A HTS assay for the detection of organophosphorus nerve agent scavengers

Article abstract of DOI:10.1002/chem.200902986

A new pro-fluorescent probe aimed at a HTS assay of scavengers is able to selectively and efficiently cleave the P-S bond of organophosphorus nerve agents and by this provides non-toxic phosphonic acid has been designed and synthesised. The previously described pro-fluorescent probes were based on a conventional activated P-Oaryl bond cleavage, whereas our approach uses a self-immolative linker strategy that allows the detection of phosphonothioase activity with respect to a non-activated P-Salkyl bond. Further, we have also developed and optimised a high-throughput screening assay for the selection of decontaminants (chemical or biochemical scavengers) that could efficiently hydrolyse highly toxic V-type nerve agents. A preliminary screening, realised on a small α-nucleophile library, allowed us to identify some preliminary "hits", among which pyridinealdoximes, α-oxo oximes, hydroxamic acids and, less active but more original, amidoximes were the most promising. Their selective phosphonothioase activity has been further confirmed by using PhX as the substrate, and thus they offer new perspectives for the synthesis of more potent V nerve agent scavengers.

Full text of DOI:10.1002/chem.200902986

DOI: 10.1002/chem.200902986
A HTS Assay for the Detection of Organophosphorus
Nerve Agent Scavengers
Ludivine Louise-Leriche,[a, b] Emilia Paˇunescu,[a, b] Gꢀraldine Saint-Andrꢀ,[c]
Rachid Baati,*[c] Anthony Romieu,[a, b] Alain Wagner,[c] and Pierre-Yves Renard*[a, b, d]
This paper is dedicated to the memory of the late Dr. Charles Mioskowski
À
Abstract: A new pro-fluorescent probe
aimed at a HTS assay of scavengers is
able to selectively and efficiently
with respect to a non-activated P
Salkyl bond. Further, we have also de-
veloped and optimised a high-through-
put screening assay for the selection of
decontaminants (chemical or biochemi-
cal scavengers) that could efficiently
hydrolyse highly toxic V-type nerve
agents. A preliminary screening, real-
ised on a small a-nucleophile library,
allowed us to identify some preliminary
“hits”, among which pyridinealdox-
imes, a-oxo oximes, hydroxamic acids
and, less active but more original, ami-
doximes were the most promising.
Their selective phosphonothioase activ-
ity has been further confirmed by using
PhX as the substrate, and thus they
offer new perspectives for the synthesis
À
cleave the P S bond of organophos-
phorus nerve agents and by this pro-
vides non-toxic phosphonic acid has
been designed and synthesised. The
previously described pro-fluorescent
probes were based on a conventional
Keywords: fluorescent probes · hy-
drolysis · nerve agent scavengers ·
organophosphonothioase activity ·
phosphorus
À
activated P Oaryl bond cleavage,
whereas our approach uses a self-im-
molative linker strategy that allows the
detection of phosphonothioase activity
of more potent
scavengers.
V
nerve agent
Introduction
ties aimed at preventing their proliferation, the ease of their
synthesis and the similarity between their synthetic precur-
sors and those of widely used pest-control agents have pre-
vented any efficient control of the use of these weapons of
mass destruction.[2] Moreover, the threat of their use
became reality as illustrated by both the 1995 Tokyo subway
strike with sarin and the spread of anthrax through the US
postal system in the fall 2001. The decontamination and de-
militarisation of OPNAs as well as the treatment of organo-
phosphorus (OP)-based pest agent poisonings (a yearly
average of 200000 lethal poisoning cases has been estimated
recently[3]) are therefore imperative. Most of the methods of
decontamination involve neutralising the nerve agents
(NAs) by using strong alkaline and/or oxidative media and
are thus incompatible with medical use and the treatment of
skin or other sensitive material.[4] In the last few years, con-
siderable work has been undertaken to develop new means
of chemical and/or biotechnological decontamination.[5]
Exceedingly toxic phosphonothioate VX (LD50 =8 mgkgÀ1
(i.v.) and 28 mgkgÀ1 (per-coetaneous, rabbit))[1] is by far the
most difficult target to deal with[4b] and so far no satisfactory
mild means of decontamination have been proposed be-
cause the hydrolytic efficiencies of the reported decontami-
Organophosphorus nerve agents (OPNAs) such as chemical
warfare agents sarin (1a), soman (1b), tabun (2) or the ex-
ceedingly toxic VX (3a) act as acetylcholine esterase
(AChE) irreversible inhibitors.[1] Despite international trea-
ˇ
[a] L. Louise-Leriche, E. Paunescu, A. Romieu, P.-Y. Renard
Equipe de Chimie Bio-Organique
COBRA—CNRS UMR 6014 & FR 3038
rue Tesniꢀre, 76130 Mont-Saint-Aignan (France)
Fax : (+33)235-52-29-59
E-mail: pierre-yves.renard@univ-rouen.fr
ˇ
[b] L. Louise-Leriche, E. Paunescu, A. Romieu, P.-Y. Renard
Universitꢁ de Rouen, IRCOF
rue Tesniꢀre, 76130 Mont Saint Aignan (France)
[c] G. Saint-Andrꢁ, R. Baati, A. Wagner
Laboratoire de Chimie des Systꢀmes Fonctionnels CNRS/UMR7199
Facultꢁ de Pharmacie, Universitꢁ de Strasbourg
BP 24, 67401 Illkirch (France)
Fax : (+33)3368-854-301
E-mail: baati@bioorga.u-strasbg.fr
[d] P.-Y. Renard
Institut Universitaire de France, 103
boulevard Saint Michel, 75005 Paris (France)
3510
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3510 – 3523

FULL PAPER
by the use of catalytic antibod-
ies.[5] Consequently, it became
of great importance to develop
HTS assays aimed at the iden-
tification of new scavengers
able to selectively and efficient-
À
À
À
ly cleave the P F, P CN or P
S bond under mild conditions.
nation agents towards V-type NAs like VX remain signifi-
cantly lower than for the less toxic G-type nerve and pest-
control agents.[6] This is mainly due to the relative stability
As far as the V agents are concerned, their hydrolysis releas-
es a thiol. Although this thiol rapidly dimerises under slight-
ly basic conditions, a popular thiol-sensitive chromogenic re-
agent such as Ellmanꢃs reagent[21] could be used in the HTS
assay. Yet, in our hands, Ellmanꢃs reagent suffers many limi-
tations, namely 1) it does not solve the issue of G agents,
2) it is not compatible with most of the thiol-containing cul-
ture media used for enzyme production and protein evolu-
tion, but most of all, 3) it has been shown not to be compati-
ble with some of the chemistry-based scavengers we have
used (see below), such as other thiols, oxidants or even
some a nucleophiles at basic pH (for example, by aromatic
nucleophilic substitution).
Reported assays for the evaluation of OP nerve agent hy-
drolysis are based either on the detection of the OP them-
selves,[22] of their degradation agents[23] or of the remaining
activity of the inhibited AChE.[24] Nevertheless, most of the
assays developed to detect enzymes displaying phosphatase
activity are based on fluorogenic, chromogenic or chemilu-
minescent substrates.[25] Some of them have been directly
applied to OP nerve agents, the leaving group being re-
placed with a phenol-based chromogenic or fluorescent unit.
The most widely used assay exploits the properties of para-
oxon, which releases yellow-coloured p-nitrophenol after hy-
drolysis (Figure 1). Soukharev and Hammond synthesised
and evaluated a stable fluorogenic substrate, the 7-diethoxy-
phosphinoyloxy-6,8-difluoro-4-methylcoumarin (DEPFMC),
for the specific detection of organophosphorus hydrolases
(OPH) enzymes that can hydrolyse NAs such as sarin or
soman (Figure 1).[26]
À
À
À
of the P S bond compared with the activated P F or P CN
bond. Because there are different pathways to VX hydroly-
sis,[4b] to avoid the formation of even more toxic species, the
reactions involved have to avoid the formation of a penta-
coordinated phosphorane intermediate. Scavengers that
could meet this requirement should act preferentially by nu-
cleophilic substitution on the phosphorus atom, similar to a
SN2P mechanism. Such scavengers could thus also act on G
agents and pest control agents. Interestingly, PhX (3b)[7] dis-
plays the same hydrolysis profile as VX (3a), whereas its
toxicity has been estimated to be two orders of magnitude
lower and in vitro AChE inhibition appears reversible.[5c]
For safety and regulatory[8] reasons, we have chosen to study
the hydrolysis of this less toxic NA to validate our strategy.
Reactive decontaminants should be characterised by the
ability to neutralise chemical warfare agents in a rapid and
safe manner, their ease of handling, stability on long-term
storage, availability and easy disposal. They should also be
environmentally benign and benefit from low cost and a
lack of corrosiveness. The scavengers so far discovered
range from metal-centred oxidation catalysts to engineered
catalytic antibodies.[5]
For now the most promising agents are still strong oxi-
dants that act as a nucleophiles,[9–12] for example, hydrogen
peroxide, peracids or oxidative chlorides or peroxides. The
phosphorolytic reactivity of o-iodosylcarboxylates and relat-
ed nucleophiles has also received a lot of interest,[13] yet
such very efficient G nerve agent scavengers are poorly
active towards V nerve agents.[4b] As far as inorganic salts
are concerned, solvent-free hydrolysis-based chemical de-
struction of various warfare agents has been described using
alumina-supported fluoride reagents[14] or nanosize inorganic
oxide particles.[15] Among the enzyme-mediated bioremedia-
tion methods recently reported, most promising are the
exogenous administration of acetyl cholinesterase or human
butyryl cholinesterase used as a stoichiometric trap.[16] Some
enzymatic catalytic hydrolysis[17] or oxidation[18] approaches
have been proposed, yet, in all these cases, the enzymatic
activity is particularly substrate-dependent, and even though
some activity against V-type nerve agents has been reported,
they are still insufficient.[4]
More recently, another 12 fluorogenic analogues of vari-
ous OPNAs were synthesised by BriseÇo-Roa et al.[27] using
3-chloro-7-oxy-4-methylcoumarin (Figure 1) to develop an
assay that would allow the screening and selection of en-
zymes that are able to efficiently hydrolyse OP pest control
The activities of such chemical or biochemical scavengers
can be easily tuned and improved through parallel or combi-
natorial synthesis and site-directed or randomised mutagen-
esis (for example, this has recently been reported for phos-
photriesterases (PTE)[19] and cholinesterases[20]) as well as
Figure 1. Reported structures of chromogenic or fluorogenic substrates.
Chem. Eur. J. 2010, 16, 3510 – 3523
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3511

P.-Y. Renard, R. Baati et al.
agents (paraoxon, parathion and dimefox) and OP warfare
agents (DFP, tabun, cyclosarin, soman and VX). However,
the fact that the phenol-based fluorophore or chromophore
(coumarin, fluorescein or p-nitrophenol) is directly linked to
the organophosphoric unit dramatically slants the result for
the most stable OP nerve agents. Under these conditions,
the detectable moiety (an acti-
genic probe. Another interesting source of inspiration was
the example of Zhu and co-workers who successfully de-
signed and synthesised new fluorogenic probes that specifi-
cally target different classes of protein phosphatases.[29]
As illustrated in Figure 2, the designed probe contains a
À
P Salkyl moiety attached to a fluorescent unit through a re-
vated phenol) is a good leaving
À
group, which activates the P O
bond rendering it more sensi-
tive to hydrolysis. Furthermore,
modification of the substrate
hampers the detection of en-
zymes, the recognition sites of
which should enclose bulky hy-
drophobic aromatic leaving
groups. Considering the in-
creased stability of the non-acti-
À
vated P Salkyl bond in V-type
nerve agents and the toxicity of
the S-alkyl phosphonothioic
acids resulting from the alterna-
Figure 2. Self-immolative spacer strategy applied to pro-fluorescent probes suitable for organophosphono-
thioase activity detection assays.
À
tive P O bond cleavage, this
type of phenol-based probe is not suitable for the detection
of enzymes or chemical agents susceptible to act as decon-
taminants or scavengers in this case.
active linker. This rational approach implies a succession of
À
chain reactions: once the P S bond is cleaved through an
SN2P nucleophilic addition reaction, the spontaneous de-
composition of the self-immolative spacer should lead to the
release of the fluorescent specie.
In this article we describe a HTS assay that allows the
identification of either chemical or biochemical scavengers
À
À
able to efficiently cleave not only activated P F or P O
As a consequence, the design of the self-immolative
linker-containing probes 4 and 5 was based on the observa-
tion that thiolates are susceptible to forming the correspond-
ing thiiranes when they are sub-
À
bonds, but also the non-activated P Salkyl bond of alkyl
phosphonothioates. This assay is based on the use of an orig-
inal pro-fluorescent probe, provided with a self-immolative
À
linker, which permits the quantification of P Salkyl bond
stituted with a labile oxygenat-
cleavage through a two-step release of a hydrosoluble 7-hy-
droxycoumarin derivative. The test has been further validat-
ed by the identification of the most active compounds
among a small focused library of nucleophiles. New oxime,
amidoxime and hydroxamic acid based scavengers able to
decontaminate V-type OP nerve agents in an aqueous envi-
ronment close to physiological pH have been identified, and
their activities and selectivities have been additionally con-
firmed by using PhX as the substrate.
ed group at the b position.[30]
Therefore the cleavage of the
À
probe P S bond (mediated by
an enzyme or by a nucleophile)
should generate an unstable 7-
(2-mercaptoethoxy)coumarin,
which might spontaneously decompose further into the cor-
responding highly fluorescent 7-hydroxycoumarin (lem
=
452 nm, F=0.76 in phosphate buffer at pH 7.4)[31] and thi-
irane (Scheme 1).
We have to be aware that the decomposition of the self-
immolative linker could lead to the formation of two prod-
Results and Discussion
Inspired by the pioneering re-
search of Reymond and co-
workers for the detection of es-
terase activity[28] and because
the targeted scavengers have to
act preferentially by nucleophil-
ic substitution on the phospho-
rus, similar to an SN2P mecha-
nism,[4b] we have proposed a
strategy for the rational design
of a new self-immolative fluoro- Scheme 1. Mechanism proposed for the decomposition of the pro-fluorogenic probe.
3512
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3510 – 3523

Organophosphorus Nerve Agent Scavengers
FULL PAPER
ucts, either the thiirane resulting from nucleophilic attack of
the thiolate on the b-carbon atom or the ethylene corre-
sponding to the b-elimination product. An alternative to
these mechanisms is a Smiles-type rearrangement,[32] but this
would involve nucleophilic attack of the thiolate upon the
carbon atom of the aromatic phenol, which is less probable.
The two first generation probes (4 and 5) were not fluores-
cent and our strategy was validated by the preliminary re-
sults obtained in a base-induced (8:2 borate buffer (pH 9.0)/
acetonitrile) hydrolysis of probe 4,[33] which proved that the
Dibenzyl 3-oxopentanedioate, isolated (92% yield) after
transesterification of the corresponding commercially avail-
able dimethyl ester, was treated with resorcinol at 808C in
the presence of samariumACTHNUGRTENUNG(III) nitrate under solventless con-
ditions,[41b] which allowed the isolation of the desired cou-
marin 7 in a moderate, yet optimised yield (55%). Phenol 7
was alkylated with O-silyl-protected 2-bromoethanol to give
ether 8 in a yield of 40%, which was further brominated in
the presence of dibromophosphorane (quantitative yield).
Finally, the brominated derivative 9 was treated with a dicy-
clohexylammonium thiophosphonate salt to give the pro-
tected probe 10 in good yield (89%).
À
product obtained from the hydrolysis of the P S bond cleav-
age is indeed 7-hydroxycoumarin. However, because both
probes 4 and 5 exhibit poor hy-
Unfortunately, none of the attempts to remove the benzyl
protecting group by hydrogenolysis was very successful.
Only a 12% conversion was obtained under pressure
(20 bar) in the presence of the Pd/C catalyst in acetic acid at
room temperature, whereas heating led to a complete degra-
drosolubility, we designed
second-generation water-solu-
ble pro-fluorogenic probe
a
6
containing an additional hydro-
philic carboxylic acid function
at the 4-position of the coumar-
À
dation of the reaction mixture (due to the fragile P S
bond), and palladium-, titanium- and platinum-based cata-
lysts were poisoned by the sulfur. Other methods for the
soft deprotection of carboxylic acid esters, such as the use of
in unit.
The first challenge was the synthesis of a suitable fluoro-
phore. Because the selective protection of the carboxylic
acid function in the presence of a phenol group (for exam-
ple, 7-hydroxycoumarin-4-acetic acid) proved problematic,
we preferred to synthesise directly the 7-hydroxycoumarin
unit bearing a suitable carboxylic ester at the 4-position.
The classic methods for the synthesis of 4-substituted cou-
marins use Pechmann,[34] Perkin,[35] Knoevenagel,[36] Refor-
matsky[37] or Wittig[38] reactions, which involve the condensa-
tion of a phenol with a b-keto ester in the presence of differ-
ent Brønsted[39] or Lewis acids.[40] Recently, solventless syn-
thetic methods have been developed and applied to Pech-
mann and Knoevenagel reactions, the condensation
reactions taking place in the presence of an appropriate cat-
bisACHTNUTGRNEUNG
(tributyl)tin oxide in an aprotic solvent,[42] also failed. Fi-
nally, the use of BCl3 in anhydrous dichloromethane at
room temperature gave satisfactory results. Purification by
reversed-phase flash column chromatography (C18 silica gel)
allowed the isolation of the fluorogenic probe 6 in a satisfac-
tory yield of 48% (overall yield of 8% for the six steps).
In contrast to the first-generation fluorogenic probes and
as expected, the introduction of the carboxylic acid function
led to a good hydrosolubility for 6. As depicted in Figure 3,
compared with 7-hydroxycoumarin-4-acetic acid, fluorogenic
probe 6 displays a very low fluorescence in water (200 mm in
borate buffer at pH 8.75), which designates it as a valid pro-
fluorescent probe candidate because it fulfils perfectly the
imposed requirements (Figure 3).
alyst (p-toluenesulfonic acid, piperidine and samariumACTHNUTRGNEU(GN III)
salts).[41] In our case the best results were obtained by using
the latter conditions, as presented in Scheme 2.
Under simulated physiological conditions, 6 proved to
have good stability even after prolonged storage. For this
Scheme 2. Preparation of fluorogenic probe 6.
Chem. Eur. J. 2010, 16, 3510 – 3523
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3513

P.-Y. Renard, R. Baati et al.
Figure 3. Comparison of the fluorescence emissions of the fluorogenic
probe 6 and 7-hydroxy coumarin-4-acetic acid (200 mm in borate buffer at
pH 8.75, excitation wavelength 370 nm).
type of thiophosphonate, it is important to avoid the use of
phosphate buffers because, as has already been repor-
ted,[5c,43] phosphate ions can also act as weak nucleophiles
leading to a significant increase in the rate of hydrolysis of
phosphonothioates and thus distort the results. That is why,
contrary to the commonly used procedures, the experiments
were performed in Tris·HCl buffer (pH 7.4). Under the con-
ditions used, after one week of storage at room temperature
À
only traces of P S bond hydrolysis were detected by HPLC
Figure 4. A) Fluorescence emission (lem =370 nm) and B) RP-HPLC elu-
tion profile (detection at lex =320 nm) for the fluorogenic probe 6 (con-
centration: 200 mm) after a) 1 and b) 10 days at 378C in 0.1m borate
buffer at the appropriate pH.
analysis and fluorescence measurements at lem =455 nm
(lex =370 nm). Considering this particular and unexpected
stability, we were concerned by the potential toxicity of
phosphonothioate 6, but inhibition experiments performed
on human recombinant AChE (at the French Ministry of
Defence Procurement Agency - DGA, Centre d’Etude du
Bouchet) revealed only a moderate inhibition activity.
of 7-(2-mercaptoethoxy)coumarin-4-acetic acid was ob-
served (below 0.5% as estimated by HPLC). Because the
pH range used in these experiments corresponds to the pKa
of the free thiol, and although we were unable to detect
either episulfur or ethylene, we concluded that these experi-
ments confirm the expected mechanism for the decomposi-
tion of the self-immolative linker: nucleophilic attack of the
thiolate anion on the carbon atom at the b position
(Scheme 1).
Detection of traces of the 7-(2-mercaptoethoxy)coumarin
intermediate also clearly shows that the limiting-step of the
whole process is this latter nucleophilic attack, and that the
stability of the intermediate thiol is the key feature in the
design of the probe. We and others[44] have already observed
that such a thiol is stable up to pH 7.5, and that its sponta-
neous decomposition is pH-dependant, the decomposition
being spontaneous above pH 8.0, which is consistent with
our results described herein.
With the pro-fluorescent probe in hand, we decided to
evaluate a library of known and potential chemical scaveng-
ers. Because most of the previously described efficient meth-
ods for the decontamination of V-type agents are based on
strong nucleophiles, and more particularly on a nucleo-
philes, we chose to focus our library on this type of com-
pound. Beyond the classic a nucleophiles, recent computa-
tional results regarding the mechanism of the nucleophilic
Subsequently, the kinetics for the hydrolysis of the fluoro-
genic probe 6 were evaluated in 0.1m borate and Tris·HCl
buffers (pH range from 7.0 to 10.0). The presence and quan-
tification of the expected hydrolysis products of 6 (7-hydrox-
ycoumarin-4-acetic acid, O-ethyl methylphosphonic acid, 7-
(2-mercaptoethoxy)coumarin-4-acetic acid and the corre-
sponding disulfide dimer) were analysed both by LC–MS
and fluorescence assays. Similar HPLC experiments per-
formed in parallel on PhX (3b) at pH>9.0 revealed signifi-
À
cant amounts of the P O bond cleavage product, which is
consistent with literature reports.[9–12] In the case of the fluo-
rogenic probe 6, no traces of phosphonothioic acid were de-
tected. As expected, the rate of hydrolysis of 6 is significant-
ly accelerated under basic pH conditions because hydroxide
ions are the active species during the spontaneous hydrolysis
of the phosphonothioates. A slow spontaneous hydrolysis of
the fluorogenic probe was detected at pH 8.0, and the cou-
marin unit showed satisfactory stability at pH<9.0 but
proved troublesome above this pH (Figure 4). The LC–MS
experiments showed that the optimal pH for the fluores-
cence experiments was between 8.5 and 8.75. Under these
slightly basic pH conditions, the only hydrolysis product of 6
detected was the expected fluorescent 7-hydroxycoumarin-
4-acetic acid; only a weak and apparently constant amount
3514
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3510 – 3523

Organophosphorus Nerve Agent Scavengers
FULL PAPER
substitution of oximes on phosphonates[45] have attracted
our attention. From these studies it was concluded that,
even if they do not act through the SN2P mechanism, no
phosphorane intermediate can be expected, and thus this
type of structure represents a basis for the construction of
our chemical library.
The reactions of various oximes with different OP ana-
logues have already been studied[46] and the reactivity of
oximes towards OPs (such as diisopropyl fluorophosphates;
DFP) was the basis of a strategy for the design of chemically
reactive colorimetric or fluorescent sensors.[47] Bromberg
and Hatton[48] used recyclable catalytic magnetic nanoparti-
rivatives (12–19), trimedoxime (TMB-4, 20), obidoxime (21)
and HI-6 (22)) already known for their ability to reactivate
OPNA-inhibited AChE. Their efficiencies were compared
with those of other oximes and strong nucleophiles such as
hydroxy- or mercaptopyridines, hydroxypyridinones, hydrox-
ylamines, pyrimidines, hydroxamic acids, quinazolines and
peracids.
cles (magnetite (Fe3O4) nanoparticles modified with
a
common antidote, 2-pralidoxime (2-PAM) or its polymeric
analogue, poly(4-vinylpyridine-N-phenacyloxime-co-acrylic
acid)) for nerve agent destruction. Oximes are well known
to act as reactivators for organophosphorus-inhibited
AChE.[49] Thus, pralidoxime or 2-PAM (an oxime reactiva-
tor) has been approved for clinical use in poisonings with in-
secticidal organophosphates, facilitating the reactivation of
phosphorylated AChE back to normal activity. However, 2-
PAM is not an effective reactivator of AChE inhibited with
nerve agents.[50]
We extended the library with oxime-related compounds
also known to act as phosphorylated cholinesterase reactiva-
tors such as hydroxamic acids.[51] In this context it is interest-
ing to note the early report of Sloan et al.[52] who found a
marked acceleration of paraoxon hydrolysis with various N-
alkylhydroxamic acids in micellar conditions.
The screening test performed with the fluorogenic sub-
strate 6 was conducted in 96 well-plates, the fluorescence
measurements being realised daily for each sample. As eval-
uated in the HTS fluorescent assay summarised in Figure 5,
most of the tested nucleophiles exhibit a weak phosphono-
thioate activity. Thus, as shown by the colour-coded pattern
(Figure 5), HOBt (65), peracids 68 and 69, iodosobenzoic
acid (IBA, 71), which according to the literature survey and
to our previous experiments give satisfactory results with G-
type agents and the HTS assay using fluorogenic probes
bearing the coumarin unit directly linked to the phospho-
rus,[13] in our case provided very disappointing results (which
were further confirmed for the hydrolysis of PhX (3b)). As
To evaluate the newly synthesised fluorogenic probe 6 we
first selected 60 potential a nucleophiles, the large majority
being commercially available products. Our library con-
tained oximes (such as pralidoxime (2-PAM, 11) and its de-
Chem. Eur. J. 2010, 16, 3510 – 3523
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3515

P.-Y. Renard, R. Baati et al.
pectedly strong hydrolytic activity: salicylohydroxamic acid
(45) and its pyridine-derived analogue (47), benzoylformal-
doxime (36) and its derivatives (37–40) and amidoxime (30).
For all the tested compounds, the fluorescence HTS assay
results were corroborated by HLPC analyses of the crude
reaction mixtures (data not shown). Except for the reactions
using fluorescent nucleophiles (such as 32 and 58), the
HPLC analyses and fluorescence yield measurements of the
released 7-hydroxycoumarin-4-acetic acid are in perfect
agreement.
The efficiency of some representative nucleophiles (11,
23, 25, 26, 30, 32, 36 and 47)
was further tested directly in
the hydrolysis of PhX (3b) at
pH 8.75 in either 0.1m borate
or Tris·HCl buffer at 378C. As
we have previously observed,[5c]
temperature has a very strong
effect on these phenylphos-
phonic esters because, under
our conditions, the initial rate
of spontaneous hydrolysis of
PhX is significantly higher at
378C than at 208C. The two
main hydrolytic pathways are
shown in Scheme 3. The reac-
tion was monitored by HPLC
as previously described.[5c] Fur-
ther LC–MS experiments al-
lowed us to identify the main
hydrolysis products: phosphonic
acid 72 (which is the targeted
product), thiol 73 and its corre-
sponding oxidised form 74 (the
formation of which is favoured
by the slightly basic conditions),
À
and the undesired P O bond
cleavage product 75. In a con-
trol experiment performed only
with Tris·HCl buffer, traces of
the Tris addition product (76)
Figure 5. Representative HTS results after 36 h of reaction of 100 mm nucleophile and 50 mm fluorogenic probe
6 in 0.1m borate buffer at pH 8.75 and 378C.
we expected, the quaternised pyridinealdoximes, which are
described as being the best OP-poisoned AChE reactivators,
display the highest phosphonothioate activity, 2-PAM (11)
being slightly better. Their nucleophilicity is high because at
pH 8.75 the oxime is totally de-
were also detected, with Tris adding to the phosphorus
atom.
To study the hydrolysis of PhX (3b) in the presence of
the nucleophiles, two possibilities were considered: monitor-
protonated. The four uncharged
oximes 25, 26, 31 and 32[42] also
gave satisfactory rates of hy-
drolysis; the difference in their
reactivity compared with the
corresponding pyridinium ana-
logues remained high, but is
lessened at a slightly basic pH
at which they are also depro-
tonated. Interestingly, other a
nucleophiles exhibited unex- Scheme 3. Pathways for the hydrolysis reaction of PhX (3b) in the presence of a nucleophile.
3516
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3510 – 3523

Organophosphorus Nerve Agent Scavengers
FULL PAPER
ing of the decrease of PhX (3b) or of the increase of the
newly formed hydrolysis product 72. The first approach
allows the reduction of toxicity to be studied directly, where-
as the second allows the confirmation of the selectivity of
Conclusion
We have developed and optimised a new HTS fluorogenic
assay that has allowed us to identify efficient and selective a
nucleophiles displaying phosphatase activity on non-activat-
ed phosphonothioate. The method used is simple and inex-
pensive to implement. This assay was further used to elabo-
rate potential scavengers of the highly toxic OP nerve
agents used as chemical warfare agents such as VX and
PhX. In this preliminary study, we have been able to identi-
fy four very simple structures: pralidoxime (11), 3-hydroxy-
2-pyridinealdoxime (32), 3-hydroxy-2-pyridinecarbohydroxa-
mic acid (47) and to a lesser extent 3-hydroxy-2-amidoxime
(30), which exhibit significant scavenging activity towards
PhX. With these interesting
À
the hydrolysis with respect to P S bond cleavage in PhX.
However, monitoring the formation of 72 could be problem-
atic with some of the nucleophiles, because in the chosen
elution system their HPLC retention times are close to the
À
retention time of 72. Because the undesired P O bond-
cleavage product (phosphonothioic acid 75) is easier to
detect, we decided to evaluate the consumption of PhX. For
the majority of the evaluated nucleophiles (the reaction con-
ditions were as follows: 1.0 mm PhX and 5.0 mm nucleophile
in 0.1m Tris·HCl buffer at pH 8.75 and 378C, see Table 1
“hits” in hand, supplementary
and complementary tests are
Table 1. Initial rates of reaction for the hydrolysis of PhX.[a]
Nuc.[b]
Structure
Initial rate [mmminÀ1
]
Nuc.[b]
Structure
Initial rate [mmminÀ1
]
presently being undertaken to
further elaborate new a nucleo-
philes structures and to apply
these scavengers to more toxic
methylphosphonates such as
VX (3a). Further tests are nec-
essary to analyse the influence
of relative concentrations and
to identify scavengers that
could be used in a wider pH
range. To put the results of this
work into perspective, this
assay could also be applied to
bioscavanger libraries elaborat-
ed for use at a physiological
pH. Interestingly, among the
new scavengers we have high-
lighted, 2-hydroxyarylaldoximes
similar to 31 and 32 have very
None
1.27
32
11
47
41.5
37.1
32.3
25
16.8
15.5
6.1
36
26
30
25.2
23
4.5
[a] Reagents and conditions: 1.0m PhX and 5.0m nucleophile in 0.1m Tris·HCl buffer at pH 8.75 and 378C.
[b] Nucleophile.
and the Supporting Information for further details), the
HPLC results were consistent with those of the HTS assay.
The two main exceptions were crowded nucleophiles such as
dipyridine oxime 26, for which access to the phosphorus
atom seems hampered by the pyridine group, and, to a
lesser extent hydroxamic acid 47, which seems slightly less
reactive with phenylphosphonic ester 3b than with methyl-
phosphonic ester 6. The two most active compounds, prali-
doxime (11) and 3-hydroxy-2-pyridinealdoxime (32) (which
could not be evaluated through the fluorescent assay be-
cause it is also fluorescent),[53] presented a very similar ini-
tial rate. Interestingly, the activity of two slightly less effi-
cient compounds with structures that are more original, hy-
droxamic acid 47 and amidoxime 30, was also confirmed.
Note that in these four cases (11, 30, 32 and 47), even after
completion of the reaction, no traces of undesired phospho-
nothioic acid 75 were detected by HPLC.
recently been used as pro-fluorescent probes for the detec-
tion of organophosphorus nerve agents by Dale and
Rebek.[47c] These hydroxy oximes have been shown to effi-
ciently react with OP nerve agent surrogates such as diiso-
propylfluorophosphate at a physiological pH to yield the
corresponding isoxazole and non-toxic phosphonic acid,
which further supports the results presented herein on their
role as potential nerve agent scavengers. In another recent
article, the mechanism of hydroxamic acids (such as the
ones we have highlighted in this article) in the dephosphory-
lation of bis(2,4-dinitrophenyl) phosphates has also been in-
vestigated.[51d]
Experimental Section
General: The chemicals used in the syntheses were from Aldrich, Acros
or Alfa-Aesar and were used without further purification. The solution
of PhX in CH3CN (20 mg/mL) was stored at À208C. PhX and phosphon-
ic acids 72 and 75 were prepared as previously described.[5c,7] All solvents
were dried following standard procedures (CH3CN: distillation over
CaH2; MeOH: distillation over MgACHTUNTRGNENUG(OMe)2 and storage over 4 ꢄ molecu-
Chem. Eur. J. 2010, 16, 3510 – 3523
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3517

P.-Y. Renard, R. Baati et al.
lar sieves; DMF: distillation over BaO and storage over 4 ꢄ molecular
sieves; DIEA and triethylamine: distillation over CaH2 and storage over
BaO; CH2Cl2: distillation over P2O5; THF: distillation over Na/benzo-
phenone; toluene: distillation over Na). Purifications by column flash
chromatography were performed on silica gel SDS Si 60 (40–63 mm).
TLC was carried out on Merck DC Kieselgel 60 F-254 aluminium sheets
and visualised by employing a short wavelength UV lamp (l=254 nm) or
by staining with a 3.5% (w/v) phosphomolybdic acid solution in absolute
ethanol.
50 mmol) in a 70% sulfuric acid aqueous solution (50 mL) at 08C. The
mixture was allowed to warm to room temperature and stirred overnight
(18 h). The resulting yellow solution was poured onto crushed ice and the
white precipitate formed was collected by filtration, washed with water,
EtOAc and diethyl ether and then dried overnight under reduced pres-
sure to afford the desired product (8.12 g, 70%) as a white solid.
1H NMR (300 MHz, [D6]DMSO): d=7.55 (d, 3J
ACHTUNGTRENNUNG
(dd, 3J(H-H)=9 Hz, 4J(H-H)=2 Hz, 1H), 6.70 (d, 3J
G
R
ACHTUNGTRENNUNG
6.19 (s, 1H), 3.81 ppm (s, 2H); 13C NMR (75 MHz, [D6]DMSO): d=
172.0, 162.8, 162.3, 156.1, 151.3, 126.9, 113.7, 112.5, 112.4, 102.9,
37.8 ppm; IR (KBr): n˜ =3267, 1702, 1618, 1560, 1373, 1328, 1251, 1214,
Reversed-phase column flash chromatography was performed on octa-
decyl-functionalised silica gel (200–400 mesh, loading 20–22%, surface
area ~550 m2 gÀ1, and mean pore size 60 ꢄ) from Aldrich.
1141, 1063, 860, 684, 617 cmÀ1
.
All aqueous buffers were prepared by using water purified with a Milli-Q
system (purified to 18.2 MWcmÀ1).
Dibenzyl 1,3-acetonedicarboxylate This intermediate was synthesised fol-
lowing an adapted literature procedure.[55] Dimethyl 1,3-acetonedicarbox-
ylate (30 mL, 204 mmol) and benzyl alcohol (43 mL, 408 mmol) were
heated at 170–1808C and the methanol formed during the reaction was
removed from the reaction mixture by distillation. When no more metha-
nol was formed, the reaction mixture was cooled to room temperature
and the residual methanol was removed under reduced pressure. The re-
sidual benzyl alcohol was then distilled from the mixture under a pres-
sure of 0.4 mmHg to give the benzyl ester (61.6 g, 92%) as a yellow oil.
Borate buffers 0.1n, pH ranging from 7.0 to 10.0: [boric acid]=0.1n ad-
justed with 1n NaOH solution.
Tris·HCl buffer 0.1n, pH 8.75: [Tris]=0.1n adjusted to pH 8.75 with 1n
HCl solution.
Tris·HCl buffer 0.1n, pH 7.4: [Tris]=0.1n adjusted to pH 7.4 with 1n
HCl solution.
Tris·HCl buffer 0.1n, 0.15n NaCl, pH 7.4: [Tris]=0.1n, [NaCl]=0.15n,
adjusted to pH 8.75 with 1n HCl solution.
1H NMR (300 MHz, CDCl3): d=7.35 (s, 10H), 5.15 (s, 4H), 3.64 ppm (s,
4H); 13C NMR (75 MHz, CDCl3): d=195.6, 166.9, 135.5, 129.0 (2C),
128.8 (2C), 128.6, 67.7, 49.3 ppm; IR (KBr): n˜ =1738, 1652, 1455, 1327,
1261, 1223, 1177, 1145, 1000, 748, 697 cmÀ1; MS (ESI): m/z: calcd for [M]:
326.16; found: 327; elemental analysis calcd (%) for C19H18O5: C 69.93,
H 5.56; found: C 69.37, H 5.86.
Tris·HCl buffer 0.01n, 0.15n NaCl, pH 7.3: [Tris]=0.01n, [NaCl]=0.15n,
adjusted to pH 7.3 with 1n HCl solution.
Phosphate buffer 0.01n, 0.015n NaCl, pH 7.4: [KH2PO4]=0.002n,
[Na2HPO4]=0.008n, [NaCl]=0.015n.
Benzyl 7-hydroxycoumarin-4-acetate (7): This intermediate was synthes-
ised using an adapted literature protocol.[56] A neat mixture of resorcinol
(7.6 g, 68.9 mmol), dibenzyl 1,3-acetonedicarboxylate (22.5 g, 68.9 mmol)
Phosphate buffer 0.01n, 0.15n NaCl, pH 7.3: [KH2PO4]=0.002n,
[Na2HPO4]=0.008n, [NaCl]=0.15n.
Melting points were recorded on a LEICA VMHB Kofler system at at-
mospheric pressure and are uncorrected. Infrared spectra were recorded
using a Perkin–Elmer FT-IR Paragon 500 spectrometer in KBr pellets
with frequencies given in cmÀ1. NMR spectra were recorded on a Bruker
AM300 spectrometer (300.15 MHz for 1H, 75.4 MHz for 13C and 121.5
for 31P). Chemical shifts (d) are expressed in parts per million (ppm) with
[D6]acetone (dH =2.05, dC =29.84, 206.26 ppm) or CDCl3 (dH =7.26, dC =
77.16 ppm) as internal references and coupling constants (J) values are
expressed in Hz. The multiplicities are abbreviated as follows: s (singlet),
d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet), quint.
(quintuplet) and m (multiplet). Analytical HPLC was performed on a
Thermo Electron Surveyor instrument equipped with a PDA detector.
High-performance liquid chromatography separation was performed on a
Thermo Hypersil GOLD C18 column (5 mm, 10ꢅ250 mm) with CH3CN
and 0.1% aqueous trifluoroacetic acid (aq. TFA, 0.1%, v/v, pH 2.0) as
eluents (100% aq. TFA (5 min), linear gradient from 0 to 40% of
CH3CN (40 min), then 40 to 60% of CH3CN (10 min)) at a flow rate of
1.0 mLminÀ1. Dual UV detection was achieved at 290 and 350 nm. GC–
MS spectra were recorded on a Thermoquest Finnigan Trace GC spec-
trometer coupled to an Auto Mass Multi III analyser using electron
impact ionisation. ESI-MS spectra and LC–MS experiments were per-
formed with a Finnigan LCQ Advantage MAX (ion trap) apparatus
equipped with an electrospray source. UV/Vis spectra were recorded on
a Varian Cary 50 scan spectrophotometer. Fluorescence spectroscopic
and samariumACTHNUTRGENUG(N III) nitrate hexahydrate (6.1 g,13.73 mmol) was heated at
808C for 18 h. The dark-red oil obtained was dissolved in ethyl acetate
(50 mL) and washed with water (50 mL) and brine (50 mL). The organic
layer was dried over anhydrous Na2SO4 and concentrated under vacuum.
The crude solid was then triturated in diethyl ether and filtered to give
coumarin derivative (7; 6.6 g, 55%) as a beige solid.
1H NMR (300 MHz, [D6]acetone): d=9.44 (s, 1H), 7.55 (d, 3J
ACHTUNGTRENNUNG
9 Hz, 1H), 7.34 (m, 5H), 6.81 (dd, 3J
(H-H)=9 Hz, 4J
E
4
6.77 (d, J
N
13C NMR (75 MHz, [D6]acetone): d=170.1, 162.4, 161.2, 156.9, 150.3,
137.3, 129.7, 129.4, 129.3, 127.9, 114.1, 114.0, 113.1, 103.9, 67.8, 38.5 ppm;
IR (KBr): n˜ =3259, 1728, 1693, 1614, 1562, 1328, 1193, 1144, 832, 740,
700 cmÀ1; MS (ESI): m/z: calcd for [M]: 310.08; found: 311 [M+H]+, 643
[2M+Na]+; elemental analysis calcd (%) for C18H14O5: C 69.67, H 4.55;
found: C 69.68, H 4.69.
(2-Bromoethoxy)(tert-butyl)dimethylsilane: This intermediate was syn-
thesised using
a
literature procedure.[57] 2-Bromoethanol (4.28 mL,
60.3 mmol) was added to a solution of tert-butyldimethylsilyl chloride
(10 g, 66.3 mmol) and imidazole (5.34 g, 78.4 mmol) in anhydrous DMF.
The reaction mixture was stirred for 18 h at room temperature. Water
(50 mL) was added and the reaction mixture was extracted twice with
DCM (50 mL). The organic layers were washed with brine, dried over an-
hydrous Na2SO4 and concentrated under vacuum. The crude product was
then purified by distillation under vacuum to give the desired product
(8.02 g, 60%) as a colourless oil.
studies were performed in
a semi-micro fluorescence cell (Hellma,
104FQS, 10ꢅ4 mm, 1400 mL) or in 96 well-plates (Corning or Nunc)
using a Varian Cary Eclipse spectrophotometer.
1H NMR (300 MHz, CDCl3): d=3.88 (t, 3J
AHCTUNGTRENNUNG
ACHTUNGERTN(NUNG H-H)=6 Hz, 2H), 3.39 (t,
3J(H-H)=6 Hz, 2H), 0.90 (s, 9H), 0.08 ppm (s, 6H); 13C NMR (75 MHz,
7-Hydroxycoumarin-4-acetic acid: 7-Hydroxycoumarin-4-acetic acid used
as a reference for the HTS assay was synthesised following an improved
literature procedure (Scheme 4).[54] 1,3-Acetonedicarboxylic acid (7.3 g,
CDCl3): d=63.9, 33.6, 26.2, 18.7, À4.9 ppm; IR (KBr): n˜ =2956, 2930,
2886, 2858, 1472, 1463, 1389, 1362, 1288, 1257, 1189, 1125, 1096, 1022,
947, 892, 838, 778, 674 cmÀ1
; elemental analysis calcd (%) for
50 mmol) was added portionwise to
a solution of resorcinol (5.5 g,
C8H19BrOSi: C 40.17, H 7.95; found: C 40.21, H 7.72.
Benzyl 7-[2-(tert-butyldimethylsilyloxy)ethoxy]coumarin-4-acetate (8).
This intermediate was synthesised using an adapted literature protocol.[58]
A
solution
of
(2-bromoethoxy)-tert-butyldimethylsilane
(1.7 g,
7.12 mmol) in acetonitrile (30 mL) was added to a suspension of coumar-
in derivative 7 (1.7 g, 5.48 mmol) and K2CO3 (2.3 g, 16.44 mmol) in anhy-
drous acetonitrile (30 mL). The reaction mixture was heated at reflux for
Scheme 4. Synthesis of 7-hydroxycoumarin-4-acetic acid.
3518
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3510 – 3523

Organophosphorus Nerve Agent Scavengers
FULL PAPER
24 h and then concentrated under vacuum. The crude was dissolved in
ethyl acetate and the organic layer obtained was washed with water,
dried over anhydrous Na2SO4 and concentrated. The product was puri-
fied by flash chromatography (2 to 10% ethyl acetate in cyclohexane) to
give the desired compound (1 g, 40%) as a yellow solid.
ACTHNGUTERNNUG
(t, 3J(H-H)=7 Hz, 3H); 13C NMR (75 MHz, [D6]acetone): d=170.6,
162.5, 160.7, 156.5, 150.1, 127.4, 114.5, 113.9, 113.2, 102.4, 69.2 (d), 61.9
(d), 37.8, 29.6, 20.9 (d), 16.6 ppm (d); 31P NMR (121.5 MHz,
[D6]acetone): d=52.9 ppm; IR (KBr): n˜ =2904, 1718, 1614, 1394, 1304,
1267, 1200, 1142, 1029, 971, 896, 749 cmÀ1; MS (ESI): m/z: found: 341
[MÀCO2ÀH]À; elemental analysis calcd (%) for C16H19O7PS: C 49.74, H
4.96, S 8.30; found: C 49.71, H 4.88, S 8.11.
1H NMR (300 MHz, [D6]acetone): d=7.63 (d, 3J
(m, 5H), 6.92 (s, 1H), 6.89 (d, 3J
(H-H)=7 Hz, 1H), 6.29 (s, 1H), 5.18 (s,
2H), 4.21 (t, 3J(H-H)=6 Hz, 2H), 4.04 (t, 3J
(H-H)=6 Hz, 2H), 4.00 (s,
ACHTUNGTREN(NUNG H-H)=7 Hz, 1H), 7.35
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
Synthesis of the new a nucleophiles
2H), 0.90 (s, 9H), 0.11 ppm (s, 6H); 13C NMR (75 MHz, [D6]acetone):
d=170.1, 163.6, 161.1, 156.9, 150.2, 137.3, 129.7, 129.4, 129.3, 127.6, 114.7,
113.8, 113.7, 102.6, 71.5, 67.8, 63.0, 38.4, 26.6, 19.3, À4.5 ppm; IR (KBr):
n˜ =2952, 2930, 2885, 2851, 1732, 1614, 1560, 1510, 1454, 1392, 1288, 1262,
1126, 1069, 1003, 962, 837, 776 cmÀ1; MS (ESI): m/z: calcd for [M]:
468.20; found: 469 [M+H]+, 937 [2M+H]+, 659 [2M+Na]+; elemental
analysis calcd (%) for C26H32O6Si: C 66.64, H 6.88; found: C 66.98, H
7.03.
2-Hydroxybenzamidoxime (30): Amidoxime (30) was obtained in one
step by the nucleophilic addition of hydroxylamine to the electrophilic
cyanide (Scheme 5).
Benzyl 7-(2-bromoethoxy)coumarin-4-acetate (9): A solution of bromine
(426 mL, 8.3 mmol) in anhydrous DCM (40 mL) was added dropwise to a
solution of triphenylphosphine (2.2 g, 8.3 mmol) in anhydrous DCM
(40 mL) until the persistence of a pale-yellow colour. A solution of the
coumarin derivative 8 (3 g, 6.4 mmol) in anhydrous DCM (10 mL) was
then added and the reaction mixture was stirred for 5 h at room tempera-
ture. The mixture was washed twice with water, dried over anhydrous
Na2SO4 and concentrated. The crude product was triturated in cold etha-
nol and filtered to give the desired product (2.6 g, 97%) as a pale-yellow
solid.
Scheme 5. Synthesis of 2-hydroxybenzamidoxime (30).
A
solution of hydroxylamine hydrochloride (278 mg, 4 mmol) and
Na2CO3 (212 mg, 2 mmol) in water (3 mL) was added to a solution of 2-
hydroxybenzonitrile (119 mg, 1 mmol) in ethanol (0.5 mL) and the mix-
ture obtained was heated at 808C for 16 h. The reaction mixture was al-
lowed to cool to room temperature and then was extracted with ethyl
acetate. The organic phase was dried over Na2SO4 and concentrated. The
crude was purified by column chromatography using cyclohexane/ethyl
acetate (40:60) as eluent to provide the desired compound as a beige
solid (138 mg, 91%).
1H NMR (300 MHz, [D6]acetone): d=7.64 (d, 3J
(m, 5H), 6.95 (s, 1H), 6.92 (d, 3J
(H-H)=7 Hz, 1H), 6.31 (s, 1H), 5.18 (s,
2H), 4.51 (t, 3J(H-H)=5 Hz, 2H), 4.00 (s, 2H), 3.84 ppm (t, 3J
(H-H)=
ACHTUNGTREN(NUNG H-H)=7 Hz, 1H), 7.35
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
5 Hz, 2H); 13C NMR (75 MHz, [D6]acetone): d=170.0, 162.7, 161.0,
156.8, 150.1, 137.3, 129.7, 129.5, 129.4, 127.8, 114.7, 114.2, 113.7, 102.8,
69.8, 67.8, 38.5, 30.5 ppm; IR (KBr): n˜ =3063, 1722, 1618, 1396, 1330,
1296, 1274, 1238, 1190, 1149, 1065, 1024, 963, 853, 742 cmÀ1; MS (ESI):
m/z: calcd for [M]: 416.03; 417–419 [M+H]+, 857–859 [2M+Na]+; ele-
mental analysis calcd (%) for C20H17BrO5: C 57.57, H 4.11; found: C
57.54, H 4.14.
M.p. 978C; 1H NMR (300 MHz, MeOD): d=7.50 (dd, 3J
3J(H-H)=8 Hz, 1H), 7.22 (td, 3J(H-H)=1.5 Hz, 3J
(H-H)=8 Hz, 1H),
6.86 ppm (t, J
(H-H)=8 Hz, 2H); 13C NMR (50 MHz, MeOD): d=158.7,
ACHTUNGTREN(NUNG H-H)=1.5 Hz,
A
E
ACHTUNGTRENNUNG
3
AHCTUNGTRENNUNG
155.2, 131.4, 126.5, 119.7, 117.8, 116.2 ppm; IR (KBr): n˜ =3367, 2360,
2342, 1645, 1254 cmÀ1; MS (ESI): m/z: calcd for [M]: 152.15; found: 153.
Benzyl 7-{2-[ethoxyACHTUNGTRENNUNG(methyl)phosphinoylsulfanyl]ethoxy}coumarin-4-ace-
3-Hydroxy-2-pyridinealdoxime (32): This compound was synthesised in
two steps starting from commercially available 2-hydroxymethyl-3-hy-
droxypyridine, which was first oxidised in the presence of MnO2 to give
the corresponding aldehyde in a modest yield (22%). This aldehyde was
then transformed under classic conditions into the corresponding aldox-
ime 32 in a yield of 62% (Scheme 6).
tate (10) Coumarin derivative 9 (2.3 g, 5.5 mmol) and O-ethyl methyl-
phosphonothioate (2.3 g, 7.2 mmol) were dissolved in toluene (60 mL)
and the mixture was heated at reflux for 3 h. Once the reaction evolution
monitored by 31P NMR analysis showed completion, the reaction mixture
was filtered, the precipitate was washed with diethyl ether and the filtrate
was concentrated under vacuum. After purification by flash chromatogra-
phy (DCM/acetone/MeOH, 92:2.5:0.5), the desired product (2.35 g,
89%) was obtained as a yellow oil.
1H NMR (300 MHz, [D6]acetone): d=7.61 (d, 3J
(m, 5H), 6.95 (s, 1H), 6.92 (d, 3J
(H-H)=9 Hz, 1H), 6.29 (s, 1H), 5.17 (s,
2H), 4.35 (m, 2H), 4.12 (m, 2H), 3.99 (s, 2H), 3.30 (m, 2H), 1.80 (d, 2J-
(H-P)=16 Hz, 3H), 1.30 ppm (t, 3J(H-H)=7 Hz, 3H); 13C NMR
ACHTUNGTREN(NUNG H-H)=9 Hz, 1H), 7.35
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
(75 MHz, [D6]acetone): d=170.1, 162.8, 161.1, 156.8, 150.1, 137.3, 129.8,
129.5, 129.4, 127.7, 114.9, 114.1, 113.6, 102.8, 69.6, 67.9, 62.3, 38.5, 30.1,
21.4, 17.0 ppm; 31P NMR (121.5 MHz, [D6]acetone): d=53.0 ppm; IR
(KBr): n˜ =2985, 2938, 1731, 1715, 1621, 1618, 1393, 1301, 1266, 1219,
1144, 1030, 961, 883, 743 cmÀ1; MS (ESI): m/z: calcd for [M]: 476.11; 477
[M+H]+, 499 [M+Na]+, 975 [2M+Na]+; elemental analysis calcd (%)
for C23H25O7PS: C 57.98, H 5.29; found: C 58.05, H 5,37.
Scheme 6. Synthesis of 3-hydroxy-2-pyridinealdoxime (32).
A solution of 3-hydroxy-2-(hydroxymethyl)pyridine hydrochloride (2 g,
10.52 mmol, 85%) in MeOH (10 mL) was neutralised with KOH
(590 mg, 10.5 mmol) in a salt-ice bath for 1 h and the precipitated KCl
was removed by filtration. The filtrate was concentrated to dryness under
vacuum. The free 3-hydroxy-2-(hydroxymethyl)pyridine was suspended
in DCM (20 mL) and treated with active MnO2 (5 g, 58%). The reaction
mixture was heated at reflux for 5 h and was then allowed to cool to
room temperature overnight. The crude mixture was filtered and the
MnO2 cake was washed with DCM (2ꢅ40 mL). The filtrate was concen-
trated to dryness and the crude product was purified by flash chromatog-
raphy on silica gel with cyclohexane/ethyl acetate (90:10 to 80:20) as
eluent to give the desired product (3-hydroxy-2-pyridinecarbaldehyde) as
a pale-green solid (288 mg, 22%).
7-{2-[EthoxyACHTUNGTRENNUNG(methyl)phosphinoylsulfanyl]ethoxy}coumarin-4-acetic acid
(6): Boron trichloride (18.4 mL, 1m in DCM) was added dropwise to a
cooled solution (08C) of the coumarin derivative 10 (2.2 g, 4.61 mmol) in
anhydrous DCM (20 mL) and the reaction mixture was stirred at room
temperature for a further 2 h. The reaction was quenched by the addition
of water/MeOH (10 mL/10 mL) and concentrated under reduced pres-
sure. The crude product was purified by reversed-phase silica gel chroma-
tography (MeOH/H2O (0.1% TFA), 10:90 to 35:65) to give the desired
product (860 mg, 48%) as a white solid.
1H NMR (300 MHz, [D6]acetone): d=7.71 (d, 3J
(H-H)=9 Hz, 1H), 7.02
3
3
(d, J
G
1H NMR (300 MHz, CDCl3): d=10.71 (s, 1H), 10.05 (s, 1H), 8.32 (dd, J-
2H), 3.92 (s, 2H), 3.30 (m, 2H), 1.80 (d, 2J
A
A
N
(H-H)=9 Hz, 3J
ACHUTGTNRNEUNG ACHTUNGTRENNUNG(H-H)=
Chem. Eur. J. 2010, 16, 3510 – 3523
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3519

P.-Y. Renard, R. Baati et al.
3
4
(H-H)=1 Hz, 1H); 13C NMR
4 Hz, 1H), 7.33 ppm (dd, JACTHNUTRGENN(UG H-H)=9 Hz, JCAHTUNGTRENNGUN
(75 MHz, CDCl3): d=198.8, 158.7, 142.6, 136.8, 130.1, 126.1 ppm; IR
(KBr): n˜ =1681, 1578, 1467, 1353, 1320, 1233, 1149, 1113, 1063, 871, 809,
646 cmÀ1; MS (ESI): m/z: calcd for [M]: 123.03; found: 124; elemental
analysis calcd (%) for C6H5NO2: C 58.54, H 4.09, N 11.38; found: C
58.59, H 4.04, N 11.39.
Hydroxylamine hydrochloride (104 mg, 1.5 mmol) and sodium acetate
(204 mg, 1.5 mmol) were added to a solution of 3-hydroxy-2-pyridinecar-
baldehyde (123 mg, 1 mmol) in dry ethanol (2.5 mL).The mixture was
stirred at room temperature overnight and then concentrated. The crude
product was diluted in EtOAc (10 mL), washed with water (2ꢅ10 mL),
dried over Na2SO4 and purified by flash column chromatography on
silica gel with cyclohexane/EtOAc (80:20 to 70:30) as eluent to give the
desired product as a white solid (86 mg, 62%).
Scheme 8. Synthesis of the 2,6-pyridinedicarbohydroxamic acid (44).
Pyridine-2,6-dicarboxylic acid (1 g, 6 mmol) was dissolved in MeOH
(12 mL). Concentrated H2SO4 (2 mL) was added and the mixture was
heated at reflux for 48 h. After cooling to room temperature, the mixture
was concentrated under vacuum, diluted in H2O (50 mL) and neutralised
by the addition of solid K2CO3. The solution was extracted with EtOAc
(2ꢅ50 mL) and the organic layers were washed with brine, dried over
Na2SO4 and concentrated to give the desired product (dimethyl 2,6-pyri-
dinedicarboxylate) as a yellow solid (820 mg, 70%).
1H NMR (300 MHz, CDCl3): d=9.84 (s, 1H), 8.48 (s, 1H), 8.45 (s, 1H),
8.22 (dd, 3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG ACTUHNGTRENNUGN
(H-H)=5 Hz, 4J(H-H)=1 Hz, 1H), 7.34 (dd, 3J
4J(H-H)=1 Hz, 1H), 7.25 ppm (dd, 3J(H-H)=9 Hz, 3J
A
A
A
1H); 13C NMR (75 MHz, [D6]acetone): d=155.1, 154.1, 142.0, 137.0,
125.6, 124.4 ppm; IR (KBr): n˜ =3075, 2994, 2874, 2737, 1447, 1319, 1278,
1183, 1020, 910, 812, 703, 656 cmÀ1; MS (ESI): m/z: calcd for [M]: 138.04;
found: 139.
1H NMR (300 MHz, CDCl3): d=8.29 (d, 3J
AHCTUNGTRENNUNG
ACHTUNGERTN(NUNG H-H)=8 Hz, 2H), 8.01 (t,
3J(H-H)=8 Hz, 1H), 4.00 ppm (s, 6H); 13C NMR (75 MHz, CDCl3): d=
165.14, 148.2, 138.5, 128.1, 53.3 ppm; IR (KBr): n˜ =1741, 1572, 1449,
1291, 1246, 1197, 1164, 1144, 1080, 995, 952, 853, 812, 757, 722, 607,
645 cmÀ1; MS (ESI): m/z: calcd for [M]: 195.05; found: 196 [M+H]+, 218
[M+Na]+; elemental analysis calcd (%) for C9H9NO4: C 55.39, H 4.65, N
7.18; found: C 55.53, H 4.59, N 7.05.
2-Oxo-2-(pyridin-2-yl)acetaldehyde oxime (37): Oximes at the a position
of a ketone were obtained by the reaction of the carbonyl compound
with an alkyl nitrite in the presence of a base.[59] Thus, the expected com-
pound 37 could be isolated by treating isoamyl nitrite with 2-acetylpyri-
dine in the presence of potassium tert-butoxide in toluene, but a poor
yield of only 10% was obtained (Scheme 7). As the quantity of the prod-
uct obtained (~135 mg) was sufficient to realise the hydrolysis tests, this
reaction was not further optimised.
Dimethyl 2,6-pyridinecarboxylate (200 mg, 1.02 mmol) was dissolved in
THF/MeOH (1.5 mL/1.5 mL). An aqueous hydroxylamine solution
(50%, 0.63 mL, 10.2 mmol) and KCN (13 mg, 0.2 mmol) were added to
the solution and the mixture was stirred at room temperature for 36 h.
The crude mixture was concentrated under vacuum and recrystallisation
from water gave the desired product as a white solid (32 mg, 16%).
1H NMR (300 MHz, [D6]DMSO): d=11.83 (s, 2H), 9.32 (s, 2H),
8.13 ppm (s, 3H); 13C NMR (75 MHz, [D6]DMSO): d=160.9, 148.3,
139.6, 123.8 ppm; IR (KBr): n˜ =3283, 3138, 2886, 1674, 1644, 1514, 1436,
1250, 1189, 1022, 1002, 887, 841, 743, 630 cmÀ1; MS (ESI): m/z: calcd for
[M]: 197.04; found: 198 [M+H]+, 220 [M+Na]+; elemental analysis calcd
(%) for C7H7N3O4: C 42.65, H 3.58, N 21.31; found: C 42.82, H 3.53, N
21.49.
Synthesis of hydroxamic acids 46 and 47: Hydroxamic acids 46 and 47
were obtained by a two-step procedure, the first step consisting of the
synthesis of the methyl esters of the starting acids by heating them in
methanol at reflux in acidic media (H2SO4),[60] followed by hydroxyami-
nation. In this manner, the corresponding two esters could be isolated in
yields of 80 and 61%, respectively (Scheme 9). The hydroxyamination re-
action was achieved by using aqueous hydroxylamine in MeOH/THF in
the presence of a catalytic amount of KCN.[61] After purification, we
were able to isolate the two hydroxamic acids 46 and 47 in yields of 56
and 16%, respectively, for the two steps.
Scheme 7. Nitrosation of 2-acetylpyridine.
2-Acetylpyridine (1 mL, 8.9 mmol) and potassium tert-butoxide (2.5 g,
22.3 mmol) were dissolved in anhydrous toluene (44 mL) at À108C.
After 15 min of stirring, isoamyl nitrite (1.42 mL, 10.7 mmol) was added
dropwise and stirring was maintained for 30 min. Iced water (40 mL) and
acetic acid (4.5 mL) were added to the reaction mixture. The solution
was then extracted with EtOAc (2ꢅ40 mL) and the combined organic
layers were washed with brine, dried over Na2SO4, concentrated and pu-
rified by silica gel chromatography with cyclohexane/EtOAc (80:20) as
eluent to give the desired product as a white solid (135 mg, 10%).
1H NMR (300 MHz, CDCl3): d=11.64 (s, 1H), 8.70 (d, 3J
ACTHUNRTGNE(GNU H-H)=5 Hz,
3
3
1H), 8.50 (s, 1H), 8.12 (d, J
G
G
ACHTUNGTRENNUNG
4J
4J
G
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
146.6, 137.7, 127.6, 124.3 ppm; IR (KBr): n˜ =3306, 1693, 1593, 1582, 1454,
1438, 1314, 1286, 1226, 1070, 982, 877, 808, 745, 695, 617 cmÀ1; MS (ESI):
m/z: calcd for [M]: 150.04; found: 151; elemental analysis calcd (%) for
C7H6N2O2: C 56.00, H 4.03, N 18.66; found: C 55.72, H 4.11, N 18.63.
Scheme 9. Synthesis of the hydroxamic acids 46 and 47.
2,6-Pyridinedicarbohydroxamic acid (44): Dihydroxamic acid 44 was syn-
thesised in two steps (Scheme 8) from the corresponding commercially
available 2,6-pyridinedicarboxylic acid. Thus, the intermediate methyl
diester could be obtained as previously described in a yield of 70%. The
reaction of this diester in the presence of hydroxylamine led to a com-
plete conversion of the starting compound, but due to the high polarity
of the obtained diacid the purification was tricky, and product 44 was fi-
nally isolated after recrystallisation in water with a poor yield of only
16%.
N-Hydroxypicolinamide (46): Picolinic acid (1 g, 8.12 mmol) was dis-
solved in MeOH (16 mL). Concentrated H2SO4 (1 mL) was added and
the mixture was heated at reflux for 24 h. After cooling to room tempera-
ture, the mixture was concentrated under vacuum, diluted in H2O
(10 mL) and neutralised by the addition of solid K2CO3. The solution was
extracted with EtOAc (2ꢅ20 mL) and the organic layers were washed
with brine, dried over Na2SO4 and concentrated to give methyl picolinate
as a yellow oil (890 mg, 80%).
3520
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3510 – 3523

Organophosphorus Nerve Agent Scavengers
FULL PAPER
1H NMR (300 MHz, CDCl3): d=8.75 (dd, 3J
G
G
(System A, injected volume=20 mL) and by fluorescence measurement
(in a fluorescence cell of 1400 mL, lex =370 nm, lem =400–600 nm, volt-
age=600 W).
3
4
1 Hz, 1H), 8.12 (dd, J
H)=8 Hz, 4J
5 Hz, 4J
(H-H)=8 Hz, J
A
ACHTUNGTRENNUNG
T
G
ACHTUNGTRENNUNG
Screening tests on the fluorogenic substrate 6
CDCl3): d=165.79, 149.9, 147.9, 137.1, 127.0, 125.2, 53.01 ppm; IR (KBr):
n˜ =3418, 2954, 1731, 1586, 1445, 1434, 1312, 1249, 1196, 1134, 1090, 751,
706 cmÀ1; MS (ESI): m/z: calcd for [M]: 137.05; found: 138.
Calibration curves: A 5 mm solution of 7-hydroxycoumarine-4-acetic acid
was prepared in 0.1n borate buffer at pH 8.75. Dilutions of this stock so-
lution were performed to obtain concentrations ranging from 1.56 to
50.0 mm. Each solution was analysed by fluorescence measurement
Methyl picolinate (50 mg, 0.36 mmol) was dissolved in THF/MeOH
(0.5 mL/0.5 mL). An aqueous hydroxylamine solution (50%, 0.24 mL,
3.6 mmol) and KCN (5 mg, 0.07 mmol) were added to the solution and
the mixture was stirred at room temperature overnight. After the addi-
tion of 10% citric acid (15 mL), the mixture was extracted with EtOAc
(2ꢅ15 mL). The organic layers were dried over Na2SO4 and concentrated
to give the desired product as a pale-rose solid (35 mg, 70%).
(300 mL in six different wells of a 96 well-plate, lex =370 nm, lem
=
470 nm, voltage=570 W, five measures per well) and by RP-HPLC
(System B, injected volume=20 mL).
Sample without nucleophile: A sample containing a solution of 6 (50 mm)
was prepared in 0.1n borate at buffer pH 8.75 (Vtot =12 mL). The sample
was thermostatted at 378C.
1H NMR (300 MHz, CD3CN): d=8.53 (dd, 3J
G
ACHTUNGTRNE(NUNG H-H)=
Samples with nucleophiles: Samples containing a solution of 6 (50 mm)
and the nucleophile (100 mm) were prepared in 0.1n borate buffer at
pH 8.75 (Vtot =12 mL). The samples were thermostatted at 378C.
1 Hz, 1H), 8.01 (dd, 3J
G
ACHTUNGTRENNUNG
3J
3J
N
4J
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
Fluorescence measurements: Each sample was analysed every 24 h in a 96
well-plate (300 mL in two different wells), lex =370 nm, lem =470 nm, volt-
age=570 W, five measures per well).
(75 MHz, CD3CN): d=162.9, 149.7, 149.4, 138.6, 127.6, 122.8 ppm; IR
(KBr): n˜ =3449, 3266, 2804, 1672, 1518, 1474, 1177, 1029, 815, 751,
632 cmÀ1; MS (ESI): m/z: calcd for [M]: 138.04; found: 139.
HPLC analysis: Each sample was analysed by HPLC (System B, injected
volume=20 mL) at the end of the experiment.
N,3-Dihydroxypicolinamide (47): 3-Hydroxypicolinic acid (500 mg,
3.6 mmol) was dissolved in MeOH (7 mL). Concentrated H2SO4 (0.5 mL)
was added and the mixture was heated at reflux for 24 h. After cooling to
room temperature, the mixture was concentrated under vacuum, diluted
in H2O (10 mL) and neutralised by the addition of solid K2CO3. The solu-
tion was extracted with EtOAc (2ꢅ20 mL) and the organic layers were
washed with brine, dried over Na2SO4 and concentrated to give methyl 3-
hydroxypicolinate as a white solid (335 mg, 61%).
Hydrolysis of PhX in the presence of nucleophiles
Calibration curve of PhX: A 1.0 mm solution of PhX was prepared in
Tris·HCl buffer (0.1n, pH 8.75). Dilutions of this solution were per-
formed to obtain concentrations ranging from 0.062 to 1.0 mm. Each solu-
tion was analysed by RP-HPLC (System C, injected volume=20 mL).
Calibration curve of 72: A 1.0 mm solution of 72 was prepared in
Tris·HCl buffer (0.1n, pH 8.75). Dilutions of this solution were per-
formed to obtain concentrations ranging from 0.062 to 1.0 mm. Each solu-
tion was analysed by RP-HPLC (System C, injected volume=20 mL)).
1H NMR (300 MHz, CDCl3): d=10.57 (s, 1H), 8.22 (dd, 3J
ACHTUNGTRENNUNG
3
4
4J
(dd, 3J
N
ACHUTGTNREN(NUG H-H)=8 Hz, JACHTNUGTRENNUNG
N
ACHTUNGTRENNUNG
(75 MHz, CDCl3): d=169.9, 158.8, 141.5, 129.9, 129.6, 126.2 ppm; IR
(KBr): n˜ =1676, 1449, 1369, 1304, 1209, 1100, 862, 807, 709, 665 cmÀ1; MS
(ESI): m/z: calcd for [M]: 153.04; found: 154; elemental analysis calcd
(%) for C7H7NO3: C 54.90, H 4.61, N 9.15; found: C 55,43, H 4.76, N
9.12.
Sample preparation: Samples containing a solution of PhX (1.0 mm) and
the nucleophile (5.0 mm) were prepared in Tris·HCl buffer (0.1n,
pH 8.75, Vtot =1 mL) for HPLC analysis. The samples were placed in the
HPLC autosampler thermostatted at 378C.
HPLC analysis: Each sample was analysed by RP-HPLC every 30 min
(for the first 3 h) and further injections were made after longer periods
of time if necessary (20 mL eluted with System C).
Methyl 3-hydroxypicolinate (150 mg, 0.98 mmol) was dissolved in THF/
MeOH (1.5 mL/1.5 mL). An aqueous hydroxylamine solution (50%,
0.6 mL, 9.8 mmol) and KCN (13 mg, 0.19 mmol) was added to the solu-
tion and the mixture was stirred at room temperature overnight. The
mixture was concentrated and 10% citric acid (20 mL) was added to the
crude product. The mixture was extracted with EtOAc (2ꢅ20 mL) and
the organic layers were dried over Na2SO4, concentrated and purified by
flash column chromatography on silica gel using cyclohexane/EtOAc
(50:50) as eluent to give the desired product as a beige solid (40 mg,
26%).
Acknowledgements
This work was supported by the Rꢁgion Haute-Normandie, the Dꢁlega-
tion Gꢁnꢁrale de l’Armement (French Ministry of Defence Procurement
Agency) for Ph. D. grants to L.L.-L. and G.S.A., and the Agence Natio-
nale pour la Recherche (ANR 06 BLAN 0163 DETOXNEURO) for a
postdoctoral grant to E.P. We thank Elisabeth Roger, Laꢆtitia Bailly and
Annick Le Boisselier (UMR 6014 CNRS) for IR measurements, mass
analyses and elemental analyses, and the company QUIDD for open
access to their HPLC instruments. Dr. Laurent Verdier (centre d’Etude
du Bouchet) is gratefully acknowledge for the pro-fluorescent probe tox-
icity evaluation. Dr. Florian Nachon and Prof. Patrick Masson (CRSSA -
Grenoble) are gratefully acknowledged for the gift of commercially avail-
able oximes, as well as Dr. Frꢁdꢁric Taran (CEA Saclay) for the gift of
hydroxypyridines, pyridine N-oxides and pyridinones.
1H NMR (300 MHz, MeOD): d=8.11 (dd, 3J
1 Hz, 1H), 7.46 (dd, 3J(H-H)=8 Hz, 4J
(H-H)=4 Hz, 1H), 7.38 ppm (dd,
3J(H-H)=8 Hz, 4J(H-H)=1 Hz, 1H); 13C NMR (75 MHz, MeOD): d=
(H-H)=4 Hz, 4J
ACHUTGTNRNEUNG AHCUTNGTREN(NUGN H-H)=
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
166.5, 158.3, 149.5, 131.9, 129.8, 127.6 ppm; MS (ESI): m/z: calcd for [M]:
154.04; found: 153.
Biochemical assays
High-performance liquid chromatography separations: Several chromato-
graphic systems were used for the analytical experiments. System A: RP-
HPLC (Thermo Hypersil GOLD C18 column, 5 mm, 4.6ꢅ150 mm) with
CH3CN and 0.1% aq. trifluoroacetic acid (aq. TFA, 0.1%, v/v, pH 2.0) as
eluents (20% CH3CN (2 min), linear gradient from 20 to 25% (5 min),
25% (10 min), linear gradient from 25 to 20% (2 min) and 20% (2 min)
at a flow rate of 1 mLminÀ1) and UV detection at 320 nm. System B:
System A with isocratic elution at 25% CH3CN (15 min). The tray,
column and oven were heated at 378C. System C: System B with isocratic
elution at 20% CH3CN (15 min) and UV detection at 264 nm.
[1] a) P. Taylor in Goodman and Gilmanꢀs The Pharmacological Basis
of Therapeutics, 10th ed. (Eds.: J. G. Hardman, L. E. Limbird, A. G.
Goodman), McGraw-Hill, New York, 2001, pp. 175–192; b) R. E.
Langford in Introduction to Weapons of Mass Destruction, Radiolog-
ical, Chemical and Biological, Wiley, New York, 2004.
Study of the influence of pH on the hydrolysis of fluorogenic substrate 6
The samples were prepared in 0.1n borate buffer with pH values ranging
from 7.0 to 10.0 (7, 7.5, 8, 8.25, 8.5, 8.75, 9, 9.5 and 10) with a substrate
concentration of 200 mm. The hydrolysis was followed by RP-HPLC
[2] Convention on the Prohibition of the Development, Production,
Stockpiling and Use of Chemical Weapons and on their Destruction;
Chem. Eur. J. 2010, 16, 3510 – 3523
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3521

P.-Y. Renard, R. Baati et al.
The Technical Secretariat of the Organization for the Prohibition of
Chemical Weapons, Den Hague, Netherlands, 1993.
[3] M. Eddleston, N. A. Bucklay, P. Eyer, A. H. Dawson, Lancet 2008,
371, 597–607.
[4] a) Y. C. Yang, J. A. Baker, J. R. Ward, Chem. Rev. 1992, 92, 1729–
1743; b) Y. C. Yang, Acc. Chem. Res. 1999, 32, 109–115.
[5] a) D. Crabb, J. DeFrank, C. Penet, Chem. Ind. 2004, 14, 15; b) L. M.
Eubanks, T. J. Dickerson, K. D. Janda, Chem. Soc. Rev. 2007, 36,
458–470; c) P. Vayron, P.-Y. Renard, F. Taran, C. Crꢁminon, Y. Fro-
bert, J. Grassi, C. Mioskowski, Proc. Natl. Acad. Sci. USA 2000, 97,
7058–7063; d) B. M. Smith, Chem. Soc. Rev. 2008, 37, 470–478;
e) S. S. Talmage, A. P. Watson, V. Hauschild, N. B. Munro, J. King,
Curr. Org. Chem. 2007, 11, 285–298; f) A. W. Reshetnyak, M. F. Ar-
mentano, N. A. Pomarenko, D. Vizzuso, O. M. Durova, R. Zigan-
shin, M. Serebryakova, V. Govorun, G. Gololobov, H. C. Morse III,
A. Friboulet, S. P. Makker, A. G. Gabibov, A. Tramontano, J. Am.
Chem. Soc. 2007, 129, 16175–16182.
D. M. Quinn, Z. Radic, P. Taylor, B. P. Doctor, Biochem. Pharmacol.
1997, 54, 269- 274; c) B. P. Doctor, A. Saxena, Chem.-Biol. Interact.
2005, 157–158, 167–171.
[17] For OPH (organophosphorous hydrolases), see: a) F. C. G. Hoskin,
J. E. Walker, W.-D. Dettbarn, J. R. Wild, Biochem. Pharmacol. 1995,
49, 711–715; b) V. K. Rastogi, J. J. Defrank, T.-C. Cheng, J. R. Wild,
Biochem. Biophys. Res. Commun. 1997, 241, 294–296; c) A. A.
Wang, W. Chen, A. Mulchandani, Biotech. Bioeng. 2005, 91, 379–
386; d) A. Singh, Y. Lee, W. J. Dressick, Adv. Mater. 2004, 16, 2112–
2115; for PTE–phosphotriesterase see : e) E. Ghanem, F. M. Raush-
el, Toxicol. Appl. Pharmacol. 2005, 207, 459–470.
[18] a) Using laccase: G. Amitai, R. Adani, G. Sod-Moriah, I. Rabino-
vitz, A. Vincze, H. Leader, B. Chefetz, L. Leibovitz-Persky, D. Frie-
sem, Y. Hadar, FEBS Lett. 1998, 438, 195–200; b) using chloroper-
oxidase (CPO) together with urea hydrogen peroxide: G. Amitai, R.
Adani, M. Hershkovitz, P. Bel, I. Rabinovitz, H. Meshulam, J. Appl.
Toxicol. 2003, 23, 225–233.
[6] a) D. P. Dumas, H. D. Durst, W. G. Landis, F. M. Raushel, J. R. Wild,
Arch. Biochem. Biophys. 1990, 277, 155–159; b) J. E. Kolakowski,
J. J. DeFrank, S. P. Harvey, L. L. Szafraniec, W. Beaudry, K. Lai,
J. R. Wild, Biocatal. Biotransform. 1997, 15, 297–312.
[7] P.-Y. Renard, H. Schwebel, P. Vayron, L. Josien, A. Valleix, C. Mios-
kowski, Chem. Eur. J. 2002, 8, 2910–2916.
[8] C.o. Disarmament, The Convention on the Prohibition of the Devel-
opment, Production, Stockpiling and Use of Chemical Weapons and
on their Destruction, 1992, CD/1170.
[19] a) E. Ghanem, F. M. Raushel, Toxicol. Appl. Pharmacol. 2005, 207,
459–470; b) E. Carletti, L. Jacquamet, M. Loiodice, D. Rochu, P.
Masson, F. Nachon, J. Enzyme Inhib. Med. Chem. 2009, 24, 1045–
1055.
[20] a) C. B. Millard, O. Lockridge, C. A. Broomfield, Biochemistry 1998,
37, 237–247; b) F. Nachon, Y. Nicolet, N. Viguiꢁ, P. Masson, C. Ju,
O. Fontecilla-Camps, Lockridge, Eur. J. Biochem. 2002, 269, 630–
637; c) P. Masson, E. Carletti, F. Nachon, Protein Pept. Lett. 2009,
16, 1215–1224.
[9] Activated hydrogen peroxides: a) Y. C. Yang, L. L. Szafraniec, W. T.
Beaudry, C. A. Bunton, J. Org. Chem. 1993, 58, 6964–6965; b) G. W.
Wagner, Y.-C. Yang, Ind. Eng. Chem. Res. 2002, 41, 1925–1928;
c) W. Wagner, L. R. Procell, Y.-C. Yang, C. A. Bunton US
6723891B1, 2004; d) G. W. Wagner, D. C. Sorrick, L. R. Procell,
M. D. Brickhouse, I. F. Mcvey, L. I. Schwartz, Langmuir 2007, 23,
1178–1186.
[10] Activated chlorides: a) Y. C. Yang, J. A. Baker, J. R. Ward, Chem.
Rev. 1992, 92, 1729–1743; b) “Decontamination”: C. G. Hurst in
Medical Aspects of Chemical and Biological Warfare (Eds.: F. R.
Sidell, E. T. Takafuji, D. R. Franz), The Borden Institute, Chap-
ter 15, 2008.
[21] G. L. Ellman, Arch. Biochem. Biophys. 1959, 82, 70–77.
[22] a) T. J. Dale, J. Rebek, Jr., J. Am. Chem. Soc. 2006, 128, 4500–4501;
b) S. Royo, R. Martꢇnez-Mꢈnez, F. Sancenon, A. M. Costero, M.
Para, S. Gil, Chem. Commun. 2007, 4839–4847; c) M. Burnworth,
S. J. Rowan, C. Weder, Chem. Eur. J. 2007, 13, 7828–7836.
[23] a) Y. R. Shu, A. K. Su, J. T. Liu, C. H. Lin, Anal. Chem. 2006, 78,
4697–4701; b) J. Wang, G. Chen, A. Muck, M. P. Chatrathi, A. Mul-
chandani, W. Chen, Anal. Chim. Acta 2004, 505, 183–187; c) A. L.
Jenkins, O. M. Uy, G. M. Murray, Anal. Comm. 1997, 34, 221–224.
[24] a) S. Jin, Z. Xu, J. Chen, X. Liang, Y. Wu, X. Qian, Anal. Chim.
Acta 2004, 523, 117–123; b) F. Arduini, A. Amine, D. Moscone, F.
Ricci, G. Palleschi, Anal. Bioanal. Chem. 2007, 388, 1049–1057.
[25] a) M. Burnworth, S. J. Rowan, C. Weder, Chem. Eur. J. 2007, 13,
7828–7836; b) S. Royo, R. Martꢇnez-MꢈÇez, F. Sancenꢉn, A. M.
Costero, M. Parra, S. Gil, Chem. Commun. 2007, 4839–4847.
[26] S. Soukharev, D. J. Hammond, Anal. Biochem. 2004, 327, 140–148.
S. Soukharev, D. J. Hammond, WO 2004/094658, 2004.
[27] L. Briseno-Roa, J. Hill, S. Notman, D. Sellers, A. P. Smith, C. M.
Timperley, J. Wetherell, N. H. Williams, G. R. Williams, A. R.
Fersht, A. D. Griffiths, J. Med. Chem. 2006, 49, 246–255.
[28] a) J.-P. Goddard, J.-L. Reymond, Trends Biotechnol. 2004, 22, 363–
370; b) J.-P. Goddard, J.-L. Reymond, Curr. Opin. Biotechnol. 2004,
15, 314–322; c) J.-L. Reymond, V. S. Fluxꢊ, N. Maillard, Chem.
Commun. 2009, 34–49.
[29] a) Q. Zhu, X. Huang, G. Y. J. Chen, S. Yao, Tetrahedron Lett. 2004,
45, 707–710; b) M. S. T. GonÅalves, Chem. Rev. 2009, 109, 190–212;
c) D. Wahler, F. Badalassi, J. L. Reymond, Chem. Eur. J. 2002, 8,
3211–3228; d) Q. Zhu, M. Uttamchandani, D. B. Li, M. L. Lesai-
cherre, S. Q. Yao, Org. Lett. 2003, 5, 1257–1260; e) D. Wahler, F.
Badalassi, P. Crotti, J. L. Reymond, Angew. Chem. 2001, 113, 4589–
4592; Angew. Chem. Int. Ed. 2001, 40, 4457–4460.
[30] a) M. Lapeyre, J. Leprince, M. Massonneau, H. Oulyadi, P.-Y.
Renard, A. Romieu, G. Turcatti, H. Vaudry, Chem. Eur. J. 2006, 12,
3655–3671; b) M. Michalska, E. Brzezinska, P. Lipka, J. Am. Chem.
Soc. 1991, 113, 7945–7951; c) C. Schultz, Bioorg. Med. Chem. 2003,
11, 885–898; d) H. Kawa, N. Ishikawa, Bull. Chem. Soc. Jpn. 1980,
53, 2097–2098; e) R. A. Boigegrain, B. Gross, Carbohydr. Res. 1975,
41, 135–142.
[11] FOxone: Y. C. Yang, F. J. Berg, L. L. Szafraniec, W. T. Beaudry,
C. A. Bunton, A. Kumar, J. Chem. Soc. Perkin Trans. 2 1997, 607–
613.
[12] Peracids : a) Y. C. Yang, L. L. Szafraniec, W. T. Beaudry, D. K.
Rohrbaugh, J. Am. Chem. Soc. 1990, 112, 6621–6627; b) T. Cas-
sagne, H.-J. Cristau, G. Delmas, M. Desgranges, C. Lion, G. Mag-
naud, E. Torreilles, D. Virieux, Heteroat. Chem. 2001, 12, 485–490.
[13] a) P. S. Hammond, J. S. Forster, C. N. Lieske, H. Dupont Durst, J.
Am. Chem. Soc. 1989, 111, 7860–7866; b) R. A. Moss, H. Morales-
Rojas, H. Zhang, B.-D. Park, Langmuir 1999, 15, 2738–2744; c) F. J.
Berg, R. A. Moss, Y.-C. Yang, H. Zhang, Langmuir 1995, 11, 411–
413; d) H. Morales-Rojas, R. A. Moss, Chem. Rev. 2002, 102, 2497–
2522; e) N. Masurier, F. Estour, M.-T. Froment, B. Lefevre, J.-C. De-
bouzy, B. Brasme, P. Masson, O. Lafont, Eur. J. Med. Chem. 2005,
40, 615–623; f) N. Masurier, O. Lafont, R. Le Provost, D. Lesur, P.
Masson, F. Djedaini-Pilard, F. Estour, Chem. Commun. 2009, 589–
591.
[14] E. Gershonov, I. Columbus, Y. Zafrani, J. Org. Chem. 2009, 74, 329–
338.
[15] a) G. W. Wagner, O. B. Koper, E. Lucas, S. Decker, K. J. Klabunde,
J. Phys. Chem. B 2000, 104, 5118; b) G. W. Wagner, P. W. Bartram,
O. B. Koper, K. J. Klabunde, J. Phys. Chem. B 1999, 103, 3225;
c) G. W. Wagner, P. W. Bartram, Langmuir 1999, 15, 8113; d) G. W.
Wagner, L. R. Procell, R. J. OꢃConnor, S. Munavalli, C. L. Carnes,
P. N. Kapoor, K. J. Klabunde, J. Am. Chem. Soc. 2001, 123, 1636;
e) G. W. Wagner, L. R. Procell, S. J. Munavalli, J. Phys. Chem. C
2007, 111, 17564; f) G. W. Wagner, Q. Chen, Y. J. Wu, J. Phys. Chem.
C 2008, 112, 11901; g) I. Columbus, D. Waysbort, L. Shamueli, I.
Nir, D. Kaplan, Environ. Sci. Technol. 2006, 40, 3952–3958.
[16] a) L. Raveh, E. Grauer, J. Grunwald, E. Cohen, Y. Ashani, Toxicol.
Appl. Pharmacol. 1997, 145, 43–53; b) A. Saxena, D. M. Maxwell,
[31] K.-I. Setsukinai, Y. Urano, K. Kikuchi, T. Higuchi, T. Nagano, J.
Chem. Soc. Perkin Trans. 2 2000, 2453–2457.
[32] P. C. H. Eichinger, J. H. Bowie, R. N. Hayes, J. Am. Chem. Soc.
1989, 111, 4224–4227.
3522
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3510 – 3523

Organophosphorus Nerve Agent Scavengers
FULL PAPER
[33] G. Roger, University of Toulouse and P. Badol, University Paris VI,
MS internship, results not shown.
[34] H. Von Pechmann, C. Duisberg, Ber. Dtsch. Chem. Ges. 1883, 16,
2119.
Opletalova, K. Kuca, Bioorg. Med. Chem. 2008, 16, 8218–8223;
c) T.-H. Kim, K. Kuca, D. Jun, Y.-S. Jung, Bioorg. Med. Chem. Lett.
2005, 15, 2914–2917; d) F. Terrier, P. Rodriguez-Dafonte, E. Le
Guꢁvel, G. Moutiers, Org. Biomol. Chem. 2006, 4, 4352–4362; e) S.
Eckert, P. Eyer, N. Herkert, R. Bumm, G. Weber, H. Thiermann, F.
Worek, Biochem. Pharmacol. 2008, 75, 698–703; f) F. Worek, H.
Thiermann, L. Szinicz, P. Eyer, Biochem. Pharmacol. 2004, 68,
[35] J. R. Jonson, Org. React. 1942, 1, 210.
[36] a) G. Brufola, F. Fringuelli, O. Piermatti, F. Pizzo, Heterocycles 1996,
43, 1257–1266; b) G. Jones, Org. React. 1967, 15, 204–599.
[37] R. L. Shriner, Org. React. 1942, 1, 1–37.
[38] a) I. Yavari, R. Hekmat-Shoar, A. Zonouzi, Tetrahedron Lett. 1998,
39, 2391–2392; b) N. S. Narasimhan, R. S. Mali, M. V. Barve, Synthe-
sis 1979, 906–909.
´
´
2237–2248; g) M. Jokanovic, M. P. Stojiljkovic, Eur. J. Pharmacol.
2006, 553, 10–17; h) C. M. Timperley, M. Bird, S. C. Heard, S.
Notman, R. W. Read, J. E. H. Tattersall, S. R. Turner, J. Fluorine
Chem. 2005, 126, 1160–1165; i) K. Musilek, K. Kuca, D. Jun, M. Do-
lezal, Curr. Org. Chem. 2007, 11, 229–238; j) J. Kassa, K. Kuca, L.
Bartosova, G. Kunesova, Curr. Org. Chem. 2007, 11, 267–283; k) J.
Bajgar, J. Fusek, K. Kuca, L. Bartosova, D. Jun, Mini-Rev. Med.
Chem. 2007, 7, 461–466; l) K. Kuca, D. Jun, K. Musilek, Mini-Rev.
Med. Chem. 2006, 6, 269–277; m) J. Wang, J. Gu, J. Leszczynski, M.
Feliks, W. A. Sokalski, J. Phys. Chem. B 2007, 111, 2404–2408; n) F.
Worek, P. Eyer, N. Aurbek, L. Szinicz, H. Thiermann, Toxicol. Appl.
Pharmacol. 2007, 219, 226–234; o) D. M. Maxwell, I. Koplovitz, F.
Worek, R. E. Sweeney, Toxicol. Appl. Pharmacol. 2008, 231, 157–
164.
[39] W.-C. Sun, K. R. Gee, R. P. Haugland, Bioorg. Med. Chem. 1998, 6,
3107–3110.
[40] a) S. P. Chavan, K. Shivasankar, R. Sivappa, R. Kale, Tetrahedron
Lett. 2002, 43, 8583–8586; b) D. Subhas Bose, A. P. Rudradas, M.
Hari Babu, Tetrahedron Lett. 2002, 43, 9195–9197; c) Y. Ma, W.
Luo, P. J. Quinn, Z. Liu, R. C. Hider, J. Med. Chem. 2004, 47, 6349–
6362.
[41] a) T. Sugino, K. Tanaka, Chem. Lett. 2001, 110–111; b) S. S. Bahe-
kar, D. B. Shinde, Tetrahedron Lett. 2004, 45, 7999–8001.
[42] a) E. G. Mata, O. A. Mascaretti, Tetrahedron Lett. 1988, 29, 6893–
6896; b) C. J. Salomon, E. G. Mata, O. A. Mascaretti, Tetrahedron
Lett. 1991, 32, 4239–4242; c) C. J. Salomon, E. G. Mata, O. A. Mas-
caretti, J. Org. Chem. 1994, 59, 7259–7266.
[43] Y. C. Yang, L. L. Szafraniec, W. T. Beaudry, D. K. Rohrbaugh, L. R.
Procell, J. B. Samuel, J. Org. Chem. 1996, 61, 8407–8413.
[44] a) A. Satyam, Bioorg. Med. Chem. Lett. 2008, 18, 3196–3199; b) S.
Far, O. Melnyk, Tetrahedron Lett. 2004, 45, 7163–7165; c) M. La-
peyre, J. Leprince, M. Massonneau, H. Oulyadi, P.-Y. Renard, A.
Romieu, G. Turcatti, H. Vaudry, Chem. Eur. J. 2006, 12, 3655–3671;
d) O. P. Varghese, W. Sun, J. Hilborn, D. A. Ossipov, J. Am. Chem.
Soc. 2009, 131, 8781–8783.
[50] J. E. Chambers, S. F. Oppenheimer, Toxicol. Sci. 2004, 77, 185–187.
[51] a) B. E. Hackley, R. Plapinger, M. Stolberg, T. Wagner-Jauregg, J.
Am. Chem. Soc. 1955, 77, 3651–3653; b) M. A. Stolberg, W. A.
Mosher, J. Am. Chem. Soc. 1957, 79, 2618–2620; c) R. Swidler,
R. E. Plapinger, G. M. Steinberg, J. Am. Chem. Soc. 1959, 81, 3271–
3274; d) M. Silva, R. S. Mello, M. A. Farrukh, J. Venturini, C. A.
Bunton, H. M. S. Milagre, M. N. Eberlin, H. D. Fiedler, F. Nome, J.
Org. Chem. 2009, 74, 8254–8260.
[52] K. B. Sloan, N. Bodor, T. Higuchi, R. Little, M.-S. Wu, J. Chem. Res.
1977, 290–291.
[53] The strong fluorescence of 3-hydroxy-2-pyridinealdoxime (32; lex
=
[45] a) I. B. Wilson, J. Biol. Chem. 1951, 190, 111–117; b) K. Kuca, J.
Cabal, J. Patocka, J. Kassa, Lett. Org. Chem. 2004, 1, 84–86; c) J.
Wang, J. Gu, J. Leszczynski, M. Feliks, A. Sokalski, J. Phys. Chem. B
2007, 111, 2404–2408; d) A. S. Khan, M. K. Kesharwani, T. Bandyo-
padhyay, B. Ganguly, J. Mol. Graphics Modell. 2009, 28, 177–182.
[46] a) G. Becker, A. Kawan, D. Gutzeit, F. Worek, L. Szinicz, Arch.
Toxicol. 2007, 81, 415–420; b) V. P. Kumar, B. Ganguly, S. Bhatta-
charya, J. Org. Chem. 2004, 69, 8634–8642; c) J. Epstein, J. J. Ka-
minski, N. Bodor, R. Enever, J. Sowa, T. Higuchi, J. Org. Chem.
1978, 43, 2816–2821; d) R. E. Plapinger, O. O. Owens, J. Org. Chem.
1956, 21, 1186–1187; e) J. C. Lamb, G. M. Steinberg, S. Solomon,
B. E. Hackley, Jr., Biochemistry 1965, 4, 2475–2484.
380 nm, lem =435 nm, F=0.93, ethanol/water 1:4, see: J. V. Ruiz,
A. G. de Torres, J. M. Cano-Pavon, Talanta 1984, 31, 29–32) and N-
hydroxy-2-oxo-6-pyridinecarboxylic acid (58) prevented any use of
the fluorescence-based HTS assay with these nucleophiles. The re-
sults given here are those of the HPLC assays.
[54] P. Heinz, P. Polczynski, J. Prakt. Chem. 1970, 312, 240–244.
[55] S. S. Bahekar, D. B. Shinde, Tetrahedron Lett. 2004, 45, 7999–8001.
[56] J. Vader, H. Sengers, A. De Groot, Tetrahedron 1989, 45, 2131–
2142.
[57] a) Q. Zhu, X. Huang, G. Y. J. Chen, S. Q. Yao, Tetrahedron Lett.
2004, 45, 707–710; b) B. Wang, X. Guan, Y. Hu, Z. Su, Adv. Polym.
Technol. 2007, 18, 529–534.
[47] a) K. J. Wallace, R. I. Fagbemi, F. J. Folmer-Andersen, J. Morey,
V. M. Lynch, E. V. Anslyn, Chem. Commun. 2006, 3886–3888;
b) K. J. Wallace, J. Morey, V. M. Lynch, E. V. Anslyn, New J. Chem.
2005, 29, 1469–1474; c) T. J. Dale, J. Rebek, Jr., Angew. Chem. 2009,
121, 7990–7992; Angew. Chem. Int. Ed. 2009, 48, 7850–7852; d) Q.
Zhu, M. Uttamchandani, D. Li, M. L. Lesaicherre, S. Q. Yao, Org.
Lett. 2003, 5, 1257–1260.
[58] a) M. G. Bock, R. M. DiPardo, B. E. Evans, K. E. Rittle, D. F.
Veber, R. M. Freidinger, J. Hirshfield, J. P. Springer, J. Org. Chem.
1987, 52, 3232–3239; b) F. M. Hauser, W. P. Ellenberger, J. Org.
Chem. 1988, 53, 1118–1121.
[59] J. Swiatek-Kozlowska, E. Gumienna-Kontecka, A. Dobosz, I. A.
Golenya, I. O. Fritsky, J. Chem. Soc. Dalton Trans. 2002, 4639–4643.
[60] H. L. Yale, Chem. Rev. 1943, 33, 209–256.
[48] L. Bromberg, T. A. Hatton, Ind. Eng. Chem. Res. 2005, 44, 7991–
7998.
[61] C. Y. Ho, E. Strobel, J. Ralbovsky, R. A. Galemmo, J. Org. Chem.
2005, 70, 4873–4875.
[49] a) G. Y. Yang, K.-A. Oh, N.-J. Park, Y.-S. Jung, Bioorg. Med. Chem.
2007, 15, 7704–7710; b) K. Musilek, J. Kucera, D. Jun, V. Dohnal, V.
Received: October 28, 2009
Published online: February 8, 2010
Chem. Eur. J. 2010, 16, 3510 – 3523
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3523