Two step synthesis of a non-symmetric acetylcholinesterase reactivator

Article abstract of DOI:10.3390/12081755

The newly developed and very promising acetylcholinesterase reactivator (E)-1-(2-hydroxyiminomethylpyridinium)-4-(4-hydroxyiminomethylpyridinium) -but-2-ene dibromide was prepared using two different pathways via a two-step synthesis involving the appropriate (E)-1-(4-bromobut-2-enyl)-2- or 4-hydroxyiminomethyl-pyridinium bromides. Afterwards, purities and yields of the desired product prepared by both routes were compared. Finally, its potency to reactivate several nerve agent-inhibited acetylcholinesterases was tested.

Full text of DOI:10.3390/12081755

Molecules 2007, 12, 1755-1761
molecules
ISSN 1420-3049
© 2007 by MDPI
www.mdpi.org/molecules
Communication
Two Step Synthesis of a Non-symmetric Acetylcholinesterase
Reactivator
Kamil Musilek 1,2, Kamil Kuca 1,3*, Vlastimil Dohnal 1,4, Daniel Jun 1,3, Jan Marek 2 and Vit
Koleckar 3
1
Department of Toxicology, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec
Kralove, Czech Republic
2
Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05
Hradec Kralove, Czech Republic
3
Center of Advanced Studies, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec
Kralove, Czech Republic
4
Department of Food Technology, Mendel University of Agriculture and Forestry Brno, Zemedelska
1, 613 00 Brno, Czech Republic
* Author to whom correspondence should be addressed; E-mail: kucakam@pmfhk.cz
Received: 25 June 2007; in revised form: 1 August 2007 / Accepted: 6 August 2007 / Published: 7
August 2007
Abstract: The newly developed and very promising acetylcholinesterase reactivator (E)-1-
(2-hydroxyiminomethylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene
dibromide was prepared using two different pathways via a two-step synthesis involving
the appropriate (E)-1-(4-bromobut-2-enyl)-2- or 4-hydroxyiminomethyl-pyridinium
bromides. Afterwards, purities and yields of the desired product prepared by both routes
were compared. Finally, its potency to reactivate several nerve agent-inhibited
acetylcholinesterases was tested.
Keywords: Quaternary; alkylation; acetylcholinesterase; reactivator; oxime; nerve agents

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Introduction
Organophosphorus nerve agents are probably the most toxic compounds prepared by the chemical
industry. Their production began in Germany prior to World War II (the organophosphate Tabun,
1936). Since World War II, many other organophosphorus derivatives have been considered as more
or less toxic compounds for military purposes. After the introduction of nerve agents, their antidotes
were also extensively developed. General treatments for nerve agent intoxication consist of
anticholinergics (mainly atropine) and acetylcholinesterase (AChE; EC 3.1.1.7) reactivators
(pralidoxime, HI-6, obidoxime) [1-3] (Figure 1).
Figure 1. Reactivators of acetylcholinesterase.
CONH2
HON=HC
CH=NOH
2 Cl
HON=HC
N
CH3
N N
CH2OCH2
CH=NOH
N
N
CH2OCH2
2 Cl
Cl
pralidoxime
HI-6
obidoxime
Unfortunately, no single compound is capable of acting as broad-spectrum AChE reactivator for
treatment of intoxications by every nerve agent [3]. Due to this, many laboratories are focused on
synthesis of such broad-spectrum reactivators [4-8]. In 2006 we prepared a promising non-symmetrical
AChE reactivator: 1-(2-hydroxyiminomethylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-
ene dibromide (designated as K053) [5]. It was prepared using two-step synthetic pathway via 1-(4-
bromobut-2-ene)-2-hydroxyiminomethylpyridinium bromide. Because of its very promising activity
and to carry out further in vitro and in vivo investigations, it was necessary to increase the yield and
purity of this compound. Owing to this fact, in this article we focus our attention on preparing this
reactivator using a reverse sequence of reaction steps. A similar approach for reactivator K027 was
previously published by our group [9]. This compound is currently under consideration as a very
promising AChE reactivator with a broad reactivation potency and relatively low toxicity [10-16].
However, it was not applicable for our current synthesis of the new reactivator K053 (3) because of
different connection chains between both the substituted pyridinium rings in each compound (a
trimethylene linker in K027 versus an (E)-but-2-ene in K053).
Results and Discussion
This work could be divided into two parts – a synthetic part (main part) and a biochemical part.
The target bisquaternary pyridinium AChE reactivator (E)-1-(2-hydroxyiminomethylpyridinium)-4-(4-
hydroxyiminomethylpyridinium)-but-2-ene dibromide (3; K053) can be prepared via two different
synthetic pathways, each involving two steps. The first step – creation of the monoquaternary
intermediate – is the overall yield limiting one because of the potential creation of byproducts. To
decrease formation of the undesired byproduct, a 5 equivalent excess of the alkylating agent was added
the solvent volume was increased (to 60 mL). The minor bisquaternary byproducts present in the

Molecules 2007, 12
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mixture, could be removed by recrystallization from acetonitrile. Results of both monoquaternizations
are summarized in Scheme 1.
Scheme 1. Preparation of oxime K053.
CH2CH=CHCH2Br
N
CH=NOH
HON=HC
N
a)
Br
30%
b)
HON=HC
1
90%
N
N
2 Br
HON=HC
CH2CH=CHCH2Br
3
d)
N
N
c)
Br
21%
95%
CH=NOH
CH=NOH
2
(a) (E)-1,4-dibromobut-2-ene (5 equiv), acetone, 50°C, 6.5 h; (b) 4-hydroxyiminomethylpyridine
(2 equiv), DMF, 60°C, 6 h; (c) (E)-1,4-dibromobut-2-ene (5 equiv), acetone, reflux, 2 h; (d) 2-
hydroxyiminomethylpyridine (2 equiv), DMF, 60°C, 10.5 h
The second step of the complete synthetic pathway – the creation of the non-symmetrical product
(3; K053) did not prove as difficult as in the case of the monoquaternary intermediates (no byproducts
are possible and therefore, the desired transformation can be carried out under more forcing reaction
conditions: 10 mL DMF, 60°C). Grey crystals were formed in the solution and then they were simply
recrystallized from acetonitrile. Results obtained for both second steps are summarized in Scheme 1.
Comparing the yields of monoquaternary intermediates, the higher yield was obtained and almost
no impurities were formed for the pyridine with an oxime group in the para-position. The lower
quaternization yield obtained for an ortho-substituted pyridine was probably due to the steric
hindrance of the oxime group, which hinders attack on the pyridine nitrogen. As mentioned, no
byproducts are possible in the second step of each sequence, but a similar yield dependency on the
substitution pattern of the pyridine was observed and a lower yield was noted (21% – presumably due
to steric hindrance in the pyridine substituted at the ortho-position), compared to the para-substituted
pyridine (90 % in the second step). If the overall yields of both synthetic pathways are compared,
synthesis via an ortho-substituted pyridine in the first step gave higher yield.
Biochemistry
In our biochemical studies, the ability of the prepared compound 3 (K053) to split
organophosphate-AChE complexes was tested using our in vitro reactivation test [19]. Our previous
results (on chlorpyrifos-inhibited AChE reactivation) were updated with results for Tabun-,
Cyclosarin- and VX-inhibited AChE reactivation (Table 1). As it is clearly presented, this oxime is
also able to reactivate all other nerve agent-inhibited AChEs tested. Maximal reactivation activity was
achieved for chlorpyrifos, the lowest for Tabun. These results are closely related to the splitting of an

Molecules 2007, 12
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enzyme-inhibitor complex. While the complex of AChE with the pesticide chlorpyrifos is easily
reactivated, the nerve agent complexes are degraded by a process called aging, during which the nerve
agent moiety undergoes a change, whereby an alkyl or amidoyl group is replaced by a hydroxyl
(negative charge) and consequently, becomes almost resistant to reactivation. Although Tabun does
not have a fast aging halftime (t1/2 = 46 h), its reactivation is poor (Table 1) [17]. The poor reactivation
can be explained by the free electron pair on the amidoyl group, which may easily interact with the
other hydrogen bonds. In contrast, Cyclosarin (t1/2 = 40 h) and VX (t1/2 ~ 48 h) are reactivated
satisfactorily, whereas Cyclosarin had the better reactivator profile with K053. The worse reactivation
of VX was probably caused by presence of an oxime in the 2-position in K053 instead of an oxime in
position four, which is required for VX poisoning [18]. The biochemical results should be further
investigated by appropriate more sophisticated methods.
Table 1. Reactivation of nerve agent-inhibited AChE by reactivator K053 (3) (source of
the enzyme – rat brain acetylcholinesterase; time of inhibition by nerve agent – 30 min;
time of reactivation – 10 min; pH – 7.6; 25°C).
Organophosphorus Resulting Enzyme-Inhibitor
% Reactivation *
% Reactivation**
Inhibitor
Chlorpyrifos
Tabun
Complex
O
P
O Et
63
9
48
0
AChE O
O
Et
O
P
O Et
Me
AChE O
Me
N
O
P
Cyclosarin
VX
31
18
40
32
O
AChE O
Me
O
P
O Et
AChE O
Me
Reactivation data are expressed as mean form three independent measurements
* concentration of reactivator 10-3 M
** concentration of reactivator 10-5 M
Conclusions
In conclusion, a new promising AChE reactivator was prepared using two different synthetic
pathways, via two different reactive monoquaternary intermediates. These intermediates could be
simply used for preparation of other reactivators derived from the structure of the oxime K053. This
procedure will be in future used for preparation of large amounts of this compound which will be
needed for further in vitro and in vivo investigations. Moreover, splitting of the bond between nerve
agents and AChE showed that this reactivator is able to reactivate not only chlorpyrifos (a pesticide),
as previously known before, but also Cyclosarin-, Tabun- and VX-inhibited AChE

Molecules 2007, 12
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Experimental
General
Solvents (acetone, DMF, MeCN) and reagents were purchased from Fluka and Sigma-Aldrich
(Czech Republic) and used without further purification. Reactions were monitored by TLC using DC-
Alufolien Cellulose F (Merck, Germany) and 5:1:2 BuOH-CH3COOH-H2O as mobile phase; detection
was by a Dragendorff reagent solution (10 mL CH3COOH, 50 mL H2O and 5 mL of basic solution
prepared by mixing of two fractions – fraction A: 850 mg Bi(NO3)3, 40 mL H2O, 10 mL CH3COOH;
fraction B: 8 g KI, 20 mL H2O). Melting points were measured on a PHMK 05 (VEB Kombinat
Nagema, Radebeul, Germany) micro heating stage and are uncorrected. NMR spectra were generally
recorded on a Varian (Palo Alto CA, US) Gemini 300 instrument (1H at 300 MHz, 13C at 75 MHz). In
all cases, the chemical shift values are reported in ppm (δ) relative to residual CHD2SO2CD3 (δ 2.50)
1
13
or D2O (δ 4.79) for the H spectra and to the DMSO-d6 solvent peak (δ 39.43) for the C spectra,
respectively. Signals are quoted as s (singlet), d (doublet), t (triplet) and m (multiplet). Mass spectra
were recorded using combination of high performance liquid chromatography and mass spectrometry.
A HP1100 HPLC system (Agilent Technologies, Waldbronn, Germany) was used. It consisted of
vacuum degasser G1322A, quaternary pump G1311A, autosampler G1313A and quadrupole mass
spectrometer MSD1456 VL equipped with electrospray ionization source. Nitrogen for the mass
spectrometer was supplied by a Whatman 75-720 nitrogen generator. Data were collected in positive
ion mode with an ESI probe voltage of 4000 V. The pressure of nebulizer gas was set up to 35 psig.
Drying gas temperature was operated at 335 °C and flow at 13 L/min.
Synthesis
Preparation of monoquaternary salts – A solution of the corresponding hydroxyiminomethylpyridine
(2.0 g, 16.4 mmol) and (E)-1,4-dibromobut-2-ene (17.51 g, 81.9 mmol) in acetone (60 mL) was stirred
at reflux for 6-6.5 h. The reaction mixture was cooled to the room temperature; the crystalline crude
product was collected by filtration, washed with acetone (3×20 mL) and recrystalized from MeCN.
Preparation of bisquaternary salts – A solution of the monoquaternary salt (0.50 g, 1.5 mmol) and
corresponding hydroxyiminomethylpyridine (2.2 mmol) in DMF (10 mL) was stirred at 60 °C for 2-
10.5 h. The reaction mixture was cooled to the room temperature and partitioned with acetone (50
mL); the crystalline crude product was collected by filtration, washed with acetone (3×20 mL) and
recrystalized from MeCN.
Product characterization data
(E)-1-(4-brombut-2-enyl)-2-hydroxyiminomethylpyridinium bromide (1). Yield 30%; m.p. 132-135°C;
1H-NMR (DMSO d6): δ 9.11 (d, 1H, J = 6.0 Hz, H-6), 8.78-8.54 (m, 2H, H-5 + -CH=NOH), 8.42 (d,
1H, J = 6.6 Hz, H-3), 8.22-8.08 (m, 1H, H-4), 6.18-6.04 (m, 1H, N-CH2-CH=), 6.03-5.82 (m, 1H,
=CH-CH2-Br), 5.53 (d, 2H, J = 5.8 Hz, N-CH2-), 4.14 (d, 2H, J = 7.4 Hz, -CH2-Br); 13C-NMR (DMSO

Molecules 2007, 12
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d6): δ 147.06, 145.82, 145.68, 141.31, 132.05, 127.85, 125.66, 121.41, 60.13, 58.13; ESI-MS: m/z
254.9 [M]+ (calculated for [C10H12N2O]+ 255.01); EA: calculated 35.74% C, 3.60% H, 8.34% N; found
36.12% C, 3.89% H, 8.79% N.
(E)-1-(4-brombut-2-enyl)-4-hydroxyiminomethylpyridinium bromide (2). Yield 95%; m.p. 187-191°C;
1H-NMR (DMSO d6): δ 9.02 (d, 2H, J = 6.0 Hz, H-2 + H-6), 8.45 (s, 1H, -CH=NOH), 8.27 (d, 2H, J =
6.0 Hz, H-3 + H-5), 6.26-6.09 (m, 2H, -CH=CH-CH2Br), 5.32 (d, 2H, J = 4.9 Hz, N-CH2-), 4.17 (d,
13
2H, J = 6.0 Hz, -CH2-Br); C-NMR (DMSO d6): δ 148.67, 145.05, 144.85, 133.72, 127.69, 124.17,
60.25, 32.01; ESI-MS: m/z 254.9 [M]+ (calculated for [C10H12N2O]+ 255.01); EA: calculated 35.74%
C, 3.60% H, 8.34% N; found 35.69% C, 3.72% H, 8.49% N.
(E)-1-(2-hydroxyiminomethylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide
(3). Yield 85%; m.p. 202-203°C; 1H-NMR (DMSO d6): δ 9.15 (d, 1H, J = 6.0 Hz, H-6), 9.02 (d, 2H, J
= 6.0 Hz, H-2´ + H-6´), 8.72-8.55 (m, 2H, H-5 + -CH=NOH), 8.51-8.36 (m, 2H, H-3 + -CH=NOH´),
8.25 (d, 2H, J = 6.0 Hz, H-3´+ H-5´), 8.20-8.10 (m, 1H, H-4), 6.37-6.22 (m, 1H, N-CH2-CH=), 6.06-
5.91 (m, 1H, =CH-CH2-N´), 5.57 (d, 2H, J = 4.7 Hz, N-CH2-), 5.32 (d, 2H, J = 6.0 Hz, -CH2-N´); 13C-
NMR (DMSO d6): δ 148.64, 147.10, 146.03, 145.73, 145.04, 144.90, 141.48, 131.20, 127.95, 127.70,
125.72, 123.98, 60.23, 58.23; ESI-MS: m/z 149.1 [M]2+ (calculated for [C8H9N2O]2+ 149.07); EA:
calculated 41.95% C, 3.96% H, 12.23% N; found 41.73% C, 4.15% H, 12.41% N.
Acknowledgements
The authors would like to thank Mrs Martina Hrabinova, Mrs. Petra Hanusova and Mr. Petr
Stodulka for their excellent technical help. This work was supported by the grant agency of Ministry of
Defence (Czech Republic) – grant no. FVZ0000604.
References
1. Marrs, T.C. Organophosphate poisoning. Pharmacol. Therap. 1993, 58, 51-66.
2. Bajgar, J. Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis,
and treatment. Adv. Clin. Chem. 2004, 38, 151-216.
3. Kuca, K.; Jun, D.; Musilek, K. Structural requirements of acetylcholinesterase reactivators.Mini-
Rev. Med. Chem. 2006, 6, 269-277.
4. Pang, YP.; Kollmeyer, TM.; Hong, F.; Lee, JC.; Hammond, PI.; Haugabouk, SP.; Brimijoin, S.
Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase
reactivators. Chem. Biol. 2003, 10, 491-502.
5. Musilek, K.; Kuca, K.; Jun, D.; Dohnal, V.; Dolezal, M.. Synthesis of the novel series of
bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity
against chlorpyrifos-inhibited acetylcholinesterase. Biorg. Med. Chem. Lett. 2006, 16, 622-627.
6. Kim, T.H.; Kuca, K.; Jun, D.; Jung, Y.S. Design and synthesis of new bis-pyridinium oximes as
cyclosarin-inhibited acetylcholinesterase reactivators. Bioorg. Med. Chem. Lett. 2005, 15, 2914-
2917.

Molecules 2007, 12
1761
7. Musilek, K.; Holas, O.; Hambalek, J.; Kuca, K.; Jun, D.; Dohnal, V.; Dolezal, M. Synthesis of
Bispyridinium Compounds bearing Propane Linker and Evaluation of their Reactivation Activity
against Tabun- and Paraoxon-Inhibited Acetylcholinesterase. Lett. Org. Chem. 2006, 3, 831-835.
8. Musilek, K.; Kuca, K; Jun, D.; Dolezal, M. Progress in synthesis of new acetylcholinesterase
reactivators during the period 1990-2004. Curr. Org. Chem. 2007, 11, 229-238.
9. Kuca, K.; Bielavsky, J.; Cabal, J., Bielavska, M. Synthesis of a potential reactivator of
acetylcholinesterase
1-(4-hydroxyiminomethylpyridinium)-3-(carbamoylpyridinium)-propane
dibromide. Tetrahedron Lett. 2003, 44, 3123-3125.
10. Kuca, K.; Kassa, J. In vitro reactivation of acetylcholinesterase using of the oxime K027. Vet.
Hum. Toxicol. 2004, 46, 15-18.
11. Kassa, J.; Kuca, K.; Cabal, J.; Paar, M. A comparison of the efficacy of new asymmetric
bispyridinium oximes (K027, K048) with currently available oximes against tabun by in vitro and
in vivo methods. J. Toxicol. Env. Health 2006, 69, 1875-1882.
12. Calic, M.; Lucic-Vrdoljak, A.; Radic, B.; Jelic, D.; Jun, D.; Kuca, K.; Kovarik, Z. In vitro and in
vivo evaluation of pyridinium oximes: mode of interaction with acetylcholinesterase, effect on
tabun- and soman-poisoned mice and their cytotoxicity. Toxicology 2006, 219, 85-96.
13. Petroianu, GA.; Arafat, K.; Kuca, K.; Kassa, J. Five oximes (K-27, K-33, K-48, BI-6 and
methoxime) in comparison with pralidoxime: in vitro reactivation of red blood cell
acetylcholinesterase inhibitied by paraoxon. J. Appl. Toxicol. 2006, 26, 64-71.
14. Petroianu, GA.; Nurulain, SM.; Nagelkerke, N.; Al-Sultan, MAH.; Kuca, K.; Kassa, J. Five
oximes (K-27, K-33, K-48, BI-6 and methoxime) in comparison with pralidoxime: survival in rats
exposed to the organophosphate paraoxon. J. Appl. Toxicol. 2006, 26, 262-268.
15. Tekes, K.; Hasan, MY.; Sheen, R.; Kuca, K.; Petroianu, G. HPLC determination of the serum
concentration of K-27, a novel oxime-type cholinesterase reactivator. J. Chromatogr. A. 2006,
1122, 84-87.
16. Lucic-Vrdoljak, A.; Calic, M.; Radic, B.; Berend, S.; Kuca, K.; Kovarik, Z. Pre-treatment with
pyridinium oximes improves antidotal therapy against tabun poisoning. Toxicology 2006, 228, 41-
50.
17. Gupta, R. Toxicology of organophosphate & carbamate compounds, In Cl Cholinesterase
inhibitors as chemical warfare agents: Community preparedness guidelines; Elsevier Academic
Press: London, 2006; pp. 47-68.
18. Kuca, K.; Jun, D.; Musilek, K.; Bajgar, J. Structure-activity relationship for the reactivators of
acetylcholinesterase inhibited by nerve agent VX. Toxicol. Lett. 2006, 164, S51-S52.
19. Kuca, K., Cabal, J. Evaluation of newly synthesized reactivators of the brain cholinesterase
inhibited by sarin-nerve agent. Toxicol. Mech. Meth. 2005, 15, 247-252.
Sample Availability: Samples of the prepared compounds are available from authors.
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