Design and synthesis of new bis-pyridinium oxime reactivators for acetylcholinesterase inhibited by organophosphorous nerve agents

Article abstract of DOI:10.1016/j.bmcl.2005.03.060

New bis-pyridinium oxime reactivators connected with a CH ₂CH₂OCH₂CH₂ linker between two pyridinium rings were designed and synthesized. In the test of their potency to reactivate AChE inhibited by cyclosarin, the bis-pyridinium oxime 6b achieved reactivation potency higher than 10% at the lower concentration 10^-4 M.

Full text of DOI:10.1016/j.bmcl.2005.03.060

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2914–2917
Design and synthesis of new bis-pyridinium oxime reactivators for
acetylcholinesterase inhibited by organophosphorous nerve agents
Tae-Hyuk Kim,a Kamil Kuca,b Daniel Junb and Young-Sik Junga,*
aMedicinal Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Yusong, Taejon 305-606, Korea
bDepartment of Toxicology, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic
Received 14 February 2005; revised 10 March 2005; accepted 16 March 2005
Available online 21 April 2005
Abstract—New bis-pyridinium oxime reactivators connected with a CH2CH2OCH2CH2 linker between two pyridinium rings were
designed and synthesized. In the test of their potency to reactivate AChE inhibited by cyclosarin, the bis-pyridinium oxime 6b
achieved reactivation potency higher than 10% at the lower concentration 10À4 M.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
In the middle of 1950s, Wilson6 reported that hydroxyl-
amine reactivated organophosphoryl-inhibited ChE
faster than water did, and later reported that an oxime,
2-PAM, was far more effective than hydroxylamine in
reactivating the enzyme.7 After thorough study of many
of these compounds, bis-pyridinium oximes such as
TMB4,8 Toxogonin,9 and HI-610 have been developed
and are used currently in many countries (Fig. 1).
Among these oxime reactivators, HI-6 is the most effec-
tive against nerve agent-inhibited ChE. The structural
characteristics of HI-6 compared with other pyridi-
nium oximes can be described in several points; (1)
Organophosphorus nerve agents such as tabun (GA),
sarin (GB), soman (GD), cyclosarin (GF), and VX are
compounds that exert their biological effects by inhibi-
tion of cholinesterases (ChE), in particular, acetylcholin-
esterase (AChE).1 Thus, the inhibition of AChE
increases the amount of acetylcholine (ACh) at central
and peripheral sites of the nerve system. After the organo-
phosphorus compounds attach to AChE to inhibit it,
AChE may be spontaneously reactivated by hydrolytic
cleavage of the organophosphoryl–AChE bond, or the
dealkylation of the organophosphoryl–AChE complex
may proceed. This is the process known as aging that
makes reactivation of AChE activity by any current
reactivator no longer possible.2 Because the nerve agents
differ in structures, their rates of spontaneous reactiva-
tion and aging differ. For example, the soman–enzyme
complex does not spontaneously reactivate; the half-life
for aging is about 2 min.3 The half-life for aging of the
sarin–enzyme complex is about 5 h, and a small percent-
age (5%) of the enzyme undergoes spontaneous reactiva-
tion.4 Therefore, soman is probably the most dangerous
organophosphorus agent among the known organo-
phosphorus nerve agents. Cyclosarin is also a highly
toxic nerve agent, which is resistant to conventional
reactivators because of the bulky cyclohexyl moiety.5
Keywords: Organophosphorus nerve agents; Bis-pyridinium oxime
reactivators; Inhibition; Acetylcholinesterase.
*
Corresponding author. Tel.: +82 42 860 7135; fax: +82 42 861
1291; e-mail: ysjung@krict.re.kr
Figure 1. Structures of currently used AChE reactivators.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.060

T.-H. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2914–2917
2915
bis-pyridinium oxime HI-6 versus mono-pyridinium
oxime 2-PAM, (2) CH2OCH2 chain between pyridinium
rings in HI-6 versus CH2CH2CH2 chain in TMB4, (3) 4-
carbamoylpyridinium in HI-6 versus 4-(hydroxyimino-
methyl)pyridinium in Toxogonin. These speculations
prompted us to design new oximes in which a longer
ether linker was introduced to connect two pyridine
rings. Moreover, a recent study suggested that the reac-
tivation activity is dependent on the number of the
methylene groups between the two quaternary nitrogens
of bis-pyridinium oximes.5,11 In order to develop new
bis-pyridinium oxime reactivators for the ChE inhibited
by organophosphorus nerve agents, we newly designed
and synthesized bis-pyridinium oxime reactivators,
involving a CH2CH2OCH2CH2 linker instead of a
CH2OCH2 chain or polymethylene chains. Afterwards,
we have tested their potency to reactivate AChE inhib-
ited by suitable nerve agent representative, cyclosarin.
amide 2 was treated with bromide 3a in MeCN at 65–
70 °C to give mono-pyridinium bromide 4 in 71% yield
(Scheme 2). However, the reaction of isonicotinamide
2 and bromide 3a in the reflux condition of MeCN pro-
vided bis-4-isonicotinamide 5 instead of mono-pyridi-
nium bromide 4 as a major product, and 81% yield of
5 was obtained when 2 equiv of the isonicotinamide 2
was reacted with 3a in MeCN for 80 h. Bromide 4 was
heated with aldoxime 1a in MeCN to give bis-pyridi-
nium 6c in 72% yield, and bis-pyridinium 6d was
obtained from bromide 4 and aldoxime 1b in 76% yield.
The newly synthesized bis-pyridinium oximes 6 involv-
ing a CH2CH2OCH2CH2 linker were identified by their
1H NMR spectra.14
3. In vitro experiment
In vitro testing of synthesized oximes involved a stan-
dard collection of experimental procedures. The whole
method is in detail described in the work of Kuca and
Kassa.15 The reactivating efficacy of oximes was evalu-
ated in 10% rat brain homogenate that was incubated
with cyclosarin (O-cyclohexylmethylfluorophosphonate)
for 30 min and then, the tested oxime of appropriate
concentrations (10À4 and 10À2 M) was added for
10 min. The activity of brain AChE was measured by
potentiostatic method with the usage of automatic titra-
tor RTS 822 (Radiometer, Denmark). The data about
initial rate of enzyme reaction with substrate made pos-
sible the calculation of percentage of increase in the
activity of reactivated enzyme in the reaction mixture.
2. Chemistry
Bis-4-pyridiniumaldoxime 6a connected with
a
CH2CH2OCH2CH2 linker between the two quaternary
nitrogens was obtained from the alkylation of the corre-
sponding aldoxime 1a with 2-bromoethylether 3a under
the reflux condition of MeCN (Scheme 1). However, bis-
2-pyridiniumaldoxime 6b was not obtained by the above
method, therefore bistriflate 3b as an alkylating reagent
was used in MeCN at room temperature. The resulting
triflate salt was subjected to ion-exchange chromato-
graphy on Dowex–Cl resin to provide 6b.12 Next, we
tried to prepare bis-pyridinium oximes from pyr-
idiniumaldoxime and isonicotinamide, connected with
a CH2CH2OCH2CH2 linker. There are two possible
methods of synthesizing the bis-pyridinium oximes.
According to previous reports,13 the reaction of isonico-
tinamide 2 with bromide 3a followed by treatment with
aldoxime 1 should be a more efficient method than the
reaction of aldoxime 1 with bromide 3a followed by
treatment with isonicotinamide 2. Therefore, isonicotin-
4. Results and discussion
We have compared reactivation potency of newly syn-
thesized oximes with two commercially available AChE
reactivators—pralidoxime and obidoxime. As it is
shown in Figure 2, both commercially available AChE
reactivators are able to reactivate cyclosarin-inhibited
Scheme 1.

2916
T.-H. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2914–2917
CONH2
CONH2
Br
Br
Br
MeCN
+
O
65-70 oC, 23h
N
Br
N
O
4 (71%)
2
3a
3a
CONH2
CONH2
MeCN
+
2
N
O
N
reflux, 80h
2Br
5 (81%)
NOH
CONH2
NOH
MeCN
4
+
reflux, 38h
N
O
N
N
2Br
6c (72%)
1a
CONH2
NOH
MeCN
NOH
+
4
N
O
N
reflux, 38h
N
2Br
6d (76%)
1b
Scheme 2.
Figure 2. Reactivation potency of newly synthesized compounds 6a–d.
AChE at high concentrations (10À2 M). Unfortunately,
their potency at the concentration 10À4 M is low. Due
to this fact, oxime 6b is the most potent AChE reactiva-
tor from the group of tested oximes. On the other hand,
currently there are many AChE reactivators better able
to reactivate AChE inhibited by cyclosarin.16 For exam-
ple, HI-6 is currently the most potent cyclosarin-inhi-
bited AChE reactivator. It is able to reactivate
cyclosarin-inhibited AChE to 95% at the concentration
10À4 M. On the other hand, its reactivation potency at
the concentration at the concentration 10À2 M is 6%.
This fact is caused by the inhibition of AChE by high
doses of HI-6.17
Our work confirms the fact that compounds with oxime
group at the position 2 on the pyridinium ring are the
most potent reactivators of cyclosarin-inhibited
AChE.18 Although these compounds were not extraor-
dinarily potent reactivators of AChE inhibited by
cyclosarin, they could be effective for reactivation of

T.-H. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2914–2917
2917
J.; Cabal, J.; Kassa, J. Bioorg. Med. Chem. Lett. 2003, 13,
3545.
AChE inhibited by other nerve agents or pesticides,
because reactivation potency of AChE reactivators
depends on the nerve agent used.16,19
14. Compound 6a: mp 195–198 °C; 1H NMR (500 MHz,
D2O): d 4.03 (t, J = 5.0 Hz, 4H, 2CH2), 4.78 (t, J = 5.0 Hz,
4H, 2CH2), 8.11 (d, J = 6.8 Hz, 4H, ArH), 8.36 (s, 2H,
N@CH), 8.71 (d, J = 6.8 Hz, 4H, ArH). Compound 6b:
mp 210–212 °C; 1H NMR (500 MHz, D2O): d 4.02 (t,
J = 4.9 Hz, 4H, 2CH2), 4.92 (t, J = 4.9 Hz, 4H, 2CH2),
8.02 (td, J = 6.9, 1.3 Hz, 2H, 2ArH), 8.40 (dd, J = 8.2,
1.1 Hz, 2H, ArH), 8.56 (t, J = 7.8 Hz, 2H, ArH), 8.64 (s,
2H, N@CH), 8.72 (d, J = 6.2 Hz, 2H, ArH). Compound
Acknowledgements
´
The authors are grateful to Mrs. M. Hrabinova for her
skilful technical assistance. This work was supported by
grants from the Ministry of Science and Technology in
Korea, and from the agency of the Ministry of defense
of the Czech republic—grant no. ONVLAJEP20031.
1
6c: mp 224–227 °C; H NMR (500 MHz, D2O): d 4.03 (t,
J = 4.9 Hz, 2H, CH2), 4.06 (t, J = 4.8 Hz, 2H, CH2), 4.78
(t, J = 4.9 Hz, 2H, CH2), 4.89 (t, J = 4.8 Hz, 2H, CH2),
8.14 (d, J = 6.6 Hz, 2H, ArH), 8.31 (d, J = 6.5 Hz, 2H,
ArH), 8.36 (s, 1H, N@CH), 8.73 (d, J = 6.7 Hz, 2H, ArH),
8.96 (d, J = 6.6 Hz, 2H, ArH). Compound 6d: mp 185–
References and notes
1
187 °C; H NMR (500 MHz, D2O): d 4.05–4.08 (m, 4H,
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