Synthesis of the novel series of bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity against chlorpyrifos-inhibited acetylcholinesterase

Article abstract of DOI:10.1016/j.bmcl.2005.10.059

Six potential AChE reactivators were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by insecticide chlorpyrifos was tested in vitro. According to the results, (E)-1-(2-hydroxyiminomethylpyridinium)-4-(4-hydroxyiminomethylpyridinium) -but-2-ene dibromide seems to be the most potent AChE reactivator. The reactivation potency of these compounds depends on structural factors such as constitution of the linking chain between both pyridinium rings, position of the oxime moiety at the pyridinium ring and presence of quaternary nitrogens.

Full text of DOI:10.1016/j.bmcl.2005.10.059

Bioorganic & Medicinal Chemistry Letters 16 (2006) 622–627
Synthesis of the novel series of bispyridinium compounds
bearing (E)-but-2-ene linker and evaluation of their
reactivation activity against chlorpyrifos-inhibited
acetylcholinesterase
a
b,
b
c
a
*
Kamil Musilek, Kamil Kuca, Daniel Jun, Vlastimil Dohnal and Martin Dolezal
a
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
b
Department of Toxicology, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
Department of Food Technology, Mendel University of Agriculture and Forestry Brno, Zemedelska 1, 613 00 Brno, Czech Republic
c
Received 27 September 2005; revised 12 October 2005; accepted 13 October 2005
Available online 8 November 2005
Abstract—Six potential AChE reactivators were synthesized using modification of currently known synthetic pathways. Their
potency to reactivate AChE inhibited by insecticide chlorpyrifos was tested in vitro. According to the results, (E)-1-(2-hydroxy-
iminomethylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide seems to be the most potent AChE reactivator.
The reactivation potency of these compounds depends on structural factors such as constitution of the linking chain between both
pyridinium rings, position of the oxime moiety at the pyridinium ring and presence of quaternary nitrogens.
ꢀ
2005 Elsevier Ltd. All rights reserved.
6
Organophosphates (OP) are used for military purposes
as nerve agents (tabun, sarin, soman or VX) and also
as pesticides in agriculture and for various purposes in
tylcholine. The reactivator is able to cleave the OP moi-
ety from inhibited AChE and thereby restore activity of
the enzyme. The effective antidotes used among these
intoxications are oxime reactivators usually in combina-
1
the industry. The threat of intoxications by these com-
1
pounds rapidly increases in relationship with menace of
terrorist attacks. The substances commonly used for
agricultural purposes are, for example, parathion
(O,O-diethyl-O-(4-nitrophenyl)thiophosphate), chlor-
pyrifos (O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)thio-
phosphate) (Fig. 1).
tion with atropine. The often used reactivators of
AChE are pralidoxime (1, 2-PAM, 2-hydroxyimino-
methyl-1-methylpyridinium chloride), trimedoxime (2,
TMB-4, 1,3-bis(4-hydroxyiminomethylpyridinium)-pro-
pane dibromide), obidoxime (3, 1,3-bis(4-hydroxy-
iminomethylpyridinium)-2-oxa-propane
HI-6 (4, 1-(2-hydroxyiminomethylpyridinium)-3-(4-car-
dichloride),
These compounds inhibit irreversibly enzyme acetylcho-
linesterase (AChE, EC 3.1.1.7) in the same course as
bamoylpyridinium)-2-oxa-propane dichloride) and
methoxime (5, MMC, bis(4-hydroxyiminomethylpyridi-
2
1,7–13
nerve agents do. Their toxic effect is based on
phosphorylation of the enzyme active site, where they
are covalently bounded on serine hydroxyl group
nium)methane dichloride) (Fig. 2).
None of known
reactivators is able to satisfactorily reactivate AChE
inhibited by various types of OP due to the broad struc-
14
tural variability of OP.
2
–5
(
Scheme 1).
The inhibition of AChE leads to hyperstimulation of
muscarinic and nicotinic receptors due to excess of ace-
CH CH O
3
Cl
2
S
CH CH O
3
S
2
P
P
CH CH2
O
O
NO
2
3
CH CH O O
Cl
3
2
Keywords: Organophosphate; Acetylcholinesterase; Reactivation;
Pesticide; Chlorpyrifos; Oxime.
N
chlorpyrifos Cl
parathion
*
Corresponding author. Tel.: +420 973 251 523; fax: +420 495 518
94; e-mail: kucakam@pmfhk.cz
0
Figure 1. Organophosphate insecticides.
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.059

K. Musilek et al. / Bioorg. Med. Chem. Lett. 16 (2006) 622–627
623
All reported compounds were prepared by alkylation of
corresponding hydroxyiminomethylpyridine derivatives.
In general, three chemical pathways were used. The sym-
metrical bispyridinium compounds 6b,c were synthe-
sized by modification of an older published system
Cl
S
AChE
OH
Cl
O
P OCH CH3
2
N
OCH CH
2
3
Cl
1
6
(
Scheme 2). A decomposition of the product in the
reaction mixture owing to the powerful reaction condi-
tions rendered it unable to apply the same procedure
to the compound 6a; therefore, the reaction temperature
was decreased to 60 ꢁC and time of reaction extended to
5 h (Scheme 2).
Cl
S
O P OCH CH
AChE
Cl
OH
2
3
The asymmetric substances were prepared using a novel
approach in two-step synthesis. At first the monoquater-
nary salts 8a,b were obtained in acetone in various
conditions depending on the position of the hydroxyim-
inomethyl group on the pyridinium ring (Scheme 3). An
improvement used to increase the yields without excess
bisquaternary product depends on the addition of the
N
OCH CH
2
3
Cl
Scheme 1. Inhibition of AChE by chlorpyrifos.
HON=HC
CH=NOH
N
5
equiv of alkylating agent to the reaction mixture.
The monoquaternary salt was easily purified by the
recrystallization from acetonitrile, where the bi-product
is almost insoluble.
HON=HC
N
CH3
N
Cl
2Br
CH CH CH
2
2
2
1
2
The second step consists in the completion of asymmet-
ric bisquaternary compounds similar to the preparation
of symmetrical substances (Scheme 4) in high yields
HON=HC
N
CH=NOH
CONH2
2
Cl
(
7a—82% and 7b—85%) according to our unsuccessful
N
CH OCH
N
CH OCH
N
CH=NOH
2Cl
2
attempts afford those from 3- or 4-monoquaternary salts
and 2-hydroxyiminomethylpyridine under the same
conditions.
2
2
2
3
4
HON=HC
N CH
2
N
CH=NOH
Cl
In vitro testing of synthesized oximes involved a stan-
dard collection of experimental procedures. The 10%
rat brain homogenate (source of AChE) in water was
inhibited by chlorpyrifos to achieve 95% inhibition of
AChE. After 30 min of incubation with chlorpyrifos,
the reactivator was added to the solution for the next
2
5
Figure 2. Currently used reactivators of AChE.
1
0 min. Activities of intact AChE (a ), inhibited AChE
0
The development and selection of new effective reactiva-
tors of AChE like antidotes of OP are very important
due to the extended usage of pesticides in agriculture
(
a ) and reactivated AChE (a ) were deduced from the
i r
influence of consumption of NaOH solution (0.01 M)
on time. The percentage of reactivation (%) was calcu-
lated from the measured data according to the
formula:
1
5
and therefore eventual intoxications of human.
Our work was focused on finding novel effective struc-
tures for the treatment of chlorpyrifos poisonings and
testing their ability in vitro.
ꢀ
ꢁ
a
r
ꢀ a
i
x ¼ 1 ꢀ a
ꢁ 100½%ꢂ
0
ꢀ a
i
CH=NOH
N
N
(
E )- BrCH CH=CHCH Br
2
2
N
CH=NOH
HON=HC
2Br
DMF; 100˚C; 2h
6
b 3,3´-CH=NOH
6
c 4,4´-CH=NOH
HON=HC
N
CH=NOH (E )- BrCH CH=CHCH
2
Br
2
N
N
DMF; 60˚C; 5h
2
Br
CH=NOH
6a
Scheme 2. The synthesis of symmetrical bispyridinium compounds.

624
K. Musilek et al. / Bioorg. Med. Chem. Lett. 16 (2006) 622–627
N
CH=NOH
Br
(E)- BrCH CH=CHCH Br
2 2
N
acetone; 50˚C; 15h
CH=NOH Br
8a
N
Br
(E)- BrCH CH=CHCH Br
2 2
N
acetone; reflux; 1.5h
Br
HON=HC
CH=NOH
8b
Scheme 3. Preparation of monoquaternary salts in unsymmetrical synthesis.
N
CH=NOH
CH=NOH
Br
N
N
N
DMF; 60˚C; 2h
CH=NOH Br
2Br
CH=NOH
8a
7a 3-CH=NOH
7b 4-CH=NOH
N
Br
CH=NOH
DMF; 100˚C;1.5h
N
N
N
CH=NOH
2Br
Br
HON=HC
HON=HC
8b
7c
Scheme 4. Completion of asymmetric bispyridinium compounds.
1
4
The whole method is described in detail in the work of
toxic effect on the patient. It means that the oxime
7b surpasses all other in vitro tested compounds (48%
reactivation) and is the most promising among all the
compounds tested. Oximes 6c, 7a,c, obidoxime (3) and
trimedoxime (2) have also satisfactory reactivation abil-
1
4
Kuca and Kassa. Pralidoxime (1), trimedoxime (2),
obidoxime (3), HI-6 (4) and methoxime (5) previously
synthesized in our laboratory were used as reference
compounds. Their purity was estimated using HPLC.
Obtained data are summarized in Table 1 and Figure 3.
ꢀ
5
ity at 10 M concentration.
The reactivation potency of tested compounds depends
4,17
Consequently, we can recommend structural factors
appropriate for reactivation of chlorpyrifos-inhibited
AChE.
1
on the structure of the OP inhibitor. The most po-
tent reactivators of chlorpyrifos-inhibited AChE seem
to be reference compounds obidoxime (3) and trim-
1
4
ꢀ
3
edoxime (2) in a concentration of 10 M. However,
the newly synthesized oxime 7b shows satisfactory reac-
tivation results at this concentration too. Unfortunately,
The oxime functional group breaks down the bond OP
inhibitor enzyme and is essential for activity of the reac-
2
0,21
tivator.
Our results confirm that position of
ꢀ
3
concentration of 10 M is not applicable for further
1
hydroxyiminomethyl group influences the reactivation
potency. While compound 6b bearing both oxime
groups in position 3 is practically ineffective, substances
4,17–19
ꢀ5
The concentration of 10 M is
in vivo testing.
more suitable from the point of view of reactivatorÕs
Table 1. Reactivation potencies (%, mean value of three independent determinations) of tested oximes (time of inhibition by chlorpyrifos—30 min;
time of reactivation by AChE reactivators—10 min; pH 8; temperature 25 ꢁC)
ꢀ
3
ꢀ5
Compound
% (10 M)
% (10 M)
Reference compounds
Pralidoxime (1)
Trimedoxime (2)
Obidoxime (3)
HI-6 (4)
38
66
63
20
45
4
38
35
11
10
Methoxime (5)
Synthesized oximes
6a
4
13
25
30
63
34
9
2
6b
6c
7a
7b
7c
38
22
48
24

K. Musilek et al. / Bioorg. Med. Chem. Lett. 16 (2006) 622–627
625
100
0
.001 M
.00001 M
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6a
6b
6c
7a
7b
7c
Sample
Figure 3. Efficacy of tested oximes in reactivation of chlorpyrifos-inhibited AChE.
6
c and 7a–c bearing at least one oxime group in position
or 4 show promising results with the surprising excep-
100
80
2
tion of compound 6a.
The quaternary nitrogen is other important factor neces-
sary for the ability of the reactivator to bind on the
6
4
2
0
0
0
20,21
anionic sites of both intact and inhibited AChE.
It
is generally known that bisquaternary substances have
a higher affinity towards AChE in comparison with
monoquaternary compounds due to the two cationic
2
2
binding domains in the molecule of AChE.
0
-8
-6
-4
-2
The length of the linking chain also influences the
3
reactivation potency. In our case, there is the same
log concentration of reactivator [M]
length of (E)-but-2-ene bridge for all compounds. This
bridge is shorter than butane and longer than propane
linker due to the known fact that a double bond is
Figure 4. Reactivation curve of HI-6 (source of the enzyme—rat brain
homogenate; AChE inhibitior—sarin; time of inhibition—30 min; time
of reactivation—10 min; pH 8; temperature—25 ꢁC).
2
3
shorter than a single bond. The optimal length of
the linking chain lies between three and four atoms
as was previously reported and synthesized com-
varies with the optimal concentration for maximal reac-
tivation potency and in the case of 6a or c the optimum
lies at lower concentrations.
3
pounds suit this purpose.
On the other hand, there is a specific kind of ÔrigidityÕ
caused by the double bond in position (E-). The only
bonds accessible for free rotation in these molecules
are methylene junctions in contrast to free rotation in
the molecules with propane 2 or 2-oxa-propane linking
chains 3 and 4. Therefore, the spatial orientation of
pyridinium rings is limited.
Five novel reactivators 6a,b and 7a,c and one cur-
rently known reactivator 6c were prepared in satisfac-
tory yields and purity. Their ability to reactivate
chlorpyrifos-inhibited AChE was measured in vitro.
The most promising compound was (E)-1-(2-hydroxy-
iminomethylpyridinium)-4-(4-hydroxyiminomethylpy-
ridinium)-but-2-ene dibromide 7b, which overlaps the
effectiveness of trimedoxime 2 and obidoxime 3. The
reactivation potency of these compounds depends on
structural factors such as position of the functional
oxime group at the pyridinium ring, presence of qua-
ternary nitrogens and the constitution of the linking
chain.
The higher reactivation activity of some compounds
(
6a,c) at lower concentrations is caused by inhibition
of the intact AChE by high affinity of some reactivators
towards AChE (in vivo overdose can occur). The mea-
surements were made for two concentrations and the
whole concentration scale is generally bell-shaped
1
4
(
Fig. 4). The reactivation process is characterized by
2
4,25
the increasing part and the decreasing part shows both
reactivation and subsequent inhibition of liberated in-
tact AChE by the reactivator itself. Every reactivator
Preparation of quaternary salts.
We had used three
synthetic pathways for the preparation of bisquaternary
aldoximes:

626
K. Musilek et al. / Bioorg. Med. Chem. Lett. 16 (2006) 622–627
(
8
4
A) A solution of the hydroxyiminomethylpyridine (1.0 g,
7. Kuca, K.; Bielavsky, J.; Cabal, J.; Bielavska, M. Tetrahe-
dron Lett. 2003, 44, 3123.
.2 mmol) and (E)-1,4-dibromobut-2-ene (8.76 g,
0.9 mmol) in acetone (30 mL) was stirred (50 ꢁC—re-
8
. Wilson, I. B.; Ginsburg, S.; Meilisch, E. K. J. Am. Chem.
Soc. 1955, 77, 4286.
flux). The reaction mixture was cooled to room tempera-
ture and the crystalline crude product was collected by
filtration, washed with acetone (2· 30 mL) and recrystal-
lized from acetonitrile.
9
. Poziomek, E. J.; Hackley, B. E.; Steinberg, G. M. J. Org.
Chem. 1958, 23, 714.
0. Krejcova, G.; Kassa, J. Toxicology 2003, 185, 129.
1
1
1. Rousseaux, C. G.; Gua, A. K. Can. J. Physiol. Pharmacol.
1
(
B) A solution of the hydroxyiminomethylpyridine (1.0 g,
989, 67, 1183.
12. Kassa, J.; Cabal, J.; Bajgar, J.; Szinicz, L. ASA Newslett.
1997, 97, 16.
8
3
1
.2 mmol) and (E)-1,4-dibromobut-2-ene (0.79 g,
.7 mmol) in DMF (10 mL) was stirred in the range 60–
00 ꢁC depending on the substance synthesized. The reac-
13. Sevelova, L.; Kuca, K.; Krejcova-Kunesova, G. Toxicol-
ogy 2005, 207, 1.
tion mixture was cooled to room temperature and por-
tioned with acetone (30 mL); the crystalline crude
product was collected by filtration, washed with acetone
(
(
iminomethylpyridinium bromide or 1-(4-bromobut-2-
enyl)-4-hydroxyiminomethylpyridinium bromide (0.5 g,
1
1
1
1
1
4. Kuca, K.; Kassa, J. J. Enzyme Inhib. Med. Chem. 2003, 18,
29.
5
5. Mattingly, J. E.; Sullivan, J. E.; Spiller, H. A.; Bosse, G.
M. J. Emerg. Med. 2003, 25, 379.
6. Patocka, J.; Bielavsky, J.; Ornst, F. FEBS Lett. 1970, 10,
2· 30 mL) and recrystallized from acetonitrile.
C) A solution of the 1-(4-bromobut-2-enyl)-2-hydroxy-
1
82.
7. Kuca, K.; Patocka, J. J. Enzyme Inhib. Med. Chem. 2004,
19, 39.
.5 mmol) and 3-hydroxyiminomethylpyridine or 4-
hydroxyiminomethylpyridine (0.27 g, 2.2 mmol) in
DMF (10 mL) was stirred (60–100 ꢁC). The reaction
mixture was cooled to room temperature and portioned
with acetone (30 mL); the crystalline crude product was
collected by filtration, washed with acetone (2· 30 mL)
and recrystallized from acetonitrile.
18. Sevelova, L.; Kuca, K.; Krejcova, G.; Vachek, J. J. Appl.
Biomed. 2004, 2, 163.
1
2
2
2
9. Cabal, J.; Kuca, K.; Kassa, J. Pharmacol. Toxicol. 2004,
9
5, 81.
0. Kim, T.-H.; Kuca, K.; Jun, D.; Jung, Y.-S. Bioorg. Med.
Chem. Lett. 2005, 15, 2914.
1. Kuca, K.; Bielavsk y´ , J.; Cabal, J.; Kassa, J. Bioorg. Med.
Chem. Lett. 2003, 13, 3545.
2. Pang, Y.-P.; Kollmeyer, T. M.; Hong, F.; Lee, J.-C.;
Hammond, P. I.; Haugabouk, S. P.; Brimijoin, S. Chem.
Biol. 2003, 10, 491.
Biochemistry. The 10% rat brain homogenate was used
as a source of AChE. The brain homogenate (0.5 mL)
was mixed with 20 lL of isopropanol solution of chlor-
pyrifos (99.2% analytical standard from Sigma–Aldrich)
and incubated at 25 ꢁC for 30 min. Three molar solution
of sodium chloride (2.5 mL) was added to the mixture
and filled to the volume of 23 mL with distilled water.
Finally, 2 mL of solution of acetylcholine iodide
23. Carey, F. A.; Sundberg, R. J. In The Structure and
Mechanisms, 4th ed.; Plenum: New York, 2000; p 13.
2
4. Solvents (acetone and DMF) and reagents were purchased
from Fluka and Sigma–Aldrich (Czech Republic), and
used without further purification. Reactions were moni-
tored by TLC using DC-Alufolien Cellulose F (Merck,
(
0.02 M) was added. The enzyme activity was measured
at pH 8.0 and temperature 25 ꢁC on autotitrator RTS
22 (Radiometer, Denmark). The same procedure was
repeated with the reactivated enzyme subjected to fur-
Germany) and mobile-phase BuOH–CH
:1:2, detection by Dragendorff reagent (solution contain-
ing 10 mL CH COOH, 50 mL H O and 5 mL of basic
3 2
COOH–H O,
5
8
3
2
solution prepared by mixing of two fractions—fraction I:
850 mg Bi(NO ) , 40 mL H O, 10 mL CH COOH; frac-
tion II: 8 g KI, 20 mL H O). Melting points were measured
1
4
ther 10 min incubation with the reactivator.
3
3
2
3
2
with a microheating stage PHMK 05 (VEB Kombinat
Nagema, Radebeul, Germany) and are uncorrected. NMR
spectra were generally recorded at Varian Gemini 300 ( H
Acknowledgments
1
1
3
3
00 MHz, C 75 MHz, Palo Alto CA, USA). In all cases,
The authors express their appreciation to Mrs. M. Hra-
binova for her technical assistance. The work was sup-
ported 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.
ONVLAJEP20031.
1
the chemical shift values for H spectra are reported in
parts per million (d) relative to residual CHD SO CD (d
2.50), shift values for C spectra are reported in parts per
million (d) relative to solvent peak dimethylsulfoxide-d
2
2
3
1
3
6
d 39.43. Signals are quoted as s (singlet), br s (broad singlet),
d (doublet), t (triplet) and m (multiplet). Mass spectra were
recorded using a combination of high performance liquid
chromatography and mass spectrometry. HP1100
HPLC system was obtained from Agilent Technologies
(Waldbronn, Germany). It consisted of vacuum degasser
G1322A, quaternary pump G1311A, autosampler G1313A
and quadrupole mass spectrometer MSD1456 VL equipped
with electrospray ionization source. Nitrogen for mass
spectrometer was supplied by 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 flowrate at 13 L/min.
References and notes
1. Kassa, J. J. Toxicol., Clin. Toxicol. 2002, 40, 803.
2. Marrs, T. C. Pharmacol. Ther. 1993, 58, 51.
3. Kuca, K.; Patocka, J.; Cabal, J. J. Appl. Biomed. 2003, 1,
207.
4
5
6
. Sultatos, L. G.; Minor, L. D.; Murphy, S. D. J. Pharma-
col. Exp. Ther. 1985, 232, 624.
. Sams, C.; Cocker, J.; Lennard, M. S. Xenobiotica 2004, 34,
8
61.
. Taylor, P. In The Pharmacological Basis of Therapeutics,
th ed.; McGraw Hill: New York, 1996; pp 161–176.
25. (E)-1,4-Bis(2-hydroxyiminomethylpyridinium)-but-2-ene
dibromide (6a). Prepared by method B. The reaction
mixture was stirred at 60 ꢁC and stopped after 5 h. Yield
9

K. Musilek et al. / Bioorg. Med. Chem. Lett. 16 (2006) 622–627
627
1
1
0
spectrum (300 MHz, DMSO-d
.78 g (46%), TLC R
f
0.2, mp decomp. 208 ꢁC. H NMR
): d (ppm) 9.08 (d, 2H,
mp 192–194 ꢁC. H NMR spectrum (300 MHz, DMSO-d ):
6
6
d (ppm) 13.09 (s, 1H, –CH@NOH), 12.84 (s, 1H,
–CH@NOH), 9.15 (d, 1H, J = 6.0 Hz, aryl), 9.02 (d, 2H,
J = 6.0 Hz, aryl), 8.72–8.55 (m, 2H, aryl + –CH@NOH),
8.51–8.36 (m, 2H, aryl + –CH@NOH), 8.25 (d, 2H,
J = 6.0 Hz, aryl), 8.20–8.10 (m, 1H, aryl), 6.37–6.22 (m,
1H, –CH@), 6.06–5.91 (m, 1H, –CH@), 5.57 (d, 2H,
J = 6.0 Hz, aryl), 8.67–8.54 (m, 4H, aryl + –CH@NOH),
8
6
.39 (d, 2H, J = 8.0 Hz, aryl), 8.17–8.09 (m, 2H, aryl),
.11–6.05 (m, 2H, –CH@), 5.57–5.53 (m, 4H, –CH –),
C NMR spectrum
2
1
3
ꢀ
1.93 (br s, 2H, –CHNOH).
(
75 MHz, DMSO-d ): d (ppm) 147.05, 145.81, 145.69,
41.28, 129.09, 127.63, 125.68, 58.11. ESI-MS: m/z 149.1
6
1
3
1
J = 4.7 Hz, –CH
2
–), 5.32 (d, 2H, J = 6.3 Hz, –CH
2
–).
C
2
+
2+
O]
[
M]
(calculated for [C
8
H
9
N
2
:
C H Br N O ): C, H, N. (E)-1,4-Bis(3-hydroxy-
149.07). Anal.
NMR spectrum (75 MHz, DMSO-d
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
6
): d (ppm) 148.64,
(
iminomethylpyridinium)-but-2-ene dibromide (6b). Pre-
1
6
18
2
4
2
2
+
2+
pared by method B. The reaction mixture was stirred at
1
149.1 [M] (calculated for [C
): C, H, N.
8 9 2
H N O] :149.07). Anal.
00 ꢁC and stopped after 2 h. Yield 1.64 g (97%), TLC R
f
2 4 2
(C16H18Br N O
1
0
.2, mp 249–250 ꢁC. H NMR spectrum (300 MHz,
): d (ppm) 12.26 (s, 2H, –CH@NOH), 9.33 (s,
H, aryl), 9.10 (d, 2H, J = 6.0 Hz, aryl), 8.77 (d, 2H,
J = 8.0 Hz, aryl), 8.40 (s, 2H, –CH@NOH), 8.26–8.15 (m,
(E)-1-(3-Hydroxyiminomethylpyridinium)-4-(4-hydroxy-
iminomethylpyridinium)-but-2-ene dibromide (7c). Pre-
pared by method C. The reaction mixture was stirred at
100 ꢁC and stopped after 1.5 h. Yield 0.66 g (97%), TLC R
0.2, mp228–230 ꢁC. H NMRspectrum(300 MHz, DMSO-
6
d ): d (ppm) 12.85 (s, 1H, –CH@NOH), 12.25 (s, 1H,
DMSO-d
6
2
f
1
2
–
H, aryl), 6.32–6.23 (m, 2H, –CH@), 5.49–5.33 (m, 4H,
1
–). C NMR spectrum (75 MHz, DMSO-d
3
CH
2
6
): d
(
ppm) 144.14, 143.89, 142.46, 141.96, 133.32, 129.64,
–CH@NOH), 9.33(s, 1H, aryl), 9.12–9.04(m, 3H, aryl), 8.77
(d, 1H, J = 8.0 Hz, aryl), 8.47 (s, 1H, –CH@NOH), 8.40 (s,
1H, –CH@NOH), 8.27 (d, 2H, J = 6.0 Hz, aryl), 8.24–8.16
(m, 1H, aryl), 6.33–6.15 (m, 2H, –CH@), 5.46–5.30 (m, 4H,
2
28.04, 61.58. ESI-MS: m/z 149.1 [M] (calculated for
+
1
[
2
+
C H N O] : 149.07). Anal. (C H Br N O ): C, H, N.
8 9 2 16 18 2 4 2
E)-1,4-Bis(4-hydroxyiminomethylpyridinium)-but-2-ene
dibromide(6c).PreparedbymethodB.Thereactionmixture
(
1
3
2 6
–CH –). C NMR spectrum (75 MHz, DMSO-d ): d (ppm)
was stirred at 100 ꢁC and stopped after 1.5 h. Yield 1.58 g
148.66, 145.13, 145.05, 144.48, 143.21, 142.54, 141.81,
133.38, 130.47, 129.70, 128.18, 124.04, 61.02, 60.28. ESI-
1
(
(
93%), TLC R
300 MHz, DMSO-d
f
0.2, mp 250–251 ꢁC. H NMR spectrum
2
MS: m/z 149.1 [M] (calculated for [C
+
2+
6
): d (ppm) 9.05 (d, 4H, J = 5.8 Hz,
8 9 2
H N O] : 149.07).
aryl), 8.46 (s, 2H, –CH@NOH), 8.26 (d, 4H, J = 6.0 Hz,
Anal. (C16 ): C, H, N.
1-(4-Bromobut-2-enyl)-2-hydroxyiminomethylpyridinium
bromide (8a). Prepared by method A. The reaction mixture
2 4 2
H18Br N O
aryl), 6.24-6.17 (m, 2H, –CH@), 5.34 (d, 4H, J = 3.3 Hz,
1
3
–
(
1
CH
75 MHz, DMSO-d
30.06, 124.04, 60.30. ESI MS: m/z 149.1 [M] (calculated
2
–), 3.41 (br s, 2H, –CH@NOH). C NMR spectrum
6
): d (ppm) 148.66, 145.11, 145.06,
was stirred at 50 ꢁC and stopped after 15 h. Yield 1.04 g
2
+
1
(38%), TLC R 0.2, mp 118–122 ꢁC. H NMR spectrum
f
2
+
for[C H N O] : 149.07). Anal. (C H Br N O ): C, H, N.
2
(300 MHz,DMSO-d ):d(ppm)9.11(d,1H,J = 6.0 Hz,aryl),
8
9
2
16 18
2
4
6
(
E)-1-(2-Hydroxyiminomethylpyridinium)-4-(3-hydroxy-
8.78–8.54 (m, 2H, aryl + –CH@NOH), 8.42 (d, 1H,
J = 6.6 Hz, aryl), 8.22–8.08 (m, 1H, aryl), 6.18–6.04 (m,
1H, –CH@), 6.03–5.82 (m, 1H, –CH@), 5.67–5.42 (m, 2H,
iminomethylpyridinium)-but-2-ene dibromide (7a). Pre-
paredbymethodC.Thereactionmixturewasstirredat60 ꢁC
and stopped after 2 h. Yield 0.56 g (82%), TLC R
f
0.2, mp
): d
ppm)13.09(s,1H,–CH@NOH),12.25(s,1H,–CH@NOH),
.28 (s, 1H, aryl), 9.13 (d, 1H, J = 6.0 Hz, aryl), 9.01 (d, 1H,
J = 6.0 Hz,aryl),8.75(d,1H,J = 8.0 Hz,aryl),8.69–8.55(m,
–CH
2
–), 4.77 (br s, 1H, –CH@NOH), 4.14 (d, 2H, J =
1
13
1
(
9
92–195 ꢁC. H NMR spectrum (300 MHz, DMSO-d
6
7.4 Hz,–CH –). CNMRspectrum(75 MHz,DMSO-d ):d
2
6
(ppm) 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]
+
+
O] : 255.01). Anal. (C10
(calculated for [C10
2
O ): C, H, N.
H
12
N
2
H
12Br
N
2 4
2
–
–
–
H, aryl + –CH@NOH), 8.48–8.33 (m, 2H, aryl +
CH@NOH), 8.23–8.11 (m, 2H, aryl), 6.40–6.24 (m, 1H,
CH@), 6.05–5.90 (m, 1H, –CH@), 5.56 (d, 2H, J = 4.7 Hz,
1-(4-Bromobut-2-enyl)-4-hydroxyiminomethylpyridinium
bromide (8b). Prepared by method A. The reaction mixture
1
3
CH
2
–), 5.36 (d, 2H, J = 6.3 Hz, –CH
): d (ppm) 147.12, 146.03, 145.73,
44.24, 143.17, 142.41, 141.85, 141.42, 133.37, 131.51,
28.11, 127.70, 125.68, 61.01, 58.24, 35.76, 34.14. ESI-MS:
2
–). C NMR spec-
was stirred at reflux and stopped after 1.5 h. Yield 2.60 g
(95%), TLC R
1
trum (75 MHz, DMSO-d
1
1
m/z 149.1 [M] (calculated for [C H N O] : 149.07). Anal.
8 9 2
(
(
iminomethylpyridinium)-but-2-ene dibromide (7b). Pre-
pared by method C. The reaction mixture was stirred at
6
6
f
0.2, mp 187–191 ꢁC. H NMR spectrum
(300 MHz, DMSO-d ): d (ppm) 12.85 (s, 1H, –CH@NOH),
6
9.02(d, 2H, J = 6.0 Hz, aryl), 8.45(s, 1H, –CH@NOH), 8.27
(d, 2H, J = 6.0 Hz, aryl), 6.26–6.09 (m, 1H, –CH@), 5.32 (d,
2
+
2+
C
16
2
H18Br N
4
O
2
): C, H, N.
2 2
2H, J = 4.4 Hz, –CH –Br), 4.17 (d, 2H, J = 5.2 Hz, –CH –
1
3
E)-1-(2-hydroxyiminomethylpyridinium)-4-(4-hydroxy-
N@). C NMR spectrum (75 MHz, DMSO-d ): d (ppm)
6
148.67, 145.05, 144.85, 133.72, 127.69, 124.17, 60.25. ESI-
MS: m/z 254.9 [M] (calculated for [C10
Anal. (C10 12Br ): C, H, N.
+
+
12 2
H N O] : 255.01).
0 ꢁC and stopped after 2 h. Yield 0.58 g (85%), TLC R
f
0.2,
H
2 4 2
N O