Synthesis of ester and amide derivatives of 1-phenyl-3-(thiophen-3-yl)-1H- pyrazole-4-carboxylic acid and study of their anticancer activity

Article abstract of DOI:10.1007/s00044-012-0317-2

A series of novel thiophene containing 1,3-diarylpyrazole derivatives were synthesized and the structures were determined by IR, ¹H-NMR, and HRMS analysis. The anticancer activity of the title compounds against MCF7, MDA-MB-231, HeLa, Raji, and HL60 human cancer cells growth were investigated by MTT assay. Interestingly, Raji and HL60 cells exhibited more sensitivity to synthesized compounds. (4-Benzyl-piperidin-1-yl)-(1-phenyl-3-thiophen-3-yl-1H- pyrazol-4-yl)-methanone (4c) possessed the highest growth inhibitory effect on Raji and HL60 cancer cells (GI₅₀ 25.2 ± 3.2 and 28.3 ± 1.53 μM, respectively). Compound 4f, 4-trifluoromethylphenyl piperazine at the amide part, also showed potent activity on Raji and HL60 cancer cell lines with a GI₅₀ value of 32.0 ± 1.27 and 36.7 ± 2.15 μM, respectively. Physicochemical properties of the synthesized compounds were evaluated in silico, and it was found that all compounds should present good passive oral absorption. All synthesized compounds demonstrated good drug-likeness values. The data suggested that these compounds might be promising for further development.

Full text of DOI:10.1007/s00044-012-0317-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3109–3118
DOI 10.1007/s00044-012-0317-2
ORIGINAL RESEARCH
Synthesis of ester and amide derivatives of 1-phenyl-3-(thiophen-
3-yl)-1H-pyrazole-4-carboxylic acid and study of their anticancer
activity
•
•
Nazan Inceler Akın Yılmaz Sultan Nacak Baytas
Received: 7 June 2012 / Accepted: 25 October 2012 / Published online: 10 November 2012
Ó Springer Science+Business Media New York 2012
Abstract A series of novel thiophene containing 1,3-di-
arylpyrazole derivatives were synthesized and the struc-
tures were determined by IR, 1H-NMR, and HRMS
analysis. The anticancer activity of the title compounds
against MCF7, MDA-MB-231, HeLa, Raji, and HL60
human cancer cells growth were investigated by MTT
assay. Interestingly, Raji and HL60 cells exhibited more
sensitivity to synthesized compounds. (4-Benzyl-piperidin-
1-yl)-(1-phenyl-3-thiophen-3-yl-1H-pyrazol-4-yl)-metha-
none (4c) possessed the highest growth inhibitory effect
on Raji and HL60 cancer cells (GI50 25.2 3.2 and
28.3 1.53 lM, respectively). Compound 4f, 4-trifluoro-
methylphenyl piperazine at the amide part, also showed
potent activity on Raji and HL60 cancer cell lines with a
GI50 value of 32.0 1.27 and 36.7 2.15 lM, respec-
tively. Physicochemical properties of the synthesized
compounds were evaluated in silico, and it was found that
all compounds should present good passive oral absorption.
All synthesized compounds demonstrated good drug-like-
ness values. The data suggested that these compounds
might be promising for further development.
Introduction
Cancer is one of the most serious threats against human
health in the world, and the clinical prognosis remains
relatively poor. Chemotherapy is a major form of cancer
treatment. However, the majority of cancers are either
resistant to chemotherapy or acquire resistance during
treatment. As a result, the design and discovery of non-
traditional, efficient, and safe chemical classes of agents
are the prime targets in contemporary medicinal chemistry
(Cozzi, 2003; Johnston et al., 2005).
Pyrazoles constitute an important heterocyclic family
containing nitrogen in their five-membered ring. Several
pyrazole derivatives have been found to possess significant
biological activities, stimulating the research activity in
medicinal chemistry and chemical biology (Silveira et al.,
1993; Sridhar et al., 2004; Nagarapu et al., 2011; Padmaja
et al., 2009; Gaston et al., 1996). Many pyrazole derivatives
are well acknowledged to possess a wide range of anticancer
bioactivities (Riyadh et al., 2010; Anzaldi et al., 2009; El-
Shafei et al., 2009; Zheng et al., 2010; Xie et al., 2010; Lian
et al., 2009). Literature survey revealed that some pyrazoles
have been implemented as antileukemic (Daidone et al.,
2004; Chou et al., 2007), antitumor (Li et al., 2006; Xia et al.,
2007), and anti-proliferative (Schenone et al., 2004) agents,
beside their capability to exert remarkable anticancer effects
through inhibiting different types of enzymes which play
important roles in cell division (Warshakoon et al., 2006;
Huang et al., 2007). Although the skeleton of pyrazole plays
an important role in its biological effects, the type of
peripheral substituents is also crucial. Wei et al. demon-
strated that ethyl 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-
pyrazole-5-carboxylate derivatives suppressed lung cancer
cell growth (Wei et al., 2006). They found that ethyl
1-arylmethyl-3-aryl-1H-pyrazole-5-carboxylate derivatives
Keywords Synthesis ꢀ Anticancer activity ꢀ
1H-Pyrazoles ꢀ Thiophene ꢀ MTT
N. Inceler ꢀ S. N. Baytas (&)
Division of Pharmaceutical Sciences, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi
University, Etiler, 06330 Ankara, Turkey
e-mail: sbaytas@gmail.com; baytas@gazi.edu.tr
A. Yılmaz
Department of Medical Biology and Genetics, Faculty of
Medicine, Gazi University, Bes¸evler, 06500 Ankara, Turkey
123

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Med Chem Res (2013) 22:3109–3118
promoted human umbilical vein endothelial cell (HUVEC)
apoptosis to a certain extent at the concentrations of
5–20 lM and the effect on the cell viability was dose
dependent (Ding et al., 2007). Pyrazole-fused heterocycles
such as dihydropyrazoloxazin-4-one and pyrazolopyrazi-
none derivatives also showed anticancer activity (Wei et al.,
2007; Xie et al., 2008; Zhang et al., 2008; Pan et al., 2008).
It is well known that the incorporation of heterocyclic
rings into prospective pharmaceutical candidates is a major
tactic to gain activity and safety advantages. Heterocyclic
rings such as thiophene are important pharmacophores in
search of molecules with antitumor activity (Micale et al.,
2002; Insuasty et al., 2008; Leite et al., 2004; Al-Obaid et al.,
2009; Basnet et al., 2010; Barresi et al., 2002). N-substituted
2-pyrazolines bearing thiophene substituent showed impor-
tant activity against different cancer cell lines with remark-
able values in leukemia, and renal and prostate cancer panels.
It is also reported that terthiophene derivatives, bioisosteres
of terpyridine, resulted considerable protein kinase C (PKC)
inhibitory activity and antitumor cytotoxicities against sev-
eral human cancer cell lines (Kim et al., 1998).
Results
Chemistry
We focused our synthetic efforts on diaryl heterocyclic ring
systems as illustrated in Scheme 1. The starting compound,
the hydrazone derivative 1, was generated by carrying out
condensation reaction in the presence of phenyl hydrazine
and acetic acid in refluxing ethanol. This hydrazone
derivative was then reacted with POCl3 and DMF resulting
in 1,3-diaryl pyrazole 2 with aldehyde group at 4 position.
1-Phenyl-3-thiophen-3-yl-1H-pyrazole-4-carbaldehyde
2
was oxidized to carboxylic acid derivative 3 in the presence
of potassium permanganate in acetone–water mixture. By
treatment of 3 with appropriate amines or phenols in the
presence of triethylamine and ethyl chloroformate, which
was used as the carboxylate activator, the amide and ester
derivatives 4 were prepared in good yield (40–88 %)
(Table 1). The chemical structures of these compounds
were elucidated by their IR, HRMS, and 1H-NMR spectral
data.
In the present study, Compounds A (Zheng et al., 2009)
and B (Insuasty et al., 2011) were considered as the parent
compounds for the design of our target compounds
(Fig. 1). In our effort to discover and develop potential new
anticancer agents, we synthesized a series of novel 1,3-
diarylpyrazole derivatives bearing thiophene functionality
and evaluated their anticancer activities. The title com-
pounds 4a–m were designed by inserting thiophene-3-yl
residue and introduction of the carboxamide side chain at 4
position of pyrazole ring. Herein, we report the synthesis
and antiproliferative activity of compounds against five
different human cancer cell lines. In silico evaluations of
title compounds are also reported.
The IR spectrum of hydrazone 1 has shown the disap-
pearance of the carbonyl peak belonging to acetyl group of
3-acetyl-thiophene and the secondary N–H stretching band
was observed at 3349 cm-1 as singlet. The IR spectra of
compound 2 exhibited a characteristic strong absorption for
aldehyde carbonyl group at 1667 cm-1. In the IR spectra of
compound 3, strong absorption band at 1690 cm-1 and
intense O–H stretching absorption in the region of
3300–2500 cm-1 for carboxylic acid were observed. Final
amide and ester derivatives exhibited a characteristic
strong absorption in the area of 1744–1615 cm-1 attribut-
able to the C=O of amide or ester group.
1
In the H-NMR spectra of compound 2, two singlets
were observed due to the aldehyde group at 10.10 ppm and
pyrazole-H5 at 8.50 ppm, each showing the integration for
one proton. In the 1H-NMR spectra of compounds 3, signal
of the carboxylic acid was not observed, but signal of the
pyrazole-H5 was seen at 8.59 ppm. In the 1H-NMR spectra
of final amide and ester derivatives 4a–m, pyrazole-H5
gave a singlet at about 9.00–8.04 ppm.
O
HO
N
H
N
N
R
N
N
N
S
O
R
A
B
Biological evaluations
Effects of the compounds on the viability of cancer
cells
O
S
N
Synthesized 1-phenyl-3-thiophen-3-yl-1H-pyrazole-4-car-
boxylic acid 3 and its amide and ester derivatives 4a–m
were screened against five human cancer cell lines
(HeLa, MCF7, MDA-MB-231, Raji, and HL60) using
3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide
N
4a-m
N
Fig. 1 Design of the title compounds
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Med Chem Res (2013) 22:3109–3118
3111
Scheme 1 a Phenyl hydrazine,
acetic acid, ethanol, 2 h reflux.
b Dimethyl formamide, POCl3,
50 °C, 4 h. c Potassium
S
a
N
b
N
S
S
CH3
HN
N
H
O
permanganate, acetone–water
(3:2), 2.5 h reflux.
O
2
1
d Appropriate amine or phenol
derivative, ethyl chloroformate,
triethylamine, dichloromethane,
rt, overnight
S
S
N
d
N
c
N
N
COR
COOH
3
4
Table 1 Characterization data of 1,3-diarylpyrazole derivatives (3, 4a–m)
S
N
N
COR
Compnd.
R
Melting point (°C) % of yield Compnd.
R
Melting point (°C)
% of yield
N
N
N
3
OH
201-203
128-130
87
40
4g
4h
243-245
41
4a
NH
O
O
153-155
49
4b
4c
4i
4j
O
O
N
O
147-149
78-80
70
61
75-77
65-68
54
88
N
O
4d
4k
CH3
N
N
110-112
179-181
143-145
42
40
43
116-118
146-148
129-131
43
56
64
4e
4f
4l
N
N
O
O
4m
F
F
N
N
F
(MTT) assay. The MTT cell proliferation assay has been
widely accepted as a reliable way to measure the cell
proliferation rate, and conversely when metabolic events
lead to apoptosis or necrosis (Holst-Hansen and Bru¨nner,
1998; Reile et al., 1990, Kueng et al., 1989; Senaratne
et al., 2000). After incubation with compounds at the
concentration of 50 lM for 48 h, the cells were treated
with MTT to measure their growth/viability (% of the
untreated control) by a spectrophotometric method as
described previously (Mosmann, 1983; Denizot and Lang,
1986). Figure 2 indicates the cytotoxic effects of the
compounds on the viability of the tested cancer cells that
were incubated for 48 h. For example, exposure of HeLa
cells to compounds 4a, 4c, 4e, and 4f at 50 lM for 48 h
resulted in cell viability decrease from 100 to 82.3, 71.8,
67.9, and 80.7 %, respectively. For MDA-MB-231 cells,
compounds 3, 4a, 4b, 4c, 4e, 4f, and 4g decreased the cell
viability from 100 to 80.9, 85.0, 77.6, 82.8, 81.4, 84.2, and
87.4 %, respectively. Furthermore, only compounds 4c and
4f exhibited poor activity toward the MCF7 cell line (cell
viability reduced from 100 to 90.7 % for 4c and from 100
to 85.9 % for 4f). On the other hand, compound 4c showed
the ever highest growth inhibitory potential against Raji
cell line. Additionally, compound 4c was able to display a
significant activity against HL60 tumor cell lines, reducing
cell viability from 100 to 39.1 %.
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Table 2 The GI50 values of the 4c and 4fa for cell viability in Raji
and HL60 cell lines
Among all compounds, 4c showed the most potent
inhibitory effect on Raji and HL60 cells growth (GI50
25.2 3.2 and 28.3 1.53 lM, respectively), followed
by 4f (GI50 32.0 1.27 and 36.7 2.15 lM, respec-
tively) (Table 2).
Cell line
GI50 (lM)
4c
4f
Raji
HL60
a
25.2 3.2
28.3 1.53
36.7 2.15
32.0 1.27
Lipinski’s rule-of-five and drug-likeness profile
Rest of the compounds have IC50 values of [100 lM. Results are
presented as mean SE; (n = 4. * P \ 0.05 vs control; ** P \ 0.01
vs control; *** P \ 0.001 vs control)
The synthesized compounds (4a–m) were submitted to an
in silico evaluation using a molecular modeling approach.
In order to predict drug-like properties of the synthesized
compounds, we analyzed these derivatives according to the
rule-of-five developed by Lipinski et al. 2001. Predictions
of ADME properties for the compounds are given in
Table 3. Calculated physicochemical properties (http://www.
molinspiration.com/cgi-bin/properties) showed that most of
the compounds fulfilled the Lipinski ‘‘rule-of-five.’’ The
compound 4f violated the criteria for molecular weight and
logP. Compounds 4c, 4i, 4j, 4k, 4l, and 4m had very large
clogP values, which might be disadvantageous with regard to
pharmacokinetic properties of these molecules in biological
systems. Rest of the compounds exhibited favorable clog P
values. The polar surface areas of the synthesized compounds
were relatively small in comparison with the average value
Discussion
Cancer is the second leading cause of death in developed
countries, accounting for nearly one in five deaths. A key
feature of cancer cells is their uncontrolled proliferation;
thus, inhibition of proliferative pathways is believed to be
an effective strategy to fight cancer and much attention has
recently been paid to the discovery and development of
new, more selective anticancer agents (Lee et al., 2007;
Kemnitzer et al., 2008; Hu et al., 2008). The design and
synthesis of new pyrazole derivatives has been a subject of
consistent interest. Pyrazole derivatives have occupied a
unique position in drug discovery due to their broad range
of biological activities, including anti-inflammatory, anti-
angiogenic, antibacterial, antimicrobial, anticancer, anti-
depressant, antioxidant, anti-influenza, and analgesic
activities (Tewari et al., 2010; Christodoulou et al., 2010;
Lv et al., 2010; Gouda et al., 2010; Chou et al., 2010;
Abdel-Aziz et al., 2009; Bandgar et al., 2009; Shih et al.,
2010; Machado et al., 2009). In the present study, 1,3-
diarylpyrazole derivatives bearing thiophene moiety
showed moderate anticancer activity. It is possible to relate
the structural characteristics of the compounds to their
´
2
˚
for acceptable drug molecules (\90A ).
Currently, there are many approaches to assess a com-
pound’s drug-likeness based on topological descriptors,
fingerprints of molecular drug-likeness structure keys
(Tetko, 2005). In this work, we used the Osiris program
(http://www.organic-chemistry.org/prog/peo) for calculat-
ing the fragment-based drug-likeness of the compounds
(Table 3). The most active compound 4c demonstrated
good drug-likeness value (=3.5).
Fig. 2 Effects of the compounds on cervical carcinoma (HeLa),
breast cancer (MDA-MB-231 and MCF7), Burkitt lymphoma (Raji),
and human promyelocytic leukemia (HL60) cells viability. HeLa,
MDA-MB-231, MCF7, Raji, and HL60 cells were treated with
compounds 3, 4a–m at concentration of 50 lM or left untreated
(control) for 48 h. Cell viability was analyzed by MTT assay and
illustrated in column figures. Results are presented as mean SE;
(n = 4; * P \ 0.05 vs control; ** P \ 0.01 vs control;
*** P \ 0.001 vs control); Compounds 4a and 4e were not tested
against MCF7, Raji, and HL60 cells due to poor solubility
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Med Chem Res (2013) 22:3109–3118
3113
Table 3 Calculated physicochemical properties of the synthesized compounds
Compound
Predicted oral bioavailability
Drug-likeness
HBAa
HBDb
M.Wc
clogPd
logSe
TPSAf
3
4
4
5
4
5
5
5
6
5
4
4
4
4
4
1
1
0
0
0
0
0
0
0
0
0
0
0
0
296.35
385.49
365.45
453.61
378.50
440.57
508.56
441.56
402.47
400.50
414.53
428.55
448.54
422.50
2.642
3.901
2.594
5.325
2.639
4.336
5.232
3.047
4.42
-3.41
-4.73
-3.11
-5.29
-2.61
-4.46
-5.23
-3.66
-4.89
-5.37
-5.74
-6.08
-6.96
-6.48
55.125
46.924
47.369
38.135
41.373
41.373
41.373
54.265
53.365
44.131
44.131
44.131
44.131
44.131
0.69
0.25
4a
4b
4c
4d
4e
4f
4g
4h
4i
1.58
3.5
6.94
7.06
-3.32
5.29
-1.83
-1.64
-2.65
-4.89
-1.42
-3.73
5.278
5.852
5.779
6.159
5.547
4j
4k
4l
4m
a
Number of hydrogen-bond acceptor
Number of hydrogen-bond donor
Molecular weight
b
c
d
e
f
Calculated lipophilicity
Solubility parameter
´
2
˚
Topological polar surface area (A )
antitumor activity. The data obtained by MTT assay
showed that amide derivatives 4a–g had inhibitory effects
on the growth of tested human cancer cells as shown in
Fig. 2. When chemical structure of the compounds are
taken into consideration, introduction of benzylpiperidin
moiety at the amide portion led to 4c with enhanced
activity against all the cancer cell lines with the best results
against Raji and HL60. The compound 4b with morpholine
was inactive against all tested tumor cell lines except
against MDA-MB-231 which showed poor inhibitor effect
on cell growth. Piperazine amide derivatives 4d, 4e, and 4g
displayed diminished activity. Interestingly, introduction of
p-fluoromethylphenyl piperazine group in amide portion
led to 4f with improved activity against especially Raji and
HL60 cells. Taken together, compound 4c was the most
effective compound in suppressing Raji and HL60 cells
growth (GI50 25.2 3.2 and 28.3 1.53 lM, respec-
tively). Ester derivatives of 1-phenyl-3-(thiophen-3-yl)-1H-
pyrazole-4-carboxylic acid 4h–m did not affect the growth
of HeLa, MCF7, MDA-MB-231, Raji, and HL60 cells.
We then determined the suitability of compounds for
anticancer purposes where a good absorption after oral
administration is obligatory. LogP, the measure of the
compound’s solubility and permeability, is believed to be
very important. Very high lipophilicity and the resulted
large logP values cause poor absorption or permeation and
should be avoided. Physicochemical properties of the
synthesized compounds were evaluated in silico, and found
that all compounds should present good passive oral
absorption. All the synthesized compounds demonstrated
good drug-likeness values.
Conclusion
A series of novel 1,3-diarylpyrazole derivatives has been suc-
cessfully synthesized. Structures of all products were deter-
mined by IR, 1H-NMR, and HRMS. We found that compounds
4c and 4f could suppress Raji and HL60 cancer cell growth.
Compound 4c bearing benzylpiperidine group in the amide
portion showed significant inhibitory effect against Raji and
HL60 cell lines. Owing to the significant results obtained,
chemical studies are being conducted to modify structures to
improve the antitumor activity of such compounds.
Experimental
Chemistry
The chemicals were purchased from the commercial
vendors and were used without purification. Thin-layer
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Med Chem Res (2013) 22:3109–3118
1-Phenyl-3-(thiophen-3-yl)-1H-pyrazole-4-carboxylic acid
3
chromatography (TLC) was performed on Merck 60F254
plates. Reactions were monitored by TLC on silica gel,
with detection by UV light (254 nm) or charring Drag-
endorff reagent (Stahl, 1969). Melting points were deter-
mined with an SMP-II Digital Melting Point Apparatus and
are uncorrected (Schorpp Geaetetechnik, Germany). IR
spectra were obtained in-house using a Perkin Elmer
Spectrum 400 FTIR/FTNIR spectrometer equipped with a
universal ATR Sampling Accessory. 1H-NMR spectra were
recorded in CDCl3 or DMSO-d6 on a Varian Mercury
400 MHz High Performance Digital FT-NMR spectrome-
ter using tetramethylsilane as the internal standard at the
NMR facility of Faculty of Pharmacy, Ankara University.
All chemical shifts were recorded as d (ppm). High-reso-
lution mass spectra data (HRMS) were collected in-house
using a Waters LCT Premier XE Mass Spectrometer (high-
sensitivity orthogonal acceleration time-of-flight instru-
ment) operating in either ESI (?) or ESI (-) methods, also
coupled to an AQUITY Ultra Performance Liquid Chro-
matography system (Waters Corporation, Milford, MA,
USA). Flash chromatography was performed with a
CombiflashÒ Rf automated flash chromatography system
with RediSep columns (Teledyne-Isco, Lincoln, NE, USA)
using dichloromethane-methanol solvent gradients.
To a solution of aldehyde (2 g, 7.86 mmol) in 100 ml
acetone–water mixture (3:2) potassium permanganate
(2.48 g, 15.7 mmol) was added and the corresponding dark
violet reaction mixture was stirred for 2.5 h to achieve full
conversion. The solvents were removed under vacuum and
the dark suspension was quenched upon addition of 20 ml
saturated sodium sulfite solution. The violet solid was fil-
tered off and washed with water. The turbid light brown
mother liquor was treated with HCl adjusting the pH to 1
and CH2Cl2 was added. Organic phase was washed with
water, dried over sodium sulfate, and evaporated under
vacuum. The precipitate obtained was purified by crystal-
lization in acetone–water mixture. IR (FT-IR/ATR):
1
1690 cm-1 (C=O), 2618 cm-1 (C=C). H-NMR (CDCl3)
d: 8.59 (1H, s, pyrazole H-5), 8.32 (1H, m, ArH), 7.79 (3H,
m, ArH), 7.51 (2H, m, ArH), 7.37 (2H, m, ArH). HRMS
C14H11N2O2S [M ? H]? Calc. 271.0541, Found m/z
271.0535.
General procedure for the preparation of amide/ester
derivatives of 1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-4-
carboxylic acid 4
N-Phenyl-N0-(1-thiophen-3-yl-ethylidene)hydrazine 1
To the solution of acid derivatives (1 mmol) triethylamine
(2 mmol) and ethyl chloroformate (1 mmol) were added,
followed by stirring at 0 °C for 30 min. After addition of
the appropriate amine or phenol derivative (1.2 mmol),
mixture was stirred for an additional 1 h at 0 °C. Then, the
reaction mixture was warmed to room temperature and
kept stirring overnight. After the solvent was evaporated
under reduce pressure, acetone was added, filtered, and
evaporated. Residue was dissolved in DCM and the organic
phase was washed with a 1 N HCl and 1 % NaHCO3
solutions and brine, dried over Na2SO4, and evaporated
under vacuum. The residue was purified by automated flash
column chromatography (CombiflashÒ Rf) using DCM-
MeOH (0–10 %) as eluents.
A solution of acetylthiophene (0.052 mol), phenyl hydra-
zine (0.058 mol), and acetic acid (2 ml, 0.035 mol) in
ethanol was stirred for 2 h at reflux, and then evaporated.
The precipitated was filtered off and dried. Yield 80 %; IR
1
(FT-IR/ATR): 3349 cm-1 (N–H), 1933 cm-1 (C=N). H-
NMR (DMSO-d6) d: 9.13 (1H, s, N–NH), 7.64 (1H, m,
ArH), 7.59 (1H, m, ArH), 7.51 (1H, m, ArH), 7.20 (4H, m,
ArH), 6.73 (1H, m, ArH), 2.23 (3H, s, C–CH3).
1-Phenyl-3-(thiophen-3-yl)-1H-pyrazole-4-carbaldehyde 2
In a dry flask, phosphoroxy chloride (POCl3) (16.77 ml,
0.18 mol) was added dropwise to an ice-cold stirred solu-
tion of hydrazone derivative (0.06 mol) in 100 ml DMF.
The reaction mixture was allowed to attain room temper-
ature, and then heated at 50 °C for about 5 h. The resulting
mixture was poured onto crushed ice, neutralized with
dilute NaOH, and left overnight. The precipitate obtained
was purified by crystallization in acetone–water mixture.
Yield 82 %, mp 124 °C (Lit. mp: 120 °C (Arbaciauskiene
et al., 2011); IR (FT-IR/ATR): 1667 cm-1 (C=O). 1H-
NMR (CDCl3) d: 10.10 (1H, s, CHO), 8.50 (1H, s, pyrazole
H-5), 8.19 (1H, m, ArH), 7.80–7.77 (2H, m, ArH), 7.70
(1H, m, ArH), 7.54–7.50 (2H, m, ArH), 7.44–7.38 (2H, m,
ArH). HRMS C14H11N2OS [M ? H]? Calc. 255.0592,
Found m/z 255.0586.
N-Benzyl-1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-4-carbox-
amide 4a IR (FT-IR/ATR): 1639 cm-1 (C=O), 3318
1
cm-1 (N–H). H-NMR (CDCl3) d: 8.45 (1H, s, pyrazole
H-5), 7.73 (2H, m, ArH), 7.68 (1H, m, ArH), 7.48–7.44
(2H, m, ArH), 7.40–7.21 (8H, m, ArH), 6.13 (1H, s, NH),
4.54 (2H, d, J = 5.6 Hz, NH–CH2). HRMS C21H18N3OS
[M ? H]? Calc. 360.1171, Found m/z 360.1162.
Morpholin-4-yl[1-phenyl-3-(thiophen-3-yl)-1H-pyrazol-4-
yl]methanone 4b IR (FT-IR/ATR): 1630 cm-1 (C=O).
1H-NMR (CDCl3) d: 8.09 (1H, s, pyrazole H-5), 7.73 (2H,
m, ArH), 7.65 (1H, m, ArH), 7.48 (1H, m, ArH), 7.47 (2H,
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3115
m, ArH), 7.40 (1H, m, ArH), 7.35 (1H, m, ArH), 3.70 (4H,
m, morpholine H-2,6), 3.29 (4H, m, morpholine H-3,5).
HRMS C18H18N3O2S [M ? H]? Calc. 340.1120, Found
m/z 340.1103.
HRMS C23H22N5OS [M ? H]? Calc. 416.1545, Found
m/z 416.1534.
4-Methoxyphenyl 1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-
4-carboxylate 4h IR (FT-IR/ATR): 1738 cm-1 (C=O).
1H-NMR (CDCl3) d: 8.67 (1H, s, pyrazole H-5), 8.39 (1H,
m, ArH), 7.83 (3H, m, ArH), 7.53 (2H, m, ArH), 7.40–7.32
(2H, m, ArH), 7.13 (2H, m, ArH), 6.95 (2H, m, ArH), 3.82
(3H, s, CH3). HRMS C21H17N2O3S [M ? H]? Calc.
377.0960, Found m/z 377.0956.
(4-Benzylpiperidin-1-yl)(1-phenyl-3-(thiophen-3-yl)-1H-pyr-
azol-4-yl)-methanone 4c IR (FT-IR/ATR): 1618 cm-1
1
(C=O). H-NMR (CDCl3) d: 8.04 (1H, s, pyrazole H-5),
7.73 (2H, m, ArH), 7.64 (1H, m, ArH), 7.50–7.44 (3H, m,
ArH), 7.36–7.23 (4H, m, ArH), 7.19 (1H, m, ArH), 7.08
(2H, m, ArH), 4.75–2.70 (4H, m, piperidin H-2,6), 2.46
(2H, s, CH2), 1.73–1.20 (4H, m, piperidin H-3,5), 0.71 (1H,
m, piperidin H-4). HRMS C26H26N3OS [M ? H]? Calc.
428.1797, Found m/z 428.1790.
4-Ethylphenyl 1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-4-
1
carboxylate 4i IR (FT-IR/ATR): 1714 cm-1 (C=O). H-
NMR (CDCl3) d: 8.67 (1H, s, pyrazole H-5), 8.39 (1H, m,
ArH), 7.81 (3H, m, ArH), 7.53–7.49 (2H, m, ArH),
7.40–7.36 (1H, t, J = 7.6 Hz, ArH), 7.32 (1H, m, ArH),
7.25 (2H, d, J = 8.8 Hz, ArH), 7.10 (2H, d, J = 8.4 Hz,
ArH), 2.69–2.63 (2H, q, J = 7.6 Hz, CH2–CH3), 1.26 (3H,
t, J = 7.2 Hz, CH2–CH3). HRMS C22H19N2O2S
[M ? H]? Calc. 375.1167, Found m/z 375.1150.
(4-Methylpiperazin-1-yl)[1-phenyl-3-(thiophen-3-yl)-1H-
pyrazol-4-yl]methanone 4d IR (FT-IR/ATR): 1619 cm-1
1
(C=O). H-NMR (CDCl3) d: 8.06 (1H, s, pyrazole H-5),
7.73 (2H, d, J = 7.2 Hz, ArH), 7.64 (1H, m, ArH),
7.50–7.44 (3H, m, ArH), 7.37 (1H, m, ArH), 7.33 (1H, m,
ArH), 3.80 (2H, m, piperazine H-5), 3.29 (2H, m, pipera-
zine H-3), 2.42 (2H, m, piperazine H-2), 2.23 (3H, s, CH3),
2.06 (2H, m, piperazine H-6). HRMS C19H21N4OS
[M ? H]? Calc. 353.1436, Found m/z 353.1432.
3-Isopropylphenyl 1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-
4-carboxylate 4j IR (FT-IR/ATR): 1744 cm-1 (C=O).
1H-NMR (CDCl3) d: 8.67 (1H, s, pyrazole H-5), 8.39 (1H,
m, ArH), 7.82 (3H, m, ArH), 7.51 (2H, m, ArH), 7.40–7.31
(3H, m, ArH), 7.13 (1H, m, ArH), 7.05 (2H, m, ArH), 2.93
(1H, m, CH–(CH3)2), 1.27 (6H, s, CH–(CH3)2). HRMS
C23H21N2O2S [M ? H]? Calc. 389.1324, Found m/z
389.1325.
[1-Phenyl-3-(thiophen-3-yl)-1H-pyrazol-4-yl)(4-phenylpi-
perazin-1-yl)methanone 4e IR (FT-IR/ATR): 1636 cm-1
1
(C=O). H-NMR (CDCl3) d: 8.11 (1H, s, pyrazole H-5),
7.73 (2H, m, ArH), 7.67 (1H, m, ArH), 7.52–7.46 (3H, m,
ArH), 7.37 (2H, m, ArH), 7.25 (2H, m, ArH), 6.90 (3H, m,
ArH), 3.93 (2H, s, piperazine H-5), 3.43 (2H, s, piperazine
H-3), 3.20 (2H, s, piperazine H-2), 2.81 (2H, s, piperazine
H-6). HRMS C24H23N4OS [M ? H]? Calc. 415.1593,
Found m/z 415.1576.
2-Isopropyl-5-methyl-phenyl 1-phenyl-3-(thiophen-3-yl)-
1H-pyrazole-4-carboxylate
4k IR
(FT-IR/ATR):
1729 cm-1 (C=O). H-NMR (CDCl3) d: 8.67 (1H, s, pyr-
azole H-5), 8.40 (1H, m, ArH), 7.82 (3H, m, ArH), 7.54
(2H, m, ArH), 7.41 (1H, m, ArH), 7.32 (1H, m, ArH), 7.23
(1H, m, ArH), 7.08 (1H, m, ArH), 6.94 (1H, s, ArH), 3.07
(1H, m, CH–(CH3)2), 2.34 (3H, s, CH3), 1.21–1.19 (6H, s,
CH–(CH3)2). HRMS C24H23N2O2S [M ? H]? Calc.
403.1480, Found m/z 403.1465.
1
[1-Phenyl-3-(thiophen-3-yl)-1H-pyrazol-4-yl)[4-(4-trifluo-
romethylphenyl)-piperazin-1-yl]-methanone 4f IR (FT-
1
IR/ATR): 1615 cm-1 (C=O). H-NMR (CDCl3) d: 8.12
(1H, s, pyrazole H-5), 7.75 (2H, d, J = 8 Hz, ArH), 7.66
(1H, m, ArH), 7.51–7.46 (5H, m, ArH), 7.38 (2H, m, ArH),
6.86 (2H, d, J = 8.4 Hz, ArH), 3.92 (2H, m, piperazine
H-5), 3.43–3.30 (4H, m, piperazine H-2,3), 2.89 (2H, m,
piperazine H-6). HRMS C25H22F3N4OS [M ? H]? Calc.
483.1466, Found m/z 483.1467.
Biphenyl-4-yl 1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-4-
1
carboxylate 4l IR (FT-IR/ATR): 1698 cm-1 (C=O). H-
NMR (CDCl3) d: 9.00 (1H, s, pyrazole H-5), 8.70 (1H, m,
ArH), 8.13 (3H, m, ArH), 7.94–7.89 (2H, m, ArH),
7.89–7.69 (8H, m, ArH), 7.58–7.49 (2H, m, ArH). HRMS
C26H19N2O2S [M ? H]? Calc. 423.1167, Found m/z
423.1174.
[1-Phenyl-3-(thiophen-3-yl)-1H-pyrazol-4-yl)(4-pyridin-4-
yl-piperazin-1-yl)-methanone
4g IR
(FT-IR/ATR):
1630 cm-1 (C=O). 1H-NMR (CDCl3) d: 8.28 (2H, d,
J = 6.8 Hz, pyridine H-2,6), 8.12 (1H, s, pyrazole H-5),
7.74 (2H, m, ArH), 7.66 (1H, m, ArH), 7.50–7.46 (3H, m,
ArH), 7.38–7.32 (2H, m, ArH), 6.58 (2H, d, J = 6.8 Hz,
pyridine H-3,5), 3.88–2.99 (8H, m, piperazine H-2,3,5,6).
Naphthalen-2-yl 1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-
4-carboxylate 4m IR (FT-IR/ATR): 1732 cm-1 (C=O).
1H-NMR (CDCl3) d: 8.96 (1H, s, pyrazole H-5), 8.61 (1H,
d, J = 2.8 Hz, ArH), 8.13–8.03 (6H, m, ArH), 7.89 (1H, d,
123

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Med Chem Res (2013) 22:3109–3118
J = 2.4 Hz, ArH), 7.76–7.69 (4H, m, ArH), 7.63–7.55
(3H, m, ArH). HRMS C24H17N2O2S [M ? H]? Calc.
397.1011, Found m/z 397.1008.
Statistical significance of the effects of compounds to
the cell viability was analyzed by one-way ANOVA and
Tukey’s post hoc test using SigmaStat v3.5 software.
P values \0.05 were considered to be statistically signifi-
cant. (* P B 0.05, ** P B 0.01, *** P B 0.001).
Anticancer activity
Lipinski’s rule-of-five and drug-likeness profile
Cell lines and cell culture
In order to explore the bioavailability of synthesized deriv-
atives, theoretical calculations were carried out to predict
some physicochemical properties of the synthesized com-
pounds. The bioavailability of the compounds was assessed
using ADME (absorption, distribution, metabolism, and
elimination) prediction methods. In particular, we calculated
the compliance of compounds to the Lipinski’s rule-of-five
(Lipinski et al., 2001). Lipinski’s ‘‘rule-of-five’’ and the later
addition of other parameters such as polar surface area
(PSA) (Ertl et al., 2000) describes molecular properties
important for a drug pharmacokinetics in the human body.
This approach has been widely used as a filter for substances
that would likely be further developed in drug design pro-
grams. Poor absorption and permeation are more likely to
occur when there are more than five hydrogen-bond donors
(HBD), more than ten hydrogen-bond acceptors (HDA), the
molecular mass (MM) is greater than 500, or the log P value
(clogP) is greater than five. The topological polar surface
The human cancer cell lines, cervical carcinoma (HeLa),
estrogen receptor positive breast carcinoma (MCF7),
estrogen receptor negative breast carcinoma (MDA-MB-
231), Burkitt’s lymphoma (Raji), and human promyelo-
cytic leukemia (HL60), were obtained from ATCC.
Cytotoxicity assay
MDA-MB-231 and MCF7 cells were cultured in DMEM;
whereas, HeLa, HL60, and Raji cells were grown in RPMI-
1640 medium in a humidified atmosphere containing 5 %
CO2 at 37 °C. Both DMEM and RPMI-1640 medium were
supplemented with 10 % fetal bovine serum (FBS),
200 mM L-glutamine, 100 IU/ml penicillin, and 100 lg/ml
streptomycin (all from Hyclone Laboratories, Logan, UT,
USA). Cell viability was determined using MTT (3-[4,5-
˚
area (TPSA) should be smaller than 90 A (Brueggemeier
dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium
bro-
mide) assay (Cell Proliferation Kit I, Roche, Germany).
Briefly, cells were seeded in a 96-well plate at 10.000 cells
per well and cultured for overnight in growth medium
containing 1 % FBS. Then cells were treated with 50 lM
of the test compounds for 48 h. As a solvent control, cells
were also treated with dimethyl sulfoxide (DMSO) at a
final concentration of 0.1 %. At the end of the incubation
time, MTT reagent at the final concentration of 0.5 mg/ml
was added to each well and incubated for an additional 4 h.
After formation of blue formazan crystals, medium con-
taining MTT was discarded and DMSO was added to the
wells to dissolve crystals. The absorbance of samples was
measured with Spectra Max M3 micro plate reader
(Molecular Devices, Sunnyvale, CA, USA) at a wavelength
of 570 nm. Average absorbance values from quadruplicate
replicates per test compound and solvent control (DMSO)
were calculated. Mean solvent control values were set to
100 % of viability and then the effects of the test com-
pounds to the cell viability were calculated by comparing
mean values obtained from compound treated culture wells
with those of the solvent controls. As the compounds
exhibited to reduction on cell viability more than 50 % at
50 lM, the GI50 values were also calculated from the
concentration-response curves using the PRISM 5,
GraphPad Software (GraphPad Prism Version 5.04, 2010).
et al., 2005). Molecules violating more than one of these
rules may have problems with bioavailability.
Acknowledgments This investigation was partially supported by
Scientific Research Grant 02/2011-08, awarded by Gazi University
BAP.
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