Antimicrobial and Physicochemical Characterizations of Thiosemicarbazide and S-Triazole Derivatives

Article abstract of DOI:10.1080/10426507.2014.902831

Two series of thiosemicarbazide derivatives and three series of s-triazole derivatives have been synthesized. All of these compounds were tested for their in vitro antibacterial activity against Gram-positive and Gram-negative bacterial strains. Among tested thiosemicarbazide derivatives, the best bioactivity was detected for two 1-formylthiosemicarbazides with 3-/4-tolyl substitution (1 l, 1 m) (MICs range between 31.25 and 250 μg/mL). All tested s-triazole derivatives exhibited lower antibacterial activity than their acyclic precursors.

Full text of DOI:10.1080/10426507.2014.902831

This article was downloaded by: [McMaster University]
On: 16 February 2015, At: 10:43
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Phosphorus, Sulfur, and Silicon and the
Related Elements
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gpss20
Antimicrobial and Physicochemical
Characterizations of Thiosemicarbazide
and S-Triazole Derivatives
Edyta Kuśmierza, Agata Siweka, Urszula Kosikowskab, Anna Malmb,
Tomasz Plecha, Andrzej Wróbelc & Monika Wujeca
a Department of Organic Chemistry, Medical University, Chodźki 4a,
20-093 Lublin, Poland
b Department of Pharmaceutical Microbiology, Medical University,
Click for updates
Chodźki 1, 20-093 Lublin, Poland
c History of Medical Sciences Department, Medical University,
Szkolna 18, 20-124 Lublin, Poland
Accepted author version posted online: 11 Jun 2014.Published
online: 25 Sep 2014.
To cite this article: Edyta Kuśmierz, Agata Siwek, Urszula Kosikowska, Anna Malm, Tomasz Plech,
Andrzej Wróbel & Monika Wujec (2014) Antimicrobial and Physicochemical Characterizations of
Thiosemicarbazide and S-Triazole Derivatives, Phosphorus, Sulfur, and Silicon and the Related
Elements, 189:10, 1539-1545, DOI: 10.1080/10426507.2014.902831
To link to this article: http://dx.doi.org/10.1080/10426507.2014.902831
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,

systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Phosphorus, Sulfur, and Silicon, 189:1539–1545, 2014
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2014.902831
ANTIMICROBIAL AND PHYSICOCHEMICAL
CHARACTERIZATIONS OF THIOSEMICARBAZIDE
AND S-TRIAZOLE DERIVATIVES
Edyta Kus´mierz,1 Agata Siwek,1 Urszula Kosikowska,2 Anna
Malm,2 Tomasz Plech,1 Andrzej Wro´bel,3 and Monika Wujec1
1Department of Organic Chemistry, Medical University, Chodz´ki 4a, 20-093
Lublin, Poland
2Department of Pharmaceutical Microbiology, Medical University, Chodz´ki 1,
20-093 Lublin, Poland
3History of Medical Sciences Department, Medical University, Szkolna 18, 20-124
Lublin, Poland
GRAPHICAL ABSTRACT
Abstract Two series of thiosemicarbazide derivatives and three series of s-triazole derivatives
have been synthesized. All of these compounds were tested for their in vitro antibacterial activity
against Gram-positive and Gram-negative bacterial strains. Among tested thiosemicarbazide
derivatives, the best bioactivity was detected for two 1-formylthiosemicarbazides with 3-/4-
tolyl substitution (1 l, 1 m) (MICs range between 31.25 and 250 μg/mL). All tested s-triazole
derivatives exhibited lower antibacterial activity than their acyclic precursors.
Keywords Antibacterial activity; s-triazole; structure-activity relationship (SAR); thiosemi-
carbazide derivatives; physicochemical properties
Received 21 November 2013; accepted 6 March 2014.
Address correspondence to Monika Wujec, Department of Organic Chemistry, Medical University, Chodz´ki
4a, 20-093 Lublin, Poland. E-mail: monika.wujec@am.lublin.pl
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
1539

´
1540
E. KUSMIERZ ET AL.
NCS
NaOH
R
R
R
R1
R1
R1
3a-n, 4a-n, 5a-n
(
)
1a-n. 2a-n
(
)
R= H(1,3), CH3(2,4), Ph(5); R1= H(a), 2-F(b), 3-F(c), 4-F(d), 2-Cl(e), 3-Cl(f), 4-Cl(g), 2-
Br(h), 3-Br(i), 4-Br(j), 2-CH3(k), 3-CH3(l), 4-CH3(m), 4-OCH3 (n)
Scheme 1
INTRODUCTION
Organosulfur compounds are defined as organic molecules containing one or more
carbon-sulfur bonds. Among them, thiosemicarbazide derivatives and products of their
alkaline cyclocondensation, s-triazoles, possess interesting biological activities, such as
anti-inflammatory,1–3 antibacterial,4–8 and antifungal.9,10 The difficulty of treating bacterial
infection caused by multidrug-resistant bacteria continues to provide impetus to search for
new compounds with promising antimicrobial potency. In continuation of our ongoing in-
terests in the development of new antimicrobial agents,11,12 two series of thiosemicarbazide
derivatives and their heterocyclocondensation products s-triazoles were synthesized and
their antibacterial potency has been evaluated. Since one of the most important issues in
the search for new chemical compounds with an expected activity is to identify the relation
between their chemical structure and biological response, the second part of this paper was
dedicated to investigate and search for correlation between biological activities of studied
compounds and their physicochemical properties.
RESULTS AND DISCUSSION
The title thiosemicarbazide derivatives (series 1 and 2) were synthesized based on
known procedures11,13 (Scheme 1) and their antimicrobial potency has been evaluated
against a panel of Gram-positive and Gram-negative strains by determining compound
concentrations compared to the MIC and MBC. Minimal inhibitory concentrations (MICs)
were defined as the minimum concentration of the compound which inhibits the growth of
tested microorganism and the results are presented in Table S.1 in Supplemental Materials.
Minimum bactericidal concentration (MBC) was defined as the lowest concentration of
the compound which inhibits the growth of tested microorganism by 99.9% kill after 24 h
of incubation. Bacteriostatic activity was defined as a ratio of MBC to MIC of >4 while
bactericidal effect was defined as a ratio of MBC to MIC of ≤4.
As seen from results collected in Table S.1, thiosemicarbazide derivatives with formyl
moiety at N1 position (series 1) were definitely more active than 1-acetylthiosemicarbazide
derivatives (series 2), although the structures of the thiosemicarbazides from series 1
and 2 are very similar (see Figure 1 for two selected compounds). Within series of

THIOSEMICARBAZIDES AND S-TRIAZOLES AS ANTIBACTERIAL AGENTS
1541
Figure 1 Comparison of geometries of two selected 4-phenylthiosemicarbazides 1a (sticks) and 2a (balls
and sticks); remaining thiosemicarbazides (series 1 and 2) share very similar geometry of molecule.
1-formylthiosemicarbazide derivatives, the best bioactivity was noted for two deriva-
tives with electronic donating methyl group; 3-methylphenyl derivative (1l) (MICs range
31.25–125 μg/mL) and 4-methylphenyl derivative (1 m) (MICs range 62.5–250 μg/mL).
Besides, within series 1, with the exception of 2-chlorophenyl derivative (1e), all com-
pounds with ortho-substitution were inactive. These results suggest that both electronic
and steric affects of substituents play important role in the molecular recognition process
between a ligand and a biological target while geometry of molecule is only the secondary
parameter. To test the effect of a geometry of thiosemicarbazide core on biological response,
cyclic derivatives of 1 and 2 with s-triazole ring (series 3 and 4) were synthesized based on
know procedure14 (Scheme 1) and their bioactivity was tested. As can be seen from results
shown in Table S.1 in Supplemental Materials, all s-triazoles 3 and 4 possessed lower
antibacterial potency than their acyclic precursors 1 and 2. The same trend was observed
when bioactivity of previously described 1-benzoylthiosemicarbazides11 and their cyclic
derivatives s-triazoles (series 5) was compared, unambiguously confirming the importance
of NH-NH-CS-NH core on antibacterial activity of thiosemicarbazide derivatives.
In the second step of this study some lipophilic, electronic, and structural parameters
as partition cofficient (log p), surface area (SA), volume (V), refractivity (Rf), polarizability
(α), dipole moment (μ), hydration energy (EH), the highest occupied molecular orbital
energy (EHOMO), the lowest unoccupied molecular orbital energy (ELUMO), the difference
between HOMO and LUMO energy levels (HLG), hardness (η) obtained from the equa-
tion η = (ELUMO – EHOMO)/2, Mulliken electronegativity (χ) obtained from the equation
χ = −(EHOMO + ELUMO)/2, total energy (ET), binding energy (EB), isolated atomic en-
ergy (EIA), electronic energy (EE), core-core interaction (IC-C), heat of formation (HF)
were calculated with hope to obtain some correlations between the observed bioactivity of
thiosemicrabazide derivatives (1 and 2) and their physicochemical properties. As presented
in Table S.2 in Supplemental Materials, the most active tolyl derivatives (1l, 1 m) had
the highest values of EH than remaining thiosemicarbazides. Also, they showed relatively
higher values of EIA, ET and relatively lower values of EB than remaining ones. Within

´
E. KUSMIERZ ET AL.
1542
series of s-triazoles, the most active 5j presents the highest values of surface area (SA),
polarizability (α), refractivity (Rf) and heat of formation (HF).
EXPERIMENTAL
Chemistry
All reagents and solvent were purchased from either Lancaster or Sigma Aldrich
and used without further purification. Melting points were determined on a Fisher-Johns
blocks and are presented without corrections. Elemental analyses were performed on AMZ
1
851 CHX analyzer and the results were within 0.4% of the theoretical value. The H
NMR spectra were recorded on a Bruker Avance 300 MHz instrument using DMSO-d6 as
a solvent and TMS as an internal standard. The progress of the reaction was monitored on
Merck 60F254 silica gel plates and visualized by UV irradiation (254 nm).
General Procedures for the Preparation of Thiosemicarbazide Deriva-
tives (Series 1 and 2). A reaction mixture of appropriate hydrazide (0.01 mol) and
aryl isothiocyanate (0.01 mol) in anhydrous EtOH (25 mL) was heated under reflux for
10–30 min and progress of the reaction was monitored by thin layer chromatography. Next,
the solution was cooled and the solid formed (of white color) was filtered off, washed with
diethyl ether, dried and crystallized from ethanol. The structures of compounds 1a–1g,
1j–1n, 2a–2g, 2i–2n are known (CAS registry numbers), however there is no reference
reporting their preparation or physico-chemical characterization, therefore their data have
been maintained in the paper. Characterization data for representative compounds (1h, 1i,
2h) are given below. Characterization data for remaining thiosemicarbazide derivatives
1a–1g, 1j–1n, 2a–2g, 2i–2n are presented in Supplemental Materials.
4-(2-Bromophenyl)-1-formylthiosemicarbazide (1h). Yield: 89%. Mp.
1
220◦C. H NMR (DMSO-d6) δ (ppm): 7.22–7.73 (m, 4H, CHarom); 8.12 (s, 1H, formyl);
9.53 (s, 1H, NH); 9.98 (s, 1H, NH); 10.21 (s, 1H, NH). IR (KBr, ν, cm−1): 3360, 2826,
1680, 1543, 1478, 1380. Anal. Calc. for C8H8BrN3OS (274.14): C 35.05, H 2.94, N 15.33.
Found: C 35.07, H 2.91, N 15.32.
4-(3-Bromophenyl)-1-formylthiosemicarbazide (1i). Yield: 86%. Mp.
1
185◦C. H NMR (DMSO-d6) δ (ppm): 7.51–8.07 (m, 4H, CHarom); 8.14 (s, 1H, formyl);
9.64 (s, 1H, NH); 9.84 (s, 1H, NH); 10.12 (s, 1H, NH). IR (KBr, ν, cm−1): 3365, 2840,
1678, 1540, 1470, 1380. Anal. Calc. for C8H8BrN3OS (274.14): C 35.05, H 2.94, N 15.33.
Found: C 35.06, H 2.95, N 15.30.
1-Acetyl-4-(2-bromophenyl)thiosemicarbazide (2h). Yield: 81%. Mp.
162-163◦C. 1H NMR (DMSO-d6) δ (ppm): 1.97 (s, 3H, acetyl); 7.25-7.77 (m, 4H, CHarom);
9.54 (s, 1H, NH); 9.72 (s, 1H, NH); 10.01 (s, 1H, NH). IR (KBr, ν, cm−1): 3365, 2830,
1680, 1540, 1470, 1378. Anal. Calc. for C9H10BrN3OS (288.16): C 37.51, H 3.50, N
14.58. Found: C 37.53, H 3.49, N 14.57.
General Procedure for the Preparation of s-triazoles (series 3–5).
Thiosemicarbazide derivatives 1 and 2 (0.01 mol) were dissolved in 2% solution of sodium
hydroxide (20 mL) and heated under reflux for 2–3 h. After cooling, the solution was
neutralized with dilute hydrochloric acid. The precipitate (of white color) was filtered off
and then crystallized from 95% ethanol.
The structures of compounds 3a – 3n, and 4a – 4n are known (CAS registry numbers),
however, there is no reference reporting their preparation or physico-chemical character-
ization, therefore their data have been maintained in the paper. Characterization data for

THIOSEMICARBAZIDES AND S-TRIAZOLES AS ANTIBACTERIAL AGENTS
1543
representative compounds (3a, 4a, 5a) are given below. Characterization data for remaining
s-triazoles 3b–3n, 4b–4n, 5b–5n are presented in the Supplemental Materials.
4-Phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3a). CAS Registry Num-
ber: 5373-72-8.
Yield: 71%. Mp. 172◦C. 1H NMR (DMSO-d6) δ (ppm): 7.09–7.48 (m, 5H, CHarom);
8.12 (s, 1H, CH), 13.90 (1s, 1H, NH). IR (KBr, ν, cm−1): 3315, 2826, 1575, 1543, 1218,
746. Anal. Calc. for C8H7N3S (177.27): C 54.22, H 3.98, N 23.71. Found: C 54.21, H 3.97,
N 23.70.
5-Methyl-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (4a). CAS Reg-
istry Number: 6232-82-2.
1
Yield: 89%. Mp. 205-206◦C. H NMR (DMSO-d6) δ (ppm): 1.88 (s, 3H, CH3);
7.24–7.87 (m, 5H, CHarom); 13.74 (1s, 1H, NH). Anal. Calc. for C9H9N3S (191.25): C
56.52, H 4.74, N 21.97. Found: C 56.53, H 4.73, N 21.96.
4,5-Diphenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (5a). CAS Registry
Number: 6596-82-3.
1
Yield: 93%. Mp. 291–292◦C. H NMR (DMSO-d6) δ (ppm): 7.20–7.86 (m, 10H,
CHarom); 13.67 (1s, 1H, NH). Anal. Calc. for C14H11N3S (253.32): C 66.38, H 4.38, N
16.59. Found: C 66.37, H 4.39, N 16.60.
Antibacterial Assay
The antimicrobial activity of the compounds was tested on the Gram-positive strains:
Staphylococcus aureus (ATCC 25923, MICROBANK14001), Staphylococcus epidermidis
ATCC 12228, Bacillus subtilis ATCC 6633, Micrococcus luteus ATCC 10240 and on
the Gram-negative bacteria: Eschericha coli ATCC 25922, Klebsiella pneumoniae ATTC
13883, Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 9027.
Microbial suspensions were prepared in sterile saline (0.85% NaCl) with an optical
density of 0.5 McFarland standard—150 × 106 CFU/mL (CFU—colony forming units). All
stock solutions of the tested compounds were dissolved in dimethyl sulfoxide (DMSO). The
medium with DMSO at the final concentration and without the tested compounds served
as control - no microbial growth inhibition was observed. Initially, antibacterial activity of
thiosemicarbazides was screened on the basis of growth inhibition zone (giz) using the agar
dilution method on the basis of the growth inhibition on the Mueller-Hinton agar to which
the tested compounds at concentration 1000 μg/mL were added. For compounds showing
the inhibitory effect on the growth of tested bacteria, monitored as an appearance of giz,
the MICs were determined using broth microdilution method.15 Commercially available
ampicillin was used as the reference drug for comparison under identical experimental
conditions. MBC was determined by a broth microdilution technique by plating out the
contents of wells (20 mL), that showed no visible growth of bacteria, on to Muellere Hinton
agar plates and incubating at 35◦C for 18 h. Both MIC and MBC values were given in
μg/mL. The results were summarized in Table S.1 in Supplemental Materials.
Computational Details
Conformational search was performed at molecular mechanics level with OPLS force
field as implemented in HyperChem version 7.5.16 The most stable structures obtained were
subsequently optimized to the closest local minimum at the semiempirical level using PM3

´
E. KUSMIERZ ET AL.
1544
parametrizations. Convergence criteria were set to 0.1 and 0.01 kcal·mol−1Å−1 for OPLS
and PM3 calculations, respectively.
CONCLUSION
In this contribution we have synthesized two series of thiosemicarbazides and three
series of s-triazoles to evaluate their antibacterial activity and to study their physicochem-
ical properties. Although the results of antibacterial screening were not impressive, some
indications for a molecular basis of antibacterial activity of thiosemicarbazide derivatives
have been identified from SAR studies and can be summarized as follows: (i) geometry
of molecule as well as electronic and steric affects of substituents play important role
in the molecular recognition process between a 4-arylthiosemicarbazide and a biological
target (ii) physicochemical parameters, such as EH, EIA, ET, EB are directly correlate with
antibacterial potency of 1-formylthiosemicarbazides.
FUNDING
The project was supported by the Research Grant for Young Scientists and Grant No.
UMO-2012/05/D/NZ7/02278.
SUPPLEMENTAL MATERIAL
Supplemental data for this article can be accessed on the publisher’s website.
www.tandfonline.com/gpss
Tables S1 and S2 are available online in Supplemental Materials.
REFERENCES
1. Raman, K.; Singh, H. K.; Salzman, S. K.; Parmar, S. S. J. Pharm. Sci. 1993, 82, 167-169.
˙
2. Ku¨c¸u¨kgu¨zel, S¸. G.; Ku¨c¸u¨kgu¨zel, I.; Tatar, E.; Rollas, S.; S¸ahin, F.; Gu¨llu¨ce, M.; De Clercq, E.;
Kabasakal, L. Eur. J. Med. Chem. 2007, 42, 893-901.
3. Labanauskas, L.; Udrenaite, E.; Gaidelis, P.; Brukstus, A. Farmaco 2004, 59, 255-259.
4. Plech, T.; Wujec, M.; Siwek, A.; Kosikowska, U.; Malm, A. Eur. J. Med. Chem. 2011, 46,
241-248.
5. Siwek, A.; Staczek, P.; Stefanska, J. Eur. J. Med. Chem. 2011, 46, 5717-5726.
6. Arafa, W. A. A. J. Heterocycl. Chem. 2010, 47, 1109-1115.
7. Colanceska-Ragenovic, K.; Dimova, V.; Kakurinov, V.; Molnar, D. G.; Buzarovcka, A. Molecules
2001, 6, 815-824.
8. Siwek, A.; Wujec, M.; Stefan´ska, J.; Paneth, P. Phosphorus Sulfur Silicon Relat. Elem., 2009,
184, 3149-3159.
9. Kokil, G. R.; Rewatkar, P. V.; Gosain, S.; Aggarwal, S.; Verma, A.; Kalra A.; Thareja, S. Lett.
Drug. Des. Discov. 2013, 7, 46-49.
10. Wei, Q. L.; Zhang, S. S.; Gao, J.; Li, W. H.; Xu, L. Z.; Yu, Z. G. Bioorg. Med. Chem. 2006, 14,
7146-7153.
11. Kusmierz, E.; Siwek, A.; Kosikowska, U.; Malm, A.; Wujec, M. Lett. Drug. Des. Discov. 2012,
9, 828-832.
12. Kusmierz, E.; Siwek, A.; Kosikowska, U.; Malm, A.; Stefanska, J.; Dzitko, K.; Wujec, M. Lett.
Drug. Des. Discov. 2013, 10, 95-101.

THIOSEMICARBAZIDES AND S-TRIAZOLES AS ANTIBACTERIAL AGENTS
1545
13. Plech, T.; Wujec, M.; Siwek, A.; Kosikowska, U.; Malm, A. Eur. J. Med. Chem. 2011, 46,
241-248.
14. Siwek, A.; Wawrzycka-Gorczyca, I.; Dobosz, M. Ann. Pol. Chem. Soc. 2005, 1, 164-168.
15. CLSI, Performance Standards for Antimicrobial Susceptibility Testing; Eighteenth International
Supplement. CLSI Document M7-MIC. Clinical Laboratory Standards Institute, Wayne, PA,
2008.
16. HyperChem 7.5, HyperCube Inc., Gainsville, FL, USA, 2007.