Synthesis and evaluation of novel analogues of vitamin B6 as reactivators of tabun and paraoxon inhibited acetylcholinesterase

Article abstract of DOI:10.1016/j.cbi.2010.02.004

A series of novel pyridinium oximes was prepared by reactions of quaternization of pyridoxal oxime with substituted phenacyl bromides in acetone at room temperature. The structures of compounds were determined according to the data obtained by IR spectros

Full text of DOI:10.1016/j.cbi.2010.02.004

Chemico-Biological Interactions 187 (2010) 234–237
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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Synthesis and evaluation of novel analogues of vitamin B6 as reactivators of
tabun and paraoxon inhibited acetylcholinesterase
Dajana Gasˇo-Sokacˇ a,b,∗, Maja Katalinic´ c, Zrinka Kovarikc, Valentina Busˇic´ a, Spomenka Kovacˇ a,b
a Department of Chemistry, J.J. Strossmayer University of Osijek, Kuhacˇeva 20, HR-31000 Osijek, Croatia
b Faculty of Food Technology, J.J. Strossmayer University of Osijek, Kuhacˇeva 20, HR-31000 Osijek, Croatia
c Institute for Medical Research and Occupational Health, Ksaverska c. 2, P.O. Box 291, HR-10001 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 February 2010
A series of novel pyridinium oximes was prepared by reactions of quaternization of pyridoxal oxime with
substituted phenacyl bromides in acetone at room temperature. The structures of compounds were deter-
mined according to the data obtained by IR spectroscopy, mass spectrometry, 1H and 13C nuclear magnetic
resonance spectroscopy as well as by elemental analysis. We tested pyridoxal oxime (1) and five pre-
pared oximes in 1 mM concentration as reactivators of human erythrocytes acetylcholinesterase (AChE)
inhibited by organophosphorus compounds tabun and paraoxon: 1-phenacyl-3-hydroxy-4-hydro-
xyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide (2), 1-(4ꢀ-chlorophenacyl)-3-hydroxy-
4-hydroxyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide (3), 1-(4ꢀ-fluorophenacyl)-3-
hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide (4), 3-hydroxy-4-
hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4ꢀ-methylphenacyl)pyridinium bromide (5), 3-
hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4ꢀ-methoxyphenacyl)pyridinium bro-
mide (6). However, tested oximes were not efficient in reactivation of either tabun or paraoxon inhibited
AChE. The maximum restored enzyme activity in 24 h was below 25%. Therefore, this class of compounds
cannot be considered as potential improvement in a search for new and more efficient antidotes against
OP poisoning.
Keywords:
Acetylcholinesterase
Tabun
Paraoxon
Pyridoxal oxime
Reactivation
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
[4]. Unfortunately, none of the currently used oximes is sufficiently
effective against all AChE inhibitors, there is no single AChE reacti-
The organophosphorus (OP) compounds are widely used in
agriculture as insecticides (parathion, paraoxon, malathion), in
industry and technology as softening agents, additives to lubri-
cants, and in military technology as chemical warfare agents (sarin,
soman, cyclosarin, tabun, VX agent) [1]. They are extremely potent
inhibitors of the enzyme acetylcholinesterase (AChE; EC 3.1.1.7)
that is responsible for the termination of the action of acetylcholine
(ACh) at cholinergic synapses. Knowledge of the basic mechanism
of toxicity of organophosphorus compounds allowed the develop-
ment of pharmacologically based medical countermeasures. The
current standard treatment for poisoning with highly toxic OPs usu-
ally consist of combined administration of anticholinergic drugs
(preferably atropine), and an AChE reactivators (called oximes)
[2,3]. The reactivation of inhibited AChE depends not only on the
inhibitors used but also on the chemical structure of the reactivator
vator having the ability to sufficiently reactivate inhibited enzyme
regardless of the chemical structure of the inhibitor [5]. Besides
acting as reactivators, oximes are also reversible inhibitors of AChE
and therefore they could protect the catalytic site against phos-
phorylation due to a direct competition between the oxime and
the phosphorylating agent [6–9].
Commonly used reactivators are characterised by the presence
of several structural features: functional oxime group, quaternary
nitrogen group and different length of linking chain between two
pyridinium rings in the case of bispyridinium reactivators. In the
present study, we have synthesized five new oximes, as derivatives
of vitamin B6, and tested them as reactivators of AChE inhibited by
tabun or paraoxon.
2. Materials and methods
2.1. Preparation of the new oximes
∗
Corresponding author at: Department of Chemistry, J.J. Strossmayer University
Preparation of the new oximes 2–6 is shown in Scheme 1. Pyri-
doxal oxime 1 was prepared according to the described procedures
[10]. Solvents and reagents were purchased from Fluka and Aldrich
of Osijek, Kuhacˇeva 20, HR-31000 Osijek, Croatia. Tel.: +385 31 224 327;
fax: +385 31 207 115.
E-mail address: dajana.gaso@ptfos.hr (D. Gasˇo-Sokacˇ).
0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.02.004

D. Gasˇo-Sokacˇ et al. / Chemico-Biological Interactions 187 (2010) 234–237
235
and used without further purification. IR spectra were measured
2.1.4. 3-Hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-
on Paragon 500 FT-IR spectrophotometer with KBr pellets. 1H NMR
and 13C NMR spectra were measured on a Varian XL-GEM 300 spec-
trophotometer in DMSO-d6 solutions and chemical shifts reported
in ı values downfield from TMS as an internal standard. Mass spec-
tra were recorded using API2000 LC/MS/MS System from Applied
Biosystems/MDS SCIEX.
methyl-1-(4ꢀ-methylphenacyl) pyridinium bromide
(5)
M.p. after crystallization from methanol 240–241.5 ◦C. IR (KBr):
3388, 3040–2861, 1686, 1643–1571, 1257, 1087–980 cm−1 1H
.
NMR (DMSO-d6) ı 13.02 (bs, 1H, NOH), 8.66 (bs, 1H, OH), 8.59 (s,
1H, H-6), 8.01 (d, J = 8.13 Hz; 2H, H-3ꢀꢀ, H-5ꢀꢀ), 7.99 (d, J = 8.12 Hz, 2H,
H-2ꢀꢀ, H-6ꢀꢀ), 6.60 (s, 2H, CH2CO), 4.77 (s, 2H, CH2OH), 3.44 (bs, 1H,
CH2OH), 2.51 (s, 3H, CH3), 2.45 (s, 3H, CH3). 13C NMR (DMSO-d6)
ı 189.89, 152.54, 145.56, 145.51, 139.71, 137.35, 135.11, 132.28,
130.50, 128.18, 64.52, 58.53, 13.37. Anal. calc. for C17H19N2O4Br
(M = 395.2478 g mol−1): C 51.66, H 4.85, N 7.09; found: C 51.65, H
4.83, N 7.27%. MS m/z: 395.2 (100%), 313.0 (44.37%), 295.5 (21.83%),
264.8 (22.54%), 162.1 (33.09%), 132.9 (22.54%). Yield 0.49 g (62%).
The synthesis of quaternary salts 2–6: pyridoxal oxime (1)
(0.36 g; 2 mmol) was dissolved in acetone (300 mL) and stirred
(20 min) at 50 ◦C. The reaction mixture was cooled to room temper-
ature, and substituted phenacyl bromides (R = H, Cl, F, CH3, OCH3)
were added (2 mmol). The reaction mixture was stirred for 24 h,
and left in dark for 3 weeks at room temperature. The crystalline
crude product was collected by filtration under reduced pressure
and recrystallized. The purity of all compounds was determined by
1H and 13C NMR, MS and elemental analysis.
2.1.5. 3-Hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-
methyl-1-(4ꢀ-methoxyphenacyl) pyridinium bromide
(6)
2.1.1. 1-Phenacyl-3-hydroxy-4-hydroxyiminomethyl-5-
hydroxymethyl-2-methylpyridinium bromide
(2)
M.p. after crystallization from ethyl-acetate 267–268 ◦C. IR
(KBr): 3381, 3041–2839, 1677, 1641–1516, 1243, 1047–978 cm−1
.
1H NMR (DMSO-d6): 13.00 (bs, 1H, NOH), 12.01 (bs, 1H, OH), 8.66
(s, 1H, H-6), 8.58 (s, 2H, CH2CO), 8.08 (d, J = 8.89 Hz; 2H, H-3ꢀꢀ, H-5ꢀꢀ),
7.19 (d, J = 8.94 Hz; 2H, H-2ꢀꢀ, H-6ꢀꢀ), 4.80 (m, 2H, CH2OH), 3.90 (s,
3H, OCH3), 3.44 (bs, 1H, CH2OH), 2.50 (s, 3H, CH3). 13C NMR (DMSO-
d6): 189.89, 152.54, 145.56, 145.51, 139.71, 137.35, 135.11, 132.28,
130.50, 128.18, 64.52, 58.53, 13.37. Anal. calc. for C17H19N2O5Br
(M = 411.2472 gmol−1): C 49.65, H 4.66, N 6.81, Br 19.43; found:
C 49.79, H 4.46, N 6.83, Br 19.35%. MS m/z: 411.2 (96.5%); 331.0
(3.49%); 329.35 (100%); 311.0 (17.48%); 299.1 (42.65%); 134.0
(57.34%); 106.0 (60.84%). Yield 0.55 g (67%).
M.p. after crystallization from methanol 250–251 ◦C. IR (KBr):
3276, 3096–2730, 1700, 1641–1449, 1228, 1084–978 cm−1 1H
.
NMR (DMSO-d6) ı 13.00 (bs, 1H, NOH), 11.96 (bs, 1H, OH), 8.62
(s, 1H, H-6), 8.11 (d, J = 7.34 Hz, 2H, H-3ꢀꢀ, H-5ꢀꢀ), 8.10 (d, J = 7.26 Hz,
2H, H-2ꢀꢀ, H-6ꢀꢀ), 7.80 (s, 1H, H-4ꢀꢀ), 6.64 (s, 2H, CH2CO), 4.80 (m,
2H, CH2OH), 3.43 (bs, 1H, CH2OH), 1.91 (s, 3H, CH3). 13C NMR
(DMSO-d6) ı 190.70, 152.57, 145.56, 145.43, 137.33, 135.11, 129.05,
128.53, 128.15, 64.51, 58.52, 13.29. Anal. calc. for C16H17N2O4Br
(M = 381.22 g mol−1): C 50.41, H 4.49, N 7.35, Br 20.96; found: C
50.58, H 4.33, N 7.48, Br 20.93%. MS m/z: 381.0 (83%); 299.3 (100%);
281.4 (52.45%); 269.2 (37.06%); 161.0 (47.55%); 106.0 (31.46%).
Yield 0.29 g (38%).
2.2. Reactivation of inhibited acetylcholinesterase
2.1.2. 1-(4ꢀ-Chlorophenacyl)-3-hydroxy-4-hydroxyiminomethyl-
5-hydroxymethyl-2-methylpyridinium bromide
(3)
The reactivation efficacy of the pyridoxal oxime (1) and newly
synthesized compounds 2–6 (cf. Scheme 1) were evaluated accord-
ing to previously published procedure [11]. The source of AChE
was native intact human erythrocytes; the final dilution dur-
ing enzyme assay was 400-fold. All experiments were done in
0.1 M phosphate buffer, pH 7.4, at 25 ◦C and the substrate was
acetylthiocholine, ATCh (1 mM final concentration). The enzyme
activity was measured spectrophotometrically according to the
Ellman procedure [12] with the thiol reagent DTNB (5,5ꢀ-dithiobis-
2-nitrobenzoic acid), final concentration 0.3 mM. The increase in
absorbance was read at 436 nm. All spectrophotometric measure-
ments were performed on a CARY 300 spectrophotometer (Varian
Inc., Australia).
The undiluted erythrocyte AChE was incubated with 5 ␮M
tabun (O-ethyl-N,N-dimethylphosphoramidocyanidate) or 1 ␮M
paraoxon (O,O-diethyl-O-(4-nitrophenyl)phosphate) up to 60 min,
achieving 95–100% inhibition. The excess of organophosphorus
compound was extracted with a 5-fold volume of hexane. Incuba-
tion mixture was diluted 10-times with 0.1 M phosphate buffer, pH
7.4, containing the oxime to start the reactivation. Final oxime con-
centrations used for the reactivation of inhibited AChE was 1 mM.
After a given time of reactivation aliquots were diluted 40-times in
buffer, DTNB and ATCh (acetylthiocholine iodide, 1.0 mM final con-
centration) were added, and the enzyme activity was measured. An
equivalent sample of uninhibited enzyme was diluted to the same
extent as the inhibited AChE, and control activity was measured
in the presence of oxime at concentrations used for reactivation.
Both activities of control and reactivation mixture were corrected
for oxime-induced hydrolysis of ATCh. Under these conditions, the
enzyme activity in the absence of the oxime was stable and no spon-
taneous reactivation of the phosphorylated enzyme took place.
M.p. after crystallization from methanol 230–233 ◦C. IR (KBr):
3384, 3069–2984, 1688, 1641–1592, 1261, 1053–1002 cm−1 1H
.
NMR (DMSO-d6) ı 13.01 (bs, 1H, NOH), 8.67 (bs, 1H, OH), 8.59 (s,
1H, H-6), 8.12 (d, J = 8.63 Hz, 2H, H-2ꢀꢀ, H-6ꢀꢀ), 7.77 (d, J = 8.61 Hz,
2H, H-3ꢀꢀ, H-5ꢀꢀ), 6.66 (s, 2H, CH2CO), 5.84 (bs, 1H, CH2OH),
4.81 (s, 2H, CH2OH), 2.51 (s, 3H, CH3). 13C NMR (DMSO-d6) ı
189.86, 152.56, 145.53, 145.49, 139.69, 137.32, 135.08, 130.46,
129.16, 128.18, 64.47, 58.51, 13.34. Anal. calc. for C16H16N2O4BrCl
(M = 415.66 g mol−1): C 46.23, H 3.88; N 6.74; found: C 46.34, H 3.86,
N 6.72%. MS m/z: 415.0 (54.16%), 335.2 (43.05%), 333.0 (100%), 302.8
(39.58%), 164.1 (36.11%). Yield 0.55 g (66%).
2.1.3. 1-(4ꢀ-Fluorophenacyl)-3-hydroxy-4-hydroxyiminomethyl-
5-hydroxymethyl-2-methylpyridinium bromide
(4)
M.p. after crystallization from ethyl-acetate 225–225.5 ◦C. IR
(KBr): 3384, 3069–2923, 1688, 1641–1592, 1261, 1053–1002 cm−1
.
1H NMR (DMSO-d6) ı 11.45 (bs, 1H, NOH), 8.54 (bs, 1H, OH),
8.49 (s, 1H, H-6), 8.17 (d, J = 8.03 Hz, 2H, H-2ꢀꢀ, H-6ꢀꢀ), 7.47 (d,
J = 8.05 Hz, 2H, H-3ꢀꢀ, H-5ꢀꢀ), 6.26 (s, 2H, CH2CO), 5.38 (bs, 1H,
CH2OH), 4.61 (s, 2H, CH2OH), 2.24 (s, 3H, CH3). 13C NMR (DMSO-
d6) ı 190.27, 165.61, 147.02, 144.94, 137.70, 130.73, 135.11, 132.28,
130.50, 125.79, 63.84, 60.42, 12.81. Anal. calc. for C16H16BrFN2O4
(M = 399.21 g mol−1): C 48.14, H 4.04, N 7.02; found: C 47.53, H
4.22, N 6.95%. MS m/z: 399.2 (53.52%), 319.1 (9.86%), 317.25 (100%),
299.1 (30.28%), 287.3 (36.62%), 137.0 (32.39%), 133.0 (36.62%). Yield
0.23 g (36%).

236
D. Gasˇo-Sokacˇ et al. / Chemico-Biological Interactions 187 (2010) 234–237
Scheme 1. Synthesis of the oximes 2–6 from pyridoxal oxime (1).
Table 1
Reactivation of inhibited human erythrocyte AChE by pyridoxal oxime (1) and oximes 2–6.
Oxime (1 mM)
Tabun-inhibited AChE
Reactmax (%)
Paraoxon-inhibited AChE
Reactmax (%)
Time of Reactmax (h)
Time of Reactmax (h)
1
2
3
4
5
6
<10
<10
<10
<10
<10
<10
23
23
23
23
23
23
<15
23
23
23
23
23
23
15
10
20
23
20
3. Results and discussion
matic rings which makes the structure less flexible and therefore
inappropriate for accommodation within the active site, as it was
shown with similar compounds [16]. Also, vicinal groups to oxime
moiety could influence the pKa value of the oxime group lowering
its nucleophilicity at the pH 7.4 and most certainly present steric
hindrances that prevent efficient orientation of the oxime group
towards the phosphorylated serine [9]. Therefore, this class of com-
pounds cannot be considered as potential improvement in a search
for new and more efficient antidotes against OP poisoning.
Pyridinium, imidazolium and quinuclidinium oximes are pre-
pared in laboratories in Croatia since the middle of 1970s [5]. The
majority of prepared compounds are mono- and bis-pyridinium
oximes. Numerous attempts had been made to improve the anti-
dotal properties of the conventional mono- and bis-pyridinium
oximes by modifying their structure. Pyridoxal oxime, a deriva-
tive of B6 vitamin, is suitable to be incorporated into a reactivator
molecule and can be used for synthesis of compounds structurally
similar to common antidotes. Very little data seem to be available
on the reactivating property of this group of compounds. Milatovic´
et al. [13] synthesized three novel compounds, derivatives of pyri-
doxal oxime, which combine the structural features of pyridoxal
oxime and toxogonine, which were tested as reactivators of AChE
inhibited by sarin, soman, tabun and VX. Their reactivating potency
was compared with the reactivating potency of the conventional
antidotes Toxogonin and PAM-2. They showed reactivating potency
comparable with that of the conventional antidotes. This was a
reason why we in the present study synthesized five new oximes
derivatives of pyridoxal oxime and tested them as reactivators of
tabun or paraoxon inhibited AChE.
Conflicts of interest
None.
Acknowledgment
This work was supported by the Ministry of Science, Education
and Sports of the Republic of Croatia (No. 022-0222148-2889; 291-
0580000-0169, 073-0731674-0841).
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