Synthesis and antimicrobial evaluation of some novel 1,2,4-triazole and 1,3,4-thiadiazole derivatives

Article abstract of DOI:10.1007/s00044-012-0302-9

This study presents the synthesis and spectral analysis of new derivatives of 1,2,4-triazole-3-thione and 1,3,4-thiadiazole. New compounds were prepared by cyclization reaction of acyl thiosemicarbazide derivatives in the presence of alkaline and acidic media. All synthesized compounds were screened for their in vitro antibacterial activity by using the agar dilution technique. Six of the compounds had potential activity against Gram-positive bacteria (minimal inhibitory concentration [MIC] = 15.63-500 μg/mL). Some compounds showed good activity especially against Bacillus subtilis ATCC 6633 (MIC = 15.63-250 μg/mL), Staphylococcus aureus ATCC 25923 (MIC = 31.25-250 μg/mL), and Micrococcus luteus ATCC 10240 (MIC = 125-250 μg/mL).

Full text of DOI:10.1007/s00044-012-0302-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3134–3147
DOI 10.1007/s00044-012-0302-9
ORIGINAL RESEARCH
Synthesis and antimicrobial evaluation of some novel
1,2,4-triazole and 1,3,4-thiadiazole derivatives
•
Łukasz Popiołek Urszula Kosikowska
•
•
Liliana Mazur Maria Dobosz Anna Malm
•
Received: 12 March 2012 / Accepted: 24 October 2012 / Published online: 11 November 2012
ꢀ The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract This study presents the synthesis and spectral
analysis of new derivatives of 1,2,4-triazole-3-thione and 1,3,
4-thiadiazole. New compounds were prepared by cyclization
reaction of acyl thiosemicarbazide derivatives in the presence
of alkaline and acidic media. All synthesized compounds were
screened for their in vitro antibacterial activity by using the agar
dilution technique. Six of the compounds had potential activity
against Gram-positive bacteria (minimal inhibitory concentra-
tion [MIC] = 15.63–500 lg/mL). Some compounds showed
good activity especially against Bacillus subtilis ATCC 6633
(MIC = 15.63–250 lg/mL), Staphylococcus aureus ATCC
25923 (MIC = 31.25–250 lg/mL), and Micrococcus luteus
ATCC 10240 (MIC = 125–250 lg/mL).
in various applications, such as pharmaceuticals, propel-
lants, explosives, and pyrotechnics.
The recent literature is enriched with progressive find-
ings about the synthesis and pharmacological action of
triazole and thiadiazole derivatives. Heterocycles bearing
1,2,4-triazole and 1,3,4-thiadiazole moiety are reported to
show a broad spectrum of biologic activity such as anal-
gesic (Turan-Zitouni et al., 1999), antiphlogistic (Harish
et al., 2008; El Shehry et al., 2010; Schenone et al., 2006),
anticonvulsant (Dogan et al., 2002; Almasirad et al., 2004),
antitumor (Duran et al., 2002; Kumar et al., 2010), anti-
viral (Al-Soud et al., 2004), antifungal (Collin et al., 2003;
Wei et al., 2006), antibacterial (Ulusoy et al., 2001; Gu¨l-
erman et al., 2001; Padmavathi et al., 2009; Demirbas
et al., 2009; Liesen et al., 2010), and antitubercular action
Keywords Antimicrobial activity ꢀ
ˇ
´
1,2,4-Triazole derivatives ꢀ 1,3,4-Thiadiazole derivatives
(Klimesova et al., 2004; Gadad et al., 2004; Shiradkar
et al., 2007). A large number of ring systems containing
triazoles and thiadiazoles have been incorporated into a
wide variety of therapeutically interesting drug candidates.
Some of them are approved as drugs, for example,
alprazolam (Pick, 1997), etizolam (Shiroki et al., 1976), or
vibunazole (Holmwood et al., 1982). Vorozole, letrozole,
and anastrozole are non-steroidal drugs used for the treat-
ment of cancer (Clemons et al., 2004). Triazoles are also
used as intermediates for the synthesis of antifungal agents
such as fluconazole, voriconazole, and itraconazole (Bailey
et al., 1990; McGinnis et al., 1997).
Introduction
For the last few decades, there has been a tremendous
growth of research in the synthesis of nitrogen and sulfur
containing heterocyclic derivatives because of their utility
Ł. Popiołek (&) ꢀ M. Dobosz
Department of Organic Chemistry, Faculty of Pharmacy,
Medical University, Lublin, Poland
e-mail: lukasz.popiolek@umlub.pl
In continuation of our research program on the synthesis
of 1,2,4-triazole and 1,3,4-thiadiazole compounds exhibit-
ing biologic activity, it was thought to be interesting to
synthesize new antimicrobial agents, especially when the
development of resistance of pathogenic bacteria toward
available antibiotics is rapidly becoming a major world-
wide problem. The designing of new compounds to deal
with resistant bacteria has become one of the most
U. Kosikowska ꢀ A. Malm
Department of Pharmaceutical Microbiology, Faculty
of Pharmacy, Medical University, Lublin, Poland
L. Mazur
Department of General and Coordination Chemistry, Faculty
of Chemistry, Maria Curie-Sklodowska University,
Lublin, Poland
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Med Chem Res (2013) 22:3134–3147
3135
important areas of antibacterial research today. In addition,
primary and opportunistic microbial infections continue to
increase rapidly because of the increased number of
immunocompromised patients.
In three cases, the cyclization reaction of thiosemicarba-
zide derivatives 4j–l in alkaline media was accompanied by
hydrolysis. The [(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfa-
nyl] acetic acid 8 was obtained in cyclization of 4-ethox-
ycarbonyl-1-substituted thiosemicarbazide 4j. This compound
was described earlier, but it was obtained in a different way
(Kaplaushenko et al., 2008). The cyclization in alkaline
media of the thiosemicarbazide which contains the ethoxy-
carbonylmethyl group 4k and benzoyl 4l in the fourth posi-
tion led us to obtain substituted 1,2,4-triazole-3-thione
derivatives 9, 10. These compounds were subjected to the
reaction with pyrrolidine and formaldehyde to get new
N-substituted 1,2,4-triazole-3-thione derivatives 11, 12.
The thiosemicarbazide derivatives 4a–i were also sub-
mitted to the cyclization reaction in acidic media. In this
way, we were able to obtain new compounds which consist
of 1,2,4-triazole-3-thione and 1,3,4-thiadiazole system, that
is (5-aminosubstituted)-2-{[(4,5-diphenyl-4H-1,2,4-triazol-
3-yl)sulfanyl]methyl}-1,3,4-thiadiazole 6a–i. Afterward,
the derivatives of N,N-disubstituted acetamide 7a–i were
obtained by the acylation reaction of 2,5-disubstituted-
1,3,4-thiadiazoles 6a–i with acetic anhydride.
Keeping in mind the above facts, we designed and
synthesized series of some new 1,2,4-triazole-3-thione and
1,3,4-thiadiazole derivatives and evaluated their in vitro
antibacterial activity.
Results and discussion
Chemistry
The substituted 1,2,4-triazole and 1,3,4-thiadiazole deriv-
atives are generally obtained by the cyclization reaction of
thiosemicarbazide derivatives, which is dependent not only
on the pH of the medium, but also on the nature of sub-
stituents in thiosemicarbazide derivatives (Dobosz and
Pachuta-Stec, 1995, 1996). The presence of alkaline media
usually promotes the reaction of cyclization to obtain 1,2,
4-triazole systems, whereas in acidic media, 1,3,4-thiadia-
zole derivatives were obtained.
The mechanism of cyclization of thiosemicarbazide was
investigated earlier (Siwek and Paneth, 2007). It was
proved that the direction of cyclization is dependent on the
nature of substituents and acidic or alkaline media (Siwek
et al., 2010).
4,5-Diphenyl-4H-1,2,4-triazole-3-thione 1 was a starting
material for the synthesis of new compounds, which consist
of two 1,2,4-triazole systems or 1,2,4-triazole and 1,3,
4-thiadiazole systems connected with the S-methylene
group. Compound 1 was obtained by the cyclization
reaction of 1,4-diphenyl thiosemicarbazide in alkaline
media. In the next step, compound 1, which can exist in
two tautomeric forms, was submitted to the reaction with
ethyl bromoacetate in the presence of sodium ethanolate.
The reaction let us obtain ethyl 2-[(4,5-diphenyl-4H-1,2,4-
triazol-3-yl)sulfanyl] acetate (2). The direction of this
reaction to form a thio derivative of compound 1 was
revealed and confirmed by X-ray crystallography (Dobosz
et al., 1996). The mechanism of this reaction as a nucle-
ophilic substitution on the sulfur atom had been studied and
investigated earlier (Wujec and Paneth, 2007).
The structure of all obtained compounds was confirmed
1
by elementary analysis, IR and H NMR spectra. Some of
the compounds were also submitted to 13C NMR and MS
spectra analyses. The crystal structure of the representative
compound 2 was determined by the single-crystal X-ray
analysis. The reactions were performed according to
Schemes 1 and 2.
In the IR spectra of the thiosemicarbazide derivatives 4a–l,
the following characteristic absorption bands were observed:
about 1,700 cm-1 corresponding to the C=O group and in the
range of 1,300 cm-1 corresponding to the C=S group. Com-
pounds which consist of two 1,2,4-triazole systems 5a–i, 9, 10
had absorption bands: about 1,300 cm-1 (C=S group), about
1,500 cm-1 (C–N group), in the range of 1,600 cm-1 (C=N
group), andabout3,100–3,200 cm-1 (NH group). Then, in the
IR spectra of the new derivatives of 1,3,4-thiadiazole 6a–i, the
following characteristic absorption bands were observed: in
the range of 1,500 cm-1 corresponding to the C–N group and
in the range of 1,600 cm-1 corresponding to the C=N group
and about 3,200 cm-1 for the NH group. Compounds 7a–i, 11
had a characteristic absorption band at about 1,700 cm-1 for
the C=O group.
Subsequently, compound 2 was converted to hydrazide
3 in reaction with 100 % hydrazine hydrate. Then, reac-
tions of hydrazide 3 with various isothiocyanates were
performed in two ways.
All new thiosemicarbazide derivatives 4a–l were obtained
by heating reactants in an oil bath; temperatures were
selected experimentally (t = 50–110 ꢁC). Thiosemicarba-
zide derivatives 4a, c, d were products of the reaction of
hydrazide 3 with appropriate isothiocyanates in the pres-
ence of diethyl ether carried in room temperature.
A new group of compounds, which consist of two 1,2,
4-triazole-3-thione derivatives 5a–i, were acquired in
cyclization reaction with 2 % aqueous solution of sodium
hydroxide of new acyl thiosemicarbazide derivatives 4a–i.
1H NMR spectra of the thiosemicarbazide derivatives
4a–l show three proton signals typical for the NH group in
the d 8.32–12.87 ppm range, whereas for the new com-
pounds consisting of two 1,2,4-triazole system 5a–i, 9, 10,
123

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Med Chem Res (2013) 22:3134–3147
Scheme 1 Synthesis of new derivatives of thiosemicabrazide, 1,2,4-triazole-3-thione and 1,3,4-thiadiazole
one proton signal of the NH group was observed in the
d 13.62–14.13 ppm range. The 1,3,4-thiadiazole deriva-
tives 6a–i had one typical proton signal of the NH group in
the d 9.35–10.47 ppm range. Derivatives of N,N-disubsti-
tuted acetamide 7a–i had one proton signal of the CH3
group in the d 2.06–2.16 ppm range. Compound 11 had one
proton signal for the OH group (d 13.68 ppm) and for the
pyrrolidine substituent. Similarly, 4,5-disubstituted-2-
(pyrrolidin-1-ylmethyl)-1,2,4-triazole-3-thione 12 had one
typical proton signal for the NH group (d 14.68 ppm) and
for the pyrrolidine substituent.
thioacetate unit intersects that of the 1,2,4-triazole ring at the
angle of 81.4(1)ꢁ. The carbonyl C2=O2 group in 2 is cis ori-
ented with respect to the thioether S1 atom. What is more, it
seems to be preferred in thioacetate derivatives in the solid state
(CSD, V.5.33, Allen, 2002). The geometric parameters of the
ester group are within normal ranges (International Tables for
Crystallography, 1995). Likewise, the S1–C3 and S1–C1 dis-
˚
tances, being of 1.738(2) and 1.789(3) A, are in agreement with
the single thioether C–S bonds. The most characteristic feature
of the crystal of 2 is the presence of centrosymmetric molecular
dimers. The ‘‘head-to-head’’ oriented molecules within the
i
˚
Compound 2 crystallizes in the monoclinic space group P21/
n with one molecule in the asymmetric unit of the crystal. The
diffraction study confirmed that the molecule contained the
1,2,4-triazole ring, substituted at C3, N4, and C5 atoms by
thioacetate moiety and two phenyl rings, respectively (Fig. 1).
The chain of atoms from S1 to ethyl C4 is almost planar
dimer form short S1ꢀꢀꢀO2 [3.268(3) A; (i) 1 - x, -y, -z]
contacts which might be attractive in their nature (Ramasubbu
and Parthasarathy, 1989).
Microbiology
˚
(rmsd = 0.006 A); a higher twist (4.56ꢁ) is observed around the
C4–O1 bond in the solid state. The best plane of the atoms of
On the basis of the preliminary results obtained by the agar
dilution method, it was shown that some of the newly
123

Med Chem Res (2013) 22:3134–3147
3137
Scheme 2 Synthesis of new derivatives of 1,2,4-triazole-3-thione
synthesized compounds had the potential activity against
reference strains of Gram-positive bacteria. None of the
compounds had inhibitory effect on the Gram-negative bac-
teria growth.
the benzyl group in 1,3,4-thiadiazole derivative yielded
active compounds endowed with a wide spectrum of anti-
microbial activities.
The compounds 4l and 6h with potential activity against
the reference strains of Gram-positive bacteria may be
regarded as precursor compounds for searching for new
derivatives showing antimicrobial activity against patho-
genic (e.g. S. aureus) or opportunistic (e.g. S. epidermidis,
M. luteus, B. subtilis, or B. cereus) bacteria.
According to Table 1, on the basis of minimal inhibitory
concentration (MIC) values obtained by the broth microdilution
method, it was shown that the highest activity had compound 4l
with MIC = 31.25 lg/mL against Staphylococcus aureus
ATCC 25923, MIC = 125 lg/mL against Staphylococcus
epidermidis ATCC 12228, Bacillus cereus ATCC 10876, and
Micrococcus luteus ATCC 10240 or MIC = 250 lg/mL against
S. aureus ATCC 6538 and Bacillus subtilis ATCC 6633. Com-
pound 6h was also active especially against B. subtilis ATCC
6633 with MIC = 15.63 lg/mL and with MIC = 125 lg/mL
against M. luteus ATCC 10240 or MIC = 250 lg/mL against S.
aureus ATCC 25923.
Experimental
Chemistry
Melting points were determined in Fisher–Johns blocks
(Pittsburgh, US) and presented without any corrections.
The IR spectra (m, cm-1) were recorded in KBr tablets
using a Specord IR-75 spectrophotometer (Germany). The
NMR spectra were recorded on a Bruker Avance 300
apparatus (Bruker BioSpin GmbH, Rheinstetten/Karlsruhe,
Germany) in dimethyl sulfoxide (DMSO)-d6 with TMS as
the internal standard, and chemical shifts are given in ppm
(d-scale). The MS spectra were recorded on a Thermo-
The somewhat lower activity against reference strains of
Gram-positive bacteria was shown by compound 5c (MIC val-
ues from 250 to 1,000 lg/mL). According to our results, MICs of
cefuroxime, which has been extensively used to treat bacterial
infections, were 0.24–1.95 lg/mL for Staphylococcus species
and 0.49–62.5 lg/mL for the other Gram-positive bacteria.
With our research, it has been established that the
introduction of the benzoyl group in thiosemicarbazide and
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Med Chem Res (2013) 22:3134–3147
Fig. 1 Molecular structure of 2
with atom-labeling scheme.
Displacement ellipsoids are
drawn at the 50 % probability
level. Selected bond distances
´
˚
(A): C3–S1 1.738(2), C1–S1
1.789(3), C1–C2 1.494(4),
C2–O1 1.321(3), C2–O2
1.191(3), C4–O1 1.460(3)
Table 1 The inhibitory
activities of newly synthesized
compounds against Gram-
positive bacteria on the basis of
MIC (lg/mL) values
Species:
Compounds
Sa25923
MIC (lg/mL)
Sa6538
Se12228
Bs6633
Bc10876
Ml10240
4c
[1000
500
[1000
1000
[1000
250
[1000
500
1000
500
1000
1000
500
1000
1000
1000
125
4i
determined by broth
microdilution method
4j
[1000
31.25
1000
500
1000
125
500
4l
250
125
5b
1000
250
1000
500
1000
500
1000
1000
500
1000
250
Bold values indicate the lowest
MIC nd Not determined,
Sa25923 S. aureus ATCC
25923, Sa6538 S. aureus ATCC
6538, Se12228 S. epidermidis
ATCC 12228, Bs6633 B.
subtilis ATCC 6633, Bc10876
B. cereus ATCC 10876,
Ml10240 M. luteus ATCC
10240
5c
5d
1000
1000
1000
[1000
250
[1000
[1000
1000
1000
nd
[1000
[1000
1000
[1000
500
[1000
[1000
[1000
[1000
15.63
0.49
[1000
[1000
1000
[1000
125
5g
500
5h
[1000
[1000
nd
5i
6h
Cefuroxime
0.49
1.95
0.24
62.5
0.49
Finnigan Trace DSQ GC MS apparatus (Waltham, Mas-
sachusetts, US). Chemicals were purchased from Merck
Co., or Lancaster and used without further purification.
The purity of the obtained compounds was checked by
TLC on aluminum oxide 60 F254 plates (Merck Co., White-
house Station, New Jersey, US), in a CHCl3/C2H5OH (10:1,
v/v) solvent system with UV visualization (k = 254 nm).
Elemental analysis of the obtained compounds was
performed for C, H, N, S. The maximum percentage dif-
ferences between calculated and found values for each
element were within the error and amounted to 0.4 %.
restraints/parameters 3930/0/217 (Rint = 0.04), final R indices
(I[2r(I)): R1 = 0.0548, wR2 = 0.0888, R indices (all data):
R1 = 0.1867, wR2 = 0.1202, largest diff. peak and hole: 0.16
-3
˚
and -0.17 e A
.
Single-crystal diffraction data were measured at room
temperature on an Oxford Diffraction Xcalibur diffrac-
tometer with the graphite-monochromated Mo Ka radiation
(k = 0.71073). The programs CrysAlis CCD and CrysAlis
Red (Oxford Diffraction, Xcalibur CCD System, 2006)
were used for data collection, cell refinement, and data
reduction. The intensity data were corrected for Lorentz
and polarization effects. The structure was solved by direct
methods using SHELXS-97 and refined by the full-matrix
least-squares on F2 using the SHELXL-97 (Sheldrick,
2008). All non-hydrogen atoms were refined with aniso-
tropic displacement parameters. All H-atoms were posi-
tioned geometrically and allowed to ride on their parent
atoms with Uiso(H) = 1.2 Ueq(C).
Crystal data for 2
C18H17N3O2S, colorless prism, 0.45 9 0.29 9 0.14 mm3,
˚
monoclinic, P21/n, a = 11.692(1) A, b = 9.414(1) A,
˚
3
˚
˚
c = 15.740(2) A, b = 100.24(1)ꢁ, V = 1,704.9(3) A , Z = 4,
dcalc = 1.322 g cm-3, l = 0.205 mm-1, GooF = 0.977, data/
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Med Chem Res (2013) 22:3134–3147
3139
Crystallographic data have been deposited with the
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax:
?44 1223 366033; e-mail: deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk) and are available on request,
quoting the deposition number CCDC 860357.
21.52; S, 9.82; found: C, 59.10; H, 4.63; N, 21.49; S, 9.78. IR
(KBr), m (cm-1): 3105 (CH aromatic), 2980, 1423 (CH ali-
phatic), 1698 (C=O), 1611 (C=N), 1522 (C–N), 699 (C–S).
1H NMR (DMSO-d6) d (ppm): 3.91 (s, 2H, CH2), 4.31 (s, 2H,
NH2), 7.31–7.57 (m, 10H, 10ArH), 9.40 (brs, 1H, NH).
Ethyl 2-[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)
sulfanyl]acetate (2)
Derivatives of thiosemicarbazide (4a–l)
General method (for compounds 4a–l)
Method A
A mixture of 3.25 g (10 mmol) of hydrazide (3) and
10 mmol appropriate isothiocyanate was heated in an oil
bath at 50–110 ꢁC for 8–20 h. The product was washed
with diethyl ether to remove unreacted isothiocyanate.
Then it was filtered, dried, and crystallized from ethanol
4a–c, d, g–l, butanol 4e, or methanol 4f.
0.23 g (10 mmol) of sodium was added to 5 mL of anhy-
drous ethanol. The solution was placed in a three-necked
flask equipped with reflux condenser and closed with a tube
of CaCl2 and mercury stirred. The content was mixed till the
sodium dissolved completely and then 2.53 g (10 mmol) of
4,5-diphenyl-4H-1,2,4-triazole-3-thione (1) 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 of anhydrous ethanol was
added and heated for 1 h. The mixture was filtered of inor-
ganic compounds. After cooling, the precipitate was filtered
and crystallized from ethanol.
Method B (for compounds 4a, c, d)
10 mmol of appropriate isothiocyanate was added to 3.25 g
(10 mmol) of hydrazide 3 in 10 mL of anhydrous diethyl
ether. The mixture, placed in a conical bulb, was mixed for
5 min and left in room temperature for 24 h. The precipi-
tation of thiosemicarbazide 4a, c, d was filtered, dried, and
crystallized from ethanol. The obtained compounds had the
same melting points as the compounds obtained by the
general method.
Method B
2.53 g (10 mmol) of 4,5-diphenyl-4H-1,2,4-triazole-3-
thione (1) was dissolved in 10 mL of N,N-dimethylform-
amide. Then, 1 g of potassium carbonate and 1.22 mL of
ethyl bromoacetate were added to the solution. The content
of the flask was refluxed for 2 h. The mixture was filtered
of inorganic compounds. Then, the distilled water was
added and the precipitated compound was filtered, dried,
and crystallized from ethanol.
4-Ethyl-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} thiosemicarbazide (4a) Yield: 94.0 %. Tempera-
ture of reaction: 70 ꢁC for 8 h, mp: 205–207 ꢁC (dec.).
Analysis for C19H20N6OS2 (412.53); calculated: C, 55.32;
H, 4.89; N, 20.37; S, 15.54; found: C, 55.23; H, 4.88; N,
20.43; S, 15.59. IR (KBr), m (cm-1): 3199 (NH), 3101 (CH
aromatic), 2974, 1453, 741 (CH aliphatic), 1699 (C=O),
1607 (C=N), 1519 (C–N), 1329 (C=S), 691 (C–S). 1H
NMR (DMSO-d6) d (ppm): 1.12 (t, J = 9 Hz, 3H, CH3),
3.51–3.60 (q, J = 7.5 Hz, J = 7.5 Hz, 2H, CH2), 3.90 (s,
2H, CH2), 7.34–7.57 (m, 10H, 10ArH), 8.32, 9.33, 10.25
(3brs, 3H, 3NH). 13C NMR d (ppm): 14.61 (CH3), 30.75 (–
S–CH2–), 33.90 (–CH2–CH3), 126.42, 127.68, 127.95,
128.79, 130.07, 130.11 (10CH aromatic), 130.33, 133.65
(2C aromatic), 152.08 (C–S), 154.59 (C-3 triazole), 166.82
(C=O), 181.23 (C=S). MS m/z (%): 412 (M?, 2), 397 (3),
335 (2), 325 (5), 294 (26), 253 (61), 252 (100), 194 (21),
180 (20), 149 (20), 118 (23), 104 (25), 91 (44), 77 (79).
Yield: 67.8 %, mp: 92–94 ꢁC (dec.). Analysis for
C18H17N3O2S (339.41); calculated: C, 63.70; H, 5.05; N,
12.38; S, 9.45; found: C, 63.92; H, 5.03; N, 12.41; S, 9.48.
IR (KBr), m (cm-1): 3091 (CH aromatic), 2955, 1422 (CH
1
aliphatic), 1701 (C=O), 1611 (C=N), 676 (C–S). H NMR
(DMSO-d6) d (ppm): 1.19 (t, J = 6 Hz, 3H, CH3), 4.09 (s,
2H, CH2), 4.11–4.17 (q, J = 5 Hz, J = 5 Hz, 2H, CH2),
7.31–7.58 (m, 10H, 10ArH).
[(4,5-Diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetohydrazide (3)
0.5 mL of 100 % hydrazine hydrate was added to 3.39 g
(10 mmol) of compound 2 in 10 mL of anhydrous ethanol.
The mixture was left at room temperature for 24 h. The
precipitation of hydrazide 3 was filtered, dried, and crys-
tallized from ethanol.
4-Allyl-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} thiosemicarbazide (4b) Yield: 90.7 %. Tempera-
ture of reaction: 55 ꢁC for 12 h, mp: 192–194 ꢁC (dec.).
Analysis for C20H20N6OS2 (424.54); calculated: C, 56.58;
H, 4.75; N, 19.79; S, 15.10; found: C, 56.53; H, 4.76; N,
19.81; S, 15.14. IR (KBr), m (cm-1): 3218 (NH), 3078 (CH
Yield: 91.4 %, mp: 196–198 ꢁC (dec.). Analysis for
C16H15N5OS (325.39); calculated: C, 59.06; H, 4.65; N,
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Med Chem Res (2013) 22:3134–3147
aromatic), 2963, 1431, 761 (CH aliphatic), 1705 (C=O),
1603 (C=N), 1511 (C–N), 1351 (C=S), 686 (C–S). 1H
NMR (DMSO-d6) d (ppm): 3.95 (s, 2H, CH2), 4.13 (d,
J = 5 Hz, 2H, CH2), 5.02–5.13 (dd, J = 5 Hz, J = 5 Hz,
2H, =CH2), 5.79–5.90 (m, 1H, CH), 7.35–7.57 (m, 10H,
10ArH), 8.45, 9.45, 10.33 (3brs, 3H, 3NH).
55.80; H, 3.87; N, 16.98; S, 12.95; Cl, 9.16; found: C, 55.83;
H, 3.88; N, 16.93; S, 12.90. IR (KBr), m (cm-1): 3202 (NH),
3093 (CH aromatic), 2983 (CH aliphatic), 1705 (C=O), 1608
1
(C=N), 1338 (C=S), 688 (C–S). H NMR (DMSO-d6) d
(ppm): 3.98 (s, 2H, CH2), 7.31–7.56 (m, 14H, 14ArH), 9.81,
9.88, 10.46 (3brs, 3H, 3NH). 13C NMR d (ppm): 34.39 (–S–
CH2–), 121.95, 125.63, 128.40, 128.56, 128.76, 129.37,
129.54, 130.08 (14CH aromatic), 128.60, 130.19, 133.65,
137.92 (4C aromatic), 151.63 (C–S), 154.30 (C-3 triazole),
166.85 (C=O), 180.84 (C=S).
4-Cyclohexyl-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sul-
fanyl]acetyl} thiosemicarbazide (4c) Yield: 64.5 %.
Temperature of reaction: 50 ꢁC for 12 h, mp: 188–190 ꢁC
(dec.). Analysis for C23H26N6OS2 (466.62); calculated: C,
59.20; H, 5.62; N, 18.01; S, 13.74; found: C, 59.35; H, 5.63;
N, 17.95; S, 13.70. IR (KBr), m (cm-1): 3208 (NH), 3109
(CH aromatic), 2987, 1424, 753 (CH aliphatic), 1699
(C=O), 1595 (C=N), 1519 (C–N), 1331 (C=S), 689 (C–S).
1H NMR (DMSO-d6) d (ppm): 1.01–1.72 (m, 10H, 5CH2
cyclohexane), 3.87 (s, 2H, CH2), 4.31 (m, 1H, CH cyclo-
hexane), 7.28–7.56 (m, 10H, 10ArH), 8.71, 9.35, 10.20
(3brs, 3H, 3NH).
4-(4-Methoxyphenyl)-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} thiosemicarbazide(4g) Yield: 95.2 %.
Temperature of reaction: 60 ꢁC for 18 h, mp:172–174 ꢁC(dec.).
Analysis for C24H22N6O2S2 (490.60); calculated: C, 58.75; H,
4.52; N, 17.13; S, 13.07; found: C, 58.97; H, 4.51; N, 17.18; S,
13.10. IR (KBr), m (cm-1): 3198 (NH), 3102 (CH aromatic),
2988, 1452, 759 (CH aliphatic), 1710 (C=O), 1605 (C=N), 1519
(C–N), 1329 (C=S), 693 (C–S). 1H NMR (DMSO-d6) d (ppm):
3.74 (s, 3H, CH3), 3.99 (s, 2H, CH2), 6.90 (d, J = 6 Hz, 2H,
2ArH), 7.32–7.56 (m, 10H, 10ArH), 7.57 (d, J = 6 Hz, 2H,
2ArH), 9.61, 9.66, 10.40 (3brs, 3H, 3NH).
4-Phenyl-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} thiosemicarbazide (4d) Yield: 91.0 %. Tempera-
ture of reaction: 50 ꢁC for 15 h, mp: 178–180 ꢁC (dec.).
Analysis for C23H20N6OS2 (460.57); calculated: C, 59.98; H,
4.38; N, 18.25; S, 13.92; found: C, 60.03; H, 4.38; N, 18.30; S,
13.96. IR (KBr), m (cm-1): 3205 (NH), 3114 (CH aromatic),
2978 (CH aliphatic), 1705 (C=O), 1610 (C=N), 1516 (C–N),
1337 (C=S), 685 (C–S). 1H NMR (DMSO-d6) d (ppm): 4.00
(s, 2H, CH2), 7.12–7.51 (m, 15H, 15ArH), 9.38, 9.76, 10.47
(3brs, 3H, 3NH). 13C NMR d (ppm): 34.55 (–S–CH2–),
125.23, 125.79, 126.45, 127.77, 127.92, 128.09, 128.75,
130.07, 130.15 (15CH aromatic), 130.36, 133.78, 139.09 (3C
aromatic), 151.75(C–S), 154.48(C-3triazole), 166.95(C=O),
180.98 (C=S). MS m/z (%): 460 (M?, 1), 383 (1.2), 325 (13),
294 (20), 252 (60), 194 (10), 180 (10), 149 (8), 135 (74), 131
(5), 104 (25), 91 (33), 77 (100).
4-Benzyl-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} thiosemicarbazide (4h) Yield: 95.0 %. Tempera-
ture of reaction: 50 ꢁC for 12 h, mp: 176–180 ꢁC (dec.).
Analysis for C24H22N6OS2 (474.60); calculated: C, 60.74;
H, 4.67; N, 17.71; S, 13.51; found: C, 60.77; H, 4.66; N,
17.78; S, 13.55. IR (KBr), m (cm-1): 3209 (NH), 3087 (CH
aromatic), 2971, 1439 (CH aliphatic), 1700 (C=O), 1611
(C=N), 1520 (C–N), 1351 (C=S), 689 (C–S). 1H NMR
(DMSO-d6) d (ppm): 3.90 (s, 2H, CH2), 4.84 (s, 2H, CH2),
7.15–7.54 (m, 15H, 15ArH), 8.82, 9.54, 10.41 (3brs, 3H,
3NH). 13C NMR d (ppm): 33.68 (–S–CH2–), 46.62 (–CH2–
), 126.47, 127.12, 127.46, 127.83, 128.16, 128.51, 128.83,
129.83, 130.04 (15CH aromatic), 133.71, 134.71, 139.34
(3C aromatic), 151.95 (C–S), 154.32 (C-3 triazole), 166.79
(C=O), 182.09 (C=S).
4-(4-Bromophenyl)-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} thiosemicarbazide (4e) Yield: 88.3 %.
Temperature of reaction: 110 ꢁC for 16 h, mp: 188–190 ꢁC
(dec.). Analysis for C23H19BrN6OS2 (539.47); calculated:
C, 51.21; H, 3.55; N, 15.58; S, 11.88; Br, 14.81; found: C,
51.27; H, 3.54; N, 15.61; S, 11.92. IR (KBr), m (cm-1):
3213 (NH), 3116 (CH aromatic), 2972 (CH aliphatic), 1703
(C=O), 1600 (C=N), 1341 (C=S), 690 (C–S). 1H NMR
(DMSO-d6) d (ppm): 3.97 (s, 2H, CH2), 7.29–7.55 (m,
14H, 14ArH), 9.79, 9.82, 10.46 (3brs, 3H, 3NH).
4-(4-Methoxybenzyl)-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]acetyl} thiosemicarbazide (4i) Yield: 97.4 %.
Temperature of reaction: 50 ꢁC for 14 h, mp: 176–178 ꢁC
(dec.). Analysis for C25H24N6O2S2 (504.63); calculated: C,
59.50; H, 4.79; N, 16.65; S, 12.71; found: C, 59.61; H,
4.78; N, 16.68; S, 12.75. IR (KBr), m (cm-1): 3222 (NH),
3102 CH (aromatic), 2973, 1448, 767 (CH aliphatic), 1697
(C=O), 1599 (C=N), 1514 (C–N), 1349 (C=S), 680 (C–S).
1H NMR (DMSO-d6) d (ppm): 3.76 (s, 3H, CH3), 4.01 (s,
2H, CH2), 4.74 (s, 2H, CH2), 6.86–7.64 (m, 14H, 14ArH),
8.33, 9.55, 10.44 (3brs, 3H, 3NH).
4-(4-Chlorophenyl)-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)
sulfanyl]acetyl} thiosemicarbazide (4f) Yield: 97.8 %.
Temperature of reaction: 100 ꢁC for 16 h, mp: 180–184 ꢁC
(dec.). Analysis for C23H19ClN6OS2 (495.02); calculated: C,
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Med Chem Res (2013) 22:3134–3147
3141
4-Ethoxycarbonyl-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)
sulfanyl]acetyl} thiosemicarbazide (4j) Yield: 98.6 %.
Temperature of reaction: 55 ꢁC for 14 h, mp: 178–180 ꢁC
(dec.). Analysis for C20H20N6O3S2 (456.54); calculated: C,
52.62; H, 4.41; N, 18.41; S, 14.05; found: C, 52.76; H,
4.42; N, 18.44; S, 14.01. IR (KBr), m (cm-1): 3219 (NH),
3105 (CH aromatic), 2973, 1452, 765 (CH aliphatic), 1728
(C=O acidic), 1699 (C=O), 1608 (C=N), 1511 (C–N), 1338
(C=S), 691 (C–S). 1H NMR (DMSO-d6) d (ppm): 1.22
(t, J = 5 Hz, 3H, CH3), 4.09 (s, 2H, CH2), 4.12–4.21 (q,
J = 7.5 Hz, J = 7.5 Hz, 2H, CH2), 7.28–7.56 (m, 10H,
10ArH), 11.07, 11.38, 11.51 (3brs, 3H, 3NH).
found: C, 57.67; H, 4.59; N, 21.33; S, 16.21. IR (KBr), m
(cm-1): 3135 (NH), 3085 (CH aromatic), 2958, 1422, 758
(CH aliphatic), 1600 (C=N), 1502 (C–N), 1350 (C=S), 692
1
(C–S). H NMR (DMSO-d6) d (ppm): 1.22 (t, J = 5 Hz,
3H, CH3), 3.91–3.97 (q, J = 5 Hz, J = 5 Hz, 2H, CH2),
4.39 (s, 2H, CH2), 7.27–7.54 (m, 10H, 10ArH), 13.62 (s,
1H, NH). MS m/z (%): 394 (M?, 0.2), 365 (0.1), 339
(0.12), 264 (0.1), 253 (64), 252 (68), 194 (21), 149 (33),
128 (16), 118 (37), 104 (10), 91 (58), 77 (100).
4-Allyl-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfa-
nyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5b) Yield:
90.5 %, mp: 207–208 ꢁC (dec.). Analysis for C20H18N6S2
(406.53); calculated: C, 59.10; H, 4.46; N, 20.67; S, 15.77; found:
C, 58.96; H, 4.45; N, 20.64; S, 15.74. IR (KBr), m (cm-1): 3185
(NH), 3091 (CH aromatic), 2989, 1450, 756 (CH aliphatic), 1604
(C=N), 1510 (C–N), 1343 (C=S), 684 (C–S). 1H NMR (DMSO-
d6)d(ppm):4.44 (s, 2H, CH2), 4.69–4.71(d, J = 5 Hz,2H,CH2),
5.24–5.41 (dd, J = 5 Hz, J = 5 Hz, 2H, =CH2), 5.82–5.93 (m,
1H, CH), 7.37–7.62 (m, 10H, 10ArH), 13.81 (brs, 1H, NH).
4-Ethoxycarbonylmethyl-1-{[(4,5-diphenyl-4H-1,2,
4-triazol-3-yl)sulfanyl]acetyl}
thiosemicarbazide
(4k)
Yield: 91.9 %. Temperature of reaction: 50 ꢁC for 14 h, mp:
188–190 ꢁC (dec.). Analysis for C21H22N6O3S2 (470.57);
calculated: C, 53.60; H, 4.71; N, 17.86; S, 13.63; found: C,
53.46; H, 4.72; N, 17.90; S, 13.67. IR (KBr), m (cm-1): 3211
(NH), 3096 (CH aromatic), 2975, 1464, 758 (CH aliphatic),
1737 (C=O acidic), 1703 (C=O), 1611 (C=N), 1511 (C–N),
1344 (C=S), 686 (C–S). 1H NMR (DMSO-d6) d (ppm): 1.16
(t, J = 5 Hz, 3H, CH3), 3.96 (s, 2H, CH2), 4.02–4.11 (q,
J = 7.5 Hz, J = 7.5 Hz, 2H, CH2), 4.30 (s, 2H, CH2),
7.33–7.58 (m, 10H, 10ArH), 8.78, 9.69, 10.47 (3brs, 3H,
3NH).
4-Cyclohexyl-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfa
-nyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5c) Yield:
62.4 %, mp: 186–188 ꢁC (dec.). Analysis for C23H24N6S2
(448.61); calculated: C, 61.58; H, 5.39; N, 18.73; S, 14.29;
found: C, 61.37;H, 5.38;N, 18.68; S, 14.32. IR (KBr), m(cm-1):
3175 (NH), 3088 (CH aromatic), 2963, 1449, 759 (CH ali-
phatic), 1611 (C=N), 1505 (C–N), 1339 (C=S), 684 (C–S). 1H
NMR (DMSO-d6) d (ppm): 1.05–1.73 (m, 10H, 5CH2 cyclo-
hexane), 4.04 (s, 2H, CH2), 4.45 (m, 1H, CH cyclohexane),
7.29–7.56 (m, 10H, 10ArH), 14.13 (brs, 1H, NH).
4-Benzoyl-1-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfa-
nyl]acetyl} thiosemicarbazide (4l) Yield: 96.8 %. Tem-
perature of reaction: 50 ꢁC for 20 h, mp: 180–182 ꢁC (dec.).
Analysis for C24H20N6O2S2 (488.58); calculated: C, 59.00; H,
4.13; N, 17.20; S, 13.12; found: C, 58.95; H, 4.12; N, 17.26; S,
13.08. IR (KBr), m (cm-1): 3176 (NH), 3088 (CH aromatic),
2979, 1449 (CH aliphatic), 1746 (C=O acidic), 1703 (C=O),
1608 (C=N), 1509 (C–N), 1311 (C=S), 681 (C–S). 1H NMR
(DMSO-d6) d (ppm): 4.15 (s, 2H, CH2), 7.35–7.96 (m, 15H,
15ArH), 11.33, 11.77, 12.87 (3brs, 3H, 3NH).
4-Phenyl-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
(5d) Yield:
methyl}-4H-1,2,4-triazole-3(2H)-thione
76.9 %, mp: 209–210 ꢁC (dec.). Analysis for C23H18N6S2
(442.56); calculated: C, 62.42; H, 4.10; N, 18.99; S, 14.49;
found: C, 62.28; H, 4.09; N, 18.93; S, 14.51. IR (KBr), m
(cm-1): 3175 (NH), 3090 (CH aromatic), 2972 (CH ali-
phatic), 1598 (C=N), 1505 (C–N), 1326 (C=S), 684 (C–S).
1H NMR (DMSO-d6) d (ppm): 4.14 (s, 2H, CH2), 7.12–7.59
(m, 15H, 15ArH), 13.86 (brs, 1H, NH). 13C NMR d (ppm):
26.22 (–S–CH2–), 125.61, 128.44, 128.55, 128.63, 128.74,
129.23, 129.41, 129.58, 130.11 (15CH aromatic), 138.23,
146.83, 148.15 (3C aromatic), 150.65 (C-30 triazole), 153.33
(C–S), 166.98 (C-3 triazole), 167.42 (C=S). MS m/z (%): 442
(M?, 2), 306 (1), 294 (1), 252 (98), 194 (23), 149 (18), 127
(14), 118 (44), 104 (8), 91 (27), 77 (100).
Derivatives of 4,5-disubstituted-1,2,4-triazole-3(2H)-
thione (5a–i)
General procedure
A mixture of thiosemicarbazide 4a–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 formed precipitate
was filtered and crystallized from ethanol 5c, d, h, i,
butanol 5b, e, f, or methanol 5a, g.
4-(4-Bromophenyl)-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione
4-Ethyl-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
methyl}-4H-1,2,4-triazole-3(2H)-thione
(5e)
(5a) Yield:
Yield: 97.2 %, mp: 210–212 ꢁC (dec.). Analysis for
C23H17BrN6S2 (521.45); calculated: C, 52.98; H, 3.29; N,
87.6 %, mp: 214–216 ꢁC (dec.). Analysis for C19H18N6S2
(394.52); calculated: C, 57.84; H, 4.60; N, 21.30; S, 16.25;
123

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Med Chem Res (2013) 22:3134–3147
16.12; S, 12.30; Br, 15.32; found: C, 52.93; H, 3.28; N,
16.15; S, 12.32. IR (KBr), m (cm-1): 3178 (NH), 3102 (CH
aromatic), 2965, 1448 (CH aliphatic), 1609 (C=N), 1504
13.14. IR (KBr), m (cm-1): 3174 (NH), 3071 (CH aro-
matic), 2982, 1453, 764 (CH aliphatic), 1612 (C=N), 1510
1
(C–N), 1358 (C=S), 673 (C–S). H NMR (DMSO-d6) d
(ppm): 3.71 (s, 3H, CH3), 4.33 (s, 2H, CH2), 5.20 (s, 2H,
1
(C–N), 1367 (C=S), 688 (C–S). H NMR (DMSO-d6) d
(ppm): 4.17 (s, 2H, CH2), 7.14–7.46 (m, 14H, 14ArH),
CH2), 6.83–7.52 (m, 14H, 14ArH), 13.82 (brs, 1H, NH).
13.89 (brs, 1H, NH).
Derivatives of 2,5-disubstituted-1,3,4-thiadiazole (6a–i)
Method A (for compounds 6a–i)
4-(4-Chlorophenyl)-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione
(5f)
Yield: 96.0 %, mp: 118–120 ꢁC (dec.). Analysis for
C23H17ClN6S2 (477.00); calculated: C, 57.91; H, 3.59; N,
17.62; S, 13.44; Cl, 7.43; found: C, 57.85; H, 3.58; N,
17.65; S, 13.41. IR (KBr), m (cm-1): 3143 (NH), 3088 (CH
aromatic), 2985, 1459 (CH aliphatic), 1601 (C=N), 1500
10 mmol of 4-substituted-1-{[(4,5-diphenyl-4H-1,2,4-tria-
zol-3-yl)sulfanyl]acetyl} thiosemicarbazide 4a–i was dis-
solved in 10–20 mL diluted sulfuric acid and stirred in a
closed bulb for 1 h. Subsequently, the solution was poured
out on crushed ice (50 g) and stirred until the ice was
completely dissolved. Later, the solution was neutralized
with ammonium hydroxide. The precipitate that formed
was filtered, dried, and crystallized from ethanol 6a, c, d,
g–i or butanol 6b, e, f.
1
(C–N), 1361 (C=S), 690 (C–S). H NMR (DMSO-d6) d
(ppm): 4.17 (s, 2H, CH2), 7.22–7.58 (m, 14H, 14ArH),
13.89 (brs, 1H, NH).
4-(4-Methoxyphenyl)-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-
(5g)
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione
Yield: 98.3 %, mp: 206–208 ꢁC (dec.). Analysis for
C24H20N6OS2 (472.58); calculated: C, 60.99; H, 4.26; N,
17.78; S, 13.57; found: C, 61.16; H, 4.25; N, 17.71; S,
13.61. IR (KBr), m (cm-1): 3164 (NH), 3094 (CH aromatic),
2969, 1441 (CH aliphatic), 1612 (C=N), 1506 (C–N), 1319
Method B (for compounds 6a, d)
20 mL of 10 % ethanolic solution of hydrochloric acid was
added to thiosemicarbazide 4a, d and the reaction mixture
was heated under reflux for 1 h. Subsequently, the solution
was left at room temperature for 24 h. The precipitate
formed was separated by filtration, dried, and crystallized
from ethanol.
1
(C=S), 691 (C–S). H NMR (DMSO-d6) d (ppm): 3.80 (s,
3H, CH3), 4.14 (s, 2H, CH2), 7.03–7.59 (m, 14H, 14ArH),
13.82 (brs, 1H, NH). 13C NMR d (ppm): 27.55 (–S–CH2–),
55.56 (CH3), 114.56, 125.67, 127.62, 128.33, 128.67,
129.32, 129.55, 130.11 (14CH aromatic), 133.71, 134.62,
139.61, 159.81 (4C aromatic), 150.51 (C-30 triazole),
154.73 (C–S), 168.42 (C-3 triazole), 168.61 (C=S). MS m/
z (%): 472 (M?, 0.07), 457 (0.03), 440 (0.02), 339 (0.21),
306 (0.05), 294 (0.11), 268 (11), 252 (59), 209 (7), 194 (15),
149 (12), 135 (86), 118 (13), 104 (10), 91 (27), 77 (100).
Method C (for compounds 6e, f)
A mixture of 10 mmol of thiosemicarbazide 4e, f in 10 mL
of anhydrous acetic acid was refluxed for 1 h. Subse-
quently, the solution was left at room temperature for 12 h.
The precipitate that formed was separated by filtration,
dried, and crystallized from butanol.
4-Benzyl-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
(5h) Yield:
5-Aminoethyl-2-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sul-
fanyl]methyl}-1,3,4-thiadiazole (6a) Yield: 81.3 %, mp:
168–170 ꢁC (dec.). Analysis for C19H18N6S2 (394.52);
calculated: C, 57.84; H, 4.60; N, 21.30; S, 16.25; found: C,
57.69; H, 4.58; N, 21.26; S, 16.21. IR (KBr), m (cm-1):
3244 (NH), 3071 (CH aromatic), 2944, 1458, 733 (CH
methyl}-4H-1,2,4-triazole-3(2H)-thione
79.0 %, mp: 136–140 ꢁC (dec.). Analysis for C24H20N6S2
(456.58); calculated: C, 63.13; H, 4.41; N, 18.41; S, 14.04;
found: C, 63.26; H, 4.42; N, 18.35; S, 14.08. IR (KBr), m
(cm-1): 3155 (NH), 3091 (CH aromatic) 2961, 1453, 762
(CH aliphatic), 1609 (C=N), 1508 (C–N), 1342 (C=S), 677
1
aliphatic), 1602 (C=N), 1506 (C–N), 671 (C–S). H NMR
1
(C–S). H NMR (DMSO-d6) d (ppm): 4.29 (s, 2H, CH2),
5.24 (s, 2H, CH2), 7.22–7.53 (m, 15H, 15ArH), 13.86 (brs,
(DMSO-d6) d (ppm): 1.13 (t, J = 7.5 Hz, 3H, CH3),
3.21–3.27 (q, J = 5 Hz, J = 5 Hz, 2H, CH2), 4.57 (s, 2H,
CH2), 7.17–7.70 (m, 10H, 10ArH), 9.35 (brs, 1H, NH).
1H, NH).
4-(4-Methoxybenzyl)-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione
5-Aminoallyl-2-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sul-
fanyl]methyl}-1,3,4-thiadiazole (6b) Yield: 68.9 %, mp:
208–210 ꢁC (dec.). Analysis for C20H18N6S2 (406.53);
calculated: C, 59.09; H, 4.46; N, 20.67; S, 15.77; found: C,
59.22; H, 4.45; N, 20.65; S, 15.73. IR (KBr), m (cm-1):
(5i)
Yield: 98.5 %, mp: 118–120 ꢁC (dec.). Analysis for
C25H22N6OS2 (486.61); calculated: C, 61.70; H, 4.56; N,
17.27; S, 13.18; found: C, 61.61; H, 4.55; N, 17.25; S,
123

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3143
3256 (NH), 3083 (CH aromatic), 2955, 1489, 741 (CH
1
aliphatic), 1610 (C=N), 1503 (C–N), 679 (C–S). H NMR
17.78; S, 13.57; found: C, 60.89; H, 4.26; N, 17.75; S, 14.55.
IR (KBr), m (cm-1): 3211 (NH), 3038 (CH aromatic), 2982,
1451 (CH aliphatic), 1600 (C=N), 1502 (C–N), 692 (C–S). 1H
NMR (DMSO-d6) d (ppm): 3.71 (s, 3H, CH3), 4.65 (s, 2H,
CH2), 6.89–7.78 (m, 14H, 14ArH), 10.07 (brs, 1H, NH).
(DMSO-d6) d (ppm): 3.87 (s, 2H, CH2), 4.12 (d, J = 5 Hz,
2H, CH2), 5.02–5.13 (dd, J = 5 Hz, J = 5 Hz, 2H, =CH2),
5.79–5.88 (m, 1H, CH), 7.40–8.56 (m, 10H, 10ArH), 10.13
(brs, 1H, NH).
5-Aminobenzyl-2-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-1,3,4-thiadiazole (6h) Yield: 70.2 %,
mp: 146–148 ꢁC (dec.). Analysis for C24H20N6S2 (456.58);
calculated: C, 63.13; H, 4.41; N, 18.41; S, 14.04; found: C,
63.05; H, 4.39; N, 18.36; S, 14.09. IR (KBr), m (cm-1):
3272 (NH), 3042 (CH aromatic), 2934, 1458 (CH ali-
5-Aminocyclohexyl-2-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-1,3,4-thiadiazole (6c) Yield: 75.6 %, mp:
172–174 ꢁC (dec.). Analysis for C23H24N6S2 (448.61); cal-
culated: C, 61.58; H, 5.39; N, 18.73; S, 14.30; found: C,
61.61; H, 5.37; N, 18.76; S, 14.27. IR (KBr), m (cm-1): 3190
(NH), 3093 (CH aromatic), 2972, 1467, 749 (CH aliphatic),
1
phatic), 1601 (C=N), 1512 (C–N), 686 (C–S). H NMR
1
(DMSO-d6) d (ppm): 4.11 (s, 2H, CH2), 4.73 (s, 2H, CH2),
7.34–7.62 (m, 15H, 15ArH), 10.47 (brs, 1H, NH).
1620 (C=N), 681 (C–S). H NMR (DMSO-d6) d (ppm):
1.1–1.65 (m, 10H, 5CH2 cyclohexane), 3.03 (m, 1H, CH
cyclohexane), 4.22 (s, 2H, CH2), 7.33–8.06 (m, 10H,
10ArH), 10.16 (brs, 1H, NH).
[5-Amino-(4-methoxybenzyl)]-2-{[(4,5-diphenyl-4H-1,2,
4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole
(6i)
Yield: 71.4 %, mp: 218–220 ꢁC (dec.). Analysis for
C25H22N6OS2 (486.61); calculated: C, 61.70; H, 4.56; N, 17.27;
S, 13.18; found: C, 61.77; H, 4.55; N, 17.23; S, 13.22. IR (KBr),
m (cm-1): 3268 (NH), 3095 (CH aromatic), 2955, 1420, 765
(CH aliphatic), 1598 (C=N), 1508 (C–N), 690 (C–S). 1H NMR
(DMSO-d6) d (ppm): 3.68 (s, 3H, CH3), 3.98 (s, 2H, CH2), 4.44
(s, 2H, CH2), 6.86–7.64 (m, 14H, 14ArH), 10.44 (brs, 1H, NH).
5-Aminophenyl-2-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-1,3,4-thiadiazole (6d) Yield: 50.9 %,
mp: 192–198 ꢁC (dec.). Analysis for C23H18N6S2 (442.60);
calculated: C, 62.42; H, 4.10; N, 19.00; S, 14.49; found: C,
62.36; H, 4.09; N, 18.97; S, 14.53. IR (KBr), m (cm-1):
3199 (NH), 3011 (CH aromatic), 2968 (CH aliphatic), 1610
1
(C=N), 1504 (C–N), 683 (C–S). H NMR (DMSO-d6) d
(ppm): 4.02 (s, 2H, CH2), 6.98–7.54 (m, 15H, 15ArH),
Derivatives of N,N-disubstituted acetamide (7a–i)
General method (for compounds 7a–i)
10.42 (brs, 1H, NH).
[5-Amino-(4-bromophenyl)]-2-{[(4,5-diphenyl-4H-1,2,
4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole
(6e)
A mixture of 10 mmol of appropriate 2,5-disubstituted-1,
3,4-thiadiazole 6a–i in 5 mL of acetic anhydride was heated
under reflux for 2 h. Distilled water was added to the reaction
mixture and it was allowed to cool. The resulting precipitate
was filtered and washed with distilled water. The residue was
purified by recrystallization from ethanol.
Yield: 89.4 %, mp: 203–205 ꢁC (dec.). Analysis for
C23H17BrN6S2 (521.45); calculated: C, 52.98; H, 3.29; N,
16.12; S, 12.30; Br, 15.32; found: C, 52.73; H, 3.27; N,
16.15; S, 12.27. IR (KBr), m (cm-1): 3167 (NH), 3110 (CH
aromatic), 2954, 1441 (CH aliphatic), 1602 (C=N), 680 (C–
S). 1H NMR (DMSO-d6) d (ppm): 4.22 (s, 2H, CH2),
6.89–7.65 (m, 14H, 14ArH), 10.23 (brs, 1H, NH).
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
1,3,4-thiadiazol-2-yl)-N-ethylacetamide
(7a) Yield:
[5-Amino-(4-chlorophenyl)]-2-{[(4,5-diphenyl-4H-1,2,
4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole
75.6 %, mp: 182–184 ꢁC (dec.). Analysis for C21H20N6OS2
(436.55); calculated: C, 57.78; H, 4.62; N, 19.25; S, 14.69;
found: C, 57.81; H, 4.61; N, 19.28; S, 14.69. IR (KBr),
m (cm-1): 3091 (CH aromatic), 2922, 1467, 742 (CH ali-
phatic), 1701 (C=O), 1610 (C=N), 1512 (C–N), 692 (C–S).
1H NMR (DMSO-d6) d (ppm): 1.31 (t, J = 7.5 Hz, 3H,
CH3), 2.15 (s, 3H, CH3), 3.65–3.70 (q, J = 5 Hz, J = 5 Hz,
2H, CH2), 4.44 (s, 2H, CH2), 7.33–8.04 (m, 10H, 10ArH).
(6f)
Yield: 94.7 %, mp: 215–218 ꢁC (dec.). Analysis for
C23H17ClN6S2 (477.00); calculated: C, 57.91; H, 3.59; N,
17.62; S, 13.44; Cl, 7.43; found: C, 57.71; H, 3.60; N, 17.58;
S, 13.39. IR (KBr), m (cm-1): 3245 (NH), 3065 (CH aro-
matic), 2977 (CH aliphatic), 1611 (C=N), 1506 (C–N), 695
1
(C–S). H NMR (DMSO-d6) d (ppm): 3.89 (s, 2H, CH2),
7.39–7.64 (m, 14H, 14ArH), 10.36 (brs, 1H, NH).
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
1,3,4-thiadiazol-2-yl)-N-allylacetamide
(7b) Yield:
[5-Amino-(4-methoxyphenyl)]-2-{[(4,5-diphenyl-4H-1,2,
4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole
62.1 %, mp: 212–214 ꢁC (dec.). Analysis for
C22H20N6OS2 (448.56); calculated: C, 58.91; H, 4.49; N,
18.74; S, 14.30; found: C, 58.94; H, 4.51; N, 18.76; S,
(6g)
Yield: 53.6 %, mp: 152–154 ꢁC (dec.). Analysis for
C24H20N6OS2 (472.58); calculated: C, 60.99; H, 4.26; N,
123

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Med Chem Res (2013) 22:3134–3147
14.28. IR (KBr), m (cm-1): 3122 (CH aromatic), 2978,
1492, 742 (CH aliphatic), 1708 (C=O), 1614 (C=N), 1515
for C26H22N6O2S2 (514.62); calculated: C, 60.68; H, 4.31;
N, 16.33; S, 12.46; found: C, 60.64; H, 4.29; N, 16.37; S,
12.45. IR (KBr), m (cm-1): 3067 (CH aromatic), 2987,
1452 (CH aliphatic), 1710 (C=O), 1611 (C=N), 1508 (C–
N), 679 (C–S). 1H NMR (DMSO-d6) d (ppm): 2.09 (s, 3H,
CH3), 3.78 (s, 3H, CH3), 3.87 (s, 2H, CH2), 7.09–8.50 (m,
14H, 14ArH).
1
(C–N), 688 (C–S). H NMR (DMSO-d6) d (ppm): 2.11 (s,
3H, CH3), 4.27 (s, 2H, CH2), 4.35 (d, J = 5 Hz, 2H, CH2),
5.14–5.18 (dd, J = 5 Hz, J = 5 Hz, 2H, =CH2), 5.81–5.86
(m, 1H, CH), 7.34–8.07 (m, 10H, 10ArH).
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
1,3,4-thiadiazol-2-yl)-N-cyclohexylacetamide (7c) Yield:
87.5 %, mp: 193–195 ꢁC (dec.). Analysis for C25H26N6OS2
(490.64); calculated: C, 61.20; H, 5.34; N, 17.13; S, 13.07;
found: C, 61.22; H, 5.32; N, 17.16; S, 13.05. IR (KBr),
m (cm-1): 3108 (CH aromatic), 2988, 1487, 755 (CH ali-
phatic), 1705 (C=O), 1603 (C=N), 1506 (C–N), 674 (C–S).
1H NMR (DMSO-d6) d (ppm): 1.36–1.84 (m, 10H, 5CH2
cyclohexane), 2.14 (s, 3H, CH3), 3.64 (m, 1H, CH cyclo-
hexane), 4.26 (s, 2H, CH2), 7.33–8.05 (m, 10H, 10ArH).
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
(7h) Yield:
1,3,4-thiadiazol-2-yl)-N-benzylacetamide
73.4 %, mp: 156–158 ꢁC (dec.). Analysis for C26H22N6OS2
(498.62); calculated: C, 62.63; H, 4.45; N, 16.85; S, 12.86;
found: C, 62.67; H, 4.48; N, 16.81; S, 12.84. IR (KBr), m
(cm-1): 3076 (CH aromatic), 2965, 1468 (CH aliphatic),
1713 (C=O), 1614 (C=N), 1523 (C–N), 695 (C–S). 1H NMR
(DMSO-d6) d (ppm): 2.06 (s, 3H, CH3), 4.26 (s, 2H, CH2),
4.75 (s, 2H, CH2), 7.19–8.36 (m, 15H, 15ArH).
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
methyl}-1,3,4-thiadiazol-2-yl)-N-phenylacetamide
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
1,3,4-thiadiazol-2-yl)-N-(4-methoxybenzyl)acetamide (7i)
Yield: 69.4 %, mp: 201–203 ꢁC (dec.). Analysis for
C27H24N6O2S2 (528.65); calculated: C, 61.34; H, 4.58; N,
15.90; S, 12.13; found: C, 61.37; H, 4.59; N, 15.89; S,
12.16. IR (KBr), m (cm-1): 3103 (CH aromatic), 2967,
1461, 756 (CH aliphatic), 1704 (C=O), 1607 (C=N), 1514
(7d)
Yield: 67.7 %, mp: 209–211 ꢁC (dec.). Analysis for
C25H20N6OS2 (484.59); calculated: C, 61.96; H, 4.16; N,
17.34; S, 13.23; found: C, 61.95; H, 4.08; N, 17.31; S,
13.26. IR (KBr), m (cm-1): 3098 (CH aromatic), 2978 (CH
aliphatic), 1699 (C=O), 1602 (C=N), 1509 (C–N), 694 (C–
1
S). H NMR (DMSO-d6) d (ppm): 2.12 (s, 3H, CH3), 4.22
(s, 2H, CH2), 7.16–7.92 (m, 15H, 15ArH).
1
(C–N), 697 (C–S). H NMR (DMSO-d6) d (ppm): 2.16 (s,
3H, CH3), 3.77 (s, 3H, CH3), 4.24 (s, 2H, CH2), 4.31 (s,
2H, CH2), 6.88–7.71 (m, 14H, 14ArH).
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
methyl}-1,3,4-thiadiazol-2-yl)-N-(4-bromophenyl)acetamide
(7e) Yield: 84.6 %, mp: 222–224 ꢁC (dec.). Analysis for
C25H19BrN6OS2 (563.49); calculated: C, 53.29; H, 3.40; N,
14.91; S, 11.38; Br, 14.18; found: C, 53.33; H, 3.38; N,
14.95; S, 11.36. IR (KBr), m (cm-1): 3123 (CH aromatic),
2974, 1467 (CH aliphatic), 1712 (C=O), 1621 (C=N), 1509
[(4,5-Diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl] acetic acid
(8) Compound 8 was obtained using the same method as
described earlier for derivatives 5a–i. That is, a mixture of
thiosemicarbazide 4j (10 mmol) and 20 mL of 2 % aque-
ous solution of sodium hydroxide was refluxed for 2 h.
Then, the solution was neutralized with diluted hydro-
chloric acid and the formed precipitate was filtered and
crystallized from ethanol.
1
(C–N), 684 (C–S). H NMR (DMSO-d6) d (ppm): 2.15 (s,
3H, CH3), 4.25 (s, 2H, CH2), 7.27–7.94 (m, 14H, 14ArH).
Yield: 70.3 %, mp: 248–249 ꢁC (dec.). Analysis for
C16H13N3O2S (311.36); calculated: C, 61.72; H, 4.21; N,
13.49; S, 10.30; found: C, 61.59; H, 4.19; N, 13.54; S,
10.28. IR (KBr), m (cm-1): 3079 (CH aromatic), 3045
(OH), 2982 (CH aliphatic), 1702 (C=O), 1599 (C=N), 688
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
methyl}-1,3,4-thiadiazol-2-yl)-N-(4-chlorophenyl)acetamide
(7f) Yield: 59.8 %, mp: 229–231 ꢁC (dec.). Analysis for
C25H19ClN6OS2 (519.04); calculated: C, 57.85; H, 3.69; N,
16.19; S, 12.36; Cl, 6.83; found: C, 57.81; H, 3.65; N,
16.22; S, 12.37. IR (KBr), m (cm-1): 3090 (CH aromatic),
2980, 1451 (CH aliphatic), 1695 (C=O), 1601 (C=N), 1521
1
(C–S). H NMR (DMSO-d6) d (ppm): 4.04 (s, 2H, CH2),
7.28–7.61 (m, 10H, 10ArH), 12.97 (s, 1H, OH).
1
(C–N), 689 (C–S). H NMR (DMSO-d6) d (ppm): 2.15 (s,
3H, CH3), 4.24 (s, 2H, CH2), 7.26–7.91 (m, 14H, 14ArH).
4-Carboxymethyl-5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (9) Com
-pound 9 was obtained using the same method as described
earlier for derivatives 5a–i. That is, a mixture of thiosemi-
carbazide 4k (10 mmol) and 20 mL of 2 % aqueous solution
of sodium hydroxide was refluxed for 2 h. Then, the solution
N-(5-{[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]
methyl}-1,3,4-thiadiazol-2-yl)-N-(4-methoxyphenyl)acetam-
ide (7g) Yield: 62.8 %, mp: 174–176 ꢁC (dec.). Analysis
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Med Chem Res (2013) 22:3134–3147
3145
was neutralized with diluted hydrochloric acid and the formed
precipitate was filtered and crystallized from ethanol.
Yield: 97.2 %, mp: 157–159 ꢁC (dec.). Analysis for
C19H16N6O2S2 (424.50); calculated: C, 53.76; H, 3.80; N,
19.80; S, 15.11; found: C, 53.88; H, 3.81; N, 19.74; S,
15.47. IR (KBr), m (cm-1): 3228 (NH), 3095 (OH), 3062
(CH aromatic), 2991 (CH aliphatic), 1713 (C=O), 1605
(C=N), 1504 (C–N), 1343 (C=S), 681 (C–S). 1H NMR
(DMSO-d6) d (ppm): 4.42 (s, 2H, CH2), 4.78 (s, 2H, CH2),
7.27–7.56 (m, 10H, 10ArH), 13.80 (s, 1H, OH), 14.13 (brs,
1H, NH).
were added. The mixture was stirred for 2 h at room
temperature. After that, distilled water was added and the
precipitate that formed was filtered, washed with distilled
water, and crystallized from ethanol.
Yield: 74.8 %, mp: 224–226 ꢁC (dec.). Analysis for
C22H23N7S2 (449.59); calculated: C, 58.77; H, 5.16; N,
21.81; S, 14.26; found: C, 58.79; H, 5.14; N, 21.83; S,
12.24. IR (KBr), m (cm-1): 3290 (NH), 3098 (CH aro-
matic), 2978, 1482 (CH aliphatic), 1623 (C=N), 1522 (C–
N), 1341 (C=S), 685 (C–S). 1H NMR (DMSO-d6) d (ppm):
1.67–1.73 (m, 4H, 2CH2), 2.32 (t, J = 5 Hz, 2H, CH2),
2.77 (t, J = 5 Hz, 2H, CH2), 4.05 (s, 2H, CH2), 4.68 (s, 2H,
CH2), 7.36–8.35 (m, 10H, 10ArH), 14.68 (brs, 1H, NH).
5-{[(4,5-Diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
2,5-dihydro-4H-1,2,4-triazole-3(2H)-thione (10) Com-
pound 10 was obtained using the same method as described
earlier for derivatives 5a–i. That is, a mixture of thiosem-
icarbazide 4l (10 mmol) and 20 mL of 2 % aqueous
solution of sodium hydroxide was refluxed for 2 h. Then,
the solution was neutralized with diluted hydrochloric acid
and the formed precipitate was filtered and crystallized
from ethanol.
Microbiology
Materials and methods
All synthesized compounds were preliminarily tested for
their in vitro antibacterial activity against Gram-positive
and -negative reference bacterial strains and next by the
broth microdilution method against the selected bacterial
strains.
Yield: 78.9 %, mp: 210–212 ꢁC (dec.). Analysis for
C17H14N6S2 (366.46); calculated: C, 55.72; H, 3.85; N,
22.93; S, 17.50; found: C, 55.58; H, 3.83; N, 23.01; S,
17.46. IR (KBr), m (cm-1): 3256 (NH), 3079 (CH aro-
matic), 2956, 1461 (CH aliphatic), 1603 (C=N), 1510 (C–
N), 1329 (C=S), 695 (C–S). 1H NMR (DMSO-d6) d (ppm):
4.04 (s, 2H, CH2), 7.29–7.92 (m, 10H, 10ArH), 13.33 (s,
1H, NH), 14.15 (brs, 1H, NH).
Panel reference strains of aerobic bacteria from the
American Type Culture Collection, including six Gram-
positive bacteria, S. aureus ATCC 25923, S. aureus ATCC
6538, S. epidermidis ATCC 12228, B. subtilis ATCC 6633,
B. cereus ATCC 10876, M. luteus ATCC 10240, and four
Gram-negative bacteria, Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabilis
ATCC 12453, Pseudomonas aeruginosa ATCC 9027, were
used. Microbial suspensions with an optical density of 0.5
McFarland standard 150 9 106 CFU/mL (CFUs—colony
forming units) were prepared in sterile 0.85 % NaCl. All
stock solutions of the tested compounds were prepared in
DMSO. The medium with DMSO at the final concentration
and without the tested compounds served as the control—
no microbial growth inhibition was observed.
[3-{[(4,5-Diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
1-(pyrrolidin-1-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-
triazol-4-yl]acetic acid (11) To a solution of 10 mmol of
compound 9 in ethanol, pyrrolidine (10 mmol) and form-
aldehyde (0.2 mL) were added. The mixture was stirred for
2 h at room temperature. After that, distilled water was
added and the precipitate that formed was filtered, washed
with distilled water, and crystallized from ethanol.
Yield: 66.8 %, mp: 173–175 ꢁC (dec.). Analysis for
C24H25N7O2S2 (507.63); calculated: C, 56.78; H, 4.96; N,
19.31; S, 12.63; found: C, 56.80; H, 4.97; N, 19.34; S,
12.66. IR (KBr), m (cm-1): 3100 (OH), 3069 (CH aro-
matic), 2962 (CH aliphatic), 1715 (C=O), 1611 (C=N),
1514 (C–N), 1367 (C=S), 692 (C–S). 1H NMR (DMSO-d6)
d (ppm): 1.66–1.72 (m, 4H, 2CH2), 2.29 (t, J = 5 Hz, 2H,
CH2), 2.68 (t, J = 5 Hz, 2H, CH2), 4.27 (s, 2H, CH2), 4.58
(s, 2H, CH2), 4.69 (s, 2H, CH2), 7.47–8.08 (m, 10H,
10ArH), 13.68 (s, 1H, OH).
Preliminary antimicrobial potency in vitro of the tested
compounds was screened using the agar dilution method on
the basis of the bacterial growth inhibition on the Mueller–
Hinton agar containing the compounds at a concentration
of 1,000 lg/mL. The plates were poured on the day of
testing. 10 lL of each bacterial suspension was put onto the
prepared solid media. The plates were incubated at 37 ꢁC
for 18 h (Bourgeois et al., 2007).
The antibacterial activity in vitro of the potentially
active compounds was determined by the broth microdi-
lution method on the basis of MIC, usually defined as the
lowest concentration of the compound at which there was
no visible growth of microorganisms (White et al., 2002).
Determination of the MIC value was achieved by the broth
microdilution method according to a CLSI (Clinical and
5-{[(4,5-Diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-
2-(pyrrolidin-1-ylmethyl)-2,4-dihyro-3H-1,2,4-triazole-3-
thione (12) To a solution of 10 mmol of compound 10 in
ethanol, pyrrolidine (10 mmol) and formaldehyde (0.2 mL)
123

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Dobosz M, Pachuta-Stec A (1996) Synthesis of new derivatives of
3-benzyl-D2-1,2,4-triazoline-5-thione and 5-benzyl-1,3,4-thiadi-
azole. Acta Pol Pharm 53:123–131
Dobosz M, Pitucha M, Wujec M (1996) The reactions of cyclization
of thiosemicarbazide derivatives to 1,2,4-triazole or 1,3,4-
thiadiazole system. Acta Pol Pharm 53:31–38
Dogan HN, Duran A, Rollas S, Sener G, Uysal MK, Gu¨len D (2002)
Synthesis of new 2,5-disubstituted-1,3,4-thiadiazoles and pre-
liminary evaluation of anticonvulsant and antimicrobial activi-
ties. Bioorg Med Chem 10:2893–2898
Duran A, Dogan HN, Rollas S (2002) Synthesis and preliminary
anticancer activity of new 1,4-dihydro-3-(3-hydroxy-2-naph-
Laboratory Standards Institute) recommendation with some
modifications (2008). The 96-well microplates were used;
198 lL of Mueller–Hinton broth with a series of twofold
dilutions of the tested compound in the range of the final
concentrations from 0.24 to 1,000 lg/mL was inoculated
with 2 lL of microbial suspension (total volume per each
well—200 lL). After incubation (at 35 ꢁC for 18 h), spec-
trophotometric measurements of optical density (OD600) of
the bacterial cultures with the tested compounds were
performed in order to determine MIC. OD600 of bacterial
cultures in the medium without the tested compounds was
used as a control. The blank control wells with twofold
dilution of each of the tested compounds added to the
Mueller–Hinton broth without bacterial suspension were
incubated under the same conditions. Cefuroxime,
belonging to the second generation of cephalosporins, was
used as a control antimicrobial agent.
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˘
Gu¨lerman NN, Dogan HN, Rollas S, Johansson C, C¸ elik C (2001)
Conflict of interest The authors declare no conflict of interest.
Synthesis and structure elucidation of some new thioether
derivatives of 1,2,4-triazoline-3-thiones and their antimicrobial
activities. Farmaco 56:953–958
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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