Design, synthesis and evaluation of new α-nucleophiles for the hydrolysis of organophosphorus nerve agents: Application to the reactivation of phosphorylated acetylcholinesterase

Article abstract of DOI:10.1016/j.tet.2011.05.130

A series of new α-nucleophiles including oximes and amidoximes have been synthesized, and their ability to efficiently and selectively cleave the P-S bond of organophosphorus nerve agents has been evaluated. The relationship between the chemical structure

Full text of DOI:10.1016/j.tet.2011.05.130

Tetrahedron 67 (2011) 6352e6361
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design, synthesis and evaluation of new
a
-nucleophiles for the hydrolysis
of organophosphorus nerve agents: application to the reactivation
of phosphorylated acetylcholinesterase
a
a
b,c
ꢀ
ꢀ a
Geraldine Saint-Andre , Maria Kliachyna , Sanjeevarao Kodepelly , Ludivine Louise-Leriche
,
,
c
,
e
,
d
a,
a
Emilie Gillon d, Pierre-Yves Renard b
, Florian Nachon , Rachid Baati , Alain Wagner
Laboratoire de Chimie des Systemes Fonctionnels CNRS/UMR7199, Faculte de Pharmacie, Universite de Strasbourg, BP 24, 67401 Illkirch, France
Equipe de Chimie Bio-Organique, COBRA-CNRS UMR 6014 & FR 3038, rue Tesniere, 76130 Mont-Saint-Aignan, France
Universite de Rouen, IRCOF, rue Tesniere, 76130 Mont Saint Aignan, France
*
*
a
ꢁ
ꢀ
ꢀ
b
c
ꢁ
ꢀ
ꢁ
d
ꢀ
ꢀ
ꢀ
ꢀ
Cellule enzymologie, Departement de Toxicologie, Institut de Recherche Biomedicale des Armees, 24, Avenue des Maquis du Gresivaudan, BP87, 38702 La Tronche, France
e Institut Universitaire de France, 103, Boulevard Saint Michel, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 April 2011
Received in revised form 23 May 2011
Accepted 31 May 2011
Available online 13 June 2011
A series of new
ability to efficiently and selectively cleave the PeS bond of organophosphorus nerve agents has been
evaluated. The relationship between the chemical structure of the -nucleophiles and their reactivity
towards PhX hydrolysis is reported. A significant effect induced by an ortho-hydroxyl group of aryl- and
pyridyl-oximes, amidoximes on their organo-phosphono-thioase reactivity was discovered. The evalu-
a-nucleophiles including oximes and amidoximes have been synthesized, and their
a
ation of the initial rates of PhX hydrolysis reaction in the presence of
a-nucleophiles allowed the
Keywords:
discovery of new uncharged molecules with increased reactivity compared to positively charged 2-
pralidoxime used as antidote. The mechanism of their action was studied in details by kinetic analysis,
HPLC and LCeMS methods. The most promising structures were then considered as potent in vitro re-
activators of phosphylated acetylcholinesterase (AChE), which was confirmed by the preliminary results
of AChE reactivation initially inhibited by VX.
a
-Nucleophiles
ortho-Hydroxyl effect
Hydrolysis
Organophosphorus nerve agents
Organo-phosphono-thioase activity
Reactivation of acetylcholine esterase
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
treatments of the OP poisoning as well as prevention of their tox-
icity remains a challenging problem.
Chemical warfare agents (CWA) (sarin, soman, cyclosarin, tabun,
methylphosphonothioate VX) as well as insecticides (paraoxon,
parathion, tetraethyl pyrophosphate (TEPP)) are extremely toxic
organophosphorus compounds (Fig. 1). Their mechanism of action
consists of the irreversible inhibition of acetylcholinesterase
(AChE).1 Being phosphylating agents, these compounds react with
a serine hydroxyl group in the active site of AChE, which leads to
the irreversible inhibition. This event results in the accumulation of
neurotransmitter acetylcholine at nerve synapses, which leads to
nervous and respiratory failure and eventually can kill in minutes.2
Due to the easy access to CWA and the similarity between their
chemical precursors and commonly used pest-control agents, any
efforts to control the proliferation of the chemical weapons were
unsuccessful.3 Therefore, the development of effective organo-
phosphorus nerve agent (OPNAs) decontaminants and medical
a
-Nucleophiles including H2O2, activated hydrogen peroxides,4
oxidative chlorides5 and peracids6 are the most commonly used
decontaminants of organophosphorus nerve agents. ortho-Iodoso-
benzoates and their derivatives have also been shown to effectively
hydrolyze organophosphorus compounds, such as sarin, soman, ta-
bun and paraoxon (but not VX).7 An interesting approach consists in
the use of inorganic substances for OP agents cleavage, for example,
alumina supported fluoride reagents8 and nanosized metal oxides
particles.9 Another approach to the destruction of chemical warfare
agents and their poisoning treatment involves the use of enzymes,
such as organophosphate hydrolases,10 PTEephosphotriesterase,11
laccase12 and chloroperoxidase (CPO).13 New concept towards the
safe organophosphorus chemical weapons neutralization consists of
the use of catalytic antibodies,14 which have been found to hydrolyze
VX and PhX.15 However, despite all the existing methods mentioned
above the development of more effective tools for the detoxification
of dangerous V-type OP nerve agents remains a challenging task.
Between all the CWA methylphosphonothioate VX exhibits
* Corresponding authors. Fax: þ33 235 52 29 59 (P. -Y. R), þ33 3368 854 301
(R.B.); e-mail addresses: pierre-yves.renard@univ-rouen.fr (P.-Y. Renard), baati@
bioorga.u-strasbg.fr (R. Baati).
the highest toxicity (LD50¼8
m m
g kgꢀ1 (iv) and 28 g kgꢀ1 (per-
cutaneous, rabbit)).1 Moreover, VX is the most persistent
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.05.130

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G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
6353
O
P
O
O
P
O
O
P
O
O
P
N
O
P
F
F
N
F
O
C N
O
S
R
1
3
2
4
5a: VX R = CH3
Cyclosarin
Sarin
Soman
5b
Tabun
: PhX R = Ph
O
S
O
P
O
P
O
P
O
NO2
O
P
O
NO2
O
O
O
O
O
O
O
7
8
6
Paraoxon
Parathion
Fig. 1. Structures of organophosphorus CWA and insecticides.
TEPP
organophosphorus agent due to its low volability and Henry’s law
constants.16 Additionally, this compound is the most resistant to
water hydrolysis,17 which can be explained by its low hydro-
solubilisation and the presence of a quite stable PeS bond. On the
other hand, when VX hydrolysis is performed in neutral conditions
the reaction is quite selective and leads to relatively nontoxic
phosphonic acid 9,18 whereas in basic media, besides the main
hydrolytic pathway A, pathway B competes resulting in formation
of exceedingly toxic phosphothioate 11 (Scheme 1).19 Therefore, the
development of decontaminants able to selectively cleave the PeS
bond of V-type of OPN agents is of paramount importance.
2. Results and discussions
2.1. Screening of the
a
-nucleophiles in PhX hydrolysis
Among the known
a-nucleophiles, oximes were proved to be
effective decontaminants of OPNAs.22 The computational studies of
the mechanism of the reaction of oximes with phosphonates23
showed no possible formation of the phosphorane intermediate,
which is important for the selective hydrolysis of V-type nerve
agents. Moreover, oximes were shown to reactivate the phosphy-
lated AChE.24 As we showed recently through the use of a
Path A
O
O
OH
HS
+
P
N
neutral conditions
O
P
R
R
O
S
N
10
9
Path B
5a
: VX R = CH3
O
P
R
+
S
HO
OH
N
5b: PhX R = Ph
basic conditions
11
Scheme 1. Hydrolytic pathways of VX and PhX in different conditions.
It is interesting tonote, that phenylphosphonothioate 5b (PhX),20
which is the aromatic derivative of VX 5a, exhibits the same hy-
drolysis profile as VX, while its toxicity is lower and the inhibition of
AChE in vitro is pseudo-irreversible.15 For safety and regulatory
reasons21 we decided to study the hydrolysis of less toxic PhX.
preliminary high-throughput screening assay, aryl- and pyridyl-
amidoximes, and hydroxamic acids also exhibited quite promising
organo-phosphono-thioase activity.25
In order to investigate the influence of the
a-nucleophiles
structure on their hydrolytic activity, libraries of known and orig-
inal molecules, such as oximes, amidoximes and hydroxamic acids
The present work is focused on the development of new a-nu-
cleophiles able at performing two complementary reactions: (1)
the selective PeS bond cleavage of V-type organophosphorus nerve
agents (OPNAs), to be used as decontaminants and (2) the reac-
tivation of AChE inhibited by VX, to be used as curative treatment.
In this article we describe the study of the relationship between the
were evaluated in the hydrolysis of PhX. The screening test of the a-
nucleophiles efficacy for this reaction was performed by means of
HPLC monitoring the consumption of PhX 5b during the hydrolysis.
For all evaluated nucleophiles the reaction conditions were the
following: 5 equiv of hydrolyzing agent were used in the presence
of 1 equiv of PhX 5b in 0.1 N Tris/HCl buffer at pH¼8.5 and at
20e22 ꢁC. In all the cases the reactions were performed during 2 h
(Scheme 2).
structure of the
a-nucleophiles and their organo-phosphono-
thioase activity, as well as our investigations on the mechanism
of their reaction with PhX.
O
pH = 8.5
O
P
R
O
S
O
OH
HS
+
nucleophile
(5eq)
P
Ph
N
+
N
Tris.HCl
buffer
9
10
5b: PhX
(
1 eq)
Scheme 2. Screening test of the a-nucleophiles in PhX hydrolysis.

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6354
G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
Table 1 (continued)
2.1.1. Aryl and pyridinaldoximes. 2-Pralidoxime 12 (2-PAM), which
is known as antidote for organophosphorus pesticides was chosen
as the reference compound.26 Compound 12 hydrolyzes quantita-
tively PhX in our reaction conditions, demonstrating the high re-
activity of positively charged pyridinium oxime (Table 1).
Structure
PhX hydrolyzed, %
55
NOH
OH
27
HO
HO
Table 1
NOH
Activity of benzaldoximes in PhX hydrolysis reaction
OH
Structure
PhX hydrolyzed, %
100
28
80
OH
12
N
N
OH
OH
Cl
OH
29
30
84
22
N
N
N
13
14
11
60
N
N
OH
OH
OH
OH
N
OH
OH
N
15
16
3
2
O
For uncharged ortho monosubstituted aryl oximes 13e22 bear-
ing different electron-donating and electron-withdrawing groups,
it was found that the electronic effects did not impact significantly
the hydrolytic activity. For example, benzaldoximes 15, 16 and 17
substituted in ortho-position with methoxy, amino and dimethy-
lamino groups (donors by mesomeric effect) showed slightly lower
hydrolytic activity towards PhX than nonsubstituted oxime 13
(Table 1). Aryl oximes 18 and 19 bearing a nitro or fluoro electron-
withdrawing group in ortho-position (Table 1) as well as oxime 21
(Table 1) exhibited almost similar hydrolytic activity to oxime 13.
Very interestingly, benzaldoximes 14 and 20 (Table 1) having
a hydroxyl or a carboxyl group in ortho-position showed enhanced
activity towards hydrolysis of PhX as compared to 13, which can be
explained by the intramolecular hydrogen bonding between hy-
droxyl group and nitrogen atom of oxime.27 This hydrogen bonding
was shown by Shimizu to decrease the pKa of oximes bearing ortho-
hydroxyl group in comparison with nonsubstituted ones (for
example, for 14 pKa¼9.3 compared to 13 with pKa¼10.9).28 It is
suggested that lower pKa results in the increase of the oximate
concentration leading to the observed enhanced hydrolytic activity.
This fact is in accordance with PhX hydrolysis data obtained for
compounds 22 and 23 bearing one or two hydroxyl group in meta
position to the oxime function, and whose efficacies are in the same
order of magnitude as nonsubstituted oxime 13 (Table 1). Identi-
cally, preventing the H-bonding by the substitution the ortho-hy-
droxyl functionality by a methoxy group in compound 15, leads, as
could be expected, to the inactivation of the nucleophile towards
the hydrolysis of PhX (Table 1). The reactivity of other di- and tri-
substituted ortho-hydroxyl aryl oxime was then envisioned. Com-
pound 24 possessing two hydroxyl groups in ortho-position to the
oxime function shows the same activity as nucleophile 14 bearing
one hydroxyl group in the ortho-position (Table 1), this result
suggests that the second hydroxyl function does not influence the
oxime pKa. Other ortho-hydroxyloximes 25, 26 and 27 substituted
with additional hydroxyl group in position 3, 4 and 5, respectively,
exhibit quite comparable activity as oxime 14 (Table 1). Only in the
case of compound 28 (Table 1) the significant increase of hydrolytic
activity in comparison with oxime 14 was observed. This increased
hydrolytic reactivity might be ascribed to the additional electronic
donatingeffectof thethird OH group inpara positioncompared to24
and 26. Very interestingly, the same effect of ortho-hydroxyl group
was also observed for uncharged pyridinealdoxime derivatives.
Indeed, pyridinealdoxime 29 bearing OH function in ortho-position
N
NH2
N
OH
17
18
3
N(Me)2
OH
N
15
NO2
OH
OH
N
F
19
20
5
N
42
CO2H
OH
N
21
22
7
B(OH)2
OH
N
13
OH
NOH
23
11
HO
OH
NOH
OH
HO
24
25
61
73
NOH
OH
OH
NOH
OH
26
41
OH

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G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
6355
exhibited significantly higher hydrolytic activity than non-
substituted compound 30 (Table 1). It is interesting to note, that the
pyridine ring contributes to decrease the oxime pKa function of
compounds 29 (pKa¼8.2ꢂ0.09, see ESD) and 30 (pKa¼9.85ꢂ0.05),29
leading to increased reactivities compared to aryl oximes 14 and 13,
respectively. Having identified the positive ortho-hydroxyl effect of
aryl- and pyridylbenzaldoximes on their hydrolytic activity towards
PhX, wedecidednext tostudy theinfluenceof thenatureof the other
substituents in the aromatic ring of ortho-hydroxybenzaldoximes.
Accordingly, ortho-hydroxylbenzaldoximes 31e39 containing
electron-donating and electron-withdrawing groups were studied
in order to evaluate their reactivity. It is assumed that the presence
of electron-withdrawing groups in ortho- and para-positions to
the hydroxyl function decreases the pKa of the phenol facilitating
H-bonding with the free electron pair of the nitrogen atom. These
events result in increased oximate concentration leading to in-
creased hydrolytic activities.
14. It is interesting to note that oximes bearing halogen atom in
para-position position to the hydroxyl group (31 and 33) showed
higher organo-phosphono-thioase activity than ortho-substituted
ones 32 and 34. The introduction of NO2 group in ortho- or para-
position to hydroxyl function for compounds 36 (pKa¼6.27ꢂ0.02,
see ESD) and 37 (pKa¼6.7)29 is supposed to significantly decrease
their pKa. Therefore, under the standard experimental conditions
(pH¼8.5) compounds 36 and 37 exist almost totally in the pheno-
late form. This situation is not optimum for the formation of hy-
drogen bond with the nitrogen atom of the oxime and,
consequently, these molecules exhibit lower hydrolytic activity
towards PhX (Table 2). In contrast, benzaldoximes 38 and 39
substituted in ortho- or para-position to the hydroxyl function with
electron-donating methyl and methoxy groups correspondingly
exhibited similar activities as nonsubstituted ortho-hydrox-
ylbenzaldoxime 14 (Table 2).
For example, benzaldoximes 31e35 (Table 2) substituted with
halogen atoms showed slightly higher (34 and 35) to better (31 and
33) hydrolytic activities than nonsubstituted ortho-hydroxyloxime
2.1.2. Aryl and pyridinamidoximes. As we reported recently, benza-
lamidoximes were found to have a quite promising organo-phos-
phono-thioase activity towards PhX.25 Inspired by our results
obtained for aryl- and pyridylaldoximes series we decided to eval-
uatetheinfluenceofortho-hydroxyl group andthepyridineringonto
the hydrolytic activity of amidoximes. We discovered that, as ob-
served for arylaldoxime 14 (Table 1), amidoxime 41 (Table 3,
pKa¼8.88ꢂ0.6, see ESD) bearing hydroxyl group in ortho-position to
the nucleophilic function, has a significantly higher hydrolytic
Table 2
Activity of ortho-hydroxylbenzaldoximes in PhX hydrolysis
Structure
PhX hydrolyzed, %
83
NOH
OH
31
Table 3
F
Activity of amidoximes in PhX hydrolysis
NOH
Structure
PhX hydrolyzed, %
25
OH
32
33
60
77
N
40
F
OH
OH
OH
NH2
NOH
OH
N
OH
41
42
43
91
100
100
Cl
NH2
NOH
OH
N
OH
34
35
66
63
Cl
OH NH2
NOH
OH
OH
Cl
OH
N
OH
OH
Cl
NH2
NOH
OH
N
N
36
37
22
16
44
45
46
25
100
9
NH2
NO2
NOH
OH
OH
N
N
OH
NH2
O2N
OH
NOH
N
OH
N
N
OH
38
39
55
60
I
NH2
Me
OMe
N
NOH
47
29
OH
OH
(continued on next page)
NH2
MeO

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G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
6356
Table 3 (continued )
The comparison of PhX hydrolysis results obtained for aldox-
imes and amidoximes species allowed us to conclude that in all the
cases amidoximes (40, 41, 42 and 43) exhibit higher activity than
aldoximes (13, 14, 24 and 25) with a similar structure.
Structure
PhX hydrolyzed, %
15
N
N
48
49
HO
N
OH
2.1.3. Hydroxamic acids. Finally, the reactivity of hydroxamic acid
was also investigated. All the hydroxamic acids evaluated 50e54
showed good to excellent results for the hydrolysis of PhX. In
NH2
NH2
OH
N
contrast to the
a-nucleophiles mentioned before (oximes and
18
NH2
amidoximes) the presence of hydroxyl group in ortho-position to
the hydroxamic function does not affect significantly the reactivity
towards PhX hydrolysis, however an increase of reactivity has been
observed for pyridyl hydroxamic acids. For example, compounds 50
and 51 exhibit a comparable hydrolytic activity for PhX (Table 4).
However, for pyridyl derivative 52 the presence of the hydroxyl
group in ortho-position to the hydroxamic function increases sig-
nificantly the percentage of hydrolyzed PhX in comparison with
compound 53 (Table 4). For compounds 51 and 52, the activation
mode of the nucleophilic function by the hydroxyl group is not
completely understood yet and would need further studies.
N
HO
activity towards PhX than nonsubstituted compound 40 (Table 3).
Identically, ortho-hydroxylsubstituted amidoximes 42 and 43 (Table
3) bearing an additional hydroxyl group showed a dramatic increase
of theorgano-phosphono-thioaseactivityascomparedto40. Indeed,
due to intramolecular H-bonding between the amidoxime function
and the OH group, a quantitative hydrolysis of PhX took place within
2 h of reaction for these compounds. At this stage of our study we
have identified new uncharged aryl derivatives capable to hydrolyze
efficiently and quantitatively PhX as does pyridinium 2-pralidoxime
12, a known antidote. In connectionwith these results it appears that
Table 4
Activity of hydroxamic acids in PhX hydrolysis
a
-hydroxybenzalamidoximes have superior reactivity towards PhX
Structure
PhX hydrolyzed, %
85
than -hydroxybenzaldoximes. We further discovered that -hy-
a
a
droxyl effect is also effective for pyridylamidoxime derivative 45
(Table 3, 100% PhX hydrolysis) compared to 44 (Table 3, 25% PhX
hydrolysis). Similarly to oxime functionality it is suggested that the
increased reactivity observed for hydroxy aryl- and pyridyl-
amidoxime is ascribed to the existence of an intramolecular hydro-
gen bonding between the hydroxyl group and the nitrogen atom of
amidoxime group, which results in decrease of pKa (for example, for
45 pKa¼7.96ꢂ0.1 compared to 44 with pKa¼12.06ꢂ0.03, see ESD).
These hypotheses are in accordance with results obtained for com-
pound 46, which showed low activity towards PhX hydrolysis (Table
3). X-ray analysis of 46 revealed that the compound exists in zwit-
terion form in which the hydroxyl group is totally deprotonated,
preventing any activation of the nucleophilic function by intra-
molecular H-bonding (see X-ray crystal structure in Fig. 2, ESD).
H
N
50
OH
OH
O
OH
H
51
52
83
N
O
OH
H
100
N
N
N
OH
OH
O
H
N
53
54
75
O
N
H
N
H
N
100
HO
OH
O
O
2.1.4. Kinetics and mechanistic insights. The screening of oximes,
amidoximes and hydroxamic acid towards selective PhX hydrolysis
allowed us to identify new structurally simple uncharged mole-
cules with high organo-phosphono-thioase activity. These mole-
cules are able to quantitatively and selectively hydrolyze PhX in 2 h
at room temperature, as the well known pralidoxime. We decided
next to perform kinetic studies with the most reactive molecules
identified.
The initial rates of PhX hydrolysis reaction for nucleophiles 12,
28, 29, 41, 43, 45, 52 and 54 selected by the preliminary screening
were evaluated (see ESD). It was found, that amidoxime 43 shows
the highest initial rate, molecules 41 and 52 have almost the same
values as the reference compound 2-pralidoxime 12 (Table 5). It
should be mentioned, that amidoxime 45 exhibited such a high PhX
hydrolytic activity, that it was not possible to evaluate the initial
rate for it in the standard conditions of the test (1.0 mM PhX and
5.0 mM nucleophile in mixture acetonitrile/0.1 M Tris/HCl buffer
11/89 at pH 8.5 and 25 ꢁC).
Fig. 2. X-ray structure of amidoxime 46.
The low reactivity of amidoxime 47 (29% hydrolysis of PhX)
bearing an ortho-methoxy group is also in agreement with our
findings (Table 3). Compound 48 possessing two amidoxime
functions and 49 bearing both amidoxime and oxime functions
showed low activity, which proves the absence of any additive ef-
fect (Table 3). It was found that in contrast to oximes the pyridine
ring does not affect significantly the organo-phosphono-thioase
activity of amidoxime derivatives. For example, pyridylamidoxime
44 (Table 3) showed the same reactivity as arylamidoxime 40 (Table
3), but in the case of compound 45 the replacement of benzene ring
with pyridine leads to the increase of the hydrolytic activity in
comparison with 41.

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G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
6357
Table 5
Initial rates of PhX hydrolysis
profile of pralidoxime 12 is more or less linear, the kinetic profile of
amidoxime 45 shows a plateau reminiscent of a possible inhibition
(see ESD). It was thus supposed, that, contrary to pralidoxime 12 for
which the initial nucleophilic addition rate yielding the phos-
phoryloxime is predominant, reaction of amidoxime 45 with PhX
includes two stages of kinetic significance: (i) formation of the in-
termediate phosphorylamidoxime and (ii) its hydrolysis (Scheme
3). Inhibition of nucleophile 45, which was observed at 40 ꢁC
allowed us to assume that the rate of the intermediate formation
rises more quickly than the rate of its hydrolysis. The decrease of
the pH from 8.5 to 7.55 results in significant decrease of the initial
rate of the reaction (Table 6). The kinetic study of PhX hydrolysis by
amidoxime 45 also allowed us to conclude that this compound is
not really catalytic, since there is no turnover.
In order to explain the results of the kinetic analysis obtained for
amidoxime 45 the reaction of PhX with pyridinaldoxime 30 was
studied by HPLC and LCeMS. The analysis of reaction mixture by
LCeMS allowed to identify four peaks with [MþH]þ 123, 187, 330
and 291, which correspond to pyridinaldoxime 30, phosphonic acid
9, PhX 5b, and the intermediate phosphoryloxime 55, respectively
(Scheme 3).
The study of the reaction by HPLC proved the formation of the
intermediate (phosphoryloxime 55), which is quite stable and hy-
drolyzes slowly to phosphonic acid 9. Thus, the process consists of
two stages, which have different rates. In order to evaluate the
stability of the intermediate the reaction was studied at different
temperatures. It was found, that stability of phosphoryloxime de-
creases with elevation of the temperature. For example, at 20 ꢁC
phosphonic acid started to form after 8 h of reaction, and even at
40 ꢁC the reaction was quite slow (5 h), which proves that pyr-
idinaldoxime 30 does not exhibit any catalytic activity.
The reaction of amidoxime 45 with PhX (45/PhX¼0.4/1) was
further studied by LCeMS and 31P NMR. Thus, in the LCeMS
spectrum of the reaction mixture were detected the following
compounds: PhX 5b, phosphonic acid 9, aminothiol 10, Tris and
intermediate phosphorylamidoxime 56. The presence in LCeMS
spectrum of peak with [Mþ1]þ¼136, which was assigned to iso-
xasole structure 57 allowed to assume, that phosphorylamidoxime
56 in basic conditions undergoes intramolecular cyclization
(Scheme 4). The results obtained are in good correlation with the
31P NMR data. Thus, in the 31P NMR spectrum of the reaction
mixture peak at 49.1 corresponds to PhX, peak at 29.8 to phos-
phonic acid 9 and 15.1 to phosphorylamidoxime 56.
Entry
1
Structure
Initial rate,
30
mmol/min
NOH
N
12
28
Cl
NOH
OH
HO
2
22
OH
OH
NOH
3
4
19
29
N
OH
N
31.3
OH
OH
OH
NH2
41
43
OH
OH
N
5
6
38.1
28.6
NH2
OH
H
N
N
O
52
H
N
H
N
7
8
14.7
HO
N
OH
45
54
O
O
OH
>40a
N
N
OH
NH2
Reagents and conditions: 1.0 mM PhX and 5.0 mM nucleophile in mixture aceto-
nitrile/0.1 M Tris HCl buffer 11/89 at pH 8.5 and 25ꢁ C.
a
To high to be accurately measured.
In order to understand the origin of this high hydrolytic activity,
the kinetic profile of compound 45 was studied at different PhX/
nucleophile ratios, temperature, pH and compared with reference
compound pralidoxime 12 (see ESD). It was then found, that initial
rate for amidoxime 45 is higher than for pralidoxime 12. The dif-
ferent types of curves obtained for nucleophiles 45 and 12 evidence
the different mechanisms of PhX hydrolysis. While the kinetic
The cyclization of phosphorylamidoxime 56 explains the ab-
sence of the catalytic activity of amidoxime 45. This fact is in ac-
cordance with results obtained by Rebeck, which showed, that in
basic conditions
b-hydroxy oxime reacts with OP nerve agent with
similar cyclization of the OP ester to the isoxasole,30 and also with
k1
N
N
N
+
O
S
N
OH
+
HS
O
P O
O
N
P
O
N
10
55 intermediate
k2
30
5b: PhX
H2O
+
Nu
H
HO
O
P
O
9
Scheme 3.

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G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
6358
Table 6
This means that much higher concentrations of compounds 29 and
Initial rates of PhX hydrolysis mmol/min
45 are required to reach the desirable reactivation of enzyme. Nev-
ertheless, the results obtained for the reactivation of VX poisoned
AChE are quite promising regarding that Kd of the nucleophiles can
be further improved by structural modifications increasing the af-
finity of the nucleophiles to the inhibited enzyme.
PhX/nucleophile ratio pH 8.5, 25 ꢁC
pH 8.5, 40 ꢁC
pH 7.55, 25 ꢁC
1/0.2
1/1
1/0.4 1/0.2 1/0.4 1/0.2
OH
N
37.5
8
2
4.5
0.7
10.9
5.3
5.2
2.7
2.1
0.2
N
OH
NH2
3. Conclusions
45
NOH
In conclusion, the new uncharged nucleophiles, such as oximes
and amidoximes show a comparable and in some cases increased
reactivity towards PhX hydrolysisin comparisonwith 2-pralidoxime
have been described. Among them 3-hydroxy-pyridylamidoxime
was found to be the most reactive towards PhX hydrolysis. The
N
4.4
Cl
12
OH
O
O
P
CH3CN/Tris·HCl buffer, pH 8.5, 25 C
O
P
N
+
N
N
N
O
O
HO
O
NH2
NH2
9
56
57
Scheme 4. Intramolecular cyclization of phosphorylamidoxime.
the results we observed with akin
b
-hydroxy oximes.24p,25 It should
kinetic analysis showed that 3-hydroxy-pyridyl-amidoxime reacts
with PhX much faster than 2-pralidoxime but due to the isoxasole
derivative formation this compound does not show any catalytic
activity. The preliminary results of the VX poisoned AChE reac-
be mentioned, that amidoxime 45 due to the cyclization of the
intermediate 56 possess the privilege of preventing the recapture
phenomenon in comparison with oximes known antidotes against
OP poisoning. Oximes or amidoximes that do not possess an OH
group in the beta position, after reactivation of AChE form an ester
with organophosphorus agents, which in its turn exhibit powerful
AChE inhibition.31
tivation allowed to find very promising neutral a-nucleophiles with
high reactivation rate constant. The synthesis and evaluation of the
reactivators with improved affinity towards poisoned enzyme is in
progress in our laboratories.
2.2. Poisoned AChE reactivation
4. Experimental section
Considering that pralidoxime, HI-6 and other pyridiniumaldox-
imes are the best known poisoned AChE reactivators to date, we
wondered whether the synthesized nucleophiles could also be used
as poisoned AChE reactivators, which would make them valuable
dual nerve agents poisoning countermeasures: (1) through the hy-
drolysis of the nerve agent and (2) as reactivator of the poisoned
4.1. General materials and methods
The chemicals used in the synthesis were purchased from
Aldrich, Acros, Alfa-Aesar or ABCR and used without further puri-
fication. The solution of PhX in CH3CN (20 mg/mL) and the solu-
tions of VX in isopropanol (1 mM and 5 mM) were stored at ꢀ20 ꢁC.
PhX,20 phosphonic acid 9,32 17,33 21,34 23,35 29,25 47,36 5225 and
5325 were prepared as previously described. The following solvents
were distilled from the indicated drying agents: CH2Cl2 (CaH2), THF
AChE. Therefore the ability of the most active a-nucleophiles 25, 28,
29, 41 and 45 to reactivate the human AChE poisoned with VX was
studied by spectrophotometry using Ellman’s reagent and com-
pared with pralidoxime 12. It was found that compounds 25, 28 and
41 do not showsufficient in vitro reactivation of AChE poisoned with
VX (Table 7, entries 4e6). Whereas oxime 29 and amidoxime 45
(Table 7, entries 2 and 3) exhibited high reactivation, especially 29,
which reactivation rate constant (kr) was higher than the reference
compound pralidoxime 12. However, these nucleophiles exhibited
much lower affinity to the inhibited enzyme in comparison with 12,
this low affinity being due to their high dissociation constants (Kd).
ꢂ
(Na), CH3CN (CaH2) or dried over 4 A molecular sieves. Thin layer
chromatographies (TLC) were carried out using Merck silica gel
plates 0.25 nm (Kieselgel 60 F254, 40e60 mm, 230e400 mesh
ASTM). TLC plates were visualized using a combination of UV lamps
(256, 365 nm), followed by detection with p-anisaldehyde, ceric
ammonium molybdate or ninhydrin. Flash chromatographies were
performed using silica gel Merck Kieselgel (40e60 mm, 230e
400 mesh ASTM) according to a standard procedure. All aqueous
buffers were prepared by using water purified with a Milli-Q sys-
tem (purified to 18.2 MW cmꢀ1).
Tris/HCl buffer 0.1 N, 0.15 N NaCl, pH 8.5: [Tris]¼0.1 N, [NaCl]¼
0.15 N, adjusted to pH 8.5 with concentrated HCl solution.
Tris/HCl buffer 0.1 N, 0.15 N NaCl, pH 7.55: [Tris]¼0.1 N, [NaCl]¼
0.15 N, adjusted to pH 7.5 with concentrated HCl solution.
Tris/HCl buffer 0.05, pH 8: [Tris]¼0.05 N, [NaCl]¼0.15 N, ad-
justed to pH 8 with concentrated HCl solution.
Table 7
Specific reactivation rate constant at a given concentration of reactivator(kobs),
reactivation rate constant (kr) and dissociation constant (Kd)a
Compound
Entry
kobs, minꢀ1
kr, minꢀ1
Kd, m
Mꢀ1
12
29
45
25
28
41
1
2
3
4
5
0.080 ꢂ 0.005 at 1 mM
0.03 ꢂ 0.01 at 1 mM
0.010 ꢂ 0.002 at 10 mM
NA
0.06 ꢂ 0.01
0.5 ꢂ 0.1
215 ꢂ 75
30 ꢂ 10ꢃ103
30 ꢂ 20ꢃ103
0.08 ꢂ 0.1
0.005 ꢂ 0.001 at 10 mM
Phosphate buffer 0.1 N pH 7: [NaHPO4]¼0.1 N, [Na2PO4]¼0.1 N.
6
0.004 ꢂ 0.002 at 10 mM
1H NMR, 13C NMR and 31P NMR spectra were recorded on
!
kr
a
kobs
¼
, Rdreactivator concentration.
Brucker DPX200, DPX300, DPX400 and DPX500. Chemical shifts (
are quoted in parts per million and are calibrated relative to solvent
d)
Kd
R
1
þ

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G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
6359
residual peaks. Multiplicities are reported as follows: s¼singlet,
(300 MHz, CD3OD):
d
¼6.71 (t, 1H, J¼8 Hz), 6.80 (dd, J¼8, 1.5 Hz),
d¼doublet, t¼triplet, m¼multiplet, br¼broad. Coupling constant
are reported in Hertz. Mass spectra were recorded on an MS/MS
high resolution Micromass ZABSpecTOF spectrometry. Infrared
spectra of samples were measured using a Nicolet 380 FI IR Spec-
trometer from Thermo Electron Corporation as a CH3Cl solution or
solid on a diamond plate. UV-visible spectra were obtained on
KRONTON Instruments UVIKON 943 and UVIKON 933. Absorption
cells (Hellma 100eOS 10 mm).
7.00 ppm (dd, 1H, J¼8, 1.5 Hz). 13C NMR (75 MHz, CD3OD):
d
¼116.23, 117.05, 117.17, 119.53, 146.85, 146.97, 155.46 ppm. IR
(cmꢀ1): 3354, 2487, 1622, 1583, 1462. MS (ESI): m/z: calcd for [M]:
168.15; found: 169 [MþH]þ.
4.2.7. 2-Hydroxypyridylamidoxime (45). Obtained with yield 82%
as white needle like crystals after filtration. 1H NMR (400 MHz,
DMSO-d6):
d
¼6.36 (br s, 2H), 7.29e7.34 (m, 2H, J¼3.8 Hz), 8.11 (dd,
1H, J¼3.8, 2.1 Hz), 10.25 (s, 1H), 12.06 (s, 1H). 13C NMR (100 MHz,
4.2. General experimental procedure for oximes and
amidoximes synthesis
CD3OD):
d¼125.11, 126.17, 133.88, 140.39, 155.03, 155.67 ppm. IR
(cmꢀ1): 3428, 2849, 1653, 1594, 1567, 1390, 939. MS (ESI): m/z:
calcd for [M]: 153.14; found 154 [MþH]þ.
Hydroxylamine hydrochloride (4 mmol) and Na2CO3 (2 mmol)
were mixed together in water (3 mL) until the solid dissolves and
the gas emission stops. This aqueous solution was added to the
corresponding aldehyde or nitrile (1 mmol) in absolute ethanol
(0.5 mL).The mixture was heated at 70 ꢁC for 2e24 h. The reaction
mixture was then allowed to cool down to room temperature. If the
compound precipitated, the solid was filtered off, washed with cold
water and dried under vacuum. Otherwise the reaction mixture was
extracted with ethyl acetate and dried over Na2SO4. After removal of
the solvent the compound was purified by column chromatography
(cyclohexane/ethylacetate)toaffordthedesiredoximeoramidoxime.
4.2.8. N-Methyl-3-hydroxy-2-pyridylamidoxime iodide (46). N-
methyl-2-cyano-3-hydroxypyridine iodide (221 mg, 0.84 mmol)
and hydroxylamine (50 wt % in H2O, 5 equiv) in methanol were
refluxed for 18 h. The reaction mixture was allowed to cool down at
room temperature and was extracted with ethyl acetate, dried over
Na2SO4. After purification by column chromatography (silica gel,
DCM/MeOH¼90/10) pale orange compound 46 was obtained with
yield 45%.
1H NMR (300 MHz, DMSO-d6):
d
¼3.95 (s, 3H), 5.90 (br s, 2H),
6.95 (d, 1H, J¼9 Hz), 7.20e7.25 (dd, 1H, J¼9, 5 Hz), 7.34 (d, 1H,
J¼5 Hz), 9.66 (br s, 1H). 13C NMR (125 MHz, DMSO-d6):
¼45.57,
d
4.2.1. 2,6-Dihydroxybenzaldoxime (24). Obtained with 45% yield
from 2,6-dihydroxy-benzaldehyde37 using the general procedure
and purification by flash chromatography (cyclohexane/ethyl ace-
126.29, 133.85, 136.06, 146.23 ppm. MS (ESI): m/z: calcd for [M]:
167.17; found 168 [M]þ.
The final atomic coordinates and crystallographic data for
molecule 46 have been deposited at the Cambridge Crystallo-
graphic Data Centre (12 Union Road, CB2 1EZ, UK, fax: þ44 1223
336033; e-mail: deposit@ccdc.cam.ac.uk) and are available on re-
quest quoting the deposition number CCDC 818187.
tate 30/70). 1H NMR (300 MHz, CD3OD):
d
¼6.32 (d, 2H, J¼8 Hz),
7.01 (t, 1H; J¼8 Hz), 8.56 (s, 1H). 13C NMR (75 MHz, CD3OD):
d
¼106.62, 107.53, 132.20, 148.51, 158.91 ppm. IR (cmꢀ1):
3500e2500 (br), 1622, 1467, 1227. MS (ESI): m/z: calcd for [M]:
153.14; found: 154 [MþH]þ.
4.2.9. N0-Hydroxy-4-((hydroxyimino)methyl)benzimidamide
(49). Obtained with yield 60% from 4-cyanobenzaldehyde ac-
cordingly to the general procedure and purified by crystallization
4.2.2. 2,4,6-Trihydroxybenzaldoxime (28). Obtained with yield 50%
as orange powder after purification by column chromatography
(cyclohexane/ethyl acetate¼1/1). 1H NMR (300 MHz, CD3OD):
from CH2Cl2. 1H NMR (300 MHz, DMSO-d6):
d
¼5.82 (s, 2H), 7.57 (d,
d
¼5.84 (s, 2H), 8.43 (s, 1H). 13C NMR (125 MHz, CD3OD):
¼95.47,
d
2H, J¼8 Hz), 7.69 (d, 2H, J¼8 Hz), 8.14 (s, 1H), 9.71 (s, 1H),
99.69, 107.18, 148.76, 160.15, 161.86 ppm. IR (cmꢀ1): 3364, 1507,
11.28 ppm (s, 1H). 13C NMR (75.5 MHz, DMSO-d6):
d¼125.61,
1015. MS (ESI): m/z: calcd for [M]: 169.13; found: 170 [MþH]þ.
126.09, 133.43, 133.97, 147.74, 150.35 ppm. IR (cmꢀ1): 3333, 1648,
1435, 1289, 916. MS (ESI) m/z: calcd for [M]: 179.18; found: 180
[MþH]þ.
4.2.3. 4,6-Dichloro-2-hydroxybenzaldoxime
(35). Obtained
as
a white solid after filtration with yield 93%. 1H NMR (400 MHz,
CD3OD):
d
¼6.91 (s, 1H), 7.02 (s, 1H), 8.62 ppm (s, 1H). 13C NMR
(100 MHz, CD3OD):
d
¼115.01, 116.76, 121.46, 135.46, 136.98, 148.61,
4.3. Hydrolysis of PhX in the presence of nucleophiles
160.54 ppm. IR (cmꢀ1): 3363, 2988, 1605, 1556, 1408. MS (ESI): m/z:
calcd for [M]: 206.03; found 206 [M]þ.
High-performance liquid chromatography separations were
performed on Schimadzu 10 AVP HPLC (column Agilent Zorbax SB-
4.2.4. 3-Nitro-2-hydroxybenzaldoxime (36). Obtained with yield
CN, 5
m
m, 4.6ꢃ150 mm) with 0.012% (v/v) trifluoroacetic acid CH3CN/
81% after filtration. 1H NMR (400 MHz, CD3OD):
d
¼7.04 (t, 1H,
0.008% (v/v) trifluoroacetic acid H2O as the eluent [75% H2O
(2.5 min), linear gradient from 75% to 40% (2.5e5.00 min), 40%
(5.00e10.00 min), linear gradient from 40% to 75% (10.00e
11.00 min), 75% (11.00e15.00 min)] at a flow rate 1 mL/min. UV de-
J¼8 Hz), 7.83 (dd,1H, J¼8,1.6 Hz), 8.00 (dd,1H, J¼8,1.6 Hz), 8.41 ppm
(s,1H). 13C NMR (75 MHz, CD3OD):
d
¼120.42,123.48,126.93,135.12,
137.83, 147.80, 152.90 ppm. IR (cmꢀ1): 3411, 2536, 1622, 1526, 739.
MS (ESI): m/z: calcd for [M]: 182.13; found 183 [MþH]þ.
tection was achieved at 263 nm. The volume injected was 20 mL.
4.2.5. 2,6-Dihydroxybenzamidoxime (42). Obtained with yield 60%
from 2,6-dihydroxybenzonitrile38 using the general procedure and
purification by flash chromatography (cyclohexane/ethyl acetate
4.3.1. Screening tests. Samples preparation: Samples containing
1 mM of PhX (50 L of 100 mM stock solution in CH3CN) and nu-
m
cleophile 5 mM were prepared in CH3CN (500
mL) and Tris/HCl
40/60). 1H NMR (300 MHz, CD3OD):
d
¼6.33 (d, 2H, J¼8 Hz),
buffer (0.1 N pH 8.5, VTOT¼5 mL).
6.98 ppm (m, 3H, J¼8 Hz). 13C NMR (75 MHz, CD3OD):
d
¼108.5,
HPLC analysis: Each sample was analysed by HPLC at t¼0 and
t¼2 h. The PhX peaks areas (rtz8 min) were measured.
110.6, 132.9, 148.2, 158.4 ppm. IR (cmꢀ1): 3505, 3333, 1630, 1254.
MS (ESI): m/z: calcd for [M]: 168.15; found: 169 [MþH]þ.
4.3.2. Kinetic studies. Samples preparation: Samples containing
4.2.6. 2,3-Dihydroxybenzamidoxime (43). Obtained with yield 70%
as whitish solid using the general procedure and purification by
flash chromatography (cyclohexane/ethyl acetate 40/60). 1H NMR
1 mM of PhX (50
cleophile 5 mM, 1 mM, 0.4 mM or 0.2 mM were prepared in CH3CN
(500 L) and Tris/HCl buffer (0.1 N pH 8.5 or 0.1 N pH 7.55,
mL of 100 mM stock solution in CH3CN) and nu-
m

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G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
6360
Mass Destruction, Radiological, Chemical and Biological; Wiley: New York, NY,
2004.
2. (a) Marrs, T. C. Pharmacol. Ther. 1993, 58, 51e66; (b) Sidell, F. R.; Borak, J. Ann.
Emerg. Med. 1992, 21, 865e871.
3. Convention on the Prohibition of the Development, Production, Stockpiling and Use
of Chemical Weapons and on their Destruction; the Technical Secretariat of the
Organization for the Prohibition of Chemical Weapons; Den Hague: Netherlands,
1993.
VTOT¼5 mL).The samples were dispatched in HPLC samples and put
into the HPLC auto-sampler.
Forthe experimentat40 ꢁC,thesamplesweremaintained at40 ꢁC
in an oil bath, and the injections were done manually every 15 min.
HPLC analysis: Each sample was analysed by HPLC every 15 min.
The PhX peaks areas (rtz8 min) were measured.
4. (a) Yang, Y. C.; Szafraniec, L. L.; Beaudry, W. T.; Bunton, C. A. J. Org. Chem. 1993,
58, 6964e6965; (b) Yang, Y. C.; Berg, F. J.; Szafraniec, L. L.; Beaudry, W. T.;
Bunton, C. A.; Kumar, A. J. Chem. Soc., Perkin Trans. 2 1997, 607e613; (c) Wagner,
G. W.; Yang, Y.-C. Ind. Eng. Chem. Res. 2002, 41, 1925e1928; (d) Wagner, G. W.;
Procell, L. R.; Yang, Y.-C.; Bunton, C. A. U.S. Patent 6,723,891B1, 2004; (e)
Wagner, G. W.; Sorrick, D. C.; Procell, L. R.; Brickhouse, M. D.; Mcvey, I. F.;
Schwartz, L. I. Langmuir 2007, 23, 1178e1186.
5. (a) Yang, Y. C.; Baker, J. A.; Ward, J. R. Chem. Rev. 1992, 92, 1729e1743; (b)
‘Decontamination’: C. G. Hurst in Medical Aspects of Chemical and Biological
Warfare; Sidell, F. R., Takafuji, E. T., Franz, D. R., Eds.; The Borden Institute:
Washington, DC, 2008, Chapter 15.
4.4. In vitro reactivation of VX-inhibited human AChE
ˇ
Recombinant human AChE (hAChE) was produced and purified
as previously described.39 VX was from DGA maõtrise NRBC (Vert le
Petit, France). All chemicals were from Sigma.
Stock solution of VX was 5 mM in isopropanol. The nerve agents
were further diluted in MeOH to low mM concentrations. The in-
hibition of hAChE was performed in phosphate buffer (0.1 M, pH
7.0, 0.1% BSA) at 25 ꢁC. An approximatively stoichiometric amount
of VX was chosen to attain an inhibition plateau >90% in about 1 h
while carefully avoiding 100% inhibition. Under these conditions,
there is no inhibitor left that can affect the reactivation rate mea-
surements and ageing of the conjugate is negligible (half-life is 7.9
days, data not shown).
6. (a) Yang, Y. C.; Szafraniec, L. L.; Beaudry, W. T.; Rohrbaugh, D. K. J. Am. Chem. Soc.
1990, 112, 6621e6627; (b) Cassagne, T.; Cristau, H.-J.; Delmas, G.; Desgranges,
M.; Lion, C.; Magnaud, G.; Torreilles, E.; Virieux, D. Heteroat. Chem. 2001, 12,
485e490.
7. (a) Hammond, P. S.; Forster, J. S.; Lieske, C. N.; Durst, H. D. J. Am. Chem. Soc. 1989,
111,
7860e7866; (b) Moss, R. A.; Morales-Rojas, H.; Zhang, H.; Park, B.-D.
Langmuir 1999, 15, 2738e2744; (c) Berg, F. J.; Moss, R. A.; Yang, Y.-C.; Zhang, H.
Langmuir 1995, 11, 411e413; (d) Morales-Rojas, H.; Moss, R. A. Chem. Rev. 2002,
102, 2497e2522; (e) Ramaseshan, R.; Sundarrajan, S.; Yingjun, L.; Barhate, R. S.;
Lada, N. L.; Ramakrishna, S. Nanotechnology 2006, 17, 2947e2953; (f) Masurier,
N.; Estour, F.; Froment, M.-T.; Lefevre, B.; Debouzy, J.-C.; Brasme, B.; Masson, P.;
Lafont, O. Eur. J. Med. Chem. 2005, 40, 615e623.
8. Gershonov, E.; Columbus, I.; Zafrani, Y. J. Org. Chem. 2009, 74, 329e338.
9. (a) Wagner, G. W.; Koper, O. B.; Lucas, E.; Decker, S.; Klabunde, K. J. J. Phys. Chem.
B 2000, 104, 5118e5123; (b) Wagner, G. W.; Bartram, P. W.; Koper, O. B.; Kla-
bunde, K. J. J. Phys. Chem. B 1999, 103, 3225e3228; (c) Wagner, G. W.; Bartram,
P. W. Langmuir 1999, 15, 8113e8118; (d) Wagner, G. W.; Procell, L. R.; Connor, R.
J. O.; Munavalli, S.; Carnes, C. L.; Kapoor, P. N.; Klabunde, K. J. J. Am. Chem. Soc.
2001, 123, 1636e1644; (e) Wagner, G. W.; Procell, L. R.; Munavalli, S. J. J. Phys.
Chem. C 2007, 111, 17564e17569; (f) Wagner, G. W.; Chen, Q.; Wu, Y. J. J. Phys.
Chem. C 2008, 112, 11901e11906; (g) Columbus, I.; Waysbort, D.; Shamueli, L.;
Nir, I.; Kaplan, D. Environ. Sci. Technol. 2006, 40, 3952e3958.
10. (a) Hoskin, F. C. G.; Walker, J. E.; Dettbarn, W.-D.; Wild, J. R. Biochem. Pharmacol.
1995, 49, 711e715; (b) Rastogi, V. K.; Defrank, J. J.; Cheng, T.-C.; Wild, J. R. Bi-
ochem. Biophys. Res. Commun. 1997, 241, 294e296; (c) Wang, A. A.; Chen, W.;
Mulchandani, A. Biotechnol. Bioeng. 2005, 91, 379e386; (d) Singh, A.; Lee, Y.;
Dressick, W. J. Adv. Mater. 2004, 16, 2112e2115.
11. Ghanem, E.; Raushel, F. M. Toxicol. Appl. Pharmacol. 2005, 207, 459e470.
12. Amitai, G.; Adani, R.; Sod-Moriah, G.; Rabinovitz, I.; Vincze, A.; Leader, H.; Chefetz,
B.; Leibovitz-Persky, L.; Friesem, D.; Hadar, Y. FEBS Lett. 1998, 438, 195e200.
13. Amitai, G.; Adani, R.; Hershkovitz, M.; Bel, P.; Rabinovitz, I.; Meshulam, H. J.
Appl. Toxicol. 2003, 23, 225e233.
OP-inhibited hAChE was incubated at 25 ꢁC with different con-
centrations of reactivator at 25 ꢁC in phosphate buffer (0.1 M, pH
7.0, 0.1% BSA). The final concentrations of compounds used for VX-
hAChE reactivation were: 1, 3, 10 and 20 mM or 0.1, 0.5, 1 and 2 mM
for 2-PAM. 50-ml aliquots were transferred to 1-mL cuvettes at time
intervals ranging from 1 min to 10 min depending on the reac-
tivation rate for measurement of hAChE activity using 0.75 mM
propionylthiocholine in Ellman’s buffer (phosphate 0.1 M, pH 7.0,
0.1% BSA, 0.5 mM DTNB, 25 ꢁC).40 The same procedure was applied
to the control containing the uninhibited enzyme and reactivator.
The enzyme activity in the control remained constant during the
experiment. The percentage of reactivated enzyme (%Ereact) was
calculated as the ratio of the recovered enzyme activity and activity
in the control. The dissociation constant KD of inhibited enzy-
meereactivator complex (E-PR) and the reactivity rate constant kr
were calculated by non-linear fit using the standard concentration-
dependent reactivation equation derived from the following
scheme:
14. (a) Smith, B. M. Chem. Soc. Rev. 2008, 37, 470e478; (b) Eubanks, L. M.; Dick-
erson, T. J.; Janda, K. D. Chem. Soc. Rev. 2007, 36, 458e470; (c) Vayron, P.; Re-
nard, P.-Y.; Valleix, A.; Mioskowski, C. Chem.dEur. J. 2000, 6, 1050e1063; (d)
Jovic, F.; Louise, L.; Mioskowski, C.; Renard, P.-Y. Tetrahedron Lett. 2005, 46,
6809e6814.
KD
kr
E-P þ R#E-PR/E þ P ꢀ R
ꢀ
ꢁ
ꢀ
15. Vayron, P.; Renard, P.-Y.; Taran, F.; Creminon, C.; Frobert, Y.; Grassi, J.; Mios-
kr½Rꢄ
obs$t
%Ereact ¼ 100$ 1 ꢀ ek
and kobs
¼
kowski, C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7058e7063.
16. Bromberg, L.; Schreuder-Gibson, H.; Creasy, W. R.; McGarvey, D. J.; Fry, R. A.;
Hatton, T. A. Ind. Eng. Chem. Res. 2009, 48, 1650e1659.
KD þ ½Rꢄ
Acknowledgements
17. Munro, N. B.; Talmage, S. S.; Griffin, G. D.; Waters, L. C.; Watson, A. P.; King, J. F.;
Hauschild, V. Environ. Health Perspect. 1999, 107, 933e974.
18. Yang, Y.-C.; Szafraniec, L. L.; Beaudry, W. T.; Rohrbaugh, D. K.; Procell, L. R.;
Samuel, J. B. J. Org. Chem. 1996, 61, 8407e8413.
19. Wagner, G. W.; O’Connor, R. J.; Edwards, J. L.; Brevett, C. A. S. Langmuir 2004, 20,
The DGA and the ANR (Detoxneuro ANR-06-BLAN-163 and
ReAChE ANR-09-BLAN-192) are gratefully acknowledged for finan-
cial support to G.S.A., L.L.L., S.K. and F.N., M.K., respectively. We also
thank Dr. Patrick Wehrung, Pascale Buisine and Cyril Antheaume
(UdS, Faculty of Pharmacy, Service Commun d’Analyse) for mass and
NMR analysis. We are grateful to Dr. Lydia Brelot (Institut de Chimie
UMR 7177 CNRS-ULP, Service de Radiocristallographie) for assis-
tance with an X-ray diffraction experiment.
7146e7150.
20. Renard, P.-Y.; Schwebel, H.; Vayron, P.; Josien, L.; Valleix, A.; Mioskowski, C.
Chem.dEur. J. 2002, 8, 2910e2916.
21. C.o. Disarmament, The Convention on the Prohibition of the Development,
Production, Stockpiling and Use of Chemical Weapons and on their Destruction,
1992, CD/1170.
22. (a) Becker, G.; Kawan, A.; Gutzeit, D.; Worek, F.; Szinicz, L. Arch. Toxicol. 2007, 81,
415e420; (b) Kumar, V. P.; Ganguly, B.; Bhattacharya, S. J. Org. Chem. 2004, 69,
8634e8642; (c) Epstein, J.; Kaminski, J. J.; Bodor, N.; Enever, R.; Sowa, J.; Hi-
guchi, T. J. Org. Chem. 1978, 43, 2816e2821; (d) Plapinger, R. E.; Owens, O. O. J.
Org. Chem. 1956, 21, 1186e1187; (e) Lamb, J. C.; Steinberg, G. M.; Solomon, S.;
Hackley, B. E., Jr. Biochemistry 1965, 4, 2475e2484.
Supplementary data
23. (a) Wilson, I. B. J. Biol. Chem. 1951, 190, 111e117; (b) Kuca, K.; Cabal, J.; Patocka,
J.; Kassa, J. Lett. Org. Chem. 2004, 1, 84e86; (c) Wang, J.; Gu, J.; Leszczynski, J.;
Feliks, M.; Sokalski, A. J. Phys. Chem. B 2007, 111, 2404e2408; (d) Khan, A. S.;
Kesharwani, M. K.; Bandyopadhyay, T.; Ganguly, B. J. Mol. Graphics Modell. 2009,
28, 177e182.
24. (a) Yang, G. Y.; Oh, K.-A.; Park, N.-J.; Jung, Y.-S. Bioorg. Med. Chem. 2007, 15,
7704e7710; (b) Musilek, K.; Kucera, J.; Jun, D.; Dohnal, V.; Opletalova, V.; Kuca,
K. Bioorg. Med. Chem. 2008, 16, 8218e8223; (c) Kim, T.-H.; Kuca, K.; Jun, D.;
Jung, Y.-S. Bioorg. Med. Chem. Lett. 2005, 15, 2914e2917; (d) Terrier, F.;
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.05.130.
References and notes
1. (a) Taylor, P. In Goodman and Gilman’s the Pharmacological Basis of Therapeutics,
10th ed.; Hardman, J. G., Limbird, L. E., Goodman, A. G., Eds.; McGraw-Hill: New
York, NY, 2001; pp 175e192; (b) Langford, R. E. In Introduction to Weapons of

ꢀ
G. Saint-Andre et al. / Tetrahedron 67 (2011) 6352e6361
6361
ꢀ
Rodriguez-Dafonte, P.; Le Guevel, E.; Moutiers, G. Org. Biomol. Chem. 2006, 4,
28. (a) Hayashi, I.; Ogihara, K.; Shimizu, K. Bull. Chem. Soc. Jpn. 1983, 56, 2432e2437;
(b) Hayashi, I.; Shimizu, K. Bull. Chem. Soc. Jpn. 1983, 56, 3197e3198.
29. Ashani, Y.; Cohen, S. J. Med. Chem. 1971, 14, 621e626.
30. Dale, T. J.; Rebek, J., Jr. Angew. Chem. 2009, 121, 7990e7992; Angew. Chem., Int.
Ed. 2009, 48, 7850e7852.
31. Luo, C.; Saxena, A.; Ashani, Y.; Leader, H.; Radic, Z.; Taylor, P.; Doctor, B. P. Chem.-
Biol. Interact. 1999, 119e120, 129e135.
32. Parsy, C.C.; Alexandre, F.-R.; Bonnaterre, F.; Surleraux, D. Patent WO 2010/
4352e4362; (e) Eckert, S.; Eyer, P.; Herkert, N.; Bumm, R.; Weber, G.; Thier-
mann, H.; Worek, F. Biochem. Pharmacol. 2008, 75, 698e703; (f) Worek, F.;
Thiermann, H.; Szinicz, L.; Eyer, P. Biochem. Pharmacol. 2004, 68, 2237e2248;
ꢀ
ꢀ
(g) Jokanovic, M.; Stojiljkovic, M. P. Eur. J. Pharmacol. 2006, 553, 10e17; (h)
Timperley, C. M.; Bird, M.; Heard, S. C.; Notman, S.; Read, R. W.; Tattersall, J. E.
H.; Turner, S. R. J. Fluorine Chem. 2005, 126, 1160e1165; (i) Musilek, K.; Kuca, K.;
Jun, D.; Dolezal, M. Curr. Org. Chem. 2007, 11, 229e238; (j) Kassa, J.; Kuca, K.;
Bartosova, L.; Kunesova, G. Curr. Org. Chem. 2007, 11, 267e283; (k) Bajgar, J.;
Fusek, J.; Kuca, K.; Bartosova, L.; Jun, D. Mini-Rev. Med. Chem. 2007, 7, 461e466;
(l) Kuca, K.; Jun, D.; Musilek, K. Mini-Rev. Med. Chem. 2006, 6, 269e277; (m)
Worek, F.; Eyer, P.; Aurbek, N.; Szinicz, L.; Thiermann, H. Toxicol. Appl. Phar-
macol. 2007, 219, 226e234; (n) Maxwell, D. M.; Koplovitz, I.; Worek, F.;
Sweeney, R. E. Toxicol. Appl. Pharmacol. 2008, 231, 157e164; (o) Kuca, K.; Cabal,
J.; Jun, D.; Musilek, K.; Soukup, O.; Pohanka, M.; Pejchal, J.; Oh, K.-A.; Yang,
G.-Y.; Jung, Y. eS. Environ. Toxicol. Pharmacol. 2010, 30, 85e87; (p) Mercey, G.;
ꢀ
090976A1, 2010.
33. Gale, D. J.; Wilshire, J. F. K. Aust. J. Chem. 1975, 28, 2447e2458.
34. Luo, Z.; Slee, D.; Tellew, J. E.; Williams, J.; Zhang, X. Patent WO 2005/063755 A1,
2005, p 86.
35. Panzella, L.; De Lucia, M.; Amalfitano, C.; Pezzella, A.; Evidente, A.; Napolitano,
A.; d’Ischia, M. J. Org. Chem. 2006, 71, 4246e4254.
36. Vourloumis, D.; Takahashi, M.; Winters, G.; Zhou, J.; Duchene, R. U.S. Patent
2004/015249, 2004, p 179.
ꢀ
Verdelet, T.; Saint-Andre, G.; Gillon, E.; Wagner, A.; Baati, R.; Jean, L.; Nachon F.;
Renard, P. Y. Chem. Commun. (Camb.) 2011, 5295e5297.
37. Bajwa, N.; Jennings, M. P. J. Org. Chem. 2006, 71, 3646e3649.
38. Amaranatha Reddy, R.; Sadashiva, B. K. Liq. Cryst. 2003, 30, 273e283.
ꢃ
ꢀ
€
25. Louise-Leriche, L.; Paunescu, E.; Saint-Andre, G.; Baati, R.; Romieu, A.; Wagner,
A.; Renard, P.-Y. Chem.dEur. J. 2010, 16, 3510e3523.
39. Carletti, E.; Li, H.; Li, B.; Ekstrom, F.; Nicolet, Y.; Loiodice, M.; Gillon, E.; Froment,
M. T.; Lockridge, O.; Schopfer, L. M.; Masson, P.; Nachon, F. J. Am. Chem. Soc.
26. Chambers, J. E.; Oppenheimer, S. F. Toxicol. Sci. 2004, 77, 185e187.
27. (a) Blatt, A. H.; Russell, L. A. J. Am. Chem. Soc. 1936, 58, 1903e1908; (b) Free-
dmann, H. H. J. Am. Chem. Soc. 1961, 83, 2900e2905.
2008, 130, 16011e16020.
40. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Phar-
macol. 1961, 7, 88e95.