Synthesis, characterization and biological evaluation of some novel sulfonylthiosemicarbazides

Article abstract of DOI:10.1080/10426507.2019.1633320

A series of thiosemicarbazides were synthesized and structurally characterized by spectroscopic techniques (NMR, FT-IR) besides elemental analysis. These compounds were evaluated for their cytotoxicity against human breast cancer cell line MCF7 and prostate cancer cell line PC3 and nonmalignant fibroblast L929 cell line by MTT assay. Among the compounds, N-[2-(4-chlorophenyl)ethyl]-2-[(4-methylphenyl)sulfonyl]hydrazinecarbothioamide (3d) and 2-[(4-methylphenyl)sulfonyl]-N-[4-(trifluoromethoxy)phenyl]hydrazinecarbothioamide (3f) were found to display significant cytotoxicity with IC₅₀ of 13.87 μM (against PC3 cell line) and 1.47 μM (against MCF7 cell line), respectively. These compounds were non-cytotoxic to normal cell line with IC₅₀>100 μM. Western blotting studies demonstrated that compound 3f induced apoptosis and caused cell death in the MCF7 and PC3 cell lines via an increase in Bax protein expression and a slight decrease in Bcl-2 protein expression. The gene expression ratio Bax/Bcl-2 showed the induction of mitochondrial apoptosis in cancer cell lines. All of synthesized compounds have also been tested for antioxidant activity and all compounds achieved strong inhibition of the DPPH radical. These findings showed that compound 3f, displays potential to be further explored in the development of new anticancer agents.

Full text of DOI:10.1080/10426507.2019.1633320

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: https://www.tandfonline.com/loi/gpss20
Synthesis, characterization and
biological evaluation of some novel
sulfonylthiosemicarbazides
Sevil Şenkardeş, İhsan Han, Hakan Hançer, Mürüvvet Abbak, Özge Çevik & Ş.
Güniz Küçükgüzel
To cite this article: Sevil Şenkardeş, İhsan Han, Hakan Hançer, Mürüvvet Abbak, Özge Çevik
&
Ş. Güniz Küçükgüzel (2019): Synthesis, characterization and biological evaluation of some
novel sulfonylthiosemicarbazides, Phosphorus, Sulfur, and Silicon and the Related Elements, DOI:
0.1080/10426507.2019.1633320
1
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
https://doi.org/10.1080/10426507.2019.1633320
Synthesis, characterization and biological evaluation of some novel
sulfonylthiosemicarbazides
a
b
a
c
d
a
_
€
Sevil S¸ enkarde s¸
, Ihsan Han , Hakan Han c¸ er , M u€ r u€ vvet Abbak , Ozge C¸ evik , and S¸ . G u€ niz K u€ c¸ u€ kg u€ zel
a
b
_
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Haydarpa s¸ a, Istanbul, Turkey; Department of
c
Pharmaceutical Chemistry, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey; Scientific Technology Research and Application Centre,
Adnan Menderes University, Aydın, Turkey; Department of Biochemistry, School of Medicine, Adnan Menderes University, Aydın, Turkey
d
ABSTRACT
ARTICLE HISTORY
A series of thiosemicarbazides were synthesized and structurally characterized by spectroscopic
techniques (NMR, FT-IR) besides elemental analysis. These compounds were evaluated for their
cytotoxicity against human breast cancer cell line MCF7 and prostate cancer cell line PC3 and non-
malignant fibroblast L929 cell line by MTT assay. Among the compounds, N-[2-(4-chloropheny-
l)ethyl]-2-[(4-methylphenyl)sulfonyl]hydrazinecarbothioamide (3d) and 2-[(4-methylphenyl)sulfonyl]-
N-[4-(trifluoromethoxy)phenyl]hydrazinecarbothioamide (3f) were found to display significant cyto-
toxicity with IC50 of 13.87 lM (against PC3 cell line) and 1.47 lM (against MCF7 cell line), respect-
ively. These compounds were non-cytotoxic to normal cell line with IC50>100lM. Western
blotting studies demonstrated that compound 3f induced apoptosis and caused cell death in the
MCF7 and PC3 cell lines via an increase in Bax protein expression and a slight decrease in Bcl-2
protein expression. The gene expression ratio Bax/Bcl-2 showed the induction of mitochondrial
apoptosis in cancer cell lines. All of synthesized compounds have also been tested for antioxidant
activity and all compounds achieved strong inhibition of the DPPH radical. These findings showed
that compound 3f, displays potential to be further explored in the development of new anticancer
agents.
Received 21 February 2019
Accepted 14 June 2019
KEYWORDS
Sulfonyl derivatives;
anticancer activity;
apoptosis; DPPH;
thiosemicarbazide
Abbreviations: ACN: Acetonitrile; Bax: Bcl-2-associated X protein; BCA: Bicinchoninic acid; Bcl-2: B-
cell lymphoma 2; BSA: Bovine serum albumin; FBS: Fetal bovine serum; GAPDH: Glyceraldehyde 3-
phosphate dehydrogenase; HMBC: Heteronuclear multiple bond correlation; HPLC: High perform-
ance liquid chromatography; DMSO: Dimethyl sulfoxide; DPPH: 2,2-diphenyl-1-picrylhydrazyl; mp:
Melting point; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS: phosphate-
buffered saline; RIPA: Radio immunoprecipitation assay; SDS: Sodium dodecyl sulfate; THF:
Tetrahydrofuran; TMS: Tetramethylsilane; TLC: Thin layer chromatography
GRAPHICAL ABSTRACT
Introduction
of drug resistance appear as a serious problem in the field
[
3,4]
of cancer.
Hence, drug researchers focus on the develop-
Globally, cancer is a potentially fatal disease characterized by
broken down control mechanisms that regulate cell prolifer-
ation and differentiation. Surgery, radiotherapy, chemo-
therapy, hormone and immune therapies are being used for
cancer treatment. Among them, chemotherapy is a wide-
spread and effective cancer treatment that uses powerful
ment of safer and more effective compounds for can-
cer treatment.
Targeting apoptosis is an indispensable approach in can-
cer therapy. Apoptosis (programmed cell death) is an
important part of the normal development and plays a crit-
ical role for maintaining homeostasis between the rates of
[
1]
[
2]
drugs to kill cancer cells. Drug toxicities and development
CONTACT Sevil S¸ enkarde s¸
Istanbul, Turkey.
sevil.aydin@marmara.edu.tr
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Haydarpa s¸ a,
_
This work was partly presented in 11th Joint Meeting on Medicinal Chemistry 2019.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
Supplemental data for this article is available online at https://doi.org/10.1080/10426507.2019.1633320.
ß 2019 Taylor & Francis Group, LLC

2
S. S¸ ENKARDE S¸ ET AL.
[
5]
cell proliferation and cell death. A lot of cancer cells div-
ide and grow uncontrollably because of inactivating muta-
tions in genes encoding pro-apoptotic proteins or through
[
6]
upregulation of anti-apoptotic proteins. Many antitumor
compounds exert cytotoxic effects on malignant cells by
inducing apoptosis so apoptosis have provided a new oppor-
tunity for anticancer drug discovery.
Free radicals can be described as atoms, molecules, or
[
7]
ions with unpaired electrons. These highly reactive and
unstable radicals play an important role in the body with
various chemical and biological reactions . As a result of
these reactions, many diseases may occur including cardio-
Scheme 1. The synthetic route for compounds 3a–j.
[
8,9]
vascular diseases, diabetes, cancer and AIDS.
Antioxidant
1
compounds that can scavenge free radicals have some
In the H-NMR spectra of synthesized compounds, there
[
10]
important functions in the improvement of these diseases
are not any peaks belonging -NH2 which concerning to
and have become a mainstay in pharmaceutical applications. –SO NHNH functional group. Besides, new NH protons of
2
2
Scientific evidence suggests that antioxidants reduce the risk thiosemicarbazide group were observed at 7.93–10.00 ppm
for such chronic diseases and may protect against cancer and three NH protones which prove the presence of com-
1
through different mechanisms involving up-regulation of pounds were detected in H-NMR spectrum and the infor-
[
23,24]
antioxidant enzymes, repair of nuclear DNA, and mation given is supported by the literature.
apoptosis.
The
[
11]
-C(¼S)-N-H proton of 3a occurs an upfield signal at
Nitrogen and sulfur containing organic compounds have 7.93 ppm due to the electron-donating alkyl group. All the
been used to create important medicinal agents like sulfona- title compounds exhibit a multiplet in a region of d
[
12–14]
mides and thiazines.
Among them, thiosemicarbazides 6.84–7.78 ppm owing to benzene protons. In the 13C-NMR
are important intermediate compounds because of their spectra of compounds (3a–j) show the C ¼ S signal at d
reported biological activities and used in the synthesis of 180.62-183.72 ppm, corresponding to thiosemicarbazide
[
25,26]
heterocyclic compounds, such as 1,2,4-triazoles, 1,3,4-thia- moieties.
[
15–18]
diazoles and 1,3,4-oxadiazoles derivatives.
lar, compounds bearing sulfonyl group at the
thiosemicarbazide side chain possess diverse pharmaco-
In particu-
The HMBC spectrum of 3j was chosen a prototype to
confirm the structure and connectivity (Table S1 and Figure
S32 in Supplemental Materials). The aromatic tertiary and
quaternary carbons are established through the connectiv-
ities between the carbon and its neighboring proton by
using a long–range correlated HMBC experiment. The
measured HMBC correlations (see Table S1) confirmed that
definitely: for tolyl moiety the strong couplings are H1–C2,
H1–C3, H1–C4, H1–5, and H3–C2, H3–C4 and H3–C5. For
the substituted aromatic ring, there are strong HMBC corre-
lations between H8–C9, H8–C10 and H8–C11 and H9–C11.
a
[
19]
logical activities which include antitrombotic,
antimyco-
[
20]
[21]
bacterial,
and antibacterial,
properties. However, there
is no report about anticancer activity of these compounds in
the literature. Based on the above findings, it deemed of
interest to design and synthesize a series of novel com-
pounds based on N-substituted-sulfonylthiosemicarbazide
derivatives to assess their cytotoxic and radical scavenging
profiles as described in this research work.
1
In particular, three of H signals at 7.70 ppm, 7.74 ppm and
7
.76 ppm are very close and HMBC spectrum helped us to
Results and discussion
assign the signal to individual atoms.
Synthesis
Anticancer activity
Scheme 1 outlines the synthetic procedures of compounds
3
a–j. Compound 2, 4-methylbenzensulfonyl hydrazide was
MTT assay was performed to determine the antiproliferative
effects of compounds 3a–j on PC3 (androgen-independent)
human prostate and MCF7 (includes oestrogene and proges-
treated with different isothiocyanates in acetonitrile to afford
the corresponding thiosemicarbazides 3a–j in good yields.
All the newly synthesized compounds were characterized by
27
terone receptors) breast cancer cell lines. The anticancer
IR and NMR spectroscopy as well as elemental analysis, as agent cisplatin was used as a positive control. L929 mouse
listed in the Experimental Section.
fibroblast cell line was chosen as an example of normal cells
The infrared spectra of 3a–j showed the sulfonylhydra- to understand toxicity. Among all the compounds, com-
zine moiety by the presence of absorptions at pound 3d with (4-chlorophenyl)ethyl substitution exhibited
ꢀ
1
3
142–3302 cm , typical for the group -NH-NH .Their spec- the highest potency against PC3 cell line with IC50 values of
2
tra revealed characteristic absorption bands at 1317–1357 13.86 mM, whereas compound 3f with 4-(trifluoromethoxy)-
ꢀ
1
and 1141–1182 cm attributed to SO group. Also, the sig- phenyl substitution showed highest potency against MCF7
2
nals from the group C ¼ S are observed in the range cell line with IC50 of 1.47 mM, a superior values than the
ꢀ
21,22]
1
1
225–1261 cm . These are consistent with the litera- standart cisplatin. However, the rest of compounds demon-
[
ture data.
strated moderate to low activities.

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
The selectivity index values (SI) (IC50 normal cell/IC50 Bcl-2 may contribute to the caspase-3 activation in mito-
[
30]
cancer cell) of the compounds 3a–j, on L929 mouse fibro- chondria.
According to results in Figures S2 and S3,
blast cells were investigated using MTT test. The results are compound 3f could triggered mitochondrial apoptosis in
summarized in Table S2 (Supplemental Materials). PC3 and MCF-7 cells by upregulating Bax/Bcl-2 ratio which
Compounds 3a and 3b presented the same toxicity in L929 are responsible for caspase-3 activation.
and cancerous cells while compounds 3d, 3e, 3f and 3g pre-
sented a selectivity index higher than one, indicating a con-
siderable safety profile. In particular, compound 3f showed
Free radical-scavenging ability of compounds
the most potent effects against both cancer cell lines with
high selectivity with 12.05 and 215.49 SI values for PC3 and
MCF7 cancer cell lines, respectively. Because of these results,
compound 3f was chosen as a prototype for the fur-
ther studies.
DPPH method is a quick, simple and cheap method to
measure the antioxidant activity of compounds in vitro. The
antioxidant or DPPH free-radical scavenging activity of all
the synthesized compounds were performed using DPPH
method and the results were found in Table S3. Ascorbic
acid was used as a positive control for comparison. The
lower IC50 values in the DPPH test indicate high antioxidant
activities. According to the results, all compounds tested
Morphological characterization of apoptosis
Herein, compound 3f was chosen as a prototype for anti- showed a strong DPPH radical scavenging activity compared
cancer mechanism of action study on PC3 and MCF7 cell to that of antioxidant ascorbic acid ranged from 0.80 to 2.
models. When PC3 and MCF7 cells were incubated with 64 lM. Compound 3j gave an IC50 value of 0.80 ± 0.01 lM,
IC50 values of 3f for 12, 24, and 48 h, these results suggested while ascorbic acid gave IC50 value of 2.88 ± 0.19 lM. The
that compound 3f decreased the viability of PC3 and MCF7 highest activity of compound 3j, may be due to the cyano
cells in a time-dependent manner. Cancer cells were treated group attached to the phenyl ring. The cyano group pres-
with IC50 values of 3f, and then cellular morphology was ence on the phenyl ring which is a strong electron-with-
observed under an inverted microscope.
drawing group, had a great impact on the activity. CN
substituent polarizes the p-electrons of the phenyl ring,
which increases the conjugated NH group acidity, exceeding
The results in crystal violet stained MCF7 and PC3 cells
clearly showed a reduction in cells numbers with IC50 con-
centration of compound 3f compared to the untreated con-
trol (Figure S1, Supplemental Materials). Figure S1 showed
that 3f induced remarkable changes of cellular morphology,
such as cell shrinkage and loss of cellular architecture in
both cell lines. These observations indicated that compound
[
31]
the acidity of the other NH groups in the compound.
Experimental
Materials and methods
3f inhibits cellular proliferation by inducing cell death and
All materials used were obtained from Sigma Aldrich (St.
Louis, USA). Melting points were determined using the
Electrothermal IA9300 apparatus and are uncorrected. FT-
IR spectra were recorded on a Schimadzu FT-IR-8400S spec-
these cells were undergoing apoptosis. The process of apop-
tosis is characterized by many morphological and biochem-
ical changes.
1
trometer. H and 13C-NMR spectra were recorded on
Western blotting
Bruker 300 MHz Ultrashield TM spectrometer. Elemental
analyses were determined by CHNS-932 (LECO). The liquid
chromatographic system consists of an Agilent Technologies
Different molecular mechanisms of anticancer agents
induced apoptosis in cancer cells were reported, including
[
28]
1
100 series instrument equipped with a quaternary solvent
regulation of apoptotic proteins such as Bax and Bcl-2.
delivery system and a model Agilent series G1315 A photo-
diode array detector. The chromatographic data were col-
lected and processed using Agilent Chemstation Plus
software. Chromatographic separation was performed at
ambient temperature using a reverse phase Zorbax C8
To characterize the signaling pathways in 3f-induced apop-
tosis, the expression levels of Bcl-2 and Bax in 3f treated
PC3 and MCF-7 cells were analyzed by Western blotting
(Figures S2 and S3, Supplemental Materials). Bcl-2 protein
expression was slightly suppressed by 1.47 lM 3f in MCF7
and 26.3 lM in PC3 cells following a 24 h treatment com-
pared with the control group while the expression of Bax
was increased at same concentration. GAPDH gene used as
an internal control.
(
4.0 ꢁ 250 mm) column. All experiments were performed
using ACN-H O (v/v, 70/30) mobile phase with UV detec-
2
tion at 230 nm. The Supplemental Materials contains 1H
13
and
C NMR and IR spectra of the products 3
(Figures S4–S34).
Bax is a protein that promotes apoptosis by mitochon-
drial dysfunction and is controlled by the antiapoptotic Bcl-
2
protein. Conversely, Bcl-2 is responsible for the release of
General procedure for the synthesis of p-toluenesulfonyl
hydrazide (2)
cytochrome C into the cytosol, prevents apoptosis by inhib-
iting the activity of Bax.
[
29]
The balance Bcl-2 and Bax
determines can determine the cellular fate and the ratio of A mixture of p-toluensulfonyl chloride (1) (1.0 eq.) and
ꢂ
Bax/Bcl-2 accelerates apoptosis via mitochondrial apoptotic hydrazine hydrate (3.0 eq.) was stirred at 0–5 C for 1–2 h
pathway. Literature survey demonstrated that, higher Bax/ in THF (20 mL). The reaction progress and completion were

4
S. S¸ ENKARDE S¸ ET AL.
studied using TLC. After completion of the reaction, excess (DMSO-d /TMS) d ppm: 21.56 (CH ), 120.70 (Ar-CH),
6
3
THF was distilled off. The product was subsequently washed 124.63 (Ar-C), 125.96 (Ar-CH), 128.12 (Ar-CH), 128.63
[
32,33]
with water, dried, and recrystallized from ethanol.
(2 Ar-CH), 130.04 (2 Ar-CH), 130.23 (Ar-CH), 135.24 (Ar-
C), 140.93 (Ar-C), 144.42 (Ar-C), 181.20 (C ¼ S); Anal. calcd
for C H BrN O S (400.313): C, 42.02; H, 3.53; N, 10.50;
14
14
3
2 2
General procedure for the synthesis N-substituted-2-[(4-
methylphenyl)sulfonyl]hydrazine carbothioamides (3a–j)
S, 16.02 Found: C, 42.52; H, 3.59; N, 9.85; S, 15.91.
A solution 4-methylbenzenesulfonylhydrazide (2) (10 mmol)
N-[2-(4-chlorophenyl)ethyl]-2-[(4-methylphenyl)sulfonyl]
hydrazinecarbothioamide (3d)
in acetonitrile is mixed with a solution of substituted iso-
[
34]
thiocyanate (10 mmol) in acetonitrile
and refluxed for
3
–4 h and the reaction mixture poured into ice cold water to
ꢂ
ꢀ1
1
Yield 85%; m.p. 210 C; HPLC t (min.): 4.55; IR: ꢀ/cm :
R
get precipitate of derivatives which was filtered dried and
recrystallized from ethanol.
3
336, 3173 (N-H); 1357 and 1168 (SO ); 1247 (C ¼ S); H-
2
NMR (300 MHz), (DMSO-d /TMS) d ppm: 2.41 (s, 3H, Ar-
6
CH ), 2.73 (t, 2H, CH ); 3.60 (t, 2H, CH ); 7.23 (d, 2H,
3
2
2
N-(cyclohexylmethyl)-2-[(4-methylphenyl)sulfonyl]
hydrazinecarbothioamide (3a)
J¼ 8.4 Hz, Ar-H); 7.35 (d, 2H, J¼ 8.4Hz, Ar-H); 7.42 (d, 2H,
J¼ 8.1Hz, Ar-H); 7.70 (d, 2H, J¼ 8.4 Hz, Ar-H); 8.17 (s, 1H,
CSNH-Ar), 9.36 (s, 1H, SO NH-NH-CS), 9.80 (s, 1H, SO NH-
ꢂ
2
2
Yield 78%; m.p. 196-198 C; HPLC t (min.): 4.68; IR:
R
NH-CS); 13C-NMR (75 MHz) (DMSO-d /TMS) d ppm: 21.53
ꢀ
1
6
ꢀ
(
/cm : 3346, 3165 (N-H); 1352 and 1166 (SO ); 1246
2
1
(CH ), 34.32 (CH ), 45.30 (CH ), 128.46 (2 Ar-CH), 128.76
3 2 2
C ¼ S); H-NMR (300 MHz), (DMSO-d /TMS) d ppm: 0.8-
6
(2 Ar-CH), 130.02 (2 Ar-CH), 130.95 (2 Ar-CH), 131.21 (Ar-C),
1
3
2
.65 (m, 11H, cyclohexyl hydrogens), 3.05 (s, 3H, Ar-CH ),
3
1
35.32 (Ar-C), 138.68 (Ar-C), 144.16 (Ar-C), 182.07 (C ¼ S);
.24 (t, 2H, N-CH ), 7.41 (d, 2H, J ¼ 8.1Hz, Ar-H), 7.70 (d,
2
Anal. calcd for C H ClN O S (383.916): C, 50.06; H, 4.73;
16
18
3 2 2
H, J ¼ 8.4Hz, Ar-H), 7.93 (t, 1H, CSNH-CH ), 9.25 (s, 1H,
2
N, 10.95; S, 16.70 Found: C, 49.59; H, 4.78; N, 10.73; S, 16.16.
SO NH-NH-CS), 9.78 (s, 1H, SO NH-NH-CS); 13C-NMR
2
2
(
(
75 MHz) (DMSO-d /TMS) d ppm: 21.56 (CH ), 25.89
6 3
2CH ), 26.56 (CH ), 30.64 (2CH ), 37.20 (CH), 50.10 (N-
2
2
2
2
-[(4-methylphenyl)sulfonyl]-N-[4-(trifluoromethyl)phenyl]
CH ), 128.43 (2Ar-CH), 130.01 (2Ar-CH), 135.36 (Ar-C),
1
2
hydrazinecarbothioamide (3e)
44.13 (Ar-C), 182.10 (C ¼ S); Anal. calcd for C H N O S
15
23 3 2 2
ꢂ
ꢀ1
1
(341.492): C, 52.76; H, 6.79; N, 12.30; S, 18.78 Found: C, Yield 70%; m.p. 173-175 C; HPLC t (min.): 4.31; IR: ꢀ/cm :
R
5
2.43; H, 6.72; N, 12.14; S, 18.75.
3315, 3238 (N-H); 1317 and 1157 (SO ); 1257 (C ¼ S); H-
2
NMR (300 MHz), (DMSO-d /TMS) d ppm: 2.37 (s, 3H, Ar-
6
CH ), 7.40 (d, 2H, J¼ 8.1 Hz, Ar-H); 7.65 (s, 4H, Ar-H); 7.75
3
N-(2-bromophenyl)-2-[(4-methylphenyl)sulfonyl]
hydrazinecarbothioamide (3b)
(
d, 2 H, J¼ 8.4Hz, Ar-H); 9.95 (bs, 1H, CSNH-Ar), 10.02 (s,
1
H, SO NH-NH-CS), 10.05 (s, 1H, SO NH-NH-CS); 13C-
2
2
ꢂ
Yield 88%; m.p. 146-148 C; HPLC t (min.): 3.88; IR: NMR (75 MHz) (DMSO-d /TMS) d ppm: 21.47 (CH ), 122.97
R
6
3
ꢀ
1
ꢀ
(
(
/cm : 3329, 3215 (N-H); 1323 and 1141 (SO ); 1255
2
(2 Ar-CH), 125.42 (CF , q, 1J ¼269 Hz) 125.96 (Ar-C),
3
C-F
1
C ¼ S); H-NMR (300 MHz), (DMSO-d /TMS) d ppm: 1.05
6
126.57 (2 Ar-CH), 128.57 (2 Ar-CH), 130.06 (2 Ar-CH), 135.20
(Ar-C), 143.08 (Ar-C), 144.41 (Ar-C), 181.26 (C ¼ S); Anal.
calcd for C H F N O S (389.415): C, 46.26; H, 3.62; N,
ethanol CH ), 2.41 (s, 3H, Ar-CH ), 3.45 (ethanol CH ),
3
3
2
7
1
.09-7.78 (m, 8H, Ar-H), 9.53 (s, 1H, CSNH-Ar), 9.98 (bs,
H, SO NH-NH-CS), 10.16 (s, 1H, SO NH-NH-CS); 13C-
15 14 3 3 2 2
2
2
10.79; S, 16.47 Found: C, 46.71; H, 4.02; N, 9.93; S, 15.98.
NMR (75 MHz) (DMSO-d /TMS) d ppm: 21.55 (CH ),
6
3
1
20.89 (Ar-C), 128.05 (Ar-CH), 128.24 (Ar-CH), 128.65
(
2 Ar-CH), 129.32 (Ar-CH), 130.09 (2 Ar-CH), 132.86 (Ar- 2-[(4-methylphenyl)sulfonyl]-N-[4-(trifluoromethoxy)phenyl]
CH), 135.21 (Ar-C), 137.96 (Ar-C), 144.39 (Ar-C), 181.80
hydrazinecarbothioamide (3f)
(
(
C ¼ S); Anal. calcd for C H BrN O S .1/3C H OH
1
4
14
3
2 2
2
5
ꢂ
Yield 77%; m.p. 204-206 C; HPLC t (min.): 4.33; IR:
R
415.670): C, 42.38; H, 3.88; N, 10.11; S, 16.02 Found: C,
2.67; H, 3.82; N, 9.75; S, 15.76.
ꢀ
1
ꢀ
/cm : 3319, 3238 (N-H); 1334 and 1153 (SO ); 1261
2
4
1
(
C ¼ S); H-NMR (300 MHz), (DMSO-d /TMS) d ppm: 2.38
6
(
s, 3H, Ar-CH ), 7.29 (d, 2H, J ¼ 8.1 Hz, Ar-H); 7.41 (d, 2H,
3
N-(3-bromophenyl)-2-[(4-methylphenyl)sulfonyl]
hydrazinecarbothioamide (3c)
J ¼ 8.1Hz, Ar-H); 7.45 (d, 2H, J ¼ 9Hz, Ar-H); 7.75 (d, 2H,
J ¼ 8.1Hz, Ar-H); 9.81 (s, 1H, CSNH-Ar), 9.90 (s, 1H,
ꢂ
SO NH-NH-CS), 10.01 (bs, 1H, SO NH-NH-CS); 13C-NMR
2 2
Yield 76%; m.p. 153-154 C; HPLC t (min.): 4.14; IR:
ꢀ
R
ꢀ
1
(
(
75 MHz) (DMSO-d /TMS) d ppm: 21.46 (CH ), 120.54
6 3
CF , q, 1J ¼254 Hz), 121.08 (2CH), 127.33(2CH), 128.56
/cm : 3302, 3232 (N-H); 1327 and 1157 (SO ); 1255
2
1
(
(
C ¼ S); H-NMR (300 MHz), (DMSO-d /TMS) d ppm: 2.39
3
C-F
6
s, 3H, Ar-CH ), 7.22–7.38 (m, 3H, Ar-H), 7.41 (d, 2H, (2CH), 130.07 (2CH), 135.14 (C), 144.46 (C), 138.46 (C),
3
J ¼ 8.1Hz, Ar-H), 7.52 (s, 1H, Ar-H), 7.75 (d, 2H, J ¼ 8.4Hz, 145.61 (C), 181.41 (C ¼ S); Anal. calcd for C15
H F N O S
14 3 3 3 2
Ar-H), 9.77 (s, 1H, CSNH-Ar), 9.95 (s, 1H, SO NH-NH- (405.415): C, 44.44; H, 3.48; N, 10.36; S, 15.82 Found: C,
2
CS), 10.01 (s, 1H, SO NH-NH-CS); 13C-NMR (75 MHz) 44.16; H, 3.53; N, 10.28; S, 14.82.
2

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
5
N-(2,6-difluorophenyl)-2-[(4-methylphenyl)sulfonyl]
hydrazinecarbothioamide (3g)
N-(4-cyanophenyl)-2-[(4-methylphenyl)sulfonyl]
hydrazinecarbothioamide (3j)
ꢂ
ꢂ
Yield 72%; m.p. 176-177 C; HPLC t (min.): 3.52; IR: Yield 82%; m.p. 211-212 C; HPLC t (min.): 3.40; IR:
R
R
ꢀ
1
ꢀ1
1 1
ꢀ
/cm : 3335, 3203 (N-H); 1340 and 1161 (SO ); 1259 ꢀ/cm : 3302, 3259, 3217 (N-H); 2237 (CꢃN), 1327 and
2
(
C ¼ S); H-NMR (300 MHz), (DMSO-d /TMS) d ppm: 1.03 1159 (SO ); 1257 (C ¼ S); H-NMR (600 MHz), (DMSO-d /
6
2
6
(
Ethanol CH ); 2.48 (s, 3H, Ar-CH ), 3.79 (Ethanol CH ); TMS) d ppm: 2.37 (s, 3H, Ar-CH ), 7.41 (d, 2H, J ¼ 8.4Hz,
3
3
2
3
7
(
.06-7.44 (m, 3H, Ar-H); 7.43 (d, 2H, J ¼ 8.1Hz, Ar-H); 7.75 Ar-H); 7.70 (d, 2H, J ¼ 9Hz, Ar-H), 7.74 (d, 2H, J ¼ 8.4Hz,
d, 2H, J ¼ 8.4Hz, Ar-H); 9.51 (s, 1H, CSNH-Ar), 10.04 (s, Ar-H); 7.76 (d, 2H, J ¼ 9Hz, Ar-H); 10.00 (s, 1H, CSNH-
1
H, SO NH-NH-CS), 10.09 (bs, 1H, SO NH-NH-CS); 13C- Ar), 10.08 (bs, 1H, SO NH-NH-CS), 10.13 (s, 1H, SO NH-
2
2
2
2
NMR (75 MHz) (DMSO-d /TMS) d ppm: 21.56 (CH ), NH-CS); 13C-NMR (75 MHz) (DMSO-d /TMS) d ppm:
6
3
6
2
5.95 (ethanol CH ), 62.50 (ethanol CH ), 111.86, 112.17 21.51 (CH ), 106.96 (Ar-C), 119.42 (2 Ar-CH), 125.13 (CN),
3 2 3
(
2 Ar-CH, d, 2J ¼ 23.75 Hz), 117.15 (Ar-CH), 128.53 (2 Ar- 128.58 (2 Ar-CH), 130.15 (2 Ar-CH), 132.57 (2 Ar-CH),
CF
CH), 129.20 (Ar-C, d, 2J ¼ 20.25 Hz) , 130.02 (2 Ar-CH), 135.15 (Ar-C), 143.79 (Ar-C), 144.46 (Ar-C), 181.02 (C ¼ S);
CF
1
1
35.39 (Ar-C), 144.17 (Ar-C), 157.64, 160.95 (2C-F, dd, Anal. calcd for C H N O S (346.427): C, 52.01; H, 4.07;
15 14 4 2 2
J ¼ 243 Hz, 3J ¼ 4.7Hz), 183.72 (C ¼ S); Anal. calcd for N, 16.17; S, 18.51 Found: C, 52.22; H, 4.50; N, 16.81;
CF
CF
C H F N O S .1/3 C H OH (372.755): C, 47.26; H, 4.06; S, 18.53.
1
4
13
2
3
2 2
2
5
N, 11.27; S, 17.20 Found: C, 47.44; H, 3.94; N, 11.33;
S, 17.82.
Biological assays
Cell culture and cell viability assay
2
-[(4-methylphenyl)sulfonyl]-N-(3,4,5-trimethoxyphenyl)
Cell lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA). Human breast can-
cer cell line MCF-7, human prostate cancer cell line PC3,
mouse fibroblast cell line L929 cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen)
or RPMI-1640 supplemented with 10% FBS, 2 mM l-glutam-
ine, 100 U/mL penicillin, and 100 lg/mL streptomycin and
hydrazinecarbothioamide (3h)
ꢂ
Yield 85%; m.p. 186-187 C; HPLC t (min.): 3.73; IR:
R
ꢀ
1
ꢀ
/cm : 3311, 3142 (N-H); 1345 and 1168 (SO ); 1226
2
1
(
(
C ¼ S); H-NMR (300 MHz), (DMSO-d /TMS) d ppm: 2.39
6
s, 3H, Ar-CH ), 3.64 (s, 3H, Ar-OCH ), 3.73 (s, 6H, Ar-
3
3
OCH ), 6.84 (s, 2H, Ar-H), 7.42 (d, 2H, J ¼ 8.1Hz, Ar-H);
3
ꢂ
kept in a humidified atmosphere at 37 C incubator with 5%
7
.77 (d, 2H, J ¼ 8.4Hz, Ar-H); 9.52 (s, 1H, CSNH-Ar), 9.78
CO in air. To determine the cell viability cells were plated
(s, 1H, SO NH-NH-CS), 9.96 (bs, 1H, SO NH-NH-CS);
2
2
2
4
onto 96-well plates (1 ꢁ 10 cells/well). The cells were
1
5
3C-NMR (75 MHz) (DMSO-d /TMS) d ppm: 21.51 (CH ),
6 3
6.27 (2 OCH ), 60.51 (OCH ), 102.75 (2 Ar-CH), 128.61
treated with different concentrations (0, 0.1, 1, 10, 100, and
3
3
1
000 lM) of synthesized compound (3a–j) incubated for
(
1
(
2 Ar-CH), 130.05 (2 Ar-CH), 134.96 (Ar-C), 135.08 (Ar-C),
35.27 (Ar-C), 144.33 (Ar-C), 152.47 (2 Ar-C), 180.62
C ¼ S); Anal. calcd for C H F N O S (411.495): C, 49.26;
2
4 h. After the incubation cells were washed with PBS and
added to 100 lL DMEM/RPMI-1640. A total of 10 lL of the
MTT (Vybrant, Invitrogen) labeling reagent was added to
17
21
2
3
5 2
H, 5.14; N, 10.21; S, 15.58 Found: C, 49.09; H, 5.40; N,
0.09; S, 15.50.
each well and incubated for 4 h in humidified atmosphere at
1
ꢂ
3
1
7 C incubator with 5% CO in air. After the incubation,
2
00 lL of the SDS buffer was added into each well for solu-
bilization of formazan precipitate. Then absorbance was
measured by microplate reader at 570 nm and was carried
out in triplicate of each assay.
2
-[(4-methylphenyl)sulfonyl]-N-(pyridin-3-yl)
[
27]
hydrazinecarbothioamide (3i)
ꢂ
Yield 77%; m.p. 175-176 C; HPLC t (min.): 3.19; IR:
R
ꢀ
1
ꢀ
/cm : 3313, 3142 (N-H); 1345 and 1165 (SO ); 1251
2
Cell growing assays
1
(
C ¼ S); H-NMR (600 MHz), (DMSO-d /TMS) d ppm: 2.39
6
(
s, 3H, Ar-CH ), 7.34 (m, 1H, Ar-H), 7,43 (d, 2H, Ar-H); In order to investigate the effects of compound 3f on cell
3
7
8
.74 (m, 1H, Ar-H), 7.77 (d, 2H, Ar-H), 8.32 (d, 1H, Ar-H), survival of PC3 and MCF7 cell line, cells were seeded in 24
.43 (s, 1H, Ar-H), 9.91 (s, 1H, CSNH-Ar), 10.02 (1H, well plated and incubated with compounds at a final con-
SO NH-NH-CS), 10.07 (bs, 1H, SO NH-NH-CS); 13C-NMR centration of IC50 in growth medium (without antibiotics)
2
2
(
75 MHz) (DMSO-d /TMS) d ppm: 21.53 (CH ), 123.22 (Ar- for 12 h, 24 h and 48 h. After the incubation, methylene blue
6 3
CH), 128.64 (2Ar-CH), 130.05 (2Ar-CH), 133.48 (Ar-CH), staining was used for cell survivals. The cells were extracted
35.24 (Ar-C), 136.16 (Ar-C), 144.41 (Ar-CH), 146.28 (Ar- with 1% SDS in PBS solution and stained with 0.01%
1
C), 147.25 (Ar-CH), 181.96 (C ¼ S); Anal. calcd for methylene blue solutions. The absorbance was measured at
C H N O S .1/4 C H OH (333.923): C, 48.56; H, 4.68; N, 600 nm with microplate reader. Each experiment was
1
3
14
4
2 2
2
5
1
6.78; S, 19.21 Found: C, 48.95; H, 4.99; N, 17.43; S, 19.71.
repeated three times.

6
S. S¸ ENKARDE S¸ ET AL.
Western blot analysis for protein expression
Conclusion
Cells were treated with final concentration of IC50 of synthe- In these work, new sulfonylthiosemicarbazide derivatives hav-
sized compound 3f in 6 well plates and incubated for 24 h. ing different substitution have been synthesized and their bio-
Cells were lysed using RIPA cell lysis buffer (89900, logic activities are investigated. Compound 3d with (4-
Thermo), and the protein concentrations were determined chlorophenyl)ethyl substitution exhibited the highest potency
by the BCA protein assay (Thermo Scientific). Samples were against PC3 cell line and 4-(trifluoromethoxy)phenyl substi-
denaturated with Laemli buffer and 25 lg protein was tuted compound 3f displayed good in vitro dose dependent
loaded to each well. The samples were resolved by 4–12% cytotoxic activity against PC3 and MCF7 cells. Compounds
sodium dodecyl sulfate–polyacrylamide gel electrophoresis 3d and 3f did not show any toxic effects on the L929 mouse
and were transferred to polyvinylidene fluoride membrane, fibroblasts cell line. The mechanistic studies showed that 3f
which was then blocked with 3% BSA (Capricorn Scientific, caused apoptosis of tumor cells possibly through regulation of
BSA-1T) in Tris buffered saline (TBS). The membrane was the expression levels of Bcl-2 and Bax proteins and a result of
washed twice in TBS containing Tween-20 (0.1%) and incu- the described.
bated overnight with primary antibodies (1:500 dilution
In study, an increased Bax/Bcl-2 ratio indicates the acti-
anti-Bax sc-7480, anti-Bcl2 sc-492, anti-GAPDH sc-25778, vation of mitochondrial apoptosis in the both of cells.
Santa Cruz Biotechnology, Heidelberg, Germany) and Morphological study suggested that IC50 concentration of
washed with TBS containing Tween-20 (0.1%). The mem- compound 3f changed cell morphology and induced cell
brane was washed and then incubated with horseradish per- death. Moreover, all compounds also exhibited promising
oxidase conjugated secondary antibody (anti-mouse; sc-2060 antioxidant activity against free radical production. A further
or anti-rabbit; sc-2004, Santa Cruz Biotechnology) for 2 h. study should be carried out on other cancer cell lines and
The blots were developed with chemiluminescence reagents there is a need to carry out in vivo antioxidant studies of
(sc2048, Santa Cruz Biotechnology, Texas, USA) and ana- this compounds.
lyzed with chemiluminescent imaging systems (Syngene,
Cambridge, UK). Data were analyzed using “Image J
Programme Optical Density Analysis Software” (NIH) by
Disclosure statement
measuring each band three times for quantification. Signals The authors have declared no conflict of interest.
were normalized with respect to GAPDH.
Funding
DPPH radical scavenging activity
The authors would like to thank TUBITAK [2209-A] for partially pro-
viding financial support (Project No:1919B011701846). This work was
supported by the Research Fund of Marmara University, project num-
ber: SAG-K-120917-0495.
Free radical scavenging capacity of compounds were eval-
uated according to the previously reported procedure using
[
34]
the stable DPPH.
Briefly, 10 ml of extracts in DMSO at
different concentrations were added to 190 ml methanol solu-
tion of DPPH (0.1 mM) in a well of 96-well plate. The mix-
ture was shaken vigorously and allowed to stand in the dark ORCID
at room temperature for 30 min. Absorbance readings were
Sevil S¸ enkarde ¸s
http://orcid.org/0000-0002-0523-459X
taken at 517 nm. The percent radical scavenging activity of
extracts and standard against DPPH were calculated accord-
ing to the following:
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