Synthesis and antimycobacterial activity of 1,2,4-triazole 3-benzylsulfanyl derivatives

Article abstract of DOI:10.1016/j.farmac.2004.01.006

Series of 3-benzylsulfanyl derivatives of 1,2,4-triazole and 4-methyl-1,2,4-triazole were synthesized by alkylation of starting triazole-3-thiol with appropriately substituted benzyl halide. All members of the set were evaluated for in vitro antimycobacte

Full text of DOI:10.1016/j.farmac.2004.01.006

IL FARMACO 59 (2004) 279–288
www.elsevier.com/locate/farmac
Synthesis and antimycobacterial activity of 1,2,4-triazole
3-benzylsulfanyl derivatives
Veˇra Klimešová a,*, Lenka Zahajská a, Karel Waisser a, Jarmila Kaustová b, Ute Möllmann c
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, 50005 Hradec Králové, Czech Republic
b National Reference Laboratory for Mycobacterium kansasii, Regional Institute of Hygiene, 72892 Ostrava, Czech Republic
c Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstrasse 11, 07745 Jena, Germany
Received 4 December 2003; accepted 20 January 2004
Abstract
Series of 3-benzylsulfanyl derivatives of 1,2,4-triazole and 4-methyl-1,2,4-triazole were synthesized by alkylation of starting triazole-3-
thiol with appropriately substituted benzyl halide. All members of the set were evaluated for in vitro antimycobacterial activity against
Mycobacterium tuberculosis, M. avium, and two strains of M. kansasii. The activities were expressed as the minimum inhibitory
concentration. The compounds exhibited only a moderate or slight antimycobacterial activity. Minimum inhibitory concentrations fall into a
range of 32->1000 µmol/l. The most active substances bear two nitro groups or a thioamide group on the benzyl moiety. As regards the
cytotoxicity effect, the evaluated compounds can be considered as moderately toxic.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Antimycobacterial activity; 3-benzylsulfanyl-1,2,4-triazole; 3-benzylsulfanyl-4-methyl-1,2,4-triazole
1. Introduction
plex) produce severe disseminated infections. These strains
represent a great health danger because of their resistance
against current drugs, attacking, first of all, immunocompro-
mised persons [4].
In spite of an enormous amount of work done in under-
standing the genome sequence of mycobacteria, no antituber-
culotic specific drug has been discovered in over 30 years.
Hence, there is a pressing need to develop novel chemothera-
peutic agents and to change current drug regimens in order to
shorten the lengthy treatment, to minimize the resistance
problem in mycobacterial strains, and/or to improve the treat-
ment of latent TB infection.
To pursue this goal, our research efforts are directed to
find new chemical classes of antimycobacterially active
agents. The methods of investigation of structure-activity
relationships (SAR) enabled us to find some new pharma-
cophores of the above-mentioned activity [5].A lot of studies
were carried out on heterocyclic systems bearing an alkylsul-
fanyl group as a pharmacophore [6]. Various heterocyclic
rings were taken as a ground to constitute large series of
compounds, e.g. tetrazole [7], pyrazine [8], quinoxaline [9],
quinazoline [10]. Agents with higher activity were recorded
among purine [11], benzimidazole [12], 5-methyl-
benzimidazole [13], benzoxazole, benzothiazole [14], and
Worldwide, tuberculosis (TB) still remains a major public
health problem. The World Health Organisation (WHO) esti-
mates that one third of the population is infected with latent
Mycobacterium tuberculosis and approximately 3 million
people per year decease of illnesses caused by this bacillus
[1].
The treatment is complicated by a long-term administra-
tion of few antituberculotic agents, which intensifies drug
side effects and, by non-compliance of patients, often results
in the development of multidrug resistant strains [2]. In
addition, the disease often attacks immunoaltered individuals
- TB is together with mycoses the most common complica-
tion and the cause of death in AIDS patients [3]. A great
problem is also migration of inhabitants from the areas with a
higher incidence of TB to the regions with a favourable
epidemiologic situation. The current TB treatment is not
satisfactorily effective in the eradication of latent TB infec-
tion. Except M. tuberculosis, ever frequently appearing non-
tuberculous mycobacteria (for example the M. avium com-
* Corresponding author.
E-mail address: klimeso@faf.cuni.cz (V. Klimešová).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.01.006

280
V. Klimešová et al. / IL FARMACO 59 (2004) 279–288
Scheme 1
(a) Na, CH3OH, DMF; (b) 4j, KMnO4, CH3COOH
R
X-CH2
H
N
N
N
3
N
b
N
N
N
N
N
a
R
S
SO2
R
SH
R1
CH3
1 R=H
2 R=CH3
4a-z, aa R=H
8a-f R=CH3
7
4
a
b
c
d
e
f
g
h
i
R1
4
j
k
l
m
n
o
p
q
r
R1
4
s
t
u
v
w
x
y
z
R1
8
a
b
c
d
e
f
R1
H
4-CH3
3-CH3
4-OCH3
3-OCH3
4-NO2
3-NO2
2-NO2
2-Cl-6-F
3,4-Cl2
3,4-F2
H
4-Cl
3-Cl
2-Cl
4-F
3-F
2-F
4-Br
3-Br
3,5-(NO2)2
2,4-(NO2)2
2-F-6-NO2
4-CF3
4-Cl
3-Br
3,5-(NO2)2
2,4-(NO2)2
4-CN
3-CF3
3,5-(CF3)2
4-CN
aa
3-CN
pyridine [15] derivatives. Special attention was paid to their
benzylsulfanyl derivatives [12,13,14,16,17,18] and the influ-
ence of the substituents of the benzyl moiety on the activity.
QSAR calculations carried out on various types of hetero-
cycles proved that the activity is enhanced with electron-
withdrawing substituents, especially nitro, trifluoromethyl,
and thioamide groups, respectively [19].
Continuing in our antituberculotic program, we have cho-
sen 1,2,4-triazole-3-thiol (1) and 4-methyl-1,2,4-triazole-3-
thiol (2) as the basic heterocyclic moiety. We have prepared
and evaluated 37 new benzylsulfanyl derivatives possessing
different substituents on the benzyl moiety.
3-thiol (1) and 4-methyl-1,2,4-triazole-3-thiol (2), which
were alkylated with benzyl halides, as reported previously
for other heterocyclic derivatives bearing an alkysulfanyl
moiety [12,13,14,16,18].
Starting thiols 1 and 2 were converted to the correspond-
ing sodium salts by dissolving in a methanolic solution of
sodium methanolate, and the resultant salts were subjected to
a nucleophilic substitution upon an addition of various ben-
zyl halides (3). The reaction was carried out in DMF at room
temperature for 1-23 hours and yielded desired monosubsti-
tuted and disubstituted 3-benzylsulfanyl derivatives 4 and 8,
respectively. The sulfur atom in the alkylsulfanyl moiety was
subsequently subjected to oxidation. The oxidation of 4j by
potassium permanganate in acetic acid afforded sulfone de-
rivative 7. Compounds bearing a CN group served the pur-
pose of obtaining other derivatives. The carbonitrile group in
compounds 4z, 4aa, 8f was converted into the carbothioam-
ide group by the addition of hydrogen sulfide in
pyridine/triethylamine solution (5a, 5b, 9). Conversions of
2. Chemistry
The synthetic routes leading to the desired compounds are
shown in Schemes 1 and 2. The benzylsulfanyl derivatives (4,
8) were prepared from commercially available 1,2,4-triazole-
Scheme 2
(a) H2S, TEA, pyridine; (b) 4aa, NaN3, LiCl, NH4Cl, DMF
N
N
N
a
S
R
N
N
CSNH2
N
5a,b R=H
9
R=CH3
S
R
CN
H
b
N
4z,aa R=H
N
N
HN
8f
R=CH3
N
N
N
S
6
4z
4aa
4-CN
3-CN
5a
5b
4-CSNH2
3-CSNH2
8f
9
4-CN
4-CSNH2

V. Klimešová et al. / IL FARMACO 59 (2004) 279–288
281
4aa into the tetrazole derivative 6 was performed, with a
good yield (87%), carrying out the reaction with sodium
azide in the presence of lithium chloride and ammonium
chloride in DMF as a solvent.
strains. On the other hand, some antimycobacterial activity is
apparent in their 3-benzylsulfanyl derivatives. It indicates
that antimycobacterial activity is connected with the pres-
ence of a benzylsulfanyl group on the triazole ring [5]. The
activity is affected by substituents on the phenyl ring. It has
been observed that the electron-withdrawing groups reduce
the MICs values. Thus, 3-(2,4-dinitrobenzylsulfanyl)-1,2,4-
triazole (4u) and 3-(3,5-dinitrobenzylsulfanyl)-1,2,4-triazole
(4t) are the most active compounds in the set, their activities
ranging from 32 to 500 µmol/l. The activities of compounds
bearing trifluoromethyl group rank among the more active
derivatives. A promising effect was confirmed for thioamide
derivatives 5, especially the 4-substituted derivative (5a)
exhibited greater activity (MIC = 62.5-250 µmol/l).
Replacement of hydrogen atom in position 4 by a methyl
group (2, 8, 9) is connected with a decrease in activity. The
MIC values reach the highest concentrations (MIC = 500-
>1000 µmol/l). Even compounds with dinitro groups on the
benzyl moiety (8d, e) did not show any activity, whereas
dinitroderivatives of triazole (4t, u) belonged to the most
active compounds presented here. The presence of a hydro-
gen atom in position 4 is probably necessary for an interac-
tion with the receptor; any substituent forbids the formation
of a hydrogen bond or causes a sterical barrier.
1
The structures of the compounds were confirmed by H
NMR, 13C NMR, and IR spectral data, and their purity by
1
elemental analysis. H NMR spectra of all compounds dis-
play the singlet at 4.21-4.72 ppm indicating a benzylic SCH2
group, singlet at 3.36-3.47 ppm for 4-methylated triazoles,
broad singlet of hydrogen in position 5 on the triazole ring at
8.42-8.60 ppm (8.89 ppm for sulfone 7), which is sharper by
4-methylated triazoles. The spectra of all substances display
multiplets in the aromatic region indicating the presence of a
benzyl ring and the type of its substitution. Resonances of
carbothioamide group hydrogens of compounds 5 and 9 are
apparent as two broad singlets at approximately 9.85 and
9.47 ppm.
13C NMR spectra of 1,2,4-triazole derivatives lack one or
both signals of triazole ring carbons, whereas the spectra of
4-methyl-1,2,4-triazole derivatives reveal signals of all car-
bons.
The IR spectra of compounds are also in agreement with
the structures. The N-H stretching frequencies cannot be
seen due to the presence of moisture in KBr pellets. The
spectra of all alkylsulfanyl derivatives show the absorption
signals of alkyl group vibrations between 2926-3128 cm-1. H
bridges fall into the region between 2094-2559 cm-1. The
presence of a nitril group in 4z, 4aa and 8f is certified by the
characteristic frequencies at 2231, 2229, and 2232, respec-
tively. Vibrations of nitro groups reveal two characteristic
absorption maxima at 1519-1541 cm-1 and 1334-1361 cm-1,
respectively.
The other modification of the molecule was carried out on
the sulfur atom. Compounds with a sulfide moiety are gener-
ally activated in vivo by oxidation of sulfur to sulfoxides and
sulfones. Therefore we decided to prepare the sulfone deriva-
tive. This modification did not lead to increases in activity,
compound 7 turned out to be antimycobacterially inactive.
The conversion of the carbonitrile group into the tetrazole
ring did not lead to any significant enhancement of activity as
well.
The most active compounds 4i, t, and 5a (as a representa-
tive of halide, nitro, and thioamide derivatives, respectively)
and one random sample 4r were subjected to an antiprolif-
erative and cytotoxic effect assays (Table 2). The values of
GI50 (growth inhibition) express the antiproliferative effect,
the values of CC50 and CC10, respectively, express the cyto-
toxic effect of compounds. The results are reported in µg/ml
obtained from biological study and in µmol/l, for a compari-
son with MIC values. According to the values of GI and CC,
the tested compounds can be considered as moderately toxic.
In conclusion, microbiological data given for the com-
pounds presented in this paper allowed us to state that the
alkylsulfanyl group bonded to triazole ring is not suitable for
antituberculotic activity and discouraged us to continue in
the synthesis of triazole derivatives.
3. Results and discussion
The results of antimycobacterial screening of newly pre-
pared compounds 4-9 expressed as the MIC are summarized
in Table 1. 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, 2 and the standard isoniazide (INH) are also included.
The antimycobacteriological assessment revealed that the
triazole derivatives possess only a moderate or slight activity.
The MIC values are generally within the range of 32->1000
µmol/l, most often between 250-1000 µmol/l against all
evaluated strains. By comparing their MIC values with INH,
the triazole derivatives were less active against M. tubercu-
losis 331/88 and M. kansasii 6509/96 than INH. On the other
hand, in some cases, the compounds exhibited comparable or
better activities against M. kansasii 235/80 and M. avium
330/88 than those of INH.
4. Experimental
4.1. Chemistry
The starting 1,2,4-triazole-3-thiol (1) and 4-methyl-1,2,4-
triazole-3-thiol (2) are characterised by the activity within a
range of 500->1000 µmol/l. It means that these starting
triazole-3-thiols are nearly inactive against mycobacterial
Melting points were determined on a Kofler block and are
uncorrected. Analytical samples were dried over P4O10 at

282
V. Klimešová et al. / IL FARMACO 59 (2004) 279–288
Table 1
Antimycobacterial activity of compounds 1, 2, 4-9 expressed as MIC (µmol/l)
Comp.
R1
Strain
M. tuberculosis My
331/88
M. avium My 330/88
M. kansasii My 235/80
M. kansasi 6509/96
14 days
500
21 days
14 days
1000
>1000
500
21 days
1000
>1000
1000
500
7 days
1000
1000
1000
500
14 days
>1000
>1000
>1000
1000
1000
>1000
500
21 days
>1000
>1000
>1000
>1000
>1000
>1000
500
7 days
500
14 days
1000
>1000
>1000
1000
500
21 days
1000
>1000
>1000
1000
1000
1000
500
1
2
-
1000
>1000
1000
1000
1000
1000
500
-
1000
500
1000
500
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
4-Cl
3-Cl
2-Cl
4-F
3-F
2-F
4-Br
3-Br
4-CH3
3-CH3
4-OCH3
3-OCH3
4-NO2
3-NO2
2-NO2
2-Cl-6-F
3,4-Cl2
3,4-F2
500
250
500
500
250
500
500
250
500
500
1000
500
500
250
500
250
250
250
125
500
250
500
250
500
125
250
500
62.5
62.5
125
125
250
250
500
250
500
125
250
500
250
250
250
500
250
500
250
250
500
250
500
250
500
250
250
62.5
250
125
250
62.5
250
125
125
4j
4k
4l
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
>1000
1000
1000
>1000
62.5
250
500
1000
500
1000
500
1000
500
1000
500
250
500
250
125
500
1000
1000
500
500
1000
1000
1000
1000
1000
500
1000
1000
1000
1000
>1000
500
500
1000
1000
1000
1000
500
1000
1000
1000
1000
1000
500
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
4y
4z
4aa
5a
5b
6
500
500
500
500
250
250
500
1000
1000
1000
1000
1000
1000
500
500
500
500
500
250
500
250
125
500
500
250
500
500
125
500
500
500
250
500
1000
125
250
500
500
3,5-(NO2)2 62.5
2,4-(NO2)2 32
2-F-6-NO2 1000
500
32
62.5
62.5
125
62.5
250
125
62.5
500
250
250
500
250
>1000
500
>1000
250
>1000
250
1000
125
1000
250
>1000
250
1000
125
1000
250
1000
250
4-CF3
3-CF3
250
250
500
250
500
125
125
250
125
250
500
3,5-(CF3)2 125
250
500
1000
1000
1000
250
250
250
500
250
250
250
4-CN
3-CN
>1000
1000
>1000
>1000
250
500
500
1000
>1000
125
>1000
>1000
250
500
1000
>1000
125
1000
>1000
250
1000
250
1000
125
1000
62.5
1000
500
4-CSNH2 125
3-CSNH2 500
1000
>500
>500
>1000
1000
1000
>1000
>1000
>1000
500
500
1000
>500
>500
>1000
1000
1000
>1000
>1000
>1000
1000
>250
10001000
500
1000
>500
>500
>1000
1000
1000
>1000
>1000
>1000
500
500
1000
500
3-(5-tetr)
4-CH3
H
4-Cl
3-Br
>500
>500
>500
>1000
1000
1000
>1000
>1000
>1000
1000
250
>500
>500
>1000
>1000
>1000
>1000
>1000
>1000
1000
>250
>500
>500
>1000
1000
1000
>1000
>1000
>1000
1000
8
7
>500
1000
1000
500
500
500
>500
>1000
1000
1000
>1000
>1000
>1000
1000
8
8a
8b
8c
8d
8e
8f
1000
1000
500
1000
1000
500
3,5-(NO2)2 >1000
2,4-(NO2)2 >1000
>1000
>1000
>1000
500
>1000
>1000
1000
500
4-CN
1000
9
INH
4-CSNH2 250
0.5
1
>250
>250
4
Table 2
Antiproliferative (GI50) and cytotoxic (CC) activity of some compounds
Comp.
R1
L-929
GI50
[µg/ml]
>50
K-562
GI50
HeLa
CC50
CC10
[µmol/l]
>185.1
100.3
[µg/ml]
44.9
[µmol/l]
166.2
82.6
[µg/ml]
[µmol/l]
[µg/ml]
10.5
7.2
[µmol/l]
38.9
4i
3-Br
3,4-Cl2
3,5-(NO2)2
4-CSNH2
32.8
15.4
29.7
46.4
121.4
59.2
4r
4t
5a
26.1
21.5
27.7
41.9
149.0
35.8
127.7
>199.7
105.6
185.3
13.0
24.2
46.2
>50
>199.7
>50
96.7
76°C or at room temperature and 2.4-2.6 kPa for 3-8 h.
Elemental analyses were performed on a CHNS-O CE instru-
ment (FISONS EA 1110), the results are indicated by the
symbols of the elements and were within 0.4 % of theoreti-
cal values. IR spectra were obtained on a Nicolet impact
400 spectrometer in KBr pellets. NMR spectra were recorded

V. Klimešová et al. / IL FARMACO 59 (2004) 279–288
283
in DMSO-d6 solutions at ambient temperature on a Varian
Mercury-Vx BB 300 spectrometer operating at 300 MHz.
Chemical shifts were recorded as d values in ppm and were
indirectly referred to tetramethylsilane (TMS). Signal multi-
plicities are presented by: s (singlet), bs (broad singlet), d
(doublet), t (triplet), q (quartet), and m (multiplet). Coupling
constants (J) are given in Hz. Reactions were monitored and
purity of products checked by TLC (SilufolVU 254 Kavalier,
Votice, Czech Republic, and Merck TLC plates silica gel
60 F254, aluminium back) in acetone/light petroleum or ethyl
acetate/light petroleum. The plates were visualized using UV
light, iodine fumes and/or dipping in a solution of
Ce(SO4)·4H2O, H3Mo12O40P·xH2O, H2SO4, H2O and sub-
sequent heating. Preparative thin-layer chromatography was
carried out on silica gel 60 F254 (0.015-0.040 mm, Merck).
The following compounds had been described in the lit-
erature: 4a, R1 = H [20]; 4h, R1 = 4-Br [21]; 4l, R1 = 4-OCH3
[22]; 4n, R1 = 4-NO2 [21]; 8a, R1 = H [23].
225.7; m.p. 75.5-77 °C (toluene); IR (cm-1): 3113, 3089
(CH), 2916-2675 (H bridges), 1599, 1464, 1230, 1078; H
NMR d 8.45 (bs, 1H, H5), 7.45-7.42 (m, 1H, ar.), 7.35-7.25
(m, 3H, ar.), 4.33 (s, 2H, CH2); 13C NMR d 34.81, 127.29,
127.70, 128.79, 130.39, 133.01, 141.08. Anal. C9H8ClN3S
(C, H, N, S).
1
4.1.1.4. 3-(2-Chlorobenzylsulfanyl)-1,2,4-triazole (4d).
Alkylation of 1 was carried out with 2-chlorobenzyl chloride.
The reaction mixture was stirred for 2 h. Yield 69%; m.w.
225.7; m.p. 94-97 °C (toluene); IR (cm-1): 3112 (CH), 2919-
2704 (H bridges), 1446, 1275, 1173, 1051; 1H NMR d 8.47
(bs, 1H, H5), 7.49-7.42 (m, 2H, ar.), 7.32-7.20 (m, 2H, ar.),
4.42 (s, 2H, CH2); 13C NMR d 33.73, 127.45,129.50,
129.68,131.30, 133.40, 135.48. Anal. C9H8ClN3S (C, H, N,
S).
4.1.1.5. 3-(4-Fluorobenzylsulfanyl)-1,2,4-triazole (4e).
Alkylation of 1 was carried out with 4-fluorobenzyl chloride.
The reaction mixture was stirred for 5 h. Yield 72%; m.w.
209.3; m.p. 92-94 °C (toluene); IR (cm-1): 2912-2673 (H
bridges), 1608, 1510, 1462, 1263, 1230; 1H NMR d 8.44 (bs,
1H, H5), 7.43-7.36 (m, 2H, ar.), 7.10 (tt, 2H, J=8.9, 2.3 Hz,
ar.), 4.33 (s, 2H, CH2); 13C NMR d 34.75, 115.34 (d, J=21.2
Hz), 130.98 (d, J=8.3 Hz), 134.52, 161,52 (d, J=243.06 Hz).
Anal. C9H8FN3S (C, H, N, S).
4.1.1. General procedure for preparation of compounds
4a-aa and 8a-f
1,2,4-Triazole-3-thiol (1) / 4-methyl-1,2,3-triazole-3-thiol
(2) (0.69 g, 6 mmol) in dry N,N-dimethylformamide (DMF)
(3 ml) was added to a solution of sodium (0.14 g, 6 mmol) in
dry methanol (2 ml). After 10 min of stirring at room tem-
perature, benzyl halide (3) (6 mmol) was added. The result-
ant suspension was stirred with CaCl2 cap at room tempera-
ture for 1-23 h. The course of reactions was monitored by
TLC. In some cases, in order to accelerate the reaction
course, the mixture was heated at 50-80°C. The reaction
mixture was then poured into an ice bath (cca 150 ml) and left
overnight. The solid of 4 and 8 (except of 4m, 4u, 8e and 8f)
was filtered off, washed with cold water and air-dried. The
crude product was purified by crystallization. In some cases,
the preparative thin layer chromatography was used.
4.1.1.6. 3-(3-Fluorobenzylsulfanyl)-1,2,4-triazole (4f).
Alkylation of 1 was carried out with 3-fluorobenzyl chloride.
The reaction mixture was stirred for 4 h. Yield 73%; m.w.
209.3; m.p. 74.5-75 °C (toluene); IR (cm-1): 3110, 3089
1
(CH), 2918-2675 (H bridges), 1587, 1446, 1258, 1133; H
NMR d 8.46 (bs, 1H , H5), 7.36-7.27 (m, 1H, H5’), 7.22-7.16
(m, 2H, H2’,6’), 7.09-7.01 (m, 1H, H4’), 4.35 (s, 2H, CH2);
13C NMR d 34.95, 114.18 (d, J=20.9 Hz), 115.71 (d, J=21.5
Hz), 125.12 (d, J=2.6 Hz), 130.47 (d, J=8.6 Hz), 141.33 (d,
J=7.7 Hz), 162.16 (d, J=243.4). Anal. C9H8FN3S (C, H, N,
S).
4.1.1.1. 3-Benzylsulfanyl-1,2,4-triazole (4a). Alkylation of 1
was carried out with benzyl chloride. The reaction mixture
was stirred for 2.5 h. Yield 46%; m.w. 191.3; m.p. 79-81 °C
(toluene). Reference [20] gives 84-85 °C (ethanol). IR (cm-
): 3112, 3084 (CH), 2914-2673 (H bridges), 1462, 1230; 1H
4.1.1.7. 3-(2-Fluorobenzylsulfanyl)-1,2,4-triazole (4g).
Alkylation of 1 was carried out with 2-fluorobenzyl chloride.
The reaction mixture was stirred for 3 h. Yield 57%; m.w.
209.3; m.p. 94-96 °C (toluene); IR (cm-1): 3110, 3088 (CH),
1
NMR d 8.44 (bs, 1H, H5), 7.39-7.33 (m, 2H, ar.), 7.31-7.18
(m, 3H, ar.), 4.34 (s, 2H, CH2); 13C NMR d 35.63, 127.40,
128.60, 129.02, 138.14. Anal. C9H9N3S (C, H, N, S).
1
2917-2672 (H bridges), 1587, 1490, 1233; H NMR d 8.45
(bs, 1H, H5), 7.41 (td, 1H, J=7.7, 1.6 Hz, H6’), 7.34-7.26 (m,
1H, H4’), 7.22-7.07 (m, 2H, H3’,5’), 4.35 (s, 2H, CH2); 13C
NMR d 29.16, 99.90, 115.55 (d, J=21.2 Hz), 124.57 (d, J=3.4
Hz), 125.12 (d, J=14.6 Hz), 129.73 (d, J=8.3 Hz), 131.32 (d,
J=4.0 Hz ), 145.16, 160.53 (d, J=245,92 Hz). Anal.
C9H8FN3S (C, H, N, S).
4.1.1.2. 3-(4-Chlorobenzylsulfanyl)-1,2,4-triazole (4b).
Alkylation of 1 was carried out with 4-chlorobenzyl chloride.
The reaction mixture was stirred for 2 h. Yield 80%; m.w.
225.7; m.p. 131-133 °C (toluene); IR (cm-1): 3093 (CH),
2919-2706 (H bridges), 1489, 1450, 1266, 1026; 1H NMR d
8.43 (bs, 1H, H5), 7.41-7.24 (m, 4H, ar.), 4.33 (s, 2H, CH2);
13C NMR d 34.74, 128.51, 130.85, 131.93, 137.51. Anal.
C9H8ClN3S (C, H, N, S).
4.1.1.8. 3-(4-Bromobenzylsulfanyl)-1,2,4-triazole (4h).
Alkylation of 1 was carried out with 4-bromobenzyl chlo-
ride. The reaction mixture was stirred for 1 h. Yield 92%;
m.w. 270.2; m.p. 134.5-135.5 °C (ethanol/water 1/3). Refer-
ence [21] gives 139 °C. IR (cm-1): 2904-2703 (H bridges),
4.1.1.3. 3-(3-Chlorobenzylsulfanyl)-1,2,4-triazole (4c).
Alkylation of 1 was carried out with 3-chlorobenzyl chloride.
The reaction mixture was stirred for 4 h. Yield 56%; m.w.

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1
1485, 1449, 1266, 1026; H NMR d 8.47 (bs, 1H, H5),
7.49-7.44 (m AA’BB’, 2H, ar.), 7.34-7.29 (m AA’BB’, 2H,
ar.), 4.31 (s, 2H, CH2); 13C NMR d 34.81, 120.45, 131.20,
131.43, 137.93. Anal. C9H8BrN3S (C, H, N, S).
ar.), 6.95-6.89 (m, 2H, ar.), 6,79 (dd, 1H, J=8.2, 2.2 Hz, ar.),
4.30 (s, 2H, CH2), 3.70 (s, 3H, OCH3); 13C NMR d 35.60,
55.16, 112.83, 114.62, 121.20, 129.66, 139.65, 159.36. Anal.
C10H11N3OS (C, H, N, S).
4.1.1.9. 3-(3-Bromobenzylsulfanyl)-1,2,4-triazole (4i).
Alkylation of 1 was carried out with 3-bromobenzyl bro-
mide. The reaction mixture was stirred for 1.5 h. Yield 77%;
m.w. 270.2; m.p. 65-68 °C (toluene); IR (cm-1): 3104 (CH),
2918-2668 (H bridges), 1569, 1475, 1240, 1071; 1H NMR d
8.46 (bs, 1H, H5), 7.58 (t, 1H, J=1.8 Hz, ar.), 7.44-7.33 (m,
2H, ar.), 7.23 (t, 1H, J=7.9 Hz, ar.), 4.33 (s, 2H, CH2); 13C
NMR d 34.76, 121.64, 128.07, 130.17, 130.68, 131.65,
141.36. Anal. C9H8BrN3S (C, H, N, S).
4.1.1.14. 3-(4-Nitrobenzylsulfanyl)-1,2,4-triazole (4n).
Alkylation of 1 was carried out with 4-nitrobenzyl chloride.
The reaction mixture was stirred for 23 h. Yield 83%; m.w.
236.3; m.p. 176-178 °C (ethanol/water 1/1). Reference [21]
gives 180 °C. IR (cm-1): 3127, 3077 (CH), 2918-2703 (H
1
bridges), 1600, 1519 (NO2), 1346 (NO2), 1235, 1109; H
NMR d 8.46 (bs, 1H, H5), 8.16-8.10 (m AA’BB’, 2H, H3’,5’),
7.65-7.60 (m AA’BB’, 2H, H2’,6’), 4.45 (s, 2H, CH2); 13C
NMR d 34.75, 123.67, 130.21, 146.72, 146.82. Anal.
C9H8N4O2S (C, H, N, S).
4.1.1.10. 3-(4-Methylbenzylsulfanyl)-1,2,4-triazole (4j).
Alkylation of 1 was carried out with 4-methylbenzyl chlo-
ride. The reaction mixture was stirred for 5 h. Yield 86%;
m.w. 205.3; m.p. 138-141 °C (ethanol/water 3/2); IR (cm-1):
4.1.1.15. 3-(3-Nitrobenzylsulfanyl)-1,2,4-triazole (4o).
Alkylation of 1 was carried out with 3-nitrobenzyl chloride.
The reaction mixture was stirred for 23 h. Yield 85%; m.w.
236.3; m.p. 119-122 °C (toluene); IR (cm-1): 3122, 3090
(CH), 1530 (NO2), 1358 (NO2), 1277, 1225, 1080; 1H NMR
d 8.54 (s, 1H, H5), 8.25 (t, 1H, J=2.0 Hz, ar.), 8.08 (dd, 1H,
J=8.3, 1.6 Hz, ar.), 7.82 (d, 1H, J=7.7 Hz, ar.), 7.57 (t, 1H,
J=8.0 Hz, ar.), 4,47 (s, 2H, CH2); 13C NMR d 34.48, 122.28,
123.60, 130.01, 135.76, 141.25, 147.81. Anal. C9H8N4O2S
(C, H, N, S).
1
3128, 3092 (CH), 2912-2687 (H bridges), 1476, 1233; H
NMR d 8.55 (bs, 1H, H5), 7.26-7.21 (m AA’BB’, 2H, ar.),
7.11-7.04 (m AA’BB’, 2H, ar.) 4,30 (s, 2H, CH2), 2.24 (s,
3H, CH3); 13C NMR d 20.91, 35.35, 128.94, 129.17, 135.02,
136.58, 144.88. Anal. C10H11N3S (C, H, N, S).
4.1.1.11. 3-(3-Methylbenzylsulfanyl)-1,2,4-triazole (4k).
Alkylation of 1 was carried out with 3-methylbenzyl chlo-
ride. The reaction mixture was stirred for 23 h. Yield 55%;
m.w. 205.3; m.p. 55-58 °C (toluene); IR (cm-1): 3111, 3088
4.1.1.16. 3-(2-Nitrobenzylsulfanyl)-1,2,4-triazole (4p).
Alkylation of 1 was carried out with 2-nitrobenzyl chloride.
The reaction mixture was stirred for 4 h. Yield 69%; m.w.
236.3; m.p. 98-102.5 °C (ethyl acetate/light petroleum); IR
(cm-1): 3126, 3081 (CH), 2912-2677 (H bridges), 1609, 1523
1
(CH), 2919-2669 (H bridges), 1608, 1465, 1273 1226; H
NMR d 8.43 (bs, 1H, H5), 7.25-7.00 (m, 4H, ar.), 4,30 (s, 2H,
CH2), 2.25 (s, 3H, CH3); 13C NMR d 21.15, 35.64, 126.11,
128.07, 128.51, 129.62, 137.72, 137.91. Anal. C10H11N3S
(C, H, N, S).
1
(NO2), 1334 (NO2), 1232, 1079; H NMR d 8.47 (bs, 1H,
H5), 8.03-7.98 (m, 1H, ar.), 7.66-7.61 (m, 2H, ar.), 7.55-7.47
(m, 1H, ar.), 4.62 (s, 2H, CH2); 13C NMR d 32.80, 125.17,
129.08, 132.42, 133.71, 133.87, 148.35. Anal. C9H8N4O2S
(C, H, N, S).
4.1.1.12. 3-(4-Methoxybenzylsulfanyl)-1,2,4-triazole (4l).
Alkylation of 1 was carried out with 4-methoxybenzyl chlo-
ride. The reaction mixture was stirred for 5 h at room tem-
perature and 25 min at 50 °C. Yield 55%; m.w. 221.3; m.p.
98-101.5 °C (ethanol/water 2/7). Reference [22] gives 84-85
°C. IR (cm-1): 3113, 3070 (CH), 2929-2681 (H bridges),
4.1.1.17. 3-(2-Chloro-6-fluorobenzylsulfanyl)-1,2,4-triazole
(4q). Alkylation of 1 was carried out with 2-chloro-6-
fluorobenzyl chloride. The reaction mixture was stirred for
5 h. Yield 89%; m.w. 243.7; m.p. 99.5-103 °C (toluene); IR
(cm-1): 3108 (CH), 2914-2699 (H bridges), 1578, 1452,
1250, 1180, 1026; 1H NMR d 8.60 (s, 1H, H5), 7.43-7.20 (m,
2H, H3’,5’), 7.25-7.17 (m, 1H, H4’), 4.43 (s, 2H, CH2); 13C
NMR d 27.65, 114.82 (d, J=22.3 Hz), 123.24 (d, J=18.6 Hz),
125.84 (d, J=3.2 Hz), 130.26 (d, J=9.7 Hz), 134.70 (d,
J=5.2 Hz), 145.16, 160.90 (d, J=249.7 Hz). Anal.
C9H7ClFN3S (C, H, N, S).
1
1613, 1513, 1470, 1242, 1174, 1037; H NMR d 8.42 (bs,
1H, H5), 7.30-7.25 (m AA’BB’, 2H, ar.), 6.86-6.81 (m
AA’BB’, 2H, ar.), 4.29 (s, 2H, CH2), 3.70 (s, 3H, OCH3); 13
C
NMR d 35.21, 55.24, 113.99, 129.83, 130.24, 158.64. Anal.
C10H11N3OS (C, H, N, S).
4.1.1.13. 3-(3-Methoxybenzylsulfanyl)-1,2,4-triazole (4m).
Alkylation of 1 was carried out with 3-methoxybenzyl chlo-
ride. The reaction mixture was stirred for 3 h. No solid arose
after pouring into water. Thus, the mixture was evaporated in
vacuo and a crude product was purified by preparative TLC
chromatography using dichloromethane/ethyl acetate 7/3 as
an eluent. Yield 42%; m.w. 221.3; m.p. 42-46 °C; IR (cm-1):
3112 (CH), 2922-2680 (H bridges), 1600, 1490, 1268, 1153,
4.1.1.18. 3-(3,4-Dichlorobenzylsulfanyl)-1,2,4-triazole (4r).
Alkylation of 1 was carried out with 3,4-dichlorobenzyl
chloride. The reaction mixture was stirred for 4 h.Yield 97%;
m.w. 260.2; m.p. 84-86.5 °C (methanol/water); IR (cm-1):
3110, 3089 (CH), 2919-2672 (H bridges), 1471, 1230, 1134,
1033; 1H NMR d 8.57 (bs, 1H, H5), 7.63 (d, 1H, J=1.92 Hz,
1
1046; H NMR d 8.44 (bs, 1H, H5), 7.19 (t, 1H, J=8.0 Hz,

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285
H2’), 7.54 (d, 1H, J=8.51 Hz, H5’), 7.40-7.30 (m, 1H, H6’),
4.32 (s, 2H, CH2); 13C NMR d 34.02, 129.36, 129.84,
130.67, 130.94, 140.15, 145.08, 158.22. Anal. C9H7Cl2N3S
(C, H, N, S).
4.5 h. Yield 90%; m.w. 259.3; m.p. 111-113 °C
(ethanol/water 1/4); IR (cm-1): 3134, 3104 (CH), 2915-2696
(H bridges), 1618, 1478, 1327, 1274, 1162, 1124; 1H NMR d
8.57 (s, 1H, H5), 7,68-7,53 (m, 4H, ar.), 4,41 (s, 2H, CH2);
13C NMR d 34.71, 124.47 (q, J=271.7 Hz), 125.38 (q, J=3.9
Hz), 127.84 (q, J=31.2 Hz), 129.76, 143.69, 145.07, 158.33.
Anal. C10H8F3N3S (C, H, N, S).
4.1.1.19. 3-(3,4-Difluorobenzylsulfanyl)-1,2,4-triazole (4s).
Alkylation of 1 was carried out with 3,4-difluorobenzyl bro-
mide. The reaction mixture was stirred for 2.5 h. Yield 64%;
m.w. 227.2; m.p. 94-96.5 °C (chloroform/light petroleum
1/2); IR (cm-1): 3109, 3092 (CH), 2915-2672 (H bridges),
4.1.1.24. 3-(3-Trifluoromethylbenzylsulfanyl)-1,2,4-triazole
(4x). Alkylation of 1 was carried out with 3-trifluoro-
methylbenzyl chloride. The reaction mixture was stirred for
6.5 h. Yield 59%; m.w. 256.3; m.p. 59.5-62 °C (methanol/
water); IR (cm-1): 3111, 3095 (CH), 2919-2679 (H bridges),
1
1609, 1517, 1435, 1291, 1230, 1110; H NMR d 8.45 (bs,
1H, H5), 7.43 (ddd, 1H, J=11.8, 8.0, 2.2, H4’), 7.39-7.28 (m,
1H, ar.), 7.24-7.17 (m, 1H, ar.), 4.32 (s, 2H, CH2); 13C NMR
d 34.40, 117.74 (dd, J=33.7, 17.3 Hz), 125.87 (dd, J=6.3,
3.4 Hz), 136.42 (d, J=4.3 Hz), 148.76 (dd, J= 245.1,
12.6 Hz), 149.21 (dd, J=245.3, 12.6 Hz). Anal. C9H7F2N3S
(C, H, N, S).
1
1451, 1333, 1230, 1175, 1123, 1072; H NMR d 8.46 (bs,
1H, H5), 7.72 (s, 1H, H2’), 7.67 (d, 1H, J=7.4 Hz, ar.),
7.61-7.46 (m, 2H, ar.), 4.42 (s, 2H, CH2); 13C NMR d 34.86,
124.03 (q, J=3.7 Hz), 124.36 (q, J=272.3 Hz), 125.53 (q,
J=4.0 Hz), 129.21 (q, J=31.5 Hz), 129.60, 131.12, 131.14,
140.20, 146.33. Anal. C10H8F3N3S (C, H, N, S).
4.1.1.20. 3-(3,5-Dinitrobenzylsulfanyl)-1,2,4-triazole (4t).
Alkylation of 1 was carried out with 3,5-dinitrobenzyl chlo-
ride. The reaction mixture was stirred for 20 h. Yield 85%;
m.w. 281.3; m.p. 140-141 °C (ethanol/water 2/1); IR (cm-1):
3133, 3100 (CH), 2923-2749 (H bridges), 1628, 1541 (NO2),
1347 (NO2), 1277, 1079,; 1H NMR d 8.70-8.67 (m, 3H, ar.),
8.55 (bs, 1H, H5), 4.58 (s, 2H, CH2); 13C NMR d 33.74,
99.91, 117.51, 129.49, 144.05, 145.27, 147.99, 157.85. Anal.
C9H7N5O4S (C, H, N, S).
4.1.1.25. 3-(3,5-Ditrifluoromethylbenzylsulfanyl)-1,2,4-tria-
zole (4y). Alkylation of 1 was carried out with 3,5-
ditrifluoromethylbenzyl chloride. The reaction mixture was
stirred for 5.5 h. Yield 84%; m.w. 327.3; m.p. 118-119 °C
(ethyl acetate); IR (cm-1): 3140, 3104 (CH), 2920-2695 (H
1
bridges), 1624, 1484, 1375, 1277, 1168, 1129; H NMR d
8.49 (bs, 1H, H5), 8.07 (s, 2H, H2’,6’), 7.95 (s, 1H, H4’), 4.50
(s, 2H, CH2); 13C NMR d 34.29, 121.07 (m, 1C), 123.50 (q,
J=272.5 Hz), 129.88, 129.9 (m, 2C), 130.2 (q, J=32.6 Hz),
142.65, 146.25. Anal. C11H7F6N3S (C, H, N, S).
4.1.1.21. 3-(2,4-Dinitrobenzylsulfanyl)-1,2,4-triazole (4u).
Alkylation of 1 was carried out with 2,4-dinitrobenzyl chlo-
ride. The reaction mixture was stirred for 4 h. No solid arose
after pouring into water. Thus, the mixture was evaporated in
vacuo and a crude product was purified by preparative TLC
chromatography using light petroleum/ethyl acetate 3/7 as an
eluent.Yield 41%; m.w. 281.3; viscous substance; IR (cm-1):
3109 (CH), 2922-2748 (H bridges), 1608, 1534 (NO2), 1348
(NO2), 1276, 1066; 1H NMR d 8.70 (d, 1H, J=2.2, H3’), 8.47
(dd overlapped, 1H, J=8.5, 2,2 Hz, H5’), 8.46 (s overlapped,
1H, H5), 7.93 (d, 1H, J=8.5, H6’), 4.71 (s, 2H, CH2); 13C
NMR d 32.25, 120.49, 127.77, 133.96, 140.86, 146.31,
146.80, 148.35, 156.34. Anal. C9H7N5O4S (C, H, N, S).
4.1.1.26. 4-[3-(1,2,4-Triazolyl)sulfanylmethyl]benzonitrile
(4z). Alkylation of 1 was carried out with 4-bromomethyl-
benzonitrile. The reaction mixture was stirred for 4 h. Yield
74%; m.w. 216.3; m.p. 141-144 °C (toluene); IR (cm-1):
3147, 3088 (CH), 2922 (H bridges), 2231 (CN), 1606, 1479,
1365, 1273,1211, 1077; 1H NMR d 8.46 (bs, 1H, H5), 7.80-
7.70 (m AA’BB’, 2H, ar.), 7.60-7.52 (m AA’BB’, 2H, ar.),
4.40 (s, 2H, CH2); 13C NMR d 35.03, 110.03, 118.99,
129.97, 132.46, 144.59. Anal. C10H8N4S (C, H, N, S).
4.1.1.27. 3-[3-(1,2,4-Triazolyl)sulfanylmethyl]benzonitrile
(4aa). Alkylation of 1 was carried out with 3-bromomethyl-
benzonitrile. The reaction mixture was stirred for 2 h. Yield
56%; m.w. 216.3; m.p. 72.5-75.5 °C (ethyl acetate/light pe-
troleum); IR (cm-1): 3111, 3081 (CH), 2914-2672 (H
4.1.1.22. 3-(2-Fluoro-6-nitrobenzylsulfanyl)-1,2,4-triazole
(4v). Alkylation of 1 was carried out with 2-fluoro-6-
nitrobenzyl bromide. The reaction mixture was stirred for
3 h. Yield 50%; m.w. 254.2; m.p. 103-106 °C (ethyl
acetate/light petroleum 2/5); IR (cm-1): 3136, 3093 (CH),
2903-2679 (H bridges), 1536, 1523 (NO2), 1462, 1361
1
bridges), 2229 (CN), 1636, 1464, 1239, 1172; H NMR d
1
8.46 (bs, 1H, H5), 7.81 (t, 1H, J=1.6 Hz, ar.), 7.73-7.67 (m,
2H, ar.), 7.49 (t, 1H, J=7.8 Hz, ar.), 4.37 (s, 2H, CH3); 13C
NMR d 34.57, 111.38, 118.89, 129.82, 131.13, 132.53,
134.00, 140.44. Anal. C10H8N4S (C, H, N, S).
(NO2), 1260, 1190, 1087; H NMR d 8.43 (bs, 1H, H5),
7.83-7.77 (m, 1H, H5’), 7.64-7.49 (m, 2H, H3’,4’), 4.56 (s,2H,
CH2); 13C NMR d 25.78, 121.01 (d, J=19.5 Hz), 121.18,
121.86 (d, J=17.8 Hz), 130.91 (d, J=9.4 Hz), 149.51, 160.51
(d, J=249.64 Hz). Anal. C9H7FN4O2S (C, H, N, S).
4.1.1.28. 3-Benzylsulfanyl-4-methyl-1,2,4-triazole (8a).
Alkylation of 2 was carried out with benzyl chloride. The
reaction mixture was stirred for 2 h. Yield 70%; m.w. 205.3;
m.p. 79-82 °C (toluene). Reference [23] gives 84-85 °C from
4.1.1.23. 3-(4-Trifluoromethylbenzylsulfanyl)-1,2,4-triazole
(4w). Alkylation of 1 was carried out with 4-trifluoro-
methylbenzyl bromide. The reaction mixture was stirred for

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V. Klimešová et al. / IL FARMACO 59 (2004) 279–288
benzene, hexane. IR (cm-1): 3104 (CH), 1520, 1453, 1413,
1210, 1071; 1H NMR d 8.52 (s, 1H, H5), 7.30-7.23 (m ,5H,
ar.), 4.29 (s, 2H, CH2), 3.36 (s, 3H, CH3); 13C NMR d 30.79,
37.59, 127.67, 128.67, 129.06, 137.54, 146.40, 148.68. Anal.
C10H11N3S (C, H, N, S).
into water. Then, the mixture was concentrated to dryness in
vacuo. The residue was suspended in water (15 ml) and
extracted three times into 15 ml of ethyl acetate. The organic
layer was dried over Na2SO4, concentrated, and the crude
product was further purified by crystallization using ethyl
acetate. Yield 67%; m.w. 230.3; m.p. 116-117 °C (ethyl
acetate); IR (cm-1): 3126, 2925, 2232 (CN), 1606, 1518,
1419, 1198, 1157; 1H NMR d 8.52 (s, 1H, H5), 7.78-7.72(m
AA’BB’, 2H, ar.), 7.51-7.46 (m, AA’BB’, 2H, ar.), 4.38 (s,
2H, CH2), 3.42 (s, 3H, CH3); 13C NMR d 30.88, 36.63,
110.28, 118.93, 130.07, 132.53, 143.79, 146.58, 148.23.
Anal. C11H10N4S (C, H, N, S).
4.1.1.29. 3-(4-Chlorobenzylsulfanyl)-4-methyl-1,2,4-tria-
zole (8b). Alkylation of
2 was carried out with
4-chlorobenzyl chloride. The reaction mixture was stirred at
room temperature for 2 h and than at 70 °C for 1.5 h. Yield
68%; m.w. 239.7; m.p. 75-76.5 °C (toluene); IR (cm-1): 3128,
2926 (CH), 1644, 1493, 1413, 1359, 1199; 1H NMR d 8.53
(s, 1H, H5), 7.36-7.27 (m, 4H, ar.), 4.30 (s, 2H, CH2), 3.41 (s,
3H, CH3); 13C NMR d 30.86, 36.52, 128.59, 130.94, 132.24,
136.84, 146.48, 148.46. Anal. C10H10ClN3S (C, H, N, S).
4.1.2. General procedure for preparation of compounds
5a, 5b and 9
Dry hydrogen sulphide was passed through a solution of a
cyano compound (4z, 4aa, 8f) (7 mmol) dissolved in a
mixture of dry pyridine (10 ml) and triethylamine (1 ml). The
reaction mixture was maintained at room temperature or
heated for 5-8 h. The mixture was then poured into crushed
ice, the precipitated product was filtered off, washed with
cold water, and air-dried. The crude product was purified by
crystallization or by preparative thin layer chromatography.
4.1.1.30. 3-(3-Bromobenzylsulfanyl)-4-methyl-1,2,4-tria-
zole (8c). Alkylation of
2 was carried out with
3-bromobenzyl bromide. The reaction mixture was stirred at
room temperature for 2 h and then at 80 °C for 1.5 h. Yield
48%; m.w. 284.2; m.p. 75-77 °C (ethyl acetate/light petro-
1
leum); IR (cm-1): 3126 (CH),1567, 1509, 1420, 1196; H
NMR d 8.54 (s, 1H, H5), 7.52-7.50 (m, 1H, ar.), 7,46-7.42 (m,
1H, ar.), 7.32-7.21 (m, 2H, ar.), 4.49 (s, 2H, CH2), 3.42 (s,
3H, CH3); 13C NMR d 30.85, 36.45, 121.66, 128.16, 130.43,
130.76, 131.77, 140.61, 146.51, 148.45. Anal. C10H10BrN3S
(C, H, N, S).
4.1.2.1.
4-[3-(1,2,4-Triazolyl)sulfanylmethyl]benzothio-
amide (5a). The addition of hydrogen sulphide was carried
out at room temperature for 8 h. The crude product was
purified by crystallization from ethanol. Yield 49%; m.w.
250.4; m.p. 179-182 °C (ethanol); IR (cm-1): 3286, 3190,
2918-2672, 1628, 1421, 1272, 1016; 1H NMR d 9.83 (bs, 1H,
CSNH2), 9.45 (bs, 1H, CSNH2), 8.56 (bs, 1H, H5), 7.81-7.76
(m AA’BB’, 2H, ar.), 7.38 (d, 2H, J=8.0 Hz, ar.), 4.35 (s, 2H,
CH2); 13C NMR d 35.11, 127.59, 128.48, 138.50, 141.71,
199.86. Anal. C10H10N4S2 (C, H, N, S).
4.1.1.31. 3-(3,5-Dinitrobenzylsulfanyl)-4-methyl-1,2,4-tria-
zole (8d). Alkylation of 2 was carried out with 3,5-
dinitrobenzyl chloride. The reaction mixture was stirred for
2.5 h. Yield 42%; m.w. 295.3; m.p. 92-94 °C (ethyl acetate);
IR (cm-1): 3102 (CH), 1541 (NO2), 1469, 1346 (NO2), 1204,
1075; 1H NMR d 8.71 (s, 1H, H4’), 8.66 (s, 2H, H2’,6’), 8,54
(s, 1H, H5), 4.60 (s, 2H, CH2), 3.40 (s, 3H, CH3); 13C NMR d
30.98, 35.09, 117.74, 129.67, 143.03, 146.75, 148.01,
148.10. Anal. C10H9N5O4S (C, H, N, S).
4.1.2.2.
3-[3-(1,2,4-Triazolyl)sulfanylmethyl]benzothio-
amide (5b). Addition of hydrogen sulphide was carried out at
room temperature for 5 h. No solid arose after pouring the
reaction mixture into water. The mixture was extracted with
ethyl acetate. The organic layer was dried over Na2SO4 and
the solvent removed. The product was air-dried and purified
by TLC preparative chromatography using light
petroleum/acetone 2/1 as an eluent. Yield 61%; m.w. 250.4;
m.p.90-93 °C (light petroleum/acetone 2/1); IR (cm-1): 3297,
3181-2857, 1629, 1444, 1384, 1319,1277, 1236; 1H NMR d
9.87 (bs, 1H, CSNH2), 9.49 (bs, 1H, CSNH2), 8.42 (s, 1H,
H5), 7.92-7.89 (m, 1H, H2’), 7.71-7.65 (m, 1H, ar.), 7.51-7.45
(m, 1H, ar.), 7.31 (t, 1H, J=7.8 Hz, H5’), 4.37 (s, 2H, CH2);
13C NMR d 35.39, 125.81, 128.19, 128.49, 131.68, 138.13,
139.95, 146.62, 200.19. Anal. C10H10N4S2 (C, H, N, S).
4.1.1.32. 3-(2,4-Dinitrobenzylsulfanyl)-4-methyl-1,2,4-tria-
zole (8e). Alkylation of 2 was carried out with 2,4-
dinitrobenzyl chloride. The reaction mixture was stirred for
3 h. No solid arose after pouring into water. Thus, the mixture
was evaporated in vacuo and a crude product was purified by
preparative TLC chromatography using light petroleum/
acetone 2/1 as an eluent. Yield 67%; m.w. 295.3; m.p. 129-
132 °C; IR (cm-1): 3115 (CH), 1607, 1537 (NO2), 1434, 1348
(NO2), 1202, 1064; 1H NMR d 8.74 (d, 1H, J=2.2, H3’), 8.55
(s, 1H, H5), 8.48 (dd, 1H, J=8.5, 2.5, H5’), 7.85 (d, 1H, J=8.5,
H6’), 4.70 (s, 2H, CH2), 3.47 (s, 3H, CH3); 13C NMR d 30.98,
33.83, 120.68, 128.00, 134.18, 140.02, 146.83, 147.03,
147.89, 147.99. Anal. C10H9N5O4S (C, H, N, S).
4.1.2.3. 4-[3-(4-Methyl-1,2,4-triazolyl)sulfanylmethyl]ben-
zothioamide (9). Addition of hydrogen sulphide was carried
out at room temperature for 4.5 h and then at 60 °C for 1.5 h.
The crude product was purified by crystallization from etha-
nol. Yield 60%; m.w. 264.4; m.p.179-182 °C (ethanol); IR
4.1.1.33. 4-[3-(4-Methyl-1,2,4-triazolyl)sulfanylmethyl]ben-
zonitrile (8f). Alkylation of 2 was carried out with
4-bromomethylbenzonitril. The reaction mixture was stirred
for 3.5 h. No solid arose after pouring the reaction mixture

V. Klimešová et al. / IL FARMACO 59 (2004) 279–288
287
(cm-1): 3247, 3100, 1630, 1519, 1423, 1201; 1H NMR d 9.85
(bs, 1H, CSNH2), 9.49 (bs, 1H, CSNH2), 8.52 (s, 1H, H5),
7.82-7.77 (m AA’BB’, 2H, ar.), 7.35-7.29 (m AA’BB’, 2H,
ar.), 4.34 (s, 2H, CH2), 3.41 (s, 3H, CH3); 13C NMR d 30.88,
36.82, 127.64, 128.59, 138.61, 141.00, 146.50, 148.50,
199.71. Anal. C11H12N4S2 (C, H, N, S).
1000, 500, 250, 125, 62.5, 32, 16, 8, 4, and 2 µmol/l. MICs
values were determined after incubation at 37°C for 7, 14,
and 21 days. MIC was the lowest concentration of a sub-
stance at which the inhibition of growth of mycobacteria
occurred.
4.1.6. Toxicology
4.1.3. Preparation of 3-[3-(5-tetrazolyl)benzylsulfanyl]-
1,2,4-triazole (6)
The target compounds were assayed against cell lines
K-562 and L-929 for their antiproliferative effects, and
against HeLa for their cytotoxic effects. The cells were incu-
bated with ten concentrations of the test compounds.
For each antiproliferative assay with L-929 and K-562,
approximately 10,000 cells were seeded with a 0.1 ml RPMI
1640 culture medium (GIBCO BRL 21875-034), containing
10 % heat inactivated fetal bovine serum (GIBCO BRL
10500-064), 100 U/ml penicillin G-sodium salt/100 µg/ml
streptomycin-sulfate (GIBCO BRL 15140-114), but without
HEPES, into 96-well microplates. The plates were previ-
ously prepared with ten dilutions of test substances in 0.1 ml
RPMI 1640 medium.
For a cytotoxic assay with HeLa, approximately
10,000 cells were seeded with 0.1 ml RPMI 1640 culture
medium per well of the 96-well microplates. HeLa cells were
preincubated for 48 hours without the test substances. The
dilutions of the compounds were carried out carefully on the
monolayers of HeLa cells after the preincubation time.
A mixture of 4aa (0.36 g, 2 mmol), sodium azide (0.32 g,
5 mmol), lithium chloride (0.07 g, 2 mmol), and ammonium
chloride (0.09 g, 2 mmol) was dissolved in dry DMF (3 ml)
and heated at 115-120 °C for 15.5 h. After cooling, water
(8 ml) was added and pH was brought to 7 with diluted HCl.
The precipitated solid was filtered off and air-dried. The
crude product was purified by preparative TLC using
methanol/chloroform 1/9 as the mobile phase. Yield 87%;
m.w. 259.3; m.p.186-190 °C (chloroform/methanol 9/1); IR
(cm-1): 3130, 3081, 2919-2521, 1616, 1557, 1479, 1276,
1176, 1090; 1H NMR d 8.56 (bs, 1H, H5), 8.09-8.06 (m, 1H,
H2’), 7,88 (dt, 1H, J=7.4, 1.5 Hz, H4’), 7.61-7.48 (m, 2H,
H
5’,6’), 4.44 (s, 2H, CH2); 13C NMR d 35.12, 124.49, 125.94,
127.52, 129.72, 131.88, 140.02, 145.10, 155.52. Anal.
C10H9N7S (C, H, N, S).
4.1.4. Preparation of 3-(4-methylbenzylsulfonyl)-1,2,4-
triazole (7)
To a stirred solution of 4j (0.40 g, 2 mmol) in conc. acetic
acid (4.0 ml), 2 ml 7% aq. solution of potassium permanga-
nate was gradually added. The mixture was stirred at 40-60
°C for 1.5 h, then at room temperature overnight. The mix-
ture was discoloured with saturated solution of sodium
sulfite, filtered and concentrated in vacuo. The residue was
suspended in ethanol (100 ml), filtered and ethanol was
removed in vacuo. The oil product was solidified by addition
of 10 ml of water. The obtained precipitate was filtered off
and purified by crystallization from H2O. Yield 32%; m.w.
237.3; m.p.187-189.5 °C (water); IR (cm-1): 3282, 3128
(CH), 2918, 1512, 1326 (SO2), 1252, 1140 (SO2); 1H NMR d
8.89 (s, 1H, H5), 7.14-7.06 (m, 4H, ar.), 4.72 (s, 2H, CH2),
2.26 (s, 3H, CH3); 13C NMR d 20.97, 59.52, 124.63, 129.18,
131.25, 138.19, 146.27. Anal. C10H11N3O2S (C, H, N, S).
Acknowledgements
This work was supported by the Grant Agency of the
Czech Republic (grant No. 203/02/0082) and by the Ministry
of Education of the Czech Republic (project No. MSM
111600001). We would like to thank Mrs D. Karlícˇková
(Department of Pharmaceutical Chemistry and Drug Con-
trol, Faculty of Pharmacy, Charles University) for perform-
ing elemental analyses, Mrs J. Žižková for recording the IR
spectra, Mr P. Jancˇárek for recording NMR spectra, Assoc.
Prof. J. Kuneš for recording NMR spectra and his help with
their interpretation (Department of Inorganic and Organic
Chemistry, Faculty of Pharmacy, Charles University), and
Mrs. B. Janícˇková and Mrs. M. Malíková for antimycobacte-
rial evaluation (National Reference Laboratory for Mycobac-
terium kansasii, Regional Institute of Hygiene).
4.1.5. Microbiology
In vitro antimycobacterial activity of the compounds was
evaluated against Mycobacterium tuberculosis CNCTC My
331/88, M. avium CNCTC My 330/88, M. kansasii CNCTC
My 235/80, and M. kansasii 6509/96, using the micromethod
for the determination of the minimum inhibitory concentra-
tion (MIC). All strains were obtained from the Czech Na-
tional Collection of Type Cultures (CNCTC) with the excep-
tion of M. kansasii 6509/96, which was a clinical isolate. The
antimycobacterial activities of the compounds were deter-
mined in a Šula semisynthetic medium (SEVAC, Prague).
The compounds were added to the medium in dimethylsul-
foxide solutions. The following concentrations were used:
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