New benzimidazole derivatives as antimycobacterial agents

Article abstract of DOI:10.1016/S0014-827X(02)01218-1

A set of 2-alkylsulfanyl derivatives of 5-methylbenzimidazole was synthesized and evaluated for antimycobacterial activity. The structures of the compounds were confirmed by ¹H NMR and IR data, and their purity by elemental analysis. Antimycobacterial activities against Mycobacterium tuberculosis and nontuberculous mycobacteria were expressed as the minimum inhibitory concentration. The substances exhibited significant antimycobacterial activity, in particular against both strains of Mycobacterium kansasii. The effect of the most active compound in the set, 3,5-dinitro derivative 3t, exceeded that of the standard isoniazide against M. kansasii and Mycobacterium avium. Copyright

Full text of DOI:10.1016/S0014-827X(02)01218-1

Il Farmaco 57 (2002) 259–265
www.elsevier.com/locate/farmac
New benzimidazole derivatives as antimycobacterial agents
a,
b
Veˇra Klimesˇova´ *, Jan Kocˇ´ı a, Karel Waisser a, Jarmila Kaustova´
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles Uni6ersity, 50005 Hradec Kra´lo6e´, Czech Republic
b National Reference Laboratory for Mycobacterium kansasii, Regional Institute of Hygiene, 72892 Ostra6a, Czech Republic
Received 22 November 2001; accepted 13 January 2002
Abstract
A set of 2-alkylsulfanyl derivatives of 5-methylbenzimidazole was synthesized and evaluated for antimycobacterial activity. The
1
structures of the compounds were confirmed by H NMR and IR data, and their purity by elemental analysis. Antimycobacterial
activities against Mycobacterium tuberculosis and nontuberculous mycobacteria were expressed as the minimum inhibitory
concentration. The substances exhibited significant antimycobacterial activity, in particular against both strains of Mycobacterium
kansasii. The effect of the most active compound in the set, 3,5-dinitro derivative 3t, exceeded that of the standard isoniazide
´
against M. kansasii and Mycobacterium a6ium. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Antimycobacterial activity; 2-Alkylsulfanyl derivatives; 5-Methylbenzimidazole-2-thiol; Mycobacterium tuberculosis; Nontuberculous
mycobacteria
atom in various heterocycles as a potential pharma-
cophore for antimycobacterial activity. In our previous
work, we reported that a significant antimycobacterial
activity was observed in a large series of alkylsulfanyl
derivatives of pyridine [5–7]. The most promising sub-
stances were 4-benzylsulfanyl derivatives of pyridine-2-
carbonitrile/2-carbothioamide [8]. The significant
activity of the prepared compounds prompted us to
search for new substances containing heterocyclic moi-
eties other than pyridine but bearing an alkylsulfanyl
moiety in the molecule. Using several published studies,
we chose benzimidazole because of its broad spectrum
of activities (antibacterial, antifungal [9], antihelminthic
[10], antiparasitic [11], and especially a promising tuber-
culostatic activity [12–15]) and interesting chemical
properties of the benzimidazole ring.
In order to enhance the activity of benzimidazole, we
tried to substitute the hydrogen atoms at 5- and 2-posi-
tions. Thus, we synthesized derivatives of benzimida-
zole in which the CH3 group on the benzimidazole ring
at 5-position is maintained while the benzyl moiety on
the sulfur atom at 2-position is further modified by
groups with electron-accepting (NO2, CN, CF3) and
electron-donating (Cl, F, Br, OCH3) properties in order
to optimize the lipophilic and steric characteristic.
In the present paper, we wish to report the synthesis
and antimycobacterial activity of 2-alkylsulfanyl
1. Introduction
Contrary to general expectations, the incidence of
mycobacterial disease has significantly increased world-
wide since 1990. Not only has there been a resurgence
of tuberculosis, which nowadays still remains a major
public health problem, but other mycobacteria, espe-
cially the Mycobacterium a6ium complex, have emerged
as important pathogens due mainly to the AIDS epi-
demic [1]. The prevalence of HIV infection, and the
emergence of drug-resistant and multi-drug-resistant
strains of Mycobacterium tuberculosis are contributing
to the worsening impact of the disease [2]. The World
Health Organization (WHO) estimates that one-third of
the population is infected with M. tuberculosis, the
agent that causes pulmonary tuberculosis, and that 3
million people per year decease because of this illness.
There is therefore a pressing need to develop novel
chemotherapeutic agents to hinder the emergence of
resistance and, ideally, shorten the duration of therapy
of this disease [3,4].
Extensive development of the structure–activity rela-
tionship (QSAR) at our department revealed an alkyl-
sulfanyl group bound to an electron-deficient carbon
* Correspondence and reprints.
E-mail address: klimeso@faf.cuni.cz (V. Klimesˇova´).
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(02)01218-1

260
V. Klimesˇo6a´ et al. / Il Farmaco 57 (2002) 259–265
derivatives of 5-methylbenzimidazole and a comparison
with the activity of a previously synthesized set of
benzimidazoles.
band between 3500 and 3220 cm−1 was particularly
characteristic. The CH3 group exhibited four characte-
ristic vibrations. The bands at 1380 and 1450 cm−1 were
assigned to symmetric and asymmetric deformations
and peaks at 2870 and 2960 cm−1 to stretching symmet-
ric and asymmetric vibrations. The interpretation of
nitro derivatives displayed bands at 1350 and 1530
cm−1. Compounds having the CꢀN group (3z, 3aa)
exhibited characteristic frequencies at 2229 and 2231
cm−1 and the diagnostic bands of the carbothioamide
function (CꢂS stretching vibration) in derivatives 4a and
4b were at 1274 and 1273 cm−1, respectively.
2. Chemistry
2-Benzylsulfanyl derivatives of 5-methylbenzimida-
zole (3, 4) were synthesized as outlined in Scheme 1. The
commercially available 5-methyl benzimidazole-2-thiol
(1) serves as a convenient starting material in the
syntheses and it is alkylated with benzyl halides (2).
The reaction depicted in Scheme 1 was used for the
preparation of benzylsulfanyl derivatives 3. Starting
thiol 1 was converted to the corresponding sodium salt
by dissolving in a methanolic solution of sodium
methanolate, and the resultant salt was subjected to a
nucleophilic substitution upon an addition of alkyl
halides 2. The treatment of 1 with various benzyl
chlorides or bromides 2 was carried out in N,N-
dimethylformamide (DMF) at room temperature and
yielded various monosubstituted and disubstituted 2-
benzylsulfanyl derivatives 3. Compounds bearing a CꢀN
(3z, 3aa) group were further transformed into the corre-
sponding thioamide (4a, 4b) through reaction with hy-
drogen sulfide in the presence of triethylamine and
pyridine.
The structures of the compounds were confirmed by
1H NMR and IR spectral data, and their purity by
elemental analysis. The singlet of the benzylic CH2S
group at 4.47–4.90 ppm and the singlet of CH3 group
at 2.24–2.38 ppm was observed in the 1H NMR spectra
of 5-methylbenzimidazole derivatives 3, 4. The spectra
of all substances displayed multiplets in the aromatic
region indicating the presence of the benzimidazole ring
and double doublet at 6.93–6.95 ppm belonging to H at
position 6 of the benzimidazole core. In the spectra of
carbothioamides 4a and 4b, NꢁH resonances were ap-
parent between 9.4 and 9.9 ppm.
Chemical and physical data of compounds 3 and 4 are
described in Tables 1 and 2.
3. Experimental
3.1. Chemistry
Melting points were determined on a Kofler block
and are uncorrected. Analytical samples were dried over
P4O10 at 82 or 61 °C and 2.4–2.6 kPa for 8–10 h.
Elemental analyses were performed on a CHNS-O CE
instrument (FISONS EA 1110) and were within 90.4%
of the calculated values. IR spectra were obtained on a
Nicolet Impact 400 spectrometer in KBr pellets. NMR
spectra were recorded in DMSO-d6 solutions at ambient
temperature on a Varian Mercury-Vx BB 300 spectro-
meter operating at 300 MHz. Chemical shifts were
recorded as l values in ppm and were indirectly referred
to tetramethylsilane (TMS). Coupling constants (J) are
given in Hz. Reactions were monitored and the purity of
products checked by TLC (Silufol UV 254 Kavalier,
Votice, Czech Republic, and Merck TLC plates silica
gel 60 F254, aluminum back) in acetone–light petroleum.
The plates were visualized using UV light, iodine fumes
and/or dipping in
a solution of Ce(SO4)2·4H2O,
H3Mo12O40P·xH2O, H2SO4, H2O and subsequent heat-
ing. Preparative thin-layer chromatography was carried
out on silica gel 60 F254 (0.015–0.040 mm, Merck).
The IR spectra of compounds 3, 4 were also in
agreement with the structures. The NꢁH absorption
Scheme 1. (a) Na, CH3OH, DMF; (b) H2S, TEA, pyridine.

V. Klimesˇo6a´ et al. / Il Farmaco 57 (2002) 259–265
261
Table 1
Chemical and physical data of compounds 3 and 4
Comp. no.
R
Formula
M.w.
Yield (%)
M.p. (°C)
3a
3b
3c
3d
3e
3f
3g
3h
3I
3j
3k
3l
3m
3n
3o
3p
3q
3r
H
4-Cl
3-Cl
2-Cl
4-F
3-F
2-F
4-Br
C
C
15H14N2S
15H13ClN2S
254.4
288.8
288.8
288.8
272.3
272.3
272.3
333.3
333.3
268.4
268.4
284.4
284.4
299.4
299.4
299.4
306.8
323.2
290.3
344.3
344.3
317.3
322.6
322.6
390.4
279.4
279.4
313.4
313.4
66
66
63
71
75
78
66
58
69
66
83
67
69
80
22
43
73
67
68
37
56
57
90
87
85
83
80
81
94
142.5–147
178–181
142–145
127–131
145.5–149.5
142–147
113–115
194–197 [15]
144.5–148
149.5–157
135.5-142
144.5–151.5
122–127
163–172
159.5–161.5
130–142
139.5–144
125–128 [15]
133–141
132–138
179–188
75–85
C15H13ClN2S
C15H13ClN2S
C15H13FN2S
C15H13FN2S
C15H13FN2S
C15H13BrN2S
3-Br
C15H13BrN2S
4-CH3
3-CH3
4-OCH3
3-OCH3
4-NO2
3-NO2
2-NO2
2,6-Cl,F
3,4-(Cl)2
3,4-(F)2
3,5-(NO2)2
2,4-(NO2)2
2,6-F,NO2
4-CF3
3-CF3
3,5-(CF3)2
4-CN
C16H16N2S
C16H16N2S
C16H16N2OS
C16H16N2OS
C
15H13N3O2S
C15H13N3O2S
C15H13N3O2S
C15H12ClFN2S
C15H12Cl2N2S
C15H12F2N2S
C15H12N4O4S
C15H12N4O4S
C15H12FN3O2S
3s
3t
3u
3v
3w
3x
3y
3z
3aa
4a
4b
C
16H13F3N2S
135–156
89–94
C16H13F3N2S
C17H12F6N2S
C16H13N3S
C
C
116–120
197–201
136–142
101–111
75–85
3-CN
4-CSNH2
3-CSNH2
16H13N3S
16H15N3S2
C16H15N3S2
Lit. [15] gives 189 °C (3h), 197 °C (3r).
3.1.1. General procedure for the preparation of
compounds 3a–z, aa
onto crushed ice with intensive stirring, the precipitated
product was filtered off, washed with cold water, and
air-dried. The residue was purified by preparative thin-
layer chromatography using acetone–light petroleum
(1:1, 1:2) as the eluent to give the yellow needles of pure
product 4 in 81–94% yield (Tables 1 and 2).
5-Methylbenzimidazole-2-thiol 1 (6 mmol) in dry
N,N-dimethylformamide (8 ml) was added to a solution
of sodium (6 mmol) in dry methanol (2.5 ml). After 10
min of stirring at room temperature, benzyl halide 2 (6
mmol) was added in 2–3 portions, and the resultant
suspension was stirred for 2–8 h. The reaction mixture
was then poured into an ice bath and left overnight.
The solid was filtered off, washed with cold water
(2×30 ml) and air-dried. The crude product was dis-
solved in acetone, filtered, adhered to silica gel and
purified by TLC preparative thin-layer chromatography
using acetone–light petroleum (1:2, 1:3, 1:4, 1:5, 1:6) as
the eluent to give white or brownish needles of pure
compounds 3 in 22–90% yields (Tables 1 and 2).
3.2. Antimycobacterial acti6ity
In vitro antimycobacterial activity of the compounds
was evaluated against M. tuberculosis CNCTC My
331/88, Mycobacterium kansasii CNCTC My 235/80,
M. kansasii 6509/96, and M. a6ium CNCTC My 330/88
using the micromethod for the determination of the
minimum inhibitory concentration (MIC). All strains
were obtained from the Czech National Collection of
Type Cultures (CNCTC) with the exception of M.
kansasii 6509/96, which was a clinical isolate. Antimy-
cobacterial activities of the compounds were deter-
3.1.2. General procedure for the preparation of
compounds 4a–4b
&
Dry hydrogen sulfide was passed through a solution
of a cyano compound (3z, 3aa) (2 mmol) dissolved in a
mixture of dry pyridine (7 ml) and triethylamine (0.7
ml). The reaction mixture was maintained at room
temperature for 4–5 h and then heated at 45 °C for an
additional hour. After cooling, the mixture was poured
mined in a Sula semisynthetic medium (SEVAC,
Prague). The compounds were added to the medium in
dimethylsulfoxide solutions. The following concentra-
tions were used: 500, 250, 125, 62, 32, 16, 8, 4, and 2
mmol/l. MIC values were determined after incubation at
37 °C for 7, 14, and 21 days. MIC was the lowest

262
V. Klimesˇo6a´ et al. / Il Farmaco 57 (2002) 259–265
Table 2
1H NMR and IR spectroscopic data of the compounds 3 and 4
Comp. no.
NMR l (ppm)
IR w, l (cm−1
)
3a
3b
3c
3d
2.37 (3H, s), 4.53 (2H, s), 6.93 (1H, dd, J1=1.4 Hz, J2=8.2 Hz), 7.05–7.55 (7H,
m), 12.43 (1H, bs)
2.37 (3H, s), 4.52 (2H, s), 6.93 (1H, dd, J1=1.3 Hz, J2=8.2 Hz), 7.10–7.50 (2H, m 3444 (NꢁH), 3062–2684 (H-bridges),
overlapped), 7.30–7.40 (2H, m AA%BB%), 7.40–7.50 (2H, m AA%BB%), 12.44 (1H, bs) 2981, 2851, 1441, 1396 (CH3)
2.37 (3H, s), 4.53 (2H, s), 6.93 (1H, dd, J1=1.7 Hz, J2=8.0 Hz), 7.15–7.45 (5H,
m), 7.50–7.55 (1H, m), 12.45 (1H, bs)
3432 (NꢁH), 3063–2645 (H-bridges),
2972, 2858, 1442, 1395 (CH3)
3432 (NꢁH), 3066–2737 (H-bridges),
2996, 2860, 1437, 1392 (CH3)
3432 (NꢁH), 3058–2722 (H-bridges),
2.37 (3H, s), 4.61 (2H, s), 6.94 (1H, dd, J1=1.4 Hz, J2=8.3 Hz), 7.05–7.40 (4H,
m), 7.48 (1H, dd, J1=1.8 Hz, J2=7.5 Hz), 7.56 (1H, dd, J1=2.1 Hz, J2=7.0 Hz), 2996, 2860, 1444, 1395 (CH3)
12.46 (1H, bs)
3e
3f
2.37 (3H, s), 4.52 (2H, s), 6.93 (1H, d, J=8.2 Hz), 7.05–7.20 (3H, m), 7.40–7.60
(3H, m), 12.43 (1H, bs)
2.37 (3H, s), 4.54 (2H, s), 6.93 (1H, dd, J1=1.0 Hz, J2=8.3 Hz), 7.00–7.10 (1H,
m), 7.20–7.60 (5H, m), 12.45 (1H, bs)
3447 (NꢁH), 3043–2684 (H-bridges),
2989, 2858, 1441, 1399 (CH3)
3427 (NꢁH), 3053–2686 (H-bridges),
2976, 2852, 1446, 1396 (CH3)
3g
3h
2.37 (3H, s), 4.55 (2H, s), 6.94 (1H, d, J=8.3 Hz), 7.00–7.55 (6H, m), 12.46 (1H, 3432 (NꢁH), 3038–2618 (H-bridges),
bs) 2991, 2864, 1441, 1397 (CH3)
2.36 (3H, s), 4.50 (2H, s), 6.93 (1H, dd, J1=1.1 Hz, J2=8.2 Hz), 7.10–7.45 (2H, m 3428 (NꢁH), 3061–2637 (H-bridges),
overlapped), 7.30–7.40 (2H, m overlapped AA%BB%), 7.40–7.50 (2H, m AA%BB%),
12.43 (1H, bs)
2981, 2858, 1439, 1396 (CH3)
3I
3j
2.37 (3H, s), 4.52 (2H, s), 6.93 (1H, dd, J1=1.0 Hz, J2=8.1 Hz), 7.20–7.40 (3H,
m), 7.40–7.45 (2H, m), 7.66 (1H, t, J=1.8 Hz), 12.45 (1H, bs)
2.24 (3H, s), 2.37 (3H, s), 4.48 (2H, s), 6.93 (1H, dd, J1=1.4 Hz, J2=8.2 Hz),
7.15–7.45 (2H, m overlapped), 7.05–7.15 (2H, m AA%BB%), 7.25–7.35 (2H, m
overlapped AA%BB%), 12.40 (1H, bs)
3428 (NꢁH), 3065–2638 (H-bridges),
2989, 2861, 1440, 1394 (CH3)
3425 (NꢁH), 3023–2731 (H-bridges),
2980, 2861, 1438, 1389 (CH3)
3k
3l
2.25 (3H, s), 2.37 (3H, s), 4.49 (2H, s), 6.93 (1H, bd, J=8.2 Hz), 7.00–7.10 (1H,
m), 7.10–7.30 (4H, m), 7.30–7.50 (1H, m), 12.42 (1H, bs)
2.37 (3H, s), 3.69 (3H, s), 4.47 (2H, s), 6.80–6.90 (2H, m AA%BB%) 6.93 (1H, dd,
3432 (NꢁH), 3039–2641 (H-bridges),
2980, 2861, 1440, 1393 (CH3)
3424 (NꢁH), 3062–2637 (H-bridges),
J1=1.7 Hz, J2=8.5 Hz), 7.25–7.40 (2H, m overlapped AA%BB%), 7.10–7.50 (2H, m 2996, 2859, 1441, 1397 (CH3), 1177,
overlapped), 12.40 (1H, bs)
1033 (CꢁO ArꢁOꢁCH3)
3m
3n
3o
3p
2.37 (3H, s), 3.68 (3H, s), 4.49 (2H, s), 6.80 (1H, ddd, J1=1.1 Hz, J2=2.5 Hz,
J3=8.2 Hz), 6.90–7.05 (3H, m), 7.15–7.25 (1H, m overlapped), 7.20 (1H, t
overlapped, J=7.8 Hz), 7.32 (1H, bd, J=8.2 Hz), 12.42 (1H, bs)
2.36 (3H, s), 4.65 (2H, s), 6.93 (1H, dd, J1=1.1 Hz, J2=8.2 Hz), 7.10–7.40 (2H,
m), 7.60–7.70 (2H, m AA%BB%), 8.10–8.20 (2H, m AA%BB%) 12.47 (1H, bs)
3432 (NꢁH), 3061–2610 (H-bridges),
2998, 2858, 1466, 1403 (CH3), 1171,
1042 (CꢁO ArꢁOꢁCH3)
3427 (NꢁH), 3074–2797 (H-bridges),
2978, 2860, 1441, 1397 (CH3), 1345,
1519 (NO2)
2.36 (3H, s), 4.66 (2H, s), 6.93 (1H, dd, J1=1.4 Hz, J2=8.2 Hz), 7.10–7.45 (2H,
m), 7.57 (1H, t, J=7.8 Hz), 7.89 (1H, dm, J=7.8 Hz), 8.08 (1H, ddd, J1=1.1 Hz, 2984, 2865, 1440, 1397 (CH3), 1352,
3424 (NꢁH), 3043–2799 (H-bridges),
J2=2.5 Hz, J3=8.2 Hz), 8.36 (1H, t, J=2.0 Hz), 12.46 (1H, bs)
2.36 (3H, s), 4.81 (2H, s), 6.93 (1H, dd, J1=1.4 Hz, J2=8.2 Hz), 7.10–7.25 (1H,
m), 7.25–7.40 (1H, m), 7.51 (1H, td, J1=1.4 Hz, J2=7.7 Hz), 7.64 (1H, td,
J1=1.4 Hz, J2=7.6 Hz), 7.75 (1H, dd, J1=1.7 Hz, J2=7.7 Hz), 8.02 (1H, dd,
J1=1.4 Hz, J2=8.2 Hz), 12.41 (1H, bs)
1528 (NO2)
3428 (NꢁH), 3068–2858 (H-bridges),
2923, 2858, 1442, 1395 (CH3), 1342,
1524 (NO2)
3q
3r
2.38 (3H, s), 4.64 (2H, d, J=1.7 Hz), 6.95 (1H, dd, J1=1.4 Hz, J2=8.2 Hz),
7.05–7.30 (2H, m), 7.30–7.55 (3H, m), 12.54 (1H, bs)
2.36 (3H, s), 4.52 (2H, s), 6.93 (1H, dd, J1=1.9 Hz, J2=8.2 Hz), 7.15–7.40 (2H,
3432 (NꢁH), 3041–2718 (H-bridges),
2922, 2862, 1453, 1395(CH3)
3432 (NꢁH), 3061–2629 (H-bridges),
m), 7.41 (1H, dd, J1=8.3 Hz, J2=2.0 Hz), 7.54 (1H, d, J=8.2 Hz), 7.72 (1H, d, 2973, 2861, 1440, 1396 (CH3)
J=2.2 Hz), 12.45 (1H, bs)
3s
3t
2.37 (3H, s), 4.51 (2H, s), 6.93 (1H, dd, J1=1.1 Hz, J2=8.2 Hz), 7.05–7.45 (4H,
m), 7.51 (1H, ddd, J1=2.2 Hz, J2=8.0 Hz, J3=11.8 Hz), 12.44 (1H, bs)
2.36 (3H, s), 4.76 (2H, s), 6.93 (1H, dd, J1=1.1 Hz, J2=8.2 Hz), 7.05–7.45 (2H,
m), 8.66 (1H, t, J=2.2 Hz), 8.81 (2H, d, J=2.2 Hz), 12.47 (1H, bs)
3428 (NꢁH), 3035–2615 (H-bridges),
2996, 2856, 1437, 1400 (CH3)
3427 (NꢁH), 3100–2872 (H-bridges),
2996, 2872, 1439, 1388 (CH3), 1344,
1541 (NO2)
3u
3v
2.35 (3H, s), 4.90 (2H, s), 6.92 (1H, dd, J1=1.7 Hz, J2=8.3 Hz), 7.00–7.55 (2H,
m), 8.05 (1H, d, J=8.6 Hz), 8.46 (1H, dd, J1=2.5 Hz, J2=8.6 Hz), 8.69 (1H, d, 2950, 2869, 1440, 1385 (CH3), 1344,
J=2.5 Hz), 12.45 (1H, bs)
2.36 (3H, s), 4.79 (2H, s), 6.93 (1H, dd, J1=1.7 Hz, J2=8.0 Hz), 7.10–7.45 (2H,
m), 7.50–7.75 (2H, m), 7.83 (1H, dt, J1=1.4 Hz, J2=7.9 Hz), 12.44 (1H, bs)
3427 (NꢁH), 3114–2869 (H-bridges),
1533 (NO2)
3432 (NꢁH), 3099–3058 (H-bridges),
2923, 2865, 1464, 1385 (CH3), 1351,
1533 (NO2)
3w
3x
2.36 (3H, s), 4.61 (2H, s), 6.93 (1H, dd, J1=1.1 Hz, J2=8.3 Hz), 7.05–7.55 (2H,
m), 7.65 (4H, bs)
3432 (NꢁH), 3069–2807 (H-bridges),
2999, 2864, 1440, 1395 (CH3), 1122,
1160, 1276 (CF3)
3432 (NꢁH), 3069–2807 (H-bridges),
2927, 2865, 1450, 1395 (CH3), 1125,
1167, 1276 (CF3)
2.36 (3H, s), 4.61 (2H, s), 6.93 (1H, dd, J1=1.5 Hz, J2=8.1 Hz), 7.15–7.40 (2H,
m), 7.45–7.65 (2H, m), 7.73 (1H, bd, J=7.8 Hz), 7.83 (1H, bs)

V. Klimesˇo6a´ et al. / Il Farmaco 57 (2002) 259–265
263
Table 2 (Continued)
Comp. no.
NMR l (ppm)
IR w, l (cm−1
)
3y
2.36 (3H, s), 4.68 (2H, s), 6.93 (1H, dd, J1=1.5 Hz, J2=8.0 Hz), 7.21 (1H, bs),
7.31 (1H, bd, J=8.3 Hz), 7.95 (1H, bs), 8.20 (2H, bs)
3440 (NꢁH), 3054–2649 (H-bridges),
2988, 2865, 1442, 1399 (CH3), 1134,
1178, 1279 (CF3)
3z
2.36 (3H, s), 4.60 (2H, s), 6.93 (1H, dd, J1=1.4 Hz, J2=8.2 Hz), 7.00–7.55 (2H,
m), 7.55–7.65 (2H, m AA%BB%), 7.70–7.80 (2H, m AA%BB%) 12.46 (1H, bs)
3428 (NꢁH), 3053–2626 (H-bridges),
2925, 2862, 1441, 1398 (CH3), 2229
(CꢀN)
3aa
4a
2.37 (3H, s), 4.57 (2H, s), 6.93 (1H, dd, J1=1.6 Hz, J2=8.2 Hz), 7.15–7.40 (2H,
m), 7.49 (1H, t, J=7.7 Hz), 7.70 (1H, dt, J1=1.4 Hz, J2=7.7 Hz), 7.77 (1H, dt, 2923, 2862, 1438, 1397 (CH3), 2231
J1=1.4 Hz, J2=7.7 Hz), 7.90 (1H, bt, J=1.1 Hz), 12.45 (1H, bs)
2.37 (3H, s), 4.55 (2H, s), 6.93 (1H, dd, J1=1.7 Hz, J2=8.2 Hz), 7.10–7.40 (2H,
3428 (NꢁH), 3061–2614 (H-bridges),
(CꢀN)
3408 (NꢁH), 3288–3191 (H-bridges),
m), 7.40–7.50 (2H, m AA%BB%), 7.75–7.85 (2H, m AA%BB%), 9.44 (1H, bs), 9.83 (1H, 2923, 2857, 1436, 1385 (CH3), 1274
bs), 12.45 (1H, bs) (CꢂS ꢁNHꢁCꢂS)
2.37 (3H, s), 4.57 (2H, s), 6.93 (1H, dd, J1=1.7 Hz, J2=8.0 Hz), 7.15–7.40 (2H, m 3424 (NꢁH), 3297–3184 (H-bridges),
overlapped), 7.32 (1H, t overlapped, J=7.8 Hz), 7.55 (1H, dt, J1=1.5 Hz, J2=7.8 2923, 2857, 1433, 1387 (CH3), 1273
Hz), 7.69 (1H, dt, J1=1.5 Hz, J2=7.8 Hz), 7.99 (1H, t, J=1.5 Hz), 9.49 (1H, bs), (CꢂS ꢁNHꢁCꢂS)
9.87 (1H, bs), 12.41 (1H, bs)
4b
concentration of a substance at which the inhibition of
the growth of mycobacteria occurred.
activities ranging from 4 to 32 mmol/l. The activity
exceeded that of INH against both strains of M.
kansasii, against M. tuberculosis it was 4-fold less than
that of INH. The compound was only moderately
active (MIC=32 mmol/l) against M. a6ium, but ex-
ceeded the potency of INH. The presence of the nitro
group in positions 2 and 4 (3u) resulted in a slight
decrease in activity (MIC=8–62 mmol/l). Displace-
ment of the nitro group in position 2 led to compound
3n, which exhibited a considerable decrease in activity
against M. tuberculosis (MIC=32 mmol/l), on the other
hand the activity against M. a6ium increased (MIC=8
mmol/l). A promising effect was revealed for thioamide
derivatives (4). The 4-substituated derivative (4a) had a
greater activity (MIC=8–16 mmol/l) than its 3-substi-
tuated analog (4b) with MIC=62–125 mmol/l.
In a previous communication [16], we reported the
synthesis and biological evaluation of a series of ben-
zimidazole derivatives. The study revealed that the
newly prepared set of 5-methylbenzimidazole deriva-
tives displayed a better activity than that of the benzi-
midazole analog. The MIC values for the set of 5-
methylbenzimidazoles and benzimidazoles varied be-
tween 4–62 and 8–125 mmol/l, respectively. The mono-,
dinitro- and thioamide derivatives were the most active
compounds in both sets.
4. Results and discussion
The results of biological evaluation of newly pre-
pared benzylsulfanyl derivatives 3 and 4 are summa-
rized in Table 3. The values of antimycobacterial
activity are expressed as the MIC. In several cases
(denoted \), the MIC could not be determined due to
limited solubility of the compounds in the testing
medium. For the sake of comparison, the MIC values
of 1 and the standard isoniazide (INH) are also in-
cluded. All of these compounds (1, 3, 4) displayed in
vitro activity against all mycobacterial strains tested.
The MIC values are within a range of 4–500 mmol/l,
most often between 4 and 62 mmol/l. By comparing
their MIC values with INH, the compounds under
study were less active against M. tuberculosis 331/88
than INH, and all but one (3t) against M. kansasii
6509/96. In contrast to INH, the derivatives were com-
parably active against both strains of M. kansasii, with
better activity against M. kansasii 235/80. The com-
pounds also possessed a more pronounced effect than
INH against M. a6ium 330/88.
The starting 5-methylbenzimidazole-2-thiol (1) is
characterized by the activity within a range of \250–
500 mmol/l. Comparisons of the MIC values of 1 to
those of the benzylsulfanyl derivative (3a) indicate that
the antimycobacterial activity is connected with the
presence of the benzyl moiety at 2-position of the
5-methylbenzimidazole ring. It was expected that a
further modification of the benzyl moiety by electron-
withdrawing groups might improve the activity. Thus,
the 2-(3,5-dinitrobenzylsulfanyl)-5-methylbenzimidazol
(3t) was the most active compound in the set, having
On the basis of the pharmacophore analysis and
knowledge about the relationship between the structure
and antimycobacterial activity of pyridine [7,8,17,18]
and benzimidazole derivatives [16], we postulated that
alkylsulfanyl moiety is responsible for the observed
antituberculotic activity. This finding is in agreement
with the presented results. In addition, the activity of
compound 4 is linked to the presence of a thioamide
group, which is supposed to also be the pharmacophore
of antimycobacterial activity.

264
V. Klimesˇo6a´ et al. / Il Farmaco 57 (2002) 259–265
Table 3
Antimycobacterial activity of compounds 1, 3, 4 expressed as MIC (mmol/l)
Comp. No.
Strains
M. tuberculosis My 332/88
M. kansasii My 235/80
M. kansasii My 6509/96
M. a6ium My 330/88
14 days
21 days
7 days
14 days
21 days
7 days
14 days
21 days
14 days
21 days
1
3a
3b
3c
3d
3e
500
62
32
62
32
32
32
62
32
32
\32
32
32
62
32
62
62
16
32
32
4
\250
62
500
16
16
16
8
16
32
8
500
62
32
32
32
62
32
32
\32
16
32
32
32
62
16
32
125
32
16
32
4
8
125
32
62
62
\32
125
8
500
125
62
32
32
62
62
32
\32
32
\32
32
\62
125
32
\62
\125
62
500
16
4
4
4
8
8
4
8
4
4
4
16
16
4
8
62
8
500
32
16
16
16
32
16
16
16
16
16
16
32
32
8
500
62
16
16
16
32
32
32
32
16
32
32
32
32
16
32
125
16
16
16
4
500
62
32
32
32
32
62
62
\32
32
32
62
62
62
8
\500
\62
32
32
62
32
32
62
62
\32
62
\32
62
\32
62
32
\62
62
32
\32
\32
4
8
125
62
32
62
62
62
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
125
\62
62
\32
62
\62
\32
16
\62
125
16
\32
\62
32
\125
\125
62
62
125
32
16
8
8
16
16
16
8
16
125
16
8
16
4
8
\62
32
62
62
\32
125
8
16
125
16
8
16
4
32
62
8
32
32
4
32
4
4
2
8
32
32
32
62
125
32
32
62
32
62
16
62
250
3s
3t
3u
3v
3w
3x
3y
3z
3aa
4a
4b
INH
8
8
8
62
32
32
32
32
62
8
\125
32
\32
32
32
32
\32
125
8
62
32
32
62
\32
125
8
125
32
62
62
\32
125
8
32
62
62
\62.5
8
62
125
\62
125
8
62
500
125
16
125
500
32
1
62
1
16
500
32
500
16
4
32
8
62
8
&
The most active compound 3t was also subjected to
an antiproliferative effect assay against the K-562 and
L-929 cell lines. The values expressed as GI50 (growth
inhibition) are 7.1 and 12.0 mg/ml. The cytotoxic effect
was evaluated on HeLa cells. The cytotoxic concentra-
tion expressed as CC50 (CC5) was 14.0 (3.9) mg/ml.
According to the values of GI and CC, compound 3t
can be considered as moderately toxic.
Zizˇkova´ and P. Jancˇa´rek (Department of Inorganic and
Organic Chemistry, Faculty of Pharmacy, Charles Uni-
versity) for recording the IR spectra and NMR spectra,
respectively. The authors’ thanks are due to Dr. U.
Mo¨llmann (Hans-Kno¨ll-Institut fu¨r Naturshoff-
Forschung, Jena, Germany) for providing the cytotoxi-
city data.
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