Synthesis and in vitro antimicrobial activity of new 4-phenyl-5-methyl-4H-1,2,4-triazole-3-thione derivatives

Article abstract of DOI:10.3109/14756366.2011.653352

This study presents the synthesis, spectral analysis and antimicrobial evaluation of a new series of substituted 1,2,4-triazole (5a-i) and 1,3,4-thiadiazole derivatives (9a, c, g, h). New compounds were obtained by cyclization reaction of acyl thiosemicarbazide derivatives in the presence of alkaline and acidic media. All synthesized compounds were screened for their in vitro antimicrobial activities. Nine of the compounds had potential activity against Gram-positive bacteria (MIC = 3.91-500 μg/mL). Some compounds showed good activity especially against: Micrococcus luteus ATCC 10240 (MIC = 3.91-31.25 μg/mL), Bacillus subtilis ATCC 6633 (MIC = 15.63-62.5 μg/mL), and Staphylococcus aureus ATCC 25923 (MIC = 15.63-125 μg/mL).

Full text of DOI:10.3109/14756366.2011.653352

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 479–488
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.653352
RESEARCH ARTICLE
Synthesis and in vitro antimicrobial activity of new 4-phenyl-5-
methyl-4H-1,2,4-triazole-3-thione derivatives
Łukasz Popiołek1, Urszula Kosikowska2, Maria Dobosz1, and Anna Malm2
1Department of Organic Chemistry and 2Department of Pharmaceutical Microbiology, Medical University,
Lublin, Poland
Abstract
This study presents the synthesis, spectral analysis and antimicrobial evaluation of a new series of substituted 1,2,4-
triazole (5a–i) and 1,3,4-thiadiazole derivatives (9a, c, g, h). New compounds were obtained by cyclization reaction
of acyl thiosemicarbazide derivatives in the presence of alkaline and acidic media. All synthesized compounds were
screened for their in vitro antimicrobial activities. Nine of the compounds had potential activity against Gram-positive
bacteria (MIC=3.91–500 µg/mL). Some compounds showed good activity especially against: Micrococcus luteus ATCC
10240 (MIC=3.91−31.25 µg/mL), Bacillus subtilis ATCC 6633 (MIC=15.63− 62.5 µg/mL), and Staphylococcus aureus
ATCC 25923 (MIC=15.63−125 µg/mL).
Keywords: Antimicrobial activity, 1,2,4-triazole, 1,3,4-thiadiazole derivatives, MIC, MBC
Introduction
In the view of these facts and as a continuation of our
research on the biological properties of triazole and thia-
diazole we have designed and synthesized series of new
potentially active 1,2,4-triazole and 1,3,4-thiadiazole and
evaluation of their in vitro antibacterial activity.
A wide variety of heterocyclic systems have been explored
for developing pharmaceutically important molecules.
Among them the derivatives of 1,2,4-triazole and 1,3,4-
thiadiazole have played an important role in the medici-
nal chemistry. ese heterocycles have been found to
possess a wide spectrum of biological activities such
Experimental
as analgesic1, antifungal2,3, antibacterial4–8, antiviral9,10
antiphlogistic11–14 and antituberculous15 action.
,
Chemistry
e treatment of infectious diseases still remains an
important and challenging problem because of a com-
bination factors including emerging infectious diseases
and increasing number of multi-drug resistant microbial
pathogens witch particular relevance. In spite of the large
number of antibiotics and chemotherapeutics available
for medical use, the emergence of old and new antibiotics
resistant bacterial strains in the last decades constitutes a
substantial need for the new class of antimicrobial agents.
Triazole and thiadiazole in particular substituted
1,2,4-trazoles and 1,3,4-thiadiazole are among the vari-
ous heterocycles that have received the most attention
during last decades as potential antimicrobial agents.
Melting points were determined in Fisher-Johns blocks
(Pittsburgh, US) and presented without any corrections.
e IR spectra (ν, cm−1) were recorded in KBr tablets
using a Specord IR-75 spectrophotometer (Germany).
1
e H NMR spectra were recorded on a Bruker Avance
300 apparatus (Bruker BioSpin GmbH, Rheinstetten/
Karlsruhe, Germany) in DMSO-d6 with TMS as internal
standard. e 13C NMR spectra were recorded on a Bruker
Avance 300. Chemical shifts are given in ppm (δ-scale).
e MS spectra were recorded on a ermo-Finnigan
Trace DSQ GC MS apparatus (Waltham, Massachusetts,
US). Chemicals were purchased from Merck Co. or
Lancaster and used without further purification.
Address for Correspondence: Łukasz Popiołek, Department of Organic Chemistry, Medical University, 4A Chodźki Str., 20-093 Lublin,
Poland. E-mail: lukasz.popiolek@umlub.pl
(Received 25 August 2011; revised 25 November 2011; accepted 16 December 2011)
479

480 Ł. Popiołek et al.
e purity of obtained compounds was checked
by TLC on aluminium oxide 60 F254 plates (Merck
Co. Whitehouse Station, New Jersey US), in a CHCl3/
C2H5OH (10:1, v/v) solvent system with UV visualiza-
tion (λ = 254 nm).
diethyl ether to remove unreacted isothiocyanate. en
it was filtered off, dried and crystallized from: ethanol
(4a, 4b, 4c, 4f, 4g, 4h, 4j, 4k, 4l), butanol (4i) or metha-
nol (4d, 4e).
Elemental analysis of the obtained compounds was
performed for C, H, N, S. e maximum percentage dif-
ferences between calculated and with found values for
each element were within the error and amounted to
0.4%.
Method B (for compounds 4a, 4f, 4h, 4j)
10 mmol of appropriate isothiocyanate was added to
2.63 g (10 mmol) of hydrazide (3) in 10 mL of anhydrous
diethyl ether. e mixture placed in a conical bulb was
mixed for 5 min and left in room temperature for 24
hrs. e precipitation of thiosemicarbazide (4a, 4f, 4h,
4j) was filtered off, dried and crystallized form ethanol.
Obtained compounds had the same melting points as the
compounds obtained by the Method A.
Ethyl [(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetate (2)
0.23 g (10 mmol) of sodium was added to 5 mL of anhy-
drous ethanol. e solution was placed in a three-necked
flask equipped with reflux condenser and closed with
a tube of CaCl2 and mercury stirred. e content was
mixed till sodium dissolved completely and then 1.91 g
(10 mmol) of 4-phenyl-5-methyl-4H-1,2,4-triazole-3-thi-
one (1) was added. en 1.22 mL ethyl bromoacetate was
added drop by drop. e content of the flask was mixed
for 4 hrs and left at room temperature for 12 hrs. en
10 mL of anhydrous ethanol was added and heated for
1 hr. e mixture was filtered off inorganic compounds.
After cooling the precipitate was filtered off and crystal-
lized from ethanol.
Yield: 55.6%; M. p. 72–74°C. IR (KBr), ν (cm−1): 3109
(CH aromatic), 2935, 1444 (CH aliphatic), 1698 (C=O),
1597 (C=N), 682 (C-S). 1H NMR (DMSO-d6) δ (ppm): 1.16
(t, J= 7 Hz, 3H, CH3), 2.19 (s, 3H, CH3), 3.99 (s, 2H, CH2),
4.03–4.12 (q, J= 7,5 Hz, 2H, CH2,), 7.42–7.64 (m, 5H, 5CH
aromatic). Analysis for C13H15N3O2S (277.34); Calculated:
C, 56.30; H, 5.45; N, 15.15; S, 11.56; Found: C, 56.11; H,
5.43; N, 15.21; S, 11.60.
4-Phenyl-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)
sulfanyl]acetyl} thiosemicarbazide (4a)
IR (KBr), ν (cm−1): 3195 (NH), 3098 (CH aromatic), 2985,
1428 (CH aliphatic), 1701 (C=O), 1608 (C=N), 1518 (C-N),
1338 (C=S), 675 (C-S). 1H NMR (DMSO-d6) δ (ppm): 2.19
(s, 3H, CH3), 3.86 (s, 2H, CH2), 7.11-7.59 (m, 10H, 10CH
aromatic), 9.77, 9.82, 10.35 (3s, 3H, 3NH). 13C NMR δ
(ppm): 10.97 (CH3), 34.78 (-S-CH2-), 125.04, 125.62,
127.15, 127.97, 128.99, 130.08 (10CHaromatic), 133.15,
139.02 (2Caromatic), 149.36 (C-S), 152.58 (C-3 triazole),
166.99 (C=O), 180.64 (C=S). MS m/z (%): 398 (M+, 0.05),
383 (1), 307 (0.05), 263 (7), 232 (16), 190 (100), 158 (2),
149 (9), 135 (48), 118 (8), 93 (11), 91 (17), 77 (52). Analysis
for C18H18N6OS2 (398.50); Calculated: C, 54.25; H, 4.55;
N, 21.09; S, 16.09; Found: C, 54.30; H, 4.54; N, 21.03; S,
16.11.
4-(4-Bromophenyl)-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]acetyl} thiosemicarbazide (4b)
IR (KBr), ν (cm−1): 3243 (NH), 3098 (CH aromatic), 2952
(CH aliphatic),1710 (C=O), 1611 (C=N), 1351 (C=S), 687
(C-S). 1H NMR (DMSO-d6) δ (ppm): 2.19 (s, 3H, CH3), 4.09
(s, 2H, CH2), 7.21-7.65 (m, 9H, 9CH aromatic), 9.87, 9.96,
10.38 (3s, 3H, 3NH). Analysis for C18H17BrN6OS2 (477.40);
Calculated: C, 45.28; H, 3.59; N, 17.60; S, 13.43; Found: C,
45.44; H, 3.60; N, 17.59; S, 13.40.
[(4-Phenyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetohydrazide (3)
0.5 mL of 100% hydrazine hydrate was added to 2.77g (10
mmol) compound (2) in 10 mL of anhydrous ethanol.
e mixture was put to refrigerator for 24 hrs. After that,
the precipitation of hydrazide (3) was filtered off, dried
and crystallized form ethanol.
Yield: 72.8%; M. p. 86–88°C. IR (KBr), ν (cm−1): 3089
(CH aromatic), 2955, 1444 (CH aliphatic), 1711 (C=O),
4-(4-Chlorophenyl)-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]acetyl} thiosemicarbazide (4c)
1
1601 (C=N), 1503 (C-N), 688 (C-S). H NMR (DMSO-d6)
IR (KBr), ν (cm−1): 3211 (NH), 3113 (CH aromatic), 2973
(CH aliphatic), 1695 (C=O), 1603 (C=N), 1334 (C=S), 676
(C-S). 1H NMR (DMSO-d6) δ (ppm): 2.19 (s, 3H, CH3), 3.80
(s, 2H, CH2), 7.20-7.73 (m, 9H, 9CH aromatic), 9.86, 9.97,
10.39 (3s, 3H, 3 NH). Analysis for C18H17ClN6OS2 (432.95);
Calculated: C, 49.93; H, 3.96; N, 19.41; S, 14.81; Found: C,
50.01; H, 3.95; N, 19.42; S, 14.85.
δ (ppm): 2.19 (s, 3H, CH3), 3.79 (s, 2H, CH2), 4.21 (s, 2H,
NH2), 7.35-7.64 (m, 5H, 5CH aromatic), 9.30 (s, 1H, NH).
Analysis for C11H13N5OS (263.32); Calculated: C, 50.17; H,
4.98; N, 26.60; S, 12,18; Found: C, 50.04; H, 4.96; N, 26.51;
S, 12,16.
Derivatives of 4-substituted-1-{[(4-phenyl-5-
methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}
thiosemicarbazide
4-(4-Methoxyphenyl)-1-{[(4-phenyl-5-methyl-4H-1,2,4-
triazol-3-yl)sulfanyl]acetyl} thiosemicarbazide (4d)
Method A (for compounds 4a–4l)
IR (KBr), ν (cm−1): 3197 (NH), 3110 (CH aromatic), 2965,
1439 (CH aliphatic), 1705 (C=O), 1607 (C=N), 1512 (C-N),
1346 (C=S), 679 (C-S). 1H NMR (DMSO-d6) δ (ppm): 2.18
(s, 3H, CH3), 3.73 (s, 3H, CH3), 3.75 (s, 2H, CH2), 6.79-7.65
A mixture of hydrazide (3) 2.63g (10 mmol) and 10 mmol
appropriate isothiocyanate was heated in an oil bath
at 45-95°C for 8-16 hrs. e product was washed with
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and in vitro antimicrobial activity of triazoles 481
(m, 9H, 9CH aromatic), 9.61, 9.68, 10.32 (3s, 3H, 3NH).
Analysis for C19H20N6O2S2 (428.53); Calculated: C, 53.25;
H, 4.70; N, 19.61; S, 14.96; Found: C, 53.08; H, 4.71; N,
19.55; S, 14.92.
Hz, 2H, CH2), 5.02-5.13 (dd, 2H, = CH2), 5.78-5.89 (m,
1H, CH), 7.43-7.62 (m, 5H, 5CH aromatic), 8.45, 9.40,
10.25 (3s, 3H, 3NH). Analysis for C15H18N6OS2 (362.47);
Calculated: C, 49.70; H, 5.00; N, 23.18; S, 17.69; Found: C,
49.67; H, 4.98; N, 23.13; S, 17.73.
4-Benzyl-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)
sulfanyl]acetyl} thiosemicarbazide (4e)
4-Ethoxycarbonyl-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]acetyl} thiosemicarbazide (4j)
IR (KBr), ν (cm−1): 3199 (NH), 3109 (CH aromatic), 2978,
1429 (CH aliphatic), 1708 (C=O), 1608 (C=N), 1517 (C-N),
1336 (C=S), 684 (C-S). 1H NMR (DMSO-d6) δ (ppm): 2.14
(s, 3H, CH3), 3.91 (s, 2H, CH2), 4.83 (s, 2H, CH2), 7.22-7.69
(m, 10H, 10CH aromatic), 8.84, 9.55, 10.40 (3s, 3H, 3NH).
Analysis for C19H20N6OS2 (412.53); Calculated: C, 55.32; H,
4.89; N, 20.37; S, 15.54; Found: C, 55.49; H, 4.90; N, 20.44;
S, 15.59.
IR (KBr), ν (cm−1): 3194 (NH), 3095 (CH aromatic), 2983,
1448, 762 (CH aliphatic), 1722 (C=O acidic), 1697 (C=O),
1
1603 (C=N), 1511 (C-N), 1338 (C=S), 691 (C-S). H NMR
(DMSO-d6) δ (ppm): 1.21 (t, J= 7.5 Hz, 3H, CH3), 2.18 (s,
3H, CH3), 3.81 (s, 2H, CH2), 4.11-4.20 (q, J= 7.5 Hz, 2H,
CH2), 7.32-7.71 (m, 5H, 5CH aromatic), 10.41, 10.88,
11.38 (3s, 3H, 3NH). Analysis for C15H18N6O3S2 (394.47);
Calculated: C, 45.67; H, 4.60; N, 21.30; S, 16.26; Found: C,
45.53; H, 4.61; N, 21.33; S, 16.31.
4-(4-Methoxybenzyl)-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]acetyl} thiosemicarbazide (4f)
IR (KBr), ν (cm−1): 3212 (NH), 3092 (CH aromatic),
2983, 1442, 752 (CH aliphatic), 1708 (C=O), 1598 (C=N),
4-Ethoxycarbonylmethyl-1-{[(4-phenyl-5-methyl-4H-1,2,4-
triazol-3-yl)sulfanyl]acetyl} thiosemicarbazide (4k)
1
1508 (C-N), 1346 (C=S), 683 (C-S). H NMR (DMSO-
IR (KBr), ν (cm−1): 3191 (NH), 3089 (CH aromatic),
2965, 1455, 761 (CH aliphatic), 1740 (C=O acidic), 1708
(C=O), 1610 (C=N), 1499 (C-N), 1349 (C=S), 687 (C-S).
1H NMR (DMSO-d6) δ (ppm): 1.16 (t, J = 5 Hz, 3H, CH3),
2.18 (s, 3H, CH3), 3.71 (d, J = 5 Hz, 2H, CH2,), 3.87 (s,
2H, CH2), 4.05-4.08 (q, J = 2,5 Hz, 2H, CH2), 7.43-7.59
(m, 5H, 5CH aromatic), 8.82, 9.66, 10.45 (3s, 3H, 3NH).
Analysis for C16H20N6O3S2 (408.50); Calculated: C, 47.04;
H, 4.93; N, 20.57; S, 15.70; Found: C, 47.09; H, 4.92; N,
20.59; S, 15.68.
d6) δ (ppm): 2.18 (s, 3H, CH3), 3.74 (s, 3H, CH3), 3.90
(s, 2H, CH2), 4.75 (s, 2H, CH2), 6.84–7.67 (m, 9H, 9CH
aromatic), 8.78, 9.49, 10.37 (3s, 3H, 3NH). Analysis for
C20H22N6O2S2 (442.56); Calculated: C, 54.28; H, 5.01; N,
18.99; S, 14.49; Found: C, 54.07; H, 5.03; N, 19.01; S,
14.52.
4-Cyclohexyl-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)
sulfanyl]acetyl} thiosemicarbazide (4g)
IR (KBr), ν (cm−1): 3198 (NH), 3079 (CH aromatic),
2977, 1444, 763 (CH aliphatic), 1701 (C=O), 1591 (C=N),
4-Benzoyl-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)
sulfanyl]acetyl} thiosemicarbazide (4l)
1
1520 (C-N), 1341 (C=S), 681 (C-S). H NMR (DMSO-d6)
δ (ppm): 1.15-1.71 (m, 10H, 5CH2 cyclohexyl), 2.18 (s,
3H, CH3), 3.68 (s, 2H, CH2), 4.14 (s, 1H, CH cyclohexyl),
7.45-7.75 (m, 5H, 5CH aromatic), 9.23, 9.30, 10.10 (3s, 3H,
3NH). Analysis for C18H24N6OS2 (404.55); Calculated: C,
53.44; H, 5.98; N, 20.77; S, 15.85; Found: C, 53.63; H, 5.96;
N, 20.73; S, 15.81.
IR (KBr), ν (cm−1): 3155 (NH), 3098 (CH aromatic), 2980,
1451 (CH aliphatic), 1748 (C=O acidic), 1705 (C=O),
1
1611 (C=N), 1515 (C-N), 1324 (C=S), 671 (C-S). H NMR
(DMSO-d6) δ (ppm): 2.20 (s, 3H, CH3), 3.95 (s, 2H, CH2),
7.38-7.99 (m, 10H, 10CH aromatic), 11.12, 11.31, 11.75 (3s,
3H, 3NH). Analysis for C19H18N6O2S2 (426.51); Calculated:
C, 53.50; H, 4.25; N, 19.70; S, 15.03; Found: C, 53.58; H,
4.26; N, 19.63; S, 15.08.
4-Ethyl-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} thiosemicarbazide (4h)
IR (KBr), ν (cm−1): 3211 (NH), 3099 (CH aromatic),
2984, 1445, 751 (CH aliphatic), 1695 (C=O), 1604 (C=N),
Derivatives of 4-substituted-5-{[(4-phenyl-5-methyl-
4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4H-1,2,4-
triazole-3(2H)-thione (5a–5i)
1
1513 (C-N), 1345 (C=S), 671 (C-S). H NMR (DMSO-d6)
δ (ppm): 1.10 (t, J= 7.5 Hz, 3H, CH3), 2.23 (s, 3H, CH3),
3.51-3.55 (q, J= 5 Hz, 2H, CH2), 3.72 (s, 2H, CH2), 7.45-7.62
(m, 5H, 5CH aromatic), 8.35, 9.30, 10.18 (3s, 3H, 3NH).
Analysis for C14H18N6OS2 (350.46); Calculated: C, 47.98; H,
5.18; N, 23.98; S, 18.30; Found: C, 48.13; H, 5.16; N, 24.06;
S, 18.35.
General procedure
A mixture of thiosemicarbazide (4a–4i) (10 mmol) and
20–40 mL of 2% aqueous solution of sodium hydroxide
was refluxed for 2h. en, the solution was neutralized
with diluted hydrochloric acid and the formed precipi-
tate was filtered off and crystallized from: ethanol (5a,
5b, 5e, 5f, 5h), butanol (5c, 5i) or methanol (5d, 5g).
4-Allyl-1-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]
acetyl} thiosemicarbazide (4i)
4-Phenyl-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5a)
IR (KBr), ν (cm−1): 3208 (NH), 3101 (CH aromatic), 2984,
1426, 759 (CH aliphatic), 1699 (C=O), 1600 (C=N), 1501
(C-N), 1341 (C=S), 679 (C-S). 1H NMR (DMSO-d6) δ
(ppm): 2.26 (s, 3H, CH3), 3.84 (s, 2H, CH2), 4.16 (d, J= 5
IR (KBr), ν (cm−1): 3156 (NH), 3103 (CH aromatic), 2955,
1449 (CH aliphatic), 1599 (C=N), 1511 (C-N), 1335 (C=S),
679 (C-S). 1H NMR (DMSO-d6) δ (ppm): 2.23 (s, 3H, CH3),
© 2013 Informa UK, Ltd.

482 Ł. Popiołek et al.
4.09 (s, 2H, CH2), 7.30-7.64 (m, 10H, 10CH aromatic),
13.90 (s, 1H, NH). 13C NMR δ (ppm): 10.91 (-CH3), 27.28
(-S-CH2-), 128.21, 128.68, 128.95, 129.36, 129.61, 129.94
(10CHaromatic), 132.94, 133.16 (2Caromatic), 147.46 (C-3 triaz-
ole), 148.14 (C-3` triazole), 153.06 (C-S), 168.24 (C=S).
MS m/z (%): 380 (M+, 0.03), 348 (0.04), 326 (0.05), 313
(1), 268 (9), 204 (1), 190 (100), 158 (1.5), 149 (19), 135 (2),
118 (5), 91 (7), 77 (19). Analysis for C18H16N6S2 (380.49);
Calculated: C, 56.82; H, 4.24; N, 22.09; S, 16.85; Found: C,
56.69; H, 4.23; N, 22.02; S, 16.91.
Analysis for C20H20N6OS2 (424.54); Calculated: C, 56.58; H,
4.75; N, 19.79; S, 15.10; Found: C, 56.55; H, 4.73; N, 19.82;
S, 15.03.
4-Cyclohexyl-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5g)
IR (KBr), ν (cm−1): 3164 (NH), 3068 (CH aromatic), 2989,
1455, 764 (CH aliphatic), 1610 (C=N), 1509 (C-N), 1335
(C=S), 670 (C-S). 1H NMR (DMSO-d6) δ (ppm): 1.04-1.34
(m, 10H, 5CH2 cyclohexyl), 2.16 (s, 3H, CH3), 4.35 (s,
2H, CH2), 4.80 (s, 1H, CH cyclohexyl), 7.31-7.68 (m, 5H,
5CH aromatic), 13.63 (s, 1H, NH). Analysis for C18H22N6S2
(386.54); Calculated: C, 55.93; H, 5.74; N, 21.74; S, 16.59;
Found: C, 55.71; H, 5.72; N, 21.78; S, 16.55.
4-(4-Bromophenyl)-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5b)
IR (KBr), ν (cm−1): 3174 (NH), 3111 (CH aromatic), 2979,
1449 (CH aliphatic), 1610 (C=N), 1500 (C-N), 1322 (C=S),
688 (C-S). 1H NMR (DMSO-d6) δ (ppm): 2.17 (s, 3H,
CH3), 4.06 (s, 2H, CH2), 7.27-7.62 (m, 9H, 9CH aromatic),
13.87 (s, 1H, NH). Analysis for C18H15BrN6S2 (459.38);
Calculated: C, 47.06; H, 3.29; N, 18.29; S, 13.96; Found: C,
46.88; H, 3.29; N, 18.34; S, 14.01.
4-Ethyl-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]
methyl}-4H-1,2,4-triazole-3(2H)-thione (5h)
IR (KBr), ν (cm−1): 3155 (NH), 3064 (CH aromatic), 2966,
1449, 762 (CH aliphatic), 1608 (C=N), 1505 (C-N), 1346
1
(C=S), 682 (C-S). H NMR (DMSO-d6) δ (ppm): 1.12 (t,
J= 7.5 Hz, 3H, CH3), 2.28 (s, 3H, CH3), 3.39-3.48 (q, J= 7.5
Hz, 2H, CH2), 4.38 (s, 2H, CH2), 7.27-7.66 (m, 5H, 5CH
aromatic), 13.66 (s, 1H, NH). Analysis for C14H16N6S2
(332.45); Calculated: C, 50.58; H, 4.85; N, 25.28; S, 19.29;
Found: C, 50.62; H, 4.83; N, 25.33; S, 19.33.
4-(4-Chlorophenyl)-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5c)
IR (KBr), ν (cm−1): 3165 (NH), 3100 (CH aromatic), 2956,
1443 (CH aliphatic), 1611 (C=N), 1504 (C-N), 1312 (C=S),
683 (C-S). 1H NMR (DMSO-d6) δ (ppm): 2.21 (s, 3H,
CH3), 4.10 (s, 2H, CH2), 7.14-7.73 (m, 9H, 9CH aromatic),
13.90 (s, 1H, NH). Analysis for C18H15ClN6S2 (414.93);
Calculated: C, 52.19; H, 3.64; N, 20.25; S, 15.45; Found: C,
52.29; H, 3.63; N, 20.28; S, 15.42.
4-Allyl-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]
methyl}-4H-1,2,4-triazole-3(2H)-thione (5i)
IR (KBr), ν (cm−1): 3181 (NH), 3099 (CH aromatic), 2984,
1456, 753 (CH aliphatic), 1603 (C=N), 1501 (C-N), 1325
1
C=S, 679 C-S. H NMR (DMSO-d6) δ (ppm): 2.19 (s, 3H,
4-(4-Methoxyphenyl)-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5d)
IR (KBr), ν (cm−1): 3144 (NH), 3093 (CH aromatic), 2945,
1466 (CH aliphatic), 1602 (C=N), 1504 (C-N), 1328 (C=S),
683 (C-S). 1H NMR (DMSO-d6) δ (ppm): 2.16 (s, 3H, CH3),
3.79 (s, 3H, CH3), 4.00 (s, 2H, CH2), 6.82-7.59 (m, 9H,
9CH aromatic), 13.79 (s, 1H, NH). Analysis for C19H18N6S2
(410.51); Calculated: C, 55.59; H, 4.42 N, 20.47; S, 15.62;
Found: C, 55.80; H, 4.43; N, 20.51; S, 15.68.
CH3), 4.26 (s, 2H, CH2), 4.58-4.60 (d, J=5 Hz, 2H, CH2),
4.95-5.14 (dd, 2H, =CH2), 5.66-5.83 (m, 1H, CH), 7.32-7.63
(m, 5H, 5CH aromatic), 13.71 (s, 1H, NH). Analysis for
C15H16N6S2 (344.46); Calculated: C, 52.30; H, 4.68; N, 24.40;
S, 18.62; Found: C, 52.44; H, 4.69; N, 24.49; S, 18.66.
[(4-Phenyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl] acetic
acid (6)
Compound (6) was obtained using the same method
described earlier for derivatives (5a–5i). at is, a mix-
ture of thiosemicarbazide (4j) (10 mmol) and 20 mL of
2% aqueous solution of sodium hydroxide was refluxed
for 2 hrs. en, the solution was neutralized with diluted
hydrochloric acid and the formed precipitate was filtered
off and crystallized from ethanol.
4-Benzyl-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5e)
IR (KBr), ν (cm−1): 3150 (NH), 3097 (CH aromatic), 2981,
1439, 765 (CH aliphatic), 1602 (C=N), 1501 (C-N), 1333
1
(C=S), 679 (C-S). H NMR (DMSO-d6) δ (ppm): 2.23 (s,
Yield: 72.8%; M. p. 232-234°C. IR (KBr), ν (cm−1): 3111
(CH aromatic), 3023 (OH), 2967, 1423 (CH aliphatic),
3H, CH3), 4.26 (s, 2H, CH2), 5.28 (s, 2H, CH2), 7.18-7.66
(m, 10H, 10CH aromatic), 13.89 (s, 1H, NH). Analysis for
C19H18N6S2 (394.52); Calculated: C, 57.84; H, 4.60; N, 21.30;
S, 16.25; Found: C, 57.75; H, 4.61; N, 21.24; S, 16.21.
1
1720 (C=O), 1597 (C=N), 698 (C-S). H NMR (DMSO-d6)
δ (ppm): 2.56 (s, 3H, CH3), 4.21 (s, 2H, CH2), 7.34-7.74
(m, 5H, 5CH aromatic), 12.15 (s, 1H, OH). Analysis for
C11H11N3O2S (249.29); Calculated: C, 53.00; H, 4.45; N,
16.86; S, 12.86; Found: C, 53.13; H, 4.46; N, 16.89; S, 12.82.
4-(4-Methoxybenzyl)-5-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (5f)
IR (KBr), ν (cm−1): 3179 (NH), 3079 (CH aromatic), 2974,
1446, 759 (CH aliphatic), 1614 (C=N), 1512 (C-N), 1329
4-Carboxymethyl-5-{[(4-phenyl-5-methyl-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 earlier for derivatives (5a-5i). at is, a mixture
1
(C=S), 669 (C-S). H NMR (DMSO-d6) δ (ppm): 2.30 (s,
3H, CH3), 3.78 (s, 3H, CH3), 4.40 (s, 2H, CH2), 5.21 (s, 2H,
CH2), 6.80-7.68 (m, 9H, 9CH aromatic), 13.93 (s, 1H, NH).
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and in vitro antimicrobial activity of triazoles 483
of thiosemicarbazide (4k) (10 mmol) and 20 mL of 2%
aqueous solution of sodium hydroxide was refluxed for
2 hrs. en, the solution was neutralized with diluted
hydrochloric acid and the formed precipitate was filtered
off and crystallized from ethanol.
1H NMR (DMSO-d6) δ (ppm): 2.27 (s, 3H, CH3), 4.63 (s,
2H, CH2), 7.41-7.67 (m, 9H, 9CH aromatic), 10.13 (s, 1H,
NH). Analysis for C18H15ClN6S2 (414.93); Calculated: C,
52.10; H, 3.64; N, 20.25; S, 15.45; Found: C, 52.08; H, 3.65;
N, 20.20; S, 15.49.
IR (KBr), ν (cm−1): 3280 (NH), 3066 (OH), 3044 (CH
aromatic), 2980, 1444 (CH aliphatic), 1711 (C=O), 1602
(C=N), 1501 (C-N), 1355 (C=S), 677 (C-S). 1H NMR
(DMSO-d6) δ (ppm): 2.40 (s, 3H, CH3), 4.52 (s, 2H, CH2),
4.85 (s, 2H, CH2), 7.44-7.78 (m, 5H, 5CH aromatic), 9.33
(s, 1H, OH), 13.93 (s, 1H, NH). Analysis for C14H14N6O2S2
(362.43); Calculated: C, 46.39; H, 3.89; N, 23.19; S, 17.69;
Found: C, 46.44; H, 3.90; N, 23.28; S, 17.72.
5-Aminocyclohexyl-2-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (9g)
IR (KBr), ν (cm−1): 3276 (NH), 3099 (CH aromatic), 2975,
1460, 766 (CH aliphatic), 1608 (C=N), 1509 (C-N), 677
1
(C-S). H NMR (DMSO-d6) δ (ppm): 1.12-1.85 (m, 10H,
5CH2 cyclohexyl), 2.22 (s, 3H, CH3), 3.74 (s, 2H, CH2), 4.17
(s, 1H, CH cyclohexyl), 7.35-7.69 (m, 5H, 5CH aromatic),
10.10 (s, 1H, NH). Analysis for C18H22N6S2 (386.54);
Calculated: C, 55.93; H, 5.74; N, 21.74; S, 16.59; Found: C,
56.06; H, 5.76; N, 21.66; S, 16.63.
5-{[(4-phenyl-5-methyl-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 earlier for derivatives (5a–5i). at is, a mix-
ture of thiosemicarbazide (4l) (10 mmol) and 20 mL of
2% aqueous solution of sodium hydroxide was refluxed
for 2 hrs. en, the solution was neutralized with diluted
hydrochloric acid and the formed precipitate was filtered
off and crystallized from ethanol.
5-Aminoethyl-2-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-3-yl)
sulfanyl]methyl}-1,3,4-thiadiazole (9h)
IR (KBr), ν (cm−1): 3289 (NH), 3077 (CH aromatic), 2944,
1433, 779 (CH aliphatic), 1605 (C=N), 1502 (C-N), 671
(C-S). 1H NMR (DMSO-d6) δ (ppm): 1.20 (t, J= 7.5 Hz, 3H,
CH3), 2.27 (s, 3H, CH3), 3.24-3.30 (q, J= 5 Hz, 2H, CH2),
3.86 (s, 2H, CH2), 7.35–7.75 (m, 5H, 5CH aromatic), 10.17
(s, 1H, NH). Analysis for C14H16N6S2 (332.45); Calculated:
C, 50.57; H, 4.85; N, 25.28; S, 19.29; Found: C, 50.38; H,
4.84; N, 25.25; S, 19.33.
IR (KBr), ν (cm−1): 3198 (NH), 3082 (CH aromatic),
2989, 1432 (CH aliphatic), 1612 (C=N), 1505 (C-N), 1343
1
(C=S), 688 (C-S). H NMR (DMSO-d6) δ (ppm): 2.15 (s,
3H, CH3), 3.97 (s, 2H, CH2), 7.16-7.57 (m, 5H, 5CH aro-
matic), 13.65 (s, 1H, NH), 13.84 (s, 1H, NH). Analysis for
C12H12N6S2 (304.39); Calculated: C, 47.35; H, 3.97; N, 27.61;
S, 21.07; Found: C, 47.51; H, 3.98; N, 27.72; S, 21.02.
Microbiology
Materials and methods
ApanelreferencestrainsofaerobicbacteriafromAmerican
Type Culture Collection, including 4 Gram-positive bacte-
ria - Staphylococcus aureus ATCC 25923, Staphylococcus
epidermidis ATCC 12228, Bacillus subtilis ATCC 6633,
Micrococcus luteus ATCC 10240 and 4 Gram-negative bac-
teria – Escherichia coli ATCC 25922, Klebsiella pneumoniae
ATCC 13883, Proteus mirabilis ATCC 12453, Pseudomonas
aeruginosa ATCC 9027, were used. Microbial suspen-
sions with an optical density of 0.5 McFarland standard
– 150×106 CFU/mL (CFU – colony forming units) were
prepared in sterile 0.85% NaCl. All stock solutions of the
tested compounds were prepared in DMSO (dimethyl sul-
foxide). e medium with DMSO at the final concentration
and without the tested compounds served as control - no
microbial growth inhibition was observed.
Derivatives of (5-aminosubstituted)-2-{[(4-phenyl-5-
methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-1,3,4-
thiadiazole (9a, 9c, 9g, 9h)
General method (for compounds 9a, 9c, 9g, 9h)
10 mmol of 4-substituted-1-{[(4-phenyl-5-methyl-4H-1-
,2,4-triazol-3-yl)sulfanyl]acetyl} thiosemicarbazide (4a,
4c, 4g, 4h) was dissolved in 10–20 mL diluted sulphuric
acid and stirred in a closed bulb for 1hr. After that 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. Formed
precipitate was filtered off, dried, and crystallized from:
ethanol (9a, 9h), butanol (9c) or methanol (9g).
(5-Aminophenyl)-2-{[(4-phenyl-5-methyl-4H-1,2,4-triazol-
3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (9a)
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 concentration 1000 µg/mL. e plates were poured on
the day of testing. 10 μL of each bacterial suspension was
put onto the prepared solid media. e plates were incu-
bated at 37°C for 18 hrs.
IR (KBr), ν (cm−1): 3237 (NH), 2999 (CH aromatic), 2980,
1
1448 (CH aliphatic), 1620 (C=N), 1499 (C-N), 695 (C-S). H
NMR (DMSO-d6) δ (ppm): 2.20 (s, 3H, CH3), 4.56 (s, 2H,
CH2), 6.89–7.59 (m, 10H, 10CH aromatic), 10.31 (s, 1H, NH).
Analysis for C18H16N6S2 (380.49); Calculated: C, 56.82; H, 4.24;
N, 22.09; S, 16.85; Found: C, 56.76; H, 4.23; N, 22.16; S, 16.89.
e antibacterial activity in vitro of the potentially
active compounds were determined by broth microdilu-
tion method on the basis of minimal inhibitory concentra-
tion (MIC), usually defined as the lowest concentration
of the compound at which there was no visible growth of
[5-Amino-(4-chlorophenyl)]-2-{[(4-phenyl-5-methyl-4H-1,2,4-
triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (9c)
IR (KBr), ν (cm−1): 3198 (NH), 3076 (CH aromatic), 2934,
1455 (CH aliphatic), 1598 (C=N), 1503 (C-N), 687 (C-S).
© 2013 Informa UK, Ltd.

484 Ł. Popiołek et al.
microorganisms. e 96-well microplates were used; 198
µL of Mueller-Hinton broth without or with a series of
two-fold dilution of the tested compound in the range of
final concentrations from 3.91 to 1000 μg/mL (0.24 to 1000
μg/mL for 9a, c, g, h) was inoculated with 2 µL of micro-
bial suspension (total volume per each well - 200 µL).
After incubation (at 35°C for 18h), spectrophotometric
measurements of optical density (OD600) of the bacterial
cultures with or without the tested compounds were per-
formed in order to determine MIC. e blank control wells
with two-fold dilution of each of the tested compounds
added to Mueller-Hinton broth without bacterial suspen-
sion were incubated under the same conditions. For some
substances with promising antibacterial activity the values
of minimal bactericidal concentration (MBC), defined as
the lowest concentration of each compound that resulted
in >99.9% reduction in CFU (colony forming units) of
the initial inoculum, was also determined. e MBC was
calculated using broth microdilution technique by plating
out onto Mueller Hinton agar plates the contents of wells
(20 µL) that showed no visible bacterial growth after pre-
vious incubation under the conditions mentioned above;
incubation of plates was conducted at 35°C for 18h. Both
MIC and MBC values were given in μg/mL. On the basis of
MBC/MIC ratio, bactericidal (MBC/MIC ≤ 4) or bacterio-
static (MBC/MIC > 4) effect was assessed16,17. Cefuroxime
(belonging to 2nd generation of cephalosporins) and gen-
tamicin was used as control antimicrobial agent at final
concentration from 0.063 to 500 µg/mL.
Results and discussion
Chemistry
e common method for the preparation of 1,2,4-triazole
and 1,3,4-thiadiazole is the cyclization reaction of thi-
osemicarbazide derivatives. In earlier papers18,19 it was
Scheme 1. Synthesis of new thiosemicarbazide, 1,2,4-triazole and 1,3,4-thiadiazole derivatives.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and in vitro antimicrobial activity of triazoles 485
Table 1. Physicochemical data and synthesis parameters for thiosemicarbazide derivatives (4a–4l).
Temp. of
reaction (°C) reaction (hrs)
Time of
Molecular
weight
Compound
R
Yield (%)
92.7
M. p. (°C)
176–178
Molecular formula
C18H18N6OS2
4a
50
95
80
50
45
50
50
10
398.50
477.40
432.95
428.53
412.53
442.56
404.55
4b
4c
4d
4e
4f
89.6
86.8
98.3
97.8
77.8
83.9
116–118
128–130
130–132
168–170
152–154
151–153
14
14
12
12
12
10
C18H17BrN6OS2
C18H17ClN6OS2
C19H20N6O2S2
C19H20N6OS2
C20H22N6O2S2
C18H24N6OS2
4g
4h
4i
–C2H5
82.5
83.4
75.5
75.1
98.1
162–164
163–165
75–77
60
50
50
50
50
8
350.46
362.47
394.47
408.50
426.51
C14H18N6OS2
C15H18N6OS2
C15H18N6O3S2
C16H20N6O3S2
C19H18N6O2S2
–CH2–CH=CH2
–COOC2H5
12
14
16
16
4j
4k
4l
–CH2COOC2H5
88–90
68–70
stated that the reaction of cyclization was affected not
only by pH of the medium but also by the nature of sub-
stituents in thiosemicarbazide derivatives.
Cyclization of thiosemicarbazide in the presence of
alkaline media usually promotes reaction to obtain 1,2,4-
triazole system, while in acidic media 1,3,4-thiadiazole
derivatives were obtained20.
converted to hydrazide (3) in reaction with 100% hydra-
zine hydrate.
Reactionsofhydrazide(3)withvariousisothiocyanates
were carried out in two ways. All new thiosemicarbazide
derivatives (4a–4l) were obtained by heating substracts
in an oil bath, temperatures were selected experimen-
tally (t = 45–95°C). iosemicarbazide derivatives (4a, 4f,
4h, 4j) were products of the reaction hydrazide (3) with
appropriate isothiocyanates in the presence of diethyl
ether.
New thiosemicarbazide derivatives (4a–4i) in cycl-
ization reaction with 2% aqueous solution of sodium
hydroxide lead to a new group of 1,2,4-triazole deriva-
tives (5a–5i).
e hydrolysis was observed in the case of cyclization
of 4-ethoxycarbonyl-1-substituted thiosemicarbazide
(4j) in alkaline media, which led to obtain [(4-phenyl-5-
methyl-4H-1,2,4-triazol-3-yl)sulfanyl]aceticacid(6).is
compound was described earlier22, but it was obtained in
a different way. e cyclization in alkaline media of thi-
osemicarbazide which contains ethoxycarbonylmethyl
group (4k) and benzoyl (4l) in the 4 position led to obtain
1,2,4-triazole derivatives (7,8).
By the cyclization in alkaline media of 1-methyl-4-
phenyl thiosemicarbazide, 4-phenyl-5-methyl-4H-1-
,2,4-triazole-3-thione (1) was obtained. is compound,
can exist in two tautomeric forms, was a starting mate-
rial for synthesis of new derivatives, which consist of
two 1,2,4-triazole systems or 1,2,4-triazole and 1,3,4-
thiadiazole systems connected with S-methylene bridge.
Depending on the conditions used, 4-phenyl-5-methyl-
4H-1,2,4-triazole-3-thione (1) can lead either to the S- or
N-derivatives. In this paper the reaction of this compound
with ethyl bromoacetate was investigated (2). Basing on
the results of the previous papers, the elemental and
spectral analysis as well as on the X-ray crystallography
of similar compound, it was revealed and confirmed that
the reaction leads to the formation of S-derivative21.
Reaction with ethyl bromoacetate in presence of
sodium ethanolate gave ethyl [(4-phenyl-5-methyl-4-
H-1,2,4-triazol-3-yl)sulfanyl] acetate (2), which was
e cyclization reaction in acidic media of new
derivatives of thiosemicarbazide enabled us to
© 2013 Informa UK, Ltd.

486 Ł. Popiołek et al.
Table 2. Physicochemical data of 1,2,4-triazole and 1,3,4-thiadiazole derivatives (5a–5i, 7, 8, 9a, c, g, h).
Compound
R
Yield (%)
81.2
M. p. (°C)
122–124
Molecular weight Molecular formula
5a
380.49
459.38
414.93
410.51
394.52
424.54
386.54
C18H16N6S2
C18H15BrN6S2
C18H15ClN6S2
C19H18N6OS2
C19H18N6S2
5b
5c
5d
5e
5f
81.6
65.2
62.6
83.6
83.4
80.2
106–108
124–126
162–164
122–124
79–81
Br
C20H20N6OS2
C18H22N6S2
5g
114–116
5h
5i
7
–C2H5
85.9
39.5
87.8
28.1
78.9
145–147
158–160
226–228
105–107
112–114
332.45
344.46
362.43
304.39
380.49
C14H16N6S2
C15H16N6S2
C14H14N6O2S2
C12H12N6S2
C18H16N6S2
–CH2–CH=CH2
–CH2COOH
–H
8
9a
9c
9g
9h
52.6
62.2
67.8
106–108
122–124
138–140
414.93
386.54
332.45
C18H15ClN6S2
C18H22N6S2
C14H16N6S2
–C2H5
obtain new derivatives of 1,3,4-thiadiazole, that is:
(5-aminosubstituted)-2-{[(4-phenyl-5-methyl-4H-
1,2,4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole
(9a, 9c, 9g, 9h).
in the δ 8.35-11.75 ppm range. Whereas for the new com-
pounds consisting of two 1,2,4-triazole system (5a–5i, 7,
8) one proton signal of the NH group was observed in the
δ 13.63-13.93 ppm range. e 1,3,4-thiadiazole deriva-
tives (9a, 9c, 9g, 9h) had one typical proton signal of NH
group in the δ 10.10–10.31 ppm range.
e reactions were performed according to
Scheme 1. Physicochemical data and synthesis param-
eters for thiosemicarbazide derivatives (4a–4l) are
presented in the Table 1. e Table 2 contains physi-
cochemical data for 1,2,4-triazole and 1,3,4-thiadiazole
derivatives (5a–5i, 7, 8, 9a, 9c, 9g, 9h).
e structure of the obtained compounds was con-
1
firmed by elementary analysis, IR and H NMR spectra.
Some of the compounds were also submitted to 13C NMR
and MS spectra analysis.
In the IR spectra of the compounds which consist of two
1,2,4-triazole system (5a–5i, 7, 8), the following character-
istic absorption bands were observed: about 1500cm−1
corresponding to C-N group and in the range of 1600cm−1
– corresponding to C=N group. en in the IR spectra of
the new derivatives of 1,3,4-thiadiazole (9a, 9c, 9g, 9h) the
following characteristic absorption bands were observed:
in the range of 1500cm−1 - corresponding to C-N group and
in the range of 1600cm−1 – corresponding to C=N group.
1H NMR spectra of the thiosemicarbazide derivatives
(4a–4l) show three proton signals typical for the NH group
Microbiology
According to the preliminary results obtained by agar
dilution method, nine of newly synthesized compounds
possessed potential activity against Gram-positive bacte-
ria. None of the compounds had inhibitory effect on the
Gram-negative bacteria growth.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and in vitro antimicrobial activity of triazoles 487
Table 3. e influence of newly synthesized compounds on the growth of Gram-positive bacteria of on the basis of MIC (µg/mL) or MBC
(µg/mL) values obtained by the broth microdilution method.
Species
Sa25923
Se12228
Bs6633
Ml10240
Compound
MIC
1000
31.25
31.25
1000
1000
500
1000
>1000
125
1000
15.63
1000
1000
>1000
1000
125
MBC
nd
MIC
MBC
nd
MIC
1000
31.25
31.25
1000
1000
1000
1000
1000
15.63
1000
15.63
500
MBC
nd
MIC
250
7.8
MBC
nd
4a
4b
4c
4e
4f
4g
4h
4i
4l
5a
5b
5c
5e
5f
5g
9a
1000
31.25
62.5
1000
>1000
1000
1000
>1000
62.5
1000
31.25
>1000
1000
>1000
1000
>1000
31.25
0.49
1000
1000
nd
>1000
>1000
nd
62.5
500
nd
250
125
nd
15.63
1000
1000
125
1000
>1000
31.25
1000
15.63
7.8
1000
>1000
1000
125
3.91
0.49
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
>1000
>1000
nd
>1000
>1000
nd
>1000
250
nd
>1000
>1000
nd
>1000
>1000
nd
>1000
>1000
nd
31.25
>1000
nd
62.5
62.5
nd
1000
500
nd
nd
nd
nd
nd
nd
1000
62.5
31.25
0.49
nd
nd
>1000
1000
nd
>1000
1000
nd
>1000
31.25
nd
>1000
250
nd
9c
15.63
0.49
Cefuroxime
Gentamicin
0.015
nd
0.06
nd
0.015
nd
0.12
nd
Abbreviations: nd - not determined, Sa25923 - Staphylococcus aureus ATCC 25923, Se12228 - Staphylococcus epidermidis ATCC 12228,
Bs6633 - Bacillus subtilis ATCC 6633, Ml10240 - Micrococcus luteus ATCC 10240.
On the basis of MIC and MBC values obtained by
broth microdilution method it was shown that antibacte-
rial activity was species-dependent (Table 3). e most
sensitive bacteria to newly synthesized derivatives were
Bacillus subtilis ATCC 6633 (MIC = 15.63–62.5 µg/mL),
Micrococcus luteus ATCC 10240 (MIC = 3.91–125 µg/mL)
and Staphylococcus aureus ATCC 25923 (MIC = 15.63–125
µg/mL).
e compounds 4b, 4c, 4l, 5b, 9c had good activ-
ity against nonpathogenic Bacillus subtilis ATCC 6633
(MIC = 15.63–31.25 µg/mL, MBC = 31.25–500 µg/mL),
while compounds 4b, 4c, 4l, 5b, 5c, 9c− against oppor-
tunistic Micrococcus luteus ATCC 10240 (MIC = 7.8–31.25
µg/mL, MBC = 31.25− >1000 µg/mL).
bactericidal effect against Bacillus subtilis ATCC 6633
(MIC = MBC = 31.25 µg/mL, MBC/MIC = 1).
In our experiments MICs of available antibiotics such
as cefuroxime and gentamicin, that had been extensively
used to treat of bacterial infections, were also estimated.
MIC values for reference strains of the tested bacteria
were 0.015 to 0.12 µg/mL for gentamicin and 0.49 µg/mL
for cefuroxime.
e compounds 4b, 4c, 4l, 5b, 5c, 9a, 9c with good
activity against the reference Gram-positive bacteria may
be regarded as precursor compounds for searching new
derivatives showing better antimicrobial activity against
pathogenic (e.g. Staphylococcus aureus) or opportunis-
tic (e.g. Staphylococcus epidermidis, Micrococcus luteus,
Bacillus subtilis) bacteria. Today due to several serious
problems such as growing drug resistance of bacteria or
undesirable side effects of drugs it is very important to
find new substances with antimicrobial activity.
e
highest
activity
against
pathogenic
Staphylococcus aureus ATCC 25923 and opportunistic
Staphylococcus epidermidis ATCC 12228 had 4b, 4c, 4l,
5b, 9a, 9c compounds (MIC = 15.63–62.5 µg/mL, MBC
≥ 1000 µg/mL).
According to our results, the potentially active com-
pounds had mainly bacteriostatic effect (MBC/MIC ratio
from 8 to ≥32); bactericidal were only compounds 5b
against B. subtilis ATCC 6633 or M. luteus ATCC 10240
(MBC/MIC = 2) and 4b against B. subtilis ATCC 6633
(MBC/MIC = 2).
Among all compounds the highest activity had 9c
with bacteriostatic effect against Micrococcus luteus
ATCC 10240 (MIC = 3.91 µg/mL, MBC = 250 µg/mL,
MBC/MIC = 64), Staphylococcus aureus ATCC 25923
(MIC = 15.63 µg/mL, MBC = 1000 µg/mL, MBC/MIC = 64),
Staphylococcus epidermidis ATCC 12228 (MIC = 31.25
µg/mL, MBC = 1000 µg/mL, MBC/MIC = 32) and with
Declaration of interest
e authors declare no conflict of interest.
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