grenoble; Université Joseph Fourier,
F-38000, Grenoble, France
c Normandie University, COBRA, UMR 6014 and FR 3038; University of Rouen; INSA of Rouen; CNRS, 1 rue Tesniere 76821 Mont-Saint-Aignan, Cedex, France
d Département de Toxicologie, Institut de Recherche Biomédicale des Armées BP7391993 Brétigny/s/Orge, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new uncharged functional acetylcholinesterase (AChE) reactivators including heterodimers of
tetrahydroacridine with 3-hydroxy-2-pyridine aldoximes and amidoximes has been synthesized. These
novel molecules display in vitro reactivation potencies towards VX-, tabun- and paraoxon-inhibited
human AChE that are superior to those of the mono- and bis-pyridinium aldoximes currently used
against nerve agent and pesticide poisoning. Furthermore, these uncharged compounds exhibit a
broader reactivity spectrum compared to currently approved remediation drugs.
Received 27 November 2013
Received in revised form
3 March 2014
Accepted 14 March 2014
Available online 15 March 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Organophosphorus nerve agents
Reactivation of acetylcholinesterase
Pyridinaldoximes
Oximes
Hybrids
Tacrine
1. Introduction
Depending on the class of OP and on the administrated dose, death
can occur within minutes [2].
Organophosphorus compounds (OP) include the extremely
toxic chemical warfare agents (CWA) (sarin, soman, cyclosarin,
tabun, methylphosphonothioate VX) and pesticides (paraoxon,
parathion, tetraethyl pyrophosphate (TEPP)) (Fig. 1). Their acute
toxicity results from the irreversible inhibition of acetylcholines-
terase (AChE) through phosphylation of its catalytic serine [1].
Accumulation of neurotransmitter acetylcholine (ACh) at cholin-
ergic synapses ensues, leading to nervous and respiratory failures.
Due to the similarity between the chemical precursors of CWA
and pesticides, and to the relatively simple chemistry involved in
their synthesis, efforts to control the proliferation of these agents
have proved of limited success [3]. Illustrative examples include the
terrorist attack in the Tokyo subway in 1995, the bombing of Kurd
civilians during the IraqeIran war in 1988, and that of civilians in
Syria, as reported in August 2013. Additionally, despite the inter-
national efforts aimed at regulating and lessening the use of these
environmentally toxic compounds, ca. 100 different OP are still
used intensively as pest control agents, with only anecdotal
monitoring. This results in about 3,000,000 acute intoxications per
year, 200,000 of which lead to death [4,5]. Therefore, the devel-
opment of effective measures to counteract OP poisoning remains a
challenging issue to protect and treat both civilian and military
populations [6].
* Corresponding authors.
E-mail addresses: martin.weik@ibs.fr (M. Weik), pierre-yves.renard@univ-
rouen.fr (P.-Y. Renard), florian@nachon.net (F. Nachon), rachid.baati@unistra.fr
(R. Baati).
http://dx.doi.org/10.1016/j.ejmech.2014.03.044
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

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M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
Fig. 1. Structures of organophosphorus CWA and pesticides.
The current treatment against OP poisoning consists in the
that exhibited by 2-PAM [9]. Connection of an a-ketoaldoxime
administration of a combination of atropine (antimuscarinic agent)
and diazepam (anticonvulsant drug), to limit convulsions, and of a
standard pyridinium oxime (pralidoxime, trimedoxime, HI-6, obi-
doxime, and HLö-7, Fig. 2) to reactivate AChE. Oximes exert their
action on OP-inhibited AChE by attacking the phosphorus atom of
the phosphylated serine, yielding to the removal of the phospho-
nate and recovery of the enzyme’s catalytic activity. To this end,
pyridinium oximes must display high nucleophilicity, which is
generally attained by the formation of an oximate anion at physi-
ological pH. As of today, however, not a single oxime has proven
equally effective against all types of OP-inhibited AChE, and most
are ineffective for the reactivation of tabun-inhibited hAChE [6].
Another weakness of currently approved pyridinium aldoximes
is their difficulty in crossing the bloodebrain barrier (BBB) owing to
the permanent charge carried by the oximate [7]. For example, it
was estimated using in vivo rat brain microdialysis coupled with
HPLC/UV, that the BBB penetration of the most commonly used
oxime, 2-PAM, is only 10% [8]. Therefore, oximes reactivating AChE
in the peripheral nervous system are not effective in the brain and,
consequently, do not protect against the neurological effects of OP-
exposure.
function to a piperidine-derived peripheral site ligand (PSL)
allowed increasing the affinity for AChE, resulting in higher reac-
tivation rates of sarin- and VX- inhibited AChE. Yet, these com-
pounds were still less efficient than currently used pyridinium
oximes towards the latter, and totally inefficient towards tabun-
inhibited AChE [10]. Hydroxyimino acetamides 1aeb showed
in vitro reactivation efficacy superior to MINA and DAM, but
remained less effective than 2-PAM, except for cyclosarin [11].
Likewise, amidine-oxime reactivators 2a,b were less potent than
2-PAM in reactivating AChE in vitro [12]. Low reactivation potency
of these new uncharged reactivators is likely due to their higher
pKa, which results in inefficient deprotonation at physiological pH
and thus, in a reduced nucleophilicity (for instance, for MINA,
pKa ¼ 8.3) [13].
Recently we found that 3-hydroxy-2-pyridine aldoximes 3 and
amidoxime 4 (Fig. 4) exhibited high reactivation first order rate
constant towards VX-inhibited hAChE (kr ¼ 0.5 ꢀ 0.1 minꢁ1 and
0.08ꢀ 0.1 minꢁ1, respectively)[14,15], i.e. values either similarorone
order of magnitude larger than that displayed by 2-PAM
(kr ¼ 0.06 ꢀ 0.01 minꢁ1). Owing to the lower affinity of 3 and 4 for
VX-hAChE (second order reactivation rate constants kr2 ¼ kr/KD are
0.015 and 0.0026 mMꢁ1 minꢁ1 for 3 and 4, respectively, to compare
to 0.28 mMꢁ1 minꢁ1 for 2-PAM), much higher concentrations are
however required to reach the desirable reactivation of the enzyme.
We have shown that connection of 3-hydroxy-2-
pyridinaldoxime reactivators with a PSL ligand, such as phenyl-
To overcome this obstacle, we recently reported the synthesis
of a series of uncharged oxime-based compounds, both able to
cross the BBB and to reactivate OP-inhibited AChE in the CNS [6].
Monoisonitrosoacetone (MINA) and diacetylmonooxime (DAM)
respectively bearing the
a-ketoaldoxime and ketoxime moieties
(Fig. 3) were also reported to cross the BBB, yet their in vitro
tetrahydroisoquinoline (PIQ) [16], results in
a dramatically
reactivation potency towards OP-inhibited AChE was lower than
improved affinity for the enzyme and, consequently, in increased
Fig. 2. Structures of developed pyridinium aldoxime reactivators.

M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
457
Fig. 3. Neutral reactivators: a-ketoaldoximes, ketoxime, hydroxyimino acetamides, and amidine-oximes.
Fig. 4. 3-hydroxy-2-pyridine aldoxime, 3-hydroxy-2-pyridine amidoxime, and phenyl-tetrahydroisoquinoline-3-hydroxy-2-pyridine aldoxime AChE reactivators.
reactivation potency towards OP-inhibited hAChE [17]. Recently,
similar beneficial effects have been reported for tryptoline-3-
hydroxypyridine-2-aldoxime hybrids for the reactivation of OP-
inhibited h-BChE [17c]. PIQ-functionalized 3-hydroxy-2-pyridine
aldoximes indeed displayed reactivation potency either equalling
or exceeding those of HI-6 and obidoxime. For example, 5a was 2.3
and 2.8 times more efficient than obidoxime and HI-6, respectively,
and 5 times more potent than trimedoxime, the best known reac-
tivator of tabun-inhibited hAChE (kr2 ¼ 3.4 mMꢁ1 minꢁ1 for 5a, as
compared to 0.7 mMꢁ1 minꢁ1 for trimedoxime).
One of the important drawbacks when using pyridinium
aldoximes (especially for pyridinium-4-aldoximes) is that the
Fig. 5. Structures of novel uncharged AChE hybrid reactivators investigated.

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M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
moiety would bind to the peripheral site. Obviously, the length
of the linker had to be optimized so that the oxime moiety
could approach the enzyme’s phosphylated serine without un-
binding from the peripheral site. In silico optimization of the
linker-length was performed to maximize the reactivation po-
tency of the bifunctional reactivators, and is described in Sec-
tion 3.
Fig. 6. General structure of targeted hetero-bifunctional reactivators.
reactivation products, phosphyloximes, can themselves display
high affinity for the enzyme, and thus prolong AChE inhibition
(recapture phenomenon) [18]. In the case of oximes 3, 5a, 5b, as
3. Synthesis of the hybrid molecules
well as in that of amidoxime 4, the presence of the 3-OH group in a-
3.1. Synthesis of 1,2,3,4-tetrahydro-9-aminoacridine
hydroxypyridine amidoximes
a-
position of the oxime/amidoxime moiety allows subsequent
intramolecular cyclisation, resulting in isoxasole formation and
thus in the bypass of this possible complication [14,15].
The 4-substituted
a-hydroxypyridine amidoxime 6 was syn-
In the following, we describe the rational design, synthesis and
in vitro evaluation of a new class of uncharged reactivator mole-
cules. Their design is based on a 1,2,3,4-tetrahydroacridine (tacrine)
substructure, able to bind to the peripheral site of AChE [19,20],
coupled with various linkers to either 3-hydroxy-2-pyridine
aldoxime or 3-hydroxy-2-pyridine amidoxime (Fig. 5).
thesized starting from commercially available 3-hydroxy-2-cyano-
pyridine 16 (Scheme 1). The introduction of the ortho-directing N-
(diethylamino)carbamate group (both as ortho directing group for
the metallation, and as further phenol protecting group) was ach-
ieved in standard conditions allowing to obtain compound 17 with
a good yield (95%). Subsequent ortho-lithiation and substitution
with iodine gave the key building block 18. The Sonogashira
coupling with alkyne 19 [26] proceeded cleanly and allowed pro-
ducing the hybrid compound 20 with 87% yield. Reduction of the
alkyne 20 in the presence of Pd/C yielded the corresponding
saturated compound 21. Attempts to selectively deprotect the N-
(diethylamino)carbamate group of 21 in either acidic or basic
conditions proved unsuccessful, due to the competing hydrolysis or
degradation of the cyano group. After a set of screening conditions,
this critical reaction was successfully achieved upon the treatment
of 21 with a large excess (30 equiv.) of hydroxylamine. Under these
conditions, the N-(diethylamino)carbamate was removed in a one-
pot process, and the cyano group was concomitantly converted to
the desired amidoxime 6. Thus, the final hybrid molecule 6 was
synthesized in a convergent 5-steps protocol with 13% overall yield.
2. Results and discussion
2.1. Rational design of bifunctional reactivator molecules
Acetylcholinesterase (AChE) features an active site buried at the
bottom of a deep and narrow gorge [21]. At the entrance of the
latter, a peripheral binding site serves as a transient binding loca-
tion for substrates and products en route to/from the active site
[22], but also as an anchor point for ligands [23]. Bifunctional in-
hibitors have been developed that display higher affinity, and
which are based on two moieties binding at either site that are
connected by a flexible linker [19,20,24]. By analogy, we designed
bifunctional reactivators that comprise a peripheral site ligand
(PSL) connected to a reactivator function by a covalent linker
(Fig. 6). In addition to increasing the affinity of the reactivator for
the enzyme, the use of a PSL further allows preventing unproduc-
tive binding to the active site as it was, for example, observed with
2-PAM [25].
The synthesis of a-hydroxyamidoxime tetrahydroacridine con-
jugate 7 is shown in Scheme 2. It differs from that of conjugate 6 in
that the substitution on the pyridine ring was performed using the
optimized modular approach described previously for the Sono-
gashira coupling of bromopyridine 22 [27] with alkyne 19 (Scheme
2). Indeed, this coupling worked nicely and gave 23 in excellent
yield (86%). The one-pot alkyne reduction and O-debenzylation
proceeded cleanly to give nitrile 24, which was directly reacted
In practice, we used as a starting model the X-ray crystal
structure of Torpedo californica (Tc)AChE in complex with a bis-
tacrine inhibitor [19], which allowed predicting how the tacrine
Scheme 1. Synthesis of amidoxime 6.

M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
459
Scheme 2. Synthesis of amidoxime 7.
with excess of hydroxylamine hydrochloride to give expected
amidoxime 7 with 45% overall yield over the 3 steps.
3.2. Synthesis of 1,2,3,4-tetrahydro-9-aminoacridine
hydroxypyridine aldoximes
a-
Docking studies (unpublished results) suggested that the pres-
ence of a tertiary amine in the linker could increase the affinity of
the molecule for AChE -and thus presumably enhance its reac-
tivation efficacy -owing to the amine protonation at physiological
For the synthesis of a-hydroxypyridine oxime hybrids, we first
started with the synthesis of 3-hydroxypyridine aldoxime with the
linker in position 4, using the common intermediate 21. For these
molecules, we privileged the one-pot carbamate group removal and
methanolysis of cyano- function to give methyl ester 30 with a
reasonable yield of 42% (Scheme 4). Next, 30 was converted to 31
via a three-step [17] sequence that included: (a) TBDMS protection
of phenol function; (b) reduction of methyl ester to aldehyde with
DIBAL-H; (c) and subsequent deprotection of the silyl-oxy function
using TBAF (33% over three steps). Reaction of the resulting alde-
hyde 31 with NH2OH resulted in the formation of the desired
pyridinaldoxime 10, with 7% overall yield.
pH that would allow cation-
in AChE gorge [28]. To verify this hypothesis, we therefore prepared
hybrids 8 and 9. The synthesis of these -hydroxypyridine ami-
p interaction with the tyrosine residues
a
doxime hybrids connected with tetrahydroacridine by NeMe linker
was based on the reductive amination of aldehyde 25 (obtained by
ortho-lithiation and subsequent formylation of 17) with amines 26
and 27 [29] (Scheme 3). The treatment of 28 and 29 with excess
hydroxylamine gave
a-hydroxypyridine amidoximes 8 and 9 in 13%
and 19% overall yields, respectively, over the 3 steps syntheses.
Scheme 3. Synthesis of amidoximes 8 and 9.

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M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
Scheme 4. Synthesis of oxime 10.
The 5- and 6-substituted
a
-hydroxypyridine oximes analogues
compound 48. Contrary to 35e37, the attempts to reduce the triple
11e13 were obtained using the modular synthetic approach, con-
sisting in the Sonogashira coupling reaction of 6-bromopyridine 32
[17] or 5-bromopyridine 33 [30] with alkynes 19 or 34 [31],
respectively, followed by reduction of the alkyne and finally O-
debenzylation. The subsequent three-step transformation of
methyl ester to aldehyde, ensued by reaction with hydroxylamine
bond of 48 using Pd/C or Pd(OH)2/C failed, possibly due to the
poisoning of catalyst by the reaction with thioether groups. The use
of only 1.5 equiv of PtO2 yet resulted in successful hydrogenation,
allowing to obtain compound 49 with 64% yield. The further three-
step sequence comprising protection of the phenol group, reduc-
tion of methyl ester with DIBAL-H into aldehyde, and the following
deprotection using TBAF generated the desired aldehyde 50, with
42% yield. Finally, reaction of 50 with NH2OH resulted in the for-
mation of pyridinaldoxime 14, with 6% overall yield.
(Scheme 5), yielded the desired
in 15, 28 and 18% yields.
a-hydroxypyridine oximes 11e13
3.3. Synthesis of 1,2,3,4-tetrahydro-9-thioacridine
hydroxypyridine aldoxime
a-
4. Docking and molecular dynamics simulations
In order to tune the affinity of hybrid reactivator for hAChE, the
structure of the PSL was changed by the substitution of NH at po-
sition 9 of 1,2,3,4-tetrahydroacridine with a sulphur atom. In short,
conjugate 14 bearing 1,2,3,4-tetrahydro-9-thioacridine as PSL was
Ternary complexes between VX-inhibited hAChE and 3-
hydroxypyridine aldoxime with 2, 3, 4 (compound 12) or 5 (com-
pound 11) carbons linker were investigated by molecular docking
and molecular dynamics simulations (see Supporting Information).
Flexible docking experiments were undertaken, in which the side
chains of active site gorge residues key to the binding of the reac-
tivators were allowed to wander from their native position (see
synthesized according to Scheme
6 by alkylating 1,2,3,4-
tetrahydro-9-thioacridine 44 [32] with 45 to give 46, which was
then used for Sonogashira coupling reaction with 47 to provide
Scheme 5. Synthesis of oximes 11, 12, and 13.

M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
461
Scheme 6. Synthesis of oxime 14.
Supporting Information). In all four docking experiments,
a
oxygen closer to the phosphorus atom and more frequently (Fig. 8),
suggesting that a 4-carbon linker between the PSL and the aldox-
ime is optimum for reactivation.
conformational change was observed in the side chain of
peripheral-site Trp286 (Fig. 7), faithfully reproducing the crystal-
lographic observation from the bis-tacrine/TcAChE complex (PDB
code 2CEK) [19]. This conformational change results in a
pep
5. In vitro reactivation of VX-, tabun and POX-inhibited
sandwiching of the tacrine moiety between the aromatic side
chains of Tyr72 and Trp286. The scoring function of the docking
algorithm yielded similar binding energies for the 4 molecules of
about e 9 kcal/mol, indicating that the linker is sufficiently long to
prevent unproductive binding of the reactivators in the gorge of the
inhibited enzyme.
The structures resulting from the 4 independent dockings were
then subjected to molecular dynamics (MD) simulations for 5 ns at
a temperature of 300 K. For each, the distance between the
aldoxime oxygen and the VX-AChE phosphorus atom was moni-
tored along the simulation trajectory and the distributions of dis-
tances were analyzed (Fig. 8). The shorter the distance, the higher
the probability to form a covalent bond between the reactivator
oxygen and the phosphorus atom, that is, the more likely should
reactivation occur at a fast rate. Molecule 12 brings the aldoxime
hAChE
The abilities of amidoximes 6e9, as well as of aldoximes 10e14
to reactivate in vitro VX-, tabun- and ethyl-paraoxon inhibited
hAChE were investigated by spectrophotometry using the Ellman’s
reaction. Reactivation kinetics were compared to those of known
reactivators, including 2-PAM, obidoxime, HLö-7, trimedoxime
(TMB4) and HI-6. 6-Substituted
and 4-substituted -hydroxypyridine oxime 10 were both totally
inefficient in reactivating either VX-inhibited hAChE or tabun-
inhibited hAChE. (Table 1)
a-hydroxypyridine amidoxime 7
a
In contrast, 4-substituted tetrahydroacridine a-hydroxypyridine
amidoximes 6 (C5 alkyl linker), 8 and 9 (NeMe linkers) displayed
improved affinity and higher reactivation efficiencies (kr2) towards
VX-inhibited hAChE than both 2-PAM and trimedoxime. While they
remained less efficient than obidoxime, HLö-7, and HI-6 in reac-
tivating VX-inhibited hAChE, it is noteworthy that these com-
pounds are the first amidoxime-based reactivators that are efficient
towards VX-inhibited hAChE. Quite remarkably, aldoximes 11 and
12 substituted in position 6 of the pyridine ring by the C5 and C4
alkyl linker proved more efficient in reactivating VX-inhibited
hAChE than 2-PAM, obidoxime, trimedoxime, and HI-6. Although
oximes 11 and 12 do not surpass the reactivating efficiency of HLö-
7, nor that of our previously described compound 5a, they repre-
sent an interesting new starting point for further improvement. It
must be stressed, in particular, that since they neither bear a per-
manent charge nor a tertiary amine, they should be able to cross the
BBB quite efficiently.
Also noteworthy is the fact that oxime 12 is able to reactivate
tabun-inhibited hAChE (Table 2), 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. In fact, compound 12 exhibited higher in vitro
reactivation potency than all quaternary pyridinium aldoximes
described to date. Despite a lower reactivation rate constant (kr)
compared to obidoxime and trimedoxime, compound 12 was 4-fold
more efficient than trimedoxime, the best pyridinium aldoxime
reactivator of tabun-inhibited hAChE. Again, this increase in
Fig. 7. Docked conformation of molecule 12 (yellow) at the peripheral site of VX-
hAChE. The docked conformations of the flexible residues are shown in cyan and the
native conformations (pdb code 1b41) in blue. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

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M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
Fig. 8. Distribution of distances between the aldoxime oxygen and the VX phosphorus analyzed along the simulation trajectory for the four different linker lengths (KM 2: eCH2e
CH2e; KM 3: eCH2eCH2-CH2e, KM 4: eCH2eCH2eCH2eCH2e, 12; KM 5: eCH2eCH2eCH2eCH2eCH2e, 11).
Table 1
efficiency is likely to be ascribed to its significantly greater affinity
towards the tabun-hAChE conjugate (KD). The better reactivity of
compound 12 on both VX- and tabun-inhibited hAChE, as
compared to compound 11, suggest that a 4-carbon linker is opti-
mum for both a good binding to the inhibited enzyme and an ac-
curate orientation of the oxime function needed for displacing of
the phosphyl or phosphoramidyl moiety.
As mentioned already, most organophosphate intoxication re-
sults from pesticide poisoning, which involves the formation of a
diethylphosphoryl conjugate of hAChE. Testing molecules for their
ability to reactivate ethylparaoxon-inhibited hAChE, therefore, al-
lows evaluating them for their general efficacy against pesticide
poisoning. Interestingly, compounds 11 and 12 proved more effi-
cient than obidoxime and HLö-7 towards paraoxon-inhibited
hAChE, (Table 3). Much like the trend observed in the case of VX,
decreasing the linker length from 5 to 4 carbons led to a 1.5-fold
increase in kr.
We note that compounds 7, 10, 11 and 12 are also able to inhibit
native hAChE, with IC50 similar to tacrine and tacrine-pyridyl het-
erodimer 51 (Table 4). While these inhibitory properties could be
envisaged as a drawback of our strategy aiming at increasing the
efficacy of reactivators by increasing their affinities for the target
enzyme, we argue conversely. Indeed, it remains to be proven that
significant reversible inhibition of AChE at a concentration similar
to that used to reactivate of OP-inhibited AChE is really problematic
in vivo. Also, it was shown that reversible inhibitors have a pro-
tective effect on AChE, preventing its phosphylation by transiently
occupying the active site [35].
Interestingly, 4-substituted pyridine aldoxime 10 showed no
reactivation of VX-, tabun- and paraoxon-inhibited hAChE and a
higher inhibition compared to tacrine. This is in strong contrast
with 6-substituted compound 11 and suggests that 4-substitution
of pyridine ring favours the binding of tacrine moiety to the
In vitro reactivation of VX-hAChE.
b
Reactivator
kr (minꢁ1
)
KD
(
m
M)
kr2 (mMꢁ1 minꢁ1
)
2-PAM
Obidoxime
HLö-7
Trimedoxime
HI-6
6
8
0.06 ꢀ 0.01
0.60 ꢀ 0.05
0.49a
215 ꢀ 75
54 ꢀ 12
7.8a
0.28
11
63a
ndc
ndc
0.50 ꢀ 0.02c
0.44 ꢀ 0.15
50 ꢀ 26
9
ndc
ndc
0.65c
0.6
1.1
13
0.0094 ꢀ 0.0004
0.032 ꢀ 0.001
0.56 ꢀ 0.12
0.72 ꢀ 0.07
15 ꢀ 2
30 ꢀ 3
41 ꢀ 11
31 ꢀ 6
9
11
12
13
14
22
ndd (kobs ¼ 0.0038 ꢀ 0.00006 minꢁ1 at 1
m
M)
M)
ndd (kobs ¼ 0.0076 ꢀ 0.0005 minꢁ1 at 200
m
a
From the ref. [33].
kr2 ¼ kr/KD.
b
c
If [reactivator] ꢂ KD, then there is a linear dependence between kobs and
[reactivator]: kobs ¼ (kr/KD) [reactivator]. In this case, kr and KD cannot be deter-
mined, but kr2 ¼ kr/KD is the slope of the line.
d
Not determined if kobs is <0.01 minꢁ1 at practical concentration.
Table 2
In vitro reactivation of tabun-hAChE.
a
Reactivator
kr (minꢁ1
)
KD
(
m
M)
k )
r2 (mMꢁ1 minꢁ1
Obidoxime
HLö-7
Trimedoxime
8
9
11
12
0.04 ꢀ 0.006
0.020 ꢀ 0.0007
0.085 ꢀ 0.005
250 ꢀ 110
106 ꢀ 15
145 ꢀ 25
0.16
0.2
0.7
Less than 10% reactivation at 100
m
M in 2 h
0.0075 ꢀ 0.0005
0.021 ꢀ 0.001
30 ꢀ 6
0.27
3
7.1 ꢀ 1.5
a
kr2 ¼ kr/KD.

M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
463
Table 3
conditions unless otherwise indicated. The chemicals used in the
synthesis and solvents were purchased from Aldrich, Acros, Alfa-
Aesar or ABCR and used without further purification. Thin layer
chromatographies (TLC) were carried out using Merck silica gel
plates (silica gel 60 F254). TLC plates were visualized using UV light
(254 nm), followed by detection with cerium sulphate and ninhy-
drin. The column chromatographies were performed using Merck
silica gel 60 (0.063e0.200 mm, 70e230 mesh, ASTM) according to a
standard procedure. Analytical HPLC chromatography (purity
>95%) were performed using SPD-20A Shimadzu apparatus
equipped with a Zorbax 300SB-C18 column, using a mixture of
acetonitrile and water as eluent (0.1% TFA). 1H NMR and 13C NMR
spectra were recorded on Brucker DPX300, DPX400 and DPX500.
In vitro reactivation of paraoxon-hAChE.
b
Reactivator
kr (minꢁ1
)
KD
(
m
M)
k )
r2 (mMꢁ1 minꢁ1
Obidoxime
HLö-7
Trimedoxime
8
9
11
12
0.81a
32.2a
20a
3
0.63 ꢀ 0.04
210 ꢀ 31
0.34 ꢀ 0.02a
47.8 ꢀ 6.9a
17a
Less than 10% reactivation at 100
m
M in 2 h
ndd (kobs ¼ 0.0043 ꢀ 0.0004 minꢁ1 at 100
mM)
20.9
31
0.0228 ꢀ 0.0006
0.111 ꢀ 0.002
1.1 ꢀ 0.1
3.6 ꢀ 0.2
cNot determined if kobs is <0.01 minꢁ1 at practical concentration.
a
From the ref. [34].
kr2 ¼ kr/KD.
b
Chemical shifts (d) are quoted in parts per million and are calibrated
catalytic site of the enzyme, rather than at its peripheral site. The
substitution of tacrine with thiotacrine as PSL resulted in three
order of magnitude lower inhibition of native hAChE by model
compound 52 and 14, as compared to compounds 51 and 11,
respectively. However, oxime 14 exhibited very poor reactivation of
VX-hAChE, demonstrating that the key to the improvement of the
reactivation potency is finding the optimal balance between affinity
of the bifunctional reactivator for the enzyme and positioning of
the oxime function relative to the phosphorus atom of the OP-
hAChE adduct.
relative to solvent residual peaks. Multiplicities are reported as
follows: s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet,
q ¼ quartet, qui ¼ quintet. Coupling constant are reported in Hertz.
Mass spectra were recorded on an MS/MS high resolution Micro-
mass ZABSpecTOF spectrometer.
7.2. General procedures and analytical data of key compounds 35,
36, 38, 39, 41, 42, 11, and 12
General procedure 1 for the Sonogashira coupling reaction
(synthesis of compounds 20, 23, 35, 36, and 37). The flask con-
taining solution of iodo- or bromopyridine (1 equiv) in THF/Et3N
was evacuated and filled with Ar three times before the addition of
catalysts Pd(PPh3)4 (0.1 equiv) and CuI (0.2 equiv). After degazation
with Ar the mixture was stirred at the room temperature for 5 min,
then the degazed solution of alkyne (1.1 equiv) was added and the
reaction mixture was stirred during 20 h at the room temperature.
After concentration at reduced pressure the residue was purified by
the column chromatography.
General procedure 2 for N-(diethylamino)carbamate group
removal and nitrile conversion to amidoxime (synthesis of com-
pounds 6, 8, and 9). The solution of 21, 28 or 29 (1 equiv),
NH2OH.HCl (30 equiv), and pyridine (30 equiv) in 2 mL of ethanol
was refluxed during 14 h. The solvent was removed in vacuo, the
residue was subjected to preparative HPLC chromatography.
General procedure 3 for the one-pot alkyne reduction and O-
debenzylation (synthesis of compounds 24, 38, 39, and 40). To the
degazed solution of alkyne (1 equiv) in ethyl acetate or methanol
Pearlman’s catalyst Pd(OH)2/C (20%, moisture 50%, 0.3 equiv) was
added. After the degazation the solution was bubbled with H2. The
reaction mixture was stirred at RT under H2 (1 atm) during 24 h.
6. Conclusion
We have described the synthesis and the in vitro biological
properties of
charged AChE reactivators, including differently substituted
tacrine -hydroxypyridine oximes and amidoximes. We demon-
a series of novel, promising, non-permanently
a
strated that the amidoxime function can be used as a reactivator
function, and allows producing hybrid molecules that equal or
surpass currently approved mono- and bis-pyridinium oximes,
both in terms of reactivation efficiency and broadness of their ac-
tivity spectrum. While it remains to be evaluated if the molecules
are centrally active, or require further modifications to be so, this
new family of oximes holds great promise for the medical treat-
ment of OP-poisoning.
7. Experimental part
7.1. General materials and methods
All reactions were carried out in a flame dried glassware under
an argon atmosphere with dry solvents, under anhydrous
Table 4
Inhibition of hAChE.
Compound
Tacrine
IC50
(
m
M)
Compound
IC50 (mM)
0.20 ꢀ 0.02 (Human erythrocyte AChE)a
12
1.40 ꢀ 0.03 (hAChE)
0.15 ꢀ 0.01 (hAChE)
13
0.163 (hAChE)
7
0.26 ꢀ 0.01 (hAChE)
130 ꢀ 15 (hAChE)
10
11
0.077 ꢀ 0.003 (hAChE)
0.26 ꢀ 0.01 (hAChE)
14
307 (hAChE)
a
From ref. [36].

464
M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
The solution was filtered through celite, the solvent was evapo-
rated, and the product was dried in vacuo.
7.2.3. 3-Hydroxy-6-{5-[(1,2,3,4-tetrahydroacridin-9-yl)amino]
pentyl}pyridine-2-carbaldehyde (41)
General procedure 4 for the three-step transformation of the
methyl ester into aldehyde (synthesis of compounds 31, 41, 42, 43
and 50). The solution of methyl ester 30, 38, 39, 40 or 49 (1 equiv),
imidazole (4.15 equiv), and TBDMSCl (2.4 equiv) in dry DMF was
stirred at the room temperature under argon during 2 h. Ethyl ac-
etate was added, and the organic phase was washed with water 3
times, dried over Na2SO4 and concentrated in vacuum. After drying
in vacuum the compound obtained was subjected without purifi-
cation to the following reduction of methyl ester with DIBAL-H. To
the solution of the compound obtained (1 equiv) in dry CH2Cl2
at ꢁ78 ꢃC DIBAL-H (1 M solution in CH2Cl2, 2 equiv) was added
dropwise. The reaction mixture was stirred at ꢁ78 ꢃC for 12 min,
then it was quenched with MeOH, and the cooling bath was
removed. When the mixture was warmed to the room temperature,
the organic phase was washed with aqueous solution of NaOH
(1 M), dried over Na2SO4 and concentrated under reduced pressure.
After drying in vacuum the product obtained was subjected to the
following reaction with TBAF. To the solution of the compound
obtained (1 equiv) in dry THF at 0 ꢃC TBAF (1 M solution in THF,
1.1 equiv) was added, and the reaction mixture was stirred at this
temperature for 1 h. After removal of the solvent under reduced
pressure the residue was purified by the column chromatography
(silica gel).
Compound 41 was obtained by the general procedure 4 from 38
with yield 37% (over three steps). Rf ¼ 0.23 (ethyl acetate/
MeOH ¼ 10/3). 1H NMR (400 MHz, CDCl3,
d): 1.40e1.48 (2H, m),
1.67e1.79 (4H, m), 1.85e1.90 (4H, m), 2.64 (2H, m), 2.76 (2H, t,
J ¼ 7.7 Hz), 3.05 (2H, m), 3.52 (2H, t, J ¼ 7.3 Hz), 7.23 (1H, d,
J ¼ 8.7 Hz), 7.25 (1H, d, J ¼ 8.7 Hz), 7.29e7.33 (1H, m, ArH), 7.51e
7.55 (1H, m, ArH), 7.92e7.95 (2H, m), 9.99 (1H, s, CHO). 13C NMR
(100 MHz, CDCl3, d): 22.73; 23.11; 24.88; 26.70; 29.48; 31.76; 33.48;
37.28; 49.45; 115.48; 123.10; 124.02; 126.64; 127.99; 129.01;
129.88; 131.06; 135.95; 151.45; 154.66; 157.23; 157.88; 198.83.
HRMS (ESI, m/z): calcd. for [M þ H]þ, C24H28N3O2, 390.21815;
found, 390.21892.
7.2.4. 2-[(1E)-(hydroxyimino)methyl]-6-{5-[(1,2,3,4-
tetrahydroacridin-9-yl)amino]pentyl}pyridin-3-ol (11)
Compound 11 was obtained accordingly to the general proce-
dure 5 using 41 (0.050 g, 0.13 mmol, 1 equiv), NH2OH.HCl (0.013 g,
0.19 mmol, 1.5 equiv), CH3COONa (0.016 g, 0.19 mmol, 1.5 equiv),
and 1.2 mL of ethanol. After evaporation of the solvent and purifi-
cation by column chromatography (ethyl acetate/MeOH ¼ 8/2)
product was obtained with yield 55%. Rf ¼ 0.44 (ethyl acetate/
MeOH ¼ 2/1). 1H NMR (400 MHz, CD3OD,
d): 1.37e1.44 (2H, m,
CH2), 1.68e1.76 (2H, qui, J ¼ 7.4 Hz, CH2), 1.77e1.85 (2H, qui,
J ¼ 7.4 Hz, CH2), 1.90e1.94 (4H, qui, J ¼ 3.3 Hz, CH2), 2.63 (2H, m,
CH2), 2.70 (2H, t, J ¼ 7.4 Hz, CH2), 3.00 (2H, m, CH2), 3.87 (2H, t,
J ¼ 6.9 Hz, CH2), 7.08 (1H, d, J ¼ 8.4 Hz, ArH), 7.20 (1H, d, J ¼ 8.4 Hz,
ArH), 7.50e7.54 (1H, m, ArH), 7.76e7.81 (2H, m, ArH), 8.17 (1H, s,
General procedure 5 for synthesis of oximes 10e14. The solution
of aldehyde 31, 41, 42, 43 or 50 (1 equiv), NH2OH.HCl (1.5 equiv),
and CH3COONa (1.5 equiv) in dry ethanol was stirred during 8 h.
After concentration under reduced pressure the crude product was
purified by the column chromatography or preparative HPLC.
N]CH), 8.30 (1H, d, J ¼ 8.7 Hz). 13C NMR (100 MHz, CD3OD,
d):
22.31; 23.30; 25.25; 27.05; 30.30; 30.53; 30.91; 31.42; 37.56; 117.95;
121.70; 125.51; 126.08; 126.18; 133.42; 136.35; 152.90; 153.85;
154.49. MS m/z (ESI) calcd. for C24H29N4O2 [M þ H]þ, 405.22; found,
405.2.
7.2.1. Methyl 3-(benzyloxy)-6-{5-[(1,2,3,4-tetrahydroacridin-9-yl)
amino]pent-1-yn-1-yl}pyridine-2-carboxylate (35)
Compound 35 was obtained accordingly to the general proce-
dure 1 for Sonogashira coupling using 32 (0.147 g, 0.46 mmol,
1 equiv), 19 (0.132 g, 0.5 mmol, 1.1 equiv), Pd(PPh3)4 (0.041 g,
0.046 mmol, 0.1 equiv), CuI (0.017 g, 0.09 mmol, 0.2 equiv), Et3N
(3 mL), and THF (6 mL). The crude product was purified by the
column chromatography (silica gel, gradient from ethyl acetate to
ethyl acetate/MeOH ¼ 8/2, Rf ¼ 0.24). Yield 180 mg, 78%. 1H NMR
7.2.5. Methyl 3-(benzyloxy)-6-{4-[(1,2,3,4-tetrahydroacridin-9-yl)
amino]but-1-yn-1-yl}pyridine-2-carboxylate (36)
Compound 36 was obtained accordingly to the general proce-
dure 1 for Sonogashira coupling reaction using 32 (0.292 g,
0.91 mmol, 1 equiv), 34 (0.250 g, 1 mmol, 1.1 equiv), Pd(PPh3)4
(0.081 g, 0.091 mmol, 0.1 equiv), CuI (0.035 g, 0.18 mmol, 0.2 equiv),
Et3N (6 mL), and THF (12 mL). The crude product was purified by
the column chromatography (silica gel, gradient from ethyl acetate/
MeOH ¼ 10/1 to ethyl acetate/MeOH ¼ 8/2), Rf ¼ 0.43 (ethyl ace-
(300 MHz, CDSl3,
d): 1.76e1.84 (4H, m), 1.87e1.97 (2H, qui,
J ¼ 6.7 Hz), 2.49 (2H, t, J ¼ 6.7 Hz), 2.64 (2H, m), 3.03 (2H, m), 3.67
(2H, t, J ¼ 6.9 Hz), 3.88 (3H, s, CH3O), 5.14 (2H, s, PhCH2O), 7.18e7.38
(8H, m, ArH), 7.48 (1H, t, J ¼ 7.6 Hz), 7.90e7.93 (2H, m). 13C NMR
(100 MHz, CDCl3,
d): 17.29; 22.79; 23.13; 25.05; 29.98; 33.79; 48.42;
tate/MeOH ¼ 4/1). Yield 0.440 g, 98%. 1H NMR (400 MHz, CDCl3,
d):
52.88; 71.10; 80.64; 88.84; 120.24; 121.96; 122.93; 124.19; 127.12;
128.37; 128.47; 128.63; 128.75; 128.83; 128.95; 130.11; 132.24;
132.34; 135.23; 135.66; 146.91; 150.97; 153.20; 158.23; 164.99.
1.82e1.89 (4H, m), 2.67 (2H, t, J ¼ 6.3 Hz), 2.79 (2H, t, J ¼ 5.9 Hz),
3.06 (2H, t, J ¼ 5.9 Hz), 3.70e3.75 (2H, dt, J ¼ 6.4 Hz), 3.95 (3H, s),
4.48 (1H, t, J ¼ 6.4 Hz, NH), 5.19 (2H, s, OCH2Ph), 7.25 (1H, d,
J ¼ 8.8 Hz), 7.28 (2H, d, J ¼ 8.8 Hz), 7.31e7.42 (6H, m, ArH), 7.52e
7.56 (1H, m), 7.97 (2H, t, ArH, J ¼ 8.3 Hz). 13C NMR (100 MHz, CDCl3,
7.2.2. Methyl 3-hydroxy-6-{5-[(1,2,3,4-tetrahydroacridin-9-yl)
amino]pentyl}pyridine-2-carboxylate (38)
d): 22.10; 22.59; 22.99; 24.93; 29.89; 33.29; 47.34; 52.93; 71.10;
Compound 38 was obtained accordingly to the general proce-
dure 3 for the reduction of the triple bond and O-debenzylation
using 35 (0.180 g, 0.35 mmol), Pd(OH)2/C (20%, 0.075 g, 0.11 mmol,
0.3 equiv), and 2.5 mL of MeOH. Yield 0.140 g, 94%. Rf ¼ 0.34
77.43; 81.98; 86.71; 116.94; 120.15; 121.92; 123.00; 124.56; 127.14;
127.66; 128.51; 128.97; 129.30; 130.09; 134.74; 135.59; 140.67;
146.04; 151.08; 153.37; 157.78; 164.98. HRMS (ESI, m/z): calcd. for
C
31H30N3O3 [M þ H]þ, 492.22871; found, 492.22851.
(CH2Cl2/MeOH ¼ 10/1). 1H NMR (300 MHz, CDCl3,
d): 1.40e1.50 (2H,
m), 1.67e1.78 (4H, m), 1.87e1.92 (4H, m), 2.63 (2H, m), 2.78 (2H, t,
J ¼ 7.9 Hz), 3.11 (2H, m), 3.56 (2H, t, J ¼ 7.2 Hz), 4.00 (3H, s, CH3O),
7.23 (1H, d, J ¼ 8.6 Hz), 7.28 (1H, d, J ¼ 8.6 Hz), 7.31e7.36 (1H, m),
7.2.6. Methyl 3-hydroxy-6-{4-[(1,2,3,4-tetrahydroacridin-9-yl)
amino]butyl}pyridine-2-carboxylate (39)
Compound 39 was obtained accordingly to the general proce-
dure 3 using 36 (0.43 g, 0.87 mmol), Pd(OH)2/C (20%, 0.184 g,
0.26 mmol, 0.3 equiv), and 3 mL of MeOH. Yield 0.34 g, 96%.
7.54e7.59 (1H, m), 7.95 (1H, d, J ¼ 8 Hz), 8.05 (1H, d, J ¼ 8.6 Hz). 13
C
NMR (100 MHz, CDCl3, d): 20.87; 22.11; 23.78; 26.38; 28.60; 29.62;
31.09; 37.02; 48.61; 53.55; 110.88; 115.99; 121.66; 124.19; 125.49;
127.88; 129.65; 132.63; 139.27; 152.03; 153.45; 155.55. HRMS (ESI,
Rf ¼ 0.43 (CH2Cl2/MeOH ¼ 10/1). 1H NMR (300 MHz, CD3OD,
d):
1.67e1.74 (4H, m), 1.87e1.91 (4H, m), 2.68 (2H, t, J ¼ 5.3 Hz), 2.72
(2H, t, J ¼ 7 Hz), 2.96 (2H, t, J ¼ 5.5 Hz), 3.62 (2H, t, J ¼ 6.7 Hz), 3.96
(3H, s, CH3O), 7.23 (1H, d, J ¼ 8.6 Hz), 7.27 (2H, d, J ¼ 8.6 Hz), 7.34e
m/z): calcd. for
420.22836.
C
25H30N3O3 [M
þ
H]þ, 420.22871; found,

M. Kliachyna et al. / European Journal of Medicinal Chemistry 78 (2014) 455e467
465
7.40 (1H, m), 7.57e7.62 (1H, m), 7.74 (1H, d, J ¼ 8.4 Hz), 8.10 (1H, d,
7.3.3. Reactivation of hAChE inhibited by OPs
J ¼ 8.4 Hz). 13C NMR (100 MHz, CD3OD,
d
): 23.25; 23.87; 25.96;
Hlö-7 was from DGA maîtrise NRBC (Vert le Petit, France), HI-6
from Pharmacie Centrale des Armées (Orléans, France) and obi-
doxime was from CRSSA (Lyon, France; synthesized by Bernard
Desiré). OP-inhibited hAChE was incubated at 37 ꢃC with at least 4
concentrations of oxime in phosphate buffer (0.1 M, pH 7.4, 0.1%
BSA). The final concentration of MeOH in the incubation mix was
below 2% and had no influence on the enzyme stability. At time
intervals ranging from 1 to 10 min depending on the reactivation
28.22; 31.31; 32.84; 37.27; 53.34; 115.73; 120.15; 125.07; 125.29;
125.74; 128.52; 130.46; 130.65; 131.09; 145.48; 154.49; 154.54;
156.94; 158.65; 170.62. HRMS (ESI, m/z): calcd. for C24H28N3O3
[M þ H]þ, 406.21306; found, 406.21307.
7.2.7. 3-Hydroxy-6-{4-[(1,2,3,4-tetrahydroacridin-9-yl)amino]
butyl}pyridine-2-carbaldehyde (42)
Compound 42 was obtained from 39 accordingly to the general
procedure 4 after purification by the column chromatography
(silica gel, ethyl acetate/MeOH ¼ 8/2) with yield 54% (over 3 steps).
rate, 10-mL aliquots of each solutions containing the different con-
centrations of oxime were transferred to cuvettes containing 1 mM
acetylthiocholine in 1 mL Ellman’s buffer (phosphate 0.1 M, pH 7.4,
0.1% BSA, 0.5 mM DTNB, 25 ꢃC) for measurement of hAChE activity.
The enzyme activity in the control (uninhibited enzyme þ oxime)
remained constant during the experiment. Oximolysis was
undetectable.
The percentage of reactivated enzyme (%Ereact) was calculated as
the ratio of the recovered enzyme activity and activity in the con-
trol. The apparent reactivation rate kobs for each oxime concentra-
tion, the dissociation constant KD of inhibited enzymeeoxime
complex (EePOx) and the maximal reactivation rate constant kr,
were calculated by non-linear fit with ProFit (Quantumsoft) using
the standard oxime concentration-dependent reactivation equa-
tion derived from the following scheme:
Rf ¼ 0.21 (ethyl acetate/MeOH ¼ 7/3). 1H NMR (400 MHz, CDCl3,
d):
1.66e1.75 (2H, m), 1.78e1.89 (6H, m), 2.67 (2H, m), 2.79 (2H, t,
J ¼ 7 Hz), 3.04 (2H, m), 3.52 (2H, t, J ¼ 7 Hz), 7.20 (1H, d, J ¼ 8.6 Hz),
7.24 (1H, d, J ¼ 8.6 Hz), 7.31 (1H, td, J ¼ 6.8 Hz, J ¼ 1 Hz), 7.53 (1H, td,
J ¼ 6.8 Hz, J ¼ 1 Hz), 7.89e7.93 (2H, m), 9.98 (1H, s, CH]O). 13C NMR
(100 MHz, CDCl3, d): 22.89; 23.20; 25.01; 26.96; 31.37; 33.92; 36.98;
49.32; 116.06; 120.29; 122.94; 123.95; 126.69; 128.68; 129.87;
136.00; 147.27; 150.99; 154.29; 157.29; 158.43; 198.74. HRMS (ESI,
m/z): calcd. for C23H26N3O2 [M þ H]þ, 376.20250; found, 376.20201.
7.2.8. 2-[1-(Hydroxyimino)methyl]-6-{4-[(1,2,3,4-
tetrahydroacridin-9-yl)amino]butyl}pyridin-3-ol (12)
Compound 12 was obtained accordingly to the general proce-
dure 5 using 42 (0.090 g, 0. 24 mmol, 1 equiv), NH2OH.HCl (0.025 g,
0.36 mmol, 1.5 equiv), CH3COONa (0.029 g, 0.36 mmol, 1.5 equiv),
and 2.5 mL of ethanol. Compound 12 was purified by the column
chromatography (silica gel, CH2Cl2/MeOH ¼ 8/2) with yield 55%.
KD
kr
E ꢁ P þ Ox%E ꢁ POx
E þ POx
ƒƒƒƒ!
ꢀ
ꢁ
kr½Oxꢄ
%Ereact ¼ 100$ 1 ꢁ ek
and kobs
¼
obs$t
Rf ¼ 0.6 (ethyl acetate/MeOH ¼ 2/1). 1H NMR (400 MHz, CD3OD,
d):
KD þ ½Oxꢄ
1.80e1.83 (4H, m, CH2CH2CH2CH2), 1.90e1.95 (4H, m,
CH2CH2CH2CH2), 2.62 (2H, t, J ¼ 5.5 Hz, CH2), 2.71 (2H, t, J ¼ 6.4 Hz,
CH2), 2.98 (2H, m, CH2), 3.87 (2H, t, J ¼ 5.6 Hz, CH2), 7.04 (1H, d,
J ¼ 8.4 Hz, ArH), 7.09 (1H, d, J ¼ 8.4 Hz); 7.47e7.51 (1H, m, ArH),
7.71e7.79 (2H, m, ArH), 8.08 (1H, s, CH]N), 8.24 (1H, d, J ¼ 8.8 Hz).
Detailed reactivator concentrations used for each set of experi-
ments are given in the Supplementary Information.
7.4. Molecular modelling
13C NMR (100 MHz, CD3OD,
d): 22.04; 23.09; 25.03; 27.55; 29.84;
30.60; 37.11; 113.35; 117.54; 121.08; 125.43; 125.83; 126.12; 126.19;
133.51; 136.24; 140.59; 152.44; 152.75; 153.65; 154.12; 157.25.
HRMS (ESI, m/z): calcd. for C23H27N4O2 [M þ H]þ 391.21340; found,
391.21337.
Flexible dockings have been performed using autodock vina
[37] and by preparing the system in PyMOL (Schrödinger) using the
plug-in developed by Daniel Seeliger (http://wwwuser.gwdg.de/
wdseelig/adplugin.html). VX-hAChE was constructed from the
apo form (pdb code 4EY4) by homology to the mAChE-VX structure
(pdb code 2Y2U), keeping in the active site all the usually conserved
water molecules. Residues in the gorge (Tyr72, Asp74, Trp86,
Tyr124, Ser125, Trp286, Tyr337, Phe338, Tyr341) have been chosen
as flexible, along with the ethyl group of VX. A docking box of
60 ꢅ 60 ꢅ 60 Å was chosen, centered at the bottom of the gorge
between Tyr124 and Trp86. Ligands were built and optimized from
SMILEs string using Phenix elbow [38]. The default parameter set of
Autodock vina was used to generate 9 docking poses per molecule.
Molecular dynamics simulations were carried out using
GROMACS 4.5.6 [39] and the Amber99sb forcefield [40]. The to-
pological description of each KM molecule was built using acpype
and the general amber forcefield [41]. The hAChE-VX complex in
the conformation obtained from flexible dockings together with
crystal water molecules and the different KM molecules, was
immersed in a periodic water box of cubic shape with a minimal
7.3. Biological essays
7.3.1. IC50 measurements
Recombinant hAChE was produced and purified as previously
described (see reference: http://www.ncbi.nlm.nih.gov/pubmed/
18975951). Oximes were dissolved in MeOH to make 5- or 10-
mM stock solution. Recombinant hAChE activity was measured
spectrophotometrically (absorbance at 412 nm) in the presence of
various concentrations of oximes in 1 mL Ellman’s buffer (phos-
phate 0.1 M, pH 7.4, 0.1% BSA, 5% MeOH, 0.5 mM DTNB, 25 ꢃC).
Measurements were performed at least in duplicate for each con-
centration tested. The concentration of oxime producing 50% of
enzyme inhibition was determined by non-linear fitting using
ProFit (Quantumsoft) using the standard IC50 equation:
%
Activity ¼ 100*IC50/(IC50 þ [Ox]).
ꢀ
distance of 10 A to any edge and periodic boundary conditions. The
7.3.2. Inhibition of hAChE by OPs
box was solvated using the TIP3P solvation model and chloride and
sodium counter ions were added to neutralize the simulation sys-
tem. After energy minimization using a 500-step steepest decent
method, the system was subjected to equilibration at 1 bar for 50 ps
under the conditions of position restraints for heavy atoms. The
solvent, the counter ions, and the protein were coupled separately
to a temperature bath at 300 K. The LennardeJones interactions
were cutoff at 1.4 nm. The long-range electrostatic interactions
VX, tabun were from DGA maîtrise NRBC (Vert le Petit, France).
Paraoxon-ethyl was purchased from SigmaeAldrich. Stock solution
of VX and tabun were 5 mM in isopropanol. The inhibition of
120
mM hAChE is realized with a 5-fold excess of OPs and was
performed in tris buffer (20 mM, pH 7.4, 0.1% BSA) at 25 ꢃC. After a
20-min incubation, inhibited hAChE was desalted on PD-10 column
(GE Healthcare).

466
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ꢀ
and was kept constant using a Berendsen thermostat [42] (with a
coupling time constant of 0.1 ps). Pressure with a reference value of
1 bar was controlled by a Berendsen barostat (with a coupling time
constant of 1 ps and a compressibility of 4.5 105 bar). Full MD
simulation was performed for 5 ns at 300 K, using 2 fs timesteps. All
bond lengths were constrained using the LINCS algorithm allowing
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Acknowledgements
The ANR (Detoxneuro ANR-06-BLAN-163, ReAChE ANR-09-
BLAN-192), DGA (DGA/STTC PDH-2-NRBC-4-C-403, DGA-REI
2009-34-0023) and DTRA (HDTRA1-11-C-0047) are gratefully
acknowledged for financial support. This work has been partially
supported by INSA Rouen, Rouen University, CNRS, Labex SynOrg
(ANR-11-LABX-0029) and région Haute-Normandie (CRUNCh
network). We also thank Dr. Patrick Wehrung, Pascale Buisine and
Cyril Antheaume (UdS, Faculty of Pharmacy, Service Commun
d’Analyse) for mass and NMR analyses.
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(b) G. Mercey, J. Renou, T. Verdelet, M. Kliachyna, R. Baati, E. Gillon,
M. Arboleas, M. Loiodice, F. Nachon, L. Jean, P.-Y. Renard, Phenyl-
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