Synthesis and antimicrobial activities of new pyridinium and benzimidazolium chlorides

Article abstract of DOI:10.1016/S0223-5234(01)01226-0

A novel class of pyridinium and benzimidazolium chloride has been obtained in high yield. The antimicrobial activities of three homologous series of pyridinium and benzimidazolium chlorides against cocci, rods, fungi and bacillus have been measured. The antimicrobial activities of N,N′-bis[3-(1-alkoxymethyl)pyridinium chloride]methylenediamines, 1-undecyloxymethyl-3-(1-benzimidazolmethylamino)pyridinium, 1-undecyloxymethyl- and 1-dodecyloxymethyl-3-[1(benzotriazol-1-yl)methylamino]pyridinium chlorides exhibited strong activity and wide antibacterial spectra similar to the activity of benzalkonium chloride.

Full text of DOI:10.1016/S0223-5234(01)01226-0

Eur. J. Med. Chem. 36 (2001) 313–320
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01226-0/FLA
Original article
Synthesis and antimicrobial activities of new pyridinium and benzimidazolium
chlorides
Juliusz Pernaka*, Jarostaw Rogozaa, Ilona Mirskab
;
aPoznan´ University of Technology, Skłodowskiej-Curie 2, 60-965 Poznan´, Poland
bK. Marcinkowski University of Medical Sciences, Sieroca 10, 61-771 Poznan´, Poland
Received 3 July 2000; revised 6 February 2001; accepted 8 February 2001
Abstract – A novel class of pyridinium and benzimidazolium chloride has been obtained in high yield. The antimicrobial activities
of three homologous series of pyridinium and benzimidazolium chlorides against cocci, rods, fungi and bacillus have been measured.
The antimicrobial activities of N,N%-bis[3-(1-alkoxymethyl)pyridinium chloride]methylenediamines, 1-undecyloxymethyl-3-(1-benzim-
idazolmethylamino)pyridinium, 1-undecyloxymethyl- and 1-dodecyloxymethyl-3-[1(benzotriazol-1-yl)methylamino]pyridinium chlo-
´
rides exhibited strong activity and wide antibacterial spectra similar to the activity of benzalkonium chloride. © 2001 Editions
scientifiques et me´dicales Elsevier SAS
Antimicrobial activities / Benzalkonium chloride / Benzimidazolium chlorides / Pyridinium chlorides / Bispyridinium chlorides
1. Introduction
merous studies of the synthesis and antimicrobial
characteristics of different novel QAC have been pub-
lished recently [6–15].
The present paper reports on the synthesis and
antimicrobial activities of new pyridinium, bispyri-
dinium and benzimidazolium chlorides as potential
novel biocides.
Pyridinium compounds are an important class of
chemicals used widely as biocides, cationic surfac-
tants, drugs, and herbicides. The formal positive
charge on the nitrogen atom of these compounds
possesses many unique properties, which have been
utilized in numerous applications. The first in 1915
Jacobs and Heidelberg [1, 2] published synthesis and
antimicrobial activity of the quaternary ammonium
compounds (QAC). In 1935, Domagk [3] disclosed
the antibacterial activity of the long-chain QAC. Fol-
lowing Domagk’s publication, a large number of ap-
plication areas were developed for QAC as biocides
with a wide range of antimicrobial spectra. Disinfec-
tants based on QAC are widely used in the hospital
environment and the food industry. Resistance to
disinfectants based on QAC is, according to a recent
report [4], a potential problem in the food processing
industry. QAC are studied all the time for use against
various illnesses among other things malaria [5]. Nu-
2. Chemistry
1-Alkoxymethyl-(3-nicotionylaminomethyl)benzi-
midazolium (2), 1-alkoxymethyl-3-(1-benzimidazol-
methylamino)pyridinium
[1-(benzotriazol-1-yl)methylamino]pyridinium
(4),
1-alkoxymethyl-3-
(7)
chlorides and N,N%-bis[3-(1-alkoxymethyl)pyridinium
chloride]methylenediamine (5) listed in tables I–III
were synthesized by the method shown in figure 1.
N-Mannich bases (1, 3, 6) were synthesized in the
reaction of azole with 3-substituted pyridine. N-(1-H-
Benzimidazolmethyl)-3-pyridinecarboxamide (1) is a
new compound, which was obtained in a one-pot
condensation reaction of benzimidazole–formalde-
hyde–nicotinamide. This amide was characterized by
1H- and 13C-NMR. The NH proton resonated at 9.93
* Corresponding author
E-mail address: juliusz.pernak@put.poznan.pl (J. Pernak).

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J. Pernak et al. / European Journal of Medicinal Chemistry 36 (2001) 313–320
Table
III. 1-Alkoxymethyl-3-[1-(benzotriazol-1-yl)methyl-
ppm as a triplet and the methine protons appeared at
5.83 ppm as a doublet with a coupling constant in the
range of 5.6 and 6.0 Hz, respectively. The chemical
shift of the carbonyl carbon appeared at 165.3 ppm.
3-(Benzotriazol-1-ylmethylamino)pyridine (6) was
prepared earlier by the condensation of 3-aminopy-
ridine with 1-(hydroxymethyl)benzotriazole [16]. We
obtained the same compound by direct condensation
of benzotriazole–formaldehyde–nicotinamide. For
synthesized of 3-(benzotriazol-1-ylmethylamino)-
amino]pyridinium chlorides (7) prepared
Chloride
R
Yield (%)
M.p. a (°C) Purity (%)
7a
7b
7c
7d
7e
7f
C3H7 80
C7H9 80
C5H11 82
C6H13 81
C7H15 78
132–134
139–140
139–141
146–148
133–135
132–134
134–136
138–140
135–136
134–136
99
99
99
99
99
99
C8H
C9H
80
80
17
7g
7h
7i
19
C10H21 78
C11H23 77
C12H25 78
7j
Table I. 1-Alkoxymethyl-3-(nicotionylaminomethyl)benzimi-
dazolium chlorides (2) prepared
pyridine (3) we used the microwave technique with
success.
Chloride
R
Yield (%)
M.p. a (°C) Purity (%)
All chlorides prepared (2, 4, 5 and 7) were very
hygroscopic. The chemical purity was determined by
a direct 2-phase titration procedure for chlorides 2, 5
and 7 with alkyl long chain length (6–12 carbon
atoms), presented in tables I–III. 1-Alkoxymethyl-3-
(1-benzimidazolmethylamino)pyridinium chlorides (4)
were destroyed in acidic medium which is a two-phase
titration method.
2a
2b
2c
2d
2e
2f
2g
2h
2i
C2H5 85
C3H7 85
C4H9 85
C5H11 80
C6H13 80
152–156
150–154
133–137
130–134
117–121
121–125
118–122
114–117
110–114
123–126
119–123
95
96
95
96
95
97
94
C7H
C8H
C9H
84
80
75
15
17
19
C10H21 75
C11H23 75
C12H25 70
2j
2k
3. Antimicrobial activity
a Solvent for recrystallization CHCl3–CH3COOC2H5.
All synthesized pyridinium and benzimidazolium
chlorides (2, 4, 5 and 7) were tested for antimicrobial
activity against cocci, rods, fungi and bacillus.
Table II. 1-Alkoxymethyl-3-(1-benzimidazolmethylamino)-
pyridinium chlorides (4) and N,N%-bis[3-(1-alkoxymethyl)-
pyridinium chloride]methylenediamines (5) prepared
Chloride
R
Yield (%)
M.p. a (°C) Purity (%)
4. Results and discussion
4a
4b
4c
4d
4e
4f
C3H7 82
C4H9 75
C5H11 73
C6H13 70
C7H15 67
C8H17 70
C9H19 66
C10H21 62
C11H23 65
C12H25 63
C9H19 85
C10H21 83
C11H23 84
C12H25 87
143–145
145–147
144–146
144–146
148–150
141–143
145–147
146–148
146–147
144–147
68–71
70–73
70–73
69–72
The quaternization of the three prepared N-Man-
nich bases (1, 3 and 6) was run with an electrophile
like chloromethyl alkyl ether to give good yields of
three different final chlorides — benzimidazolium
(2), bispyridinium (5) and pyridinium (7). ROCH2Cl
is an excellent reagent but very easily hydrolyzed. In
this situation, reaction should be conducted under
strictly anhydrous conditions. This quaternization de-
pends on the substituent in the three-position of the
pyridine ring. The strong electron-withdrawing group
deactivates the nitrogen atom in pyridine and in this
situation, the nitrogen atom N-3 in benzimidazole is
nucleophilic enough to react with ROCH2Cl. 1-
Alkoxymethyl-3-(1-benzimidazolmethylamino)pyri-
4g
4h
4i
4j
5a
5b
5c
5d
198
198
198
198
a Solvent for recrystallization MeOH–CH3COCH3 (4a–g),
H2O (4h–j and 5a–d).

J. Pernak et al. / European Journal of Medicinal Chemistry 36 (2001) 313–320
315
Figure 1. Synthesis of compounds 2, 4, 5, 7.
dinium chloride (4) was converted into N,N%-bis[3-(1-
alkoxymethyl)pyridinium chloride]methylenediamine
(5). The formation of bispyridinium is probably the
result of the attack of HCl on the N-3 of the benzim-
idazole ring to give an intermediate as shown in figure
2, which is unstable and quickly converted to bispyri-
dinium chloride to replace the benzimidazole moiety.
In this reaction benzimidazole is a good living group
in acidic solution. N,N%-Bis(3-pyridine)methylnedia-
mine is a well known compound [17], which was
prepared by imines as an intermediate. Probably, the
iminium salt is the intermediate in the synthesis of
bispyridinium chloride [18].
(J=5.4 Hz) for chlorides 5. The chemical shift of the
characteristic carbon a between the azole ring and
amide or amine fell in the region of 55.10–50.70.
Minimum inhibitory concentration (MIC) and min-
imum bactericidal or fungicidal concentration (MBC)
values, determined for 11 benzimidazolium chlorides
(2a–k) and for 24 pyridinium chlorides (4a–j, 5a–d
and 7a–j) are given in tables IV–VI. The calculated
average MIC values for all the strains are presented in
figure 3 as a relationship between the alkyl chain
length and antimicrobial activity. As shown by the
results, all the chlorides studied are active against
The newly prepared compounds were characterized
1
by their H- and 13C-NMR spectra and by elemental
analyses. The NH proton resonated at 11.30–11.20
for chlorides 2, at 9.10–8.95 for chlorides 4, at 9.55–
9.45 for chlorides 5 and at 10.31–9.47 for chlorides 7
as a triplet with a coupling constant in the range 5.7,
6.9, 6.0 and 6.1 Hz, respectively. The methine protons
(NCH2N) appear as a doublet at 6.38–5.90 (J=6 Hz)
for chlorides 2, 4 and 7 and as a triplet at 4.85
Figure 2. Intermediate backing to bispyridinium chloride.

316
J. Pernak et al. / European Journal of Medicinal Chemistry 36 (2001) 313–320
Table IV. The MIC values a of 1-alkoxymethyl-3-(nicotionylaminomethyl)benzimidazolium chlorides (2)
Strains
Chlorides
2a 2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
Cocci
Micrococcus luteus
Staphylococcus
aureus
MIC \1443 \1387 1335
1287
311
621
300
600
290
581
141
281
545
273
132
132
257
514
MIC \1443 \1387 \1335 1287
Enterococcus faecalis MIC \1443 \1387 \1335 1287
311
155
300
75
290
72
70
70
34
273
66
265
257
257
Moraxella catarrhalis MIC
1443
1387
668
322
Rods
Escherichia Coli
Proteus 6ulgaris
Klebsiella
MIC \1443 \1387 \1335 \1287
MIC \1443 \1387 \1335 \1287
1242
1242
1200
1200
1161
1161
1161
562
562
1125
1091
545
545
529
1028
1058 \1028
529 1028
MIC \1443 \1387 \1335 \1287 \1242 \1200
Pneumoniae
Pseudomonas
aeruginosa
MIC \1443 \1387 \1335 \1287 \1242 \1200 \1161 \1125 \1091 \1058 \1028
Fungi
Candida Albicans
Rhodotorula rubra
MIC \1443 \1387 1335
MIC \1443 \1387 1335
1287
1287
1242
1242
1200
1200
1161
581
562
562
1091
1091
265 \1028
265
1028
Bacillus
Bacillus Subtilis
MIC \1443 \1387 1335
1287
311
300
145
70
545
33
514
a In mM, the number of microorganisms in mL range from 104 to 105.
cocci, rods, fungi and bacillus. Their activities are
greatly affected by an alkyl chain length in the
alkoxymethyl substituent and a kind of quaternary
ammonium moieties in a molecule. The same correla-
tion is observed for the MIC and the MBC values.
The favorite alkyl group is the one which contains
9–12 carbon atoms. In comparison with commer-
cially available benzalkonium chloride (BAC–Aldrich
product, in which R represents a mixture of alkyls
The most active chlorides 4i, 5b and 7i were tested
against Mycobacterium tuberculosis strain H37Rv. It
was found, in the TAACF screening program, that
they are active (MIC<6.25 mg/mLand Inh.>92%) and
are potential compounds for use in the treatment of
mycobacterial infections.
from C8H17 to C18H37) 1-alkoxymethyl-3-nicotionyl-
aminomethyl)benzimidazolium chlorides (2) are only
slightly effective. The most active chlorides against
the studied microorganisms are N,N%-bis[3-(1-
alkoxymethyl)pyridinium chloride]methylenediamines
(5) and 1-undecyloxymethyl-3-(1-benzimidazolmethyl-
amino)pyridinium (4i), 1-undecyloxymethyl- and
1-dodecyloxymethyl-3-[1-(benzotriazol-1-yl)methyl-
amino]pyridinium (7i and 7j) chlorides. The activity
of chlorides 5, 4i, 7i and 7j are similar to the activity
of BAC. It is known that compounds that possess two
quaternary ammonium moieties in the molecule are
strongly active against bacteria and fungi. New obser-
vation is that pyridinium salts of formula weight,
higher than 445, with four and five nitrogen atoms are
potential new biocides.
5. Experimental protocols
5.1. Chemistry
Melting points were measured on a Kofter hot stage
apparatus and are uncorrected. H- and 13C-NMR spec-
1
tra were recorded with a Varian model XL 300 spec-
trometer with TMS as standard. Elemental analyses
CHN were done at the A. Mickiewicz University, Poz-
nan´. For all the synthesized compounds, satisfactory
microanalyses were obtained C 0.37; H 0.35; and N
0.31. ROCH2Cl was prepared via chloromethylation of
alcohol. Pyridinium and benzimidazolium chlorides ob-
tained were determined by two-phase back titration of
sodium dodecyl sulfate with Hyamine 1622 using di-
imidium bromide and erioglaucine indicators.

Table V. The MIC and MBC values a of 1-alkoxymethyl-3-(1-benzimidazolmethylamino)pyridinium chlorides (4) and N,N%-bis[3-(1-alkoxymethyl)-pyridinium chlo-
ride]methylenediamines (5)
Strains
Chlorides
b
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
5a
5b
5c
5d
BAC
7
Cocci
M. luteus
MIC
48
46
44
166
334
10.5
21
10
38
74
148
148
19
19
9
35
7
7
6.5 12
12
MBC
MIC
MBC
MIC
MBC
93
752
1504
752
46
722
1443
722
172
1386
1386
1386
1386
20
77
37
72
144
37
18
18
32
18
18
68
68
13
13
26
13
26
12
12
25
12
25
24
24
46
12
46
16
5
5
3
3
S. aureus
1335
1335
668
322
322
80
14
53
7
311
40
154
273
8.5
8.5
S. epidermidis
1504
1443
1335
160
74
144
7
Rods
E. Coli
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
376
752
1504
1504
752
1504
1504
3008
752
361
722
1443
1443
722
1443
722
2886
722
693
2774
2774
2774
693
1386
1386
5547
1386
1386
1335
1335
1335
1335
668
668
334
668
1335
1335
644
644
644
644
322
644
322
644
322
322
311
311
621
621
154
154
154
311
311
311
300
600
600
1200
149
300
149
300
300
300
290
290
1161
1161
72
144
37
144
290
290
70
70
281
281 1090
70 135
140 1090
140
140
545
545
545
27
27
214
214
53
53
27
27
27
27
26
52 195
101 195
204 195 1495
13
51
26
26
51
25
46
46
187
7
10
54
205
16
16
11
11
54
205
S. marcescens
P. 6ulgaris
48
97
97
97
97
97
93
187
46
46
93
K. Pneumoniae
P. aeruginosa
135
545
140 1090
140 1090
1504
1443
101
93
Fungi
C. albicans
MIC \12030
MBC \12030
MIC \12030
MBC \12030
\11544
\11544
\11544
\11544
\11096
\11096
\11096
\11096
5340
10681
2670
2574
2574
1287
1287
1242
1242
621
1200
1200
300
290
290
72
70
70
32
32
17.5
13.5
27
7
13 195
13 195
93
187
6
7
16
11
11
17.5
17.5
17.5
R. rubra
13
13
6
12
2670
621
300
72
27
6
Bacillus
B. subtilis
MIC
1504
1443
2774
1335
644
154
149
72
32
135
14
13
25
46
11
MBC
a In mM, the number of microorganisms in mL range from 104 to 105.
b Benzalkonium chloride.

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J. Pernak et al. / European Journal of Medicinal Chemistry 36 (2001) 313–320
Table VI. The MIC and MBC values a of 1-alkoxymethyl-3-[1-(benzotriazol-1-yl)methylamino]pyridinium chlorides (7)
Strains
Chlorides
b
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
BAC
Cocci
M. luteus
MIC
1499
1439
1383
1383
333
666
666
1332
21.3
42.6
321
321
321
642
20.5
82.2
154
154
40.0 38.3
79.3 38.3
19.8 B19.2
76.6
76.6
74.2
35.9
17.4
7
16
5
5
3
MBC \1499 \1439
MIC
MBC \1499 \1439 \1383
74.2
37.1
74.2
18.5
35.9
35.9
71.8
18.0
35.9
69.6
34.8
34.8
17.4
34.8
S. aureus
1499 \1439 \1383
S. epidermidis MIC
374
750
173
719
88.5
88.5
MBC
79.3 76.6 144
3
Rods
E. coli
MIC \1499 \1439 \1383
MBC \1499 \1439 \1383
1332
1332
642
642
79.3 76.6
79.3 76.6
74.2
74.2
144
579
144
290
144
144
71.8
71.8
281
281
71.8
281
139
281
139
139
135
7
10
54
205
16
16
11
11
54
205
135
272
272
S. marcescens MIC \1499 \1439 \1383 \1332 \1284
MBC \1499 \1439 \1383 \1332 \1284
P. 6ulgaris
310
299
599
149
599
299
599
620
310
620
620
620
MIC
MBC
96.0
375
92.0
360
346
692
165
1332
642
1284
1284
69.6
272
135
272
135
135
K. pneumoniae MIC \1499 \1439 \1383 \1332
MBC \1499 \1439 \1383 \1332 \1284
P. aeruginosa MIC \1499 \1439
1383
1332
642
1284
79.3 76.6
74.2
144
MBC \1499 \1439 \1383 \1332
154
599
Grzyby
C. albicans
MIC \1499 \1439 \1383 \1332 \1284
MBC \1499 \1439 \1383 \1332 \1284 \1239
620
299
599
149
153
144
290
74.2
139
561
35.9
281
69.6
272
17.4
7
16
11
11
R. rubra
MIC \1499 \1439 \1383
1332
642
642
310
310
MBC \1499 \1439 \1383 \1332
74.2
17.4
Laseczki
B. subtilis
MIC
MBC \1499
1499
719
1439
1383
1383
165
165
642
642
154
154
76.6
76.6
74.2
74.2
35.9
71.8
17.4
34.8
11
11
a In mM, the number of microorganisms in mL range from 104 to 105.
b Benzalkonium chloride.
5.1.1. 1-Alkoxymethyl-3-(nicotionylaminomethyl)-
benzimidazolium chlorides (2)
Nicotinamide (30.5 g, 0.25 mol), paraformaldehyde
(7.5 g, 0.25 mol), benzimidazole (29.5 g, 0.25 mol) and a
few drops of conc. sulfuric acid were refluxed in toluene
(500 mL) for 48 h with water removed azeotropically by
a Dean–Stark apparatus. Then the toluene was evapo-
rated under reduced pressure (60°C/30 Torr) and the
residue was recrystallized from MeOH. N-(1 H-benzimi-
dazolmethyl)-3-pyridinecarboxamide was obtained in
90% yield (m.p.=218–220°C). The obtained compound
(0.01 mol) was dissolved in anhydr. DMF and the
corresponding ROCH2Cl (0.01 mol) was added. The
mixture was stirred at r.t. for 1 h. DMF was removed
under reduced pressure, the product was washed with
warm hexane and recrystallized.
Figure 3. Relation between alkyl chain length and antimicro-
bial activity.

J. Pernak et al. / European Journal of Medicinal Chemistry 36 (2001) 313–320
319
5.1.2. Chloride (2k)
5.1.6. Chloride (5c)
1H-NMR (DMSO-d6) l ppm=11.25 (t, J=6 Hz,
NH), 10.38 (s, 1H), 9.22 (s, 1H), 8.80 (d, J=4.2 Hz,
1H), 8.58 (d, J=5.8 Hz, 1H), 8.52 (d, J=6.7 Hz, 1H),
8.08 (d, J=5.8 Hz, 1H), 7.74 (m, 2H), 7.60 (t, J=7.7
Hz, 1H), 6.08 (d, J=7.4 Hz, NCH2N), 6.06 (s,
NCH2O), 3.57 (t, J=6.3 Hz, 2H), 1.45 (m, 2H), 1.13
(m, 18H), 0.85 (t, J=6.6, 3H). 13C-NMR l ppm=165.8
(CO), 152.2, 148.3, 143.9, 136.2, 130.6, 130.5, 128.2,
127.0, 123.9, 114.6, 114.2, 76.7 (NCH2O), 69.0, 51.8
(NCH2N), 31.3, 29.0, 28.98, 28.92, 28.89, 28.7, 28.0,
28.5, 25.2, 22.1, 14.0.
1H-NMR (CDCl3) l ppm=9.51 (t, J=6.0 Hz, 2NH),
9.38 (s, 2H), 8.16 (d, J=8.5 Hz, 2H), 7.97 (d, J=6.0
Hz, 2H), 7.65 (t, J=8.5 Hz, 2H), 6.07 (s, 4H), 4.87 (t,
J=5.5, 2H), 3.68 (t, J=6.4 Hz, 4H), 1.61 (m, 4H), 1.32
(m, 32H), 0.88 (t, J=6.6 H2, 6H). 13C-NMR l ppm=
146.7, 130.7, 127.5, 127.0, 125.8, 88.83 (NCH2O), 71.7,
50.0 (NCH2N), 31.7, 29.4, 29.3, 29.1,28.95, 25.6, 22.5,
13.9.
5.1.7. 1-Alkoxymethyl-3-[1-(benzotriazol-1-yl)-
methylamino]pyridinium chloride (7)
3-Aminopyridine (9.4 g, 0.1 mol) formaldehyde (pow-
der, 3 g, 0.1 mol) and benzotriazole (11.9 g, 0.1 mol)
was refluxed in EtOH (100 mL). After cooling to r.t.,
H2O (50 mL) was added and the product was collected
by filtration and recrystallized from EtOH–H2O. The
product was 3-(benzotriazol-1-ylmethylamino)pyridine
(6, m.p. 153–154°C). The obtained compounds (0.01
mol) were dissolved in anhydr. acetone and the corre-
sponding ROCH2Cl was added. The mixture was stirred
at r.t. for 10 min. The solid product removed and
recrystallized from H2O.
5.1.3. 1-Alkoxymethyl-3-(1-benzimidazolmethylamino)-
pyridinium chlorides (4)
A mixture of equimolar amounts (0.1 mol) of 3-
aminopyridine, paraformaldehyde and benzimidazole
was heated between 110–90°C in a microwave reactor
(Plazmatronika S.A., Wrocław, Poland). The reaction
was carried out under atmospheric pressure with power
850 W and for 10 min. After being cooled to r.t., the
crude product was recrystallized from EtOH–H2O to
afford the desired 3-(benzimidazolmethylamino)-
pyridine: yield 80%; m.p. 160–162°C, lit. m.p. 163–
164°C [19]. The synthesized 3-substituted pyridine (0.01
mol) was dissolved in anhydrous acetone adding an
equimolar amount of the appropriate ROCH2Cl. The
mixture was stirred for 10–15 min at r.t. The precipitate
solid was removed and recrystallized.
5.1.8. Chloride (7b)
1H-NMR (DMSO-d6) l ppm=9.55 (t, J=6.1 Hz,
NH), 8.70 (s, 1H), 8.44 (d, J=5.8 Hz, 1H), 8.27 (d,
J=8.2 Hz, 1H), 8.17 (d, J=8.7 Hz, 1H), 8.04 (d,
J=8.5 Hz, 1H), 7.92 (t, J=8.5 Hz, 1H), 7.58 (t, J=7.7
Hz, 1H), 7.4 (t, J=7.6 Hz, 1H), 6.38 (d, J=6.0 Hz,
NCH2N), 5.84 (s, NCH2O), 3.47 (t, J=6.5 Hz, 2H),
1.45 (m. 2H), 1.20 (m, 2H), 0.75 (t, J=7.3 Hz, 3H).
13C-NMR l ppm=146.2, 145.5, 132.3, 132.1, 128.6,
128.0, 127.7, 126.7, 124.5, 119.3, 111.5, 88.6 (NCH2O),
69.9, 54.8 (NCH2N), 30.6, 18.3, 13.4.
5.1.4. Chloride (4f)
1H-NMR (DMSO-d6) l ppm=8.98 (t, J=6.9 Hz,
NH), 8.75 (s, 1H), 8.69 (s, 1H), 8.36 (d, J=5.8 Hz, 1H),
8.08 (d, J=8.7 Hz, 1H), 7.87 (t, J=8.5 Hz, 1H), 7.64
(d, J=7.7, 1H), 7.25 (m, 2H), 5.91 (d, J=6.9 Hz,
NCH2N), 5.52 (s, NCH2O), 3.45 (t, J=6.4 Hz, 2H),
1.45 (m, 2H), 1.22 (m, 10 H), 0.84 (t, J=6.8 Hz, 3H).
13C-NMR l ppm=146.0, 144.0, 143.5, 133.0, 131.8,
128.5, 128.0, 126.6, 122.6, 122.1, 119.4, 111.4, 88.6
(NCH2O), 70.1, 51.9 (NCH2N), 31.2, 28.6, 25.2, 22.1,
14.0.
5.2. Antimicrobial characteristic
The following microorganisms were used: Micrococ-
cus luteus ATCC 9341, Staphylococcus epidermidis
ATCC 12228, Staphylococcus aureus ATCC 6538, Pseu-
domonas aeruginosa ATCC 15442, Proteus vulgaris
NCTC 4635, Klebsiella pneumoniae ATCC 4352, Es-
cherichia coli NCTC 8196, Serratia marcescens ATCC
8100, Candida albicans ATCC 10231, Rhodotorula rubra
PhB and Bacillus subtilis ATCC 6633. The R. rubra was
taken from the Department of Pharmaceutical Bacteri-
ology, K. Marcinkowski University of Medical Sciences,
Poznan´. Antimicrobial activity was determined by the
5.1.5. N,N%-Bis[3-(1-alkoxymethyl)pyridinium
chloride]methylenediamine (5)
1-Alkoxymethyl-3-(1-benzimidazolmethylamino)pyri-
dinium chloride (4) (0.01 mol) after standing in HCl
solution (40 mL of H2O and 10 mL of concentrated
HCl) at r.t. slowly changes into N,N%-bis[3-(1-
alkoxymethyl)pyridinium
chloride]methylenediamine.
After 5 h the precipitate was filtered and recrystallized.

320
J. Pernak et al. / European Journal of Medicinal Chemistry 36 (2001) 313–320
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