Synthesis and In Vitro Evaluation of New Thiosemicarbazone Derivatives as Potential Antimicrobial Agents

Article abstract of DOI:10.1155/2016/1692540

In an effort to develop potent antimicrobial agents, new thiosemicarbazone derivatives were synthesized via the reaction of 4-[4-(trifluoromethyl)phenyl]thiosemicarbazide with aromatic aldehydes. The compounds were evaluated for their inhibitory effects o

Full text of DOI:10.1155/2016/1692540

Hindawi Publishing Corporation
Journal of Chemistry
Volume 2016, Article ID 1692540, 7 pages
http://dx.doi.org/10.1155/2016/1692540
Research Article
Synthesis and In Vitro Evaluation of New Thiosemicarbazone
Derivatives as Potential Antimicrobial Agents
Zafer AsJm KaplancJklJ,1 Mehlika Dilek AltJntop,1 Belgin Sever,1
Zerrin Cantürk,2 and Ahmet Özdemir1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eski¸sehir, Turkey
2Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Anadolu University, 26470 Eski¸sehir, Turkey
Correspondence should be addressed to Zafer Asım Kaplancıklı; zakaplan@anadolu.edu.tr
Received 16 December 2015; Revised 10 January 2016; Accepted 13 January 2016
Academic Editor: Giuseppe Gumina
Copyright © 2016 Zafer Asım Kaplancıklı et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
In an effort to develop potent antimicrobial agents, new thiosemicarbazone derivatives were synthesized via the reaction of 4-[4-
(trifluoromethyl)phenyl]thiosemicarbazide with aromatic aldehydes. e compounds were evaluated for their inhibitory effects on
pathogenic bacteria and yeasts using the CLSI broth microdilution method. Microplate Alamar Blue Assay was also carried out to
determine the antimycobacterial activities of the compounds against Mycobacterium tuberculosis H37Rv. Among these derivatives,
compounds 5 and 11 were more effective against Enterococcus faecalis (ATCC 29212) than chloramphenicol, whereas compounds
1, 2, and 12 and chloramphenicol showed the same level of antibacterial activity against E. faecalis. Moreover, compound 2 and
chloramphenicol exhibited the same level of antibacterial activity against Staphylococcus aureus. On the other hand, the most potent
anticandidal derivatives were found as compounds2 and 5. ese derivatives and ketoconazole exhibited the same level of antifungal
activity against Candida glabrata. According to the Microplate Alamar Blue Assay, the tested compounds showed weak to moderate
antitubercular activity.
1. Introduction
as allergic reactions to fungal proteins and toxic reactions to
fungi toxins and mycoses pose a constant and serious threat
to human life. Infections caused by Candida species represent
the main reason of opportunistic fungal infections world-
wide. Candidemia and other forms of invasive candidiasis
are important causes of the increasing rate of morbidity and
mortality. Candida albicans is the most common etiological
agent of candidiasis and Candida glabrata is a growing
concern in clinical settings because it causes mucosal and
systemic bloodstream infections [7–12].
e World Health Organization (WHO) evaluates tuber-
culosis as one of the deadliest global diseases associated
with the occurrence of multiple drug resistant strains of
Mycobacterium tuberculosis discovered by Robert Koch in
1882. Mycobacterium tuberculosis H37Rv, the pathogenic
agent of tuberculosis, is recognized to reproduce in host
cells by pulling through host cell defenses supplied by
macrophages and dendritic cells. In 2013, approximately
9 million people were diagnosed with active tuberculosis
roughout history, it has been a major worldwide problem
to treat microbial diseases caused by bacteria and fungi due
to impetuous development of resistance to antibacterial and
antifungal drugs. Antimicrobial resistance is observed in
pathogenic bacteria before exposure to antimicrobial agent or
related to uncontrolled infection afer a patient receives that
antimicrobial. Bacteria could be resistant inherently staying
in variable temperatures and in highly acidic or alkaline
conditions or could develop resistance by de novo mutation
or through the transmission of resistance genes from other
organisms. Gram-positive bacterial infections particularly
caused by Staphylococcus aureus and Enterococcus spp. rep-
resent a major health problem hampering the therapeutic
efficacy of antibacterial drugs due to their frequent multidrug
resistance [1–6].
In the past two decades, the incidence of fungal infections
has gone up all over the world. Fungal infections classified

2
Journal of Chemistry
H
N
on an IRPrestige-21 Fourier Transform Infrared spectropho-
NH2
1
tometer (Shimadzu, Tokyo, Japan). H NMR spectra were
O
N
recorded on a Bruker spectrometer (Bruker, Billerica, MA,
USA). Mass spectra were recorded on an Agilent LC-MSD-
Trap-SL Mass spectrometer (Agilent Technologies, Palo Alto,
CA, USA). Elemental analyses (C, H, and N) were performed
on a Perkin Elmer EAL 240 elemental analyzer (Perkin-
Elmer, Norwalk, CT, USA) and the results were within 0.4%
of the theoretical values. in Layer Chromatography (TLC)
S
N
H
Figure 1: iacetazone.
was performed on TLC Silica gel 60 F aluminium sheets
254
and 1.5 million deaths attributed to this disease. Diagnosis
and treatment are crucial for tuberculosis, particularly in
high-risk populations. Early diagnosis of tuberculosis with
radiography and computed tomography methods is essential
for effective treatment and leads to a reduced onward trans-
mission of tuberculosis. Despite the discovery of tuberculin
test and Bacillus-Calmette Gue´rin (BCG) vaccine and some
main drugs called streptomycin, isoniazid, and rifampicin for
the treatment of tuberculosis, it has remained one of the most
dangerous diseases especially associated with mycobacterial
resistance in chemotherapy and the presence of coinfectious
diseases such as acquired immune deficiency syndrome
(AIDS) [8–18].
iosemicarbazones have been investigated for medic-
inal studies for a long while due to their wide range of
biological activities including antineoplastic, antimycobac-
terial, antibacterial, antifungal, antiviral, and antimalarial
effects and versatility as nitrogen and sulfur donors allowing
them to bring on a great variety of coordination modes.
iosemicarbazone is also known as an iron-chelating group,
bonding the sulfur and azomethine nitrogen atoms. e
complexes of nitrogen and sulfur atoms with metal ions may
be considered potential biological agents. In recent years,
many thiosemicarbazone derivatives have been synthesized
and evaluated for their antibacterial activity. e current
use of these agents in bacterial infections has led to the
development of novel antibacterial drugs [19–25].
iacetazone, p-acetamidobenzaldehyde thiosemicarba-
zone, is one of the oldest and cheapest agents used as
a second line drug for tuberculosis treatment (Figure 1).
is agent is estimated to show antimycobacterial activity
inhibiting mycolic acid biosynthesis. iacetazone shows
only bacteriostatic activity and develops resistance easily
during tuberculosis therapy. It is partly cross-resistant to
ethionamide and it is forbidden to be used in patients
with human immunodeficiency virus (HIV) owing to high
frequency of Stevens-Johnson syndrome [26–28].
On the basis of aforementioned findings, herein, we
synthesized a new series of thiosemicarbazone derivatives
and investigated their in vitro antimicrobial and antimy-
cobacterial effects.
(Merck, Darmstadt, Germany) to check the purity of the
compounds.
2.2. General Procedure for the Synthesis of the Compounds
2.2.1. 4-[4-(Trifluoromethyl)phenyl]thiosemicarbazide. A mix-
ture of 4-(trifluoromethyl)phenyl isothiocyanate (0.1 mol)
and hydrazine hydrate (0.2 mol) in ethanol (30 mL) was
stirred at room temperature for 4 h and then filtered. e
residue was crystallized from ethanol [29].
2.2.2. 4-[4-(Trifluoromethyl)phenyl]-1-(substituted benzyli-
dene)thiosemicarbazides (1–12). A mixture of 4-[4-(trifluoro-
methyl)phenyl]thiosemicarbazide (0.01 mol) and aromatic
aldehyde (0.01 mol) was refluxed in ethanol for 6 h, filtered,
and crystallized from ethanol [29].
2.2.3. 4-[4-(Trifluoromethyl)phenyl]-1-(benzylidene)thiosemi-
carbazide (1). IR ]max (cm−1): 3313.71 (N-H stretching),
3140.11 (aromatic C-H stretching), 1614.42, 1539.20, 1504.48
(C=N, C=C stretching and N-H bending), 1327.03, 1278.81,
1099.43 (C-N, C=S stretching and aromatic C-H in plane
bending), 846.75, 754.17, 686.66 (aromatic C-H out of plane
bending); 1H NMR (500 MHz, DMSO-ꢀ6) ꢁ (ppm): 7.43–7.45
(m, 3H, aromatic protons), 7.72 (d, ꢂ ꢃ =0 Hz, 2H, aromatic
protons), 7.89–7.91 (m, 4H, aromatic protons), 8.19 (s, 1H,
CH=N), 10.29 (s, 1H, N-H), 12.03 (s, 1H, N-H); MS (ESI)
(ꢄꢅ/): (M + H)+ 324; Anal. Calcd for C H F N S: C, 55.72;
15 12
3
3
H, 3.74; N, 13.00; Found: C, 55.70; H, 3.76; N, 13.00.
2.2.4. 4-[4-(Trifluoromethyl)phenyl]-1-(4-fluorobenzylid-
ene)thiosemicarbazide (2). IR ]max (cm−1): 3288.63 (N-H
stretching), 3153.61 (aromatic C-H stretching), 2983.88 (C-
H stretching), 1616.35, 1600.92, 1544.98, 1504.48 (C=N, C=C
stretching and N-H bending), 1319.31, 1269.16, 1232.51, 1192.01,
1161.15, 1109.07, 1064.71, 1014.56 (C-N, C=S stretching and
aromatic C-H in plane bending), 831.32 (aromatic C-H out
of plane bending); 1H NMR (500 MHz, DMSO-ꢀ6) ꢁ (ppm):
7.62–7.63 (m, 2H, aromatic protons), 7.72 (d, ꢂ ꢃ =0 Hz, 2H,
aromatic protons), 7.89 (d, ꢂ ꢃ =0 Hz, 2H, aromatic protons),
7.97–8.01 (m, 2H, aromatic protons), 8.18 (s, 1H, CH=N), 10.31
(s, 1H, N-H), 12.03 (s, 1H, N-H); MS (ESI) (ꢄꢅ/): (M + H)+
342; Anal. Calcd for C H F N S: C, 52.78; H, 3.25; N, 12.31;
15 11
4
3
2. Experimental Section
Found: C, 52.80; H, 3.24; N, 12.30.
2.1. Chemistry. All reagents were purchased from commer-
cial suppliers and were used without further purification.
Melting points (Mp) were determined on an Electrothermal
9100 melting point apparatus (Weiss-Gallenkamp, Loughbor-
ough, UK) and are uncorrected. IR spectra were recorded
2.2.5. 4-[4-(Trifluoromethyl)phenyl]-1-(4-chlorobenzylid-
ene)thiosemicarbazide (3). IR ]max (cm−1): 3329.14 (N-H
stretching), 3134.33 (aromatic C-H stretching), 2980.02 (C-
H stretching), 1616.35, 1593.20, 1541.12, 1489.05 (C=N, C=C

Journal of Chemistry
3
stretching and N-H bending), 1321.24, 1274.95, 1109.07,
1085.92, 1062.78, 1012.63 (C-N, C=S stretching and aromatic
C-H in plane bending), 823.60 (aromatic C-H out of plane
C-H stretching), 2978.09 (C-H stretching), 1606.70, 1541.12,
1490.97 (C=N, C=C stretching and N-H bending), 1246.02,
1207.44, 1165.00, 1107.14, 1060.85, 1028.06, 1016.49 (C-N, C=S
stretching and aromatic C-H in plane bending), 829.39,
bending); 1H NMR (500 MHz, DMSO-ꢀ6) ꢁ (ppm): 7.49–7.51
(m, 2H, aromatic protons), 7.73 (d, ꢂ ꢃ =0 Hz, 2H, aromatic
protons), 7.88 (d, ꢂ ꢃ =0 Hz, 2H, aromatic protons), 7.94–7.96
792.74 (aromatic C-H out of plane bending); 1H NMR
(500 MHz, DMSO-ꢀ6) ꢁ (ppm): 3.80 (s, 3H, OCH ), 6.98–
3
(m, 2H, aromatic protons), 8.16 (s, 1H, CH=N), 10.34 (s, 1H,
7.00 (m, 2H, aromatic protons), 7.71 (d, ꢂ ꢃ =0 Hz, 2H,
N-H), 12.07 (s, 1H, N-H); MS (ESI) (ꢄꢅ/): (M + H)+ 358;
aromatic protons), 7.83–7.86 (m, 2H, aromatic protons), 7.91
(d, ꢂ ꢃ =0 Hz, 2H, aromatic protons), 8.05 and 8.14 (2s, 1H,
CH=N), 10.22 and 10.35 (2s, 1H, N-H), 11.77 and 11.92 (2s,
1H, N-H); MS (ESI) (ꢄꢅ/): (M + H)+ 354; Anal. Calcd for
C H F N OS: C, 54.38; H, 3.99; N, 11.89; Found: C, 54.37;
Anal. Calcd for C H ClF N S: C, 50.36; H, 3.10; N, 11.74;
15 11
3
3
Found: C, 50.35; H, 3.10; N, 11.75.
16 14
3
3
2.2.6. 4-[4-(Trifluoromethyl)phenyl]-1-(4-bromobenzylid-
ene)thiosemicarbazide (4). IR ]max (cm−1): 3329.14 (N-H
stretching), 3142.04 (aromatic C-H stretching), 2981.95 (C-H
stretching), 1614.42, 1589.34, 1539.20, 1521.84, 1487.12 (C=N,
C=C stretching and N-H bending), 1323.17, 1273.02, 1207.44,
1165.00, 1107.14, 1062.78, 1006.84 (C-N, C=S stretching and
aromatic C-H in plane bending), 840.96, 819.75, 808.17
H, 3.98; N, 11.91.
2.2.10. 4-[4-(Trifluoromethyl)phenyl]-1-(4-methylbenzylid-
ene)thiosemicarbazide (8). IR ]max (cm−1): 3309.85 (N-H
stretching), 3140.11 (aromatic C-H stretching), 2978.09 (C-
H stretching), 1616.35, 1541.12, 1492.90 (C=N, C=C stretching
and N-H bending), 1325.10, 1278.81, 1109.07, 1062.78 (C-N,
C=S stretching and aromatic C-H in plane bending), 806.25
1
(aromatic C-H out of plane bending); H NMR (500 MHz,
DMSO-ꢀ6) ꢁ (ppm): 7.62–7.64 (m, 2H, aromatic protons), 7.73
(d, ꢂ ꢃ = Hz, 2H, aromatic protons), 7.88 (d, ꢂ ꢃ = Hz, 4H,
aromatic protons), 8.15 (s, 1H, CH=N), 10.34 (s, 1H, N-H),
12.07 (s, 1H, N-H); MS (ESI) (ꢄꢅ/): (M + H)+ 403; Anal.
Calcd for C H BrF N S: C, 44.79; H, 2.76; N, 10.45; Found:
1
(aromatic C-H out of plane bending); H NMR (500 MHz,
DMSO-ꢀ6) ꢁ (ppm): 2.33 (s, 3H, CH ), 7.25 (d, ꢂ ꢃ =0 Hz, 2H,
3
aromatic protons), 7.72 (d, ꢂ ꢃ =0 Hz, 2H, aromatic protons),
7.79 (d, ꢂ ꢃ =0 Hz, 2H, aromatic protons), 7.90 (d, ꢂ ꢃ =0 Hz,
2H, aromatic protons), 8.06 and 8.15 (2s, 1H, CH=N), 10.26
and 10.38 (2s, 1H, N-H), 11.81 and 11.96 (2s, 1H, N-H); MS
(ESI) (ꢄꢅ/): (M + H)+ 338; Anal. Calcd for C H F N S: C,
15 11
3
3
C, 44.78; H, 2.74; N, 10.46.
2.2.7.
4-[4-(Trifluoromethyl)phenyl]-1-(4-nitrobenzylid-
16 14
3
3
ene)thiosemicarbazide (5). IR ]max (cm−1): 3307.92 (N-H
stretching), 3116.97 (aromatic C-H stretching), 2980.02 (C-H
stretching), 1614.42, 1541.12, 1516.05 (C=N, C=C stretching
and N-H bending), 1330.88, 1317.38, 1188.15, 1105.21, 1066.64
(C-N, C=S stretching and aromatic C-H in plane bending),
837.11 (aromatic C-H out of plane bending); 1H NMR
(500 MHz, DMSO-ꢀ6) ꢁ (ppm): 7.75 (d, ꢂ ꢃ = Hz, 2H,
aromatic protons), 7.87 (d, ꢂ ꢃ =0 Hz, 2H, aromatic protons),
8.18–8.20 (m, 2H, aromatic protons), 8.24–8.27 (m, 3H,
CH=N, aromatic protons), 10.48 (s, 1H, N-H), 12.28 (s,
1H, N-H); MS (ESI) (ꢄꢅ/): (M + H)+ 369; Anal. Calcd for
C H F N O S: C, 48.91; H, 3.01; N, 15.21; Found: C, 48.90;
56.96; H, 4.18; N, 12.46; Found: C, 56.95; H, 4.17; N, 12.45.
2.2.11. 4-[4-(Trifluoromethyl)phenyl]-1-(4-isopropylbenzylid-
ene)thiosemicarbazide (9). IR ]max (cm−1): 3290.56 (N-H
stretching), 3140.11 (aromatic C-H stretching), 2976.16 (C-H
stretching), 1616.35, 1541.12, 1492.90 (C=N, C=C stretching
and N-H bending), 1323.17, 1276.88, 1107.14, 1064.71 (C-
N, C=S stretching and aromatic C-H in plane bending),
839.03, 817.32 (aromatic C-H out of plane bending); 1H NMR
(500 MHz, DMSO-ꢀ6) ꢁ (ppm): 1.21 (d, ꢂ ꢃ = Hz, 6H, 2CH ),
3
2.89–2.95 (m, 1H, CH, i-Pr), 7.31 (d, ꢂ ꢃ = Hz, 2H, aromatic
protons), 7.72 (d, J = 10 Hz, 2H, aromatic protons), 7.81 (d,
ꢂ ꢃ =0 Hz, 2H, aromatic protons), 7.90 (d, ꢂ ꢃ =0 Hz, 2H,
aromatic protons), 8.07 and 8.15 (2s, 1H, CH=N), 10.25 and
10.39 (2s, 1H, N-H), 11.83 and 11.97 (2s, 1H, N-H); MS (ESI)
(ꢄꢅ/): (M + H)+ 366; Anal. Calcd for C H F N S: C, 59.16;
15 11
3
4
2
H, 3.03; N, 15.20.
2.2.8. 4-[4-(Trifluoromethyl)phenyl]-1-(4-hydroxybenzylid-
ene)thiosemicarbazide (6). IR ]max (cm−1): 3367.71, 3286.70
(N-H stretching), 3165.19 (aromatic C-H stretching), 1604.77,
1541.12, 1510.26, 1494.83 (C=N, C=C stretching and N-H
bending), 1327.03, 1276.88, 1186.22, 1163.08, 1099.43, 1064.71,
1018.41 (C-N, C-O, C=S stretching and aromatic C-H in plane
18 18
3
3
H, 4.97; N, 11.50; Found: C, 59.17; H, 4.96; N, 11.51.
2.2.12. 4-[4-(Trifluoromethyl)phenyl]-1-(4-dimethylamino-
benzylidene)thiosemicarbazide (10). IR ]max (cm−1): 3275.13
(N-H stretching), 3140.11 (aromatic C-H stretching), 2978.09
(C-H stretching), 1598.99, 1548.84, 1519.91, 1492.90 (C=N,
C=C stretching and N-H bending), 1325.10, 1315.45, 1276.88,
1161.15, 1105.21, 1060.85 (C-N, C=S stretching and aromatic C-
H in plane bending), 943.19, 840.96, 810.10, 786.96 (aromatic
C-H out of plane bending); 1H NMR (500 MHz, DMSO-ꢀ6)
1
bending), 831.32 (aromatic C-H out of plane bending); H
NMR (500 MHz, DMSO-ꢀ6) ꢁ (ppm): 6.80–6.83 (m, 2H,
aromatic protons), 7.70–7.74 (m, 4H, aromatic protons), 7.92
(d, ꢂ ꢃ =0 Hz, 2H, aromatic protons), 8.09 (s, 1H, CH=N),
9.97 (s, 1H, O-H), 10.17 (s, 1H, N-H), 11.85 (s, 1H, N-H); MS
(ESI) (ꢄꢅ/): (M + H)+ 340; Anal. Calcd for C H F N OS:
15 12
3
3
C, 53.09; H, 3.56; N, 12.38; Found: C, 53.10; H, 3.55; N, 12.37.
ꢁ (ppm): 2.97 (s, 6H, N(CH ) ), 6.72 (d, ꢂ ꢃ =0 Hz, 2H,
3 2
aromatic protons), 7.68–7.71 (m, 4H, aromatic protons), 7.94
2.2.9. 4-[4-(Trifluoromethyl)phenyl]-1-(4-methoxybenzylid-
(d, ꢂ ꢃ =0 Hz, 2H, aromatic protons), 8.07 (s, 1H, CH=N),
ene)thiosemicarbazide (7). IR ]max (cm−1): 3142.04 (aromatic
10.12 (s, 1H, N-H), 11.79 (s, 1H, N-H); MS (ESI) (ꢄꢅ/): (M +

4
Journal of Chemistry
H)+ 367; Anal. Calcd for C H F N S: C, 55.73; H, 4.68; N,
Table 1: e yields and melting points (Mp) of the thiosemicar-
bazone derivatives (1–12).
17 17
3
4
15.29. Found: C, 55.72; H, 4.69; N, 15.26.
Compound
1
R
H
Yield (%)
80
Mp (∘C)
198
2.2.13. 4-[4-(Trifluoromethyl)phenyl]-1-(4-trifluoromethyl-
benzylidene)thiosemicarbazide (11). IR ]max (cm−1): 3132.40
(aromatic C-H stretching), 2985.81, 2883.58 (C-H stretching),
1598.99, 1541.12, 1492.90 (C=N, C=C stretching and N-H
bending), 1394.53, 1319.31, 1064.71 (C-N, C=S stretching and
aromatic C-H in plane bending), 825.53 (aromatic C-H out
of plane bending); 1H NMR (500 MHz, DMSO-ꢀ6) ꢁ (ppm):
7. 76 (dd, ꢂ = 10 Hz, 20 Hz, 4H, aromatic protons), 7.88 (d,
ꢂ ꢃ = Hz, 2H, aromatic protons), 8.14 (d, ꢂ = 10 Hz, 2H,
aromatic protons), 8.24 (s, 1H, CH=N), 10.42 and 10.62 (2s,
1H, N-H), 12.02 and 12.18 (2s, 1H, N-H); MS (ESI) (ꢄꢅ/): (M
+ H)+ 392; Anal. Calcd for C H F N S: C, 49.11; H, 2.83; N,
2
F
82
169
3
4
5
6
7
Cl
Br
NO
OH
OCH
87
90
94
72
78
79
72
83
211
224
233
204
200
203
180
214
205
232
2
3
8
CH
CH(CH )
N(CH )
3
9
10
11
12
3
2
3
2
CF
CN
80
96
16 11
6
3
3
10.74; Found: C, 49.10; H, 2.82; N, 10.76.
2.2.14. 4-[4-(Trifluoromethyl)phenyl]-1-(4-cyanobenzylid-
ene)thiosemicarbazide (12). IR ]max (cm−1): 3309.85 (N-
H stretching), 3116.97 (aromatic C-H stretching), 2978.09,
2883.58 (C-H stretching), 2220.07 (C≡N stretching), 1541.12,
1521.84, 1498.69 (C=N, C=C stretching and N-H bending),
1325.10, 1269.16, 1190.08, 1166.93, 1105.21, 1087.85, 1066.64,
1016.49 (C-N, C=S stretching and aromatic C-H in plane
bending), 921.97, 831.32 (aromatic C-H out of plane bending);
1H NMR (500 MHz, DMSO-ꢀ6) ꢁ (ppm): 7.74 (d, ꢂ ꢃ = Hz,
2H, aromatic protons), 7.86–7.90 (m, 4H, aromatic protons),
8.12 (d, ꢂ ꢃ =0 Hz, 2H, aromatic protons), 8.20 (s, 1H, CH=N),
10.43 (s, 1H, N-H), 12.22 (s, 1H, N-H); MS (ESI) (ꢄꢅ/): (M +
H)+ 349; Anal. Calcd for C H F N S: C, 55.17; H, 3.18; N,
of the compounds and rifampicin (Sigma, R3501, China) were
used: 1.5625–800 ꢆg/mL. All black, clear-bottomed, 96-well
plates (Corning 3340, USA) were incubated at 37∘C in 5%
CO for 7 days. On day 7 of the incubation, freshly prepared
2
1 : 1 mixture of Alamar Blue reagent (1 : 10 dilution, Invitrogen,
1025, USA) and 10% Tween 80 was added to one well among
the positive controls. e plates were further incubated at
37∘C for 24 h. If the contents of the well turned pink, the
reagent mixture was added to all the wells of the microplate.
16 11
3
4
16.08; Found: C, 55.15; H, 3.19; N, 16.07.
3. Results and Discussion
e synthesis of new thiosemicarbazone derivatives
(1–12) was performed as described in Scheme 1. 4-[4-
(Trifluoromethyl)phenyl]thiosemicarbazide was synthesized
via the reaction of 4-(trifluoromethyl)phenyl isothiocyanate
with hydrazine hydrate. e treatment of 4-[4-(trifluoro-
methyl)phenyl]thiosemicarbazide with aromatic aldehydes
afforded the thiosemicarbazone derivatives (1–12). e yields
and melting points (Mp) of the compounds were given in
Table 1.
2.3. Microbiology
2.3.1. In Vitro Evaluation of Antimicrobial Activity. e
microbiological assay was carried out according to the CLSI
reference M7-A7 broth microdilution method as described
previously [30]. Chloramphenicol and ketoconazole were
used as reference agents.
Compounds 1–12 were investigated for their in vitro
growth inhibitory activity against pathogenic bacteria and
fungi. Microorganisms used in this study were as follows:
Staphylococcus aureus (ATCC 25923), Enterococcus faecalis
(ATCC 29212), Enterococcus faecalis (ATCC 51922), Listeria
monocytogenes (ATCC 1911), Klebsiella pneumoniae (ATCC
700603), Pseudomonas aeruginosa (ATCC 27853), Escher-
ichia coli (ATCC 35218), Escherichia coli (ATCC 25922),
Candida albicans (ATCC 90028), Candida glabrata (ATCC
90030), Candida krusei (ATCC 6258), and Candida parapsilo-
sis (ATCC 22019).
e structures of new compounds were confirmed by
spectroscopic data and elemental analysis. In the IR spectra
of compounds 1–12, the N-H stretching bands were observed
in the region 3367–3275 cm−1. e aromatic C-H stretching
vibrations gave rise to bands at 3165–3116 cm−1. C=N, C=C
stretching and N-H bending bands were observed in the
region 1616–1487 cm−1. In the IR spectra of compound 12, the
stretching band for C≡N group occurred at 2220.07 cm−1.
1
In the H NMR spectra of compounds 1–12, the signal
due to the CH=N proton was observed in the region 8.0–
8.3 ppm. e N-H protons appeared in the region 10–13 ppm.
In the 1H NMR spectra of some compounds, N-H and CH=N
protons gave rise to two singlet peaks in accordance with
the presence of the ꢇ and ꢈ isomers [31]. Other aromatic
and aliphatic protons were observed at expected regions. e
mass spectral data of the synthesized compounds were found
in full agreement with the proposed structures.
2.3.2. Microplate Alamar Blue Assay (MABA) for Antitu-
bercular Activity. Mycobacterium tuberculosis H37Rv (ATCC
27294) was provided from American Type Culture Collection
(ATCC) cell bank. e cells were grown in ATCC® Medium
1395: Middlebrook 7H9 broth with ADC enrichment at 37∘C
for 30 days. e turbidity of the cultures was adjusted to
McFarland standard number 1. e following concentrations

Journal of Chemistry
5
N
C
S
H
N
H
N
NH2
i
S
F3C
CF3
ii
H
N
H
N
N
S
F3C
R
1–12
Scheme 1: e synthetic route for the preparation of the thiosemicarbazone derivatives (1–12). Reagents and conditions: (i) NH NH ⋅H O,
2
2
2
ethanol, rt, 4 h; (ii) ArCHO, ethanol, reflux, 6 h.
Table 2: Antibacterial activity of the thiosemicarbazone derivatives (MIC values in ꢆg/mL).
Compound
1
2
3
A
B
C
D
E
F
G
H
I
400
100
400
400
400
400
400
400
400
400
400
400
100
Nt
200
200
400
400
100
400
400
400
400
400
100
200
200
Nt
400
1600
400
400
400
400
400
400
400
400
400
400
200
Nt
400
1600
800
400
400
400
800
800
400
400
800
400
100
Nt
400
800
400
400
400
400
400
400
400
400
400
400
200
Nt
400
1600
400
400
400
400
400
400
400
400
400
400
200
Nt
400
1600
400
800
400
400
800
800
800
400
400
400
100
Nt
400
400
400
400
400
400
400
400
400
400
400
400
25
200
400
400
400
400
400
200
200
200
200
200
200
Nt
4
5
6
7
8
9
10
11
12
Chloramphenicol
Rifampicin
Nt
25
A: S. aureus (ATCC 25923), B: E. faecalis (ATCC 29212), C: E. faecalis (ATCC 51922), D: L. monocytogenes (ATCC 1911), E: K. pneumoniae (ATCC 700603), F:
P. aeruginosa (ATCC 27853), G: E. coli (ATCC 35218), H: E. coli (ATCC 25922), and I: M. tuberculosis H37Rv.
Nt: not tested.
e compounds were tested in vitro against a number
of Gram-positive and Gram-negative bacteria and yeasts
using broth microdilution method. Based on this assay, the
minimum inhibitory concentrations (MICs) of the com-
pounds were determined. e observed MIC values were in
the range of 100–1600 ꢆg/mL. In general, the tested com-
pounds exhibited more potent inhibitory activity towards
Gram-positive bacteria compared to Gram-negative bacteria
(Table 2).
Compounds 1, 2, and 12 and chloramphenicol showed
the same level of antibacterial activity against E. faecalis
(ATCC 29212) with a MIC value of 200 ꢆg/mL, whilst com-
pounds 5 and 11 were more effective against E. faecalis than
chloramphenicol. Generally electron withdrawing groups
such as nitro and trifluoromethyl enhanced antibacterial
activity, whereas electron donating substituents decreased
antibacterial activity against E. faecalis.
In addition, in vitro antimycobacterial effects of the
compounds were investigated against the drug resistant M.
tuberculosis H37Rv strain using Microplate Alamar Blue
Among these derivatives, fluorosubstituted compound 2
exhibited the highest antibacterial activity against S. aureus
with a MIC value of 100 ꢆg/mL when compared with chlo-
ramphenicol (MIC = 100 ꢆg/mL). is outcome pointed
out the importance of fluorine substituent for antibacterial
activity against S. aureus.
Assay (MABA). Compounds 1, 7, 8, 9, 10, 11, and 12 exhibited
antimycobacterial activity with a MIC value of 200 ꢆg/mL,
whereas other derivatives showed antimycobacterial activity
with a MIC value of 400 ꢆg/mL.

6
Journal of Chemistry
Table 3: Anticandidal activity of the thiosemicarbazone derivatives
(MIC values in ꢆg/mL).
3
4
5
6
7
8
9
10
11
12
Conflict of Interests
e authors have declared no conflict of interests.
Compound
1
2
A
B
C
D
400
400
400
400
400
400
400
400
400
400
400
400
200
400
200
400
400
200
400
400
400
400
400
400
400
200
200
200
400
200
200
200
200
200
200
200
400
400
3.125
200
200
200
200
200
200
200
200
200
200
200
200
50
Acknowledgment
is study was supported by Anadolu University Scientific
Research Projects Commission under Grants nos. 1404S137
and 1409S386.
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In the present paper, new thiosemicarbazone derivatives were
synthesized and evaluated for their inhibitory effects on
pathogenic bacteria including M. tuberculosis H37Rv. e
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In general, the tested compounds exhibited more potent
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