Synthesis and antimicrobial and pharmacological properties of new thiosemicarbazide and 1,2,4-triazole derivatives

Article abstract of DOI:10.1002/jhet.563

Reaction of 4-phenyl-4H-1,2,4-triazole-3-thione with ethyl bromoacetate has led to the formation of ethyl [(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetate 1, the structure of which was confirmed by X-ray analysis. In the next reaction with 80% hydrazide hydrate, appropriate hydrazide 2 was obtained, which in reaction with isothiocyanates was converted to new acyl derivatives of thiosemicarbazides 3a-3h. The cyclization of these compounds in alkaline media has led to formation of new derivatives of 5-{[(4-phenyl-4H-1,2,4-triazole-3-yl) sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thiones 4a-4g, 4j. The structure of the compounds was confirmed by elementary analysis and IR, ¹H-NMR, ¹³C-NMR, and MS spectra. Compounds 3a-3h and 4a-4g were screened for their antimicrobial activities, and the influence of the compounds 4a, 4b, and 4e-4g on the central nervous system of mice in behavioral tests was examined.

Full text of DOI:10.1002/jhet.563

March 2011
Synthesis and Antimicrobial and Pharmacological Properties of
New Thiosemicarbazide and 1,2,4-Triazole Derivatives
339
Łukasz Popiołek,a* Maria Dobosz,a Anna Chodkowska,b
b
Ewa Jagiełło-Wojtowicz, Urszula Kosikowska, Anna Malm, Liliana Mazur,
c
c
d
´
d
´
and Zofia Rza˛czynska
aDepartment of Organic Chemistry, Medical University, 6 Staszica Str., 20-081 Lublin, Poland
b
´
Department of Toxicology, Medical University, 8 Chodzki Str., 20-093 Lublin, Poland
c
´
Department of Pharmaceutical Microbiology, Medical University, 1 Chodzki Str,
20-093 Lublin, Poland
dDepartment of General and Coordination Chemistry, Faculty of Chemistry,
Maria Curie-Sklodowska University, M. Curie-Sklodowska Sq. 3, 20-031 Lublin, Poland
*E-mail: lukasz.popiolek@umlub.pl
Received October 28, 2009
DOI 10.1002/jhet.563
Published online 16 December 2010 in Wiley Online Library (wileyonlinelibrary.com).
Reaction of 4-phenyl-4H-1,2,4-triazole-3-thione with ethyl bromoacetate has led to the formation of ethyl
[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetate 1, the structure of which was confirmed by X-ray analysis.
In the next reaction with 80% hydrazide hydrate, appropriate hydrazide 2 was obtained, which in reaction
with isothiocyanates was converted to new acyl derivatives of thiosemicarbazides 3a–3h. The cyclization
of these compounds in alkaline media has led to formation of new derivatives of 5-{[(4-phenyl-4H-1,2,4-
triazole-3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thiones 4a–4g, 4j. The structure of the compounds
1
was confirmed by elementary analysis and IR, H-NMR, 13C-NMR, and MS spectra. Compounds 3a–3h
and 4a–4g were screened for their antimicrobial activities, and the influence of the compounds 4a, 4b, and
4e–4g on the central nervous system of mice in behavioral tests was examined.
J. Heterocyclic Chem., 48, 339 (2011).
obtained, whereas in acidic media 1,3,4-thiadiazole
derivatives were obtained [5].
INTRODUCTION
Cyclization of thiosemicarbazide derivatives leads to
1,2,4-triazole derivatives in alkaline media [1,2], where as
in acidic media [3,4] to 1,3,4-thiadiazole derivatives.
In previous papers [2–5], it was stated that the reaction
of cyclization was affected not only by pH of the medium
but also by the nature of substituents in thiosemicarbazide
derivatives. The course of cyclization in alkaline and
acidic media for thiosemicarbazide derivatives of formic,
benzoic, and nicotinic acids was investigated [5].
Cyclization of thiosemicarbazide derivatives of formic
and nicotinic acids in alkaline and in acidic media leads
to 1,2,4-triazole derivatives. In the cyclization reaction
of benzoic acid thiosemicarbazide derivatives in the
presence of alkaline media 1,2,4-triazole system was
4-Phenyl-4H-1,2,4-triazole-3-thione, which can exist
in two tautomeric forms (Scheme 1), was a starting ma-
terial for synthesis.
In various reactions, depending on the conditions used,
this compound can lead either to the S- or N-derivatives.
In this article, the reaction of 4-phenyl-4H-1,2,4-triazole-
3-thione with ethyl bromoacetate was investigated. Based
on the results of the previous papers [5,6], the elemental
and spectral analysis as well as the X-ray crystallography,
it was revealed and confirmed that reaction leads to the
formation of S-derivative 1 (Fig. 1).
This compound was converted to hydrazide 2, then
reaction
in
with
isothiocyanates
appropriate
C
V
2010 HeteroCorporation

´
340
Ł. Popiołek, M. Dobosz, A. Chodkowska, E. Jagiełło-Wojtowicz, U. Kosikowska, A. Malm,
´
L. Mazur, and Z. Rza˛czynska
Vol 48
Scheme 1. Thiol/thione tautomerism forms of 4-phenyl-4H-1,2,4-tria-
zole-3-thiol.
Figure 1. Molecular structure with atom numbering scheme for com-
pound 1. Displacement ellipsoids are drawn at the 50% probability level.
thiosemicarbazide derivatives were obtained 3a–3h. Cy-
clization of these compounds in alkaline media leads to
new derivatives of 1,2,4-triazole 4a–4g.
The reactions were performed according to Scheme 2,
and the substituents are presented in Table 1.
show analgesic [8], antifungal [9], antibacterial [10–12],
antiphlogistic [13], and antituberculous [14,15] action.
Pharmacological experiments (on CNS on mice) were
carried out for compounds 4a, 4b, and 4e–4g.
All tested compounds were screened for in vitro anti-
bacterial activity by the agar well diffusion method, and
for agents 3d, 3f, 3g, and 3h showing potential inhibi-
tory effect on the growth of bacteria minimal inhibitory
Depending on the nature of substituents, the 1,2,4-tri-
azole derivatives show various pharmacological activ-
ities. Some of them, obtained in previous papers and
investigated on experimental animals, had potential
action on central nervous system (CNS) [7]. Others
Scheme 2. Synthesis of new thiosemicarbazide and 1,2,4-triazole derivatives.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

March 2011
Synthesis, Antimicrobial and Pharmacological Properties of New
Thiosemicarbazide and 1,2,4-Triazole Derivatives
341
Table 1
led to formation of 4-carboxymethyl-5-{[(4-phenyl-4H-
1,2,4-triazol-3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-
thione 4j.
Substituents of compounds 3a–4j.
Compounds
R
Compounds
R
The hydrolysis was also observed in the case of cycliza-
tion of 4-ethoxycarbonyl-1-{[(4-phenyl-4H-1,2,4-triazol-
3-yl)sulfanyl]acetyl}thiosemicarbazide 3i in alkaline media,
which led to formation of [(4-phenyl-4H-1,2,4-triazol-
3-yl)sulfanyl] acetic acid 5. This compound was described
earlier [18,19], but it was obtained in a different way.
The test of cyclization of 1-{[(4-phenyl-4H-1,2,4-
triazol-3-yl)sulfanyl]acetyl}-1-phenyl thiosemicarbazide
3a in acidic media (acetic acid) gave the derivative with
1,2,4-triazole system 4a.
3a, 4a
3f, 4f
3b, 4b
3c, 4c
3d, 4d
3e, 4e
3g, 4g
C2H5
3h
CH2COOC2H5
The structure of the obtained compounds was con-
1
firmed by elementary analysis, IR, and H-NMR spectra.
Some of them were also submitted to 13C-NMR and MS
spectral analysis.
3i
COOC2H5
In the IR spectra of 1,2,4-triazole system 4a–4g, 4j, the
following characteristic absorption bands were observed:
1513–1530 cmꢁ1 corresponding to CAN group and in the
range of 1608–1624 cmꢁ1 corresponding to C¼¼N group.
1H-NMR spectra of the thiosemicarbazide derivatives 3a–
3i show three proton signals typical for the NH group in the
d 8.30–10.45 ppm range. Whereas for the new compounds
of 1,2,4-triazole system 4a–4g, 4j one proton signal of the
NH group was observed in the d 13.63–13.99 ppm range.
4j
CH2COOH
concentration (MIC) values were estimated by microdi-
lution technique [16,17].
Table 2
Antinociceptive activity compounds 4a, 4b, 4e and 4f in the writhing
syndrome test in mice.
RESULTS AND DISCUSSION
4-Phenyl-4H-1,2,4-triazole-3-thione was the starting
material for synthesis of new derivatives, which consist
of two 1,2,4-triazole systems connected with S-methyl-
ene group.
Part of
LD50
Mean writhing
number
Inhibition
(%)
Compound
Control 4a
–
47.9 6 4.7
41.4 6 6.2
15.5 6 4.5**
12.0 6 5.0**
57.2 6 5.1
–
14
0.025
0.05
0.1
This compound was obtained (using the method
described earlier) by cyclization of 1-formyl-4-phenyl
thiosemicarbazide in alkaline media. Reaction with ethyl
bromoacetate in the presence of sodium ethanolate gave
ethyl [(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl] acetate 1
(Fig. 1), which was converted to hydrazide 2 on reaction
with 80% hydrazine hydrate.
68**
75**
–
Control 4b
–
0.00625
0.0125
0.025
0.05
0.1
58.2 6 6.2
–
26.4 6 8.4**
28.1 6 5.6**
10.0 6 4.4**
10.9 6 4.3**
64.8 6 5.9
54**
51**
82.5**
81**
–
Control 4e
Control 4f
–
Reactions of hydrazide 2 with aliphatic, aromatic,
ethoxycarbonyl, and ethoxycarbonylmethyl isothiocyanates
were carried out by heating substrates in an oil bath; tem-
peratures were selected experimentally (t ¼ 40–95ꢀC).
New thiosemicarbazide derivatives 3a–3g in cycliza-
tion reaction with 2% aqueous solution of sodium hy-
droxide leads to a new group of 5-{[4-phenyl-4H-1,2,4-
triazol-3-yl)sulfanyl]methyl}-4H-1,2,4-triazol-3(2H)-thione
4a–4g derivatives.
0.0125
0.025
0.05
0.1
62.0 6 6.5
4
47.8 6 8.8*
29.1 6 5.5**
5.6 6 1.8**
46.8 6 2.6
26*
55**
91**
–
–
0.0125
0.025
0.05
0.1
40.7 6 2.3
13**
24*
27*
44**
35.5 6 4.3*
34.3 6 5.9*
25.9 6 3.2**
Compounds were given 30 min before the test.
The cyclization of thiosemicarbazide in alkaline
media, which contain ethoxycarbonylmethyl group 3h,
was accompained by hydrolysis of ester group and
% of inhibition obtained by comparison with control group.
* P < 0.05 vs. the control group.
** P < 0.001.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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Ł. Popiołek, M. Dobosz, A. Chodkowska, E. Jagiełło-Wojtowicz, U. Kosikowska, A. Malm,
´
L. Mazur, and Z. Rza˛czynska
Vol 48
Table 3
The influence of assayed 3d, 3f, 3g, 3h compounds on the growth of gram-positive bacteria recorded as the average diameter of the growth
inhibition zone (including 8 mm of the well) in the agar well diffusion technique and on the basis of MIC values in the broth microdilution
method (OD600).
3d
3f
3g
3h
Zone of growth
MIC
Zone of growth
MIC
Zone of growth
MIC
Zone of growth
MIC
inhibition (mm) (mg/L) inhibition (mm) (mg/L) inhibition (mm) (mg/L) inhibition (mm) (mg/L)
Species
Staphylococcus aureus
ATCC 25923
Staphylococcus aureus
ATCC 6538
Staphylococcus epidermidis
ATCC 12228
8
8
>1000
>1000
>1000
17
15
17
500
125
250
17
8
500
>1000
500
16
8
1000
>1000
1000
15
17
16
Bacillus subtilis ATCC 6633
Bacillus cereus ATCC 10876
Micrococcus luteus
ATCC 10240
25
17
22
1000
>1000
1000
8
16
30
500
250
125
54
11
50
500
>1000
250
32
9
1000
>1000
250
50
13C-NMR spectra were performed for compounds 3a,
3f, 3g, 4a, 4f, and 4g, whereas MS spectra for 3f, 3g,
4a, 4f, and 4g compounds.
tory zone from 11 to 54 mm, MIC ¼ 500 or >1000
mg/L), and 3h (inhibitory zone from 9 to 32 mm, MIC
¼ 1000 or >1000 mg/L). For comparison, MIC values
for reference strains of tested bacteria were 0.015–0.49
mg/L for gentamicin and 0.49–1.95 mg/L for
cefuroxime.
The behavioral study showed that the compounds 4a,
4b, 4e, and 4f weakly affected the CNS of mice. Com-
pound 4g was without effect. The compounds 4a, 4b,
4e, and 4f showed antinociceptive properties (Table 2).
The most active compounds were 4b in doses of
0.0125–0.1 LD50 and 4e in doses of 0.025–0.1 lethal
dose (LD)50. These doses induced a decrease (by 54–
81% and 26–91%, respectively) in a number of mice
exhibiting pain reactivity in the ‘‘writhing syndrome’’
test. In the remaining tests, none of the compounds pro-
duced a statistically significant effect. In our research,
we have shown that the antinociceptive activity of 4b
(4-(4-methoxyphenyl)-5-{[(4-phenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione) and
It was found that the most effective compound against
Gram-positive microorganisms was 3f with diameter of
the growth inhibition zone from 8 to 30 mm at 5000
mg/L concentration and MIC values from 125 to 500
mg/L (Table 3). No activity of the tested compounds
against Gram-negative bacteria or fungi was found.
Our results should be of value to further detailed stud-
ies on the biological activity of this group of com-
pounds. Especially 3f agent described in this article
could be regarded as leading structure at seeking of the
compounds with increased antibacterial activity against
potentially pathogenic or opportunistic Gram-positive
bacteria, including Staphylococci (coagulase-positive S.
aureus and coagulase-negative S. epidermidis).
4e
(5-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-
methyl}-4-cyclohexyl-4H-1,2,4-triazole-3(2H)-thione) is
interesting and should be examined in more detail.
Among Gram-positive species of bacteria, the most
sensitive to all of the assayed compounds was Micro-
coccus luteus American Type Culture Collection
(ATCC) 10240 (growth inhibition zone from 22 to 50
mm, MIC ¼ 125–1000 mg/L). The growth of Staphylo-
coccus aureus ATCC 25923, S. aureus ATCC 6538,
and Staphylococcus epidermidis ATCC 12220 were
totally or partially inhibited by the following com-
pounds: 3f (inhibitory zone from 15 to 17 mm, MIC
125–500 mg/L), 3g (inhibitory zone from 8 to 17 mm,
MIC 500–1000 mg/L), and 3h (inhibitory zone from 8
to 16 mm, MIC ¼ 1000 or >1000 mg/L), similarly
growth of Bacillus subtilis ATCC 6633 and B. cereus
ATCC 10876 by compounds 3d (inhibitory zone from
17 to 25 mm, MIC ¼ 1000 or >1000 mg/L), 3g (inhibi-
EXPERIMENTAL
Chemistry. Melting points were determined in Fisher-Johns
(Pittsburgh, PA) blocks and presented without any corrections.
The IR spectra (m, cmꢁ1) were recorded in KBr tablets using a
Specord IR-75 spectrophotometer (Germany). The 1H-NMR
spectra were recorded on a Bruker Avance 300 apparatus
(Bruker BioSpin GmbH, Rheinstetten/Karlsruhe, Germany) in
dimethyl sulfoxide (DMSO)-d6 with tetramethylsilane (TMS)
as internal standard. The 13C-NMR spectra were recorded on a
Bruker Avance 300. Chemical shifts are given in ppm
(d scale). The MS spectra were recorded on a ThermoFinnigan
Trace TSQGC MS apparatus (Waltham, MA). Chemicals were
purchased from Merck Co. (Whitehouse Station, NJ) or Lan-
caster (Windham, NH) and used without further purification.
Journal of Heterocyclic Chemistry
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Synthesis, Antimicrobial and Pharmacological Properties of New
Thiosemicarbazide and 1,2,4-Triazole Derivatives
343
The purity of obtained compounds was checked by thin
layer chromatography (TLC) on aluminum oxide 60 F254
plates (Merck Co.), in a CHCl3/C2H5OH (10:1, v/v) solvent
system with UV visualization (k ¼ 254 nm).
1-{[(4-Phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}-4-(p-tol-
yl)thiosemicarbazide (3c). Yield: 3.52 g, (88.5%). Tempera-
ture of reaction: 80ꢀC for 12 h. m.p.: 188–190ꢀC. For
C18H18N6OS2 (398.50) calculated: C: 54.2%, H: 4.55%, N:
21.1%; found: C: 54.18%, H: 4.56%, N: 21.11%. 1H-NMR
(DMSO-d6) d (ppm): 2.28 (s, 3H, CH3); 3.98 (s, 2H, CH2);
7.11–7.38 (dd, 4H, 4-CH3C6H4, J ¼ 9 Hz); 7.52–7.62 (m, 5H,
5CHar); 8.89 (s, 1H, CH); 9.67, 9.70, 10.41 (3s, 3H, 3NH).
4-Benzyl-1-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]ace-
tyl}thiosemicarbazide (3d). Yield: 3.73 g (93.9%). Tempera-
ture of reaction: 40ꢀC for 12 h. m.p.: 164–166ꢀC. For
C18H18N6OS2 (398.50) calculated: C: 54.2%, H: 4.55%, N:
21.1%; found: C: 54.18%, H: 4.53%, N: 21.09%. 1H-NMR
(DMSO-d6) d (ppm): 3.96 (s, 2H, CH2); 4.79 (s, 2H, CH2);
7.18–7.64 (m, 10H, 10CHar); 8.86 (s, 1H, CH); 8.81, 9.51,
10.37 (3s, 3H, 3NH).
Ethyl [(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl] acetate
(1). Sodium [0.23 g (0.01 mol)] was added to 5 mL of anhy-
drous ethanol, placed in a three-necked flask equipped with
reflux condenser closed with a tube of CaCl2 and mercury stir-
rer. The content was mixed till sodium dissolved completely
and then 1.77 g (0.01 mol) of 4-phenyl-4H-1,2,4-triazole-3-thi-
one was added. Then 1.22 mL ethyl bromoacetate was added
drop by drop. The content of the flask was mixed for 4 h and
left at room temperature for 12 h. Then 10 mL anhydrous
ethanol was added and heated for 1 h. The mixture was filtered
off inorganic compounds. After cooling, the precipitate was fil-
tered off and crystallized from ethanol.
4-Cyclohexyl-1-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl}thiosemicarbazide (3e). Yield: 3.29 g, (84.5%). Temper-
ature of reaction: 70ꢀC for 12 h. m.p.: 166–168ꢀC. For
C17H22N6OS2 (390.53) calculated: C: 52.24%, H: 5.67%, N:
21.51%; found: C: 54.26%, H: 5.65%, N: 21.50%. 1H-NMR
(DMSO-d6) d (ppm): 1.07–1.76 (m, 10H, 5CH2); 3.86 (s, 2H,
CH2); 4.08 (s, 1H, CH); 7.52–7.69 (m, 5H, 5CHar); 8.94 (s,
1H, CH); 9.26, 9.52, 10.16 (3s, 3H, 3NH).
Yield: 1.73 g, (65.9%). m.p.: 66–68ꢀC. For C12H13N3O2S
(263.31) calculated: C: 54.76%, H: 4.97%, N: 15.96%; found:
C: 54.73%, H: 4.95%, N: 15.95%. IR (KBr): 3098 (CHar),
2913, 1417 (CHal), 1735 (C¼¼O), 1556 (C¼¼N). 1H-NMR
(DMSO-d6): 1.17 (t, 3H, CH3, J ¼ 7.2 Hz); 4.09 (s, 2H, CH2);
4.06–4.13 (q, 2H, CH2, J ¼ 7.2 Hz); 7.51–7.65 (m, 5H,
5CHar); 8.87 (s, 1H, CH).
[(4-Phenyl-4H-1,2,4-triazol-3-yl)sulfanyl] acetohydrazide
(2). Hydrazine hydrate (0.63 mL of 80%) was added to 2.63 g
(0.01 mol) of compound 1 in 5 mL of anhydrous ethanol. The
mixture was left at room temperature for 24 h. The precipitate
of hydrazide 2 was filtered off, dried, and crystallized form
ethanol.
Yield: 1.94 g, (78%). m.p 132–134ꢀC. For C10H11N5OS
(249.29) calculated: C: 48.14%, H: 4.44%, N: 28.08%; found:
1
C: 48.16%, H: 4.41%, N: 28.05%. H-NMR (DMSO-d6): 3.89
(s, 2H, CH2); 4.29 (s, 2H, NH2); 7.51–7.61 (m, 5H, 5CHar);
8.86 (s, 1H, CH); 9.33 (s, 1H, NH).
Derivatives of 1-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl}thiosemicarbazide (3a–3i)
4-(4-Bromophenyl)-1-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sul-
fanyl]acetyl}thiosemicarbazide (3f). Yield: 3.67 g, (79.2%).
Temperature of reaction: 95ꢀC for 12 h. m.p.: 178–180ꢀC. For
C17H15N6BrOS2 (463.37) calculated: C: 44.02%, H: 3.25%, N:
18.13%; found: C: 44.0%, H: 3.26%, N: 18.15%. 1H-NMR
(DMSO-d6) d (ppm): 3.97 (s, 2H, CH2); 7.51–7.62 (m, 9H,
9CHar); 8.90 (s, 1H, CH); 9.81, 9.88, 10.45 (3s, 3H, 3NH).
13C-NMR: 34.7 (CH2); 125.2, 125.4, 129.6, 129.9, 130.8
(5ꢂCHar); 117.5, 133.1, 138.4 (3ꢂCar); 145.4 (CHtriazole);
149.2 (AN¼¼CASA); 166.9 (C¼¼O); 180.8 (C¼¼S). MS m/e
(%): 463 (Mþ, 0.1); 287 (0.7); 249 (3.1); 213 (80); 190 (7);
176 (100); 157 (16); 134 (20); 104 (12); 91 (27); 77 (35).
4-Ethyl-1-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]ace-
tyl}thiosemicarbazide (3g). Yield: 2.56 g, (76.3%). Tempera-
ture of reaction: 50ꢀC for 12 h. m.p.: 171–173ꢀC. For
C13H16N6OS2 (336.43) calculated: C: 46.37%, H: 4.79%, N:
24.97%; found: C: 46.38%, H: 4.80%, N: 24.98%. 1H-NMR
(DMSO-d6) d (ppm): 1.11 (t, 3H, CH3, J ¼ 6 Hz); 3.50–3.59 (q,
2H, CH2, J ¼ 6.6 Hz); 3.90 (s, 2H, CH2); 7.53–7.65 (m, 5H,
5CHar); 8.94 (s, 1H, CH); 8.30, 9.32, 10.25 (3s, 3H, 3NH). 13C-
NMR: 14.6 (CH3); 34.1 (ASACH2A); 38.6 (ACH2ACH3); 125.3,
129.7, 129.9 (3ꢂCHar); 133.1 (Car); 145.5 (CHtriazole); 149.5
(AN¼¼CASA); 166.7 (C¼¼O); 181.2 (C¼¼S). MS m/e (%): 336
(Mþ, 1); 321 (0.5); 257 (3); 249 (2); 218 (20); 204 (7); 190 (6);
176 (100); 149 (7); 135 (5); 104 (10); 91 (18); 87 (24); 77 (30).
4-Ethoxycarbonylmethyl-1-{[(4-phenyl-4H-1,2,4-triazol-3-
General procedure. A mixture of hydrazide (2) 2.49 g (0.01
mol) and 0.01 mol appropriate isothiocyanate was heated in an
oil bath at 40–95ꢀC for 12 h. The product was washed with
diethyl ether to remove unreacted isothiocyanate and with
water to remove unreacted hydrazide (2). Then it was filtered
off, dried, and crystallized from ethanol.
1-{[(4-Phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}-4-phe-
nyl thiosemicarbazide (3a). Yield: 3.51 g, (91.5%). Tempera-
ture of reaction: 50ꢀC for 12 h. m.p.: 168–170ꢀC. For
C17H16N6OS2 (384.48) calculated: C: 53.06%, H: 4.19%, N:
21.85%; found: C: 53.09%, H: 4.20%, N: 21.87%. 1H-NMR
(DMSO-d6): 3.99 (s, 2H, CH2); 7.13–7.65 (m, 10H, 10CHar);
8.90 (s, 1H, CH); 9.74, 9.76, 10.43 (3s, 3H, 3NH). 13C-NMR:
34.8 (CH2); 125.2, 125.4, 128.0, 129.6, 129.9 (6ꢂCHar);
133.2, 139.0 (2ꢂCar); 145.4 (CHtriazole); 149.2 (N¼¼CASA);
166.9 (C¼¼O); 180.8 (C¼¼S).
yl)sulfanyl]acetyl} thiosemicarbazide (3h). Yield: 2.57
g
(65.2%). Temperature of reaction: 40ꢀC for 12 h. m.p.: 109–
112ꢀC. For C15H18N6O3S2 (394.47) calculated: C: 45.67%, H:
4.60%, N: 21.30%; found: C: 45.65%, H: 4.61%, N: 21.28%.
1H-NMR (DMSO-d6) d (ppm): 1.19 (t, 3H, CH3, J ¼ 7.2 Hz);
3.97 (s, 2H, CH2); 4.07–4.14 (q, 2H, CH2); 4.24 (d, 2H, CH2);
7.46–7.65 (m, 5H, 5CHar); 8.91 (s, 1H, CH); 8.86, 9.68, 10.46
(3s, 3H, 3NH).
1-{[(4-Phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}-4-(4-methox-
yphenyl)thiosemicarbazide (3b). Yield: 3.68 g, (88.9%). Tem-
perature of reaction: 50ꢀC for 12 h. m.p.: 174–176ꢀC. For
C18H18N6O2S2 (414.50) calculated: C: 52.11%, H: 4.37%, N:
20.27%; found: C: 52.10%, H: 4.39%, N: 20.28%. 1H-NMR
(DMSO-d6) d (ppm): 3.74 (s, 3H, CH3); 3.98 (s, 2H, CH2);
6.90–7.35 (dd, 4H, 4-CH3OC6H4, J ¼ 6 Hz); 7.52–7.61 (m,
5H, 5CHar); 8.89 (s, 1H, CH); 9.63, 9.65, 10.38 (3s, 3H,
3NH).
4-Ethoxycarbonyl-1-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sul-
fanyl]acetyl}thiosemicarbazide (3i). Yield: 2.85 g, (75.1%).
Temperature of reaction: 45ꢀC for 12 h. m.p.: 172–174ꢀC. For
C14H16N6O3S2 (380.44) calculated: C: 44.16%, H: 4.23%, N:
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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Ł. Popiołek, M. Dobosz, A. Chodkowska, E. Jagiełło-Wojtowicz, U. Kosikowska, A. Malm,
´
L. Mazur, and Z. Rza˛czynska
Vol 48
22.08%; found: C: 44.15%, H: 4.21%, N: 22.07%. 1H-NMR
(DMSO-d6) d (ppm): 1.23 (t, 3H, CH3, J ¼ 6.9 Hz); 4.09 (s, 2H,
CH2); 4.14–4.21 (q, 2H, CH2, J ¼ 6.9 Hz); 7.51–7.64 (m, 5H,
5CHar); 8.89 (s, 1H, CH); 9.96, 11.06, 11.37 (3s, 3H, 3NH).
Derivatives of 5-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-
methyl}-4H-1,2,4-triazole-3(2H)-thione (4a–4g, 4j)
General procedure. A mixture of thiosemicarbazide (3a–3h;
0.01 mol) and 20 mL of 2% aqueous solution of sodium hy-
droxide was refluxed for 2 h. Then, the solution was neutral-
ized with diluted hydrochloric acid, and the formed precipitate
was filtered off and crystallized from ethanol.
5-{[(4-Phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4-phenyl-
4H-1,2,4-triazole-3(2H)-thione (4a). Yield: 3.12 g, (85.3%).
m.p.: 160–162ꢀC. For C17H14N6S2 (366.46) calculated: C:
55.68%, H: 3.85%, N: 22.93%; found: C: 55.69%, H: 3.85%,
N: 22.91%. IR (KBr, cmꢁ1): 3110 (CHar); 2918, 1458 (CHal);
1608 (C¼¼N); 1513 (CAN). 1H-NMR (DMSO-d6) d (ppm):
4.12 (s, 2H, CH2); 7.25–7.58 (m, 10H, 10CHar); 8.89 (s, 1H,
CH); 13.83 (s, 1H, NH). 13C-NMR: 27.8 (CH2); 128.2, 129.1,
129.3, 129.5, 129.6, 129.7 (6ꢂCHar); 133.1 (2ꢂCar); 145.8
(CHtriazole); 147.1 (CH2AC); 148.0 (CAS); 168.2 (C¼¼S). MS
m/e (%): 366 (Mþ, 0.2); 266 (0.15); 222 (0.04); 190 (0.54);
176 (75); 149 (14); 135 (8); 104 (9); 91 (28); 77 (100).
5-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4-
(4-methoxyphenyl)-4H-1,2,4-triazole-3(2H)-thione (4b). Yield:
3.58 g, (90.1%). m.p.: 168–170ꢀC. For C18H16N6OS2 (396.49)
calculated: C: 54.48%, H: 4.06%, N: 21.19%; found: C:
54.48%, H: 4.08%, N: 21.17%. IR (KBr, cmꢁ1): 3095 (CHar);
2922, 1461 (CHal); 1618 (C¼¼N); 1515 (CAN). 1H-NMR
(DMSO-d6) d (ppm): 3.81 (s, 3H, CH3); 4.10 (s, 2H, CH2);
7.03 (d, 2H, 2CHar, 4-CH3OC6H4, J ¼ 9 Hz); 7.16 (d, 2H,
2CHar, 4-CH3OC6H4, J ¼ 9 Hz); 7.35–7.59 (m, 5H, 5CHar);
8.89 (s, 1H, CH); 13.77 (s, 1H, NH).
culated: C: 45.8%, H: 2.93%, N: 18.86%; found: C: 45.8%,
H: 2.92%, N: 18.88%. IR (KBr, cmꢁ1): 3088 (CHar); 2924,
1
1470 (CHal); 1624 (C¼¼N); 1527 (CAN). H-NMR (DMSO-d6)
d (ppm): 4.14 (s, 2H, CH2); 7.23 (d, 2H, 2CHar, 4-BrC6H4,
J ¼ 8.7 Hz); 7.35–7.59 (m, 5H, 5CHar); 7.72 (d, 2H, 2CHar,
4-BrC6H4, J ¼ 8.7 Hz); 8.89 (s, 1H, CH); 13.87 (s, 1H, NH).
13C-NMR: 27.8 (CH2); 125.4, 129.5, 129.7, 130.4, 132.3
(5ꢂCHar); 122.9, 132.4, 133.0 (3ꢂCar); 145.8 (CHtriazole);
147.0 (ACH2AC); 147.9 (CAS); 168.1 (C¼¼S). MS m/e (%):
445 (Mþ, 0.03); 366 (0.01); 287 (0.01); 268 (0.2); 257 (0.03);
213 (0.07); 190 (0.17); 176 (75); 149 (15); 135 (9); 104 (10);
91 (28); 77 (100).
4-Ethyl-5-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
4H-1,2,4-triazole-3(2H)-thione (4g). Yield: 2.93 g, (94.5%).
m.p.: 184–186ꢀC. For C13H14N6S2 (318.42) calculated: C:
48.98%, H: 4.43%, N: 26.38%; found: C: 48.99%, H: 4.44%,
N: 26.39%. IR (KBr, cmꢁ1): 3101 (CHar); 2922, 1463 (CHal);
1612 (C¼¼N); 1530 (CAN). 1H-NMR (DMSO-d6) d (ppm):
1.36 (t, 3H, CH3, J ¼ 9 Hz); 4.15–4.22 (q, 2H, CH2, J ¼ 7.2
Hz); 4.53 (s, 2H, CH2); 7.37–7.61 (m, 5H, 5CHar); 9.00 (s,
1H, CH); 13.63 (s, 1H, NH). 13C-NMR: 13.1 (CH3); 27.4
(ASACH2A); 38.5 (ACH2ACH3); 125.8, 129.6, 129.7
(3ꢂCHar); 133.1 (Car); 145.9 (CHtriazole); 147.3 (ACH2AC);
148.1 (CAS); 166.7 (C¼¼S). MS m/e (%): 318 (Mþ, 25); 302
(1); 290 (2); 258 (2.5); 218 (3); 204 (12); 190 (3); 176 (100);
149 (9); 135 (6); 104 (8); 91 (11); 77 (25).
4-Carboxymethyl-5-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfa-
nyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (4j). Yield: 2.71
g, (77.8%). m.p.: 123–125ꢀC. For C13H12N6O2S2 (348.40) cal-
culated: C: 44.81%, H: 3.47%, N: 24.12%; found: C: 44.82%,
1
H: 3.47%, N: 24.11%. H-NMR (DMSO-d6) d (ppm): 4.26 (s,
2H, CH2); 4.64 (s, 2H, CH2); 7.35–7.62 (m, 5H, 5CHar); 8.84
(s, 1H, CH); 10.52 (s, 1H, OH); 13.83 (s, 1H, NH).
5-{[(4-Phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4-
(4-tolyl)-4H-1,2,4-triazole-3(2H)-thione (4c). Yield: 3.52
g, (92.7%). m.p.: 166–168ꢀC. For C18H18N6S2 (380.49) calcu-
lated: C: 56.77%, H: 4.23%, N: 22.08%; found: C: 56.78%, H:
4.22%, N: 22.08%. IR (KBr, cmꢁ1): 3095 (CHar); 2922, 1459
(CHal); 1610 (C¼¼N); 1526 (CAN). 1H-NMR (DMSO-d6) d
(ppm): 2.37 (s, 3H, CH3); 4.10 (s, 2H, CH2); 7.10–7.32 (dd,
4H, 4CHar, 4-CH3C6H4, J ¼ 8.1 Hz); 7.34–7.58 (m, 5H,
5CHar); 8.89 (s, 1H, CH); 13.80 (s, 1H, NH).
4-Benzyl-5-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
4H-1,2,4-triazole-3(2H)-thione (4d). Yield: 3.61 g, (95.1%).
m.p.: 130–132ꢀC. For C18H16N6S2 (380.49) calculated: C:
56.77%, H: 4.23%, N: 22.08%; found: C: 56.77%, H: 4.22%,
N: 22.07%. IR (KBr, cmꢁ1): 3088 (CHar); 2926, 1459 (CHal);
1615 (C¼¼N); 1529 (CAN). 1H-NMR (DMSO-d6) d (ppm):
4.31 (s, 2H, CH2); 5.26 (s, 2H, CH2); 7.19–7.56 (m, 10H,
10CHar); 8.90 (s, 1H, CH); 13.83 (s, 1H, NH).
4-Cyclohexyl-5-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-
methyl}-4H-1,2,4-triazole-3(2H)-thione (4e). Yield: 3.32 g,
(89.4%). m.p.: 102–104ꢀC. For C17H20N6S2 (372.51) calculated:
C: 54.76%, H: 5.38%, N: 22.55%; found: C: 54.75%, H: 5.37%,
N: 22.55%. IR (KBr, cmꢁ1): 3100 (CHar); 2918, 1455 (CHal);
1610 (C¼¼N); 1527 (CAN). 1H-NMR (DMSO-d6) d (ppm):
1.06–1.75 (m, 10H, 5CH2); 3.58 (s, 2H, CH2); 4.44 (s, 1H, CH);
7.37–7.64 (m, 5H, 5CHar); 8.95 (s, 1H, CH); 13.95 (s, 1H, NH).
4-(4-Bromophenyl)-5-{[(4-phenyl-4H-1,2,4-triazol-3-yl)sul-
fanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (4f). Yield: 3.72
g, (83.6%). m.p.: 147–149ꢀC. For C17H13BrN6S2 (445.46) cal-
[(4-Phenyl-4H-1,2,4-triazol-3-yl)sulfanyl] acetic acid (5). Com-
pound 5 was obtained using the same method described earlier
for derivatives 4a–4g, 4j. That is, a mixture of thiosemicarba-
zide (3i; 0.01 mol) and 20 mL of 2% aqueous solution of so-
dium hydroxide was refluxed for 2 h. Then, the solution was
neutralized with diluted hydrochloric acid and the formed pre-
cipitate was filtered off and crystallized from ethanol.
Yield: 1.88 g, (80.2%). m.p.: 184–186ꢀC. For C10H9N3O2S
(235.26) calculated: C: 51.0%, H: 3.58%, N: 17.87%; found:
1
C: 51.0%, H: 3.6%, N: 17.9%. H-NMR (DMSO-d6): 4.04 (s,
2H, CH2); 7.49–7.64 (m, 5H, 5CHar); 8.86 (s, 1H, CH); 12.91
(s, 1H, OH).
X-ray crystallography. Crystal data for compound 1: space
˚
˚
group, P21/n, a ¼ 7.726(1) A, b ¼ 19.313(3) A, c ¼ 9.222(2)
3
ꢀ
˚
˚
A, b ¼ 113.92(2) , V ¼ 1257.9(4) A , Z ¼ 4, dcalc ¼ 1.390 g
cmꢁ3, l ¼ 0.255 mmꢁ1
.
A crystal with approximate dimensions of 0.41 ꢂ 27 ꢂ 0.14
mm3 was mounted on a glass fiber in a random orientation.
Single-crystal diffraction data were measured at room tempera-
ture in the x/2y mode on the Oxford Diffraction Xcalibur
diffractometer using the graphite-monochromated MoKa radia-
tion. The stability of intensities was monitored by measure-
ment of three standards for every 100 reflections. Crystal struc-
ture was solved by direct methods using SHELXS97 [20] and
refined by the full-matrix least-squares on F2 using the
SHELXL97 [21]. All non-hydrogen atoms were refined with
anisotropic displacement parameters. Hydrogen atoms were
positioned geometrically and allowed to ride on their parent
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

March 2011
Synthesis, Antimicrobial and Pharmacological Properties of New
Thiosemicarbazide and 1,2,4-Triazole Derivatives
345
atoms, with Uiso(H) ¼ 1.2 Ueq(C). Final discrepancy factors
are R1 ¼ 0.0438, wR2 ¼ 0.0954 for I > 2r(I), GOOF ¼
The total immobility time of mice was measured during the
last 4 min of the 6-min test [26].
ꢁ3
˚
1.019, Dqmin, max ¼ ꢁ0.22/0.21 e A
.
Thiopental sleeping time. Thiopental (75 mg/kg i.p.) was
given 30 min after the injection of the all compounds in a
dose of 0.1 of their LD50. The sleeping time of mice (from
disappearance to return of the righting reflex) was measured.
Antinociceptive activity. Pain reactivity was measured by the
writhing syndrome test in mice according to Witkin et al. [27].
Thirty minutes after the injection of compounds, 4a (0.025–0.1
LD50), 4b (0.00625–0.1 LD50), 4e and 4f (0.0125–0.1 their
LD50), 4g (0.1 LD50), the animals were injected with 0.6% acetic
acid. The number of writhing episodes was counted for 30 min.
Pentetrazole-induced seizures. Thirty minutes after adminis-
tration of compounds in a dose of 0.1 their LD50, the animals
were injected subcutaneously (sc) with pentetrazole (110 mg/
kg) and were observed during 30 min. The number of mice
developing clonic and tonic seizures as well as mortality was
recorded in that period.
Pharmacology. The experiments were carried out on male
Albino-Swiss mice weighing 20–24 g. The animals were
housed in colony cages with free access to food (standard lab-
oratory pellets, Bacutil Motycz, Poland) and tap water and
maintained in the natural light–dark cycle. The tested groups,
consisting of eight animals, were randomly assigned. The
experiments were performed between 8:00 a.m. and 2:00 p.m.
The investigated compounds (4a, 4b, 4e, 4f, and 4g) were
administered intraperitoneally (i.p.) in doses equivalent to
0.00625, 0.0125, 0.025, 0.05, and 0.1 of their LD50 as suspen-
sions in 1% Tween 80 in a volume 10 cm3/kg at 30 min
before tests. Control mice received the equivalent volume of
solvent. The Bioethical Committee of Lublin Medical Univer-
sity approved all experimental procedures applied in this study.
The following behavioral tests were performed.
Chimney test. The effect of above investigated compounds
in a dose of 0.1 of their LD50 on motoring impairment was
quantified with the chimney test [22]. All animals were per-
tained 24 h before the test. The mice were inserted into a plas-
tic tube (25 cm long, 3 cm in inner diameter). When the
mouse reached the end, the tube was positioned upright, and
motor impairment was indicated when the animal was unable
to climb backward up the tube within 60 s. It was quantified
as the percentage of animals that failed to complete the test.
Body temperature. The rectal body temperature in mice
(Ellab thermometer) was recorded 15, 30, 45, 60, 90, and 120
min after the administration of the investigated compounds in
a dose of 0.1 of their LD50.
Head twitches. Head-twitch responses induced by L-5-
hydroxytryptophan (L-5-HTP) were measured in mice accord-
ing to Corne et al. [28]. The compounds in a dose of 0.1 of
their LD50 were injected 30 min before L-5-HTP (165 mg/kg).
The number of head-twitch episodes of mice was counted dur-
ing 60 min after the injection of L-5-HTP.
Microbiology. The reference strains of ATCC, including six
Gram-positive bacteria (S. aureus ATCC 25923, S. aureus
ATCC 6538, S. epidermidis ATCC 12220, B. subtilis ATCC
6633, B. cereus ATCC 10876, M. luteus ATCC 10240), four
Gram-negative bacteria (Escherichia coli ATCC 25922, Kleb-
siella pneumoniae ATCC 13883, Proteus mirabilis ATCC
12453, and Pseudomonas aeruginosa ATCC 9027), and three
strains of yeasts belonging to Candida spp. (C. albicans
ATCC 10231, C. albicans ATCC 2091, C. parapsilosis ATCC
22019) were used.
Microbial suspensions were prepared in sterile saline (0.85%
NaCl) with an optical density of McFarland standard 0.5 (150 ꢂ
106 colony forming units per mL). All stock solutions of the
tested compounds were dissolved in DMSO. The medium con-
taining DMSO at the final concentrations and without any of
tested compounds served as control, and no microbial growth in-
hibition was observed. Gentamicin and cefuroxime were used as
control antimicrobial agents. Mueller-Hinton agar or Mueller-
Hinton agar supplemented with 2% glucose was used for exami-
nation of antibacterial or antifungal activity, respectively.
In the first step, all tested compounds were screened for in
vitro antimicrobial activity by the agar well diffusion method
based on the appearance of the growth inhibition zone sur-
rounding the well (d ¼ 8 mm) containing the tested chemicals
at 5000 mg/L concentration (80 lL/well separately). The ster-
ile swabs were used to spread the microbial suspensions onto
the medium surface. The plates were preincubated at room
temperature for 1.5 h to allow the diffusion of solution into
the medium and then were incubated at 37ꢀC for 18 h (for
bacteria) or at 30ꢀC for 24–48 h (for fungi).
Hole board test. To determine the effects of the compounds
on the explorative activity, the hole board test of Boissier et al.
[23] was taken. The mice were placed on the board 30 min after
administration of compounds in a dose of 0.1 of their LD50. The
number of holes explored was counted for 5 min.
Four plate test. Anxiolytic activity was measured by the
‘‘four plate’’ test in mice according to Aron et al. [24]. Thirty
minutes after the injection of compounds in a dose of 0.1 of their
LD50, the number of punished crossings was counted for 1 min.
Passive avoidance task. The passive avoidance task is con-
sidered to be a measure of long-term memory in rodents [25].
On the first day, 30 min after injection of compounds in a
dose of 0.1 of their LD50, the animals were placed in an illu-
minated box (10 ꢂ 13 ꢂ 15 cm3) connected to a dark box (25
ꢂ 20 ꢂ 15 cm3), equipped with an electric grid floor. Having
entered the dark box, the mice were punished by an electric
foot shock (0.6 mA, 2 s). Animals not entering the dark box
within 60 s were excluded from the subsequent experiment.
The next day (24 h later), animals were again placed individu-
ally in the illuminated box and observed up to 3 min. The
time from placement in the illuminated box to entry into the
dark box was taken as an indication of the degree of long-term
memory impairment. In the control group, the animals did not
enter the dark compartment within 3 min.
Subsequently, MIC of the compounds 3d, 3f, 3g, and 3h,
showing some antibacterial activity using the agar well diffu-
sion method, was estimated by microdilution technique [opti-
cal density (OD)600] and Mueller-Hinton broth containing from
1.95 to 1000 mg/L of the tested agents. MIC is usually defined
as the lowest concentration of compound at which there was
no visible growth of tested microorganisms. Microdilution
Forced swimming test. Thirty minutes after the injection of
compounds in a dose of their LD50, the mice were individually
placed and forced to swim in a glass cylinder (27 ꢂ 16 cm2)
containing 15 cm of water (25ꢀC). A mouse was considered
immobile when it floated in the water in an upright position
and made only small movements to keep its head above water.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

´
346
Ł. Popiołek, M. Dobosz, A. Chodkowska, E. Jagiełło-Wojtowicz, U. Kosikowska, A. Malm,
´
L. Mazur, and Z. Rza˛czynska
Vol 48
†
[11] Ulusoy, N.; Gu¨rsoy, A.; Otu¨k G. Il Farmaco 2001, 56, 947.
[12] Demirayak, Sꢀ.; Benkli, K.; Gu¨ven, K. Eur J Med Chem
broth technique was developed using 96-well microplates,
which were inoculated with a 1:10 diluted microbial suspen-
sion of optical density of 0.5 McFarland standard. About
20 lL of bacterial suspension was put into 180 lL of medium
containing twofold dilution of the tested compounds. After
2000, 35, 1037.
˘
[13] Palaska, E.; Sꢀahin, G.; Kelicen, P.; Tugba, D.; Altinok, G.
Il Farmaco 2002, 57, 101.
ˇ ´ ´ ´
[14] Klimensova, V.; Zahajska, L.; Waisser, K.; Kaustova, J.;
incubation (at 37ꢀC for 18 h), the optical density (OD600
)
Mo†llam, U. Il Farmaco 2004, 59, 279.
[15] Wujec, M.; Swatko-Ossor, M.; Mazur, L.; Rza˛czynska,
measurements were determined for bacterial culture in broth
medium, and the MIC values were determined by comparison
with the bacterial growth in control (compound free) medium.
´
Z.;Siwek A. J Heterocycl Chem 2008, 45, 1893.
´
[16] Pitucha, M.; Polak, B.; Swieboda, R.; Kosikowska, U.;
Malm, A. Z Naturforsch B. J Clin Sci 2009, 64b, 570.
[17] CLSI. Performance Standards for Antimicrobial Susce-
ptibility Testing; Eighteenth International Supplement. CLSI doc-
ument M7-MIC; Clinical Laboratory Standards Institute: Wayne, PA,
2008.
Acknowledgment. Cambridge Crystallographic Data Centre
(CCDC) 748859 contains the supplementary crystallographic
data for this article. Copies of the data can be obtained free of
charge from CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
(fax: þ44-1223-336-033; e-mail: deposit@ccdc. cam.ac.uk).
[18] Zotta, V.; Gasmet, A. Farmacia (Bucharest, Romania) 1963,
11, 731.
[19] Kaplaushenko, A. H.; Panasenko, O. I.; Knish, E. H.;
Shcherbina, R. O. Farm Zh (Kiev, Ukraine) 2008, 2, 67.
REFERENCES AND NOTES
˘
[1] Gu¨lerman, N. N.; Dogan, H. N.; Rollas, S.; Johansson, C.;
[20] Sheldrick, G. M. SHELXS97, Program for a Crystal
¨ ¨
Structure Solution; University of Gottingen: Gottingen, Germany,
C¸ elik, C. Il Farmaco 2001, 56, 953.
[2] Pitucha, M.; Wujec, M.; Dobosz, M. J Chin Chem Soc
2007, 54, 69.
1997.
[21] Sheldrick, G. M. SHELXL97, Program for the Refinement
¨
of a Crystal Structure from Diffraction Data; University of Gottingen:
[3] Dobosz, M.; Sikorska, M. Acta Pol Pharm 1994, 51, 377.
[4] Dobosz, M.;Pachuta A. Acta Pol Pharm 1996, 52, 123.
[5] Dobosz, M.; Pitucha, M.; Wujec, M. Acta Pol Pharm 1996,
53, 31.
¨
Gottingen, Germany, 1997.
[22] Boissier, J. R.; Tardy, J.; Diverres, J. C. Med Exp 1960,
3, 81.
[6] Maliszewska-Guz, A.; Wujec, M.; Pitucha, M.; Dobosz, M.;
´
Chodkowska, A.; Jagiełło-Wojtowicz, E.; Mazur, L.; Kozioł, A. E.
[23] Boissier, J. R.; Simon, P. Physiol Behav 1967, 2, 447.
[24] Aron, C.; Simon, P.; Larousse, C.; Boissier, J. R. Neuro-
pharmacology 1971, 10, 459.
Collect Czech Chem Commun 2005, 70, 51.
´
[7] Dobosz, M.; Struga, M.; Chodkowska, A.; Jagiełło-Wojtowicz,
[25] Venault, P.; Chapouthier, G.; de Carvalho, L. P.; Simiand, J.;
Morre, M.; Dodd, R. H.; Rossier, J. Nature (London) 1986, 321, 864.
[26] Porsolt, R. D.; Bertin, A.; Deniel, M.; Jalfre, M. Arch Int
Pharmacodyn Ther 1977, 229, 327.
E.; Ste˛pniak, K.; Kozioł, A. E. Acta Pol Pharm 2002, 59, 281.
[8] Turan-Zitouni, G.; Kaplancikli, Z. A.; Erol, K.; Kilic¸, F. S.
Il Farmaco 1999, 54, 218.
[9] Collin, X.; Sauleau, A.; Coulon, J. Biorg Med Chem Lett
2003, 13, 2601.
[27] Witkin, L.; Heubner, G.; Galdi, F.; O0Keefe, E.; Spitaletta,
F.; Plumer, A. J Pharmacol Exp Ther 1951, 132, 400.
[28] Corne, S. J.; Pickering, R. W.; Warner, B. T., Br J Pharma-
col 1963, 20, 106.
[10] Pitucha, M.; Wujec, M.; Dobosz, M.; Kosikowska, U.;
Malm, A. Acta Pol Pharm 2005, 62, 443.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet