Synthesis and in vitro evaluation of donepezil-based reactivators and analogues for nerve agent-inhibited human acetylcholinesterase

Article abstract of DOI:10.1039/c5ra25477a

Poisoning by organophosphorus nerve agents and pesticides is a serious public and military health issue with over 200 000 fatalities annually worldwide. Conventional emergency treatment consists of rapid administration of atropine and pyridinium oxime as an antidote. The reactivation of acetylcholinesterase (AChE) in the central nervous system (CNS) by the oxime is inefficient due to the fact that positively charged pyridiniums do not readily cross the blood brain barrier (BBB). Herein, we described the synthesis and in vitro evaluation of four donepezil-based non quaternary reactivators. The compounds 1-4 have been prepared in 7-8 linear steps in 1-9% overall yields and oximes 1-3 show better ability (8 fold higher) than pralidoxime to reactivate VX-inhibited human AChE (VX-hAChE). Besides, oxime 2 is 5 to 11 fold more efficient than pralidoxime and HI-6 respectively for the reactivation of VX-inhibited human butyrylcholinesterase (VX-hBChE).

Full text of DOI:10.1039/c5ra25477a

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Page 1 of 27
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Synthesis and in vitro evaluation of donepezil-based
reactivators and analogues for nerve agent-inhibited
human acetylcholinesterase
1
,§
2
1,§
1,§
2
Julien Renou , José Dias , Guillaume Mercey , Tristan Verdelet , Catherine Rousseau ,
2
2
2
3
AnneꢀJulie Gastellier , Mélanie Arboléas , Mélanie TouvreyꢀLoiodice , Rachid Baati , Ludovic
1
2
1
Jean* , Florian Nachon , PierreꢀYves Renard*
1
Normandie Univ, COBRA, UMR CNRS 6014 & FR 3038; UNIV Rouen; INSA Rouen; CNRS, IRCOF,
Rue Tesnières, 76821 MontꢀSaintꢀAignan Cedex, France, Eꢀmail: ludovic.jean@univꢀrouen.fr;
1
pierreꢀyves.renard@univꢀrouen.fr
2
Département de Toxicologie et Risque Chimique, Institut de Recherche Biomédicale des Armées, BP
7
3, 1 place du Général Valérie André, 91993 Brétigny/s/Orge, France
3
§
Université de Strasbourg, UMR CNRS 7515 ICPEES, 25 Rue Becquerel, 67087 Strasbourg, France
These authors have contributed equally to this work
Keywords:
oxime,
reactivator,
acetylcholinesterase,
nerve
agent,
donepezil,
butyrylcholinesterase
Abstract
Poisoning by organophosphorus nerve agents and pesticides is a serious public and military
health issues with over 200 000 fatalities annually worldwide. Conventional emergency
treatment consists of rapid administration of atropine and pyridinium oxime as antidote. The
reactivation of acetylcholinesterase (AChE) in the central nervous system (CNS) by the
oxime is inefficient due to the fact that positively charged pyridiniums do not cross readily the
blood brain barrier (BBB). Herein, we described the synthesis and in vitro evaluation of four
donepezilꢀbased non quaternary reactivators. The compounds 1ꢀ4 have been prepared in 7ꢀ
8
linear steps in 1ꢀ9% overall yields and oximes 1ꢀ3 show a better ability (8 fold higher) than
pralidoxime to reactivate VXꢀinhibited human AChE (VXꢀhAChE). Besides, oxime 2 is 5 to 11
fold more efficient than pralidoxime and HIꢀ6 respectively for the reactivation of VXꢀinhibited
human butyrylcholinesterase (VXꢀhBChE).
1

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Introduction
Organophosphorus compounds in the form of pesticides or nerve agents are a serious health
problem worldwide. Pesticides exposure is one of the most common causes of poisoning
throughout the world (over 200 000 deadly intoxications yearly) and organophosphorus nerve
agents (OPNA) present a persistent threat to the population as a consequence of armed
conflicts (e.g. Gulf War) and terrorist attacks (e.g., subway attacks in Japan in 1995 and
more recently during the civil war in Syria) despite an international convention prohibiting the
1
use of chemical weapons (Figure 1).
Figure 1 Structures of pesticides (A) and OPNA (B)
The acute toxicity of OPNA results from irreversible inhibition of AChE (EC 3.1.1.7), a key
enzyme in neurotransmission, via the formation of a covalent PꢀO bond at the catalytic
serine. Inhibition of AChE leads to the accumulation of acetylcholine neurotransmitter (ACh)
in the synaptic cleft causing among other symptoms, seizures and respiratory arrest leading
2
, 3
to death. The current urgency treatment of OPNA poisoning is based on the administration
of a cocktail of three components: an antimuscarinic agent (e.g. atropine), an anticonvulsant
drug (e.g. diazepam) and mono or bispyridinium AChE reactivator (e.g. pralidoxime,
obidoxime, trimedoxime) (Figure 2). The high nucleophilicity of these alphaꢀnucleophiles
allows the displacement of the phosphyl group from the catalytic serine, yielding to the
restoration of AChE activity.
2

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Figure 2 Structures of pyridinium AChE reactivators
Since the discovery of monoꢀ and bisꢀpyridinium oximes as reactivators of inhibited AChE in
950’s, many structural modifications have been realized on these structures leading to a
1
4
ꢀ7
large library of potential reactivators. However, despite hundreds of pyridinium oximes
synthesized and evaluated during the last 70 years, this family of reactivators has serious
drawbacks. Firstly, these molecules poorly cross the BBB, due to their permanent charge,
8
limiting their efficiency to reactivate AChE in the CNS. Moreover, no monoꢀ or bisꢀpyridinium
oximes are broadꢀspectrum reactivators, they have highly variable efficiency depending on
the nature of the agent. During the last decade, several groups have focused their efforts on
the development of new nonꢀpyridinium reactivators able to cross the BBB for reactivating
9
ꢀ14
central AChE.
For example, our group described the synthesis and evaluation of new
reactivator families based on 3ꢀhydroxy pyridinaldoxime attached to AChE peripheral site
ligands (e.g. phenyltetrahydroisoquinoline, tetrahydroacridine, tryptoline,ꢁ) through carbon
15ꢀ21
chains with various lengths.
Encouraged by these promising results, we envisaged to develop a new series of
®
reactivators based on Donepezil (Aricept ). Donepezil is a wellꢀknown inhibitor of AChE and
used for the treatment of Alzheimer’s disease with an inhibitory activity in the nanomolar
2
2
23, 24
range. Xꢀray structures of the complex of donepezil with human AChE has been solved.
(
Figure 3). The indanone moiety stacks against W286 at the peripheral site, while benzyl ring
stacks against W86 at the catalytic site. The nitrogen atom of the piperidine ring makes
waterꢀmediated hydrogen bond to Y341 and Y337.
3

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24
Figure 3 Donepezil-hAChE crystal structure.
Based on these structural data, we developed a series of donepezilꢀbased reactivators and
analogues by replacing the benzyl group with 3ꢀhydroxypyridinaldoxime. Four compounds
have been synthesized in order to study the structural modifications influence on the affinity
toward inhibited AChE (K ) and the reactivation efficiency (k ) (Figure 4). The role of the
D
r2
ketone and/or the piperidine ring (compounds 2-4) has also been studied.
O
O
HO
HON
N
OMe
OMe
N
OMe
OMe
N
Donepezil
1
OMe
OMe
HO
HON
O
HO
HON
N
OMe
OMe
N
N
2
3
OMe
O
OMe
N
NOH OH
4
Figure 4 Structure of the donepezilꢀbased AChE reactivator and analogues
4

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Results and discussion
The synthesis of the donepezilꢀbasedꢀ reactivator 1 (Scheme 1) started with the preparation
of the donepezil moiety 10. The first step consisted of the reduction of ethyl isonipecotate 5
4
into the corresponding alcohol by using LiAlH . The protection of the secondary amine of the
compound 6 as Bocꢀcarbamate gave compound 7 in 50% yield. Then, alcohol 7 was oxidized
into the corresponding aldehyde, which was used directly in the aldolisationꢀcrotonization
reaction with 5,6ꢀdimethoxyindanone in the presence of sodium hydroxide and a phase
transfer catalyst, to give the desired compound 8 in 57% yield over two steps. Different
reduction conditions have been tested so as to reduce the conjugated double bond to give
2
compound 9. While the assays using a palladium catalyst (e.g. Pd/C, Pd(OH) ) under
hydrogen atmosphere gave only an overꢀreduction (ketone function was converted into the
corresponding methylene group), we then evaluated the conditions reported by Keinan et al.
25
using silicon hydride in the presence of a catalytic amount of zinc chloride and Pd(PPh
3 4
) .
Under these conditions, the conjugate reduction of the ꢀunsaturated ketone 8 has been
α,β
performed in 30% yield. Finally, the deprotection of the amine 9 by treatment with
trifluoroacetic acid offered the donepezil moiety 10 in 80% yield. The second part of this
synthesis consisted on the functionalization of the reactivator function, named 3ꢀhydroxy
pyridinaldoxime. The oxidation of alcohol 11 with MnO
2
in toluene furnished the
corresponding aldehyde 12 in 76% yield. Then the protection of phenol as MOM ether has
been carried out using methyl chloromethyl ether (MOMCl) in presence of potassium
carbonate to afford 13 in 95% yield. The following steps consisted of the formation of oxime
and its protection with TBDPSiCl. Radical bromination of the benzylic position of pyridine 14
using Nꢀbromosuccinimide (NBS) followed by a nucleophilic substitution with amine 10 and
treatment with trifluoroacetic acid afforded the reactivator 1 with 0.7% yield over three steps.
It is noteworthy that the deprotection of oxime has been observed during the nucleophilic
substitution under basic conditions. The oxime 1 has been prepared in 8 linear steps from
the compounds 5 and 11 with 0.4% overall yield.
5

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Scheme 1 Reagents and conditions: a) LiAlH
4
(1.7 equiv), THF, 92% ; b) Boc
2
O (1 equiv),
THF/EtOAc, 50%; c) 1) IBX (3 equiv), EtOAc; d) 5,6ꢀdimethoxyindanone (0.9 equiv), NaOH aq. 10%,
TBAB (0.1 équiv), CH Cl , 57% (two steps); e) Ph SiH (1.8 equiv), ZnCl (0.3 equiv), Pd(PPh 1.5
mol%, CHCl , 30%; f) TFA 20% in CH Cl , 80%; g) MnO , toluene, 76%; h) MOMCl (3 equiv), K CO
4 equiv), acetone, 95%; i) NH OH.HCl (1.1 equiv), NaOAc (1.2 equiv); j) TBDPSiCl (1.5 equiv),
imidazole (1.3 equiv), CH Cl , 76% (two steps); k) Nꢀbromosuccinimide (1.1 equiv), benzoyl peroxide
0.1 equiv), CCl4, l) 10 (0.6 equiv), K CO (1.1 equiv), DMF; m) TFA 20% (v/v) in CH Cl then
2
2
2
2
2
3 4
)
3
2
2
2
2
3
(
2
2
2
(
2
3
2
2
purifications (twice) by semiꢀpreparative RPꢀHPLC, 0.7% (three steps)
The synthesis of the analogue 2 (Scheme 2) started with the preparation of the donepezil
moiety 16. Complete reduction of unsaturated ketone 8 was performed using Pd/C under
αꢀβ
hydrogen atmosphere (1 bar) to give the overꢀreduced product 15 in 85% yield. Deprotection
of amine by treatment with trifluoroacetic acid furnished the compound 16 in 75% yield.
Direct radical bromination performed on aldehyde 13 failed, leading only to degradation
products. Therefore, we decided to realize the radical bromination on the corresponding
methyl ester 17. The latter has been prepared from aldehyde 13 by treatment with iodine in
2
6
basic conditions using methanol as solvent, in 78% yield. Then, the photobromination of the
benzylic position of the ester 17 with NBS, followed by the nucleophilic substitution with 16
furnished the compound 18 in 40% yield over two steps. The last steps consisted of the
formation of oxime function and the deprotection of phenol. Reduction with DIBALꢀH,
followed by the deprotection of the phenol function using trifluoroacetic acid, and formation of
oxime allowed to afford targeted oxime 2 in 15% yield over three steps. The oxime 2 has
thus been prepared in 10 linear steps from the compounds 5 and 11 with 1% overall yield.
6

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2 2 2
Scheme 2 Reagents and conditions: a) H (1 atm), Pd/C, MeOH, 85%; b) TFA 20% (v/v) in CH Cl ,
7
1
2
5%; c) I (1.3 equiv), KOH (2.6 equiv), MeOH, 78%; d) Nꢀbromosuccinimide (1.05 equiv), hν, CCl ; e)
2
4
6 (0.85 equiv), K
2
CO
3
(3.5 equiv), CH
3 2 2
CN, 40% (two steps); f) DIBALꢀH (2 equiv), CH Cl , g) TFA
0% (v/v) in CH
Cl
2 2
; h) NH
2
OH,HCl, NaOAc, MeOH, 15% (three steps).
The synthesis of the Donepezil analogue based reactivator 3 (Scheme 3) started with the
alkylation of 5,6ꢀdimethoxyindanone 19 with propargyl bromide using lithium
diisopropylamide at ꢀ78 °C to give the desired monoꢀalkylated product 20 in 48% yield. It is
noteworthy that the starting material 19 was recovered (23%) and the diꢀalkylated product
was also isolated by flash chromatography on silica gel (15%). The second step consisted on
the Sonogashira coupling reaction between 20 and 21, which has been prepared by following
17
a reported procedure. This crossꢀcoupling reaction afforded 22 in 73% yield. The
subsequent reduction of the alkyne and deprotection of the phenol function was then
followed by a sequence of three reactions comprising (i) the protection of the phenol group
with tꢀbutyldimethylsilyl chloride (TBSCl), (ii) the reduction of the methyl ester to aldehyde
upon DIBALꢀH treatment, and finally (iii) the deprotection of the TBS ether using
tetrabutylammonium fluoride (TBAF) as fluoride source. These transformations led to
aldehyde 23 in 50% yield over four steps. During the reduction step with DIBALꢀH, the
carbonyl function on the indanone has been also reduced into the corresponding alcohol.
Therefore, an additional treatment with DessꢀMartin periodinane was required to furnish
aldehyde 24 in 72% yield. Then, the last step consisted in the formation of the oxime function
with the condensation of hydroxylamine onto the aldehyde 24. The desired oxime 3 has thus
been prepared in 8 steps from 19 with 9.5% overall yield.
7

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Scheme 3 Reagents and conditions: a) propargyl bromide (1.5 equiv), LDA (1.2 equiv), THF, 48%; b)
1 (1 equiv), Pd(PPh ) 5 mol%, CuI (10 mol%), THF/NEt (2:1 v/v), 73%; c) H (1 atm), Pearlman’s
2
3
4
3
2
catalyst (20 mol%), EtOAc; d) TBDMSiCl (1.1 equiv), imidazole (3 equiv), DMF; e) DIBALꢀH (4 equiv),
CH Cl ; f) TBAF (1.1 equiv), THF, 50% (four steps); g) Dess Martin periodinane (1 equiv), pyridine (5
equiv), CH Cl , 72%; h) NH OH.HCl (1 equiv), NaOAc (1 equiv), EtOH, 76%
2
2
2
2
2
Based on the similar synthetic scheme, the synthesis of the analogue 4 (Scheme 4) started
with the Sonogashira reaction between the alkyne 20 and bromoꢀpyridine 25, which has
2
6
been prepared by following a reported procedure. The coupling product 26 has been
obtained in 79% yield. Then, the reduction of the alkyne has been performed in a presence
of Pearlman’s catalyst under hydrogen atmosphere to afford compound 27 in quantitative
yield. The sequence of three reactions comprising the protection of the phenol group as TBS
ether, the reduction of the methyl ester to aldehyde, and the deprotection of the TBS ether
furnished the aldehyde 28 in 3% yield over three steps. During the reduction step, the
carbonyl group of the indanone has been partially reduced into the corresponding alcohol.
Yet, the two products have been separated by a flash chromatography on silica gel, and to
increase the global yields, the alcohol could have been oxidized in ketone as for compound
24. The last step consisted in the formation of the oxime by treatment with hydroxylamine
under basic conditions. The compound 4 has been synthesised in 7 steps from 19 with 1.1%
overall yield.
8

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3 4 3
Scheme 4 Reagents and conditions: a) 25 (1 equiv), Pd(PPh ) 10 mol%, CuI (20 mol%), THF/NEt
(
2:1 v/v), 79%; b) H
2
(1 atm), Pearlman’s catalyst (20 mol%), EtOAc, 99%; c ) TBDMSOTf (1.1 equiv),
2
,6ꢀlutidine (3 equiv), CH Cl ; d) DIBALꢀH (2 equiv), CH Cl ; e) TBAF (1.1 equiv), THF, 3% (three
2
2
2
2
2
steps); f) NH OH.HCl (1 equiv), NaOAc (1 equiv), EtOH, 99%
Reactivation tests
Since Donepezil is a very potent reversible inhibitor of hAChE (K
i
= 1nM), we examined if the
oxime derivatives are also inhibitors. If compounds 2, 3 and 4 are weak reversible inhibitors
of hAChE with respectively 74, 36 and 93% of residual activity at 100 ꢂM, compound 1 is
revealed to be a mild inhibitor with IC50 = 1.0±0.1 ꢂM (See Supporting Information). We
cannot anticipate that it is a drawback for a reactivator until toxicity tests are performed. The
reactivation potencies of compounds 1ꢀ4 have been determined at pH 7.4 and 37 °C, close
to physiological conditions (Table 1 and Supporting Information). The bimolecular
reactivation rate constant (k ), which reflects the potency, has been determined. When
r2
r
relevant, the reactivation rate constant (k ) and apparent dissociation constant of the
reactivator/phosphylꢀAChE complex (K ) have also been determined. The reactivation
D
parameters were compared to the reactivation constant of the marketed pyridinium
aldoximes, 2ꢀPAM and obidoxime, and to HIꢀ6 which is about to be fielded in the French
Army
.
Compounds 1ꢀ3 are one order of magnitude more efficient than 2ꢀPAM for the reactivation of
VXꢀhAChE but still inferior to obidoxime and HIꢀ6. Oxime 1 shows a similar binding affinity
toward inhibited AChE as HIꢀ6 and obidoxime (K ≈ 50 ꢂM), however kr is about 5ꢀfold lower
D
than obidoxime and HIꢀ6. Comparison of oximes 1 and 2 show that the carbonyl function on
9

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22
the indanone moiety improves the binding affinity toward VXꢀhAChE, but possibly constraint
the molecule in an orientation that results in lower kr. Decrease in K balances the decrease
in k resulting in similar kr2. Comparison of oximes 3 and 4 both devoid of the piperidine ring,
shows that position 6 onto the pyridine ring is the best position for anchoring. Indeed, oxime
is 7 fold less efficient than oxime 4. Similarly to what is observed for oxime 2, there is no
D
r
3
loss of efficiency despite the loss of affinity related to the lack of piperidine ring, because k
r
increases.
Compound 1, was also tested for in vitro reactivation of sarinꢀinhibited human AChE (sarinꢀ
hAChE) (Table 1). It is less efficient than HIꢀ6, due to a weaker affinity toward sarinꢀhAChE
(
2 fold lower than HIꢀ6) and also lower reactivation rate constant (3.4 fold lower than HIꢀ6).
Oximes 2-4 were tested for in vitro reactivation of tabunꢀ and paraoxonꢀinhibited human
AChE (tabunꢀhAChE and paraoxonꢀhAChE) (Table 1). They displayed only marginal activity
on tabunꢀhAChE. However, they appeared to be significantly active on paraoxonꢀhAChE,
being one order of magnitude more efficient than HIꢀ6, although the latter is known to be a
poor reactivator for pesticides. Surprisingly, oximes 2 and 3 have very close reactivation
parameters despite their structural difference.
A complementary approach to OPNA poisoning is a preꢀtreatment based on bioscavengers.
Bioscavengers are enzymes able to neutralize organophosphorus molecules in the
27
bloodstream before they can reach acetylcholinesterase. Human butyrylcholinesterase
(
hBChE) is the leading candidate for stoichiometric OPNA bioscavenging. Combining hBChE
2
8
to a specific reactivator can improve its scavenging efficiency. In this context, in vitro
efficiency of oxime 2 and 4 has been evaluated for the reactivation of VXꢀinhibited BChE
(
Table 1). Oxime 2 is 5 to 11 fold more efficient than pralidoxime and HIꢀ6 respectively due to
a better binding affinity to the inhibited enzyme.
r D
Table 1 Reactivation rate constant (k ) and dissociation constant (K ) for the reactivation of OPꢀ
inhibited hAChE and OPꢀinhibited hBChE.
ꢀ1
k
obs (min )
ꢀ
1
ꢀ1
ꢀ1
Oxime
(oxime
K
D
(ꢂM)
k
r
(min )
k
r2 (mM .min )
concentration ꢂM)
VX-hAChE
54±12
Obidoxime
HIꢀ6
0.39±0.04 (100)
0.45±0.06 (100)
0.015±0.001 (100)
0.10 ± 0,006 (100)
0.6±0.06
0.66±0.03
0.06±0.01
0.12±0.02
11.1
9.3
70±8
2ꢀPAM
215±75
51.4±19.1
0.28
2.3
1
10

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a
a
a
a
2
3
4
0.19±0.01 (100)
0.14±0.01 (50)
>200
>0.35
>0.14
>0.06
1.8±0.05
a
>50
2.7±0.1
a
0.038±0.002 (100)
>150
0.039±0.001
sarin-hAChE
HIꢀ6
0.69±0.06 (100)
0.11±0.01 (100)
90±34
1.17±0.19
0.34±0.05
13
2
1
175±47
paraoxon-hAChE
HIꢀ6
2
0.005±0.001 (100)
800±140
160±45
170±70
ꢀ
0.09
0.24±0.05
0.3±0.1
ꢀ
0.1
1.5
1.8
ꢀ
0.093±0.005 (100)
0.070±0.003 (50)
0.040±0.001 (100)
3
4
tabun-hAChE
HIꢀ6
2
0.055±0.004 (100)
57±22
0.074±0.010
1.3
0.001±0.0001 (100)
0.004±0.0002 (50)
0.001±0.0002 (50)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
ꢀ
ꢀ
4
VX-hBChE
2400±400
2
ꢀPAM
HIꢀ6
2
0.062±0.005 (100)
0.028±0.001 (100)
0.16±0.005 (100)
0.041±0.003 (100)
1.4±0.2
0.58
a
a
>400
>0.11
0.28±0.01
96±5
ꢀ
0.29±0.01
ꢀ
3.0
ꢀ
4
a
Not determined when [Reactivator]<<K
D
leading to a linear dependence between kobs and
[
Reactivator] : kobs=k /K *[Reactivator]. In this case, k =k /K , the slope of the line, is directly
r
D
r2
r
D
obtained by fitting.
Conclusions
A series of four donepezilꢀbased reactivators and analogues has been synthesized and
evaluated for their in vitro reactivation phosphylated acetylcholinesterase. These compounds
have been prepared in 7ꢀ8 linear steps in 0.4ꢀ9% overall yields. Oximes 1ꢀ3 show an ability
to reactivate VXꢀhAChE 8ꢀfold greater than pralidoxime. The presence of the carbonyl
function of the indanone moiety confers a good affinity toward inhibited AChE, but this is
balanced by a reduced reactivation rate. Oximes 2-4 do not readily reactivate tabunꢀhAChE,
but they proved to be relevant at reactivating paraoxonꢀhAChE. Oxime 2 is 5 to 11 fold more
efficient than pralidoxime and HIꢀ6 respectively for the reactivation of VXꢀhBChE. As with the
11

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1
8
others developed families of non quaternary reactivators (phenyltetrahydroisoquinoline and
1
9
tetrahydroacridine ), the most favourable position of the linker is the position 6 onto the
pyridine ring.
Experimental section
Chemistry
General. Column chromatography purifications were performed on Merck silica gel (40ꢀ63
ꢃm). Thinꢀlayer chromatography (TLC) was carried out on Merck DC Kieselgel 60 Fꢀ254
aluminium sheets. Compounds were visualized by illumination with a short wavelength UV
lamp (λ = 254 nm). All solvents were dried following standard procedures (CH
2 2 3
Cl , CH CN:
distillation over P , DMF: distillation over BaO under reduced pressure, THF: distillation
2
O
5
1
13
over Na/benzophenone). H and C NMR spectra were recorded on a Bruker DPX 300
spectrometer (Bruker, Wissembourg, France). Chemical shifts are expressed in parts per
1
million (ppm) from CDCl
3
(δ
Η
= 7.26, δ
C
= 77.16) and CD
3
OD (δ
Η
= 3.31, δ
C
= 49.00). J values
are expressed in Hz. Residual solvents and grease contained in NMR sample were indicated
1
13
29
on H and C spectra (see supporting information). Mass spectra were obtained with a
Finnigan LCQ Advantage MAX (ion trap) apparatus equipped with an electrospray source. All
analyses were performed in the positive mode.
All final oximes were confirmed to be of ≥ 95% purity based on HPLC analysis. Analytical
HPLC was performed on a Thermo Electron Surveyor instrument equipped with a PDA
detector under the following conditions: Thermo Hypersil GOLD C18 column (5 ꢃm, 4.6 x 100
mm) with MeOH and 0.1% aq. trifluoroacetic acid (TFA) as eluents [0.1% aq. TFA/MeOH
(
90/10) (5 min), followed by linear gradient from 10% to 100% of MeOH (45 min)] at a flow
rate of 1.0 mL/min and UV detection Max Plot 220ꢀ360 nm.
Semiꢀpreparative RPꢀHPLC was performed under the following conditions: Thermo Hypersil
3
GOLD C18 column (5 ꢂm, 21.2 x 250 mm) with CH CN and tetraethylammonium bromide
(
TEAB) (50 mM (aq), pH 7.5) as eluents (0% CH CN (5 min), followed by a gradient of 0ꢀ10%
3
CH
3
CN (5 min), then 10ꢀ90% CH
3
CN (105 min)) at a flow rate of 15 mL.minꢀ1. Double UV
detection was achieved at λ = 230 and 315 nm.
4-Piperidinemethanol (6)
To a suspension of LiAlH (2.1 g, 54.4 mmol, 1.7 equiv.) in dry THF (100 mL) at 0 °C was
4
added dropwise a solution of ethyl isonipecotate 5 (5 g, 32 mmol) in dry THF (50 mL). The
mixture was stirred overnight at rt and treated carefully with water (2.1 mL), an aqueous
12

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solution of NaOH 15% (2.1 mL) and water (6.3 mL). The resulting mixture is stirred for 1 h at
rt. The solid was filtered and the filtrate was dried over MgSO
4
. The solvent was removed
1
under reduced pressure to afford 4ꢀpiperidinemethanol 6 (3.40 g, 92%) as white solid. H
3
NMR (300 MHz, CDCl ) δ 0.90ꢀ1.03 (m, 2H), 1.42ꢀ1.64 (m, 1H), 1.66ꢀ1.70 (m, 2H), 2.50ꢀ2.59
13
(
3
m, 2H), 3.01ꢀ3.07 (m, 2H), 3.38 (d, J = 6.4 Hz, 2H). C NMR (75 MHz, CDCl ) δ 30.0, 39.1,
+
4
6.3, 67.6. MS (ESI+): m/z (%): 116 (100) [M+H] .
[
N-[(tert-Butyloxy)carbonyl]piperidin-4-yl]methyl alcohol (7)
4ꢀpiperidinemethanol 6 (3.40 g, 29.5 mmol) was stirred in EtOAc (100 mL) and THF (44 mL)
and diꢀtertꢀbutylcarbamate (6.44 g, 29.5 mmol) was slowly added and the resulting mixture
was stirred for 6 h at rt. The mixture was successively washed with an aqueous saturated
3 4
solution of NaHCO and brine. The organic layer was dried over MgSO and the solvent was
removed under reduced pressure. Chromatography on silica gel (cyclohexane/EtOAc 5:5,
1
v/v) afforded the desired compound 7 (3.2 g, 50%) as white solid. H NMR (300 MHz, CDCl
3
)
δ 1.05ꢀ1.18 (m, 2H), 1.43 (s, 9H), 1.57ꢀ1.71 (m, 3H), 1.84 (m, 1H), 2.64ꢀ2.72 (m, 2H), 3.45ꢀ
1
3
3
.49 (m, 2H), 4.08ꢀ4.11 (m, 2H). C NMR (75 MHz, CDCl
3
): δ 28.5, 28.7, 38.8, 43.8, 67.3,
30
79.4, 155.0. Spectral data are in accordance with those reported in the literature.
tert-Butyl-4-((5,6-dimethoxy-1-oxo-1H-inden-2(3H)-ylidene)methyl)piperidine-1-
31
carboxylate (8)
[
Nꢀ[(tertꢀButyloxy)carbonyl]piperidinꢀ4ꢀyl]methyl alcohol 7 (3.2 g, 14.7 mmol) and 2ꢀ
iodobenzoic acid (IBX) (12.4 g, 44 mmol, 3 equiv.) were added into EtOAc (140 mL) under
inert atmosphere. The resulting mixture was refluxed for 4 h and allowed to cool at rt. The
solid was filtered and the solvent was evaporated. The crude aldehyde was used in the next
step without further purification.
Under inert atmosphere, 5,6ꢀdimethoxyꢀindanone (2.35 g, 12.3 mmol), TBAB (394 mg, 1.25
mmol, 0.01 equiv.) and 10% aqueous NaOH (9 mL) were stirred vigorously for 30 min at rt
into CH Cl (17 mL). The crude aldehyde was added and the resulting mixture was heated at
2
2
50 °C for 5 h. After cooling at rt, the layers were separated. The aqueous layer was extracted
three times with CH Cl . The combined organic layers were washed with brine, water and
2
2
4
dried over MgSO . The solvent was evaporated under reduced pressure and the crude
product was recrystallized from isopropyl ether to afford the desired product 8 as white solid
1
(
3.3 g, 57%). H NMR (300 MHz, CDCl
3
) δ 1.41ꢀ151 (m, 2H), 1.47 (s, 9H), 1.67ꢀ1.71 (m, 2H),
2.41ꢀ2.52 (m, 1H), 2.77ꢀ2.93 (m, 2H), 3.60 (d, J = 1.7 Hz, 2H), 3.92 (s, 3H), 3.97 (s, 3H),
13
4
.11ꢀ4.16 (m, 2H), 6.62 (dt, J = 1.8, 7.7 Hz, 1H), 6.91 (s, 1H), 7.29 (s, 1H). C NMR (75
13

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MHz, CDCl
3
): δ 28.4, 29.5, 30.8, 37.2, 43.2, 56.1, 56.3, 79.6, 105.0, 107.2, 131.7, 135.9,
+
1
38.5, 144.5, 149.5, 154.8, 155.4, 192.5. MS (ESI+): m/z (%): 388 (100) [M+H] .
tert-Butyl-4-((2,3-dihydro-5,6-dimethoxy-1-oxo-1H-inden-2-yl)methyl)piperidine-1-
carboxylate (9)
To
ylidene)methyl)piperidineꢀ1ꢀcarboxylate 8 (3.26 g, 8.4 mmol), dihydrodiphenylsilane (2.74 g,
5 mmol, 1.8 equiv.) and ZnCl (350 mg, 2.6 mmol, 0.3 equiv.) in CHCl (85 mL) was added
Pd(PPh (150 mg, 0.13 mmol, 0.015 equiv.). The solution was stirred at rt for 24 h.
a
degassed
mixture
of
tertꢀbutylꢀ4ꢀ((5,6ꢀdimethoxyꢀ1ꢀoxoꢀ1Hꢀindenꢀ2(3H)ꢀ
1
2
3
3 4
)
Concentration under reduced pressure and purification on silica gel (cyclohexane/EtOAc 6:4
1
to 5:5, v/v) afforded the desired compound 9 (950 mg, 30%) as yellow solid. H NMR (300
MHz, CDCl
3
) δ 1.14ꢀ1.26 (m, 2H), 1.29ꢀ1.41 (m, 1H), 1.45 (s, 9H), 1.61ꢀ1.76 (m, 3H), 1.86ꢀ
1
2
3
.95 (m, 1H), 2.67ꢀ2.74 (m, 4H), 3.31ꢀ3.30 (m, 1H), 3.90 (s, 3H), 3.97 (s, 3H), 4.08ꢀ4.15 (m,
1
3
H), 6.86 (s, 1H), 7.17 (s, 1H). C NMR (75 MHz, CDCl
3
) δ 28.4, 31.6, 32.8, 33.3, 34.6,
8.7, 45.1, 56.1, 56.2, 79.3, 104.3, 107.3, 129.2, 129.2, 148.7, 149.4, 154.9, 155.5, 207.6.
+
+
2
MS (ESI+): m/z (%): 390 (100) [M+H] and 796 (38) [2M+H O] .
5,6-Dimethoxy-2-(piperidin-4-ylmethyl)-2,3-dihydro-1H-inden-1-one (10)
Tertꢀbutylꢀ4ꢀ((2,3ꢀdihydroꢀ5,6ꢀdimethoxyꢀ1ꢀoxoꢀ1Hꢀindenꢀ2ꢀyl)methyl)piperidineꢀ1ꢀcarboxylate
(950 mg, 2.4 mmol) was added in a 20% (v/v) solution of TFA in CH Cl (24 mL) at 0 °C.
The mixture was stirred for 4 h and was then allowed to warm at rt. TFA and CH Cl were
removed by evaporation using a water pump and the residue was dissolved into an aqueous
saturated solution of NaHCO . The aqueous layer was then extracted three times with
CH Cl and the organic layers were combined and dried over MgSO . Concentration under
9
2
2
2
2
3
2
2
4
1
reduced pressure afforded the desired product 10 as white solid (560 mg, 80%). H NMR
(
300 MHz, CDCl ) δ 1.14ꢀ1.33 (m, 3H), 1.56ꢀ1.77 (m, 3H), 1.82ꢀ1.91 (m, 1H), 2.56ꢀ2.70 (m,
3
4
1
1
H), 3.04ꢀ3.10 (m, 2H), 3.17ꢀ3.26 (m, 1H), 3.87 (s, 3H) 3.93 (s, 3H), 6.83 (s, 1H), 7.13 (s,
13
H). C NMR (75 MHz, CDCl ) δ 32.7, 33.3, 34.0, 34.7, 39.2, 45.1, 46.5, 56.1, 56.2, 104.3,
3
+
07.3, 129.3, 148.8, 149.4, 155.4, 207.8. MS (ESI+): m/z (%): 290 (100) [M+H] .
3-Hydroxy-6-methylpicolinaldehyde (12)
To a solution of 2ꢀ(hydroxymethyl)ꢀ6ꢀmethylpyridinꢀ3ꢀol 11 (2 g, 14.3 mmol) in toluene (150
mL) was added MnO (10 g), and the suspension was stirred at 80 °C until all starting
2
2
material was consumed (reaction followed by TLC). After cooling, MnO was removed by
filtration through celite and the solvent was evaporated under reduced pressure to afford the
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1
desired product 12 (1.49 g, 76%) as yellow solid. H NMR (300 MHz, CDCl
3
) δ 2.55 (s, 3H),
) δ 23.6, 126.6,
13
7
.25ꢀ7.29 (m, 2H), 10.02 (s, 1H), 10.65 (s, 1H). C NMR (75 MHz, CDCl
3
+
1
30.5, 135.7, 151.1, 157.0, 198.8. MS (ESI+): m/z (%): 138 (100) [M+H] .
3-(Methoxymethoxy)-6-methylpicolinaldehyde (13)
To a cooled (0 °C) solution of 3ꢀhydroxyꢀ6ꢀmethylpicolinaldehyde 12 (1.5 g, 10.9 mmol) and
CO (6 g, 43.4 mmol, 4 equiv.) in acetone (110 mL) at 0 °C was added dropwise MOMCl
16 mL, 32.6 mmol, 3 equiv.).The resulting mixture was stirred for 16 h at rt. Filtration of
K
2
3
(
K
2
CO
3
salt and concentration under reduced pressure afforded the good product 13 as
1
yellow liquid (1.91 g, 95%). H NMR (300 MHz, CDCl
3
) δ 2.56 (s, 3H), 3.49 (s, 3H), 5.28 (s,
1
3
2
H), 7.29 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 10.33 (s, 1H). C NMR (75 MHz,
CDCl
3
) δ 23.6, 56.6, 94.9, 124.7, 128.7, 140.5, 152.3, 154.1, 190.4. MS (ESI+): m/z (%): 182
+
(
100) [M+H] .
3-(Methoxymethoxy)-6-methylpicolinaldehyde O-tert-butyldiphenylsilyl oxime (14)
To a solution of 3ꢀ(methoxymethoxy)ꢀ6ꢀmethylpicolinaldehyde 13 (1.91 g, 10.5 mmol), in dry
EtOH (100 mL) were successively added NH OH.HCl (806 mg, 11.6 mmol, 1.1 equiv.) and
2
NaOAc (1.03 g, 12.6 mmol, 1.2 equiv.). The resulting mixture was stirred for 1 h at rt. Salts
were removed by filtration and the solution was concentrated under reduced pressure. To a
2 2
solution of crude product in dry CH Cl (160 mL) were successively added imidazole (1.3 g,
19.1 mmol, 1.9 equiv.) and TBDPSCl (4 mL, 15.3 mmol, 1.5 equiv.). The mixture was stirred
at rt for 18 h under argon atmosphere. Concentration under reduced pressure and
chromatography on silica gel (cyclohexane/EtOAc 9:1, v/v) afforded access to the desired
1
compound 14 as yellow oil (2.7 g, 76%). H NMR (300 MHz, CDCl ) δ 1.08 (s, 9H), 2.50 (s,
3
1
3
3
H), 3.27 (s, 2H), 4.96 (s, 2H), 7.26ꢀ7.40 (m, 8H), 7.71ꢀ7.80 (m, 4H), 8.64 (s, 1H). C NMR
(
75 MHz, CDCl ) δ 19.7, 27.2, 27.3, 56.2, 94.9, 127.6, 127.9, 129.7, 133.9, 134.9, 135.8,
3
+
1
40.7, 150.6, 151.7. MS (ESI+): m/z (%): 435 (100) [M+H] .
6-((4-((5,6-Dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)methyl)piperidin-1-yl)methyl)-3-
hydroxypicolinaldehyde oxime (1)
To a solution of compound 14 (1.2 g, 2.8 mmol) and NBS (541 mg, 3 mmol, 1.1 equiv.) in
CCl (30 mL) was added benzoyl peroxide (68 mg, 0.28 mmol, 0.1 equiv.). The mixture was
stirred 2 days at 75 °C. CH Cl was added and the organic layer was washed twice with
4
2
2
water, dried over MgSO
4
and concentrated under reduced pressure. To a solution of crude
product in DMF (20 mL) were added 10 (491 mg, 1.7 mmol) and K CO (708 mg, 5.13 mmol,
2
3
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3
equiv.). The solution was stirred at 60 °C for 24 h. DMF was concentrated and the crude
product was dissolved in CH Cl (15 mL). To this solution was added TFA (3 mL) and the
2
2
mixture was stirred at rt for 2 h. Solvent and the excess of TFA were removed by evaporation
under reduced pressure. Purifications (twice) by semiꢀpreparative RPꢀHPLC afforded the
1
desired compound 1 (0.97 mg, 0.7%) as yellow solid. H NMR (300 MHz, CD
3
OD) δ 1.35ꢀ
1.58 (m, 6H), 1.83ꢀ2.05 (m, 4H), 2.72ꢀ2.94 (m, 2H), 2.86ꢀ2.94 (m, 1H), 3.85 (s, 3H) 3.93 (s,
1
3
3
H), 4.20 (s, 2H), 7.05 (s, 1H), 7.13 (s, 1H), 7.38 (s, 2H), 8.37 (s,1H). C NMR (75 MHz,
OD) δ 30.2, 30.8, 31.2, 33.4, 34.1, 39.0, 46.3, 54.9, 56.5, 56.7, 61.6, 105.3, 109.0, 116.2,
20.1, 126.0, 126.9, 129.8, 138.0, 151.1, 151.3, 151.9, 157.8, 209.9. MS (ESI+): m/z (%):
CD
3
1
4
4
+
+
40 (100) [M+H] . HPLC: t
R 30 3 5
= 26.08 min, purity = 95%. HRMS (ESI+) calcd for C24H N O :
40.1938; found: 440.1942.
tert-Butyl 4-((5,6-dimethoxy-2,3-dihydro-1
15)
H-inden-2-yl)methyl)piperidine-1-carboxylate
(
To a solution of 8 (1.2 g, 3.1 mmol) in degassed methanol (50 mL) was added palladium on
carbon (10 wt%, 0.33 g, 0.31 mmol, 0.1 equiv.). The solution was bubbled with H and stirred
at rt under H atmosphere (1 atm) for 2 h. The reaction mixture was then bubbled with argon
2
2
for 15 min and palladium was removed by filtration over celite. Concentration under reduced
1
pressure afforded compound 15 (1.05 g, 85%) as a whiteꢀyellow solid. H NMR (300 MHz,
CDCl
H), 2.64ꢀ2.72 (m, 2H), 2.92ꢀ2.99 (m, 2H), 3.82 (s, 6H), 4.06ꢀ4.09 (m, 2H), 6.71 (s, 2H).
NMR (75 MHz, CDCl ) δ 28.5, 32.4, 34.8, 37.6, 39.5, 42.8, 44.1, 56.1, 79.2, 107.8, 134.8,
3
) δ 1.04ꢀ1.16 (m, 2H), 1.41ꢀ1.48 (m, 3H), 1.44 (s, 9H), 1.65ꢀ1.70 (m, 2H), 2.46ꢀ2.58 (m,
13
3
C
3
+
+
1
47.8, 154.9. MS (ESI+): m/z (%): 376 (100) [M+H] , 393 (50) [M+H O] .
2
4-((5,6-Dimethoxy-2,3-dihydro-1
H-inden-2-yl)methyl)piperidine (16)
Compound 15 (1 g, 2.7 mmol) was added to a solution of TFA in CH Cl (20% v/v, 24 mL) at
2
2
0
°C. The mixture was stirred for 1.5 h and was then allowed to warm at rt. TFA and CH
were removed by evaporation using a water pump and the residue was dissolved into an
aqueous saturated solution of NaHCO . The aqueous layer was then extracted three times
with CH Cl and the organic layers were combined and dried over MgSO . CH Cl was
2 2
Cl
3
2
2
4
2
2
removed under reduced pressure to afford the desired product 16 (550 mg, 75%) as a brown
1
solid. H NMR (300 MHz, CDCl ) δ 1.08ꢀ1.15 (m, 2H), 1.41ꢀ1.44 (m, 3H), 1.68ꢀ1.72 (m, 3H),
3
13
2
.45ꢀ2.63 (m, 5H), 2.91ꢀ2.98 (m, 2H), 3.03ꢀ3.07 (m, 2H), 3.82 (s, 6H), 6.72 (s, 2H). C NMR
(
75 MHz, CDCl ) δ 33.9, 35.0, 37.4, 39.5, 43.6, 46.9, 56.0, 107.7, 135.0, 147.7. MS (ESI+):
3
+
m/z (%): 276 (100) [M+H] .
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Methyl 3-(methoxymethoxy)-6-methylpicolinate (17)
To a cooled solution (0 °C) of 13 (1.54 g, 8.49 mmol) in MeOH (30 mL) were added a
solution of KOH (1.24 g, 22.07 mmol, 2.6 equiv.) in MeOH (30 mL) and a solution of I (2.8 g,
1.03 mmol, 1.3 equiv.) in MeOH (30 mL). The resulting mixture was allowed to warm at rt
2
1
3
and was stirred for 4 h. The reaction was quenched with a solution of NaHSO until the
brown color disappeared. MeOH was concentrated under reduced pressure and the aqueous
layer was extracted twice with EtOAc. The combined organic layers were dried over MgSO
Concentration under reduced pressure and chromatography on silica gel
4
.
(
cyclohexane/EtOAc 7:3, v/v) afforded the desired compound 17 (1.4 g, 78%) as brown solid.
1
3
H NMR (300 MHz, CDCl ) δ 2.55 (s, 3H), 3.50 (s, 3H), 3.97 (s, 3H), 5.23 (s, 2H), 7.23 (d, J =
1
3
8
.6 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H). C NMR (75 MHz, CDCl
3
) δ 23.5, 52.6, 56.3, 95.2,
125.0, 126.5, 139.2, 150.7, 151.3, 165.5.
Methyl-6-((4-((5,6-dimethoxy-2,3-dihydro-1
methoxymethoxy)picolinate (18)
H-inden-2-yl)methyl)-3-
(
To a solution of 17 (250 mg, 1.18 mmol) in CCl
4
(12 mL) was added Nꢀbromosuccinimide
221 mg, 1.24 mmol, 1.05 equiv.). The resulting mixture was irradiated with a halogen lamp
300 W) for 1 h. The organic layer was washed three times with brine, dried over MgSO and
CN (15 mL)
(490 mg, 3.54 mmol, 3.5 equiv.) and the
resulting mixture was refluxed overnight. After cooling, K CO was removed by filtration
(
(
4
concentrated under reduced pressure. To a solution of this crude product in CH
were added 16 (270 mg, 1 mmol) and K CO
3
2
3
2
3
through celite and the solvent was evaporated under reduced pressure. Purification by flash
chromatography on silica gel (EtOAc 100%) afforded desired compound 18 (190 mg, 40%)
1
as a white solid. H NMR (300 MHz, CDCl
3
) δ 1.26ꢀ1.42 (m, 5H), 1.65ꢀ1.69 (m, 2H), 2.00ꢀ
2
3
3
1
.08 (m, 2H), 2.46ꢀ2.52 (m, 3H), 2.83ꢀ2.95 (m, 4H), 3.48 (s, 3H), 3.61 (s, 2H), 3.80 (s, 6H),
13
3
.94 (s, 3H), 5.22 (s, 2H), 6.70 (s, 2H), 7.54ꢀ7.57 (m, 2H). C NMR (75 MHz, CDCl ) δ 32.5,
2.6, 34.4, 37.8, 39.5, 42.9, 52.7, 54.0, 54.1, 56.1, 56.5, 64.0, 95.2, 108.0, 128.8, 126.2,
+
35.0, 139.3, 147.8, 151.5, 152.4, 165.7. MS (ESI+): m/z (%): 485 (100) [M+H] .
6-((4-((5,6-Dimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)piperidin-1-yl)methyl)-3-
hydroxypicolinaldehyde oxime (2)
To a solution of 18 (190 mg, 0.39 mmol) in dry CH Cl (10 mL) was added DIBALꢀH (1 M in
2
2
2 2
CH Cl , 0.78 mL, 0.78 mmol, 2 equiv.) under inert atmosphere at ꢀ78 °C. The reaction
mixture was stirred at this temperature for 3 min (an extended time of reaction gave a
mixture of the corresponding aldehyde and alcohol). MeOH (0.78 mL) was added and the
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mixture was allowed to warm to rt. The organic layer was washed with an aqueous solution
of NaOH 1 M, dried over MgSO and concentrated under reduced pressure. To the obtained
residue was added a solution of TFA (20% v/v in CH Cl , 10 mL) and the mixture was stirred
for 2 h at rt. Concentration by using a water pump gave the desired intermediate. To a
solution of the crude product in MeOH (5 mL) were added NH OH.HCl (27 mg, 0.39 mmol)
4
2
2
2
and NaOAc (320 mg, 3.9 mmol, 10 equiv.) and the resulting mixture was stirred overnight at
rt. Concentration under reduced pressure and purification by flash chromatography (EtOAc
1
1
00%) afforded the desired compound 2 (25 mg, 15% over 3 steps) as a yellow solid. H
3
NMR (300 MHz, CDCl ) δ 1.20ꢀ1.50 (m, 5 H), 1.65ꢀ1.80 (m, 2H), 2.10ꢀ2.17 (m, 2H), 2.46ꢀ
2.56 (m, 3H), 2.92ꢀ3.03 (m, 4H), 3.61 (s, 2H), 3.84 (s, 6H), 6.72 (s, 2H), 7.21 (m, 2H), 8.34
13
(
s, 1H), 9.79 (bs, 1H). C NMR (75 MHz, CDCl
3
) δ 29.8, 31.7, 34.3, 37.8, 39.6, 42.6, 54.2,
56.2, 64.1, 107.9, 124.2, 125.6, 135.0, 135.8, 147.9, 153.7. MS (ESI+): m/z (%): 426 (100)
+
+
[
M+H] . HRMS (ESI+) calcd for C24
32 3 4 R
H N O : 426.2387; found: 426.2386. HPLC: t = 26.3
min, purity = 97%.
5,6-Dimethoxy-2-(prop-2-ynyl)-2,3-dihydro-1H-inden-1-one (20)
To a cooled (0 °C) solution of diisopropylamine (170 ꢂL, 1.2 mmol, 1.2 equiv.) in THF (10
mL) was added nꢀBuLi (1.6 M in hexane, 750 ꢂL, 1.2 mmol, 1.2 equiv.). The mixture was
stirred 15 min before cooled to ꢀ78 °C. A solution of 5,6ꢀdimethoxyindanone 19 (192 mg, 1
mmol) in THF solution (5 mL) was added and the reaction mixture was stirred 15 min at ꢀ78
°C. Then, propargyl bromide (170 ꢂL, 80 wt% in toluene, 1.5 mmol, 1.5 equiv.) was added
and the solution was allowed to warm at rt. The mixture was concentrated and purified by
flash chromatography on silica gel (cyclohexane/EtOAc 3:7, v/v) to give 20 (111 mg, 48%) as
1
a white solid. H NMR (300 MHz, CDCl
3
) δ 1.87 (t, J = 2.7 Hz, 1H), 2.44ꢀ2.56 (m, 1H), 2.73
(
ddd, J = 2.7, 4.5, 16.8 Hz, 1H), 2.78ꢀ2.87 (m, 1H), 2.97 (dd, J = 4.5, 16.8 Hz, 1H), 3.28 (dd,
13
J = 4.5, 16.8 Hz, 1H), 3.88 (s, 3H), 3.95 (s, 3H), 6.87 (s, 1H), 7.15 (s, 1H). C NMR (75 MHz,
CDCl ) δ 20.1, 31.7, 45.7, 56.0, 56.2, 69.5, 81.3, 104.2, 107.3, 129.0, 149.0, 149.4, 155.6,
3
204.8.
Methyl
3-(benzyloxy)-6-(3-(5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)prop-1-
ynyl)picolinate (22)
To a Schlenk tube were added 20 (400 mg, 1.74 mmol), bromopyridine 21 (560 mg, 1.74
17
mmol) (prepared as reported in the literature ), CuI (32 mg, 0.174 mmol, 0.1 equiv.),
Pd(PPh ) (100 mg, 0.087 mmol, 0.05 equiv.), degassed THF (5 mL) and NEt (2.5 mL). The
3
4
3
resulting mixture was stirred at rt for 15 h under argon atmosphere. After concentration under
reduced pressure, the residue was purified by flash chromatography on silica gel
18

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1
(
cyclohexane/EtOAc 1:1, v/v) to give 22 (600 mg, 73%) as a beige solid. H NMR (300 MHz,
CDCl ) δ 2.70 (dd, J = 8.4, 16.5 Hz, 1H), 2.92ꢀ3.12 (m, 3H), 3.36 (dd, J = 7.5, 17.1 Hz, 1H),
.93 (s, 3H), 3.98 (s, 3H), 3.99 (s, 3H), 5.21 (s, 2H), 6.92 (s, 1H), 7.20 (s, 1H), 7.25ꢀ7.50 (m,
3
3
7
1
1
13
3
H). C NMR (75 MHz, CDCl ) δ 21.3, 32.1, 46.0, 56.1, 56.3, 70.7, 80.2, 87.3, 104.3, 107.4,
21.6, 126.9, 128.2, 128.7, 128.9, 130.2, 135.0, 135.4, 140.0, 149.1, 149.5, 153.0, 155.7,
64.8, 204.9.
3-Hydroxy-6-(3-(1-hydroxy-5,6-dimethoxy-2,3-dihydro-1H-inden-2-
yl)propyl)picolinaldehyde (23)
To a solution of 22 (300 mg, 0.64 mmol) in degassed EtOAc (20 mL) was added Pearlman's
catalyst (182 mg, 0.13 mmol, 0.2 equiv., 20 wt% Pd, moisture 50%). The solution was
2 2
bubbled with H and the reaction was stirred at rt under H atmosphere (1 atm) for 1 h. The
mixture was filtrated through celite and concentrated under reduced pressure. To a solution
of the residue in dry DMF (3 mL) were successively added imidazole (122 mg, 1.8 mmol, 3
equiv.) and TBDMSCl (99 mg, 0.66 mmol, 1.1 equiv.). The mixture was stirred at rt for 2 h
under argon atmosphere. The organic layer was washed with brine (5 times), dried over
MgSO
4
and concentrated under reduced pressure. To a solution of the resulting residue in
Cl (10 mL) was added dropwise DIBALꢀH (2.4 mL, 1 M in CH Cl , 2.4 mmol, 4
dry CH
2
2
2
2
equiv.) at ꢀ78 °C. Then, the reaction mixture was stirred at this temperature for 10 min. The
reaction was quenched with MeOH (2.4 mL) and the mixture was allowed to warm at rt. The
4
organic layer was washed with an aqueous solution of NaOH 1 M, dried over MgSO and
concentrated under reduced pressure. Then, TBAF (660 ꢂL, 0.66 mmol, 1.1 equiv., 1 M in
THF) was added at 0 °C to the residue in dry THF (20 mL) and the mixture was stirred for 30
min at this temperature. After concentration under reduced pressure, a purification by flash
chromatography on silica gel (EtOAc/MeOH 1:1, v/v) afforded 23 (112 mg, 50%) as a
colourless oil. The product was isolated as a mixture of 2 diastereoisomers (Aꢀ75% and Bꢀ
1
2
5%). H NMR (300 MHz, CDCl ) δ 1.59ꢀ1.65 (m, 1HꢀA + 1HꢀB), 1.75ꢀ2.06 (m, 2HꢀA + 2HꢀB),
3
2.11ꢀ2.14 (m, 1HꢀA), 2.17ꢀ2.28 (1HꢀB), 2.37ꢀ2.51 (m, 1HꢀA + 1HꢀB), 2.70 (dd, J = 8.7, 15.6
Hz, 1HꢀA), 2.85ꢀ2.95 (m, 2HꢀA + 2HꢀB), 3.10 (dd, J = 8.7, 15.3 Hz, 1HꢀB), 3.88 (s, 3HꢀA +
HꢀB), 3.90 (s, 3HꢀB), 3.91 (s, 3HꢀA), 4.71 (br t, J = 5.1 Hz, 1HꢀB), 5.06 (br t, J = 5.1 Hz, 1Hꢀ
A), 6.73 (s, 1HꢀB), 6.77 (s, 1HꢀA), 6.92 (s, 1HꢀB), 6.99 (s, 1HꢀA), 7.30ꢀ7.40 (m, 2HꢀA + 2HꢀB),
3
10.07 (s, 1HꢀB), 10.09 (s, 1HꢀA), 10.73 (br s, 1HꢀA + 1HꢀB).
6-(3-(5,6-Dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)propyl)-3-hydroxypicolinaldehyde
(
24)
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To a solution of 23 (50 mg, 0.14 mmol) and pyridine (57 ꢂL, 0.7 mmol, 5 equiv.) in
dichloromethane (5 mL) under argon atmosphere was added Dess Martin periodinane (15
wt% in dichloromethane, 300 ꢂL, 0.14 mmol). The mixture was stirred for 1 h at rt and then
concentrated and purified by flash chromatography on silica gel (cyclohexane/EtOAc 7/3, v/v)
1
to give 24 (35 mg, 72%) as a white solid. H NMR (300 MHz, CDCl
3
) δ 1.48ꢀ1.54 (m, 1H),
1.74ꢀ1.89 (m, 2H), 1.90ꢀ2.03 (m, 1H), 2.58ꢀ2.94 (m, 4H), 3.17 (dd, J = 7.5, 16.8 Hz, 1H), 3.83
(
s, 3H), 3.89 (s, 3H), 6.79 (s, 1H), 7.09 (s, 1H), 7.19ꢀ7.27 (m, 2H), 9.95 (s, 1H), 10.58 (br s,
1
3
1
3
H). C NMR (75 MHz, CDCl ) δ 27.4, 31.3, 32.6, 37.2, 47.5, 56.1, 56.2, 104.3, 107.4,
126.5, 129.4, 129.9, 135.7, 149.0, 149.4, 154.4, 155.5, 157.0, 198.7, 207.4. MS (ESI+) m/z
+
(
%): 356 [M+H] .
6-(3-(5,6-Dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)propyl)-3-hydroxypicolinaldehyde
oxime (3)
To a solution of 24 (33 mg, 0.093 mmol) in absolute EtOH (5 mL) were added successively
.
HONH
2
HCl (6.5 mg, 0.093 mmol) and NaOAc (7.6 mg, 0.093 mmol). The mixture was stirred
at rt for 30 min under argon atmosphere. After concentration under reduced pressure, the
residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 1:1, v/v) to
1
give 3 (26 mg, 76%) as a white solid. H NMR (300 MHz, CDCl
3
) δ 1.43ꢀ1.56 (m, 1H), 1.77ꢀ
1.86 (m, 2H), 1.91ꢀ2.02 (m, 1H), 2.64ꢀ2.81 (m, 4H), 3.21 (dd, J = 7.5, 17.1 Hz, 1H), 3.88 (s,
3H), 3.94 (s, 3H), 6.83 (s, 1H), 7.04 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H),
13
8
.43 (s, 1H), 9.83 (bs, 2H). C NMR (75 MHz, CDCl
3
) δ 27.8, 31.2, 32.6, 37.2, 47.6, 56.1,
56.3, 104.3, 107.4, 124.4, 125.0, 129.3, 134.6, 149.3, 149.4, 152.7, 153.1, 153.9, 155.6,
+
+
2
08.0. MS (ESI+) m/z (%): 371 [M+H] . HRMS (ESI+) calcd for C H N O : 371.1607;
20 23 2 5
R
found: 371.1607. HPLC: t = 23.1 min, purity = 95%.
Methyl
3-(benzyloxy)-4-(3-(5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)prop-1-
ynyl)picolinate (26)
2
6
To a Schlenk tube were added alkyne 20 (115 mg, 0.5 mmol), bromopyridine 25 (161 mg, 1
equiv.), CuI (19 mg, 0.2 equiv.), Pd(PPh (58 mg, 0.1 equiv.) and degassed THF (5 mL)
3 4
)
and NEt (2.5 mL). The resulting mixture was stirred at rt for 15 h under argon atmosphere.
3
After concentration under reduced pressure, the residue was purified by flash
chromatography on silica gel (EtOAc/cyclohexane 2:3, v/v) to give 26 (187 mg, 79%) as a
1
yellow oil. H NMR (300 MHz, CDCl
3
)
δ
(ppm) 2.70ꢀ2.90 (m, 3 H), 3.00ꢀ3.15 (m, 1H), 3.20ꢀ
3.40 (m, 1H), 3.81 (s, 3H), 3.87 (s, 3H), 3.94 (s, 3H), 5.03 (s, 2H), 6.78 (s, 1H), 7.07 (s, 1H),
13
7
.20ꢀ7.40 (m, 5H), 7.49 (d, J = 4.8 Hz, 1H), 8.39 (d, J = 4.8 Hz, 1H). C NMR (75 MHz,
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CDCl3)
δ
(ppm) 21.6, 31.8, 45.7, 52.8, 56.1, 56.2, 66.4, 72.3, 76.3, 98.4, 104.2, 107.3, 128.2,
128.3, 128.8, 130.2, 136.5, 144,5, 148.9, 149.5, 155.8, 165.2, 204.6.
Methyl
4-(3-(5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)propyl)-3-
hydroxypicolinate (27)
To a solution of 26 (185 mg, 0.39 mmol) in degassed EtOAc (20 mL) was added Pearlman's
catalyst (112 mg, 0.2 equiv., 20% Pd, moisture 50%). The solution was bubbled with H and
the reaction was stirred at rt under H atmosphere (1 atm) for 1 hour. The mixture was
2
2
filtrated through celite and concentrated under reduced pressure to afforded 27 (185 mg,
1
9
9%) as a beige solid. H NMR (300 MHz, CDCl )
δ
(ppm) 1.39 – 1.53 (m, 1H), 1.63 – 1.76
3
(m, 2H), 1.84 – 1.97 (m, 1H), 2.56 – 2.71 (m, 4H), 3.16 (dd, J = 7.5, 17.1 Hz, 1H), 3.81 (s,
3H), 3.87 (s, 3H), 3.94 (s, 3H), 6.78 (s, 1H), 7.07 (s, 1H), 7.19 (d, J = 4.5 Hz, 1H), 8.07 (d, J
1
3
=
3
4.5 Hz, 1H), 10.80 (br s, 1H). C NMR (75 MHz, CDCl ) δ (ppm) 26.2, 29.2, 31.3, 32.5,
4
7.3, 53.1, 56.0, 56.2, 104.2, 107.3, 128.9, 129.3, 129.4, 132.0, 140.5, 141.0, 148.9, 149.3,
55.4, 157.5, 170.3, 207.3.
1
4-(3-(5,6-Dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)propyl)-3-hydroxypicolinaldehyde
(
28)
2 2
To a solution of 27 (145 mg, 0.38 mmol) in dry CH Cl (20 mL) were successively added 2,6ꢀ
lutidine (132 ꢂL, 3 equiv.) and TBDMSOTf (96 ꢂL, 1.1 equiv.). The mixture was stirred at rt
for 15 h under argon atmosphere. The organic layer was washed with water (once), dried
over MgSO
in dry CH Cl
C. Then, the reaction mixture was stirred at this temperature for 10 min. The reaction was
4
and concentrated under reduced pressure. To a solution of the resulting residue
2
2
(10 mL) was added dropwise DIBALꢀH (760 ꢂL, 1 M in CH Cl , 2 equiv.) at ꢀ78
2
2
°
quenched with MeOH (760 ꢂL) and the mixture was allowed to warm at rt. The organic layer
was washed with an aqueous solution of NaOH (1 M), dried over MgSO and concentrated
4
under reduced pressure. Then, TBAF (420 ꢂL, 1 M in THF, 1.1 equiv.) was added at 0 °C to
the residue in dry THF (20 mL) and the mixture was stirred for 30 min at this temperature.
After concentration under reduced pressure, the crude product was purified by a flash
chromatography on silica gel (cyclohexane/EtOAc 1:1 v/v) to afford the compound 28 (4.0
1
mg, 3%) as a colorless oil. H NMR (300 MHz, CDCl )
δ (ppm) 1.56 – 1.61 (m, 1H), 1.68 –
3
1.83 (m, 2H), 1.93 – 2.04 (m, 1H), 2.66 – 2.78 (m, 4H), 3.25 (dd, J = 8.4, 17.1 Hz, 1H), 3.90
(
s, 3H), 3.96 (s, 3H), 6.86 (s, 1H), 7.16 (s, 1H), 7.29 (d, J = 4.5 Hz, 1H), 8.24 (d, J = 4.5 Hz,
1
3
1
H), 10.06 (s, 1H), 10.98 (br s, 1H). C NMR (75 MHz, CDCl3) δ (ppm) 26.2, 28.8, 31.4,
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3
2.6, 47.3, 56.1, 56.3, 104.3, 107.4, 129.4, 129.9, 135.9, 140.5, 142.4, 148.9, 149.5, 155.6,
57.5, 199.0, 207.3.
1
4-(3-(5,6-Dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)propyl)-3-hydroxypicolinaldehyde
oxime (4)
To a solution of 28 (4 mg, 0.011 mmol) in absolute EtOH (5 mL) were added successively
.
HONH
2
HCl (0.78 mg, 1 equiv.) and NaOAc (0.92 mg, 1 equiv.). The mixture was stirred at rt
for 30 min under argon atmosphere. After concentration under reduced pressure, the residue
was purified by flash chromatography on silica gel (cyclohexane/EtOAc 1:1 v/v) to give 4 (4.3
1
mg, 99%) as a white solid. H NMR (300 MHz, CDCl
3
)
δ
(ppm) 1.43 – 1.57 (m 1H), 1.66 –
1.80 (m, 2H), 1.92 – 2.04 (m, 1H), 2.64 – 2.74 (m, 4H), 3.21 (dd, J = 5.1, 17.4 Hz, 1H), 3.87
(
s, 3H), 3.93 (s, 3H), 6.84 (s, 1H), 7.04 (d, J = 4.5 Hz, 1H), 7.14 (s, 1H), 8.06 (d, J = 4.5 Hz,
1
3
1
2
1
3
H), 8.42 (s, 1H), 9.81 (br s, 2H), 10.14 (s, 1H). C NMR (75 MHz, CDCl ) δ (ppm) 26.5,
9.3, 31.5, 32.6, 47.5, 56.1, 56.3, 104.3, 107.4, 125.3, 129.3, 134.8, 138.8, 140.9, 149.2,
+
49.4, 153.1, 154.0, 155.6, 208.0. MS (ESI+) m/z (%): 371 [M+H] . HRMS (ESI+) calcd for
+
C
20
H
23
N
2
O
5
: 371.1607; found: 371.1602. HPLC : t
R
= 24.8 min (purity = 93%).
Inhibition of hAChE and hBChE by OPNAs.
Recombinant hAChE and hBChE were produced and purified as previously described.
32, 33
VX and tabun were from DGA maîtrise NRBC (Vert le Petit, France). Paraoxonꢀethyl was
purchased from SigmaꢀAldrich. Isopropyl pꢀnitrophenyl methylphosphonate (INMP) was used
3
4
as surrogate of sarin. HIꢀ6 was from Pharmacie Centrale des Armées (Orléans, France). All
other chemicals including paraoxon were from Sigma. Stock solution of VX and tabun were 5
mM in isopropanol. The inhibition of 120 ꢂM hAChE or 100 ꢂM of hBChE is realized with a 5ꢀ
fold excess of OPNAs and was performed in tris buffer (20 mM, pH 7.4, 0.1% BSA) at 25 °C.
After 20 minutes incubation, inhibited hAChE or hBChE was desalted on PDꢀ10 column (GE
Healthcare).
IC50 measurements.
Compounds 1-4 were dissolved in MeOH to make 5 mM stock solutions. Recombinant
hAChE activity was measured spectrophotometrically (absorbance at 412 nm) in the
presence of various concentrations of oximes in 1 mL Ellman’s buffer (sodium phosphate 0.1
22

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M, pH 7.4, 0.1% BSA, 5% MeOH, 0.1 mg/mL DTNB, 25 °C) and 1 mM acetylthiocholine. The
concentration of compound 1 producing 50% of enzyme inhibition was determined by nonꢀ
linear fitting using ProFit 7.0 (Quantumsoft) using the standard IC50 equation:
%Activity=100*IC50/(IC50 + [Ox]).
Reactivation of hAChE and hBChE inhibited by OPNAs.
OPNAꢀinhibited hAChE was incubated at 37 °C with different concentrations of oxime in
.1 M phosphate buffer, pH 7.4, 0.1% BSA, 5% methanol. Methanol was used for complete
0
dissolution of the oximes. 10ꢀꢂl aliquots of mix was transferred to 1ꢀmL cuvettes (100ꢀfold
dilution) at time intervals ranging from 1 to 10 minutes depending on the reactivation rate, for
measurement of hAChE activity (1 mM acetylthiocholine) or hBChE activity (1 mM
butyrylthiocholine), in Ellman’s buffer (phosphate 0.1 M, pH 7.4, 0.1% BSA, 0.5 mM DTNB,
3
5
2
5 °C). The increase in absorbance at 412 nm was followed on a Uvikon 943
spectrophotometer.
The enzyme activity in the control (nonꢀinhibited enzyme) remained constant during the
experiment. To take into account the significant inhibition effect of compound 1 at the tested
concentration, the later was added in the control at a concentration matching that of the
sample. The percentage of reactivated enzyme (%Ereact) was calculated as the ratio of the
recovered enzyme activity and activity in the control. The apparent reactivation rate kobs for
each oxime concentration, the dissociation constant K of inhibited enzymeꢀoxime complex
D
(
r
EꢀPOx) and the maximal reactivation rate constant k , were calculated by nonꢀlinear fit using
ProFit 7.0 (Quantumsoft) and the standard oxime concentrationꢀdependent reactivation
equation derived from the following scheme:
Acknowledgments
This work was supported by Agence Nationale pour la Recherche (ANR_09_BLAN_0192
ReAChE program and ANRꢀ13ꢀASTRꢀ0002ꢀ02 ReCNSꢀAChE program), Defense Threat
Reduction Agency (DTRA) (contract number HDTRA1ꢀ11ꢀCꢀ0047) and Direction Générale
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de l’Armement (through Ph.D. fellowship to T.V., postdoctoral fellowship REIꢀDGA 2009ꢀ34ꢀ
023 to G.M., and BioMeDef action PDHꢀ2ꢀNRBCꢀ4ꢀCꢀ403 to F.N.). This work was partially
supported by INSA Rouen, Rouen University, Centre National de la Recherche Scientifique
CNRS) and Region HauteꢀNormandie (CRUNCh network).
0
(
Note and references
1
.
.
M. Eddleston, N. A. Buckley, P. Eyer and A. H. Dawson, Lancet, 2008, 371, 597ꢀ607.
P. Taylor, in Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12th
Edition, eds. L. Brunton, B. Chabner and B. Knollman, McGrawꢀHill, 2011, pp. 239ꢀ
2
254.
3.
R. E. Langford, Introduction to Weapons of Mass Destruction: Radiological, Chemical,
and Biological, John Wiley & Sons, 2004.
4.
5.
6.
7.
I. B. Wilson and B. Ginsburg, Biochim. Biophys. Acta, 1955, 18, 168ꢀ170.
F. Hobbiger, Br. J. Pharmacol., 1957, 12, 438ꢀ446.
F. Hobbiger, D. G. O'Sullivan and P. W. Sadler, Nature, 1958, 182, 1498ꢀ1499.
G. Mercey, T. Verdelet, J. Renou, M. Kliachyna, R. Baati, F. Nachon, L. Jean and P.
Y. Renard, Acc. Chem. Res., 2012, 45, 756ꢀ766.
8.
D. E. Lorke, H. Kalasz, G. A. Petroianu and K. Tekes, Curr. Med. Chem., 2008, 15,
743ꢀ753.
9
.
J. Kalisiak, E. C. Ralph and J. R. Cashman, J. Med. Chem., 2012, 55, 465ꢀ474.
J. Kalisiak, E. C. Ralph, J. Zhang and J. R. Cashman, J. Med. Chem., 2011, 54,
10.
3319ꢀ3330.
11.
M. C. de Koning, M. J. Joosen, D. Noort, A. van Zuylen and M. C. Tromp, Bioorg.
Med. Chem., 2011, 19, 588ꢀ594.
1
2.
3.
M. C. de Koning, M. van Grol and D. Noort, Toxicol. Lett., 2011, 206, 54ꢀ59.
Z. Radic, R. K. Sit, Z. Kovarik, S. Berend, E. Garcia, L. Zhang, G. Amitai, C. Green,
B. Radic, V. V. Fokin, K. B. Sharpless and P. Taylor, J. Biol. Chem., 2012, 287,
1
11798ꢀ11809.
14.
R. K. Sit, Z. Radic, V. Gerardi, L. Zhang, E. Garcia, M. Katalinic, G. Amitai, Z.
Kovarik, V. V. Fokin, K. B. Sharpless and P. Taylor, J. Biol. Chem., 2011, 286, 19422ꢀ
19430.
1
5.
6.
G. SaintꢀAndré, M. Kliachyna, S. Kodepelly, L. LouiseꢀLeriche, E. Gillon, P.ꢀY.
Renard, F. Nachon, R. Baati and A. Wagner, Tetrahedron, 2011, 67, 6352ꢀ6361.
L. LouiseꢀLeriche, E. Paunescu, G. SaintꢀAndre, R. Baati, A. Romieu, A. Wagner and
P. Y. Renard, Chem. Eur. J., 2010, 16, 3510ꢀ3523.
1
24

Page 25 of 27
RSC Advances
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DOI: 10.1039/C5RA25477A
1
7.
8.
G. Mercey, T. Verdelet, G. SaintꢀAndre, E. Gillon, A. Wagner, R. Baati, L. Jean, F.
Nachon and P. Y. Renard, Chem. Commun., 2011, 47, 5295ꢀ5297.
1
G. Mercey, J. Renou, T. Verdelet, M. Kliachyna, R. Baati, E. Gillon, M. Arboleas, M.
Loiodice, F. Nachon, L. Jean and P. Y. Renard, J. Med. Chem., 2012, 55, 10791ꢀ
10795.
19.
M. Kliachyna, G. Santoni, V. Nussbaum, J. Renou, B. Sanson, J.ꢀP. Colletier, M.
Arboléas, M. Loiodice, M. Weik, L. Jean, P.ꢀY. Renard, F. Nachon and R. Baati, Eur.
J. Med. Chem., 2014, 78, 455ꢀ467.
2
0.
1.
J. Renou, M. Loiodice, M. Arboleas, R. Baati, L. Jean, F. Nachon and P. Y. Renard,
Chem. Commun., 2014, 50, 3947ꢀ3950.
2
J. Renou, G. Mercey, T. Verdelet, E. Paunescu, E. Gillon, M. Arboleas, M. Loiodice,
M. Kliachyna, R. Baati, F. Nachon, L. Jean and P.ꢀY. Renard, Chem.-Biol. Interact.,
2
013, 203, 81ꢀ84.
H. Sugimoto, Y. Iimura, Y. Yamanishi and K. Yamatsu, J. Med. Chem., 1995, 38,
821ꢀ4829.
22.
4
2
3.
4.
G. Kryger, I. Silman and J. L. Sussman, Structure, 1999, 7, 297ꢀ307.
2
J. Cheung, M. J. Rudolph, F. Burshteyn, M. S. Cassidy, E. N. Gary, J. Love, M. C.
Franklin and J. J. Height, J. Med. Chem., 2012, 55, 10282ꢀ10286.
2
5.
6.
E. Keinan and N. Greenspoon, J. Am. Chem. Soc., 1986, 108, 7314ꢀ7325.
T. Verdelet, G. Mercey, N. Correa, L. Jean and P.ꢀY. Renard, Tetrahedron, 2011, 67,
2
8
757ꢀ8762.
F. Nachon, X. Brazzolotto, M. Trovaslet and P. Masson, Chem. Biol. Interact., 2013,
06, 536ꢀ544.
27.
28.
29.
30.
2
Z. Kovarik, M. Katalinić, G. Šinko, J. Binder, O. Holas, Y.ꢀS. Jung, L. Musilova, D. Jun
and K. Kuča, Chem. Biol. Interact., 2010, 187, 167ꢀ171.
G. R. Fulmer, A. J. M. Miller, N. H. Sherden, H. E. Gottlieb, A. Nudelman, B. M.
Stoltz, J. E. Bercaw and K. I. Goldberg, Organometallics, 2010, 29, 2176ꢀ2179.
S. Saitton, A. L. Del Tredici, M. Saxin, T. Stenstroem, J. Kihlberg and K. Luthman,
Org. Biomol. Chem., 2008, 6, 1647ꢀ1654.
3
1.
2.
T. G. Gant, S. Sarshar and M. M. Shahbaz, US20100143505A1, 2010.
E. Carletti, H. Li, B. Li, F. Ekstrom, Y. Nicolet, M. Loiodice, E. Gillon, M. T. Froment,
O. Lockridge, L. M. Schopfer, P. Masson and F. Nachon, J. Am. Chem. Soc., 2008,
3
130, 16011ꢀ16020.
3
3.
4.
X. Brazzolotto, M. Wandhammer, C. Ronco, M. Trovaslet, L. Jean, O. Lockridge, P.
Y. Renard and F. Nachon, FEBS J., 2012, 279, 2905ꢀ2916.
H. Ohta, T. Ohmori, S. Suzuki, H. Ikegaya, K. Sakurada and T. Takatori, Pharm.
Res., 2006, 23, 2827ꢀ2833.
3
25

RSC Advances
Page 26 of 27
View Article Online
DOI: 10.1039/C5RA25477A
35.
G. L. Ellman, K. D. Courtney, V. Andres, Jr. and R. M. FeatherꢀStone, Biochem.
Pharmacol., 1961, 7, 88ꢀ95.
26

Page 27 of 27
RSC Advances
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DOI: 10.1039/C5RA25477A
The synthesis and in vitro evaluation of four donepezil-based non quaternary reactivators are
reported. Oximes 1-3 show a better ability (8 fold higher) than pralidoxime to reactivate VX-
hAChE, and oxime 2 is 5 to 11 fold more efficient than pralidoxime and HI-6 respectively for
the reactivation of VX-hBChE.