First efficient uncharged reactivators for the dephosphylation of poisoned human acetylcholinesterase

Article abstract of DOI:10.1039/c1cc10787a

Nerve agents are highly toxic organophosphorus compounds with strong inhibition potency against acetylcholinesterase (AChE). Herein, we describe two first extremely promising uncharged reactivators for poisoned human AChE with a superior or similar in vitro ability to reactivate the enzyme as compared to that of HI-6, obidoxime, TMB-4 and HLoe-7.

Full text of DOI:10.1039/c1cc10787a

View Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 5295–5297
www.rsc.org/chemcomm
COMMUNICATION
First efficient uncharged reactivators for the dephosphylation of poisoned
human acetylcholinesterasew
c
d
Guillaume Mercey,ab Tristan Verdelet,ab Geraldine Saint-Andre, Emilie Gillon,
´
´
Alain Wagner,c Rachid Baati,c Ludovic Jean,ab Florian Nachond and Pierre-Yves Renard*abe
Received 10th February 2011, Accepted 15th March 2011
DOI: 10.1039/c1cc10787a
Nerve agents are highly toxic organophosphorus compounds
with strong inhibition potency against acetylcholinesterase
(AChE). Herein, we describe two first extremely promising
uncharged reactivators for poisoned human AChE with a
superior or similar in vitro ability to reactivate the enzyme as
¨
compared to that of HI-6, obidoxime, TMB-4 and HLo-7.
Organophosphorus-nerve agents (OPNA) act as irreversible
acetylcholinesterase inhibitors. They are used either as pest
control agents (such as parathion, chlorpyrifos and diazinon)
or as warfare agents (for highly toxic agents such as sarin,
tabun, soman and VX) (Fig. 1).1,2
Fig. 2 Structure of AChE reactivators.
OPNA inhibit acetylcholinesterase (AChE, EC 3.1.1.7) via
the formation of a covalent P–O bond at the serine hydroxyl
group within the enzyme’s active site. Inhibited enzyme is not
able to hydrolyze acetylcholine anymore and thus cannot
fulfill its essential role in neurotransmission. The resulting
accumulation of the neurotransmitter in the synaptic cleft
leads to the over-stimulation of cholinergic receptors, causing
seizures, respiratory arrest and death. Pest control agents have
been accounted for over 200 000 deadly chronic intoxication
yearly,3 thus it is of prior importance to find an efficient way to
reactivate the phosphylated enzyme, and not only for the
treatment of acute intoxications which could be caused by a
terrorist attack. Presently, a combination of an antimuscarinic
agent (e.g. atropine), AChE reactivator such as one of the
standard pyridinium oximes (pralidoxime, trimedoxime or
TMB-4, obidoxime, HI-6, HLo-7)4 and anticonvulsant drug
¨
(e.g. diazepam) has been used for the treatment of organo-
phosphate poisoning in humans (Fig. 2).2 Those highly
nucleophilic compounds add to the phosphorus atom of
the phosphylated serine, yielding to the removal of the
phosphonate from the enzyme, which thus induces the recovery
of the enzyme catalytic activity .
Fig. 1 Structure of some warfare agents.
a CNRS UMR 6014-COBRA & FR 3038, IRCOF,
Structural modifications of pyridinium oximes have been
widely described in the literature,5,6 but all existing reactivators
are permanently charged cationic compounds that poorly
cross the blood–brain barrier (BBB) and thus cannot efficiently
reactivate cholinesterases in the central nervous system
(CNS).7 For instance, HI-6 does not readily penetrate into
the CNS7b and the mean BBB penetration ratio of pralidoxime
was approximately 10%.7c Moreover, those reactivators do
not bind optimally to the AChE catalytic site,8 limiting their
global efficiency, and their activity depends on the nerve agent
used (for instance, HI-6 reactivates in vitro VX-poisoned
AChE, it has no effect onto Tabun-poisoned AChE). Another
drawback for these reactivators is that the resulting phos-
phylated oxime can act as a nerve agent since the recovered
`
Rue Lucien Tesniere, 76821 Mont Saint Aignan, France.
E-mail: pierre-yves.renard@univ-rouen.fr; Fax: +33 2 35 52 29 71;
Tel: +33 2 35 52 24 76
´
Universite de Rouen, Place Emile Blondel,
76821 Mont Saint Aignan, France
`
Laboratoire de Chimie des Systemes Fonctionnels,
CNRS UMR 7199, Faculte de Pharmacie, Universite de Strasbourg,
b
c
´
´
BP 24, 67401 Illkirch, France
´
d
Cellule enzymologie, Departement de Toxicologie,
´
´
Institut de Recherche Biomedicale des Armees, 24,
Avenue des Maquis du Gre´sivaudan, BP87, 38702 La Tronche,
France
e Institut Universitaire de France, 103, Boulevard Saint Michel,
75005 Paris, France
w Electronic supplementary information (ESI) available: Experimental
procedures, copies of spectral data of 2 and 3 and reactivation kinetics.
See DOI: 10.1039/c1cc10787a
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 5295–5297 5295

View Online
affinity towards inhibited enzyme (KD = 32 Æ 11 mM)
(Fig. 3).
In order to increase its affinity, we proposed to attach the
oxime 1 to a ligand able to bind with the peripheral site of the
enzyme. The latter lies at the entrance to the AChE gorge at
the bottom of which is placed the active site of the enzyme.
Oxime and ligand are linked through an alkyl or heteroalkyl
chain fitting in AChE gorge. A tightly balanced compromise
for AChE binding encouraged us to choose a moderate AChE
peripheral site binder such as phenyl-tetrahydroisoquinoline
411 in order to limit reversible AChE inhibition by the
resulting isoxazole. Moreover, molecular docking simulations
suggested that the best candidates for reactivation seem to be
compounds whose linker is in the meta position to the oxime
function.
Fig. 3 Structure of non-quaternary AChE reactivators.
catalytically active serine can further attack this phosphonate
(recapture phenomenon). Aiming at the development of a non-
ionic reactivator, which can more easily penetrate the BBB
than all existing reactivators by removing the permanent
positive charge, and thus increasing their lipophilicity, bind
efficiently to the AChE active site, and does not suffer from the
recapture phenomenon, we report in this article the synthesis
and evaluation of the first wide scope and uncharged AChE
reactivators with a similar or superior in vitro ability to
reactivate human AChE than HI-6, obidoxime, trimedoxime
The synthesis of these reactivators started with the
N-alkylation of phenyltetrahydroisoquinoline 412 with two
different tosylates to give compounds 5 and 6. Then, a
Sonogashira coupling reaction between alkynes and bromo-
pyridine 7 prepared in three steps from 3-hydroxypicolinic
acid13 (esterification, followed by electrophilic bromination
and protection of the hydroxyl group into benzyl ether)
afforded efficiently the desired compounds 8 and 9 in 66%
and 75% yield, respectively. Reduction of alkyne and
deprotection of the hydroxyl group then furnished compounds
10 and 11 in excellent yield. For the introduction of the
required aldehyde moiety, best results were obtained using a
sequence comprising the protection of the phenol group as
TBS ether, the reduction of methyl ester into corresponding
aldehyde by using DIBAL-H, and a subsequent deprotection
and Hlo-7.
¨
Recently, the ability of 3-hydroxy-2-pyridinealdoxime 1 to
cleave the P–S bond of OPNA was reported yielding the
corresponding isoxasole and non-toxic phosphonic acid.9,10
The rapid formation of this isoxazole through an intra-
molecular attack of the phenol onto the nitrogen atom could
thus prevent the recapture of the activated phosphonate by the
catalytically active serine. As expected from its reasonably low
pKa (8.2 Æ 0.1), non-quaternary oxime 1 showed a noticeable
ability to reactivate VX-poisoned human AChE (h-AChE)
(kr = 0.5 Æ 0.1 minÀ1, pH 7.0, 25 1C) yet with a very low
Scheme 1 Synthesis of reactivators 2 and 3. Conditions and reagents: (a) K2CO3, CH3CN, reflux, 15 h, 46% (n = 2), 71% (n = 3);
(b) 7, Pd(PPh3)4, CuI, NEt3, THF, RT, 15 h, 66% (n = 2), 75% (n = 3); (c) H2 (1 atm), Pd(OH)2, EtOAc, 15 h, RT, 92% (n = 3),
92% (n = 4); (d) 1. TBSCl, imidazole, DMF, RT, 2 h; 2. DIBAL-H, CH2Cl2, À78 1C, 10 min; 3. TBAF, THF, 0 1C, 30 min (over three steps) 53%
(n = 3), 53% (n = 4); (e) NH2OH, HCl, NaOAc, EtOH, RT, 1 h, 88% (n = 3), 71% (n = 4); (f) 1. MeOH, H2SO4 cat., reflux, 24 h, 80%; 2. Br2,
0 1C, H2O, 2 h, 53%; 3. BnBr, K2CO3, acetone, reflux, 15 h, 93%.
c
5296 Chem. Commun., 2011, 47, 5295–5297
This journal is The Royal Society of Chemistry 2011

View Online
Table 1 Reactivation rate constant (kr), dissociation constant (KD)
and bimolecular reactivation rate constant (kr2) of HI-6, obidoxime,
In conclusion, we have described two non-quaternary
pyridine aldoximes as a new extremely promising family of
AChE reactivators. They are as or more efficient than all
existing oximes to reactivate VX- and tabun-inhibited AChE.
Now, other analogues are in preparation to determine the
optimized length and position of the linker to the oxime
function. In this communication, 4 was used as a racemic
mixture, but we are aware of the plausible enzyme kinetic
effects of each enantiomers and it will be discussed in a future
paper. Moreover, in order to evaluate their ability to cross the
BBB, in vivo assays will be realized and the corresponding
results will be reported in due time.
HLo-7, trimedoxime, 2 and 3
¨
kr/minÀ1
KD/mM
kr2/mMÀ1 minÀ1
VX-hAChE
Obidoxime
0.60 Æ 0.05
0.893a
54 Æ 12
27.4a
11
32a
HLo-7
¨
Trimedoxime
HI-6
2
3
0.49a
—
7.8a
—
63a
0.50 Æ 0.02
0.44 Æ 0.15
0.82 Æ 0.16
0.35 Æ 0.05
50 Æ 26
47 Æ 20
6 Æ 2
9
17
61
Tabun-hAChE
Obidoxime
This work was supported by Direction Generale de
´ ´
0.040 Æ 0.006
0.040 Æ 0.001a
0.020 Æ 0.0007a
0.085 Æ 0.005
250 Æ 110
97 Æ 11a
106 Æ 15a
145 Æ 25
0.16
0.4a
0.2a
0.7
+
1.7
l’Armement (through PhD Fellowship to T.V. and to G.S.A.
and for post-doctoral fellowship to G.M. (REI-DGA 2009-
34-0023)), Institut Universitaire de France, Agence
Nationale pour la Recherche (through ANR_06_BLAN_0163
DETOXNEURO and ANR_09_BLAN_0192 ReAChE
HLo-7
¨
Trimedoxime
HI-6
2
3
b
+
b
b
+
0.042 Æ 0.006
0.015 Æ 0.002
b
25 Æ 9
5 Æ 2
3.4
a
programs) and the Re
´
gion Haute Normandie (Crunch
de
From ref. 14. No reactivation up to 5 mM HI-6.
program). We thank Dr Anthony Romieu (Universite
´
Rouen) for MS analyses.
gave the compounds 12 and 13 in 53% yield. A temporary
protection of the phenol group as TBS ether proved necessary
for an efficient reduction of the ester into aldehyde.
Finally, treatment of aldehyde with hydroxylamine afforded
quantitatively oximes 2 and 3 (Scheme 1).
Notes and references
1 (a) P. Taylor, in Goodman and Gilman’s The Pharmacological Basis
of Therapeutics, ed. J. G. Hardman, L. E. Limbird and
A. G. Goodman, McGraw-Hill, New York, 10th edn, 2001,
pp. 175–192; (b) R. E. Langford, Introduction to Weapons of Mass
Destruction, Radiological, Chemical and Biological, Wiley, New
York, 2004.
Oximes 2 and 3 are weak inhibitors of native hAChE with
IC50 about 100 mM (respectively, 50% inhibition at 100 mM
and 30% at 50 mM).
The determination of reactivation rate constants of VX- and
tabun-poisoned hAChE by oximes 2 and 3 under physiological
conditions showed that this new family of non-quaternary
reactivators is extremely promising. Their reactivation
efficiencies, or kr2 (kr/KD), equal and even exceed those of
2 J. Bajar, Adv. Clin. Chem., 2004, 38, 151.
3 M. Eddleston, N. A. Buckley, P. Eyer and A. H. Dawson, Lancet,
2008, 371, 597.
4 M. Jokanovic and M. P. Stojiljkovic, Eur. J. Pharmacol., 2006,
´ ´
553, 10.
5 (a) K. Musilek, O. Holas, J. Misik, M. Pohanka, L. Novotny,
V. Dohnal, V. Opletalova and K. Kuca, Chem. Med. Chem., 2010,
5, 247; (b) S. B. Bharate, L. Guo, T. E. Reeves, D. M. Cerasoli and
C. M. Thompson, Bioorg. Med. Chem., 2010, 18, 787; (c) J. Kassa,
J. Karasova, J. Bajgar, K. Kuca, K. Musilek and I. Kopelikova,
J. Enzyme Inhib. Med. Chem., 2009, 24, 1040; (d) K. Kuca,
L. Musilova, J. Palecek, V. Cirkva, M. Paar, K. Musilek,
M. Hrabinova, M. Pohanka, J. Z. Karasova and D. Jun,
Molecules, 2009, 14, 4915.
HI-6, obidoxime and HLo-7 in similar conditions, due to
¨
improved kr and/or KD (Table 1). For details, oxime 2 is
2-fold more efficient than HI-6 at reactivating VX-hAChE due
to higher kr. Oxime 3 is about 6-fold more efficient than HI-6
and obidoxime due to a better affinity and at least as efficient
6 For review: K. Musilek, K. Kuca, D. Jun and M. Dolezal, Curr.
Org. Chem., 2007, 11, 229.
as HLo-7. The most dramatic improvement is achieved for the
¨
reactivation of tabun-poisoned AChE. Oxime 2 is 10-fold
more efficient than obidoxime due solely to higher affinity.
Oxime 3, despite a 3-fold lower kr, is remarkably 20-fold more
efficient than obidoxime because of KD improving over
50-fold, and with a better affinity, oxime 3 is much more
7 (a) D. E. Lorke, H. Kalasz, G. A. Petroianu and K. Tekes, Curr.
Med. Chem., 2008, 15, 743; (b) B. P. C. Melchers, I. H. C. H.
M. Philippens and O. L. Wolthuis, Pharmacol., Biochem. Behav.,
1994, 49, 781; (c) K. Sakurada, K. Matsubara, K. Shimizu,
H. Shiono, Y. Seto, K. Tsuge, M. Yoshino, I. Sakai,
H. Mukoyama and T. Takatori, Neurochem. Res., 2003, 28, 1401.
efficient than HLo-7. Despite a lower reactivation rate
¨
8 F. Ekstrom, A. Hornberg, E. Artursson, L. G. Hammarstrom,
¨
G. Schneider and Y. P. Pang, PLoS One, 2009, 4, e5957.
´
9 L. Louise-Leriche, E. Paunescu, G. Saint-Andre, R. Baati,
A. Romieu, A. Wagner and P.-Y. Renard, Chem.–Eur. J., 2010,
16, 3510.
10 T. J. Dale and J. Rebek, Angew. Chem., Int. Ed., 2009, 48, 7850.
11 A. Krasinski, Z. Radic, R. Manetsch, J. Raushel, P. Taylor,
´ ´
K. B. Sharpless and H. C. Kolb, J. Am. Chem. Soc., 2005,
127, 6686.
12 R. K. Tiwari, D. Singh, J. Singh, A. K. Chhillar, R. Chandra and
A. K. Verma, Eur. J. Med. Chem., 2006, 41, 40.
13 J. Shira, T. Yoshikawa and H. Sugiyama, WO2007/089031 A1
(Chem. Abstr., 2007, vol. 147, p. 257652.
14 F. Worek, H. Thiermann, L. Szinicz and P. Eyer, Biochem.
Pharmacol., 2004, 68, 2237.
constant (2- and 6-fold lower), oximes 2 and 3 are 2-fold
and 5-fold more efficient than trimedoxime due to drastically
improved affinity towards tabun-poisoned hAChE.
The increase in linker length by one methylene unit has a
similar effect for the reactivation of both VX- and tabun-
poisoned hAChE: reactivity decreases whereas affinity
increases. Thus, there is a trade-off between affinity and
reactivity. This trade-off suggests that the longer linker gives
more flexibility to the reactivator, allowing better binding to
the inhibited enzyme, but in a less optimal orientation in
regard to the reactivation reaction.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 5295–5297 5297