Synthesis and anticancer activity of acyl thioureas bearing pyrazole moiety

Article abstract of DOI:10.1016/j.bmc.2013.04.021

In this work novel organic based compounds, acyl thiourea derivatives were synthesized and their anticancer activities were investigated. A new series of acyl thiourea derivatives containing pyrazole ring were prepared in good yield through one pot reaction of 4-benzoyl-1, 5-diphenyl-1H-pyrazole-3-carbonyl chloride with ammonium thiocyanate and various amines. The structures of the newly synthesized compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis. Anticancer activities of synthesized compounds were evaluated on human colon, liver and leukemia cancer cell lines. Cell culture studies have demonstrated significant toxicity of the compounds on the cell lines, and the levels of toxicity have altered in the presence of various side groups. These results confirm that novel pyrazolyl acyl thioureas derived compounds may be utilized for cancer treatment. Furthermore, these compounds have a great potential and significance for further investigations.

Full text of DOI:10.1016/j.bmc.2013.04.021

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anticancer activity of acyl thioureas bearing pyrazole
moiety
a,
b
b
c
⇑
_
Irfan Koca , Aykut Özgür , Kübra Açikalin Coßskun , Yusuf Tutar
a Bozok University, Faculty of Arts and Sciences, Department of Chemistry, Yozgat, Turkey
b Gaziosmanpaßsa University, Faculty of Natural Sciences and Engineering, Department of Bioengineering, Tokat, Turkey
c Cumhuriyet University, Faculty of Pharmacy, Department of Biochemistry, Sivas, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work novel organic based compounds, acyl thiourea derivatives were synthesized and their anti-
cancer activities were investigated. A new series of acyl thiourea derivatives containing pyrazole ring
were prepared in good yield through one pot reaction of 4-benzoyl-1, 5-diphenyl-1H-pyrazole-3-
carbonyl chloride with ammonium thiocyanate and various amines. The structures of the newly synthe-
sized compounds were confirmed by IR, 1H NMR, 13C NMR and elemental analysis. Anticancer activities of
synthesized compounds were evaluated on human colon, liver and leukemia cancer cell lines. Cell culture
studies have demonstrated significant toxicity of the compounds on the cell lines, and the levels of
toxicity have altered in the presence of various side groups. These results confirm that novel pyrazolyl
acyl thioureas derived compounds may be utilized for cancer treatment. Furthermore, these compounds
have a great potential and significance for further investigations.
Received 22 January 2013
Revised 2 April 2013
Accepted 6 April 2013
Available online xxxx
Keywords:
Pyrazole
Acyl thiourea
Anticancer activity
Liver
Ó 2013 Elsevier Ltd. All rights reserved.
Colon
Leukemia cell lines
1. Introduction
GTPases (Fig. 1).18 A new series of acylthiourea analogs inhibited
microsomal epoxide hydrolase (mEH) is a liver enzyme.19 In recent
Today, cancer is one of the biggest health problems in the
world. Especially, the number of deaths from leukemia, colon,
and liver cancer is increasing gradually worldwide. Pharmaceutical
industries spend billions of dollars for the development of effective
agents in the diagnosis and treatment of cancer each year. There-
fore, many organic based cytotoxic agents have been discovered,
and they are extensively applied for treatment of cancer.1,2
Acyl thiourea derivatives have been increasingly important with
a wide diversity of applications in heterocyclic chemistry, metal
complexes, molecular electronics and exhibit an array of biological
activities.3–7 Some of them are employed as fungicidal, antiviral,
antimicrobial,8 parasiticidal,9 antitumoral10 and pesticidal
agent.11–13 Acyl thiourea compounds cambinol and tenovin-1 are
small molecule inhibitors of the NAD+-dependent family of protein
deacetylases known as the sirtuins (Fig. 1). There is considerable
interest in inhibitors of this enzyme family due to possible applica-
tions in both cancer and neurodegenerative disease therapy.14–17
2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno
[2,3-c] pyran-3-carboxylic acid (PubChem CID 1067700) has been
reported as an inhibitor of nucleotide binding by Ras-related
years, You et al. reported that some acylthiourea compounds
showed HIV inhibition activity.20
The chemistry of pyrazole derivatives have been the subject of
medical research due to the various biological and pharmacological
properties such as antitumor,21 antimicrobial,22,23 anti-inflamma-
tory,24 antiviral,25 antifungal,26 anticonvulsant,27 analgesic
agents.28 Anticancer effects of pyrazole was first discovered half a
century ago with its high toxicity even at 0.15 mmol/kg/day
dose.29,30 In order to overcome this effect, 1-carboxamidopyrazole
and 1-thiocarbamoylpyrazole were synthesized. They showed
significant anticancer effects on animal experiments, but failed to
pass the clinical evaluation.31,32 In order to find better antitumor
agent, a large series of pyrazole derivatives were synthesized and
tested over the years. So the use of this powerful pharmacophore
is still very popular.33–35
In the present study, we synthesized novel pyrazolyl acyl thio-
ureas by the addition of various aromatic amines to 4-benzoyl-1,5-
diphenyl-1H-pyrazole-3-carbonyl isothiocyanate. The structures of
the synthesized compounds were characterized by IR, 1H NMR, 13
C
NMR and elemental analysis studies. Anticancer potential of com-
pounds was examined in human leukemia, colon, and liver cancer
cell lines. Our compounds may offer new agents for treatment of
leukemia, colon and liver cancer.
⇑
Corresponding author. Tel.: +90 354 242 1021; fax: +90 354 242 1022.
E-mail address: i_koca@yahoo.com (Irfan Koca).
_
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.021
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Irfan Koca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
(untreated cells).38,39 In order to determine the inhibitory effects
of compounds, the cell viability of control has been accepted as
100%, and the other results were proportioned according to the
control value.
2. Results and discussion
2.1. Chemistry
According to the results of cell culture studies, all compounds
showed antitumor activity at the two extreme concentrations
(10À4 and 10À5 M). Moreover, degree of the compound inhibition
level is proportional to the incubation time for 24–48 h time
frame. The results of these two concentrations were statistically
significant.
In the present work, one pot synthesis of pyrazolyl acyl thio-
ureas were accomplished as described in Scheme 1. The starting
material 4-benzoyl-1,5-diphenyl-1H-pyrazole-3-carbonyl chloride
(1) were prepared as described earlier.36
The synthesis began by treating 1 with ammonium thiocyanate.
The resulting acyl isothiocyanate was not isolated from the
mixture and was converted into the corresponding acyl thiourea
(3) by adding various amines. The reaction was carried out under
reflux condition. All crude products can be purified by column
chromatography. The target compounds were obtained in 62–
79% yields (Table 1). All the structures of newly synthesized com-
pounds were elucidated on the basis of their elemental analysis
and spectroscopic data.
The tested compound 3a, 3b, 3d, 3h, 3j and 3k showed signifi-
cant inhibition over 50% on the Jurkat cells at 24 h and 48 h (Fig. 2).
However, compound 3a, 3d and 3f showed anti-tumor activity at
24 h. Moreover, the results of compound 3c and 3e are not signif-
icant statistically. All of the compounds but 3i showed significant
anti-cancer activity at 24 h and 48 h in DLD-1 cells (Fig. 3) as
HepG2 cells. Overall, compounds inhibit HepG2 cells better than
DLD-1 and inhibit DLD-1 cells better than Jurkat cells since com-
pounds 3c, 3e, 3g, 3i, and 3k have weak anticancer activity
(Fig. 2). Compound 3i has the lowest anticancer activity among
the compounds in this study. 3i compound cannot adequately inhi-
bit DLD-1 cancer cells and ninety five percent of the cells survive in
the presence of this compound (Fig. 3). Similarly, this set of com-
pounds share significant anticancer activity in HepG2 cells (Fig. 4).
Side group of the compounds is one of the most important fac-
tors in the emergence of anticancer effect. Generally, the addition
of phenyl groups is excessively increased the anticancer effect of
compounds.30 Therefore, all of the compounds have greatly shown
anticancer activity for colon, leukemia and liver cancer cells. Com-
pounds 3e, 3f and 3g have different halogenic side groups (FÀ, ClÀ
and BrÀ respectively), but they display approximately similar anti-
cancer profiles. Compound 3a (phenyl group with no halogenic
side chain) has higher anticancer activity than compound 3e, 3f,
and 3g during 24 hours in Jurkat cell line. Out of 3e, 3f, and 3g,
3f displays similar activity to 3a. The electronegativity of F (3e)
and bulky group of Br (3g) as side chain most likely perturb the
anticancer activity of phenyl compound (3a). The Cl side group of
3f presumably shows optimal positioning and do not perturb the
activity of its phenylic body. Side chain length may enhance this
optimal positioning as revealed by 3h. Extra ring on phenylic com-
pound (3h) which cause steric hindrance may or may not facilitate
optimal positioning. 3h compound increases anticancer activity in
Jurkat cells while the same compound decreases anticancer
The IR spectra of 3a showed absorption band at 3368 cmÀ1 due
to the presence of the NH group. The absorptions of C@O and C@S
groups were observed at 1682, 1665 and 1176 cmÀ1 respectively.
The medium strong C@O band in the IR spectra of all the com-
pounds appeared at 1720–1620 cmÀ1, which is lower than that of
the ordinary carbonyl absorption (1730–1650 cmÀ1). The forma-
tion of H-bond leads an increase of their polarity, so the strength
of their double bond decreased, and absorption moved to lower
wave number.37 The 1H NMR spectra of 3a exhibited two broad sig-
nals at 12.06 and 10.02 ppm, which were assigned the two N–H
protons. The signals of aromatic H-atoms were observed as multi-
plets between 7.88 and 7.16 ppm. 13C NMR for 3a showed peaks at
d 190.99, 159.97 ppm for C@O and 178.07 ppm for C@S. C@C and
C@N signals arised at 144.52–123.10 ppm. 13C NMR spectra of
3b–3k were similar to those of 3a, except for the signals of the sub-
stituent at phenyl ring, which exhibited characteristic resonances
with appropriate chemical shifts.
2.2. Anticancer activity of new pyrazole derivatives
In this study, the anticancer activity of the 11 synthesized pyr-
azolyl acyl thiourea derivatives has been evaluated on human can-
cer cell lines, representing liver, leukemia and colon cancer (Table
2). Cells were exposed to five concentrations ranging from 10À4 to
10À8 M compounds. After 24 and 48 h, a XTT test protocol was
applied, and the results were compared with the control cells
Figure 1. Structure of some bioactive acyl thiourea derivatives.
Scheme 1. The synthesis of pyrazolyl acyl thioureas.
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Irfan Koca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Table 1
Some properties of synthesized compounds 3a–k
S
R
O
O
O
O
N
N
H
Ph
Ph
Cl
Ph
Ph
N
+
+
R-NH2
2
H
NH4NCS
N
N
N
Ph
Ph
1
3
Entry
R
Mp (°C)
Mol. Formula (Mol. wt.)
Yields
Micro analyses
Calcd (%)
Found (%)
C
H
71.69
4.41
71.61
4.46
C
30H22N4O2S
3a
3b
3c
3d
3e
3f
206
216
145
199
218
212
204
184
245
227
219
79
75
72
65
72
69
73
68
70
71
62
N
S
11.15
6.38
10.88
6.32
502.59
C
H
N
S
72.07
4.68
10.85
6.21
71.82
4.82
10.55
6.23
C
31H24N4O2S
H3C
516.61
C
H
N
S
72.07
4.68
10.85
6.21
71.83
4.65
10.58
6.11
C
31H24N4O2S
CH3
516.61
C
H
N
S
69.91
4.54
10.52
6.02
69.70
4.41
10.26
5.94
C
31H24N4O3S
H3CO
F
532.61
C
H
N
S
69.22
4.07
10.76
6.16
69.13
4.22
10.48
6.16
C
30H21FN4O2S
520.58
C
H
N
S
67.09
3.94
10.43
5.97
66.90
4.21
10.13
5.82
C
30H21ClN4O2S
Cl
537.03
C
H
N
S
61.97
3.64
9.64
5.51
62.24
3.92
9.54
5.28
C
30H21BrN4O2S
3g
3h
3i
Br
581.48
C
H
N
S
73.89
4.38
10.14
5.80
74.09
4.62
9.92
5.77
C
34H24N4O2S
552.65
C
H
N
S
68.24
4.25
12.84
5.88
68.02
4.06
12.69
5.51
C
31H23N5O3S
H2NOC
545.61
C
H
N
S
72.07
4.68
10.85
6.21
71.94
4.64
10.62
6.13
C
31H24N4O2S
3j
516.61
N
N
C
H
N
S
66.65
4.00
16.66
6.36
66.37
3.78
16.32
6.33
C
28H20N6O2S
3k
504.56
activity at HepG2 cell lines. However, there is no significant impact
of the halogenic side groups to the anticancer activity on the other
cell lines used in this study.
at doses of 10À5 M concentration in Jurkat cell lines. This unex-
pected behavior may be explained as activation of transcription
factor(s) of drug resistance mechanism at low doses of compounds
as cited in the literature. Continuing proliferation of cells was also
observed on cervical carcinoma cells as Jurkat cells.40 In this study,
it was shown that low doses of doxorubicin induce nuclear
Interestingly, addition of a single carbon to 3a (which forms 3j)
increases anticancer activity significantly at DLD-1 cell lines
(Fig. 3). 3a (10À4 M), 3b (10À4 M), 3d (10À4 M), 3h(10À4, 10À5 M),
and 3j (10À4 M, 10À5 M) showed significant anticancer activity in
Jurkat cell lines. The effect of different compounds to different
types of cancer cell lines shows the importance of chemical
structure on the cellular activity. It is likely that the pathway
causing carcinoma at each cell line is different from each other;
thus each cell line may have different targets for cancer prevention.
Therefore, the compounds in this study exhibited a variety of
impact on human colon, leukemia, and liver cell lines.
transcription factor-jB and this leads to drug resistance in SiHa
cell lines (human cervical carcinoma cells). These results help drug
design researchers to determine the effect of side groups at cellular
level and provide a starting point to design effective anticancer
compounds. Details of the unexpected behavior of these com-
pounds may be highlighted by employing molecular methods.
Currently there are several drugs developed that target protein
kinases for the treatment of different types of cancer. Small
molecule kinase inhibitors are widely used in the pharmaceutical
field such as, AxitinibÒ, PazopanibÒ, SunitinibÒ and SorafenibÒ
Surprisingly, 3a, 3d, and 3f compounds at 48 h incubation
displayed more cell viability compared to that of 24 h incubation
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Irfan Koca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 2
Anticancer activity of compounds as % cell viability after 24 and 48 h
Compounds
24 h
48 h
Jurkat cell line
DLD-1 cell line
HepG2 cell line
Jurkat cell line
DLD-1 cell line
HepG2 cell line
3a (10À4 M)
3a (10À5 M)
3b (10À4 M)
3b (10À5 M)
3c (10À4 M)
3c (10À5 M)
3d (10À4 M)
3d (10À5 M)
3e (10À4 M)
3e (10À5 M)
3f (10À4 M)
3f (10ÀÀ54M)
3g (10À5 M)
3g (10 M)
3h (10À4 M)
3h (10À5 M)
3i (10À4 M)
3i (10À5 M)
3j (10À4 M)
3j (10À5 M)
3k (10À4 M)
3k (10À5 M)
23.30 2.31
30.00 1.19
30.00 3.25
50.30 6.33
100.00 5.87
100.00 3.77
31.11 3.1
37.81 3.67
100.00 6.4
100.00 6.1
57.33 5.66
56.31 5.41
78.10 7.77
100.00 2.10
36.40 4.25
46.01 5.01
100.00 3.56
100.00 2.11
36.60 4.25
34.72 2.50
86.15 4.44
100.00 1.32
71.35 3.72
75.54 5.35
57.14 5.47
65.14 4.83
55.28 3.14
63.24 5.31
71.25 8.45
80.45 8.27
62.45 4.59
75.14 5.21
60.45 6.03
73.29 8.02
65.25 5.38
78.12 6.66
74.25 7.51
82.15 3.22
100.00 3.41
100.00 1.97
64.15 4.99
75.15 5.21
80.44 5.74
88.54 5.39
38.17 1.47
42.11 3.61
49.25 5.88
51.64 4.65
29.13 1.22
33.33 2.13
36.45 4.13
41.97 5.33
50.13 5.13
54.25 4.31
49.47 3.12
53.67 5.21
55.21 5.16
51.47 4.19
78.74 5.11
85.45 2.18
38.15 2.18
44.21 4.17
49.24 4.51
56.35 6.54
44.26 3.51
51.14 4.87
6.00 0.61
67.60 2.25
12.46 0.95
14.20 1.88
76.59 6.54
51.18 4.96
8.98 1.01
87.50 7.98
67.60 7.12
47.80 5.69
24.20 2.64
90.00 8.55
40.10 4.12
87.50 6.52
8.24 1.03
14.53 2.46
39.24 3.99
66.35 5.86
17.87 1.05
23.13 1.31
23.42 2.58
27.98 2.57
60.58 3.46
55.41 3.78
40.25 3.91
48.54 4.44
41.26 4.21
45.58 3.87
62.14 6.54
68.21 7.64
51.45 4.1
60.24 6.25
49.24 5.17
55.16 4.44
50.14 5.55
62.52 6.07
70.58 4.15
75.51 4.37
96.56 8.51
95.21 7.41
55.87 3.14
65.55 6.58
75.54 5.41
81.44 5.56
21.84 2.27
23.73 3.56
25.91 2.55
27.71 1.69
20.81 1.00
22.82 2.22
22.64 2.10
27.87 2.40
23.56 2.09
30.06 1.48
24.78 3.05
24.94 3.11
27.19 2.99
27.79 2.16
49.85 4.39
51.24 3.00
22.94 3.85
27.26 1.25
39.77 2.53
40.63 3.74
28.80 2.24
31.24 1.90
Figure 2. Antitumor activity of compounds for Jurkat cell line. (1) and (2) indicate 10À4 and 10À5 M respectively.
for renal cell carcinoma; BosutinibÒ and ImatinibÒ for chronic
myelogenous leukemia; SunitinibÒ, ImatinibÒ for gastrointestinal
stromal tumor; LapatinibÒ for breast cancer, and VemurafenibÒ
for melanoma (www.fda.gov). In addition to these drugs, an excel-
lent comprehensive review on small molecule kinase inhibitors
explains several applications of these compounds.41 Therefore,
drug researchers have been focused on small molecule kinase
inhibitors for affective cancer treatment.
zole derivatives show antiproliferative effects by inhibiting Aurora
kinase activity and growth factor-beta type I receptor kinase
respectively.30,42 We can propose inhibition of CDKs as a model
mechanism of our compounds for anticancer activities. Therefore,
we determined anticancer characteristics of eleven small pyrazole
derivative molecules in this study and we showed that these mole-
cules have potential properties to be developed as kinase inhibitors.
Anti-tumoral activities of pyrazole rings and their derivatives
have been known for many years. In pharmacy, cyclin-dependent
kinases (CDKs) are considered a potential target for the develop-
ment of anticancer drugs. CDKs are a family of protein kinases
which play key role in regulating the cell cycle. Pyrazole derivatives
generate anticancer action by the inhibition of the CDKs. CDK-med-
iated tumor cell proliferation may be inhibited in nanomolar con-
centrations of pyrazole based compounds. In the literature, 1-
carboxamidopyrazole and aryl- and heteroaryl-substituted pyra-
3. Conclusion
We have synthesized a new series of acyl thiourea derivatives
containing pyrazole ring were prepared in good yield via one pot
reaction. The structures of these compounds were confirmed by IR,
1H NMR, 13C NMR and elemental analysis. Anticancer activities of
synthesized compounds were evaluated on human leukemia, colon,
and liver cancer cell lines. As a result of the cell culture studies, all of
the compounds have shown anticancer activity for colon, leukemia
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Irfan Koca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Figure 3. Antitumor activity of compounds for DLD-1 cell line. (1) and (2) indicate 10À4 and 10À5 M respectively.
Figure 4. Antitumor activity of compounds for HepG2 cell line. (1) and (2) indicate 10À4 and 10À5 M respectively.
and liver cancer cells. In conclusion, novel pyrazolyl acyl thioureas
compounds might be potentially useful in the field of cancer treat-
ment. Finally, our new synthesized compounds can be suggested
as potent candidates for leukemia, liver and colon cancer drugs.
were supplied from ATCC (American Type Culture Collection, USA).
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum
and sterile phosphate buffer saline (PBS) were obtained from PAA
Ltd. (France). Trypsin–EDTA was from Biological Industries Ltd.
(Haemek, Israel).
L-glutamine–penicillin–streptomycin solution
was from Sigma–Aldrich (Steinheim am Albuch, Germany). XTT
reagent (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-
lium-5-carboxanilide) was obtained from Roche Diagnostic.
4. Experimental section
4.1. Chemistry
4.2. General procedure for the synthesis of compound 3
Melting points are uncorrected and recorded on Electrothermal
9200 digital melting point apparatus. Microanalyses were
performed on a Leco-932 CHNS-O Elemental Analyser. A Perkin
Elmer Spectrum Two Model FT-IR Spectrophotometer was used
for IR spectra using ATR method. The 1H and 13C NMR spectra were
measured with Bruker Avance III 400 MHz spectrometer using
CDCl3 solvent. The reactions were followed by TLC (Silica gel,
aluminium sheets 60 F254, Merck). Solvents and all other chemical
reagents were purchased from Merck, Sigma, Aldrich and Fluka.
Solvents were dried by refluxing with the appropriate drying
agents and distilled before use. DLD-1, HepG2 and Jurkat cell lines
0.387 g (1 mmol) 4-benzoyl-1,5-diphenyl-1H-pyrazole-3-car-
bonyl chloride (1) and 0.084 g (1.1 mmol) ammonium thiocyanate
in 15 mL acetone were refluxed for 30 min. The resulting solid
(NH4Cl) was removed by filtration. Then to this solution corre-
sponding amines (1 mmol) in 10 mL acetone was added drop wise,
the mixture was stirred under reflux for 3 h. The solvent was re-
moved under reduced pressure, and the residue was treated with
the 2-propanol to give crude product as a white solid. The resulting
precipitates were purified by column chromatography on silica gel
using chloroform/hexane (1:1) as eluent.
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4.2.1. 4-Benzoyl-1,5-diphenyl-N-(phenylcarbamothioyl)-1H-
pyrazole-3-carboxamide (3a)
144.57, 141.98, 138.37, 137.26, 136.14, 133.75, 132.05, 129.68,
129.63, 129.53, 129.25, 129.09, 128.88, 128.74, 128.61, 127.29,
125.50, 125.30, 123.14 (C@C and C@N). Calcd for C30H21ClN4O2S
(537.03): C, 67.09; H, 3.94; N, 10.43; S, 5.97. Found: C, 66.90; H,
4.21; N, 10.13; S, 5.82.
Yield 0.397 g, 79%, mp 206 °C. IR (ATR, cmÀ1): 3191 (b, NH), 1682,
1665 (C@O), 1176 (C@S). 1H NMR (CDCl3, ppm): 12.06 (s, 1H, NH),
10.02 (s, 1H, NH), 7.88–7.16 (m, 20H, Ar-H). 13C NMR (CDCl3,
ppm): 190.99 (C@O), 178.07 (C@S), 159.97 (C@O, amide), 144.52,
142.12, 138.39, 137.59, 137.30, 133.74, 129.64, 129.53, 129.25,
129.05, 128.77, 128.73, 128.61, 127.33, 126.81, 125.30, 124.30,
123.10 (C@C and C@N). Calcd for C30H22N4O2S (502.59): C, 71.69;
H, 4.41; N, 11.15; S, 6.38. Found: C, 71.61; H, 4.46; N, 10.88; S, 6.32.
4.2.7. 4-Benzoyl-N-(4-bromophenylcarbamothioyl)-1,5-
diphenyl-1H-pyrazole-3-carboxamide (3g)
Yield 0.342 g, 73%, mp 204 °C. IR (ATR, cm cmÀ1): 3191 (b, NH),
1682, 1665 (C@O), 1176 (C@S). 1H NMR (CDCl3, ppm): 12.08 (s, 1H,
NH), 10.03 (s, 1H, NH), 7.96–7.16 (m, 19H, Ar-H). 13C NMR (CDCl3,
ppm): 190.91 (C@O), 178.04 (C@S), 160.05 (C@O, amide), 144.58,
141.98, 138.38, 137.27, 136.67, 133.75, 131.85, 129.68, 129.63,
129.53, 129.25, 129.09, 128.74, 128.60, 128.33, 127.30, 125.74,
125.31, 123.16, 119.89 (C@C and C@N). Calcd for C30H21BrN4O2S
(581.48): C, 61.97; H, 3.64; N, 9.64; S, 5.51. Found: C, 62.24; H,
3.92; N, 9.54; S, 5.28.
4.2.2. 4-Benzoyl-1,5-diphenyl-N-(p-tolylcarbamothioyl)-1H-
pyrazole-3-carboxamide (3b)
Yield 0.386 g, 75%, mp 216 °C. IR (ATR, cmÀ1): 3191 (b, NH),
1682, 1665 (C@O), 1176 (C@S). 1H NMR (CDCl3, ppm): 11.95 (s,
1H, NH), 9.99 (s, 1H, NH), 7.87–7.16 (m, 19H, Ar-H), 2.34 (s, 3H,
CH3). 13C NMR (CDCl3, ppm): 190.98 (C@O), 178.17 (C@S), 159.92
(C@O, amide), 144.47, 142.16, 138.40, 137.31, 136.75, 135.03,
133.71, 129.64, 129.52, 129.23, 129.02, 128.72, 128.59, 127.36,
125.29, 124.36, 123.08 (C@C and C@N), 21.08(CH3). Calcd for
4.2.8. 4-Benzoyl-N-(naphthalen-1-ylcarbamothioyl)-1,5-
diphenyl-1H-pyrazole-3-carboxamide (3h)
Yield 0.376 g, 68%, mp 184 °C. IR (ATR, cmÀ1): 3130 (b, NH),
1677, 1669 (C@O), 1185 (C@S). 1H NMR (CDCl3, ppm): 11.95 (s,
1H, NH), 9.99 (s, 1H, NH), 7.87–7.16 (m, 22H, Ar-H). 13C NMR
(CDCl3, ppm): 190.99 (C@O), 179.95 (C@S), 160.25 (C@O, amide),
144.73, 142.29, 138.41, 137.45, 134.13, 133.67, 133.58, 129.71,
129.68, 129.53, 129.26, 129.07, 128.74, 128.66, 128.57, 128.48,
128.02, 127.35, 126.82, 126.34, 125.32, 125.18, 124.27, 123.11,
C31H24N4O2S (516.61): C, 72.07; H, 4.68; N, 10.85; S, 6.21. Found:
C, 71.82; H, 4.82; N, 10.55; S, 6.23.
4.2.3. 4-Benzoyl-1,5-diphenyl-N-(o-tolylcarbamothioyl)-1H-
pyrazole-3-carboxamide (3c)
Yield 0.372 g, 72%, mp 145 °C. IR (ATR, cmÀ1): 3134 (b, NH),
1674, 1662 (C@O), 1173 (C@S). 1H NMR (CDCl3, ppm): 11.70 (s,
1H, NH), 10.11 (s, 1H, NH), 7.87–7.16 (m, 19H, Ar-H), 2.24 (s, 3H,
CH3). 13C NMR (CDCl3, ppm): 190.97 (C@O), 179.25 (C@S), 160.07
(C@O, amide), 144.64, 142.26, 138.39, 137.40, 136.32, 133.82,
133.66, 130.72, 129.68, 129.65, 129.50, 129.24, 129.04, 128.72,
128.54, 127.75, 127.35, 126.58, 126.41, 125.29, 123.03 (C@C and
C@N), 17.91 (CH3). Calcd for C31H24N4O2S (516.61): C, 72.07; H,
4.68; N, 10.85; S, 6.21. Found: C, 71.83; H, 4.65; N, 10.58; S, 6.11.
121.98 (C@C and C@N). Calcd for
C34H24N4O2S (552.65): C,
73.89; H, 4.38; N, 10.14; S, 5.80. Found: C, 74.09; H, 4.62; N,
9.92; S, 5.77.
4.2.9. 4-Benzoyl-N-(4-carbamoylphenylcarbamothioyl)-1,5-
diphenyl-1H-pyrazole-3-carboxamide (3i)
Yield 0.380 g, 70%, mp 245 °C. IR (ATR, cmÀ1): 3422, 3377 (NH2),
3191(b, NH), 1682, 1665 (C@O), 1176 (C@S). 1H NMR (CDCl3, ppm):
11.95 (s, 1H, NH), 9.99 (s, 1H, NH), 7.87–7.16 (m, 19H, Ar-H). 13C
NMR (CDCl3, ppm): 190.95 (C@O), 177.80 (C@S), 168.29 (C@O,
amide), 160.07 (C@O, amide), 144.60, 141.91, 140.87, 138.35,
137.21, 133.80, 131.02, 129.69, 129.61, 129.54, 129.27, 129.11,
128.75, 128.62, 128.07, 127.25, 125.31, 123.55, 123.15 (C@C and
C@N). Calcd for C31H23N5O3S (545.61): C, 68.24; H, 4.25; N,
12.84; S, 5.88. Found: C, 68.02; H, 4.06; N, 12.69; S, 5.51.
4.2.4. 4-Benzoyl-N-(4-methoxyphenylcarbamothioyl)-1,5-
diphenyl-1H-pyrazole-3-carboxamide (3d)
Yield 0.346 g, 65%, mp 199 °C. IR (ATR, cmÀ1): 3177 (b, NH),
1678, 1669 (C@O), 1186 (C@S). 1H NMR (CDCl3, ppm): 11.89 (s,
1H, NH), 10.00 (s, 1H, NH), 7.87–6.88 (m, 19H, Ar-H), 3.81 (s, 3H,
OCH3). 13C NMR (CDCl3, ppm): 191.00 (C@O), 178.39 (C@S),
159.95 (C@O, amide), 158.18, 144.46, 142.15, 138.39, 137.30,
133.73, 130.54, 129.63, 129.52, 129.23, 129.03, 128.73, 128.61,
127.35, 126.03, 125.29, 123.08, 113.98 (C@C and C@N), 55.43
(OCH3). Calcd for C31H24N4O3S (532.61): C, 69.91; H, 4.54; N,
10.52; S, 6.02. Found: C, 69.70; H, 4.41; N, 10.26; S, 5.94.
4.2.10. 4-Benzoyl-N-(benzylcarbamothioyl)-1,5-diphenyl-1H-
pyrazole-3-carboxamide (3j)
Yield 0.367 g, 71%, mp 227 °C. IR (ATR, cmÀ1): 3246 (b, NH),
1679, 1660 (C@O), 1190 (C@S). 1H NMR (CDCl3, ppm): 10.56 (s,
1H, NH), 9.95 (s, 1H, NH), 7.82–7.13 (m, 20H, Ar-H), 4.86 (s, 2H,
CH2). 13C NMR (CDCl3, ppm): 190.97 (C@O), 179.76 (C@S), 159.81
(C@O, amide), 144.39, 142.24, 138.39, 137.22, 136.16, 133.66,
129.61, 129.51, 129.21, 128.99, 128.73, 128.69, 128.54, 127.93,
127.80, 127.36, 125.99, 122.93 (C@C and C@N), 49.60 (CH2–N).
Calcd for C31H24N4O2S (516.61): C, 72.07; H, 4.68; N, 10.85; S,
6.21. Found: C, 71.94; H, 4.64; N, 10.62; S, 6.13.
4.2.5. 4-Benzoyl-N-(4-fluorophenylcarbamothioyl)-1,5-
diphenyl-1H-pyrazole-3-carboxamide (3e)
Yield 0.375 g, 72%, mp 218 °C. IR (ATR, cmÀ1): 3146 (b, NH),
1676, 1622 (C@O), 1181 (C@S). 1H NMR (CDCl3, ppm): 11.98 (s,
1H, NH), 10.03 (s, 1H, NH), 7.87–7.04 (m, 19H, Ar-H). 13C NMR
(CDCl3, ppm): 190.96 (C@O), 178.60 (C@S), 160.93 (d,
1J = 248.0 Hz, C–F), 160.06 (C@O, amide), 144.54, 142.01, 138.37,
137.26, 133.76, 133.58, 133.55, 129.67, 129.62, 129.53, 129.26,
129.08, 128.74, 128.62, 127.29, 126.46, 126.37, 125.29, 123.12,
115.65 (d, 2J = 23.0 Hz), (C@C and C@N). Calcd for C30H21FN4O2S
(520.58): C, 69.22; H, 4.07; N, 10.76; S, 6.16. Found: C, 69.13; H,
4.22; N, 10.48; S, 6.16.
4.2.11. 4-Benzoyl-1,5-diphenyl-N-(pyrimidin-2-
ylcarbamothioyl)-1H-pyrazole-3-carboxamide (3k)
Yield 0.313 g, 62%, mp 219 °C. IR (ATR, cmÀ1): 3196 (b, NH),
1720, 1667 (C@O), 1193 (C@S). 1H NMR (CDCl3, ppm): 8.68 (s,
2H, 2 Â NH), 7.87–7.07 (m, 18H, Ar-H). 13C NMR (CDCl3, ppm):
190.81 (C@O), 176.80 (C@S), 159.02 (C@O, amide), 158.17,
157.26, 144.26, 138.71, 137.49, 133.54, 129.64, 129.57, 129.19,
128.90, 128.70, 128.51, 127.41, 125.21, 123.45, 117.18 (C@C
and C@N). Calcd for C28H20N6O2S (504.56): C, 66.65; H, 4.00;
N, 16.66; S, 6.36. Found: C, 66.37; H, 3.78; N, 16.32; S, 6.33.
4.2.6. 4-Benzoyl-N-(4-chlorophenylcarbamothioyl)-1,5-
diphenyl-1H-pyrazole-3-carboxamide (3f)
Yield 0.372 g, 69%, mp 212 °C. IR (ATR, cmÀ1): 3161 (b, NH),
1679, 1622 (C@O), 1188 (C@S). 1H NMR (CDCl3, ppm): 12.09 (s,
1H, NH), 10.04 (s, 1H, NH), 7.87–7.15 (m, 19H, Ar-H). 13C NMR
(CDCl3, ppm): 190.92 (C@O), 178.14 (C@S), 160.05 (C@O, amide),
_
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Irfan Koca et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
6. Aly, A. A.; NourEl-Din, A. M.; Gomaa, A. M.; Brown, A. B.; Fahmi, M. S. J. Chem.
Res. 2007, 439.
7. Rauf, M. K.; Din, I.; Badshah, A.; Gielen, M.; Ebihara, M.; Vos, D.; Ahmed, S. J.
4.3. In vitro studies
_
4.3.1. Cell lines and culture
Inorg. Biochem. 2009, 103(8), 1135.
8. Saeed, A.; Abbas, N.; Rafique, H.; Rashid, S.; Hameed, A. Khimiya (Sofiya
Bulgaria) 2009, 18, 152.
9. Müller, J.; Limban, C.; Stadelmann, B.; Missir, A. V.; Chirita, I. C.; Chifiriuc, M. C.;
Nitulescu, G. M. Parasitol. Int. 2009, 58, 128.
10. Faidallah, H. M.; Khan, K. A.; Asiri, A. M. J. Chem. Sci. (Bangalore, India) 2012,
124, 625.
11. Duan, L. P.; Xue, J.; Xu, L. L.; Zhang, H. B. Molecules 2010, 15, 6941.
12. Zhong, Z. M.; Xing, R.; Liu, S.; Wang, L.; Cai, S. B.; Li, P. C. Carbohydr. Res. 2008,
343, 566.
For in vitro experiments, DLD-1 (human colorectal adenocarci-
noma), HepG2 (human hepatocellular carcinoma) and Jurkat (hu-
man acute T-cell leukemia) cell lines were cultured in DMEM
(low glucose) medium with 10% fetal bovine serum, 1% l-gluta-
mine, 100 IU/mL penicillin and 10 mg/mL streptomycin. Cells were
cultivated in a humidified incubator at 37 °C within an atmosphere
containing 5% CO2.
13. Wang, Z. Y.; Wang, S.; Song, X. J.; Wang, Y. G. Chin. J. Pestic. Sci. 2005, 7, 282.
14. McCarthy, A. R.; Pirrie, L.; Hollick, J. J.; Ronseaux, S.; Campbell, J.; Higgins, M.;
Staples, O. D.; Tran, F.; Slawin, A. M. Z.; Lain, S.; Westwood, N. J. Bioorg. Med.
Chem. 2012, 20, 1779.
15. McCarthy, A. R.; Hollick, J. J.; Westwood, N. J. Semin. Cancer Biol. 2010, 20, 40.
16. Bedalov, Antonio; Gottschling, Daniel E.; PCT Int. Appl. WO 2003046207 A2
20030605, 2003.
4.3.2. Cytotoxic assay of compounds
XTT test was used to monitor pyrazolyl acyl thioureas deriva-
tives toxicity on cancer cells. Initially, the cancer cells were placed
in sterile 96-well culture plate (10 Â 104 cells in each well), and
our compounds were tested at individual experiments by 10-fold
diluting the starting mixture at five different concentration ranging
from 10À4 to 10À8 M at 24 and 48 h. At the end of these periods,
17. Lain, S.; Lane, D. Eur. J. Cancer 2003, 39, 1053.
18. Agola, J. O.; Hong, L.; Surviladze, Z.; Ursu, O.; Waller, A.; Strouse, J. J.; Simpson,
D. S.; Schroeder, C. E.; Oprea, T. I.; Golden, J. E.; Aube´, J.; Buranda, T.; Sklar, L. A.;
Wandinger-Ness, A. ACS Chem. Biol. 2012, 7, 1095.
19. Shen, W.; Fang, Y.; Tong, A.; Zhu, Q. Med. Chem. Res. 2012, 21, 4214.
20. You, J.C.; Han, G.H.; Lee, C.H.; Song, D.N.; Chung, K.H. PCT Int. Appl. WO
2011159137 A2 20111222, 2011.
50 ll XTT reagents were added to each well for determination of
living cells. The mitochondrial dehydrogenase enzymes reduce yel-
low colored tetrazolium salt (XTT) into water-soluble orange col-
ored formazan salt. After 4 h, the absorbance was measured
using micro plate reader (Thermo) at 450 nm, and then the per-
centage of cell viability was calculated.38,39
21. Suzuki, S.; Inoue, A.; 1990, JP Patent 02, 172, 988 Chem. Abstr. 1990, 218276.
22. Sridhar, R.; Paramasivam, T. P.; Sundaresan, E.; Guruswamy, S.;
Mondikalipudur, N. P.; Vaiyapuri, R. P.; Mathivanan, N. Bioorg. Med. Chem.
Lett. 2004, 14, 6035.
_
_
23. Bildirici, I.; Sßener, A.; Tozlu, I. Med. Chem. Res. 2007, 16, 418.
24. Bruno, O.; Ranise, A.; Bondavalli, F.; Schenone, P.; D’amico, M. Farmaco 1992,
47, 1225.
4.3.3. Statistical analysis
25. Janus, S. L.; Magdif, A. Z.; Erik, B. P.; Claus, N. Monatsh. Chem. 1999, 130, 1167.
26. Tiwari, R. K.; Verma, A. K.; Chhillar, A. K.; Singh, D.; Singh, J.; Sankar, V. K.;
Yadav, V.; Sharma, G. L.; Chandra, R. Bioorg. Med. Chem. 2006, 14, 2747.
27. Lepage, F.; Hublot, B. 1992, EP Patent 459887. Chem. Abstr. 1992, 116, 128917.
28. Hall, A.; Billinton, A.; Brown, S. H.; Clayton, N. M.; Chowdhury, A.; Giblin, G. M.
P.; Goldsmith, P.; Hayhow, T. G.; Hurst, D. N.; Kilford, I. R.; Naylor, A.;
Passingham, B.; Winyard, L. Bioorg. Med. Chem. Lett. 2008, 18, 3392.
29. Wilson, W. L.; Bottiglieri, N. G. Cancer Chemother. Rep. 1962, 21, 137.
30. Nitulescu, G. M.; Draghici, C.; Missir, A. V. Eur. J. Med. Chem. 2010, 45, 4914.
31. Foley, H. T.; Shnider, B. I.; Gold, G. L.; Uzer, Y. Cancer Chemother. Rep. 1965, 44, 45.
32. Ausman, R. K.; Nadler, S.; Moore, G. E. Cancer Chemother. Rep. 1963, 33, 41.
33. Bouabdallah, I.; M’Barek, L. A.; Zyad, A.; Ramdani, A.; Zidane, I.; Melhaoui, A.
Nat. Prod. Res. 2007, 21, 298.
Differences in the mean values of measured activities were
evaluated statistically using the SPSS 17.0 program (Univariate
Variance Analyses and Pearson Correlation). Probability values of
p <0.05 were considered to be significant.
Acknowledgements
We deeply appreciate expert Mehmet Gümüsß for helping on the
analysis of IR spectra. This work was funded through a seed grant
from the Turkish National Academy of Sciences (TUBA GEBIP
2008-29 for Y.T.).
34. Havrylyuk, D.; Zimenkovsky, B.; Vasylenko, O.; Zaprutko, L.; Gzella, A.; Lesyk, R.
Eur. J. Med. Chem. 2009, 44, 1396.
35. Shaharyar, M.; Abdullah, M. M.; Bakht, M. A.; Majeed, J. Eur. J. Med. Chem. 2010,
45, 114.
36. Akçamur, Y.; Penn, G.; Ziegler, E.; Sterk, H.; Kollenz, G.; Peters, K.; Peters, E. M.;
Von Schnering, H. G. Monatsh. Chem. 1986, 117, 231.
References and notes
1. Vujasinovic, I.; Paravic-Radicevic, A.; Mlinaric-Majerski, K.; Brajsa, K.; Bertosa,
B. Bioorg. Med. Chem. 2012, 20, 2101.
37. Saeed, S.; Rashid, N.; Jones, P. G.; Ali, M.; Hussain, R. Eur. J. Med. Chem. 2010, 45,
1323.
2. Pevarello, P.; Fancelli, D.; Vulpetti, A.; Amici, R.; Villa, M.; Pittala, V.; Vianello,
P.; Cameron, A.; Ciomei, M.; Mercurio, C.; Bischoff, J. R.; Roletto, F.; Varasi, M.;
Brasca, M. G. Bioorg. Med. Chem. Lett. 2006, 16, 1084.
3. Burrows, A. D.; Coleman, M. D.; Mahon, M. F. Polyhedron 1999, 18, 2665.
4. Henderson, W.; Nicholson, B. K.; Dinger, M. B.; Bennett, R. L. Inorg. Chim. Acta
2002, 338, 210.
38. Yurt Lambrecht, F.; Durkan, K.; Özgür, A.; Gündüz, C.; Avcı, C.B.; Susluer, S.Y. J.
Drug Target 2013, http://dx.doi.org/10.3109/1061186X.2012.757772.
39. Conti, C.; Proietti Monaco, L.; Desideri, N. Bioorg. Med. Chem. 2011, 19, 7357.
40. Yeh, P. Y.; Chuang, S. E.; Yeh, K. H.; Song, Y. C.; Cheng, A. L. Biochem. Pharmacol.
2003, 66, 25.
41. Zhang, J.; Yang, P. L.; Gray, N. S. Nat. Rev. Cancer 2009, 9, 28.
42. Park, H. J.; Lee, K.; Park, S. J.; Ahn, B.; Lee, J. C.; Cho, H. Y.; Lee, K. I. Bioorg. Med.
Chem. 2005, 15, 3307.
5. Aly, A. A.; Ahmed, E. K.; El-Mokadem, K. M.; Hegazy, M. A. F. J. Sulfur Chem.
2007, 28, 73.
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