Monooxime-monocarbamoyl bispyridinium xylene-linked reactivators of acetylcholinesterase - synthesis, in vitro and toxicity evaluation, and docking studies

Article abstract of DOI:10.1002/cmdc.200900455

Acetylcholinesterase (AChE) reactivators are crucial antidotes to organophosphate intoxication. A new series of 26 monooxime-monocarbamoyl xylene-linked bispyridinium compounds was prepared and tested in vitro, along with known reactivators (pralidoxime, HI-6, obidoxime, trimedoxime, methoxime, K107, K108 and K203), on a model of tabun- and paraoxon-, methylparaoxon- and DFP-inhibited human erythrocyte AChE. Although their ability to reactivate tabun-inhibited AChE did not exceed that of the previously known compounds, some newly prepared compounds showed promising reactivation of pesticide-inhibited AChE. The acute toxicity of the 0ovel compounds was also determined. Docking studies using tabun-inhibited AChE were performed for three compounds of interest. The structure-activity relationship (SAR) study confirmed the apparent influence of the xylene linkage and carbamoyl moiety on the reactivation ability and toxicity of the agents.

Full text of DOI:10.1002/cmdc.200900455

DOI: 10.1002/cmdc.200900455
Monooxime-monocarbamoyl Bispyridinium Xylene-Linked
Reactivators of Acetylcholinesterase—Synthesis, In vitro
and Toxicity Evaluation, and Docking Studies
Kamil Musilek,*[a, d] Ondrej Holas,[b] Jan Misik,[a] Miroslav Pohanka,[c] Ladislav Novotny,[c]
Vlastimil Dohnal,[d] Veronika Opletalova,[b] and Kamil Kuca[a, c, d]
Acetylcholinesterase (AChE) reactivators are crucial antidotes
to organophosphate intoxication. A new series of 26 mono-
oxime-monocarbamoyl xylene-linked bispyridinium com-
pounds was prepared and tested in vitro, along with known
reactivators (pralidoxime, HI-6, obidoxime, trimedoxime, me-
thoxime, K107, K108 and K203), on a model of tabun- and par-
aoxon-, methylparaoxon- and DFP-inhibited human erythrocyte
AChE. Although their ability to reactivate tabun-inhibited AChE
did not exceed that of the previously known compounds,
some newly prepared compounds showed promising reactiva-
tion of pesticide-inhibited AChE. The acute toxicity of the
novel compounds was also determined. Docking studies using
tabun-inhibited AChE were performed for three compounds of
interest. The structure–activity relationship (SAR) study con-
firmed the apparent influence of the xylene linkage and carba-
moyl moiety on the reactivation ability and toxicity of the
agents.
Introduction
Acetylcholinesterase (AChE; EC 3.1.1.7) plays a very important
role in human neurotransmission, degrading the neurotrans-
mitter acetylcholine within the synaptic cleft. A considerable
number of both natural and artificial AChE inhibitors have
been produced, tested and used for various purposes.[1–2] The
organophosphate inhibitors (OPIs) belong to the synthetic
class of AChE inhibitors, and many are available worldwide
(e.g., pesticides: chlorpyrifos, parathion, diazinon).[3] Some OPIs
enzyme and may additionally contain oxime reactivators (e.g.,
oxime HI-6), or other esterases (e.g., human butyrylcholinester-
ase) to scavenge the OPI.[6–8] The oxime reactivators are the
drugs of choice for the post-treatment of OPI intoxication and
are used worldwide.[5] They contain an oxime (hydroxyimino-
methyl) group that cleaves the AchE–OPI covalent bond, re-
storing enzymatic function. Pralidoxime (1; 2-hydroxyimino-
methyl-1-methylpyridinium chloride), oxime HI-6 (2; 1-(2-hy-
droxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-ox-
apropane dichloride) obidoxime (3; 1,3-bis(4-hydroxyiminome-
thylpyridinium)-2-oxapropane dichloride) trimedoxime (4; 1,3-
bis(4-hydroxyiminomethylpyridinium)propane dibromide) or
methoxime (5; 1,1-bis(4-hydroxyiminomethylpyridinium)me-
thane dichloride) are commercially available oxime reactiva-
tors.[9–11] In addition to AChE reactivators, atropine therapy is
are among the most toxic artificial compounds known and
have been used as chemical warfare agents (e.g., sarin, soman,
tabun (GA), VX).[3–4] Industrial OPIs (e.g., tri-O-cresylphosphate)
are used as plasticizers or flame retardants, while these com-
pounds are toxic, this effect does not originate from the inhibi-
tion of AChE.[5]
[a] Dr. K. Musilek, J. Misik, Dr. K. Kuca
Department of Toxicology, Faculty of Military Health Sciences
University of Defence, Trebesska 1575, Hradec Kralove (Czech Republic)
Fax: (+42)495-518-094
E-mail: musilek@pmfhk.cz
[b] O. Holas, V. Opletalova
OPIs inhibit AChE via covalent binding to the serine hydroxy
group within the active site.[3–4] When inhibited, AChE is not
able to fulfil its essential role in neurotransmission—nonde-
graded acetylcholine accumulates within the synaptic cleft re-
sulting in over-stimulation and a subsequent cholinergic crisis,
which results in serious malfunction of the breathing centre
followed by death.[3] Various therapies are used to counteract
the toxic effects of OPIs. A pretreatment therapy is used in in-
dividuals primarily exposed to OPIs (e.g., soldiers); it contains
weak AChE inhibitors (e.g., pyridostigmine) to sequester the
Department of Pharmaceutical Chemistry and Drug Control
Faculty of Pharmacy in Hradec Kralove, Charles University in Prague
Heyrovskeho 1203, 500 05 Hradec Kralove (Czech Republic)
[c] Dr. M. Pohanka, L. Novotny, Dr. K. Kuca
Centre of Advanced Studies, Faculty of Military Health Sciences
Trebesska 1575, Hradec Kralove (Czech Republic)
[d] Dr. K. Musilek, Dr. V. Dohnal, Dr. K. Kuca
Department of Chemistry, Faculty of Science, University of Jan Evangelista
Purkyne, Ceske mladeze 8, 400 96 Usti nad Labem (Czech Republic)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900455.
ChemMedChem 2010, 5, 247 – 254
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
247

K. Musilek et al.
MED
lecular tool in the design of new AChE reactivators. The xylene
linker appears to be involved in cation–p interactions within
the enzyme active site. Moreover, nonsymmetrical xylene-
linked bispyridinium bisoximes showed extended ability to re-
activate paraoxon-inhibited enzymes in vitro, although there
were inefficient against GA intoxication.[20]
Secondly, bispyridinium monooxime 8 showed the most ef-
fective reactivation of tabun-inhibited AChE in vitro among all
commercial and previously prepared compounds, regarding
activity–toxicity properties.[22] Its excellent reactivation of
tabun-inhibited AChE was also confirmed in vivo.[23] Much like
compounds 6–7, 8 also contains source of p-electrons ((E)-but-
ene linker), but only one oxime moiety. The second oxime
group was replaced by carbamoyl functional group that de-
creases the toxicity of 8 compared with compounds 3–4.[22]
Different functional groups were tested to improve the proper-
ties of compound 8, however, only the carbamoyl showed sig-
nificant reactivation ability of tabun-inhibited AchE in
vitro.[24–26]
also used to protect neurotransmission against a cholinergic
crisis, and diazepam is used as an anticonvulsant.[3]
After AChE inhibition, a process called aging takes place.
Further to the covalent binding of OPI in the active site, some
important hydrogen bonds change, and a negative charge is
formed due to partial degradation of the OPI–AChE complex.
The aged OPI–AChE complex can not be reactivated by nucle-
ophilic oxime.[12–13]
Hence, the valuable molecular features from previous experi-
ments (xylene linkage, carbamoyl functional group) were com-
bined to afford monooxime-monocarbamoyl xylene-linked
compounds.
Herein, we present detailed results on the design, synthesis
and in vitro evaluation of a new series of monooxime-mono-
carbamoyl xylene-linked bispyridinium reactivators. This series
extends previous successful compounds against tabun (GA)
and/or organophosphate pesticides, which are currently being
tested worldwide.[14–18]
The new reactivators (9–34; Figure 1, Table 1) were prepared
via a general synthetic strategy (Scheme 1).[27–28] The monoqua-
Results and Discussion
Design and synthesis of bispyridinium reactivators
Figure 1. Structure of newly prepared monooxime-monocarbamoyl xylene
linked compounds.
Bispyridinium reactivators described herein were primarily
based on promising known oximes. The molecular features of
K107 (6), K108 (7) and K203 (8) were combined to design
monooxime-monocarbamoyl xylene-linked compounds 9–34.
Table 1. Structure of newly prepared compounds.
Compd
A
Oxime Carbamoyl Compd
A
Oxime Carbamoyl
9
o
o
o
o
o
o
o
o
m
m
m
m
m
2’
2’
2’
3’
3’
4’
4’
4’
2’
2’
2’
3’
3’
2
3
4
3
4
2
3
4
2
3
4
2
3
22
23
24
25
26
27
28
29
30
31
32
33
34
m
m
m
m
p
p
p
p
p
p
p
p
p
3’
4’
4’
4’
2’
2’
2’
3’
3’
3’
4’
4’
4’
4
2
3
4
2
3
4
2
3
4
2
3
4
10
11
12
13
14
15
16
17
18
19
20
21
Firstly, symmetrical xylene-linked bispyridinium bisoximes 6–
7 showed promising reactivation of chlorpyrifos- and tabun-
inhibited AChE in vitro.[19–20] Though they were selected by in
vitro screening, they showed increased in vivo toxicity (lower
LD50 values) compared to all commercial reactivators.[21] How-
ever, the xylene linker was still considered to be a valuable mo-
ternary monooxime salts were prepared according to a litera-
ture procedure.[20] The reaction yields varied greatly (3–95%),
depending on the reactivity of the pyridine compounds with
the carbamoyl moiety. Whereas the 3- and 4-carbamoylpyri-
248
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 247 – 254

Acetylcholinesterase Reactivators
Scheme 1, however, we propose its minor reactivation ability
based on our observations below.
OPI-inhibited AChE reactivation
Known compounds (1–8) and the prepared reactivators 9–34
were assayed for their reactivation potency using human er-
ythrocyte AChE inhibited by tabun (GA) and organophosphate
pesticides: paraoxon (POX), methylparaoxon (MePOX) and
mimic agent diisopropylfluorophosphate (DFP).[29] The selected
organophosphates were chosen for testing based on their mo-
lecular divergence after AChE inhibition: dimethyl- (MePOX),
diethyl- (POX) or diisopropyl- (DFP) moiety. The screening con-
centrations were selected as in previous experiments, such
that a concentration of 100 mm should be attainable after in
vivo administration.[30] The reactivation results are shown in
Table 2.
Scheme 1. Synthesis of monooxime-monocarbamoyl xylene linked com-
pounds. Reagents and conditions: a) acetone, reflux; b) DMF, 50–1008C.
dines presented very good reactivity, 2-carbamoyl pyridine re-
acted not sufficiently due to steric hindrance close to pyridine
nitrogen. Nevertheless, the necessary amount of all com-
pounds for in vitro evaluation was obtained.
Reactivation in vitro should exceed 10% to suggest a prom-
ising compounds warranting further testing.[30] Concerning
tabun-inhibited AChE, commercial compounds 1–5 presented
the expected results.[31] While compounds 1 and 2 were almost
One planned compound, (2-carbamoyl-3’-hydroxyimino-
methyl-1,1’-(1,2-phenylenedi-methyl)bispyridinium dibromide),
could not be made by the synthetic strategy described in
Table 2. Reactivation ability of tested oximes (reactivation (%) ꢀSD).[a]
Inhibitor
GA
POX
MePOX
DFP
Reactivator
100 mm
1 mm
100 mm
1 mm
100 mm
1 mm
100 mm
1 mm
pralidoxime (1)
HI-6 (2)
obidoxime (3)
TMB-4 (4)
MMB-4 (5)
K107 (6)
K108 (7)
K203 (8)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
ꢁ4.0ꢀ1.0
0.9ꢀ2.2
ꢁ1.7ꢀ0.5
2.6ꢀ1.4
8.6ꢀ2.4
9.6ꢀ1.6
6.5ꢀ2.1
ꢁ3.0ꢀ0.6
4.0ꢀ0.5
8.3ꢀ2.3
7.3ꢀ1.6
18.7ꢀ2
5.5ꢀ0
6.6ꢀ1.5
2.6ꢀ2.8
4.8ꢀ0.6
8.6ꢀ0.5
1.8ꢀ1.8
11.4ꢀ2.5
5.6ꢀ1.3
7.1ꢀ0.6
8.5ꢀ2.6
12.5ꢀ4.7
20.0ꢀ2.4
1.6ꢀ0.4
9.5ꢀ2.3
8.4ꢀ1.3
9.9ꢀ2.9
2.9ꢀ1.2
8.3ꢀ2.4
4.7ꢀ1.7
10.3ꢀ2.0
9.9ꢀ1.1
6.0ꢀ4.3
5.2ꢀ0.5
8.9ꢀ3.5
6.9ꢀ0.7
5.3ꢀ1.7
5.3ꢀ0.7
8.9ꢀ0.5
4.5ꢀ0.9
0.2ꢀ1.7
1.8ꢀ0.8
10.9ꢀ2.9
2.5ꢀ0.9
6.6ꢀ4.2
1.3ꢀ0.5
ꢁ7.0ꢀ3.7
70.6ꢀ4.6
0.2ꢀ1.9
10.5ꢀ0.8
ꢁ2.0ꢀ1.8
71.1ꢀ4.7
17.7ꢀ1.4
0.4ꢀ1.0
7.1ꢀ0.6
0.3ꢀ0.6
42.4ꢀ4.0
35.7ꢀ0.4
ꢁ10.4ꢀ2.7
5.2ꢀ1.6
ꢁ3.8ꢀ3.5
12.3ꢀ1.0
ꢁ0.6ꢀ1.2
ꢁ0.8ꢀ1.9
15.9ꢀ2.2
9.6ꢀ3.9
5.1ꢀ1.2
7.8ꢀ0.8
17.4ꢀ1.8
ꢁ8.1ꢀ1.0
ꢁ4.0ꢀ2.9
4.1ꢀ2.3
20.1ꢀ1.1
0.9ꢀ0.7
ꢁ3.0ꢀ0.6
1.4ꢀ2.2
14.3ꢀ3.6
8.3ꢀ0.3
19.8ꢀ0.8
2.8ꢀ1.1
2.5ꢀ1.4
1.7ꢀ1.9
ꢁ2.0ꢀ2.9
ꢁ3.0ꢀ4.6
5.1ꢀ2.6
6.5ꢀ0.5
ꢁ2.0ꢀ3.1
ꢁ1.0ꢀ1.3
7.8ꢀ0.7
0ꢀ2.9
2.7ꢀ1.5
ꢁ2.5ꢀ1.2
0.9ꢀ1.6
ꢁ8.0ꢀ1.2
0.2ꢀ2.0
ꢁ0.9ꢀ0.3
0.9ꢀ0.6
5.8ꢀ1.3
52.4ꢀ4.6
62.5ꢀ3.1
0.3ꢀ0.8
21.5ꢀ3.6
30.2ꢀ1.9
1.1ꢀ0.7
ꢁ8.0ꢀ3.7
8.8ꢀ2.7
11.3ꢀ1.7
0ꢀ2.1
0.1ꢀ1.0
ꢁ0.3ꢀ0.6
ꢁ3.0ꢀ0.9
ꢁ1.5ꢀ0.9
3.1ꢀ4.0
ꢁ0.6ꢀ0.4
ꢁ6.0ꢀ3.2
5.0ꢀ3.0
ꢁ0.1ꢀ0.3
1.8ꢀ2.1
ꢁ3.5ꢀ2.1
4.9ꢀ0.6
10.8ꢀ3.1
7.8ꢀ4.6
ꢁ2.0ꢀ4.1
2.6ꢀ3.3
ꢁ6.0ꢀ1.9
2.3ꢀ0.3
3.9ꢀ0.3
39.3ꢀ1.0
0.8ꢀ1.7
43.2ꢀ1.5
1.7ꢀ1.5
3.6ꢀ2.8
ꢁ1.5ꢀ0.4
4.3ꢀ1.2
ꢁ3.2ꢀ0.6
ꢁ0.1ꢀ1.3
ꢁ2.3ꢀ2.0
0.5ꢀ0.7
ꢁ3.0ꢀ0.4
1.0ꢀ1.8
5.0ꢀ1.8
ꢁ5.0ꢀ3.6
2.2ꢀ2.3
4.0ꢀ3.0
0.3ꢀ2.7
ꢁ0.9ꢀ1.3
ꢁ1.5ꢀ2.1
ꢁ2.4ꢀ1.6
ꢁ0.7ꢀ0.3
2.2ꢀ0.3
0.8ꢀ3.1
ꢁ2.0ꢀ2.7
ꢁ4.0ꢀ1.9
ꢁ2.0ꢀ3.0
0.8ꢀ1.4
3.8ꢀ3.1
3.3ꢀ1.3
ꢁ4.0ꢀ4.5
ꢁ8.0ꢀ1.5
ꢁ2.0ꢀ1.2
ꢁ1.0ꢀ1.3
0.2ꢀ2.2
0ꢀ2.0
0.3ꢀ0.6
14.4ꢀ4.3
3.9ꢀ0.3
ꢁ6.0ꢀ0.5
ꢁ1.0ꢀ0.7
ꢁ3.0ꢀ0.5
ꢁ1.0ꢀ1.3
1.7ꢀ0.5
0.6ꢀ0.2
0.3ꢀ1.2
5.5ꢀ3.3
ꢁ5.0ꢀ2.8
ꢁ5.0ꢀ0.6
0.8ꢀ0.6
0.4ꢀ0.4
ꢁ2.3ꢀ0.9
ꢁ3.4ꢀ1.0
0.6ꢀ2.2
ꢁ4.0ꢀ3.4
7.3ꢀ1.3
ꢁ3.2ꢀ1.5
0.6ꢀ2.8
6.2ꢀ1.6
ꢁ2.0ꢀ1.6
ꢁ6.0ꢀ4.7
7.8ꢀ0.8
5.4ꢀ3.9
1.7ꢀ3.1
3.3ꢀ0.7
ꢁ4.0ꢀ1.4
ꢁ0.1ꢀ2.2
1.2ꢀ0.3
ꢁ0.8ꢀ0.6
0.3ꢀ0.4
ꢁ2.0ꢀ1.4
4.4ꢀ0.6
ꢁ6.0ꢀ1.6
ꢁ6.0ꢀ1.2
0.6ꢀ3.8
ꢁ2.0ꢀ0.2
ꢁ1.0ꢀ1.1
ꢁ4.0ꢀ2.0
0.7ꢀ0.9
1.0ꢀ0.8
10.8ꢀ2.9
8.0ꢀ1.5
16.2ꢀ3.8
20.9ꢀ2.5
16.2ꢀ0.1
22.5ꢀ1.8
18.1ꢀ1.6
15.8ꢀ0.7
10.9ꢀ3.0
ꢁ2.0ꢀ2.3
ꢁ3.0ꢀ3.8
ꢁ3.0ꢀ1.1
ꢁ1.9ꢀ0.2
0.0ꢀ1.5
ꢁ4.2ꢀ0.5
0.3ꢀ1.7
ꢁ1.0ꢀ2.2
2.6ꢀ0.1
9.2ꢀ5.0
0ꢀ1.9
0.7ꢀ0.6
11.9ꢀ2.7
22.9ꢀ3.5
10.2ꢀ0.9
10.7ꢀ1.4
7.4ꢀ1.2
3.7ꢀ1.7
ꢁ2.0ꢀ0.4
0.7ꢀ1.3
2.0ꢀ2.7
0.4ꢀ0.4
1.2ꢀ1.3
ꢁ9.2ꢀ2.1
ꢁ3.1ꢀ0.6
ꢁ3.2ꢀ1.8
ꢁ2.2ꢀ0.9
3.7ꢀ0.7
ꢁ3.1ꢀ2.4
1.2ꢀ0.3
ꢁ7.0ꢀ1.7
ꢁ7.0ꢀ1.5
ꢁ1.0ꢀ1.5
1.5ꢀ2.3
ꢁ3.0ꢀ0.8
ꢁ6.0ꢀ1.4
1.6ꢀ1.1
3.3ꢀ1.2
2.9ꢀ1.9
8.7ꢀ4.0
ꢁ4.0ꢀ4.0
ꢁ0.7ꢀ2.8
ꢁ0.3ꢀ0.8
0.6ꢀ1.4
4.5ꢀ1.8
ꢁ2.0ꢀ2.6
ꢁ0.5ꢀ5.9
ꢁ3.0ꢀ2.1
ꢁ2.0ꢀ1.6
ꢁ10.4ꢀ0.6
ꢁ1.0ꢀ0.9
ꢁ7.0ꢀ2.5
ꢁ1.1ꢀ3.8
ꢁ6.0ꢀ3.5
34
ꢁ4.5ꢀ2.4
[a] %, mean value of three independent determinations ꢀSD; time of inhibition 5 min; time of reactivation by AChE reactivators 15 min; pH 7.4; tempera-
ture 258C
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249

K. Musilek et al.
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ineffective against tabun, derivatives 3–5 showed some reacti-
vation ability. Interestingly, compound 5 showed the best in
vitro results of all commercial compounds against tabun-inhib-
ited AchE at a concentration of 100 mm. Conversely, com-
pounds 1 and 4 presented increased oximolysis—the hydroly-
sis of used substrate (thiocholine) by oxime itself that inter-
feres with reactivation for highly concentrated oximes.[32]
Known compounds 6–8 presented divergent results; although
xylene-linked compounds 6–7 showed mainly oximolysis, com-
pound 8 (K203) surpassed all commercial compounds and was
also found to be the best reactivator of tabun-inhibited AChE
among all tested derivatives.[22–23]
Table 3. Toxicity of selected compounds in mice.[a]
Compound
LD50 [mgkgꢁ1
]
pralidoxime (1)
HI-6 (2)
obidoxime (3)
TMB-4 (4)
MMB-4 (5)
K107 (6)
K108 (7)
K203 (8)
K646 (12)
263.6 (253.7–273.8)[33]
671.3 (627.4–718.3)[33]
188.4 (156.3–208.0)[33]
150.5 (142.1–159.4)[35]
641.8 (590.5-716.0)[34]
6.6 (4.4–8.0)
2.2 (0.8–3.2)
95.0 (88.4–102.2)[23]
100.3 (78.9–134.7)
[a] 95% confidence limits given in parentheses were calculated by probit-
logarithmic method.
Regarding the pesticide-inhibited AChE, known compounds
presented different reactivation ability for various pesticides.
For POX-inhibited AChE, compound 8 was the most promising
agent at both screening concentrations.[28] Against MePOX-in-
hibited AChE, reactivator 8 showed the best ability at 100 mm,
however, xylene-linked bis-oxime 7 showed the best ability at
1 mm. Concerning DFP-inhibited AChE, reactivator 3 exceeded
all other commercial compounds at both concentrations
tested.
symmetrical bisoximes was previously seen in rat.[36] In our ex-
periments, these data were confirmed for mice also, where
compounds 6–7 were the most toxic compounds (Table 3). Re-
garding the K203 (8) results in rat, the replacement of one
oxime by a carbamoyl moiety in xylene-linked compounds was
hypothesised to decrease their toxicity. This hypothesis was
supported when compound 12—a promising reactivator of
POX- and MePOX-inhibited AchE—displayed toxicity similar to
compound 8. However, the toxicity of the newly prepared
compounds in mice was found to be higher than the toxicity
of the commercial reactivators.
The newly prepared mono-oximes showed interesting differ-
ences in reactivation of tabun. Although most of them pre-
sented only oximolysis, several compounds (9, 11–12, 14, 33)
had some reactivation ability at 100 mm. Compound 9 was
found to be the best, but did not exceed the reactivator ability
of known oxime 8.
On the other hand, different mono-oximes showed im-
proved reactivation of pesticide-inhibited AChE. Namely, com-
pounds 12 and 28) exceeded all other newly prepared com-
pounds against POX intoxication at both screening concentra-
tions. Unfortunately, their ability to reactivate POX-inhibited
AChE was lower compared with the known compound 8. In
the case of MePOX-inhibited AChE, compound 12 was again
found to be the most potent among the newly prepared
agents. Moreover, the ability of compound 12 to reactivate
MePOX-inhibited AChE at 1 mm surpassed all other tested com-
pounds. Regarding DFP-inhibited AChE, all newly prepared
compounds showed minor reactivation of the enzyme, when
compared with the best commercial compound against DFP
intoxication (3).
Docking studies and SAR discussion
The docking studies were performed on three compounds (3,
8, 12) to rationalise possible interactions within tabun-inhibit-
ed AChE, the main organophosphate of interest.[23] Obidoxime
(3) was chosen as the commercial reactivator with proposed
ability to reactivate AchE inhibited by tabun.[33] Its top-scored
docking pose (ꢁ10.01 kcalmolꢁ1; Figure 2) showed mainly in-
teractions with aromatic residues of the internal cationic site
(ICS) and peripheral anionic site (PAS): namely, T-stacking with
Tyr337 (3.4 ꢁ) from the ICS and sandwiching between Trp286
(3.9 ꢁ) and Tyr124 (4.3 ꢁ) from the PAS. Moreover, the non-re-
activating oxime moiety hydrogen bonds to Ser298 (2.1 ꢁ).
Even though compound 3 was considered to be one of the
best commercial reactivators of tabun-inhibited AChE, model-
ling studies showed that the distance between the oxime
moiety and the tabun phosphorus atom is long (5.7 ꢁ). This
distance limits its reactivation ability towards GA-inhibited
AChE. This finding was also supported by in vitro reactivation
data (Table 2).
Acute toxicity evaluation
The acute toxicity of the reactivator (LD50) is considered to be
an important factor for further evaluation and development.
Literature data suggests that oxime HI-6 is the least toxic reac-
tivator among the commercial compounds (Table 3), however,
it is ineffective against tabun or organophosphate pesticides.[33]
Similarly, methoxime 5 showed low toxicity, but also low ability
to reactivate organophosphate pesticide- or DFP-inhibited en-
zymes.[34] Conversely, the more effective commercial AChE re-
activators against tabun and organophosphate pesticides 3–4
showed increased toxicity (lower LD50 values).[33,35] Interestingly,
K203 (8) was found to be more toxic than compounds 3–4 in
mice, whereas its toxicity in rat was lower compared with 3–
4.[25] Among the xylene compounds, the very high toxicity of
Oxime K203 (8) is a known, very promising reactivator of
tabun-inhibited AChE.[22–23] Its top-scored docking pose
(ꢁ9.87 kcalmolꢁ1; Figure 3) showed interactions with aromatic
residues of the ICS and PAS similar to compound 3: again, T-
stacking with Tyr337 (3.6 ꢁ) from the ICS and sandwiching be-
tween Trp286 (3.6 ꢁ) and Tyr124 (3.8 ꢁ) from the PAS. Howev-
er, unlike compound 3, the carbamoyl moiety forms a hydro-
gen bonded with Trp286 (3.4 ꢁ) and Glu285 (2.9 ꢁ). Moreover,
the p-electrons of the double bond in the connecting linker
displayed possible T-stacking to Phe297 (3.7 ꢁ) and Phe338
250
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ChemMedChem 2010, 5, 247 – 254

Acetylcholinesterase Reactivators
compound 12 became the object of interest. The top-scored
docking pose (ꢁ12.51 kcalmolꢁ1; Figure 4) showed interactions
with aromatic residues of the ICS and PAS similar to com-
pounds 3 and 8—namely, T-stacking with Tyr337 (3.0 ꢁ) from
Figure 2. Molecular docking study on obidoxime (3; in blue).
Figure 4. Molecular docking study on compound 12 (in magenta).
the ICS, sandwiching between Trp286 (3.7 ꢁ) and Tyr124
(3.5 ꢁ) from the PAS and one more T-stacking interaction be-
tween the xylene linker and Trp286 (3.6 ꢁ) from the PAS. Simi-
lar to compound 8, the carbamoyl moiety is involved in a hy-
drogen bond with Trp286 (3.8 ꢁ) and Ser298 (1.7 ꢁ and 2.0 ꢁ).
Nevertheless, the distance of the oxime moiety from the tabun
phosphorus atom (6.4 ꢁ) in presented model is too great, and
this correlates with the minor reactivation activity against
tabun-inhibited AChE in vitro.
The lack of correlation between the in vitro reactivation data
(Table 2) and docking calculations (Figure 2–4) can be ex-
plained by the chosen AChE conformation (PDB: 2JEZ), where
crystal structure data do not cover the real conformation of
the flexible protein, but only one particular conformation
found by crystallographic experiments at a given resolution.
The structure–activity relationship (SAR) properties valuable
for increased reactivation ability should be mentioned:[37] the
position and quantity of the oxime groups, structure of the
connecting linker, presence of the heteroarenium rings and
various functional groups. Concerning the oxime group, the
mono-oxime compounds with a hydroxyiminomethyl function-
ality in position 4 were formerly highlighted as required
groups for AChE reactivation after tabun and organophos-
phate pesticide intoxication reword.[22,2] The known com-
pounds fulfilled this hypothesis, especially the best compound
(8) against tabun.[22] However, the newly prepared reactivators
with oxime in position 2 or 3 (9, 11–12) showed the same or
increased reactivation ability when compared to similar com-
pounds with oxime in position 4 (14, 33). This phenomenon
was also observed for a previous series of reactivators with
xylene linkage and is apparently caused by the additional aro-
Figure 3. Molecular docking study on K203 (8; in green).
(3.7 ꢁ). Although compound 8 was found to be the best reacti-
vator of tabun-inhibited AChE from the tested series, the dis-
tance of the oxime moiety from the phosphorus atom of
tabun was found to be similar to that of compound 3 (5.8 ꢁ).
Consequently, the improved reactivation ability of compound
8 compared with 3 can be attributed to the slightly stronger
interactions with aromatic residues and stronger hydrogen
bonding of the carbamoyl moiety (Table 2).
Compound 12 was chosen for docking studies from the new
series of reactivators in order to rationalise the low reactivation
of tabun-inhibited AChE. While compound 12 showed minor
reactivation of tabun-inhibited AChE in vitro, like compounds
3 and 8, it was a promising reactivator of POX- and MePOX-in-
hibited enzymes. Hence, this lack of enzyme reactivation by
ChemMedChem 2010, 5, 247 – 254
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www.chemmedchem.org
251

K. Musilek et al.
MED
matic ring available for p–p interactions.[19–20] From this point
of view, the connecting linker plays an important role via inter-
actions with the active sites. Although m- or p-xylene linkers
(6–7) were formerly considered as more promising, o-xylene
linker (9, 12) seemed to have an impact on reactivation ability
also.[20] Such interactions were formerly hypothesised also for a
(E)-but-2-ene linker (8).[26–28] Apparently, heteroarenium (pyridi-
nium) moieties took part in the same cation–p interactions
within the enzyme active sites.[33] Conversely, the second
oxime or non-oxime moiety were responsible for hydrogen
bonding. A carbamoyl group in position 4 was formerly high-
lighted as the most promising substituent (compound 8).[22]
Regarding the newly prepared compounds (particularly 9, 11–
12, 14, and 33), there is no evidence that the carbamoyl posi-
tion was favourable for reactivation of tabun- or organophos-
phate-inhibited AChE. On the other hand, the introduction of a
carbamoyl moiety decreased the toxicity of the xylene-linked
compounds approximately twofold (Table 3).
sured on a microheating stage PHMK 05 (VEB Kombinat Nagema,
Radebeul, Germany) and were uncorrected.
NMR spectra were recorded on a Varian Gemini 300 (Palo Alto,
USA): 1H 300 MHz, 13C 75 MHz. In all cases, the chemical shift
values (d) in ppm relative to the residual solvent peaks: 1H,
CHD2SO2CD3 (d=2.50), D2O (d=4.79); 13C, [D6]DMSO (d=39.43).
Signals are quoted as s (singlet), d (doublet), t (triplet) and m (mul-
tiplet).
Mass spectra were measured on a LCQ FLEET ion trap and evaluat-
ed using Xcalibur v 2.5.0 software (both Thermo Fisher Scientific,
San Jose, USA). The sample was dissolved in deionized water
(Goro, s.r.o., Prague, Czech Republic), and injected continuously
(8 mLminꢁ1) by Hamilton syringe into an electrospray ion source.
The parameters of the electrospray were set up as follows: sheath
gas flow rate 20 arbitrary units, aux gas flow rate 5 arbitrary units,
sweep gas flow rate 0 arbitrary units, spray voltage 5 kV, capillary
temperature 2758C, capillary voltage 13 V, tube lens 100 V.
The monoquaternary monooxime salts were prepared according to
the previously published procedure.[20] Full spectral data for com-
pounds 9–34 can be found in the Supporting Information.
Conclusions
Preparation of bispyridinium salts (9–34)
A new series of monooxime-monocarbamoyl xylene-linked bis-
pyridinium compounds has been developed. Their potency
was tested on a model of tabun-, POX-, MePOX- and DFP-in-
hibited AChE in vitro. Their reactivation ability against tabun
did not exceed that of known inhibitor K203 (8), but one com-
pound was able to exceed the standard commercial reactiva-
tors (including obidoxime and trimedoxime) in the reactivation
of MePOX-inhibited AChE. In the case of POX- and DFP-inhibit-
ed AChE, the novel compounds were not able to surpass the
reactivation ability of K203 or obidoxime. The acute toxicity of
the newly prepared compounds in mice was greater than that
of the commercial compounds 3–4 and comparable with K203
(8), but improved over the formerly prepared xylene-linked
compounds (6–7). The introduction of a carbamoyl moiety de-
creased the toxicity in mice approximately twofold. The molec-
ular docking studies confirmed p–p and cation–p interactions
within the enzyme active sites. Hydrogen bonding was found
to be an important reactivator–AChE interaction also. Regard-
ing the SAR, compounds with an oxime moiety in position 2
or 3 showed increased reactivation activity against tabun- or
organophosphate pesticide-inhibited AChE in contrast with
previous findings. Apparently, the xylene linkage and carbamo-
yl moiety strongly influenced the reactivation ability of the
newly prepared compounds.
General procedure: A solution of monoquaternary salt (0.50 g,
1.3 mmol) and the corresponding carbamoylpyridine (0.30 g,
2.4 mmol) in DMF (10 mL) was stirred at the indicated temperature
for the appropriate length of time (Table 4). The reaction mixture
Table 4. Reaction conditions for the synthesis of compounds 9–34.
Compd
Temp
8C
Time
[h]
Yield
[%]
Compd
Temp
8C
Time
[h]
Yield
[%]
9
50
70
70
70
70
70
70
70
50
70
70
70
70
79
4
3
4.5
3
30.5
5.5
3.5
50
12.5
11.5
70
6
78
59
73
97
7
79
88
16
60
95
3
22
23
24
25
26
27
28
29
30
31
32
33
34
70
50
70
70
70
70
70
50
100
100
50
100
100
21.5
28
64
13
61
63
21
98
98
13
80
80
17
92
96
10
11
12
13
14
15
16
17
18
19
20
21
7.5
7.5
100
3.5
3.5
57
23
22
46.5
3
3
22.5
49
was cooled to RT, diluted with acetone (50 mL) and left overnight
at 58C. The liquid was decanted off and the solid crude product
was dissolved in MeCN (50 mL) and left at RT overnight. The crys-
talline product was collected by filtration, washed with MeCN (3ꢂ
20 mL) and recrystallized from MeCN. The purity of all compounds
was determined by NMR, ESI-MS and elemental analysis.
Experimental Section
Chemistry
Solvents (acetone, DMF, MeCN) and reagents were purchased from
Fluka and Sigma–Aldrich, and were used without further purifica-
tion. Reactions were monitored by TLC using DC-Alufolien Cellulo-
se F plates (Merck, Germany) with BuOH/CH3COOH/H2O (5:1:2) as
the mobile phase. Plates were visualised using a solution of Dra-
gendorff reagent (10 mL CH3COOH, 50 mL H2O, 5 mL stock solu-
tion: prepared from two fractions, A: 850 mg Bi(NO3)3, 40 mL H2O,
10 mL CH3COOH; B: 8 g KI, 20 mL H2O). Melting points were mea-
2-Carbamoyl-2’-hydroxyiminomethyl-1,1’-(1,2-phenylenedime-
thyl)bispyridinium dibromide (9): The reaction mixture was stirred
at 508C and stopped after 79 h (yield=6%): Rf =0.15; mp: 179–
1
1818C; H NMR (300 MHz, [D6]DMSO): d=9.19–8.95 (m, 2H, H-6,6’),
8.87–8.51 (m, 7H, -NH2, -CH=NOH, H-3,3’,5,5’), 8.38–8.15 (m, 2H,
H-4,4’), 7.47–7.23 (m, 2H, Ph), 6.69–6.25 ppm (m, 4H, Ph, -CH2);
13C NMR (75 MHz, [D6]DMSO): d=148.1, 146.3, 145.9, 141.2, 131.5,
252
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ChemMedChem 2010, 5, 247 – 254

Acetylcholinesterase Reactivators
129.1, 128.3, 125.8, 125.3, 57.7 ppm; Anal. calcd for C20H20Br2N4O2:
47.27 C, 3.97 H, 11.02 N; found: 46.82 C, 4.09 H, 11.22 N; MS
All measurements were carried out in triplicate and the reactiva-
tion data were expressed as average value ꢀ standard deviation
(SD).
(ESI+): m/z: 174.1 [M/2]2+
.
2-Carbamoyl-2’-hydroxyiminomethyl-1,1’-(1,3-phenylenedime-
thyl)bispyridinium dibromide (17): The reaction mixture was
stirred at 508C and stopped after 50 h (yield=16%): Rf =0.15; mp:
Acute toxicity evaluation
1
225–2278C; H NMR (300 MHz, [D6]DMSO): d=9.30–9.19 (m, 2H, H-
Experiments involving animals were performed in accordance with
the guidelines set forth by the Ethics Committee of the Faculty of
Military Health Sciences in Hradec Kralove (Czech Republic).
6,6’), 8.87 (s, 1H, -NH2), 8.84–8.59 (m, 4H, H-3,3’,-NH2,-CH=NOH),
8.47–8.40 (m, 1H, H-4), 8.34–8.16 (m, 3H, H-4’,5,5’), 7.52–7.25 (m,
4H, Ph), 6.12 (s, 2H, -CH2), 5.98 ppm (s, 2H, -CH2); 13C NMR
(75 MHz, [D6]DMSO): d=148.2, 147.3, 147.1, 146.7, 146.3, 146.0,
141.3, 134.8, 134.5, 129.8, 129.0, 128.8, 127.9, 127.9, 127.4, 126.1,
60.4, 59.8 ppm; Anal. calcd for C20H20Br2N4O2: 47.27 C, 3.97 H, 11.02
Female BALB/C mice (25–30 g) were purchased from Konarovice
(Czech Republic). They were kept in an air-conditioned room with
a standard 12 h light on/off protocol (light on: 07:00; light off:
19:00 h), with free access to standard food and water. The acute
toxicity of selected compounds (intramuscular administration (i.m.),
standard saline solution, dosage in mgkgꢁ1) was estimated as LD50
values for each reactivator (see Table 3). The 95% confidence limits
were determined using probit-logarithmical analysis of death oc-
curring within 24 h after i.m. administration of each oxime at 4–6
different doses with six animals per dose.[38] Abnormal behaviour
was defined as convulsions, apathy, or increased locomotion, and
appeared within a few minutes of administration (2–15 min, de-
pendent on dose). Death occurred within 2 h and survival re-
mained then same after 24 h.
N; found 47.27 C, 4.17 H, 10.71 N; MS (ESI+): m/z: 174.1 [M/2]2+
.
2-Carbamoyl-2’-hydroxyiminomethyl-1,1’-(1,4-phenylenedime-
thyl)bispyridinium dibromide (26): The reaction mixture was
stirred at 708C and stopped after 100 h (yield=21%): Rf =0.15;
1
mp: 218–2208C; H NMR (300 MHz, [D6]DMSO): d=9.29 (d, 2H, J=
6.2 Hz, H-6,6’), 8.88–8.73 (m, 4H, H-3,3’,-NH2,-CH=NOH), 8.69–8.63
(m, 2H, H-4,-NH2), 8.34–8.24 (m, 3H, H-3’,5,5’), 7.43 (s, 4H, Ph),
6.01 ppm (s, 4H, -CH2); 13C NMR (75 MHz, [D6]DMSO): d=148.4,
147.5, 147.1, 141.6, 134.8, 129.3, 128.2, 128.1, 60.6 ppm; Anal. calcd
for C20H20Br2N4O2: 47.27 C, 3.97 H, 11.02 N; found 47.01 C, 4.25 H,
10.71 N; MS (ESI+): m/z: 174.1 [M/2]2+
.
Molecular docking
Docking calculations were carried out using Autodock 4.0.1.[39] The
structure of mus musculus AChE was taken from the crystal struc-
ture (pdb code 2JEZ) and prepared using Autodock
Tools 1.5.2.[39–40] The three-dimentional affinity grid box was de-
signed to include the full active and peripheral sites of AChE. The
number of grid points in the x-, y- and z-axes was 110, 110 and
110, with grid points separated by 0.253 ꢁ. Ligands 3, 8, 12 were
drawn in ChemDraw 11.0 and minimised with UCSF Chimera 1.3
(amber force field) in charged form.[41] Docking calculations were
set to 50 runs. At the end of a calculation, Autodock performed
cluster analysis. The visualisations of enzyme–ligand interactions
(Figure 2–4) were prepared using Pymol 1.1.[42]
In vitro assay
The reactivation ability of the test compounds was measured on a
multichannel Sunrise spectrophotometer (Tecan, Salzburg, Austria).
The previously used Ellman’s procedure was slightly modified.[29]
The standard polystyrene microplates with 96 wells (Nunc, Rock-
ilde, Denmark) were chosen as reaction vessels. Human erythrocyte
AChE (Sigma–Aldrich) was used in all experiments. Pesticides para-
oxon (POX), methylparaoxon (MePOX) and diisopropylfluorophos-
phate (DFP) were purchased from Sigma–Aldrich. 50 mm phos-
phate buffer (pH 7.4) was used in all experiments.
The activity of the enzyme was adjusted to 0.002 UmLꢁ1. Enzyme
solution (15 mL), phosphate buffer (60 mL), 5,5’-dithiobis(2-nitroben-
zoic)acid (DTBN; 0.4 mgmLꢁ1, 20 mL) were combined in a well. The
enzyme was inhibited via addition of 5 mL of a solution of pesticide
in propan-2-ol: tabun (0.1 mm), POX (0.1 mm), MePOX (0.1 mm),
DFP (1 mm). Propan-2-ol was used as a control. The mixture was
left for 5 min. After inhibition, cholinesterase was reactivated by
the addition of a solution of test compound in the phosphate
buffer (100 or 1 mm). Enzyme activity was measured after 15 min
incubation via addition of 1 mm acetylthiocholine chloride (20 mL,
ATChCl). Oximolysis was determined similarly by displacing enzyme
with a solution of albumin in phosphate buffer (1 mgmLꢁ1, 15 mL).
The microplate was gently shaken prior to measurement. Absorb-
ance was measured against phosphate buffer at 412 nm. The reac-
tivation ability was calculated according to Equation (1):
Acknowledgements
The authors express their appreciation to Mrs. M. Hrabinova for
her technical assistance. The work was supported by the Ministry
of Defence (FVZ0000501) and by Ministry of Education, Youth
and Sports of Czech Republic (MSM 0021620822).
Keywords: acetylcholinesterases
organophosphates · reactivators · tabun
·
molecular modeling
·
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