Novel series of bispyridinium compounds bearing a (Z)-but-2-ene linker-Synthesis and evaluation of their reactivation activity against tabun and paraoxon-inhibited acetylcholinesterase

Article abstract of DOI:10.1016/j.bmcl.2007.03.025

Six novel AChE reactivators with a (Z)-but-2-ene linker were synthesized using the known synthetic pathways. Their ability to reactivate AChE, which had been previously inhibited by nerve agent tabun or pesticide paraoxon, was tested in vitro and compared

Full text of DOI:10.1016/j.bmcl.2007.03.025

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3172–3176
Novel series of bispyridinium compounds bearing a
(Z)-but-2-ene linker—Synthesis and evaluation of their reactivation
activity against tabun and paraoxon-inhibited acetylcholinesterase
b,c
Kamil Musilek,a,b Ondrej Holas,a Kamil Kuca,b,c, Daniel Jun, Vlastimil Dohnal,d
*
Veronika Opletalovaa and Martin Dolezala
aDepartment of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague,
Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
bCenter of Advanced Studies, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
cDepartment of Toxicology, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
dDepartment of Food Technology, Mendel University of Agriculture and Forestry Brno, Zemedelska 1, 613 00 Brno, Czech Republic
Received 19 February 2007; revised 7 March 2007; accepted 9 March 2007
Available online 13 March 2007
Abstract—Six novel AChE reactivators with a (Z)-but-2-ene linker were synthesized using the known synthetic pathways. Their abil-
ity to reactivate AChE, which had been previously inhibited by nerve agent tabun or pesticide paraoxon, was tested in vitro and
compared to pralidoxime, HI-6, obidoxime, and K075. The novel synthesized compounds were found to be ineffective against
GA-inhibited AChE but the ability of (Z)-1,4-bis(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide to reactivate paraoxon-
inhibited AChE was comparable with that of oxime K075. Notably, the oxime group in position four substantially increased the
ability of the novel compounds to reactivate paraoxon-inhibited AChE.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Acetylcholinesterase (AChE, EC 3.1.1.7) is a well-
known enzyme studied for various reasons, for example,
Alzheimer’s disease, Parkinson disease, an eco-toxico-
logy marker.1–3 The enzyme occurs throughout inverte-
brates and vertebrates species.4,5 Many inhibitors of
AChE exist both in natural and artificial compounds.5,6
The organophosphorus inhibitors of AChE (OPI) are
some of the oldest artificial inhibitors synthesized
(Fig. 1), for example the first members of this group
were synthesized as military nerve agents (NA; e.g., sar-
in, soman, tabun).5 Afterwards many similar com-
pounds with decreased toxicity were prepared. These
are currently used in agricultural production as pesti-
cides (e.g., parathion, chlorpyrifos, diazinon) or for
industrial purposes as softening agents and flame retar-
dants.7 Therapeutically, metrifonate was also proved for
treatment of Alzheimer’s disease.8
All OPI irreversibly inhibit AChE through binding to a
serine hydroxyl within the active site of the enzyme.
Subsequently, the AChE is not able to fulfill its physio-
logical role in cholinergic transmission and so leads to
over-stimulation by acetylcholine with the resultant pos-
sibility of respiratory failure and death.5
The AChE reactivators (e.g., pralidoxime, obidoxime,
HI-6; Fig. 2) in combination with atropine have been used
to counteract the poisonous effects of OPI. The reactiva-
tor is able to cleave the covalent bond between the OPI
and AChE, restoring the activity of the enzyme. This reac-
tivation process consists of an attack of the nucleophilic
oxime group (in the form of oximate anion) on the cova-
lent bond. However, there is no reactivator able to coun-
C2H5O
C2H5O
O
O
NC
O
P
P
NO2
C2H5O N(CH3)2
Keywords: Acetylcholinesterase; Reactivation; Nerve agent; Tabun;
Pesticide; Paraoxon; Reactivator; Oxime.
paraoxon
tabun
*
Figure 1. Examples of organophosphorus inhibitors of acetyl-
cholinesterase.
Corresponding author. Tel.: +420 973 251 523; fax: +420 495 518
094; e-mail addresses: kucakam@pmfhk.cz; kucakam@seznam.cz
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.025

K. Musilek et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3172–3176
3173
the hydrogen bonds of His447.12,13 Additionally, the con-
formational change of Pro338 partially closes the narrow
AChE cleft. Consequently the phosphoramidoyl group of
GA is replaced by a molecule of water and the rest of GA
molecule is coordinated in the enzyme’s cavity.
CONH2
2 Cl
HON=HC
N
CH3
N
N
CH=NOH
Cl
CH2OCH2
pralidoxime (1)
HON=HC CH=NOH
The reactivators of AChE bearing a (E)-but-2-ene linker
have been published previously.14,15 Some of these reac-
tivators (e.g., K075; Fig. 2) showed very promising
activity in the reactivation of GA in vitro and so were
subjected to further in vivo testing.16 Owing to the
in vitro results, the idea to modify the (E)-formation
of the double bond for related (Z)-but-2-ene linker
was used. Consequently, six novel reactivators (5–10)
were prepared in an appropriate yield and purity
(Fig. 3). At first, a novel synthetic approach for prepara-
tion of (Z)-1,4-dibromobut-2-ene (13) from a corre-
sponding diol was used.17 Second, monoquaternary
substances (11–12) were synthesized in the presence of
excess alkylating agent. Finally, the bisquaternary com-
pounds were produced (5–10) (Scheme 1).18 Their reac-
tivation activity was measured using a model of GA and
paraoxon-inhibited rat brain AChE.
HI-6 (2)
CH=NOH
2 Br
N
CH2CH=CHCH2
N
N
N
2 Cl
CH2OCH2
obidoxime (3)
K075 (4)
HON=HC
Figure 2. Examples of AChE reactivators used and tested.
teract the full spectrum of OPI.9 Moreover, the covalent
bond present between OPI and the enzyme is very stable
and is subjected to intramolecular modifications
called ‘aging’.10 For example, the nerve agent tabun
(GA; O-ethyl-N,N-dimethylphosphoramidocyanidate)
undergoes one of the fastest aging processes that makes
GA-inhibited AChE almost impossible for reactivation.11
Ekstro¨m et al. described this modification by changes
within the cavity of GA-inhibited AChE, especially at
In vitro testing of synthesized oximes involved standard
experimental procedures and is described in full by the
work of Kuca and Cabal.19 Briefly, a 10% rat brain
homogenate (the source of AChE) in water was inhib-
ited by GA or paraoxon. After 30 min of incubation
with the OPI moiety, this achieved 95% inhibition of
AChE. Next the reactivator was added to the solution
for a further 10 min. Activities of intact AChE (a0),
inhibited AChE (ai), and reactivated AChE (ar) were de-
duced from the rate of consumption of a NaOH solution
(0.01 M). The percentage of reactivation (%) was
calculated from the measured data according to the
formula:
2 Br
N
N
HON=HC
CH=NOH
Compound
Oxime position
2,2´-CH=NOH
3,3´-CH=NOH
4,4´-CH=NOH
2,3´-CH=NOH
2,4´-CH=NOH
3,4´-CH=NOH
5
6
7
8
9
ꢀ
ꢁ
a0 ꢀ ar
a0 ꢀ ai
x ¼ 1 ꢀ
ꢁ 100½%ꢂ
10
Pralidoxime, HI-6, obidoxime, and K075 (1–4) of HPLC
purity were synthesized in our laboratory and used as
references. Collected data are summarized in Table 1.
Figure 3. Six oxime reactivators tested against tabun and paraoxon-
inhibited AChE.
(Ph)3PBr2
Br
Br
HO
OH
MeCN; Ar; r.t.-50°C
13
N
2 Br
13
CH=NOH
N
N
HON=HC
CH=NOH
N
DMF; 50-100°C
5-10
CH=NOH
N
Br
13
DMF (MeCN)
50-100°C
N
Br
CH=NOH
HON=HC
acetone; 50°C
11-12
Scheme 1. Preparation of bisquaternary substances with (Z)-but-2-ene linker.

3174
K. Musilek et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3172–3176
Table 1. Reactivation potencies of tested oximes (%, mean value of
three independent determinations)—time of inhibition—30 min; time
of reactivation by AChE reactivators—10 min; pH 7.6; temperature
25 ꢁC
Additionally, the structural factors necessary for reacti-
vation of GA and paraoxon-inhibited AChE were deter-
mined. The main structural features which influence the
reactivation potency are the oxime functional group (its
position and amount), the quaternary heteroaromatic
ring, and the connecting linker for bisquaternary reacti-
vators.24 As previously mentioned, the GA-inhibited
AChE is subject to intramolecular changes which par-
tially close the cavity of AChE. For this reason, only
compounds with compatible shapes and relatively small
sizes can effectively counteract the exposure of GA.25 By
these means, compounds with an oxime moiety at posi-
tion four of the heteroaromatic ring are preferred (3–4).
Nevertheless, our results confirmed that other features
are also necessary for the reactivation process. While
oxime K075 with (E)-but-2-ene linker was promising
in in vitro tests, the novel reactivator 7 with (Z)-but-2-
ene linker was ineffective. This observation could be
plausibly explained by the distinct molecular shapes
arising from differences in the connecting chain
(Fig. 4). The interaction with the GA-inhibited AChE
is unknown and will be further investigated.
Inhibitor
Reactivation (%)
tabun paraoxon
Reactivator/concentration 10ꢀ3 M 10ꢀ5 M 10ꢀ3 M 10ꢀ5 M
Pralidoxime (1)
HI-6 (2)
4
2
1
1
0
1
0
0
42
35
76
60
26
5
1
2
2
1
1
1
3
0
0
4
1
1
Obidoxime (3)
K075 (4)
11
16
2
0
37
2
2
0
1
1
0
0
1
23
46
13
8
5
6
0
0
0
0
0
7
0
0
0
0
40
46
14
29
18
8
0
9
10
1
0
0
19
3
1
0
5
The required reactivation potency able to counteract the
effect of OPI in vivo should exceed 10% of reactivated
AChE in vitro.5 In the case of NA, there are only a few
reactivators able to fulfill this criterion.11 Our results for
GA-inhibited AChE confirmed this. Only two known
substances (3, 4) wereable toexceed 10% capability. How-
ever, a concentration of 10ꢀ3 M is not suitable for human
use due to the noxious effects of the reactivator itself.20
For this reason, only K075 (4) at a concentration of
10ꢀ5 M was chosen for further testing. Surprisingly, six
of the novel substances showed no reactivation ability,
not even compound 7 which differed from K075 only in
the shape of the double bond in the connecting linker.
In contrast, the reactivation efficacy of paraoxon-inhib-
ited AChE for K075 and the novel compound 7 was
comparable. Generally, the OP pesticides were weaker
inhibitors of AChE and moreover, the structural
changes in the enzyme’s cavity do not occur.26 The
novel compounds showed increasing efficacy for parao-
xon-inhibited AChE at a concentration of 10ꢀ5 M, influ-
enced by the position of the oxime moiety: position
3,30 < position 2,20 < position 2,30 < position 3,40 < po-
sition 2,40 < position 4,40. Remarkably, the oxime group
in position four on heteroaromatic ring increases the
ability of compound to reactivate paraoxon-inhibited
AChE.27,28 In addition, the novel reactivators contain
two quaternary nitrogens, which are considered to have
better affinity for the cavity of AChE.29–32 The influence
of (E)- or (Z)-but-2-ene linker will also be further
investigated.
The reactivation ability of oximes for the paraoxon-
inhibited AChE was different. First, the reactivators
developed for NA are not suitable for treatment of OP
pesticides (1–2) at concentrations applicable for human
use with exception of obidoxime (3).21 However, the
higher toxicity of obidoxime in comparison to HI-6 is
well known.22 All of our novel reactivators showed some
reactivation ability at a concentration of 10ꢀ5 M which
is more appropriate for human use. Although the con-
centration is lower, some of them (6–10) had higher
reactivation ability in contrast with concentration
10ꢀ3 M. This phenomenon can be explained as inhibi-
tion of AChE by the reactivator itself, as was previously
published.23 One novel compound (7) showed the most
promising result and was able to extend the reactivation
potency of K075 at the concentration 10ꢀ5 M.
In conclusion, six novel reactivators of AChE with (Z)-
but-2-ene linker were prepared in satisfactory yield and
purity. The ability of these six compounds together with
pralidoxime, HI-6, obidoxime, and K075 to reactivate
GA and paraoxon-inhibited AChE was measured
in vitro. Only K075 exceeded the potency of obidoxime
against GA. Compounds bearing an oxime group at
position four were found to be promising against
Figure 4. Different spatial orientation of (E)- and (Z)-1,4-bis(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide (from Chem 3D, version 9.0;
CambridgeSoft Corp.).

K. Musilek et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3172–3176
3175
MeCN (5–7). (B) Preparation of non-symmetrical salts—
A solution of the hydroxyiminomethylpyridine (1.5 g,
12.3 mmol) and (Z)-1,4-dibromobut-2-ene (7.88 g,
36.8 mmol) in acetone (30 ml) was stirred at 50 ꢁC. The
reaction mixture was cooled to the room temperature. The
crystalline crude product was collected by filtration,
washed with acetone (3· 20 ml), and recrystallized from
MeCN (11–12). A solution of the monoquaternary salt
(0.50 g, 1.5 mmol) and corresponding hydroxyiminometh-
ylpyridine (0.27 g, 2.2 mmol) in DMF (10 ml) or MeCN
(50 ml) was stirred at 50–100 ꢁC. The reaction mixture was
cooled to the room temperature and portioned with
acetone (50 ml); the crystalline crude product was col-
lected by filtration, washed with acetone (3· 20 ml), and
recrystallized from MeCN (8–10).
paraoxon-inhibited AChE at a concentration applicable
for use in vivo. Although the novel compounds were not
effective against GA-inhibited AChE, they showed
promising results in reactivation of paraoxon- inhibited
AChE.
Acknowledgments
The authors express their appreciation to Mrs. M. Hra-
binova and Ms. Petra Hanusova for their technical
assistance. The work was supported by the grant of
Grant Agency of Charles University No. 302/2005/
B-CH/FaF and by the grant of Ministry of Defence of
Czech Republic No. FVZ0000501.
(Z)-1,4-bis(2-hydroxyiminomethylpyridinium)-but-2-ene
dibromide (5). Prepared by method A. The reaction
mixture was stirred at 50 ꢁC and stopped after 8 h. Yield
0.24 g (29%), mp 219–221 ꢁC. 1H NMR spectrum
(300 MHz, D2O): d (ppm) 8.95 (d, 2H, J = 6.0 Hz, PyrH),
8.72 (s, 2H, –CH@NOH), 8.68–8.55 (m, 2H, PyrH), 8.45
(d, 2H, J = 7.8 Hz, PyrH), 8.22–8.04 (m, 2H, PyrH), 6.22–
References and notes
1. Moreira, P. I.; Zhu, X.; Nunomura, A.; Smith, M. A.;
Perry, G. Exp. Rev. Neurother. 2006, 6, 897.
2. Giacobini, E. Pharmacol. Res. 2004, 50, 433.
3. Sarkar, A.; Ray, D.; Shrivastava, A. N.; Sarker, S.
Ecotoxicology 2006, 15, 333.
4. Hyne, R. V.; Mahe, W. A. Ecotoxicol. Environ. Saf. 2003,
54, 366.
5. Bajgar, J. Adv. Clin. Chem. 2004, 38, 151.
6. Viegas, C., Jr.; Bolzani, V. S.; Barreiro, E. J.; Fraga, C. A.
Mini-Rev. Med. Chem. 2005, 5, 915.
7. Costa, L. G. Clin. Chim. Acta 2006, 366, 1.
8. Ringman, J. M.; Cummings, J. L. J. Clin. Psychiatry 1999,
60, 776.
9. Marrs, T. C. Pharmacol. Therap. 1993, 58, 51.
10. Patocka, J.; Kuca, K.; Jun, D. Acta Medica 2004, 47, 215.
11. Cabal, J.; Kuca, K.; Kassa, J. Basic Clin. Pharmacol.
Toxicol. 2004, 95, 81.
6.03 (m, 2H, –CH@), 5.71 (d, 4H, J = 4.1 Hz, –CH2–). 13
C
NMR spectrum (75 MHz, D2O): d (ppm) 146.40, 145.76,
145.21, 141.68, 127.77, 126.99, 55.58. EA: calcd 41.95% C,
3.96% H, 12.23% N; found 41.37% C, 4.14% H, 11.78% N.
ESI-MS: m/z 149.1 [M]2+ (calcd for [C8H9N2O]2+ 149.17).
(Z)-1,4-bis(3-hydroxyiminomethylpyridinium)-but-2-ene
dibromide (6). Prepared by method A. The reaction
mixture was stirred at 100 ꢁC and stopped after 2 h. Yield
0.75 g (89%), mp 230–232 ꢁC. 1H NMR spectrum
(300 MHz, D2O): d (ppm) 9.18 (s, 2H, PyrH), 8.96 (d,
2H, J = 6.0 Hz, PyrH), 8.80 (d, 2H, J = 8.0 Hz, PyrH),
8.41 (s, 2H, –CH@NOH), 8.23–8.13 (m, 2H, PyrH), 6.34–
6.23 (m, 2H, –CH@), 5.65 (d, 4H, J = 4.1 Hz, –CH2–). 13
C
NMR spectrum (75 MHz, D2O): d (ppm) 144.23, 143.89,
142.51, 141.98, 133.37, 128.08, 127.91,57.74. EA: calcd
41.95% C, 3.96% H, 12.23% N; found 41.58% C, 4.12% H,
12.00% N. ESI-MS: m/z 149.1 [M]2+ (calcd for
[C8H9N2O]2+ 149.17).
12. Ekstrom, F.; Akfur, C.; Tunemalm, A. K.; Lundberg, S.
Biochemistry 2006, 45, 74.
13. Ekstrom, F.; Pang, Y. P.; Boman, M.; Artursson, E.;
Akfur, C.; Borjegren, S. Biochem. Pharmacol. 2006, 72,
597.
14. Kuca, K.; Cabal, J.; Musilek, K.; Jun, D.; Bajgar, J.
J. Appl. Toxicol. 2005, 25, 491.
(Z)-1,4-bis(4-hydroxyiminomethylpyridinium)-but-2-ene
dibromide (7). Prepared by method A. The reaction
mixture was stirred at 100 ꢁC and stopped after 2 h. Yield
0.69 g (82%), mp 230–232 ꢁC. 1H NMR spectrum
(300 MHz, D2O): d (ppm) 8.90 (d, 4H, J = 6.0 Hz, PyrH),
8.41 (s, 2H, –CH@NOH), 8.26 (d, 4H, J = 6.0 Hz, PyrH),
6.34–6.20 (m, 2H, –CH@), 5.56 (d, 4H, J = 4.1 Hz, –CH2–).
13C NMR spectrum (75 MHz, D2O): d (ppm) 148.77,
145.74, 144.12, 127.96, 124.56, 57.00. EA: calcd 41.95% C,
3.96% H, 12.23% N; found 39.66% C, 4.55% H, 11.41% N.
ESI-MS: m/z 149.1 [M]2+ (calcd for [C8H9N2O]2+ 149.17).
(Z)-1-(2-Hydroxyiminomethylpyridinium)-4-(3-hydroxyi-
minomethylpyridinium)-but-2-ene dibromide (8). Pre-
pared by method B via (11). The reaction mixture was
stirred at 50 ꢁC and stopped after 6 h. Yield 0.44 g (65%),
mp 194–195 ꢁC. 1H NMR spectrum (300 MHz, D2O): d
(ppm) 9.14 (s, 1 H, PyrH), 9.00–8.89 (m, 2H, PyrH), 8.79
(d, 1H, J = 8.2 Hz, PyrH), 8.70 (s, 1H, –CH@NOH), 8.65–
8.56 (m, 1H, PyrH), 8.47–8.37 (m, 2H, PyrH+
–CH@NOH), 8.20-8-06 (m, 2H, PyrH), 6.27–6.08 (m,
2H, –CH@), 5.73 (d, 2H, J = 4.5 Hz, –CH2–), 5.60 (d, 2H,
J = 5.1 Hz, –CH2–). 13C NMR spectrum (75 MHz, D2O):
d (ppm) 146.27, 145.68, 145.26, 144.12, 142.39, 141.59,
133.29, 128.24, 128.00, 127.67, 127.16, 126.43, 57.65,
55.56. EA: calcd 41.95% C, 3.96% H, 12.23% N; found
40.77% C, 4.21% H, 11.61% N. ESI-MS: m/z 149.1 [M]2+
(calcd for [C8H9N2O]2+ 149.17).
15. Musilek, K.; Kuca, K.; Jun, D.; Dohnal, V.; Dolezal, M.
Bioorg. Med. Chem. Lett. 2006, 16, 622.
16. Kassa, J.; Karasova, J. Toxicology 2007, 229, 136.
17. Preparation of the connecting chain: Triphenylphosphine
dibromide (20.12 g, 47.7 mmol) was suspended in anhy-
drous MeCN (50 ml) under argon atmosphere. (Z)-but-2-
en-1,4-diol (1.87 ml, 22.7 mmol) was added in anhydrous
MeCN (20 ml) and mixture was stirred at 50 ꢁC for 3 h.
The reaction mixture was evaporated under reduced
pressure, extracted with ether (6· 50 ml). Flash chroma-
tography of evaporated extract (100% hexane to hexane–
EtOAc 7:3) was used to obtain (Z)-1,4-dibromobut-2-ene
1
(13) as pale yellow oil (7.46 g, 77%). H NMR spectrum
(300 MHz, CDCl3): d (ppm) 5.92–5.85 (m, 2H, –CH@CH–),
4.01 (AB system, 4H, JAB = JBA = 2.1 Hz). 13C NMR
spectrum (75 MHz, CDCl3): d (ppm) 129.74, 24.52.
18. Preparation of quaternary salts: (A) Preparation of
symmetrical salts—A solution of the hydroxyiminometh-
ylpyridine (0.5 g, 4.1 mmol) and (Z)-1,4-dibromobut-2-ene
(0.39 g, 1.8 mmol) in DMF (10 ml) was stirred at 50–
100 ꢁC. The reaction mixture was cooled to the room
temperature and portioned with acetone (50 ml). The
crystalline crude product was collected by filtration,
washed with acetone (3· 20 ml), and recrystallized from
(Z)-1-(2-Hydroxyiminomethylpyridinium)-4-(4-hydrox-
yiminomethylpyridinium)-but-2-ene dibromide (9). Pre-

3176
K. Musilek et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3172–3176
pared by method B via (11). The reaction mixture was
(Z)-1-(4-Bromobut-2-enyl)-4-hydroxyiminomethylpyrid-
inium bromide (12) Prepared by method B. The reaction
mixture was stopped after 2 h. Yield 3.55 g (86%), mp
175–177 ꢁC. 1H NMR spectrum (300 MHz, D2O): d (ppm)
8.82 (d, 2H,J = 6.0 Hz, PyrH), 8.38 (s, 1H, –CH@NOH),
8.22 (d, 2H, J = 6.0 Hz, PyrH), 6.42–6.28 (m, 1H, –CH@),
5.96–5.83 (m, 1H, –CH@), 5.40 (d, 2H, J = 7.1 Hz, –CH2–
N), 4.24 (d, 2H, J = 8.5 Hz, –CH2–Br). 13C NMR spec-
trum (75 MHz, D2O): d (ppm) 148.47, 145.75, 143.99,
133.50, 124.44, 124.09, 56.28. EA: calcd 35.74% C, 3.60%
H, 8.34% N; found 36.09% C, 3.74% H, 8.39% N. ESI-
MS: m/z 255.0 [M]+ (calcd for [C10H12BrN2O]+ 255.01).
19. Kuca, K.; Cabal, J. Toxicol. Mech. Method 2005, 15,
247.
stirred at 50 ꢁC and stopped after 5 h. Yield 0.43 g (63%),
mp 181–183 ꢁC. 1H NMR spectrum (300 MHz, D2O): d
(ppm) 8.95 (d, 1H, J = 6.0 Hz, PyrH), 8.88 (d, 2H,
J = 6.0 Hz, PyrH), 8.69 (s, 1H, –CH@NOH), 8.64–8.54
(m, 1H, PyrH), 8.47–8.37 (m, 2H, PyrH+ –CH@NOH),
8.26 (d, 2H, J = 6.0 Hz, PyrH), 8.15–8.05 (m, 1H, PyrH),
6.25–6.07 (m, 2H, –CH@), 5.71 (d,2H, J = 4.5 Hz, –CH2–),
5.54 (d, 2H, J = 5.1 Hz, –CH2–). 13C NMR spectrum
(75 MHz, D2O): d (ppm) 148.66, 146.28, 145.67, 145.63,
145.24, 144.00, 141.59, 128.02, 127.67, 127.16, 126.69,
124.48, 56.96, 55.54. EA: calcd 41.95% C, 3.96% H,
12.23% N; found 41.51% C, 4.13% H, 11.89% N. ESI-MS:
m/z 149.1 [M]2+ (calcd for [C8H9N2O]2+ 149.17).
(Z)-1-(3-Hydroxyiminomethylpyridinium)-4-(4-hydroxy-
iminomethylpyridinium)-but-2-ene dibromide (10). Pre-
pared by method B via (12). The reaction mixture was
stirred at 100 ꢁC and stopped after 1.5 h. Yield 0.44 g
(65%), mp 197–199 ꢁC. 1H NMR spectrum (300 MHz,
D2O): d (ppm) 9.17 (s, 1H, PyrH), 8.98–8.88 (m, 3H,
PyrH+ –CH@NOH), 8.79 (d, 1H, J = 8.1 Hz), 8.48–8.37
(m, 2H, PyrH+ –CH@NOH), 8.27 (d, 2H, J = 6.0 Hz,
PyrH), 8.21-8.13 (m, 1H, PyrH), 6.37–6.22 (m, 2H,
–CH@), 5.63 (d, 2H, J = 4.1 Hz, –CH2–), 5.58 (d, 2H,
J = 4.1 Hz, –CH2–). 13C NMR spectrum (75 MHz, D2O):
d (ppm) 148.75, 145.74, 144.25, 144.12, 142.52, 133.36,
128.12, 128.07, 127.71, 124.56, 57.71. EA: calcd 41.95% C,
3.96% H, 12.23% N; found 38.85% C, 4.26% H, 9.98% N.
ESI-MS: m/z 149.1 [M]2+ (calcd for [C8H9N2O]2+ 149.17).
(Z)-1-(4-Bromobut-2-enyl)-2-hydroxyiminomethylpyrid-
inium bromide (11). Prepared by method B. The reaction
mixture was stopped after 4 h. Yield 2.05 g (50%), mp
140–142 ꢁC. 1H NMR spectrum (300 MHz, D2O): d (ppm)
8.87 (d, 1H, J = 6.0 Hz, PyrH), 8.68 (s, 1H, –CH@NOH),
8.65–8.52 (m, 1H, PyrH), 8.41 (d, 1H, J = 7.7 Hz, PyrH),
8.15–8.01 (m, 1H, PyrH), 6.35–6.20 (m, 1H, –CH@), 5.87–
5.75 (m, 1H, –CH@), 5.55 (d, 2H, J = 6.9 Hz, –CH2–N),
4.22 (d, 2H, J = 8.5 Hz, –CH2–Br). 13C NMR spectrum
(75 MHz, D2O): d (ppm) 145.43, 141.53, 132.13, 127.78,
126.71, 124.24, 54.91, 54.89. ESI-MS: m/z 255.0 [M]+
(calcd for [C10H12BrN2O]+ 255.01).
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