Synthesis and antimicrobial properties of new thiosemicarbazide, 1,2,4-triazole, and 1,3,4-thiadiazole derivatives of sulfanylacetic acid

Article abstract of DOI:10.1080/10426507.2011.625511

1,2,4-triazole and 1,3,4-thiadiazole derivatives are still considered a viable lead structure for the synthesis of more efficient antimicrobial agents having a broad spectrum of activity. This study presents the synthesis and antimicrobial evaluation of a new series of substituted 1,2,4-triazole and 1,3,4-thiadiazole derivatives. Reaction of 4-phenyl-5-(pyridin-3-yl)-4H-1,2,4- triazole-3-thione with ethyl bromoacetate yields the corresponding ethyl acetate (1). In the subsequent reaction with 100% hydrazine hydrate, the hydrazide (2) was obtained, which was converted with isothiocyanates to new acyl derivatives of thiosemicarbazide (3a-l). The cyclization of these compounds in alkaline media resulted in the formation of new derivatives of 1,2,4-triazole (4a-i), whereas in acidic media new derivatives of 1,3,4-thiadiazole (5a,b,g) were obtained. All synthesized compounds were screened for their in vitro antimicrobial activities.

Full text of DOI:10.1080/10426507.2011.625511

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Phosphorus, Sulfur, and Silicon and the
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Synthesis and Antimicrobial Properties of
New Thiosemicarbazide, 1,2,4-Triazole,
and 1,3,4-Thiadiazole Derivatives of
Sulfanylacetic Acid
Łukasz Popiołek a , Urszula Kosikowska b , Maria Dobosz a & Anna
Malm b
a Department of Organic Chemistry, Medical University of Lublin,
Lublin, Poland
b Department of Pharmaceutical Microbiology, Medical University of
Lublin, Lublin, Poland
Available online: 31 Jan 2012
To cite this article: Łukasz Popiołek, Urszula Kosikowska, Maria Dobosz & Anna Malm (2012):
Synthesis and Antimicrobial Properties of New Thiosemicarbazide, 1,2,4-Triazole, and 1,3,4-
Thiadiazole Derivatives of Sulfanylacetic Acid, Phosphorus, Sulfur, and Silicon and the Related
Elements, 187:4, 468-481
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Phosphorus, Sulfur, and Silicon, 187:468–481, 2012
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2011.625511
SYNTHESIS AND ANTIMICROBIAL PROPERTIES
OF NEW THIOSEMICARBAZIDE, 1,2,4-TRIAZOLE,
AND 1,3,4-THIADIAZOLE DERIVATIVES
OF SULFANYLACETIC ACID
Łukasz Popiołek,1 Urszula Kosikowska,2 Maria Dobosz,1
and Anna Malm2
1Department of Organic Chemistry, Medical University of Lublin, Lublin, Poland
2Department of Pharmaceutical Microbiology, Medical University of Lublin,
Lublin, Poland
GRAPHICAL ABSTRACT
S
N
N
H
N
N
NHR
S
N
H
O
N
OH-
a
−
3
i
H+
N
N
N N
N
N
S
S
N
N
NH
N
RN
S
N
N
NHR
S
a−
4
a
b
, ,
i
5
g
=
-
e
c- ex
H
-
r
-
g
4 M OC6H4CH2
( )
h ,
-
e
R
Ph (
)
a ,
4 M OC6H4 (b) CH Ph ( ),
(d) 4 B C H
( )
e ,
Et
(f)
,
4 ClC H
,
c
,
(
),
2
6
4
6
4
=
CH2CH CH2 ( )
i
Abstract 1,2,4-triazole and 1,3,4-thiadiazole derivatives are still considered a viable lead
structure for the synthesis of more efficient antimicrobial agents having a broad spectrum
of activity. This study presents the synthesis and antimicrobial evaluation of a new series of
substituted 1,2,4-triazole and 1,3,4-thiadiazole derivatives. Reaction of 4-phenyl-5-(pyridin-3-
yl)-4H-1,2,4-triazole-3-thione with ethyl bromoacetate yields the corresponding ethyl acetate
(1). In the subsequent reaction with 100% hydrazine hydrate, the hydrazide (2) was obtained,
which was converted with isothiocyanates to new acyl derivatives of thiosemicarbazide (3a–l).
The cyclization of these compounds in alkaline media resulted in the formation of new deriva-
tives of 1,2,4-triazole (4a–i), whereas in acidic media new derivatives of 1,3,4-thiadiazole
(5a,b,g) were obtained. All synthesized compounds were screened for their in vitro antimicro-
bial activities.
Keywords Thiosemicarbazide; 1,2,4-triazole; 1,3,4-thiadiazole; antimicrobial activity; cy-
clization reaction
Received 24 May 2011; accepted 16 September 2011.
Address correspondence to Łukasz Popiołek, Department of Organic Chemistry, Medical University of
Lublin, Chodzki 4A Street, Lublin 20-093, Poland. E-mail: lukasz.popiolek@umlub.pl
468

SYNTHESIS AND ANTIMICROBIAL PROPERTIES
469
INTRODUCTION
Five-membered aromatic systems having three heteroatoms are still being studied
because of their interesting biological properties. Among them, the 1,2,4-triazole and 1,3,4-
thiadiazole derivatives possess a wide range of various pharmacological activities, which
mainly depend on the nature of the substituents. Some of them obtained in the course
of previous investigations and tested on animals showed potential action on the central
nervous system.1 Others exhibited analgesic,2 antifungal,3,4 antibacterial,5–7 antiviral,8,9
antiphlogistic,10,11 and antituberculous12 activity.
Research and development of potent and effective antimicrobial agents represents one
of the most important advances in therapeutics, not only in the control of serious infections
but also in the prevention and treatment of some infectious complications. Therefore, 1,2,4-
triazole and 1,3,4-thiadiazole derivatives are still considered a viable lead structure for the
synthesis of more efficient antimicrobial agents with a broad spectrum of activities.
The common method for the preparation of 1,2,4-triazoles and 1,3,4-thiadiazoles is
the cyclization reaction of thiosemicarbazide derivatives. In earlier papers,13–15 it was stated
that the cyclization was affected not only by pH of the medium but also by the nature of
substituents in the thiosemicarbazide derivatives. The course of the cyclization in alkaline
and acidic media for thiosemicarbazide derivatives of formic, benzoic, and nicotinic acids
was investigated.16 Cyclization of thiosemicarbazide derivatives of formic and nicotinic
acids in alkaline and in acidic media leads to 1,2,4-triazole derivatives. In alkaline media,
the cyclization of benzoic acid thiosemicarbazide derivatives results in the formation of
1,2,4-triazoles, while in acidic media, 1,3,4-thiadiazole derivatives were obtained.16
In view of the facts mentioned above and in continuation of our search for various
biologically active molecules as well as the encouraging antibacterial activity of some
1,2,4-triazole derivatives has prompted us to synthesize some novel compounds and to
carry out preliminary investigations on their antimicrobial activity.17 In this paper, we
report the synthesis and spectral studies of novel series of substituted 1,2,4-triazole and
1,3,4-thiadiazole derivatives and the evaluation of their in vitro antibacterial activity.
RESULTS AND DISCUSSION
Chemistry
4-Phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazole-3-thione, which can exist in two tau-
tomeric forms, was the starting material for the synthesis of new derivatives, which contain
two 1,2,4-triazole rings or one 1,2,4-triazole, and one 1,3,4-thiadiazole ring connected by
an S CH2 bridge. The thione was obtained using a method described earlier by cy-
clization of 1-(pyridin-3-yl)-4-phenyl thiosemicarbazide in alkaline medium. Depending
on the conditions used, further reaction of 4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazole-3-
thione can lead either to S- or to N-derivatives. We have investigated the reaction with ethyl
bromoacetate. On the basis of the results described in previous papers,18,19 elemental and
spectral analyses as well as X-ray crystallography data of similar compounds, it was found
that the reaction leads to the formation of an S-derivative.
Reaction of 4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazole-3-thione with ethyl bro-
moacetate in the presence of sodium ethanolate gave ethyl [(4-phenyl-(5-pyridin-3-yl)-
4H-1,2,4-triazol-3-yl)sulfanyl] acetate (1), which was converted to the hydrazide 2 by
reaction with 100% hydrazine hydrate (Scheme 1).

470
Ł. POPIOŁEK ET AL.
N
NH
S
N
N
Et
CO2
NHNH
CO
2
N
N
N N
100 NH NH2.H2O
S
S
r
B CH2CO2Et
%
N
2
N
a
N
EtOH
N
N
,
Et
H
O
N
N
H
S
N
1
2
RNCS
S
N
N
N
H
N
N
H
NHR
S
N
O
N
OH-
a
3
−
i
H+
N
N
N N
N
N
S
S
N
N
NH
N
RN
S
N
N
NHR
S
a
4 −i
a
b
, ,
5
g
g
=
-
e
c- ex
H
-
r
-
-
e
) 4 M
OC6 4C
,
R
Ph ( ) 4 M
H4
OC6
(
)
H Ph ( )
c ,
(
) 4 B H4 ( ) Et ( ) 4
C6
l
H4
(
H
H
(h)
,
2
a ,
b , C
d , f , C C6
e ,
2
=
2 (i)
H
C
H H
C 2C
Scheme 1
Reactions of the hydrazide 2 with aliphatic, aromatic, ethoxycarbonyl, ethoxycar-
bonylmethyl, and benzoyl isothiocyanates were carried out following two different experi-
mental procedures. The new thiosemibarbazide derivatives (3a–l) were obtained by heat◦ing
◦
the reactants in an oil bath without a solvent at temperatures between 50 C and 110 C.
The thiosemicarbazide derivatives 3a–d and 3f were prepared by reaction of the hydrazide
2 with the respective isothiocyanate in diethyl ether as solvent.
Cyclization reaction of the thiosemicarbazide derivatives 3a–i with 2% aqueous
solution of sodium hydroxide leads to the new 4H-1,2,4-triazol-3(2H)-thione derivatives
4a–i.
Hydrolysis was observed in the case of the cyclization of thiosemicarbazide 3j in
alkaline media, which resulted in the formation of [(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-
triazol-3-yl)sulfanyl] acetic acid 6. This compound was described earlier,20 but it was
obtained in a different way. The cyclization in alkaline media of the thiosemicarbazide 3k,
which contains an ethoxycarbonylmethyl group, was also accompanied by hydrolysis of
the ester group and yielded the 4H-1,2,4-triazole-3(2H)-thione 7. In the case of 4-benzoyl
thiosemicarbazide derivative 3l, the cyclization in alkaline media gave the 2,5-dihydro-4H-
1,2,4-triazole-3(2H)-thione 8 (Scheme 2).

SYNTHESIS AND ANTIMICROBIAL PROPERTIES
471
S
N
N
N N
H
N
Et
CO2
N
N
H
CO2
S
a
N
S
2
OH
%
N
N
H
H
O
N
N
6
3
j
t
SCNCO2E
Et
CO2
S
N
N
N N
H
N
N
t
a
N
SCNCH2CO2E
2
OH
%
N
N
S
S
N
N
2
H
H
NH
N
H
O
N
N
S
CO2
3k
7
SCNCOPh
S
N
N
N N
H
N
Ph
CO
N
N
N
S
S
a
N
2
OH
%
N
N
H
H
NH
HN
O
N
N
S
8
3l
Scheme 2
The cyclization reaction in acidic media of the new thiosemicarbazide derivatives
enabled us to obtain new the new 1,3,4-thiadiazoles 5a,b,g.
The reactions were performed according to Schemes 1 and 2; the substituents are
listed in Table 1. The structure of the compounds obtained was confirmed by elementary
1
analysis, IR, and H NMR spectra and for some of the compounds also by the 13C NMR
and MS spectra.
In the IR spectra of the 1,2,4-triazole compounds 4a–i, 7, and 8, characteristic absorp-
tion bands at about 1500 cm−1 corresponding to C N group and in the range of 1600 cm−1
corresponding to C N group were observed. The IR spectra of the new 1,3,4-thiadiazole
derivatives 5a,b,g displayed characteristic absorption bands in the range of 1500 cm−1
corresponding to the C N group and in the range of 1600 cm−1 corresponding to the C N
group.
1
The H NMR spectra of the thiosemicarbazide derivatives 3a–l show three proton
signals typical for the NH protons in the range of 8.4–12.9 ppm. For the new 1,2,4-triazole
compounds 4a–g, 7, and 8, the signal of the NH proton was observed at 13.6–14.3 ppm.
The 1,3,4-thiadiazole derivatives 5a,b,g showed one typical proton signal at 10.1–10.3
ppm. 13C NMR spectroscopic data were obtained for compounds 3b,c, 4a–c, and 5a, while
compounds 3a,c and 4b–d were also characterized by MS spectrometry.
Microbiology
All new compounds were screened for in vitro antibacterial activity by the agar well
diffusion method. For compounds 3b,e,g–k, and 4b,g–i, which showed potential inhibitory
effect on the growth of bacteria, minimal inhibitory concentration (MIC) values were
estimated by microdilution technique.

472
Ł. POPIOŁEK ET AL.
Table 1 Substituents of compounds 3a–l, 4a–i, and 5a,b,g
Compounds
R
Compounds
R
3a, 4a, 5a
3g, 4g, 5g
Cl
3b, 4b, 5b
3c, 4c
3h, 4h
3i, 4i
3j
OCH3
OCH3
CH2
CH2
CH2
3d, 4d
3e, 4e
COOC2H5
3k
CH2COOC2H5
Br
3f, 4f
C2H5
3l
O
According to the screening results obtained with 1000 µg/mL of the new compounds
by the method mentioned above, some of them possess activity only against the Gram-
positive bacteria tested (Table 2); no activity against Gram-negative bacteria was found. In
our experiments, MICs of available antibiotics such as gentamicin and cefuroxime, which
are extensively used to treat bacterial infections, were also estimated for comparison.
Table 2 The inhibitory activities of the synthesized compounds against Gram-positive bacteria on the basis of
MIC (µg/mL) values determined by broth microdilution method
Compound
Sa25923
Sa6538
Se12228
Bs6633
Bc10876
Ml10240
3b
3e
3g
3h
3i
3j
3k
3l
1000
125
1000
1000
>1000
500
1000
125
1000
500
1000
500
1000
500
1000
62.5
125
1000
1000
500
1000
1000
1000
>1000
>1000
250
1000
>1000
>1000
1000
1000
250
1000
>1000
1000
1000
1000
125
>1000
>1000
1000
>1000
1000
250
>1000
125
1000
125
4b
4g
4h
4i
1000
1000
>1000
>1000
0.49
250
1000
1000
>1000
>1000
0.49
1000
1000
1000
1000
0.49
1000
1000
1000
1000
31.25
0.49
125
250
1000
1000
0.49
0.12
1000
>1000
1000
1.95
Cefuroxime
Gentamicin
0.015
0.06
0.06
0.015
Sa25923, Staphylococcus aureus ATCC 25923; Sa6538, Staphylococcus aureus ATCC 6538; Se12228, Staphy-
lococcus epidermidis ATCC 12228; Bs6633, Bacillus subtilis ATCC 6633; Bc10876, Bacillus cereus ATCC 10876;
Ml10240, Micrococcus luteus ATCC 10240.

SYNTHESIS AND ANTIMICROBIAL PROPERTIES
473
On the basis of the MIC values (Table 2) obtained by broth microdilution method,
it becomes evident that the highest activity against Gram-positive bacteria is shown by
compounds 3e (MIC = 62.5–500 µg/mL) and 3l (MIC = 125–250 µg/mL). This activity
may be due to the introduction of a 4-bromophenyl (3e) and a benzoyl (3l) substituent in
the 4 position of the thiosemicarbazide derivative. Our investigations also show that the
acyclic thiosemicarbazide derivatives display a better antimicrobial activity than the cyclic
compounds for all reference strains of Gram-positive bacteria. For comparison, MIC values
for reference strains of the tested bacteria were 0.015–0.49 µg/mL for gentamicin and
0.49–31.25 µg/mL for cefuroxime.
In summary, among the tested new compounds, only 3e and 3l exhibited moderate
antibacterial activity against both pathogenic and opportunistic Gram-positive bacteria and
may be of use for searching new derivatives showing better antimicrobial activity.
EXPERIMENTAL
Chemistry
Melting points were determined in Fisher–Johns blocks and are uncorrected. The IR
spectra (ν, cm−1) were recorded in KBr tablets using a Specord IR-75 spectrophotometer.
1
The H NMR spectra were recorded with a Bruker Avance 300 apparatus in DMSO-d6
with tetramethylsilane (TMS) as an internal standard. The 13C NMR spectra were recorded
with a Bruker Avance 300 instrument. Chemical shifts are given in ppm (δ-scale). The MS
spectra were recorded with a Thermo-Finnigan Trace DSQ GC-MS apparatus. Chemicals
were purchased from Merck Co. or Lancaster and used without further purification.
The purity of the compounds obtained was checked by TLC on aluminum oxide 60
F254 plates (Merck), in the CHCl3/C2H5OH (10:1, v/v) solvent system with UV visualization
(λ = 254 nm).
Ethyl [(4-Phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl] Acetate
(1). Approximately 0.23g (10 mmol) of sodium was added to 5 mL of anhydrous ethanol
(EtOH). The solution was placed in a three-necked flask equipped with reflux condenser
and closed with a tube of CaCl2 and stirred. Stirring was continued until the sodium
dissolved completely and then 2.54 g (10 mmol) of 4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-
triazole-3-thione was added. Then, 1.22 mL of ethyl bromoacetate was added dropwise.
The content of the flask was stirred for 4 h and left at r.t. for 12 h. Then, 10 mL of anhydrous
EtOH was added and the reaction solution was heated for 1 h. From the mixture, the
inorganic compounds were removed by filtration. After cooling of the reaction mixture, the
precipitate formed was filtered off and crystallized from EtOH. Yield: 2.43 g (71.4%); mp
72 ◦C–74 ◦C (dec.). IR (KBr), ν (cm−1): 3110 (CH aromatic), 2982, 1445 (CH aliphatic),
1722 (C O), 1602 (C N), 669 (C S). 1H NMR (DMSO-d6): δ = 1.20 (t, J = 6 Hz, 3H,
CH3), 4.12 (s, 2H, CH2), 4.13 (q, J = 5 Hz, 2H, CH2), 7.37–8.58 (m, 9H, arom-H). Anal.
calcd. for C17H16N4O2S (340.4): C, 59.98; H, 4.74; N, 16.46; S, 9.42; Found: C, 59.85; H,
4.76; N, 16.41; S, 9.37%.
[(4-Phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl] Acetohydrazide
(2). Approximately 0.5 mL of 100% hydrazine hydrate was added to 3.40 g (10 mmol)
of compound 1 in 10 mL of anhydrous EtOH. The mixture was left at r.t. for 24 h. The
precipitate of the hydrazide 2 was filtered off, dried, and crystallized from EtOH. Yield:
2.94 g (90.2%); mp 202 ◦C–206 ◦C (dec.). IR (KBr), ν (cm−1): 3079 (CH aromtic), 2977,
1
1455 (CH aliphatic), 1672 (C O), 1608 (C N), 1502 (C N), 673 (C S). H NMR

474
Ł. POPIOŁEK ET AL.
(DMSO-d6): δ = 3.93 (s, 2H, CH2), 4.31 (s, 2H, NH2), 7.37–8.58 (m, 9H, arom-H), 9.38
(s, 1H, NH). Anal. calcd. for C15H14N6OS (326.4): C, 55.20; H, 4.32; N, 25.75, S, 9.82;
Found: C, 55.16; H, 4.35; N, 25.80; S, 9.77%.
Thiosemicarbazides 3a–3l
Method A (for compounds 3a–l). A mixture of hydrazide 2 3.26 g (10 mmol)
and 10 mmol of the respective isothiocyanate was heated in an oil bath at 50 ◦C–110 ◦C for
8–20 h. The product was washed with diethyl ether to remove unreacted isothiocyanate.
Then, it was separated by filtration, and dried and crystallized from EtOH (3a,c,f,h,i–l),
butanol (3b,d,e), or methanol (3g).
Method B (for compounds 3a–d,f). Approximately 10 mmol of the appropriate
isothiocyanate was added to 3.26 g (10 mmol) of hydrazide 2 in 10 mL of anhydrous diethyl
ether. The mixture was placed in a conical bulb, stirred for 5 min, and left at r.t. for 24 h.
The precipitate of the thiosemicarbazide (3a–d,f) was separated by filtration, and dried and
crystallized from EtOH. The compounds thus obtained showed the same melting points as
the compounds obtained by the Method A.
1-{[(4-Phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}-4-
phenyl Thiosemicarbazide (3a). Yield: 3.29 g (71.4%). Temperature of reaction:
◦
◦
◦
50 C for 14 h, mp 172 C–174 C (dec.). IR (KBr), ν (cm−1): 3201 (NH), 3103 (CH
aromatic), 2983 (CH aliphatic), 1701 (C O), 1611 (C N), 1501 (C N), 1322 (C S),
1
679 (C S). H NMR (DMSO-d6): δ = 3.93 (s, 2H, CH2), 7.30–8.58 (m, 14H, arom-H),
9.36 (s, 1H, NH), 9.68 (s, 1H, NH), 10.44 (s, 1H, NH). MS m/z (%): 461 (M+, 2), 383 (1),
326 (5), 295 (12), 254 (38), 253 (46), 221 (4), 195 (7), 181 (8), 149 (9), 119 (15), 105 (38),
91 (34), 78 (33), 77 (100). Anal. calcd. for C22H19N7OS2 (461.6): C, 57.25; H, 4.15; N,
21.24; S, 13.89; Found C, 57.32; H, 4.13; N, 21.28; S, 13.94%.
4-(4-Methoxyphenyl)-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} Thiosemicarbazide (3b). Yield: 4.06 g (82.6%). Temperature
◦
◦
◦
of reaction: 50 C for 16 h, mp 172 C–174 C (dec.). IR (KBr), ν (cm−1): 3208 (NH),
3092 (CH aromatic), 2973, 1442, 756 (CH aliphatic), 1702 (C O), 1601 (C N), 1520
1
(C N), 1339 (C S), 683 (C S). H NMR (DMSO-d6): δ = 3.67 (s, 3H, CH3), 4.03 (s,
2H, CH2), 6.85–8.57 (m, 13H, arom-H), 9.59 (s, 1H, NH), 9.66 (s, 1H, NH), 10.41 (s,
1H, NH). 13C NMR (DMSO-d6): δ = 34.4 ( S CH2 ), 55.2 ( OCH3), 113.2 (CH),
122.8 (CH), 122.9 (CH), 123.5 (CH), 127.3 (CH), 127.6 (CH), 130.1 (CH), 130.4 (CH),
131.7 (CH), 133.2 (C), 135.3 (C), 148.2 (C), 150.6 (C), 156.8 (C S), 165.9 (C3 triazole),
166.7 (C O), 181.1 (C S). Anal. calcd. for C23H21N7O2S2 (491.6): C, 56.19; H, 4.30; N,
19.94; S, 13.04; Found C, 55.97; H, 4.29; N, 19.92; S, 13.10%.
4-Benzyl-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} Thiosemicarbazide (3c). Yield: 3.97 g (83.5%). Temperature of reaction:
◦
◦
◦
50 C for 10 h, mp 180 C–182 C (dec.). IR (KBr), ν (cm−1): 3211 (NH), 3097 (CH
aromatic), 2981, 1447 (CH aliphatic), 1705 (C O), 1601 (C N), 1518 (C N), 1347
1
(C S), 685 (C S). H NMR (DMSO-d6): δ = 3.92 (s, 2H, CH2), 4.75 (s, 2H, CH2),
7.14–8.53 (m, 14H, arom-H), 9.36 (s, 1H, NH), 9.54 (s, 1H, NH), 10.41 (s, 1H, NH).
13C NMR (DMSO-d6): δ = 33.7 ( S CH2 ), 46.7 ( CH2 ), 123.5 (CH), 123.7 (CH),
126.6 (CH), 127.0 (CH), 127.7 (CH), 127.9 (CH), 130.1 (CH), 130.3 (CH), 130.4 (CH),
130.6 (CH), 133.3 (C), 135.4 (C), 139.4 (C), 152.6 (C S), 166.0 (C3 triazole), 166.8
(C O), 182.1 (C S). MS m/z (%): 475 (M+, 0.8), 384 (1.5), 326 (6), 295 (12), 254 (35),
253 (37), 221 (3), 195 (7), 149 (16), 119 (7), 91 (100), 78 (11), 77 (31). Anal. calcd. for

SYNTHESIS AND ANTIMICROBIAL PROPERTIES
475
C23H21N7OS2 (475.6): C, 58.08; H, 4.45; N, 20.61; S, 13.48; Found C, 57.88; H, 4.44; N,
20.59; S, 13.44%.
4-Cyclohexyl-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} Thiosemicarbazide (3d). Yield: 3.83 g (81.9%). Temperature of reaction:
◦
◦
80 C for 12 h, mp 185–187 C (dec.). IR (KBr), ν (cm−1): 3220 (NH), 3099 (CH
aromatic), 2978, 1434, 765 (CH aliphatic), 1703 (C O), 1610 (C N), 1516 (C N), 1343
(C S), 695 (C S). 1H NMR (DMSO-d6): δ = 1.03–1.79 (m, 10H, cyclohexyl-CH2), 3.93
(s, 2H, CH2), 4.12 (m, 1H, cyclohexyl-CH), 7.37–8.59 (m, 9H, arom-H), 9.29 (s, 1H, NH),
9.36 (s, 1H, CH), 10.19 (s, 1H, NH). Anal. calcd. for C22H25N7OS2 (467.6): C, 56.51; H,
5.39; N, 20.97; S, 13.71; Found C, 56.47; H, 5.34; N, 21.01; S, 13.80%.
4-(4-Bromophenyl)-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} Thiosemicarbazide (3e). Yield: 5.16 g (95.5%). Temperature of
reaction: 110 ◦C for 14 h, mp 163 ◦C–165 ◦C (dec.). IR (KBr), ν (cm−1): 3211 (NH), 3093
(CH aromatic), 2977 (CH aliphatic), 1702 (C O), 1609 (C N), 1331 (C S), 689 (C S).
1H NMR (DMSO-d6): δ = 4.01 (s, 2H, CH2), 7.36–8.58 (m, 13H, arom-H), 9.76 (s, 1H,
NH), 9.88 (s, 1H, NH), 10.47 (s, 1H, NH). Anal. calcd. for C22H18BrN7OS2 (540.5):
C, 48.89; H, 3.36; N, 18.14; S, 11.86; Br, 14.78; Found C, 48.85; H, 3.37; N, 18.20; S,
11.90%.
4-Ethyl-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} Thiosemicarbazide (3f). Yield: 3.34 g (80.9%). Temperature of reaction 60
◦C for 8 h, mp 194–196 ◦C (dec.). IR (KBr), ν (cm−1): 3117 (NH), 3092 (CH aromatic),
2972, 1447, 763 (CH aliphatic), 1701 (C O), 1602 (C N), 1511 (C N), 1334 (C S),
683 (C S). 1H NMR (DMSO-d6): δ = 1.14 (t, J = 9 Hz, 3H, CH3), 3.60 (q, J = 7.5 Hz,
2H, CH2), 3.83 (s, 2H, CH2), 7.43–8.56 (m, 9H, arom-H), 8.35 (s, 1H, NH), 9.42 (s, 1H,
NH), 10.21 (s, 1H, NH). Anal. calcd. for C18H19N7OS2 (413.5): C, 52.28; H, 4.63; N,
23.71; S, 15.51; Found C, 52.15; H, 4.62; N, 23.79; S, 15.44%.
4-(4-Chlorophenyl)-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} Thiosemicarbazide (3g). Yield: 4.59 g (92.7%). Temperature of
reaction: 100 ◦C for 16 h, mp 162 ◦C–164 ◦C (dec.). IR (KBr), ν (cm−1): 3217 (NH), 3108
(CH aromatic), 2974 (CH aliphatic), 1699 (C O), 1611 (C N), 1348 (C S), 685 (C S).
1H NMR (DMSO-d6): δ = 4.09 (s, 2H, CH2), 7.43–8.64 (m, 13H, arom-H), 9.85 (s, 1H,
NH), 9.96 (s, 1H, NH), 10.55 (s, 1H, NH). Anal. calcd. for C22H18ClN7OS2 (496.0): C,
53.27; H, 3.66; N, 19.77; S, 12.93; Cl, 7.15; Found C, 53.32; H, 3.67; N, 19.70; S, 12.99%.
4-(4-Methoxybenzyl)-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} Thiosemicarbazide (3h). Yield: 4.94 g (97.7%). Temperature
◦
◦
◦
of reaction: 55 C for 12 h, mp 196 C–198 C (dec.). IR (KBr), ν (cm−1): 3218 (NH),
3095 (CH aromatic), 2977, 1449, 757 (CH aliphatic), 1701 (C O), 1607 (C N), 1519
1
(C N), 1336 (C S), 689 (C S). H NMR (DMSO-d6): δ = 3.75 (s, 3H, CH3), 4.05 (s,
2H, CH2), 4.74 (s, 2H, CH2), 6.85–8.63 (m, 13H, arom-H), 8.67 (s, 1H, NH), 9.57 (s, 1H,
NH), 10.46 (s, 1H, NH). Anal. calcd. for C24H23N7O2S2 (505.6): C, 57.01; H, 4.58; N,
19.39; S, 12.68; Found C, 56.92; H, 4.57; N, 19.44; S, 12.72%.
4-Allyl-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} Thiosemicarbazide (3i). Yield: 3.32 g (78.2%). Temperature of reac-
tion: 50 ◦C for 12 h, mp 170 ◦C–172 ◦C (dec.). IR (KBr), ν (cm−1): 3209 (NH), 3092 (CH
aromatic), 2970, 1429, 759 (CH aliphatic), 1701 (C O), 1611 (C N), 1520 (C N), 1336
(C S), 689 (C S). 1H NMR (DMSO-d6): δ = 3.99 (s, 2H, CH2), 4.18 (d, J = 5 Hz, 2H,
CH2,), 5.02–5.13 (m, 2H, = CH2), 5.79–5.88 (m, 1H, CH), 7.40–8.56 (m, 9H, arom-H),
9.37 (s, 1H, NH), 9.47 (s, 1H, NH), 10.35 (s, 1H, NH). Anal. calcd. for C19H19N7OS2

476
Ł. POPIOŁEK ET AL.
(425.5): C, 53.63; H, 4.50; N, 23.04; S, 15.07; Found C, 53.76; H, 4.49; N, 22.96;
S, 15.00%.
4-Ethoxycarbonyl-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} Thiosemicarbazide (3j). Yield: 4.45 g (97.4%). Temperature
◦
◦
◦
of reaction: 50 C for 16 h, mp 176 C–178 C (dec.). IR (KBr), ν (cm−1): 3199 (NH),
3110 (CH aromatic), 2961, 1448, 755 (CH aliphatic), 1732 (C O acidic), 1700 (C O),
1603 (C N), 1518 (C N), 1348 (C S), 690 (C S). 1H NMR (DMSO-d6): δ = 1.26 (t,
J = 7.5 Hz, 3H, CH3), 4.04 (s, 2H, CH2), 4.10 (q, J = 7.5 Hz, 2H, CH2), 7.48–8.64 (m,
9H, arom-H), 11.13 (s, 1H, NH), 11.45 (s, 1H, NH), 11.61 (s, 1H, NH). Anal. calcd. for
C19H19N7O3S2 (457.5): C, 49.88; H, 4.18; N, 21.43; S, 14.02; Found C, 49.81; H, 4.19;
N, 21.44; S, 14.07%.
4-Ethoxycarbonylmetyl-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} Thiosemicarbazide (3k). Yield: 3.82 g (81.2%). Temperature of
reaction: 50 ◦C for 18 h, mp 184 ◦C–186 ◦C (dec.). IR (KBr), ν (cm−1): 3201 (NH), 3082
(CH aromatic), 2986, 1484, 751 (CH aliphatic), 1729 (C O acidic), 1701 (C O), 1599
1
(C N), 1499 (C N), 1349 (C S), 684 (C S). H NMR (DMSO-d6): δ = 1.20 (t, J =
7.5 Hz, 3H, CH3), 4,04 (s, 2H, CH2), 4.10 (q, J = 7.5 Hz, 2H, CH2), 4.34 (s, 2H, CH2),
7.50–8.61 (m, 9H, arom-H), 8.79 (s, 1H, NH), 9.74 (s, 1H, NH), 10.53 (s, 1H, NH). Anal.
calcd. for C20H21N7O3S2 (471.6): C, 50.94; H, 4.49; N, 20.79; S, 13.60; Found C, 51.08;
H, 4.50; N, 20.81; S, 13.54%.
4-Benzoyl-1-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} Thiosemicarbazide (3l). Yield: 4.82 g (98.4%). Temperature of reaction:
◦
◦
◦
50 C for 16 h, mp 162 C–164 C (dec.). IR (KBr), ν (cm−1): 3186 (NH), 3079 (CH
aromatic), 2976, 1456 (CH aliphatic), 1748 (C O acidic), 1708 (C O), 1609 (C N),
1
1511 (C N), 1359 (C S), 689 (C–S). H NMR (DMSO-d6): δ = 4.16 (s, 2H, CH2),
7.51–8.63 (m, 14H, arom-H), 11.39 (s, 1H, NH), 11.87 (s, 1H, NH), 12.92 (s, 1H, NH).
Anal. calcd. for C23H19N7O2S2 (489.6): C, 56.43; H, 3.91; N, 20.03; S, 13.10; Found C,
56.37; H, 3.92; N, 19.95; S, 13.03%.
4H-1,2,4-Triazole-3(2H)-thiones (4a–4i): General Procedure
A mixture of the thiosemicarbazide 3a–i (10 mmol) and 20–40 mL of 2% aqueous
solution of sodium hydroxide was refluxed for 2 h. Then, the solution was neutralized
with diluted hydrochloric acid and the precipitate formed was separated by filtration and
crystallized from EtOH (4a,b,f,h), butanol (4e,g,i), or methanol (4c,d).
5-{[(4-Phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4-
phenyl-4H-1,2,4-triazole-3(2H)-thione (4a). Yield: 3.36 g (75.8%); mp 210 ◦C–212
◦C (dec.). IR (KBr), ν (cm−1): 3169 (NH), 3079 (CH aromatic), 2955 (CH aliphatic),
1602 (C N), 1506 (C N), 1368 (C S), 689 (C S). 1H NMR (DMSO-d6): δ = 4.19 (s,
2H, CH2), 7.15–8.57 (m, 14H, arom-H), 13.98 (s, 1H, NH). 13C NMR (DMSO-d6): δ =
27.3 ( S CH2 ), 122.8 (CH), 123.4 (CH), 123.6 (CH), 127.5 (CH), 127.6 (CH), 128.2
(CH), 128.7 (CH), 129.3 (CH), 129.6 (CH), 130.3 (CH), 133.2 (C), 135.4 (C), 135.8 (C),
150.6 (C3‘ triazole), 150.9 (C–S), 152.5 (C3 triazole), 168.2 (C = S). Anal. calcd. for
C22H17N7S2 (443.5): C, 59.57; H, 3.86; N, 22.10; S, 14.46; Found C, 59.43; H, 3.85; N,
22.06; S, 14.50%.
4-(4-Methoxyphenyl)-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (4b). Yield: 4.30 g (90.9%);
◦
◦
mp 105 C–107 C (dec.). IR (KBr), ν (cm−1): 3189 (NH), 3091 (CH aromatic), 2974,

SYNTHESIS AND ANTIMICROBIAL PROPERTIES
477
1
1456 (CH aliphatic), 1610 (C N), 1500 (C N), 1355 (C S), 695 (C S). H NMR
(DMSO-d6): δ = 3.85 (s, 3H, CH3), 4.17 (s, 2H, CH2), 7.06–8.57 (m, 13H, arom-H), 13.82
(s, 1H, NH). 13C NMR (DMSO-d6): δ = 33.1 ( S CH2 ), 55.6 ( OCH3), 114.5 (CH),
123.6 (CH), 125.6 (CH), 127.6 (CH), 128.4 (CH), 128.9 (CH), 129.6 (CH), 129.8 (CH),
130.4 (CH), 133.2 (C), 135.8 (C), 136.1 (C), 159.8 (C), 150.5 (C3‘ triazole), 159.7 (C S),
168.9 (C3 triazole), 169.2 (C S). MS m/z (%): 473 (M+, 0.02), 368 (0.22), 354 (0.05), 328
(0.52), 312 (0.62), 295 (0.14), 268 (5), 254 (100), 253 (91), 221 (4), 195 (21), 168 (7), 149
(23), 119 (21), 104 (20), 91 (27), 78 (25), 77 (67). Anal. calcd. for C23H19N7OS2 (473.6):
C, 58.33; H, 4.04; N, 20.70; S, 13.54; Found C, 58.52; H, 4.03; N, 20.69 S, 13.59%.
4-Benzyl-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
◦
methyl}-4H-1,2,4-triazole-3(2H)-thione (4c). Yield: 3.46 g (75.7%); mp 103 C–
105 ◦C (dec.). IR (KBr), ν (cm−1): 3149 (NH), 3076 (CH aromatic), 2958, 1421, 757 (CH
1
aliphatic), 1611 (C N), 1506 (C N), 1351 (C S), 693 (C S). H NMR (DMSO-d6):
δ = 4.36 (s, 2H, CH2), 5.30 (s, 2H, CH2), 7.29–8.58 (m, 14H, arom-H), 13.87 (s, 1H,
NH). 13C NMR (DMSO-d6): δ = 26.4 ( S CH2), 45.9 (CH2), 122.9 (CH), 123.6 (CH),
123.9 (CH), 127.0 (CH), 127.7 (CH), 128.6 (CH), 128.7 (CH), 129.5 (CH), 130.1 (CH),
130.4 (CH), 133.0 (C), 135.3 (C), 135.8 (C), 150.3 (C3‘ triazole), 150.6 (C S), 152.4 (C3
triazole), 168.8 (C S). MS m/z (%): 457 (M+, 0.04), 368 (0.02), 344 (0.03), 312 (0.03),
295 (0.5), 268 (3), 221 (5), 195 (22), 181 (7), 168 (9), 149 (17), 119 (12), 104 (17), 91
(34), 78 (19), 77 (51). Anal. calcd. for C23H19N7S2 (457.6): C, 60.37; H, 4.18; N, 21.43;
S, 14.01; Found C, 60.60; H, 4.18; N, 21.38; S, 14.08%.
4-Cyclohexyl-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
◦
methyl}-4H-1,2,4-triazole-3(2H)-thione (4d). Yield: 3.56 g (79.3%); mp 162 C–
164 ◦C (dec.). IR (KBr), ν (cm−1): 3164 (NH), 3072 (CH aromatic), 2983, 1459, 767 (CH
aliphatic), 1606 (C N), 1503 (C N), 1344 (C S), 680 (C S). 1H NMR (DMSO-d6): δ =
1.18–1.72 (m, 10H, cyclohexyl-CH2), 4,05 (s, 2H, CH2), 4.47 (m, 1H, cyclohexyl-CH),
7.34–8.58 (m, 9H, arom-H), 13,56 (s, 1H, NH). MS m/z (%): 449 (M+, 0.2), 367 (0.03),
339 (0.08), 292 (0.06), 254 (61), 253 (60), 221 (14), 195 (15), 181 (5), 149 (26), 127
(8), 119 (23), 104 (17), 91 (32), 83 (12), 78 (41), 77 (100). Anal. calcd. for C22H23N7S2
(449.6): C, 58.77; H, 5.16; N, 21.81; S, 14.26; Found C, 58.87; H, 5.18; N, 21.76; S,
14.30%.
4-(4-Bromophenyl)-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (4e). Yield: 4.68 g (89.6%);
◦
◦
mp 150 C–152 C (dec.). IR (KBr), ν (cm−1): 3177 (NH), 3096 (CH aromatic), 2975,
1
1463 (CH aliphatic), 1605 (C N), 1508 (C N), 1324 (C S), 679 (C S). H NMR
(DMSO-d6): δ = 4.22 (s, 2H, CH2), 7.27–8.58 (m, 13H, arom-H), 13.90 (s, 1H, NH).
Anal. calcd. for C22H16BrN7S2 (522.4): C, 50.58; H, 3.09; N, 18.77; S, 12.27; Br, 15.29;
Found C, 50.64; H, 3.10; N, 18.80; S, 12.21%.
4-Ethyl-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
◦
methyl}-4H-1,2,4-triazole-3(2H)-thione (4f). Yield: 3.17 g (80.1%); mp 218 C–
220 ◦C (dec.). IR (KBr), ν (cm−1): 3145 (NH), 3081 (CH aromatic), 2962, 1429, 763 (CH
1
aliphatic), 1612 (C N), 1507 (C N), 1333 (C S), 695 (C S). H NMR (DMSO-d6):
δ = 1.04 (t, J = 7.5 Hz, 3H, CH3), 3.70 (q, J = 7.5 Hz, 2H, CH2), 4.06 (s, 2H, CH2),
7.36–8.57 (m, 9H, arom-H), 13.98 (s, 1H, NH). Anal. calcd. for C18H17N7S2 (395.5): C,
54.66; H, 4.33; N, 24.79; S, 16.21; Found C, 54.51; H, 4.34; N, 24.76; S, 16.18%.
4-(4-Chlorophenyl)-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (4g). Yield: 4.69 g (98.1%);
◦
◦
mp 118 C–120 C (dec.). IR (KBr), ν (cm−1): 3153 (NH), 3068 (CH aromatic), 2975,

478
Ł. POPIOŁEK ET AL.
1
1449 (CH aliphatic), 1598 (C N), 1507 (C N), 1314 (C S), 678 (C S). H NMR
(DMSO-d6): δ = 4.23 (s, 2H, CH2), 7.30–8.58 (m, 13H, arom-H), 13.92 (s, 1H, NH).
Anal. calcd. for C22H16ClN7S2 (478.0): C, 55.28; H, 3.37; N, 20.51; S, 13.42; Cl, 7.42;
Found C, 55.08; H, 3.36; N, 20.52; S, 13.37%.
4-(4-Methoxybenzyl)-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (4h). Yield: 3.11 g (63.8%);
◦
◦
mp 112 C–114 C (dec.). IR (KBr), ν (cm−1): 3152 (NH), 3061 (CH aromatic), 2972,
1463, 751 (CH aliphatic), 1610 (C N), 1507 (C N), 1338 (C S), 679 (C S). 1H NMR
(DMSO-d6): δ = 3.72 (s, 3H, CH3), 4.31 (s, 2H, CH2), 5.20 (s, 2H, CH2), 6.83–8.60 (m,
13H, arom-H), 13.84 (s, 1H, NH). Anal. calcd. for C24H21N7OS2 (487.6): C, 59.12; H,
4.34; N, 20.11; S, 13.15; Found C, 59.07; H, 4.33; N, 20.08; S, 13.20%.
4-Allyl-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]
methyl}-4H-1,2,4-triazole-3(2H)-thione (4i). Yield: 3.19 g (78.2%); mp 170
◦
◦C–172 C (dec.). IR (KBr), ν (cm−1): 3187 (NH), 3086 (CH aromatic), 2976, 1443,
1
749 (CH aliphatic), 1602 (C N), 1501 (C N), 1357 (C S), 678 (C S). H NMR
(DMSO-d6): δ = 4.38 (s, 2H, CH2), 4.64 (d, J = 5 Hz, 2H, CH2,), 5.18–5.25 (m, 2H, =
CH2), 5.83–5.95 (m, 1H, CH), 7.38–8.58 (m, 9H, arom-H), 13.74 (s, 1H, NH). Anal. calcd.
for C19H17N7S2 (407.5): C, 56.00; H, 4.20; N, 24.06; S, 15.74; Found C, 55.92; H, 4.24;
N, 24.10; S, 15.79%.
Synthesis of 1,3,4-thiadiazoles 5a,b,g
Method A (for compounds 5a,b,g). Approximately 10 mmol of the respective
thiosemicarbazide 3a,b,g was dissolved in 10–20 mL of diluted sulfuric acid and stirred
in a closed bulb for 1 h. Subsequently, the solution was poured on crushed ice (50 g) and
stirred until the ice was completely dissolved. The solution was neutralized with ammonium
hydroxide. The precipitate formed was separated by filtration, dried, and crystallized from
EtOH (5a,b) or butanol (5g).
Method B (for compound 5a). Around 20 mL of 10% ethanolic solution of
hydrochloric acid was added to thiosemicarbazide 3a and the reaction mixture was heated
under reflux for 1 h. Subsequently, the solution was left at r.t. for 24 h. The precipitate
formed was separated by filtration, dried, and crystallized from EtOH. The compound
obtained had the same melting point as the compound obtained by Method A.
Method C (for compound 5g). A mixture of 10 mmol of thiosemicarbazide 3g
in 10 mL of anhydrous acetic acid was refluxed for 1 h. Subsequently, the solution was left
at r.t. for 12 h. The precipitate formed was separated by filtration, dried, and crystallized
from butanol. The compound thus obtained had the same melting point as the compound
obtained by Method A.
(2-Aminophenyl)-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-1,3,4-thiadiazole (5a). Yield: 3.23 g (72.8%); mp 182 ◦C–
184 ◦C (dec.). IR (KBr), ν (cm−1): 3249 (NH), 3098 (CH aromatic), 2948 (CH aliphatic),
1
1609 (C N), 1507 (C N), 691 (C S). H NMR (DMSO-d6): δ = 4.63 (s, 2H, CH2),
7.39–8.55 (m, 14H, arom-H), 10.34 (s, 1H, NH). 13C NMR (DMSO-d6): δ = 29.4
( S CH2 ), 116.1 (CH), 120.6 (CH), 121.5 (CH), 122.2 (CH), 126.3 (CH), 128.7 (CH),
128.8 (CH), 129.0(CH), 146.9 (CH), 149.3 (CH), 131.9 (C), 134.0 (C), 139.2 (C), 149.9
(C S), 151.3 (C3 triazole), 154.3 (C2 thiadiazole), 164.1 (C5 thiadiazole). Anal. calcd. for

SYNTHESIS AND ANTIMICROBIAL PROPERTIES
479
C22H17N7S2 (443.5): C, 59.57; H, 3.86; N, 22.10; S, 14.46; Found C, 59.81; H, 3.87; N,
22.03; S, 14.51%.
2-[Amino-(4-methoxyphenyl)]-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-
triazol-3-yl) sulfanyl]methyl}-1,3,4-thiadiazole (5b). Yield: 4.16 g (87.8%); mp
192 ◦C–194 ◦C (dec.). IR (KBr), ν (cm−1): 3199 (NH), 3056 (CH aromatic), 2981, 1468
1
(CH aliphatic), 1603 (C N), 1500 (C N), 679 (C S). H NMR (DMSO-d6): δ = 3.65
(s, 3H, CH3), 4.24 (s, 2H, CH2), 7.32–8.60 (m, 13H, arom-H), 10.16 (s, 1H, NH). Anal.
calcd. for C23H19N7OS2 (473.6): C, 58.33; H, 4.04; N, 20.70; S, 13.54; Found C, 58.25;
H, 4.05; N, 20.62; S, 13.49%.
2-[Amino-(4-chlorophenyl)]-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-
triazol-3-yl) sulfanyl]methyl}-1,3,4-thiadiazole (5g). Yield: 4.27 g (89.3%); mp
◦
◦
208 C–210 C (dec.). IR (KBr), ν (cm−1): 3281 (NH), 3066 (CH aromatic), 2951 (CH
aliphatic), 1600 (C N), 1510 (C N), 692 (C S). 1H NMR (DMSO-d6): δ = 4.71 (s, 2H,
CH2), 7.35–8.58 (m, 13H, arom-H), 10.26 (s, 1H, NH). Anal. calcd. for C22H16ClN7S2
(478.0): C, 55.28; H, 3.37; N, 20.51; S, 13.42; Cl, 7.42; Found C, 55.11; H, 3.38; N, 20.56;
S, 13.48%.
[(4-Phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl] acetic acid (6).
Compound 6 was obtained using the same method described for derivatives 4a–i. A mix-
ture of thiosemicarbazide 3j (10 mmol) and 20 mL of 2% aqueous solution of sodium
hydroxide was refluxed for 2 h. Subsequently, the solution was neutralized with diluted
hydrochloric acid and the precipitate formed was separated by filtration and crystallized
◦
◦
from EtOH. Yield: 2.47 g (79.2%); mp 222 C–224 C (dec.). IR (KBr), ν (cm−1): 3106
(CH aromatic), 3099 (OH), 2983 (CH aliphatic), 1710 (C O), 1604 (C N), 669 (C S).
1H NMR (DMSO-d6): δ = 4.02 (s, 2H, CH2), 7.41–8.57 (m, 9H, arom-H), 12.68 (s, 1H,
OH). Anal. calcd. for C15H12N4O2S (312.3): C, 57.68; H, 3.87; N, 17.94; S, 10.26; Found
C, 57.90; H, 3.88; N, 17.87; S, 10.21%.
4-Carboxymethyl-5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (7). Compound 7 was obtained
using the same method described for derivatives 4a–i. A mixture of thiosemicarbazide
3k (10 mmol) and 20 mL of 2% aqueous solution of sodium hydroxide was refluxed for
2 h. Subsequently, the solution was neutralized with diluted hydrochloric acid and the
precipitate formed◦was separated by filtration and crystallized from EtOH. Yield: 1.18 g
◦
(27.8%); mp 222 C–224 C (dec.). IR (KBr), ν (cm−1): 3311 (NH), 3067 (OH), 3041
(CH aromatic), 2946, 1461, (CH aliphatic), 1699 (C O), 1600 (C N), 1502 (C N),
1
1345 (C S), 699 (C S). H NMR (DMSO-d6): δ = 4.51 (s, 2H, CH2), 4.85 (s, 2H,
CH2), 7.39–8.63 (m, 9H, arom-H), 13.85 (s, 1H, OH), 14.30 (s, 1H, NH). Anal. calcd. for
C18H15N7O2S2 (425.5): C, 50.81; H, 3.55; N, 23.04; S, 15.07; Found C, 50.64; H, 3.54;
N, 23.10; S, 15.12%.
5-{[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-2,5-
dihydro-4H-1,2,4-triazole-3(2H)-thione (8). Compound 8 was obtained using the
same method described for derivatives 4a–i. A mixture of thiosemicarbazide 3l (10 mmol)
and 20 mL of 2% aqueous solution of sodium hydroxide was refluxed for 2 h. Subsequently,
the solution was neutralized with diluted hydrochloric acid and the precipitate formed ◦was
separated by filtration and crystallized from EtOH. Yield: 2.39 g (65.0%); mp 152 C–
154 ◦C (dec.). IR (KBr), ν (cm−1): 3187 (NH), 3034 (CH aromatic), 2984 (CH aliphatic),
1597 (C N), 1505 (C N), 1322 (C S), 679 (C S). 1H NMR (DMSO-d6): δ = 3.87 (s,
2H, CH2), 7.43–8.56 (m, 9H, arom-H), 13.67 (s, 1H, NH), 13.99 (s, 1H, NH). Anal. calcd.

480
Ł. POPIOŁEK ET AL.
for C16H13N7S2 (367.4): C, 52.30; H, 3.57; N, 26.68; S, 17.45; Found C, 52.42; H, 3.58;
N, 26.61; S, 17.39%.
MICROBIOLOGY
Materials and Methods
A panel of 10 reference strains of aerobic bacteria from American Type Culture
Collection, including Gram-positive Staphylococcus aureus ATCC 25923, S. aureus ATCC
6538, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus
cereus ATCC 10876, Micrococcus luteus ATCC 10240, and Gram-negative Escherichia
coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453,
Pseudomonas aeruginosa ATCC 9027, were used. Microbial suspensions were prepared
in sterile saline (0.85% NaCl) with an optical density of 0.5 McFarland standard – 150 ×
106 CFU/mL (CFU – colony forming units). All stock solutions of the tested compounds
were dissolved in dimethyl sulfoxide (DMSO). The medium with DMSO at the final
concentration and without the tested compounds served as control – no microbial growth
inhibition was observed.
Preliminary in vitro antimicrobial potency of the tested compounds was screened
using the agar dilution method on the basis of the bacterial growth inhibition on the
Mueller–Hinton agar to which the tested compounds at concentration 1000 µg/mL were
added. The plates were poured on the day of testing. Approximately 10 µL of each bacterial
suspension was put onto the prepared solid media. The plates were incubated at 37 ◦C for
18 h.
The in vitro antibacterial activity of the potentially active tested compounds was
determined on the basis of MIC, usually defined as the lowest concentration of a compound
at which there was no visible growth of tested microorganisms. Determination of the MIC
value was achieved by broth microdilution method according to CLSI (Clinical and Lab-
oratory Standards Institute) recommendation with some modifications.21 Mueller–Hinton
broth was used with a series of two-fold dilutions of the tested substances in the range of
final concentrations from 3.91 to 1000 µg/mL. Cefuroxime (belonging to second genera-
tion of cephalosporins) and gentamicin were used as control antimicrobial agents at a final
concentration from 0.063 to 500 µg/mL.
In broth microdilution method, 96-well microplates were used; 198 µL of
Mueller–Hinton broth with or without the tested compound was inoculated with 2 µL◦of
microbial suspension (total volume per each well – 200 µL). After incubation (at 35 C
for 18 h), spectrophotometric measurements of optical density (OD600) of the bacterial
cultures with or without the tested compounds were performed in order to determine MIC.
The blank control wells with two-fold dilution of each of the tested compounds added to
Mueller–Hinton broth (total volume – 200 µL) without bacteria were incubated under the
same conditions.
REFERENCES
1. Dobosz, M.; Struga, M.; Chodkowska, A.; Jagiełło-Wo´jtowicz, E.; Stepniak, K.; Kozioł, A. E.
ꢀ
Acta Pol. Pharm. 2002, 59, 281-290.
2. Turan-Zitouni, G.; Kaplancikli, Z. A.; Erol, K.; Kilic¸, F. S. Il Farmaco 1999, 54, 218-223.

SYNTHESIS AND ANTIMICROBIAL PROPERTIES
481
3. Collin, X.; Sauleau, A.; Coulon, J. Bioorg. Med. Chem. Lett. 2003, 13, 2601-2605.
4. Wei, Q.-L.; Zhang, S.-S.; Gao, J.; Li, W.-H.; Xu, L.-Z.; Yu, Z.-G. Bioorg. Med. Chem. 2006, 14,
7146-7153.
5. Gu¨lerman, N. N.; Dog˘an, H. N.; Rollas, S.; Johansson, C.; C¸ elik, C. Il Farmaco 2001, 56,
953-958.
¨
6. Ulusoy, N.; Gu¨rsoy, A.; Otu¨k, G. Il Farmaco 2001, 56, 947-952.
7. Byrak, H.; Demirbas, A.; Demirbas, N.; Karaoglu, S. A. Eur. J. Med. Chem. 2009, 44, 4362-4366.
8. Al-Soud, Y. A.; Al-Dweri, M. N.; Al-Masoudi, N. A. Il Farmaco 2004, 59, 775-783.
9. De La Rosa, M.; Kim, H. W.; Gunic, E.; Jenket, Ch.; Boyle, U.; Koh, Y.-H.; Korboukh, I.; Allan,
M.; Zhang, W.; Chen, H.; Xu, W.; Nilar, S.; Yoa, N.; Hamatake, R.; Lang, S. A.; Hong, Z.;
Zhang, Z.; Girardet, J.-L. Bioorg. Med. Chem. Lett. 2006, 16, 4444-4449.
10. Palaska, E.; S¸ahin, G.; Kelicen, P.; Durlu, N. T.; Altinok, G. Il Farmaco 2002, 57, 101-107.
11. Kumar, H.; Javed, S. A.; Khan, S. A.; Amir, M. Eur. J. Med. Chem. 2008, 43, 2688-2698.
12. Klimesˇova´, V.; Zahajska´, L.; Waisser, K.; Kaustova´, J.; Mo¨llmann, U. Il Farmaco 2004, 59,
279-288.
13. Jadhav, S.; Rai, M.; Durrani, A.; Bembalkar, S. R. Orient. J. Chem. 2010, 26, 725-728.
14. Dobosz, M.; Pachuta-Stec, A. Acta Pol. Pharm. 1996, 53, 123-131.
15. Dobosz, M.; Pachuta-Stec, A. Acta Pol. Pharm. 1995, 52, 103-111.
16. Dobosz, M.; Pitucha, M.; Wujec, M. Acta Pol. Pharm. 1996, 53, 31-38.
17. Popiołek, Ł.; Dobosz, M.; Chodkowska, A.; Jagiełło-Wo´jtowicz, E.; Kosikowska, U.; Malm, A.;
Mazur, L.; Rzaczyn´ska, Z. J. Heterocycl. Chem. 2011, 48, 339-346.
ꢀ
18. Maliszewska-Guz, A.; Wujec, M.; Pitucha, M.; Dobosz, M.; Chodkowska, A.; Jagiełło-
Wo´jtowicz, E.; Mazur, L.; Kozioł, A. E. Collect. Czech. Chem. Commun. 2005, 70, 51-62.
19. Hawrył, A.; Popiołek, Ł.; Dobosz, M.; Pikula, E.; Waksmundzka-Hajnos, M. Acta Chromatogr.
2010, 22, 37-55.
20. Kaplaushenko, A. H.; Panasenko, O. I.; Knish, E. H.; Shcherbina, R. O. Farm. Zh. 2008, 2, 67-72.
21. CLSI. Performance Standards for Antimicrobial Susceptibility Testing (Eighteenth International
Supplement CLSI document M7-MIC); Clinical Laboratory Standards Institute: Wayne, PA,
2008.