Synthesis and antiproliferative activities of some pyrazole-sulfonamide derivatives

Article abstract of DOI:10.1007/s00044-013-0721-2

In this study, a series of pyrazole-sulfonamide derivatives were designed and synthesized from 1-(4-aminophenyl)-4-benzoyl-5-phenyl-N-(5-sulfamoyl-1,3,4- thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (1). The newly synthesized sulfonamides were characterized

Full text of DOI:10.1007/s00044-013-0721-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1278–1289
DOI 10.1007/s00044-013-0721-2
ORIGINAL RESEARCH
Synthesis and antiproliferative activities of some
pyrazole-sulfonamide derivatives
•
•
˘
˘
Samet Mert Ays¸e S¸ahin Yaglıoglu
•
˘
Ibrahim Demirtas Rahmi Kasımogulları
Received: 20 February 2013 / Accepted: 14 August 2013 / Published online: 30 August 2013
Ó Springer Science+Business Media New York 2013
Abstract In this study, a series of pyrazole-sulfonamide
derivatives were designed and synthesized from 1-(4-ami-
nophenyl)-4-benzoyl-5-phenyl-N-(5-sulfamoyl-1,3,4-thia-
diazol-2-yl)-1H-pyrazole-3-carboxamide (1). The newly
synthesized sulfonamides were characterized by FT-IR, 1H
NMR, 13C NMR, and elemental analyses. The compounds
were tested for their in vitro antiproliferative activities
against HeLa and C6 cell lines. The tests were carried out
as dose-dependent assay starting from 5 to 100 lg/mL. All
compounds showed especially cell selective effect against
rat brain tumor cells (C6). Some of the tested compounds
(3) and (7) showed promising broad spectrum antitumor
activity comparable to the activities of the commonly used
anticancer drugs, 5-fluorouracil and cisplatin.
metastasis, which is the spreading of cells to other tissues
of body via lymph or blood (Alvarez and Besa, 2000; Gao
et al., 2010; Gediya and Njar, 2009; Li et al., 2012; Yilmaz
and Ayan, 2011). Various drugs are available for the
treatment of cancer but many of them have side effects.
Because chemotherapy drugs have a wide range of non-
specific effects, there is an urgent need to develop safe and
cost effective anticancer agents (Da Settimo et al., 1998).
Sulfonamides possess many types of biological activities
such as antibacterial (Drews, 2000), hypoglycemic (Boyd,
1988), diuretic (Supuran and Scozzafava, 2000; Maren,
1976), and antiglaucoma (Supuran and Scozzafava, 2001;
Kasimogullari et al., 2009). Recently, many structurally
novel sulfonamide derivatives have been reported to show
significant antitumor activity (Abbate et al., 2004; Ismail
et al., 2006; Supuran et al., 2004). Also pyrazole-contain-
ing compounds have received considerable attention owing
to their diverse chemotherapeutic potentials (Ghorab et al.,
2012; Rostom, 2010; Lv et al., 2010; Strocchi et al., 2012;
Faidallah et al., 2007). For example, Celecoxib is a non-
steroidal anti-inflammatory drug and selective COX-2
inhibitor which includes sulfonamide and pyrazole scaf-
folds in the same structure. Recently, some studies have
been reported about antitumor effect of this drug (Harris
et al., 2000; Trifan et al., 2002; Cianchi et al., 2006;
Masferrer et al., 2000; Shaik et al., 2006). Literature survey
showed that aryl/heteroaryl sulfonamides may act as anti-
tumor agents through several mechanisms (Al-Said et al.,
2010). The most remarkable mechanism is the inhibition of
carbonic anhydrase isoenzymes (CA) (Abbate et al., 2004;
Casini et al., 2002). Previously we studied about synthesis
and biological activity of some sulfonamide containing
pyrazole-3-carboxamides (Kasimogullari et al., 2011; Sen
et al., 2013). We observed many compounds have inhibi-
tion potential on human carbonic anhydrase isoenzymes
Keywords Sulfonamide Á Pyrazole Á HeLa cell lines Á
C6 cell lines Á Antiproliferative activity
Introduction
Cancer is one of the most fatal health problems faced by
mankind today (Sondhi et al., 2010; Byakodi et al., 2012;
Schwartz and Hulka, 1990; Giri et al., 2001; Landis et al.,
1999). This is a group of diseases in which a number of
cells display uncontrolled growth, invasion, and sometimes
˘
S. Mert Á R. Kasımogulları (&)
Department of Chemistry, Faculty of Arts and Sciences,
Dumlupinar University, 43100 Kutahya, Turkey
e-mail: rahmikasimoglu@hotmail.com
˘
˘
A. S¸. Yaglıoglu Á I. Demirtas
Department of Chemistry, Faculty of Science, Cankiri Karatekin
University, 18100 Cankiri, Turkey
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Med Chem Res (2014) 23:1278–1289
1279
(CA I and CA II). These findings prompted us to synthesis
some pyrazole-sulfonamide derivatives and determine their
antiproliferative effects as in vitro.
group of (1) (Kasimogullari et al., 2011). The resonances
between *7.80 and *6.60 ppm can be assigned to aro-
matic protons. Compound (5) characterized by two broad
exchangeable singlets in the 12.79 and 7.50 ppm due to
COOH and OH groups, respectively. In the 1H NMR
spectrum of (7), NH proton belonging to hydrazinyl group
(Ar–NH–N=C) was appeared at 11.51 ppm. Based on this
data we can say that compound (7) prefers hydrazo tauto-
meric structure instead of azo form (Ar–N=N–C). In gen-
eral, CONH protons appear at around *13.7 ppm.
Compound (8) also shows a CONH peak at 10.17 ppm
attributed to acetamide proton. The other peaks appeared at
the expected chemical shifts and integral values. In addi-
In this research, we have aimed to synthesize, charac-
terize, and determine the antiproliferative effects of various
sulfonamide derivatives of pyrazole-3-carboxylic acid
against some tumour cells [brain tumor cells (C6), uterus
cancer cells (Hela)] with in vitro studies. Four of the
compounds (2–4) and (6) described here previously syn-
thesized and characterized in our laboratory and their
inhibition effects were investigated on human CA I and CA
II isoenzymes (Kasimogullari et al., 2011). Compounds (5,
7–9) were synthesized in this study are novel and were not
previously synthesized. Structural elucidation of synthe-
sized compounds has been performed by means of 1H
NMR, 13C NMR, IR, and elemental analysis.
1
tion to the H NMR spectra, 13C resonance assignments
have also been carried out. The peaks of C=O for alkyl
ketones are observed around *196.00 ppm while the
peaks of Ph-C=O and O=C–NH are around *191.00 and
*165.00 ppm, respectively. There are two interesting
peaks at 162.88 ppm corresponding to the ester C=O
belonging to (7) and at 153.77 ppm corresponding to the
carbamate C=O of (9). The peak resonances at 177.55 ppm
corresponds to the acid carbonyl of (5).
Results and discussion
Chemistry
The synthetic route used to synthesize the target 1,3,4-
thiadiazole-2-sulfonamide containing pyrazole-3-carbox-
amides (1–9) is outlined in Scheme 1. First an indol deriv-
ative (2) prepared by the cyclization reaction of (1) with
chloroacetone. Then diazotization of (1) with sodium nitrite
followed by treatment with KI afforded (3). Also some
coupling products (4–7) were obtained by the condensation
of phenol, salicylic acid, and 1,3-dicarbonyl compounds
with the diazonium salt solution of (1). Finally acetamide (8)
and carbamate (9) derivatives were synthesized from the
direct reaction of (1) with acetyl chloride and ethyl chloro-
formate in the presence of triethylamine. Analytical and
spectral data (1H NMR, 13C NMR, IR, and elemental anal-
ysis) of the synthesized compounds were in full agreement
with the proposed structures. IR spectra of the target pyra-
zole-sulfonamides showed NH and C=O stretching bands at
3,264–3,184 and 1,736–1,656 cm-1, respectively. S=O;
asymmetric and symmetric stretching bands were observed
at around *1,365 and *1,170 cm-1, respectively. The
peak appearing only in the spectrum of (5) at 3,358 cm-1 is
attributed to characteristic OH stretching. Also IR spectrum
of (5) showed a broad absorption band from 2,500 to
3,500 cm-1 due to OH stretching of COOH. The absorption
bands associated with other functional groups appeared in
the expected regions and the absorption values were con-
sistent with our previous reports and literature (Sen et al.,
2013; Jennings and Lovely, 1991; Patel and Agravat, 2009;
Kasimogullari et al., 2010; Lodeiro et al., 2003).
Biological activity
Antiproliferation activities of (2–9) were determined
against human cervix carcinoma (HeLa) and rat brain
tumor cells (C6) using the BrdU cell proliferation ELISA
assay (Demirtas et al., 2009; Demirtas and Sahin, 2013).
5-Fluorouracil and cisplatin were used as standards. The
activities of samples and standards were investigated at
eight different concentrations (5, 10, 20, 30, 40, 50, 75, and
100 lg/mL). The IC50 and IC75 values of the most effective
compounds against HeLa and C6 were given in Table 1.
Antiproliferative activity of compound (2) against HeLa
and C6 cell lines
Activity studies showed that compound (2) demonstrates
antiproliferative activity against HeLa and C6 cell lines in
a concentration-dependent manner (Fig. 1b). However,
compound (2) exhibits weak antiproliferative activity as
compare with the standards. The potency of inhibition for
HeLa cells is in the order of; 5-FU * cisplatin [ 2 and for
C6; cisplatin [ 5-FU [ 2.
Antiproliferative activity of compound (3) against HeLa
and C6 cell lines
Compound (3) exhibits antiproliferative activity against
HeLa and C6 cell lines in a concentration-dependent
manner (Fig. 2a, b). In addition, compound (3) has better
Pyrazole-sulfonamides (2–9) were characterized by
disappearance of a broad singlet at 6.51 ppm due to NH2
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Med Chem Res (2014) 23:1278–1289
N
N
N
NH2
SO2
NH2
SO2
NH2
SO2
N
N
N
O
O
O
O
O
O
S
S
S
Ph
NH
Ph
NH
Ph
NH
e
a
N
N
N
Ph
Ph
Ph
N
N
N
R
8
-
H
C
3
R
N
HN
NH2
d
-
Et
O
9
H
C
3
1
b
O
O
H
3C
8 9
,
2
c
N
NH2
SO2
N
O
O
N
S
NH2
SO2
N
Ph
NH
O
O
N
S
S
NH2
SO2
N
Ph
NH
N
O
O
Ph
R1 R2
N
Ph
NH
N
N
Ph
6
7
-
-
-
Ph Ph
r
A
N
-
C
Et
O
H CH CH
3
Ph
2
2
N
H
H
O
4
HN
N
N
N
O
O
O
5
H
COO
I
R1
R2
r
A
3
4 5
,
7
6
,
Scheme 1 Synthetic route for the target compounds. Reagents and
conditions: (a) chloroacetone, DMF, 60 °C, 72 h; (b) NaNO2/HCl,
0 °C then KI; (c) NaNO2/HCl, 0 °C then phenol or salicylic acid;
(d) NaNO2/HCl, 0 °C then 1,3-dicarbonyl compounds; (e) CH3COCl
or EtOCOCl, (Et)3N, THF, reflux, 5 h
Antiproliferative activities of compounds (6) and (7)
against HeLa and C6 cell lines
activity against HeLa to compare with cisplatin and 5-FU
on 100 lg/mL concentration. Moreover, compound (3) has
usually shown weak activity at lower concentrations. The
activity of compound (3) was determined as cell selectively
against C6 cell line and was observed higher antiprolifer-
ative activity than standards at high concentrations
(40–100 lg/mL). The potency of inhibition for HeLa is in
the order of 5-FU * cisplatin [ 3 and for C6 3 [ cis-
platin [ 5-FU.
The antiproliferative activity of compound (7) and standard
compounds are almost the same values on 40–100 lg/mL
concentration against HeLa cells. Compound (6) exhibits
the best activity on 100 lg/mL concentration against HeLa
cells. The antiproliferative activities of compounds (6) and
(7) against HeLa and C6 cell lines are in a concentration-
dependent manner (Fig. 4a, b).
According to Fig. 4b, both compounds (6) and (7) dem-
onstrate strong activities against C6 to compare with cisplatin
and 5-FU (for compound (6) on 50–100 lg/mL and for
compound (7) on 30–100 lg/mL concentrations). The
potency of inhibition for HeLa is in the order of 5-FU *
cisplatin [ 7 [ 6 and for C6, 6 * 7 [ cisplatin[ 5-FU.
Antiproliferative activity of compounds (4) and (5) against
HeLa and C6 cell lines
Compounds (4) and (5) show antiproliferative activity
against HeLa and C6 cell lines in a concentration-depen-
dent manner (Fig. 3a, b). Compound (4) has high antipro-
liferative activity at a concentration of 100 lg/mL against
HeLa. However, the other doses does not affect any
activities. Compounds (4) and (5) have higher activities
than standards against C6 cell lines on 100 lg/mL. In
addition, compound (4) also exhibits strong activity at the
concentration of 75 lg/mL against C6 cell lines. At the
same time, compound (4) has cell selective effect against
C6. The potency of inhibition for HeLa is in the order of
5-FU * cisplatin [ 4 [ 5 and for C6 cisplatin [ 5-
FU * 4 [ 5.
Antiproliferative activities of compounds (8) and (9)
against HeLa and C6 cell lines
Compound (8) exhibits weak activity against HeLa and C6
(Fig. 5a, b). Compound (9) is also exhibits weak activity
against HeLa, but the same compound shows strong activity
against C6 to compare with cisplatin and 5-FU (on
75–100 lg/mL concentrations). The potency of inhibition
for HeLa is in the order of 5-FU * cisplatin [ 9 [ 8 and for
C6, 9 [ cisplatin [ 5-FU [ 8.
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1281
Table 1 IC50 and IC75 values of pyrazole-sulfonamide derivatives (2–9)
N
NH
SO2
N
2
O
O
N
S
NH
Ph
Ph
N
R
Compounds
R
Hela
C6
IC50 (mM)
76.94
IC75 (mM)
91.61
IC50 (mM)
IC75 (mM)
86.21
2
70.07
N
H
C
3
O
H
3C
3
4
78.41
85.98
101.41
111.32
21.26
35.13
52.40
62.00
I
H
H
O
N
N
COO
5
6
118.92
67.09
126.91
92.73
84.72
45.31
101.82
64.16
N
H
O
N
O
Ph
Ph
N
NH
O
O
7
40.71
59.97
33.60
53.56
N
NH
Et
O
O
O
8
9
155.24
83.13
143.33
105.46
104.78
46.81
120.90
69.70
H
C
3
NH
O
Et
O
NH
123

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Med Chem Res (2014) 23:1278–1289
Fig. 1 Antiproliferative
activity of (2) and standards
against HeLa (a) and C6 (b) cell
line. Asterisks each substance
was tested twice in triplicates
against cell lines. Data show
average of two individual
experiments (p \ 0.01)
Fig. 2 Antiproliferative
activity of (3) and standards
against HeLa (a) and C6 (b) cell
line. Asterisks each substance
was tested twice in triplicates
against cell lines. Data show
average of two individual
experiments (p \ 0.01)
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1283
Fig. 3 Antiproliferative
activity of (4–5) and standards
against HeLa (a) and C6 (b) cell
line. Asterisks each substance
was tested twice in triplicates
against cell lines. Data show
average of two individual
experiments (p \ 0.01)
Fig. 4 Antiproliferative
activity of (6–7) and standards
against HeLa (a) and C6 (b) cell
line. Asterisks each substance
was tested twice in triplicates
against cell lines. Data show
average of two individual
experiments (p \ 0.01)
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Med Chem Res (2014) 23:1278–1289
Fig. 5 Antiproliferative
activity of (8–9) and standards
against HeLa (a) and C6 (b) cell
line. Asterisks each substance
was tested twice in triplicates
against cell lines. Data show
average of two individual
experiments (p \ 0.01)
Fig. 6 Antiproliferative
activity of (2–9) and standards
against HeLa (a) and C6 (b) cell
line. Asterisks each substance
was tested twice in triplicates
against cell lines. Data show
average of two individual
experiments (p \ 0.01)
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1285
Table 2 Selectivity index of pyrazole-sulfonamide derivatives (2–9), cisplatin and 5-FU
Compound names
Selectivity
2
3
4
5
6
7
8
9
Cisplatin
5-FU
100
75
50
40
30
20
10
5
25
21
86
86
72
63
45
46
47
10
84
81
54
50
40
32
20
12
77
24
88
84
63
56
42
9
88
88
86
85
72
30
-6
47
86
74
52
37
32
16
4
58
69
63
62
55
58
7
55
53
44
52
55
53
52
26
0.90
1.00
0.90
1.00
0.90
1.00
1.00
0.90
-2
-10
10
-25
-21
-35
-39
-41
-22
-47
-40
-37
-52
-44
-6
-2
-16
-19
2
-11
-17
-22
2
6
The comparison of antiproliferative activities
(2009) determined that carbamate derivatives are more
effective than acetamide derivatives against tumour CHO
cells. In this study we found the carbamate derivative (9) has
more antiproliferative activity than acetamide derivative (8)
against HeLa and C6 cell lines (carbamate deriva-
tive [ acetamide derivative, 83.13, 155.24 for HeLa cell line
and 46.81, 104.78 for C6 cell line, respectively) (Table 1).
The selectivity index values are given below (Table 2).
of compounds (2), (9) and related structures against HeLa
and C6 cell lines
According to Fig. 6a, b, all compounds have cell selective
effect against C6. However, compound (7) has only con-
siderable antiproliferative activities against HeLa to com-
pare with cisplatin and 5-FU. The potency of inhibition for
HeLa is in the order of 5-FU [ cisplatin [ 7 [ 3 [ 4 [
6 [ 9 [ 2 [ 5 [ 8 and for C6, 7 [ cisplatin [ 5-FU [
3 [ 4 [ 6 * 9 [ 5 [ 8 [ 2.
Experimental protocols
The structure–activity relationships (SAR) can be drawn
with a comparison between functional groups of the syn-
thesized compounds. Compound (3) contains an iodine atom
in the para position of the phenyl ring as different from the
other compounds. According to the in vitro studies it seems
that electron-donating resonance effect of this halogen atom
contributes to the antiproliferative activity (Fig. 6a, b). This
is consistent with the other results from the literature (Hou
et al., 2011; Parthiban et al., 2011). The results of the activity
study by Parthiban et al. (2011) indicate that halogen groups
have more effect than hydrogen atoms (Br [ Cl [ F [ H;
IC50 25.02, 48.97, 49.18, 112.72 against HeLa. Both (4) and
(5) consist phenolic hydroxyl groups. These phenolic
hydroxyl groups may be responsible for enhancement of
inhibitory activity. But compound (5) also includes a car-
boxylate group next to the hydroxyl group. We suppose that
carboxylate group decreases the activity. Furthermore, eth-
oxy groups existing at compounds (7) and (9) contribute
substantially to the antiproliferative activity higher than
methyl groups (at compound 8). Parthiban et al., (2011)
observed increase in the antiproliferative activity hydrogen
to isopropyl groups (isopropyl groups [ ethyl group-
s [ methyl groups [ hydrogen atoms; 49.54, 57.27, 112.72,
120.68 are the IC50 values of alkyl groups against HeLa,
respectively). These results are all consistent with our results
related to compounds (7) and (9). Also Benchabane et al.
Materials and methods
Chemical compounds used in this research were at ana-
lytical purity, and the solvents were purified by using
appropriate purifying agents and distillation. Tetrahydro-
furan (THF) was distilled from sodium/benzophenone prior
to use. All reactions were monitored by analytical thin-
layer chromatography (TLC) on 0.25 mm precoated Kie-
selgel 60F 254 plates (E. Merck Co., Darmstadt, Germany)
and compounds were visualized by TLC devices (Camag,
Upland, CA, USA) UV (254 and 366 nm). Melting points
(°C, uncorrected) were determined in open capillaries on a
Barnstead Electrothermal 9200 melting point apparatus
(Electrothermal Co, Essex, UK). Infrared (IR) spectra were
recorded on Bruker Optics, Vertex 70 Fourier Transform
Infrared Spectrometer (FT-IR) equipped with an Attenu-
ated Total Reflection (ATR) device and the data were
reported in reciprocal centimeters (cm-1) (Bruker Optik
GmbH, Ettlingen, Germany). 1H NMR and 13C NMR
spectra were recorded on BRUKER DPX-400 (400 MHz
1
for H NMR and 100 MHz for 13C NMR) spectrometers
(Bruker BioSpin GmbH, Rheinstetten, Germany). Ele-
mental analyses (C, H, and N) were performed on a Leco
CHNS-932 elemental analyser (LECO Corporation, Saint
Joseph, Michigan, USA).
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Med Chem Res (2014) 23:1278–1289
Synthesis of target molecules
(100 MHz, DMSO) d (ppm): 190.59 (C=O, benzoyl),
177.55 (C=O, acid), 165.24 (C=O, amide), 161.64 and
160.58 (thiadiazol C-2 and C-5), 144.46, 143.29, 138.63,
137.79, 134.80, 133.98, 132.14, 130.55, 130.31, 129.97,
129.78, 129.60, 129.33, 129.04, 128.99, 128.75, 128.15,
127.97, 127.35, 126.11, 122.88; anal. calcd. for
C32H22N8O7S2: C, 55.33; H, 3.19; N, 16.13; S, 9.23; found:
C, 55.25; H, 3.10; N, 16.18; S, 9.21.
Compound (1) was previously prepared and it was used as
a starting compound in this study (Kasimogullari et al.,
2011). Also the compounds 4-benzoyl-1-(2-methyl-1-(2-
oxopropyl)-1H-indol-5-yl)-5-phenyl-N-(5-sulphamoyl-1,3,
4-thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (2), 4-ben-
zoyl-1-(4-iodophenyl)-5-phenyl-N-(5-sulphamoyl-1,3,4-
thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (3), 4-benzoyl
-1-(4-((4-hydroxyphenyl)diazenyl)phenyl)-5-phenyl-N-(5-sul-
phamoyl-1,3,4-thiadiazol -2-yl)-1H-pyrazole-3-carboxamide
(4), and 4-benzoyl-1-(4-(2-(1,3-dioxo-1,3-diphenylpropan-2-
ylidene)hydrazinyl)phenyl)-5-phenyl-N-(5-sulphamoyl-1,3,
4-thiadiazol-2-yl)-1H-pyrazole-3-carboxamide (6) were
synthesized and characterized in our previous work
(Kasimogullari et al., 2011).
Ethyl 2-(2-(4-(4-benzoyl-5-phenyl-3-(5-sulfamoyl-1,3,4-
thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-
yl)phenyl)hydrazono)-3-oxohexanoate (7)
Ethyl butyryl acetate (0.16 ml, 1 mmol) was dissolved in
ethanol and cooled to 0–5 °C and added dropwise to the
diazonium salt solution of (1) which was prepared according
to the general procedure. The precipitated bright yellow
product was filtered and purified from ethanol. 550 mg,
77 %; mp 162–164 °C; IR (m, cm-1): 3184 (NH), 3003 (Ar
CH), 2968 (aliphatic CH), 1677 and 1656 (C=O), 1597–1449
(Ar C=C and C=N), 1368 and 1170 (S=O, assym. and sym.);
1H NMR (400 MHz, DMSO) d (ppm): 13.69 (br, s, 1H,
CONH), 11.51 (s, 1H, Ar–NH–N=), 8.36 (s, 2H, SO2NH2),
7.79–7.22 (m, 14H, ArH), 4.32 (q, J = 7.1 Hz, 2H, OCH2),
2.84 (t, J = 7.2 Hz, 2H, CH2CH2CH3), 1.59 (hextet,
J = 7.4 Hz, 2H, CH2CH2CH3), 1.29 (t, J = 7.0 Hz, 3H,
OCH2CH3), 0.91 (t, J = 7.4 Hz, 3H, CH2CH2CH3); 13C
NMR (100 MHz, DMSO) d (ppm): 196.46 and 190.58 (C=O,
ketone), 165.27 (C=O, amide), 162.88 (C=O, ester), 161.53
and 160.51 (thiadiazol C-2 and C-5), 61.73 (OCH2), 39.04
(CH2CH2CH3), 17.86 (CH2CH2CH3), 14.34 and 14.14
(CH3) 144.48, 143.30, 143.14, 137.76, 133.97, 132.77,
130.28, 129.93, 129.58, 129.03, 128.97, 127.93, 127.57,
122.77, 116.68, 115.57; anal. calcd. for C33H30N8O7S2: C,
55.45; H, 4.23; N, 15.68; S, 8.97; found: C, 55.34; H, 4.18; N,
15.71; S, 8.93.
General procedure for the syntheses of compounds (3)–(7)
1-(4-Aminophenyl)-4-benzoyl-5-phenyl-N-(5-sulfamoyl-
1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-carboxamide
(1)
(0.545 g, 1 mmol) was dissolved in a mixture of ethanol
(50 ml) and concentrated hydrochloric acid (8 ml) and the
solution was then cooled to 0–5 °C. Sodium nitrite
(0.083 g, 1.2 mmol) in water (10 ml) was then added to
this solution dropwise with vigorous stirring while cooling
at 0–5 °C. After dissolving an aromatic or b-dicarbonyl
compound (1 mmol) in a sufficient amount of ethanol
(1 mmol KI was dissolved in water for compound 3), the
solution was cooled and added dropwise into the already
prepared diazonium salt solution. The pH of the mixture, in
each case, was maintained at 7–8 through the coupling
process by adding aqueous sodium acetate. Stirring was
continued for 1 h at 0–5 °C and 2 h at room temperature.
The precipitated products were filtered off, washed with
water several times, dried, and recrystallized from an
appropriate solvent (see Scheme 1).
1-(4-Acetamidophenyl)-4-benzoyl-5-phenyl-N-(5-
sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3-
carboxamide (8)
5-((4-(4-Benzoyl-5-phenyl-3-(5-sulfamoyl-1,3,4-thiadiazol-
2-ylcarbamoyl)-1H-pyrazol-1-yl) phenyl)diazenyl)-2-
hydroxybenzoic acid (5)
To a stirring solution of (1) (0.545 g, 1 mmol) and trieth-
ylamine (0.138 ml, 1 mmol) in dry THF (30 ml), acetyl-
chloride (0.071 ml, 1 mmol) was added dropwise and the
reaction mixture was refluxed for 5 h. After removal of the
solvent, the crude product was obtained which was further
purified by recrystallization from ethanol–water (1:1)
mixture. 541 mg, 92 %; mp 188–190 °C; IR (m, cm-1):
3264 and 3198 (NH), 3026 (Ar CH), 2970 (aliphatic CH),
1691 and 1668 (C=O), 1605–1433 (Ar C=C and C=N),
1366 and 1167 (S=O, assym. and sym.); 1H NMR
(400 MHz, DMSO) d (ppm): 13.77 (br, s, 1H, CONH),
10.17 (br, s, 1H, CONHAc), 8.34 (s, 2H, SO2NH2),
Salicylic acid (0.138 g, 1 mmol) was dissolved in ethanol
and cooled to 0–5 °C and added dropwise to the diazonium
salt solution of (1) which was prepared according to the
general procedure. The precipitated yellow product was
filtered and purified from ethanol. 431 mg, 62 %; mp
212–214 °C; IR (m, cm-1): 3600–2800 (COOH), 3358
(OH), 3249 (NH), 3061 (Ar CH), 1689 (C=O), 1598–1449
(Ar C=C and C=N), 1363 and 1172 (S=O, assym. and
1
sym.); H NMR (400 MHz, DMSO) d (ppm): 13.72 (br, s,
1H, CONH), 12.79 (s, 1H, COOH), 8.29 (s, 2H, SO2NH2),
7.50 (s, 1H, OH), 7.80–7.24 (m, 17H, ArH); 13C NMR
123

Med Chem Res (2014) 23:1278–1289
1287
7.79–7.18 (m, 14H, ArH), 2.06 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO) d (ppm): 190.61 (C=O, benzoyl),
169.19 and 165.29 (C=O, amide), 161.40 and 160.42
(thiadiazol C-2 and C-5), 24.53 (CH3), 144.46, 142.92,
140.24, 137.66, 134.04, 133.45, 130.25, 129.91, 129.61,
129.08, 128.96, 127.88, 126.88, 122.69, 119.31; anal.
calcd. for C27H21N7O5S2: C, 55.19; H, 3.60; N, 16.69; S,
10.91; found: C, 55.05; H, 3.55; N, 16.70; S, 10.86.
(DMEM, Sigma), supplemented with 10 % (v/v) fetal
bovine serum (Sigma, Germany) and PenStrep solution
(Sigma, Germany) at 37 °C in a 5 % CO2 humidified
atmosphere. For proliferation assay, cells were plated in
96-well culture plates (COSTAR, Corning, USA) at a
density of 30,000 cells per well. Vehicle (DMSO), 5-flu-
orouracil, cisplatin, and the samples in various concentra-
tions (0–100 lg/mL) were added to each well. Cells were
than incubated for overnight before applying the BrdU cell
proliferation ELISA assay reagent (Roche, Germany)
according to manufacturer’s procedure. In brief, cells were
pulsed with BrdU labeling reagent for 4 h followed by
fixation in FixDenat solution for 30 min at room temper-
ature. Thereafter, cells were incubated with 1:100 dilution
of anti-BrdU–POD for 1.30 h at room temperature. The
amount of cell proliferation was assessed by determining
the 450 nm of the culture media after addition of the
substrate solution by using a microplate reader (Ryto,
China). Results were reported as percentage of the inhibi-
tion of cell proliferation, where the optical density mea-
sured from vehicle-treated cells was considered to be
100 % of proliferation. All assays were repeated at least
twice using against HeLa and C6 cells. Percentage of
inhibition of cell proliferation was calculated as follows:
[1 - (Atreatments/Avehicle control) 9 100.
Ethyl 4-(4-benzoyl-5-phenyl-3-(5-sulfamoyl-1,3,4-
thiadiazol-2-ylcarbamoyl)-1H-pyrazol-1-
yl)phenylcarbamate (9)
Compound (9) was synthesized from (1) (0.545 g, 1 mmol),
triethylamine (0.138 ml, 1 mmol), and ethylchloroformate
(0.095 ml, 1 mmol) as described for compound (8). The
crude product was recrystallized from ethanol. 550 mg,
89 %; mp 128–130 °C; IR (m, cm-1): 3235 (NH), 3003 (Ar
CH), 2972 (aliphatic CH), 1736 (C=O), 1602–1417 (Ar
C=C and C=N), 1367 and 1169 (S=O, assym. and sym.); 1H
NMR (400 MHz, CDCl3) d (ppm): 11.57 (br, s, 2H,
2xCONH), 7.81 (s, 2H, SO2NH2), 7.58–6.60 (m, 14H,
ArH), 4.23 (q, J = 6.5 Hz, 2H, OCH2), 1.28 (t, J = 7.1 Hz,
3H, CH3); 13C NMR (100 MHz, CDCl3) d (ppm): 191.77
(C=O, benzoyl), 164.68 (C=O, amide), 161.40 and 158.55
(thiadiazol C-2 and C-5), 153.77 (C=O, carbamate), 61.55
(OCH2), 14.50 (CH3), 144.51, 141.56, 138.87, 137.02,
133.77, 133.18, 133.04, 129.55, 129.77, 128.63, 128.46,
127.27, 125.84, 122.45, 118.86; anal. calcd. for
C28H23N7O6S2: C, 54.45; H, 3.75; N, 15.87; S, 10.38;
found: C, 54.31; H, 3.69; N, 15.91; S, 10.32.
Statistical analysis
The results of investigation in vitro are the mean SEM
of six measurements for each cell type. Differences
between treatment groups were tested way ANOVA and
p values of \0.01 and 0.05 were considered significant.
Chemicals for biological activities
Determination of IC50 and IC75 values
Cell proliferation ELISA, BrdU (colorimetric) kits were
obtained from Roche Diagnostics GmbH (Mannheim,
Germany). The antitumour drug 5-fluorouracil was pro-
vided from Sigma. Other antiproliferative chemicals used
were in analytical grade and obtained from Sigma-Aldrich,
Merck or Roche.
The half maximal inhibitory concentration (IC50) is a
measure of the effectiveness of a compound in inhibiting
biological function. In this paper, IC50 and IC75 values
were determined using ED50 plus v1.0.
Conclusions
Preparation of sample solutions
The compounds tested in this work have cell selective
effects against C6 cell line (p \ 0.01). According to the
results compound (7) especially have considerable anti-
proliferative activities against both Hela and C6 cell lines
to compare with cisplatin and 5-FU. Compounds (3) and
(4) have good antiproliferative activity against C6 at high
concentrations. Also (3) and (4) tested for the inhibition
over human cytosolic isozymes (CA I and CA II) in our
previous work as in vitro (Kasimogullari et al., 2011).
Interestingly, these two compounds showed the greatest
Stock solutions of the samples, 5-fluorouracil, cisplatin
were dissolved in DMSO and diluted Dulbecco’s modified
Eagle’s medium (DMEM; 1:20). The final concentration of
DMSO is below 1 % in all tests.
Cell culture and cell proliferation assay
HeLa (human cervix carcinoma) and C6 (rat brain tumor)
cells were grown in Dulbecco’s modified Eagle’s medium
123

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Med Chem Res (2014) 23:1278–1289
Gao J, Chen K, Xie R, Xie J, Yan Y, Cheng Z, Peng X, Chen X
(2010) In vivo tumor-targeted fluorescence imaging using near-
infrared non-cadmium quantum dots. Bioconj Chem 21:604–609
Gediya LK, Njar VCO (2009) Promise and challenges in drug
discovery and development of hybrid anticancer drugs. Expert
Opin Drug Dis 4:1099–1111
effect over CA I and CA II between the tested compounds.
According to the results these compounds (3), (4), and (7)
may seen as candidate for the inhibition of the tumor-
associated isozyme CA IX which is a recently discovered
member of the CA gene family.
Ghorab MM, Ragab FA, Heiba HI, Youssef HA, El-Gazzar MG
(2012) Synthesis of novel pyrazole and pyrimidine derivatives
bearing sulfonamide moiety as antitumor and radiosensitizing
agents. Med Chem Res 21:1376–1383
Acknowledgments This study was financially supported by the
grants from the Scientific and Research Council of Turkey (TUBI-
TAK 106T180) and the State Planning Organization (DPT
2003K120510), Turkey.
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