New Derivatives of Thiosemicarbazide and 1,2,4-Triazoline-5-thione with Potential Antimicrobial Activity

Article abstract of DOI:10.1080/10426500802208490

In the reaction of the hydrazide of (4-nitroimidazol-1-yl)acetic acid 1 with isothiocyanates, the respective thiosemicarbazide derivatives 2-6 were obtained. Further cyclization with 2% NaOH led to the formation of 3-[(4-nitroimidazol-1-yl)-methyl]-4-substituted-1,2,4-triazoline-5-thiones 7-11. The structures of all new products were confirmed by analytical and spectroscopic methods. Six compounds 2-5, 8, and 10 were screened for their in vitro activity against some species of aerobic bacteria and fungi. Compound 3 appears to be a promising precursor of agents with antibacterial activity against Micrococcus luteus.

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New Derivatives of
Thiosemicarbazide and 1,2,4-
Triazoline-5-thione with
Potential Antimicrobial Activity
Monika Wujec a , Urszula Kosikowska b , Agata Siwek
a & Anna Malm b
a Department of Organic Chemistry , Medical
University , Lublin, Poland
b Department of Pharmaceutical Microbiology ,
Medical University , Lublin, Poland
Published online: 20 Feb 2009.
To cite this article: Monika Wujec , Urszula Kosikowska , Agata Siwek & Anna Malm
(2009) New Derivatives of Thiosemicarbazide and 1,2,4-Triazoline-5-thione with
Potential Antimicrobial Activity, Phosphorus, Sulfur, and Silicon and the Related
Elements, 184:3, 559-567, DOI: 10.1080/10426500802208490
To link to this article: http://dx.doi.org/10.1080/10426500802208490
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Phosphorus, Sulfur, and Silicon, 184:559–567, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500802208490
New Derivatives of Thiosemicarbazide and
1,2,4-Triazoline-5-thione with Potential Antimicrobial
Activity
Monika Wujec,1 Urszula Kosikowska,2 Agata Siwek,1
and Anna Malm2
1Department of Organic Chemistry, Medical University, Lublin, Poland
2Department of Pharmaceutical Microbiology, Medical University,
Lublin, Poland
In the reaction of the hydrazide of (4-nitroimidazol-1-yl)acetic acid 1 with isothio-
cyanates, the respective thiosemicarbazide derivatives 2–6 were obtained. Further
cyclization with 2% NaOH led to the formation of 3-[(4-nitroimidazol-1-yl)-methyl]-
4-substituted-1,2,4-triazoline-5-thiones 7–11. The structures of all new products
were confirmed by analytical and spectroscopic methods. Six compounds 2–5, 8,
and 10 were screened for their in vitro activity against some species of aerobic bac-
teria and fungi. Compound 3 appears to be a promising precursor of agents with
antibacterial activity against Micrococcus luteus.
Keywords Antimicrobial activity; isothiocyanates; thiosemicarbazides; 1,2,4-triazoles
INTRODUCTION
1,2,4-Triazoles have been reported to exhibit antimicrobial, fungi-
cidal, anti-inflammatory, antiparasitic, insecticidal, herbicidal, an-
tiviral, antitumor, anticonvulsant, antidepressant, and hypotensive
effects and plant growth regulatory activities.1–8 Alternatively, ni-
troimidazoles such as metronidazole, ornidazole, secnidazole, and
tinidazole are widely used in the treatment of diseases caused by proto-
zoa and anaerobic bacteria.9–12 Furthermore, some 5-nitroimidazoles
have been shown to be active against Helicobacter pylori.13–14 It has
also been shown that the presence of the 4-nitroimidazole substituent
in the triazole, oxadiazole, and thiadiazole ring was the most im-
portant reason for antibacterial activity of the tested compounds.15
This article is a continuation of investigations of compounds with
Received 6 March 2008; accepted 16 May 2008.
Address correspondence to Monika Wujec, Department of Organic Chemistry, Medical
University, 6 Staszica Str., 20-081 Lublin, Poland. E-mail: monika.wujec@am.lublin.pl
559

560
M. Wujec et al.
promising antimicrobial activities.16–17 In this work, we present
new analogous of 4-nitroimidazole, 3-[(4-nitroimidazol-1-yl)methyl]-
4-substituted-1,2,4-triazoline-5-thione.15 It was very interesting to
investigate if the exchange of substituent in the position 4 of 3-[(4-
nitroimidazol-1-yl)methyl]-1,2,4-triazoline-5-thione will influence an-
timicrobial activity.
RESULTS AND DISCUSSION
We have used (4-nitroimidazol-1-yl)acetic acid hydrazide 1 as
the starting material for the synthesis of new derivatives of
1,2,4-triazole-5-thione 7–11. It was obtained in the reaction of
ethyl (4-nitroimidazol-1-yl)acetate with 80% hydrazine hydrate.
New thiosemicarbazide derivatives 2–6 were obtained by the re-
action of 1 with isothiocyanates. The conditions of the reaction
were established experimentally. Thiosemicarbazides 2–6 were sub-
jected to cyclization in 2% solution of sodium hydroxide yielding
the corresponding 3-[(4-nitroimidazol-1-yl)methyl]-4-substituted-1,2,4-
triazoline-5-thiones 7–11. The reactions were performed according to
Scheme 1.
O2N
O2N
N
N
R
NCS
N
N
CH2
C
O
NHNH
C
S
NHR
CH2
C
O
NHNH2
1
2-6
2% NaOH
O2N
O2N
N
N
N
N
N
NH
N
N
CH2
CH2
SH
S
N
R
N
R
7-11
SCHEME 1
The structures of the obtained products were confirmed by ele-
1
mental analysis as well as by IR and H NMR spectra. Substituents

Derivatives of Thiosemicarbazide and 1,2,4-Triazoline-5-thione
561
TABLE I Substituents and Yields of
Thiosemicarbazide Derivatives
Intermediate
R
Yield [%]
2
3
4
5
6
C6H5
70
75
78
75
70
4-CH3C6H4
4-CH3OC6H4
4-BrC6H4
4-IC6H4
and corresponding yields of intermediates 2–6 and products 7–
11 are collected in Tables I and II, respectively. As indicated in
Scheme 1, products of the cyclization can exist in two major tau-
tomeric forms: thiole (the structure on the left) and thione. In 1H
NMR spectra of compounds 7–11 proton signals for –NH group
in the δ (ppm) 13.10–14.02 range were observed. The above ob-
servations suggest that the compounds exist mainly in the thione
form.
The in vitro antimicrobial activities of the newly synthesized
compounds 2–5, 8, and 10 at concentrations ranging from 500
to 31.25 mg L−1 were screened using the agar dilution method
against four Gram-negative and five Gram-positive reference species
of bacteria and five species of fungi. According to our prelimi-
nary results, among the tested compounds, a few of them possessed
some antibacterial (compounds 2, 3, 10) or antifungal (compound 3)
activity.
Using agar and broth dilution methods, it was found that compounds
3 and 10 inhibited the growth of Gram-positive Micrococcus luteus
ATCC 10240 with MIC = 250 mg L−1 and MIC = 500 mg L−1, respec-
tively (Table III). Compound 2 was effective against Gram-negative
TABLE II Substituents and Yields of
1,2,4-Triazoline-5-thione Derivatives
Product
R
Yield [%]
7
8
9
10
11
C6H5
85
79
80
75
70
4-CH3C6H4
4-CH3OC6H4
4-BrC6H4
4-IC6H4

562
M. Wujec et al.
TABLE III The Percentage of Growth Inhibitory Effect of the Tested
Compounds 2, 3, and 10 at Selected Concentrations
Microorganisms
Compound
mgL−1
Se
Sa1
Sa2
Bs
Bc
Ml
Ec
Kp
Pm
Pa
2
500
250
125
62.5
31.25
500
250
125
62.5
31.25
500
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
100
67
40
14
100
73
37
30
11
100
65
58
54
49
0
0
0
0
0
68
62
60
56
53
0
0
0
0
0
36
22
12
11
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
10
0
0
0
0
0
0
0
0
250
125
62.5
31.25
0
0
Abbreviations: Se – Staphylococcus epidermidis ATCC 12228 (n = 0.488), Sa1
–
Staphylococcus aureus ATCC 25923 (n = 0.474), Sa2 – Staphylococcus aureus ATCC (n
= 0.489), Bs – Bacillus subtilis ATCC 6633 (n = 0.222), Bc – Bacillus cereus ATCC
10876 (n = 0.289), Ml – Micrococcus luteus ATCC 10240 (n = 0.306), Ec – Escherichia
coli ATCC 25922 (n = 0.570), Klebsiella pneumoniae ATCC 13883 (n = 0.378), Pm –
Proteus mirabilis ATCC 12453 (n = 0.469), Pa – Pseudomonas aeruginosa ATCC 9027
(n = 0.613), n = control growth of tested bacteria in broth without compound (OD600).
bacteria with MIC = 500 mg L-1 for E. coli ATCC 25922. Further-
more, this compound caused partial reduction of the growth (about
36–65% inhibition) of Klebsiella pneumoniae ATCC 13883 and Pro-
teus mirabilis ATCC 12453 at lower concentrations ranging from 7.81
to 250 mg L-1 without any activity against remaining Gram-negative
(Pseudomonas aeruginosa ATCC 9027) or Gram-positive (Staphylococ-
cus spp. and Bacillus spp.) bacteria (Table III).
Compound 3 showed inhibitory effect against Trichophyton men-
thagrophytes ATCC 9533 with MIC = 250 mg L−1; in addition, this
compound caused partial (about 5–70%) inhibition of the growth of
Candida spp. and Aspergillus niger ATCC 16404 at 31.25–500 mg L−1
concentrations (Figure 1).
The antimicrobial result demonstrated that it is worth obtaining
research in this group of compounds for more activity against Gram-
positive and fungi.

Derivatives of Thiosemicarbazide and 1,2,4-Triazoline-5-thione
563
FIGURE 1 The influence of compound 3 on fungi growth (Ca – Candida al-
bicans, Cp – Candida parapsilosis, Tm – Trichophyton menthagrophytes, An –
Aspergillus niger).
EXPERIMENTAL
Chemistry
Melting points were determined in Fisher-Johns blocs and are pre-
sented without any corrections. IR spectra were recorded in KBr using
Specord IR-75 spectrophotometer. The 1H NMR spectra were recorded
on a Brucker Avance 300 in DMSO-d6 with TMS as internal standard.
Chemicals were purchased from Lancaster or Merck Co. and used with-
out further purification. Purity was checked by TLC on Merck Co. plates
using aluminium oxide 60 F254 in a CHCl3/C2H5OH (10:1) solvent sys-
tem with UV visualization.

564
M. Wujec et al.
1-[(4-Nitroimidazol-1-yl)acetyl]-4-substituted
thiosemicarbazide 2–6
0.01 Mol of hydrazide of (4-nitroimidazol-1-yl)acetic acid (1) and 0.01
mol of appropriate isothiocyanate were heated at the 60–70oC for 8 h.
The product was washed with Et2O to remove the unreacted isothio-
cyanate, dried, and crystallized from EtOH (70–78%).
1-[(4-Nitroimidazol-1-yl)acetyl]-4-phenyl-thiosemicarbazide 2:. mp
140–142oC. IR (KBr) cm−1: 3200, 3060, 2980, 1460, 1700, 1552, 1210.
1H-NMR (DMSO-d6) δ: 4.95(2H, s), 7.12 (5H, m), 7.76 (1H, m), 8.28
(1H, m), 9.41 (1H, s), 9.68 (1H, s), 10.48 (1H, s). Anal. Calcd for
C12H12N6O3S: 320.328, C, 44.99; H, 3.78; N, 26.24. Found: C, 44.92;
H, 3.71; N, 26.33.
1-[(4-Nitroimidazol-1-yl)acetyl]-4-(4-tolyl)-thiosemicarbazide 3:. mp
178–180oC. IR (KBr) cm−1: 3210, 3071, 2950, 1450, 1710, 1556, 1200.
1H-NMR (DMSO-d6) δ: 2.23 (3H, s), 4.94 (2H, s), 7.08 (4H, m), 7.79
(1H, s), 8.29 (1H, m), 9.59 (2H, m), 10.43 (1H, s). Anal. Calcd for
C13H14N6O3S: 334.355 C, 46.69; H, 4.22; N, 25.14. Found: C, 46.32;
H, 4.21; N, 25.11.
4-(4-Metoxyphenyl)-1-[(4-nitroimidazol-1-yl)acetyl]
thiosemicarbazide 4. mp 192–194◦C. IR (KBr) cm−1: 3200, 3070,
2980, 1445, 1700, 1548, 1195. 1H-NMR (DMSO-d6) δ: 3.75 (3H, s),
4.94 (2H, s), 6.88 (4H, m), 7.78 (1H, s), 8.28 (1H, m), 9.56 (1H, s),
9.65(1H, s), 10.44(1H, s). Anal. Calcd for C13H14N6O4S: 350.354 C,
44.56; H, 4.03; N, 23.99. Found: C, 44.51; H, 4.02; N, 23.90.
4-(4-Bromophenyl)-1-[(4-nitroimidazol-1-yl)acetyl]
thiosemicarbazide 5. mp 213–215◦C. IR (KBr) cm−1: 3230, 3075,
2940, 1455, 1690, 1545, 1200. 1H-NMR (DMSO-d6) δ: 4.95 (2H, s),
7.39 (4H, m), 7.78 (1H, m), 8.28 (1H, m), 9.73 (1H, s), 9.86 (1H, s),
10.50 (1H, s). Anal. Calcd for C12H11BrN6O3S: 399.224 C, 36.10; H,
2.78; N, 21.05. Found: C, 36.10; H, 2.76; N, 21.00.
4-(4-Iodophenyl)-1-[(4-nitroimidazol-1-yl)acetyl]
thiosemicarbazide 6. mp 202–204◦C. IR (KBr) cm−1: 3240, 3058,
2940, 1468, 1698, 1551, 1198. 1H-NMR (DMSO-d6) δ: 4.95 (2H, s),
7.25 (4H, m), 7.77 (1H, m), 8.28 (1H, m), 9.70 (1H, s), 9.91 (1H, s),
10.49 (1H, s). Anal. Calcd for C12H11IN6O3S: 446.225 C, 32.30; H, 2.48;
N, 18.83. Found: C, 32.27; H, 2.46; N, 18.82.

Derivatives of Thiosemicarbazide and 1,2,4-Triazoline-5-thione
565
3-[(4-Nitroimidazol-1-yl)methyl]-4-substituted-1,2,4-triazoline-
5-thione 7–11
General procedure. 0.01 Mol of thiosemicarbazide 2–6 dissolved in
40-50 ml of 2% aqueous NaOH was refluxed for 2 h. After cooling, the
solution was neutralized with dilute hydrochloric acid. The precipitate
was filtered off and crystallized from EtOH (75–85%).
3-[(4-Nitroimidazol-1-yl)methyl]-4-phenyl-1,2,4-triazoline-5-thione
7. mp 309–310◦C. IR (KBr) cm−1: 3220, 3078, 2976, 1440, 1660, 1230.
1H-NMR (DMSO-d6) δ: 5.33 (2H, s), 7.29 (5H, m), 7.63 (1H, d, J=
1.41 Hz), 8.15 (1H, d, J= 1.41 Hz), 14.02 (1H, s). Anal. Calcd for
C12H10N6O2S: 302.313 C, 47.67; H, 3.33; N, 27.80. Found: C, 47.65; H,
3.30; N, 27.79.
3-[(4-Nitroimidazol-1-yl)methyl]-4-(4-tolyl)-1,2,4-triazoline-5-thione
8:. mp 292–293◦C. IR (KBr) cm−1: 3225, 3075, 2948, 1460, 1650, 1220.
1H-NMR (DMSO-d6) δ: 2.50 (3H, s), 5.31 (2H, s), 7.28, 7.36 (4H, dd,
J = 8.3 Hz), 7.65 (1H, d, J = 1.39 Hz), 8.15 (1H, d,J = 1.39 Hz), 13.98
(1H, s). Anal. Calcd for C13H12N6O2S: 316.34 C, 49.35; H, 3.82; N,
26.57. Found: C, 49.33; H, 3.74; N, 26.55.
4-(4-Methoxyphenyl)-3-[(4-nitroimidazol-1-yl)methyl]-1,2,4-
triazoline-5-thione 9. The synthesis and characterization data
were published previously.15
4-(4-Bromophenyl)-3-[(4-nitroimidazol-1-yl)methyl]-1,2,4-
triazoline-5-thione 10. mp 312–314◦C. IR (KBr) cm−1: 3240, 3070,
2953, 1432, 1678, 1220. 1H-NMR (DMSO-d6) δ: 5.39 (2H, s), 7.43
(2H, d,J = 8.58 Hz), 7.78 (2H, d,J = 8.58 Hz), 7.72 (1H, s), 8.23 (1H,
s), 13.10 (1H, s). Anal. Calcd for C12H9BrN6O2S: 381.209 C, 37.81; H,
2.38; N, 22.05. Found: C, 37.80; H, 2.29; N, 22.02.
4-(4-Iodophenyl)-3-[(4-Nitroimidazol-1-yl)methyl]-1,2,4-triazoline-
5-thione 11. mp 305◦C. IR (KBr) cm−1: 3220, 3080, 2957, 1433, 1640,
1230. 1H-NMR (DMSO-d6) δ: 5.33 (2H, s), 7.23; 7.93 (4H, dd, J = 8.58
Hz), 7.67 (1H, d, J = 1.39 Hz), 8.18 (1H, d, J = 1.39 Hz), 13.93 (1H,
s). Anal. Calcd for C12H9IN6O2S: 428.21 C, 33.66; H, 2.12; N, 19.63.
Found: C, 33.52; H, 2.11; N, 19.64.
Microbiology
The newly synthesized compounds 2–5, 8, and 10 were screened for
their antimicrobial activity against nine reference strains of aerobic
bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus epider-
midis ATCC 12 228, Bacillus subtilis ATCC 6633, Bacillus cereus

566
M. Wujec et al.
ATCC 10876, Micrococcus luteus ATCC 10240, Escherichia coli ATCC
25922, Proteus mirabilis ATCC 12453, Klebsiella pneumoniae ATCC
13883, and Pseudomonas aeruginosa ATCC 9027) and for five refer-
ence strains of fungi (Candida albicans ATCC 2091, Candida albi-
cans ATCC 10231, Candida parapsilosis ATCC 22019, Trichophyton
menthagrophytes ATCC 9533, and Aspergillus niger ATCC 16404). The
inoculum density was adjusted to 0.5 McFarland standard (150 × 106
cfu/mL) with sterile saline (0.85% NaCl) and then the suspensions were
diluted 1:10 in Mueller-Hinton broth (for bacteria) or Mueller-Hinton
broth with 2% glucose (for fungi). All stock solutions of the assayed
compounds were prepared in dimethyl sulfoxide (DMSO) with distil-
lated water (1:1). It was found that DMSO at the final concentration in
the medium had no influence on growth of the tested microorganisms.
In the first step, all of these compounds were examined in vitro for an-
tibacterial activity using an agar dilution method with Mueller-Hinton
medium containing from 31.25 to 500 mg L−1of the tested compounds
and subsequently, for potentially active 2, 3, and 10 compounds, spec-
trophotometrically (OD600) by a dilution method with Mueller-Hinton
broth containing from 7.81 to 500 mg L−1 of the compounds. The incu-
bation was carried out at 37◦C for 18 h.
Antifungal activity was examined using an agar dilution method.
Microbial suspensions were put onto Mueller-Hinton agar with 2% glu-
cose buffered at pH 5.6 containing several final concentrations of the
tested compounds (31.25–500 mg L−1). The incubation was carried out
at 30◦C for 24 and 48 h for Candida spp. and for 72–96 h for Aspergillus
niger ATCC 16404 and T. menthagrophytes ATCC 9533, depending on
the growth in control plates.
Microbial susceptibility testing was performed according to CLSI
guideline.18
The MIC (minimal inhibitory concentration) values were defined as
the lowest concentration of the compound required for 100% inhibition
of the visible growth of the tested bacteria or prominent growth inhi-
bition (approximately 80% inhibition compared to the growth in the
control plates) of fungi.
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