Synthesis, cytotoxicity, and molecular properties prediction of novel 1,3-diarylpyrazole derivatives

Article abstract of DOI:10.1007/s00044-013-0505-8

A novel combinatorial library of ester and amide derivatives of 1,3-diarylpyrazoles was designed and synthesized. Anticancer activities of these compounds were assessed against MCF7, MDA-MB-231, HeLa, Raji, and HL60 human cancer cells by MTT assay. Out of

Full text of DOI:10.1007/s00044-013-0505-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4893–4908
DOI 10.1007/s00044-013-0505-8
ORIGINAL RESEARCH
Synthesis, cytotoxicity, and molecular properties prediction
of novel 1,3-diarylpyrazole derivatives
•
Sultan Nacak Baytas Nazan Inceler
•
Akın Yılmaz
Received: 8 November 2012 / Accepted: 17 January 2013 / Published online: 31 January 2013
Ó Springer Science+Business Media New York 2013
Abstract A novel combinatorial library of ester and
amide derivatives of 1,3-diarylpyrazoles was designed and
synthesized. Anticancer activities of these compounds were
assessed against MCF7, MDA-MB-231, HeLa, Raji, and
HL60 human cancer cells by MTT assay. Out of these,
compounds 4c and 5f were found as the most promising
anticancer agents with IC50 values of 8.12 and 9.63 lM in
Raji cells, respectively. All compounds exhibited suitable
drug-like characteristics according to Lipinski’s rule.
problems completely. Due to several side effects, drug
resistance and failure of antitumor drugs in certain cases of
cancer, the design and discovery of non-traditional, efficient
and safe chemical classes of agents are prime targets in
contemporary medicinal chemistry (Cozzi, 2003; Johnston
et al., 2005). A key feature of cancer cells is their uncon-
trolled proliferation, thus, inhibition of proliferative path-
ways is believedto be an effective strategy to fight cancerand
much attention has recently been paid to the discovery and
development of new, more selective anticancer agents.
Pyrazoles constitute an important heterocyclic family
containing nitrogen in their five-membered ring. Several
pyrazole derivatives have been found to possess consider-
able biological activities, which have stimulated numerous
research activities in medicinal chemistry or chemical biol-
ogy (Silveira et al., 1993; Sridhar et al., 2004; Nagarapu
et al., 2011; Padmaja et al., 2009; Gaston et al., 1996, Baytas
et al., 2012; Banoglu et al., 2007). 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). The literature survey has 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, besides their capabilities to exert
remarkable anticancer effects through inhibiting different
types of enzymes that play important roles in cell division
(Warshakoon et al., 2006; Huang et al., 2007). Although the
skeleton of the pyrazole is an important building block in
biological effects, the type of peripheral substituents is also
crucial. The studies concerning structure–activity relation-
ships have shown that the cytotoxic potency of the com-
pounds has been highly dependent on the substitution types
and patterns on the aryl ring. Among the reported studies,
Keywords Synthesis ꢀ Anticancer activity ꢀ
1H-Pyrazoles ꢀ Pyridine ꢀ MTT
Introduction
Cancer is the second leading cause of death in developed
countries, accounting for nearly one in five deaths. Che-
motherapy is a major form of cancer treatment. However,
majority of cancers are either resistant to chemotherapy or
acquire resistance during treatment. Several important
drugs including 5-flurouracil (5FU), doxorubicin, cele-
coxib, and mitoxantrone with different structures and
mechanisms of antitumor activities, fail to end these
S. N. Baytas (&) ꢀ N. Inceler
Division of Pharmaceutical Sciences, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy,
Gazi University, 06330 Etiler, Ankara, Turkey
e-mail: sbaytas@gmail.com; baytas@gazi.edu.tr
A. Yılmaz
Department of Medical Biology and Genetics, Faculty of
Medicine, Gazi University, 06500 Bes¸evler, Ankara, Turkey
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Med Chem Res (2013) 22:4893–4908
pyrazole-5-carboxylate (Wei et al., 2006; Zheng et al.,
2010), pyrazole-5-carboxamide (Ding et al., 2009), and
pyrazole-5-carbohydrazide (Lian et al., 2009; Xia et al.,
2007; Xia et al., 2008; Zheng et al., 2009) derivatives have
been shown to have significant anticancer activities against
A549 lung cancer cell lines by inducing apoptosis or
autophagy. Wei et al. demonstrated that ethyl 1-(20-hydroxy-
30-aroxypropyl)-3-aryl-1H-pyrazole-5-carboxylate deriva-
tives suppressed lung cancer cell growth (Wei et al., 2006).
They found that ethyl 1-arylmethyl-3-aryl-1H-pyrazole-
5-carboxylate derivatives promoted human umbilical vein
endothelial cell (HUVEC) apoptosis to certain extents at
concentrations of 5–20 lM (Ding et al., 2007). Oxime-
containing pyrazole derivatives (1A in Fig. 1) were synthe-
sized by Zheng et al., and it was found that their effects on
dose- and time-dependent inhibition of proliferation were
mainly attributed to the autophagy induction in lung cancer
cells (Zheng et al., 2010). The effects of 3-aryl-1-(4-tert-
butylbenzyl)-1H-pyrazole-5-carbohydrazidehydrazonederiv-
atives on A549 cell growth were investigated and it was found
that 1B (Fig. 1) possessed the highest growth inhibitory effect
and induced apoptosis with IC50 value of 0.28 lM (Zheng
et al., 2009). It was suggested that the potency of 3-aryl-1-
arylmethyl-1H-pyrazole-5-carbohydrazide hydrazone against
A549 lung cancer cells related with the lipophilicity of the
compound and depended on the capacity of compound che-
lating metal ions, because it was recognized that metal ions
have important roles in the cell growth (Xia et al., 2008). In
another study, Huang et al. determined that N-((1,3-diphenyl-
1H-pyrazol-4-yl)methyl)aniline derivatives showed potent
anti-CDK2/cyclin E activity (Huang et al., 2012). Com-
pound 2 showed the most potent inhibition activity which
inhibited the growth of MCF7 and B16-F10 cell lines with
IC50 values of 1.88 and 2.12 lM and inhibited the CDK2/
cyclin E holoenzyme activities with IC50 of 0.98 lM
(Fig. 1).
In our previous work, 1,3-diarylpyrazole derivatives con-
taining thiophene were synthesized and the effects of the
compounds on MCF7, MDA-MB-231, HeLa, Raji, and HL60
human cancer cells growth were investigated. We found that
(4-benzyl-piperidin-1-yl)-(1-phenyl-3-thiophen-3-yl-1H-pyr-
azol-4-yl)methanone 3 (Fig. 1) possessed the highest growth
inhibitory effect on Raji and HL60 cancer cells (Inceler et al.,
2013). On the basis of all those observations, we considered
to design and synthesize a new type of 1,3-diarylpyrazole,
wherein potent 3-/4-pyridine moiety was linked to biologi-
cally active phenyl pyrazole moiety at C-3 position and dif-
ferent ester or amide groups at C-4 position on the basis of
combinatorial synthesis, which is the current trend being
practiced in most of the drug discoveries. In this article, we are
also interested in exploring the biological activities of such
molecules through structural modifications.
Results and discussions
Chemistry
We focused our synthetic efforts on diaryl heterocyclic ring
systems as illustrated in Scheme 1. First, the hydrazone
Fig. 1 Structure of the some
pyrazole derivatives with
anticancer activity (1A, 1B, and
2), the lead compound (3) and
general structure of the
R1
Cl
Cl
F
N
synthesized compounds
O
COO2Et
R3
N
Cl
N
H
N
N
N
HN
OH
N
N
N
HO
2
t-Bu
1A
1B
R2
N
5
N
4
S
O
N
O
R
N
R
Ar
R
N
N
R
N
N
O
3
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Med Chem Res (2013) 22:4893–4908
4895
Scheme 1 (a) Phenyl
hydrazine, acetic acid, ethanol,
2 h reflux; (b) Method A
dimethyl formamide, POCl3,
50 °C, 4 h; Method B dimethyl
formamide, POCl3, MW
irradiation, 210 W, 50 °C,
20 min; (c) potassium
Ar
b
N
Ar
a
N
Ar
CH3
HN
N
H
O
O
7a,b
6a,b
permanganate, acetone–water
(3:2), 1 h reflux; (d) appropriate
amine or phenol derivative,
ethyl chloroformate,
triethylamine, dichloromethane,
rt, overnight
Ar
Ar
COOH
N
c
N
d
N
N
COR
8a,b
4 Ar= 3-pyridyl
5 Ar= 4-pyridyl
derivative 6 was generated by condensing 3-/4-acetylpyri-
dine and phenylhydrazine in the presence of acetic acid in
refluxing ethanol. The IR spectra of hydrazone 6 showed
disappearance of the carbonyl peak that belonged to acetyl
group of acetylpyridine derivatives. In the IR spectra of
hydrazones 6a and 6b, secondary N–H stretching bands were
observed at 3169 and 3274 cm-1 as unspiked, respectively.
This hydrazone derivative was then reacted with phosphor-
oxy chloride (POCl3) and DMF resulting in 1,3-diaryl pyr-
azole 7 with aldehyde group at 4 position. We focused our
attention on the microwave-assisted synthesis technique
after obtaining compound 7. Microwave-assisted organic
synthesis is a technique which can be used to rapidly explore
the ‘‘chemistry space’’ and to increase the diversity of the
compounds produced. Nowadays, it is possible to perform
many of previous conventional heated reactions, using this
technique (Lidstrom et al., 2001). Microwave irradiation
dominates over usual conventional methods due to its non-
hazardous and eco-friendly nature. The microwave-induced
organic reaction received considerable attention due to its
simplicity and operational convenience. The use of micro-
wave irradiation as a heat source in synthetic chemistry
offers a promising alternative (Jyothi et al., 2007; Kalluraya
et al., 2008). So, these reactions were performed in a
microwave oven due to its low cost and availability. In a
typical experiment, in order to synthesize compound 7,
POCl3 was added dropwise to an ice-cold stirred solution of
hydrazone 6 in DMF. The reaction mixture was allowed to
attain room temperature, and then heated at 50 °C for 4 h.
Following the work-up, desired compounds were obtained.
Comparing the two methods, microwave-assisted synthesis
technique dramatically cut down reaction time (conventional
method time 4 h, microwave-assisted synthesis time
20 min), and increased product yields as shown in the
‘‘Experimental’’ section (conventional method yield 80 %,
microwave-assisted synthesis yield 92 %). The IR spectra of
compounds 7a, 7b characteristically exhibited strong
absorptions for aldehyde carbonyl group at 1673 and
1669 cm-1, respectively. The 1H-NMR spectrum of the
compounds 7a and 7b showed disappearance of the methyl
proton signal and N–N–H signal. In the 1H-NMR spectra of
compound 7, two bands were displayed due to aldehydic
C–H (at 10.06 ppm for 7a; at 10.04 ppm for 7b) and pyrazole
(at 8.58 for 7a; at 9.43 ppm for 7b), each denoting one
proton, after integration. 1-Phenyl-3-pyridin-3-/4-yl-1H-
pyrazole-4-carbaldehyde 7 was oxidized to carboxylic acid
derivative 8 in the presence of potassium permanganate in
acetone–water mixture. In the IR spectra of compounds 8a, 8b
strong absorption bands at 1678 and 1709 cm-1, respectively,
and intense O–H stretching absorption in the region of
3300–2500 cm-1 for carboxylic acid, were observed. In the
1H-NMR spectrum of compound 8a, signal of the car-
boxylic acid appeared at 12.74 ppm, but for 8b, signal of
the carboxylic acid was not observed due to proton
exchange. The signals of the pyrazole C–H were seen at
9.16 and 9.14 ppm for 8a and 8b, respectively. By treat-
ment of 8 with appropriate amines or phenols in the pres-
ence of ethyl chloroformate, which was used as the
carboxylate activator, and triethylamine in dichlorometh-
ane resulting amide and ester derivatives 4a–4m and 5a–5n
were prepared in good yield (43–82 %). Compounds were
purified by automated flash chromatography and were
checked for purity using a UPLC before they were tested in
biological assays (purity was [97 %). The structures of
these compounds were confirmed by high resolution mass
1
spectrometry (HRMS), IR and H- and 13C-NMR spectral
data. Final amide and ester derivatives characteristically
exhibited strong absorption in the area of 1597–1746 cm-1
attributable to the C=O of amide or ester groups. In the
1H-NMR spectra of final compounds 4a–4m and 5a–5n,
pyrazole C–H gave singlets in the 9.03–8.08 ppm range.
Effects of the compounds on the viability of cancer
cells
Synthesized
1-phenyl-3-(pyridine-3-/4-yl)-1H-pyrazole-
4-carboxylic acid 8a, 8b and its amide and ester derivatives
4a–4m and 5a–5n 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
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Med Chem Res (2013) 22:4893–4908
bromide (MTT) assay. The MTT cell proliferation assay is
widely accepted as a reliable method to measure the cell
proliferation rate (Holst-Hansen and Bru¨nner, 1998; Reile
et al., 1990; Kueng et al., 1989; Senaratne et al., 2000). In
order to measure the growth/viability (% of the untreated
control), a spectrophotometer was used as described previ-
ously (Mosmann, 1983; Denizot and Lang, 1986). The IC50
values (Table 1) were calculated from the concentration–
response curves by means of the PRISM 5, GraphPad Soft-
ware (GraphPad Prism Version 5.04 for Windows, 2010).
In human cervical carcinoma cells (HeLa), IC50 values
for the compounds 4c, 4f, 5c, and 5k were in the range of
25.26–33.56 lM (Table 1). Rest of the compounds did not
show 50 % inhibition in HeLa cells, even at a concentration
of 100 lM. Solubility problems occurred with compounds
4a, 4l, 4m, 5i, 5j, and 5m, hence they were not evaluated in
MCF7, Raji, and HL60 cell lines. Present study revealed
that among the breast cancer cell lines tested, human
estrogen receptor positive breast adenocarcinoma cells
(MCF7) are more sensitive to tested compounds than
estrogen-independent breast cancer cells (MDA-MB-231).
Compounds 4c, 5k, and 5n exhibited moderate activity
towards the MCF7 cell line (IC50 32.24, 32.25, and
44.53 lM, respectively). Compounds 4f, 4i, 4k, and 5c
were found less active against MCF7 cells, and their IC50
values were calculated as higher than 50 lM. Tested ester
and amide derivatives of 1-phenyl-3-(pyridine-3-/4-yl)-1H-
pyrazole-4-carboxylic acid 4a–4m and 5a–5n did not affect
the growth of MDA-MB-231 cells except for compound 5k,
which is an tert-buthylphenyl ester of pyrazol-4-carboxylate
(IC50 33.56 lM). The tested compounds were found more
effective in B lymphocyte cell line (Raji). In Raji cells, IC50
values for the compounds 4a–4m were in the range of 8.12
and 75.64 lM. Compound 4c (IC50 8.12 lM, carrying
3-pyridyl moiety on pyrazole ring and benzyl piperidine at
amide portion) followed by 5f (IC50 9.63 lM, carrying
4-pyridyl moiety on pyrazole ring and tert-buthylphenyl-
piperazine at amide portion) displayed significant antitumor
activities. Compounds 4f, 4i, 4k, 5c, 5l, and 5n also showed
significant cytotoxic activity in Raji cells (IC50 15.12, 11.57,
20.56, 21.3, 10.59, and 12.05 lM, respectively). The effi-
ciencies of compounds 4e, 4j, and 5k were found to be
moderate with IC50 values of 32.84, 42.26, and 33.27 lM,
respectively. Finally, for HL60 leukemia cell line, 4c (IC50
16.14 lM), 4f (IC50 16.91 lM), and 5c (IC50 16.35 lM)
followed by 5k (IC50 18.72 lM) and 5l (IC50 17.84 lM)
displayed significant antitumor activity at 48 h. Compounds
4e, 4i, and 4k were showed equipotent inhibitory activity
having the considerable growth inhibition at the end time
point (IC50 24.52, 23.69, and 22.65 lM, respectively).
shown better inhibitory effects on the growth of tested
human cancer cells than ester derivatives as indicated by
the results in Table 1. When chemical structures of the
compounds are taken into consideration, introduction of
benzylpiperidin moiety at amide part of pyrazole-3-car-
boxamide core in both 3- and 4-pyridin series, led to 4c and
5c with enhanced activities against all the cancer cell lines
with the best results against Raji cells. In our previous
study, 1,3-diarylpyrazole derivatives containing thiophene
moiety were synthesized and their anticancer effects were
evaluated. It was found that the (4-benzyl-piperidin-1-yl)-
(1-phenyl-3-thiophen-3-yl-1H-pyrazol-4-yl)methanone (3
in Fig. 1) possessed the highest growth inhibitory effect on
Raji and HL60 cancer cells (IC50 25.2 and 28.3 lM,
respectively). In present work, especially against Raji cells,
4c, bearing 4-benzyl-piperidin moiety, displayed inhibitory
activity on cell viability at 48 h, being the most efficient
derivative with IC50 value of 8.12 lM. Compound 5c was
also found to be very potent inhibitor on cell viability against
Raji and HL60 cancer cells (IC50 21.3 and 16.3 lM,
respectively). Taken these results into consideration, we
can conclude that 4-benzyl-piperidin moiety at amide part of
1,3-diarylpyrazole-4-carboxamide core enhances anticancer
activity, complying with our previous findings. By intro-
ducing tert-buthylphenylpiperazine to amide part, com-
pounds 4f (IC50 15.12 lM) and 5f (IC50 9.63 lM) were
obtained, and both showed significant anticancer activities.
The enhanced activities of compounds 4c, 4f, 4i, 5c, 5f, 5l,
and 5n were mainly attributed to the presence of biologically
active groups like 4-benzyl-piperidin and tert-buthylphenylpip-
erazine groups at amide part of the 1,3-diarylpyrazole-4-car-
boxamide core. For ester derivatives, 3-isopropylphenoxy,
2-isopropyl-5-methylphenoxy, and 2-naphthyloxy groups seem
effective in anticancer activities in these types of com-
pounds. This activity was attributed to the presence of
electronegative groups in the molecules which increase the
lipophilicity and affect the partitioning of molecules into
membranes and facilitate hydrophobic interactions of the
molecules with specific binding sites on either receptor or
enzymes. Many anticancer drugs are effective against MCF7
and A549 cells, through causing apoptosis. Hence like the
cytotoxic drugs, the synthesized compounds can be effective
anticancer drugs through a similar mechanism (Leong et al.,
2003; Mizutani, 2007).
Lipinski’s rule of five and drug-likeness profile
Physicochemical properties of synthesized compounds
The synthesized compounds 8a, 8b, 4a–4m, and 5a–5n
were submitted to an in silico evaluation using a molecular
modeling approach. To predict drug-like properties of
synthesized compounds, we analyzed these derivatives
It is possible to relate the structural characteristics of the
compounds to their antitumor activity. The data obtained
by MTT assay showed that the amide derivatives have
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Med Chem Res (2013) 22:4893–4908
4897
Table 1 In vitro cytotoxic activity of the synthesized compounds (8a, 8b; 4a–4m; and 5a–5n)
O
Ar
R
N
Ar = 3-Pyridine
4-Pyridine
N
Compounds Ar
R
IC50 (lM)a
HeLa
MDA-MB-231 MCF7
Raji
HL60
8a
8b
4a
3-Pyridine OH
4-Pyridine OH
3-Pyridine
[100
[100
[100
[100
[100
[100
[100
[100
NT
[100
[100
NT
[100
[100
NT
NH
N
4b
4c
4d
4e
4f
3-Pyridine
3-Pyridine
3-Pyridine
3-Pyridine
3-Pyridine
3-Pyridine
3-Pyridine
3-Pyridine
[100
[100
[100
[100
[100
O
25.26 – 2.23 [100
32.24 – 1.39 8.12 – 1.23
16.14 – 1.47
N
N
[100
[100
[100
[100
[100
[100
75.64 – 2.93 [100
N CH3
32.84 – 5.33 24.52 – 3.51
N
N
N
N
N
N
28.55 – 5.12 [100
75.34 – 3.90 15.12 – 0.88 16.91 – 0.12
F
F
F
4g
4h
4i
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
N
O
O
O
75.52 – 2.65 11.57 – 1.87 23.69 – 2.53
4j
3-Pyridine
3-Pyridine
[100
[100
[100
[100
[100
42.26 – 4.39 [100
O
O
4k
84.20 – 2.23 20.56 – 2.24 22.65 – 1.59
4l
3-Pyridine
3-Pyridine
4-Pyridine
[100
[100
[100
[100
[100
[100
NT
NT
NT
O
O
4m
5a
NT
NT
NT
[100
[100
[100
NH
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Med Chem Res (2013) 22:4893–4908
Table 1 continued
Compounds Ar
R
IC50 (lM)a
HeLa
MDA-MB-231 MCF7
[100 [100
Raji
HL60
5b
5c
4-Pyridine
[100
[100
[100
N
O
4-Pyridine
32.81 – 1.11 [100
56.42 – 1.33 21.3 – 1.03
16.35 – 0.82
N
N
5d
5e
4-Pyridine
4-Pyridine
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
N CH3
N
N
F
F
5f
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
N
N
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
NT
9.63 – 3.59
[100
[100
NT
[100
[100
[100
NT
F
5g
5h
5i
N
N
N
O
O
O
O
5j
NT
NT
NT
5k
5l
35.43 – 3.62 34.52 – 3.58
32.25 – 0.82 33.27 – 1.63 18.72 – 2.65
O
O
[100
[100
[100
[100
[100
[100
[100
10.59 – 1.77 17.84 – 3.44
5m
NT
NT
NT
O
O
5n
44.53 – 4.97 12.05 – 3.62 [100
NT not tested
Compounds with IC50 values lower than 100 lM are given in bold
a
Each experiment was independently performed four times and expressed as mean SD
according to the rule-of-five developed by Lipinski et al.
(2001). Lipinski’s ‘‘rule-of-five’’ and the later addition of
other parameters such as PSA (Ertl et al., 2000) describes
molecular properties important for a drug’s pharmacoki-
netics in the human body. PSA has been shown to be a very
good descriptor characterizing drug absorption, including
intestinal absorption, bioavailability, Caco-2 permeability,
and blood–brain barrier penetration. This approach has
been widely used as a filter for substances that are likely be
further developed in drug design programs. Log P, the
measure of the compound’s solubility and permeability, is
believed to be very important. Very high lipophilicity and
the resulted large log P values cause poor absorption or
permeation and should be avoided.
Predictions of ADME properties for these compounds
are given in Table 2. Calculated physicochemical proper-
ties (http://www.molinspiration.com) showed that most of
the compounds fulfilled the Lipinski’s ‘‘rule-of-five’’. Theo-
retically, these compounds should present good passive
oral absorption and differences in their bioactivity cannot
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Med Chem Res (2013) 22:4893–4908
4899
be attributed to this property. Compounds 4i, 4j, 4k, 4l, 5j, 5k,
5l, and 5m had very large clog P values of 5.20, 5.42, 5.15,
5.51, 4.99, 5.20, 4.91, and 5.29, respectively, which might be
disadvantageous with regard to pharmacokinetic properties of
these molecules in biological systems. The rest of the com-
pounds exhibited favorable clog P values. Along with this,
compounds 4c and 5f, which showed good antitumor
screening results (Raji cells, IC50 8.12 and 9.63 lM, respec-
tively), have optimal clog P values, compared to other com-
pounds in the series. Our results indicated that compounds
possessing lipophilicity with log P values in the range of
3.15–5.31 (4c, 4e, 4f, 4i, 4k, 5c, 5f, 5k, 5l, and 5n) demon-
strated better inhibitory effects on the growth of selected
cancer cells. Our results showed good correlations with pre-
viously published anticancer activities of similar pyrazole
derivatives in which they showed growth in inhibitory effect
on A549 lung cancer cells with log P values in the range of
3.12–4.94 (Xia et al., 2007, 2008). The total polar surface area
(TPSA) was calculated based on the methodology published
by Ertl et al. (2000) as sums of O- and N-centered polar
fragments contributions. The PSA is closely related to the
hydrogen bonding potential of a compound. The TPSAs of the
synthesized compounds were relatively small in comparison
with the average value for acceptable drug molecules
compounds demonstrated good drug-likeness values (from
8.99 to 0.32). A positive value states that the molecule con-
tains predominantly fragments whicharefrequentlypresent in
commercial drugs. Activities of all the compounds were
analyzed under the four known criteria for successful drug
activities in GPCR ligand, ion channel modulation, kinase
inhibition, and nuclear receptor ligand activities. Results for
all compounds are shown in Table 2. Values between -1.00
and 1.50 state that molecules may be active.
Moreover, we used the Osiris program for prediction of
overall toxicity of the derivatives as it may point to the
presence of some fragments generally responsible for the
mutagenic, tumorigenic, irritant, or reproductive effects in
these molecules. The toxicity risk predictor locates frag-
ments within a molecule, which indicate a potential toxicity
risk. Toxicity risk alert is an indication that the drawn
structure may be harmful concerning the risk category
specified. Only compounds containing 3-isopropyl-phenyl
residue (compounds 4i and 5j) and naphthalen-2-yl moiety
(4m and 5n) showed in silico irritant and mutagenic prop-
erties, respectively. The rest of the compounds presented low
in silico toxicity risk profile. In this study, we also examined
the drug score. The drug score combines drug-likeness,
clog P, log S, molecular weight, and toxicity risks in one
handy value, and may be used to judge the compound’s
overall potential to qualify for a drug. Our results showed
that the all synthesized compounds including the two of most
potent ones demonstrated good drug score values (Table 3).
2
˚
(\90 A ). It has to be kept in mind that log P and PSA values
are the most important two features, although not sufficient
for predicting oral absorption of a drug.
Drug-likeness, drug score and toxicity risk profile
of compounds (8a, 8b; 4a–4m; 5a–5n)
Conclusion
Currently, there are many approaches to assess a compound’s
drug-likeness based on topological descriptors, fingerprints of
molecular drug-likeness structure keys (Tetko, 2005). In this
study, we used the Osiris program (http://www.organic-
chemistry.org/prog/peo.) for calculating the physicochemical
properties (solubility, drug-likeness, and drug score) and the
toxicity risks (mutagenicity, tumorogenicity, irritation, repro-
duction) of the compounds 8a, 8b; 4a–4m; 5a–5n (Table 3).
Aqueous solubility of a compound significantly affects its
absorption and distribution characteristics. Typically, a low
solubility goes along with a bad absorption and therefore the
general, the aim is to avoid poorly soluble compounds. More
than 80 % of the drugs on the market have a (estimated)
log S value ofgreater than -4. The log S valuesof most ofour
compounds 8a, 8b; 4a–4m; 5a–5n, are around -4. Drug-
likeness may be defined as a complex balance of various
molecular properties and structural features which determine
whether a particular molecule is similar to known drugs.
These properties influence the behavior of a molecule in a
living organism, including bioavailability, transport proper-
ties, affinity to proteins, reactivity, toxicity, metabolic stabil-
ity, and many others. It is interesting that, most of our
This study demonstrates the synthesis of a novel combinatorial
library of a series of 1,3-diarylpyrazoles and the biological
evaluation of their cytotoxic activities. Physicochemical
properties of synthesized compounds were also evaluated in
silico, and it was found that all compounds should present good
passive oral absorption. All synthesized compounds demon-
strated good drug-likeness values. The preliminary antitumor
studies revealed that these agents exhibited significant antitu-
mor activity in inhibiting various human tumor cell growths.
The cytotoxic study results revealed that the compounds 4c and
5f were the most promising cytotoxic agents with IC50 values
of 8.12 and 9.63 lM, respectively, in Raji cells. The present
studies generated a series of new potent antitumor agents,
which have potential for further antitumor drug development.
Experimental
Chemistry
The chemicals were purchased from the commercial vendors
and were used without purification. Thin-layerchromatography
123

4900
Med Chem Res (2013) 22:4893–4908
Table 2 Calculated physicochemical properties of the synthesized compounds
Compounds
Predicted oral bioavailibility
Bioactivity scores
HBAa
HBDb
M.Mc
clog Pd
Volume
TPSAe
GPCRLf
ICMg
KIh
NRLi
PIj
EIk
8a
8b
4a
4b
4c
4d
4e
4f
5
5
5
6
5
6
6
6
7
6
5
5
5
5
5
5
6
5
6
6
6
7
6
5
5
5
5
5
5
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
265.2
265.2
354.4
334.3
422.5
347.4
409.5
477.5
410.4
371.3
383.4
397.4
397.4
417.4
391.4
354.4
334.3
422.5
347.4
409.5
477.2
410.4
371.3
369.4
383.4
397.4
397.4
417.4
391.4
1.76
1.54
2.78
1.00
3.73
1.05
2.74
3.64
1.45
3.77
5.20
5.42
5.15
5.51
4.90
2.56
0.78
3.51
0.83
2.52
3.42
1.23
3.55
4.41
4.99
5.20
4.91
5.29
4.68
230
230
323
301
397
321
376
407
372
328
353
369
369
374
347
323
301
397
321
376
407
372
328
336
353
369
369
374
347
68.01
68.01
59.81
60.26
51.02
54.26
54.26
54.26
67.15
66.25
57.02
57.02
57.02
57.02
57.02
59.81
60.26
51.02
54.26
54.26
54.26
67.15
66.25
57.02
57.02
57.02
57.02
57.02
57.02
-0.10
-0.15
0.00
-0.08
-0.12
-0.13
-0.22
-0.02
-0.11
-0.14
-0.07
-0.10
-0.16
-0.14
-0.07
-0.20
-0.09
-0.10
-0.017
-0.26
-0.05
-0.14
-0.16
-0.10
-0.13
-0.20
-0.15
-0.17
-0.10
-0.23
-0.12
-0.13
-0.04
-0.11
0.02
-0.16
-0.19
-0.27
-0.32
-0.20
-0.33
-0.29
-0.16
-0.29
-0.20
-0.11
-0.07
-0.12
-0.12
-0.11
-0.29
-0.34
-0.22
-0.35
-0.31
-0.17
-0.31
-0.22
-0.16
-0.13
-0.09
-0.14
-0.13
-0.13
-0.44
-0.48
-0.17
-0.22
-0.07
-0.20
-0.22
-0.18
-0.18
-0.37
-0.34
-0.32
-0.42
-0.27
-0.29
-0.20
-0.25
-0.10
-0.24
-0.25
-0.21
-0.21
-0.40
-0.36
-0.37
-0.35
-0.45
-0.30
-0.33
-0.02
-0.10
-0.12
-0.16
-0.08
-0.12
-0.16
-0.14
-0.12
-0.18
-0.15
-0.13
-0.19
-0.11
-0.10
-0.17
-0.22
-0.12
-0.18
-0.21
-0.18
-0.17
-0.23
-0.20
-0.20
-0.18
-0.24
-0.15
-0.15
0.01
0.05
0.14
-0.02
0.09
0.09
0.05
0.04
0.09
0.07
4g
4h
4i
0.09
0.08
-0.18
-0.15
-0.11
-0.18
-0.11
-0.11
-0.03
-0.03
0.11
-0.07
-0.12
-0.06
-0.18
-0.02
0.00
4j
4k
4l
4m
5a
5b
5c
5d
5e
5f
-0.03
-0.01
-0.06
0.04
0.05
0.03
0.00
0.07
0.03
5g
5h
5i
0.06
0.04
-0.21
-0.17
-0.18
-0.14
-0.21
-0.13
-0.14
-0.12
-0.17
-0.17
-0.11
-0.23
-0.05
-0.05
5j
5k
5l
5m
5n
a
Number of hydrogen-bond acceptor
Number of hydrogen-bond donor
Molecular mass
b
c
d
e
f
Calculated lipophilicity
2
Topological polar surface area (A )
˚
GPCR ligand
g
h
i
Ion channel modulator
Kinase inhibitor
Nuclear receptor ligand
Protease inhibitor
Enzyme inhibitor
j
k
(TLC) was performed on Merck 60F254 plates. Reactions were
monitored by TLC on silica gel, with detection by UV light
(254 nm) or charring Dragendorff reagent (Stahl, 1969).
Melting points were determined by 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 spectrometer
using tetramethylsilane as the internal standard at the NMR
facility of Faculty of Pharmacy, Ankara University, values are
123

Med Chem Res (2013) 22:4893–4908
4901
Table 3 Drug-likeness, drug
Compounds
Toxicity risk
MUTa
Bioavailability and drug score
score and toxicity risks of the
synthesized compounds (8a, 8b;
4a–4m; 5a–5n)
TUMb
IRRITc
REd
log Se
Drug-likeness
Drug score
8a
8b
4a
4b
4c
4d
4e
4f
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Medium
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Medium
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Medium
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Medium
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
-2.35
-2.35
-3.66
-2.04
-4.23
-1.55
-3.39
-4.17
-2.6
-0.03
-1.16
2.15
0.69
0.57
0.75
0.89
0.62
0.9
3.38
5.32
8.89
8.99
0.76
0.36
0.82
0.63
0.35
0.3
-1.46
7.22
4g
4h
4i
-3.82
-4.68
-4.96
-5.02
-5.89
-5.41
-3.66
-2.04
-4.23
-1.55
-3.39
-4.17
-2.6
0.72
-0.16
-1.73
-2.4
1.14
4j
4k
4l
0.29
0.38
0.26
0.7
4m
5a
5b
5c
5d
5e
5f
-1.15
1.24
2.61
0.87
0.62
0.9
4.59
8.16
8.27
0.76
0.33
0.82
0.55
0.51
0.3
-2.19
6.55
5g
5h
5i
-3.82
-4.31
-4.68
-4.96
5.02
-0.12
0.08
5j
-1.01
-2.48
-3.25
0.32
a
Mutagenic
5k
5l
0.28
0.28
0.34
0.23
b
Tumorigenic
c
Irritant
5m
5n
-5.89
-5.41
d
Reproductive effective
-2.06
e
Solubility
given in d (ppm) and J values are in Hz. 13C-NMR spectra were
recorded in CDCl3 or DMSO-d6 on a Bruker Ultrashield
300 MHz NMR spectrometer using tetramethylsilane as the
internal standard at the NMR facility of Gazi University, values
are given in d (ppm). High resolution mass spectra data
(HRMS) were collected in-house using a Waters LCT Premier
XE mass spectrometer (high sensitivity orthogonal acceleration
time-of-flight instrument) operating in ESI (?) method, also
coupled to an AQUITY ultra performance liquid chromatog-
raphy system (Waters Corporation, Milford, MA, USA). Flash
chromatography was performed using a CombiflashÒ Rf
automated flash chromatography system with RediSep col-
umns (Teledyne-Isco, Lincoln, NE, USA) using dichloro-
methane–methanol solvent gradients. Elemental analyzes
(C, H, N) were determined ona Leco CHNS 932instrument and
gave values within 0.4 % of the theoretical values. Micro-
wave-assisted reactions were carried out with a Milestone
MicroSYNTH Microwave Synthesis System. Calculation of
important physicochemical properties (log P, number of
hydrogen-bond donors and acceptors, and TPSA) was per-
formed using Molinspiration Cheminformatics Software at
URL http://www.molinspiration.com. Determining of toxicity
risk, drug-likeness and drug score was done using Osiris pro-
gram at URL http://www.organic-chemistry.org/prog/peo.
General procedure for the preparation of N-Phenyl-N’-
(1-pyridin-3/4-yl-ethylidene)-hydrazine (6a, 6b)
Compounds 6a, 6b were synthesized as previously
described (Lokhande et al., 2011; Rathelot et al., 2002). A
solution of acetylpyridine derivative (52 mmol), phenyl
hydrazine (6.27 g, 58 mmol), and acetic acid (2 ml,
35 mmol) in ethanol was stirred for 2 h at reflux, and then
evaporated. The precipitate was filtered off and dried.
123

4902
Med Chem Res (2013) 22:4893–4908
N-Phenyl-N’-(1-pyridin-3-yl-ethylidene)-hydrazine (6a)
mp 139–140 °C (Huck et al., 2004).
suspension obtained was quenched upon addition of 20 ml
saturated sodium sulfite solution. The violet solid was then
filtered 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.
N-Phenyl-N’-(1-pyridin-4-yl-ethylidene)-hydrazine (6b)
mp 147 °C [Lit. mp (Chu and Teague 1958): 148–149 °C].
General procedure for the preparation of 1-phenyl-3-
(pyridin-3-/4-yl)-1H-pyrazole-4-carbaldehyde
derivatives (7a, 7b)
1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxylic
acid (8a)
Method A (Rathelot et al., 2002; Bratenko et al., 2002)
Yield 82 %, mp 239–242 °C [Lit. mp (Bratenko et al.,
2001); 236–238 °C].
In a dry flask, phosphoroxy chloride (POCl3) (16.77 ml,
0.18 mol) was added dropwise to an ice-cold stirred solution of
hydrazone derivative (6a or 6b) (0.06 mol) in 100 ml DMF.
This reaction mixture was allowed to attain room temperature,
and then heated at 50 °C for about 4 h. The resulting mixture
was poured onto crushed ice, neutralized with diluted NaOH
and left overnight. The precipitate obtained was purified by
crystallization in acetone–water mixture.
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic
acid (8b)
Yield 79 %, mp 233–235 °C; IR (FTIR/FTNIR-ATR):
1
1709 cm-1 (C=O) H-NMR (DMSO-d6) d: 9.16 (1H, s),
8.67 (2H, s), 8.01 (2H, d, J = 8 Hz), 7.88 (2H, s), 7.56
(2H, t, J = 7.6 Hz), 7.42 (1H, m). HRMS C15H12N3O2
[M?H]? Calc. 266.0930, Found m/z 266.0933.
Method B
General procedure for the preparation of amide
and ester derivatives of 1-phenyl-3-(pyridin-3/4-yl)-1H-
pyrazole-4-carboxylic acids (4a–4m; 5a–5n)
In a dry flask, POCl3 (16.77 ml, 0.18 mol) was added
dropwise to an ice-cold stirred solution of hydrazone
derivative (0.06 mol) in 100 ml DMF. The reaction mix-
ture was allowed to attain room temperature, and then flask
was placed in MicroSYNTH Microwave Synthesis System
and irradiated at 210 W for 20 min while the temperature
was set to 50 °C. The resulting mixture was poured onto
crushed ice, neutralized with diluted NaOH and left
standing overnight. The yellow precipitate obtained was
purified by crystallization in acetone–water mixture.
Triethylamine (2 mmol) and ethyl chloroformate (1 mmol)
were added to the solution of acid derivatives (1 mmol) in
dichloromethane, then stirred at 0 °C for 30 min. After
adding of different amine or phenol derivatives (1.2 mmol),
the mixture was stirred for an additional 1 h at 0 °C. Then,
the reaction mixture was warmed to room temperature, kept
stirring overnight. After the solvent was evaporated under
reduced pressure, acetone was added, filtered, and evapo-
rated. The residue was dissolved in DCM and the organic
phase was washed with a 1 % NaHCO3 solution and brine,
dried over Na2SO4, and evaporated under vacuum. The final
residue was purified by flash column chromatography
(CombiflashÒRf) using DCM-MeOH (0–10 %) as eluents.
1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carbaldehyde
(7a) Method A: yield 85 %, Method B: 98 %, mp
159–160 °C (Huck et al., 2004).
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carbaldehyde
(7b) Method A: yield 12 g (80 %), Method B: yield 13.8
(92 %), mp 147–149 °C (Badadhe et al., 2011).
1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxylic
acid benzylamide (4a)
General procedure for the preparation of 1-phenyl-3-
(pyridin-3/4-yl)-1H-pyrazole-4-carboxylic acid
derivatives (8)
Yield 56 %, mp 162–164 °C; IR (FTIR/FTNIR-ATR):
1
1637 cm-1 (C=O), 3266 cm-1 (N–H). H-NMR (CDCl3)
d: 8.97 (1H, d, J = 2 Hz), 8.62–8.59 (1H, m), 8.42 (1H, s),
8.03 (1H, td, J = 2 Hz, J = 8 Hz), 7.71 (2H, d,
J = 7.6 Hz), 7.49–7.45 (2H, m), 7.37–7.22 (7H, m), 5.97
(1H, s), 4.53 (2H, d, J = 5.6 Hz). 13C-NMR (CDCl3) d:
167.8, 148.0, 147.5, 139.9, 138.0, 133.5, 133.3, 134.2,
129.5, 129.3, 128.7, 127.3, 126.9, 126.3, 124.4, 120.2,
Potassium permanganate (24 mmol) was added to a solu-
tion of aldehyde (12 mmol) in 100 ml acetone–water
mixture (3:2) 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
123

Med Chem Res (2013) 22:4893–4908
4903
114.9, 43.5, HRMS C22H19N4O [M?H]? Calc. 355.1559,
Found m/z 355.1545.
3.40–3.37 (2H, m), 2.79–2.74 (2H, m). 13C-NMR (CDCl3)
d: 169.3, 149.8, 148.0, 147.5, 139.9, 133.5, 133.3, 134.2,
129.8, 129.5, 129.3, 126.3, 124.4, 122.2, 120.2, 114.9,
114.6, 53.6, 49.5. HRMS C25H24N5O [M?H]? Calc.
410.1981, Found m/z 410.1977.
Morpholin-4-yl-[1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-
4-yl]methanone (4b)
Yield 56 %, mp 128–130 °C; IR (FTIR/FTNIR-ATR):
1607 cm-1 (C=O). 1H-NMR (CDCl3) d: 8.99 (1H, d,
J = 1.6 Hz), 8.63 (1H, dd, J = 1.6 Hz, J = 4.8 Hz), 8.16 (1H,
s), 8.06 (1H, td, J = 2 Hz, J = 8 Hz), 7.74 (2H, d, J = 8 Hz),
7.53–7.50 (2H, m), 7.39–7.34 (2H, m), 3.68–3.23 (8H, m). 13C-
NMR(CDCl3)d: 169.3, 148.0, 147.5, 139.9, 133.5, 133.3, 134.2,
129.5, 129.3, 126.3, 124.4, 120.2, 114.9, 67.3, 46.7. HRMS
C19H19N4O2 [M?H]? Calc. 335.1508, Found m/z 335.1495.
[1-Phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl]-[4-(4-
trifluoromethyl-phenyl)-piperazin-1-yl]methanone (4f)
Yield 71 %, mp 178–180 °C; IR (FTIR/FTNIR-ATR):
1619 cm-1 (C=O). 1H-NMR (CDCl3) d: 9.01 (1H, d,
J = 1.6 Hz), 8.62 (1H, dd, J = 1.6 Hz, J = 4.8 Hz), 8.19
(1H, s), 8.09 (1H, td, J = 2 Hz, J = 8 Hz), 7.75 (2H, d,
J = 7.6 Hz), 7.52–7.49 (2H, m), 7.45 (2H, d, J = 8.8 Hz),
7.39–7.35 (2H, m), 6.83 (2H, d, J = 8.8 Hz), 3.93–3.87
(2H, m), 3.40–3.28 (4H, m), 2.87–2.83 (2H, m). 13C-NMR
(CDCl3) d: 169.3, 153.2, 148.0, 147.5, 139.9, 133.5, 133.3,
134.2, 129.5, 129.3, 126.3, 126.0, 124.4, 124.1, 120.2,
114.9, 113.6, 53.6, 49.5. HRMS C26H23N5OF3 [M?H]?
Calc. 478.1855, Found m/z 478.1835.
(4-Benzyl-piperidin-1-yl)-[1-phenyl-3-(pyridin-3-yl)-
1H-pyrazol-4-yl]methanone (4c)
Yield 69 %, mp 158–160 °C; IR (FTIR/FTNIR-ATR):
1601 cm-1 (C=O). 1H-NMR (CDCl3) d: 8.99 (1H, d,
J = 2.4 Hz), 8.62 (1H, dd, J = 1.6 Hz, J = 4.8 Hz), 8.17
(1H, s), 8.07–8.03 (1H, m), 7.75 (2H, d, J = 8.4 Hz), 7.49
(2H, t, J = 8 Hz), 7.36–7.33 (2H, m), 7.27–7.23 (2H, m),
7.22–7.17 (1H, m), 7.06 (2H, d, J = 7.2 Hz), 4.73–3.61 (2H,
m), 2.69–2.42 (4H, m), 1.71–1.62 (3H, m), 1.35–0.63 (2H,
m). 13C-NMR (CDCl3) d: 173.4, 148.0, 147.5, 139.9, 139.7,
133.5, 133.3, 134.2, 129.5, 129.3, 128.8, 128.3, 126.3, 125.9,
124.4, 120.2, 114.9, 45.3, 41.8, 37.4, 29.5. HRMS
C27H27N4O [M?H]? Calc. 423.2185, Found m/z 423.2179.
[1-Phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl]-(4-
pyridin-4-yl-piperazin-1-yl)methanone (4g)
Yield 44 %, mp 196–198 °C; IR (FTIR/FTNIR-ATR):
1630 cm-1 (C=O). 1H-NMR (CDCl3) d: 9.00 (1H, d,
J = 2 Hz), 8.61 (1H, dd, J = 1.6 Hz, J = 4.8 Hz), 8.27
(2H, d, J = 6.4 Hz), 8.19 (1H, s), 8.07 (1H, td, J = 2 Hz,
J = 8 Hz), 7.76 (2H, d, J = 7.2 Hz), 7.53–7.47 (2H, m),
7.39–7.35 (2H, m), 6.56 (2H, d, J = 6.4 Hz), 3.88–2.93
(8H, m). 13C-NMR (CDCl3) d: 169.3, 152.2, 150.5, 148.0,
147.5, 139.9, 133.5, 133.3, 134.2, 129.5, 129.3, 126.3,
124.4, 120.2, 114.9, 107.2, 53.6, 49.5. HRMS C24H23N6O
[M?H]? Calc. 411.1933, Found m/z 411.1936.
(4-Methyl-piperazin-1-yl)-[1-phenyl-3-(pyridin-
3-yl)-1H-pyrazol-4-yl]methanone (4d)
Yield 78 %, mp 137–139 °C; IR (FTIR/FTNIR-ATR):
1613 cm-1 (C=O). 1H-NMR (CDCl3) d: 9.00 (1H, d,
J = 2 Hz), 8.65–8.59 (1H, m), 8.13 (1H, s), 8.08–8.03 (1H,
m), 7.78–7.73 (2H, m), 7.52–7.49 (2H, m), 7.36–7.31 (2H,
m), 3.79 (2H, s), 3.25 (2H, s), 2.41 (2H, s), 2.20 (3H, s),
1.98 (2H, s). 13C-NMR (CDCl3) d: 169.3, 148.0, 147.5,
139.9, 133.5, 133.3, 134.2, 129.5, 129.3, 126.3, 124.4,
120.2, 114.9, 51.7, 49.5, 46.8. HRMS C20H22N5O [M?H]?
Calc. 348.1824, Found m/z 348.1810.
1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxylic
acid 4-methoxy-phenyl ester (4h)
Yield 76 %, mp 158–160 °C; IR (FTIR/FTNIR-ATR):
1726 cm-1 (C=O). 1H-NMR (CDCl3) d: 9.13 (1H, d,
J = 1.6 Hz), 8.71 (1H, s), 8.65–8.61 (1H, m), 8.31–8.25
(1H, m), 7.85–7.79 (2H, m), 7.55–7.51 (2H, m), 7.43–7.32
(2H, m), 7.09–7.06 (2H, m), 6.93–6.87 (2H, m), 3.80 (3H,
s). 13C-NMR (CDCl3) d: 165.5, 157.6, 148.0, 147.5, 142.0,
139.9, 133.5, 133.3, 134.2, 129.5, 129.3, 126.3, 124.4,
122.9, 120.2, 115.5, 114.9, 55.9. HRMS C22H18N3O3
[M?H]? Calc. 372.1348, Found m/z 372.1330.
(4-Phenyl-piperazin-1-yl)-[1-phenyl-3-(pyridin-
3-yl)-1H-pyrazol-4-yl]methanone (4e)
Yield 73 %, mp 150–152 °C; IR (FTIR/FTNIR-ATR):
1597 cm-1 (C=O). 1H-NMR (CDCl3) d: 9.03 (1H, d,
J = 1.2 Hz), 8.60 (1H, dd, J = 1.6 Hz, J = 4.8 Hz), 8.17
(1H, s), 8.07 (1H, td, J = 2 Hz, J = 8 Hz), 7.75 (2H, d,
J = 8.4 Hz), 7.52–7.49 (2H, m), 7.37–7.33 (2H, m),
7.26–7.22 (2H, m), 6.88 (1H, t, J = 7.2 Hz), 6.83 (2H, d,
J = 8 Hz), 3.93–3.88 (2H, m), 3.42–3.37 (2H, m),
1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxylic
acid 3-isopropyl-phenyl ester (4i)
Yield 58 %, mp 63–65 °C; IR (FTIR/FTNIR-ATR): 1716 cm-1
(C=O). 1H-NMR (CDCl3) d: 9.14 (1H, d, J = 2.4 Hz), 8.72
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1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxylic
(1H, s), 8.65–8.61 (1H, m), 8.30 (1H, td, J = 2 Hz,
J = 7.6 Hz), 7.83 (2H, d, J = 8 Hz), 7.57–7.52 (2H, m),
7.43–7.29 (3H, m), 7.13–6.97 (3H, m), 2.94–2.91 (1H, m),
1.25 (6H, s). 13C-NMR (CDCl3) d: 165.5, 153.5, 150.7,
148.0, 147.5, 139.9, 133.5, 133.3, 134.2, 129.5, 129.3,
129.0, 126.3, 124.4, 123.2, 120.2, 119.8, 119.0, 115.5,
33.5, 23.5. HRMS C24H22N3O2 [M?H]? Calc. 384.1712,
Found m/z 384.1697.
acid naphthalen-2-yl ester (4m)
White solid. Yield 44 %, mp 184–186 °C; IR (FTIR/
FTNIR-ATR): 1727 cm-1 (C=O). 1H-NMR (CDCl3) d:
9.19–9.16 (1H, m), 8.77 (1H, s), 8.65–8.61 (1H, m), 8.29
(1H, td, J = 2 Hz, J = 8 Hz), 7.88–7.79 (6H, m), 7.64
(1H, d, J = 2.4 Hz), 7.55–7.28 (6H, m). 13C-NMR
(CDCl3) d: 165.5, 153.5, 148.0, 147.5, 139.9, 134.7, 134.2,
133.5, 133.3, 130.2, 129.5, 129.3, 129.1, 128.1, 126.9,
126.7, 126.3, 124.4, 124.0, 120.2, 117.8, 115.5, 109.7.
HRMS C25H18N3O2 [M?H]? Calc. 392.1399, Found
m/z 392.1391.
1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxylic
acid 4-tert-butyl-phenyl ester (4j)
Yield 66 %, mp 138–141 °C; IR (FTIR/FTNIR-ATR):
1744 cm-1 (C=O). 1H-NMR (CDCl3) d: 9.13 (1H, d,
J = 1.6 Hz), 8.71 (1H, s), 8.62 (1H, dd, J = 1.6 Hz,
J = 5.2 Hz), 8.29 (1H, td, J = 2 Hz, J = 8 Hz), 7.85–7.81
(2H, m), 7.55–7.48 (2H, m), 7.42–7.31 (4H, m), 7.09–7.06
(2H, m), 1.31 (9H, s). 13C-NMR (CDCl3) d: 165.5, 148.3,
148.0, 147.5, 146.5, 139.9, 133.5, 133.3, 134.2, 129.5,
129.3, 126.3, 125.6, 124.4, 121.5, 120.2, 115.5, 34.5, 31.5.
HRMS C25H24N3O2 [M?H]? Calc. 398.1869, Found
m/z 398.1854.
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic
acid benzylamide (5a)
White solid. Yield 50 %, mp 184–187 °C; IR (FTIR/
FTNIR-ATR): 1639 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.60 (2H, d, J = 6 Hz), 8.39 (1H, s), 7.72 (2H, d,
J = 7.6 Hz), 7.67 (2H, d, J = 6.4 Hz), 7.48 (2H, t,
J = 8 Hz), 7.38–7.25 (6H, m), 5.99 (1H, s, NH), 4.55 (2H,
d, J = 6 Hz). 13C-NMR (CDCl3) d: 168.1, 150.2, 140.5,
139.9, 138.2, 133.5, 129.5, 129.3, 128.9, 127.2, 126.9,
126.3, 121.5, 120.2, 114.0, 43.5. HRMS C22H19N4O
[M?H]? Calc. 355.1559, Found m/z 355.1559.
1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxylic
acid 2-isopropyl-5-methyl-phenyl ester (4k)
Colorless oil. Yield 51 %; IR (FTIR/FTNIR-ATR):
1
Morpholin-4-yl-[1-phenyl-3-(pyridin-4-yl)-1H-
1736 cm-1 (C=O). H-NMR (DMSO-d6) d: 9.49 (1H, s),
pyrazol-4-yl]methanone (5b)
9.00–8.97 (1H, m), 8.63 (1H, dd, J = 1.6 Hz, J = 4.8 Hz),
8.22 (1H, td, J = 1.6 Hz, J = 8.4 Hz), 8.06 (2H, d,
J = 7.6 Hz), 7.62–7.57 (2H, m), 7.51–7.45 (2H, m), 7.26
(1H, d, J = 8 Hz), 7.08 (1H, d, J = 7.6 Hz), 7.03–7.00
(1H, m), 2.99–2.96 (1H, m), 2.29 (3H, s), 1.09 (6H, s). 13C-
NMR (DMSO-d6) d: 165.5, 148.0, 147.5, 147.5, 139.9,
137.5, 136.3, 133.5, 133.3, 134.2, 129.5, 129.3, 126.3,
125.7, 125.3, 124.4, 122.6, 120.2, 115.5, 27.5, 23.5, 21.4.
HRMS C25H24N3O2 [M?H]? Calc. 398.1869, Found
m/z 398.1872.
White solid. Yield 50 %, mp 158–161 °C; IR (FTIR/
FTNIR-ATR): 1627 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.67 (2H, d, J = 6 Hz), 8.12 (1H, s), 7.73 (2H, d,
J = 7.2 Hz), 7.68 (2H, d, J = 6 Hz), 7.53–7.49 (2H, m),
7.37–7.33 (1H, m), 3.78–3.70 (4H, m), 3.31–3.24 (4H, m).
13C-NMR (CDCl3) d: 169.3, 150.2, 140.5, 139.9, 133.5,
129.5, 129.3, 126.3, 121.5, 120.2, 114.0, 67.3, 46.7. HRMS
C19H19N4O2 [M?H]? Calc. 335.1508, Found m/z 335.1519.
1-Phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxylic
(4-Benzyl-piperidin-1-yl)-[1-phenyl-3-(pyridin-4-yl)-
acid biphenyl-4-yl ester (4l)
1H-pyrazol-4-yl]methanone (5c)
White solid. Yield 78 %, mp 169–172 °C; IR (FTIR/
FTNIR-ATR): 1718 cm-1 (C=O). 1H-NMR (CDCl3) d:
9.19–9.16 (1H, m), 8.76 (1H, s), 8.67–8.63 (1H, m),
8.35–8.30 (1H, m), 7.87–7.83 (2H, m), 7.64–7.60 (2H, m),
7.58–7.35 (9H, m), 7.27–7.23 (2H, m). 13C-NMR (CDCl3)
d: 165.5, 148.5, 148.0, 147.5, 140.8, 139.9, 137.6, 133.5,
133.3, 134.2, 129.7, 129.5, 129.3, 128.1, 127.9, 126.3,
124.4, 122.3, 120.2, 115.5, HRMS C27H20N3O2 [M?H]?
Calc. 418.1556, Found m/z 418.1556.
White solid. Yield 72 %, mp 91–93 °C; IR (FTIR/FTNIR-
ATR): 1621 cm-1 (C=O) 1H-NMR (CDCl3) d: 8.65 (2H, d,
J = 6.4 Hz), 8.08 (1H, s), 7.73 (2H, d, J = 7.6 Hz), 7.68
(2H, d, J = 6 Hz), 7.53–7.48 (2H, m), 7.37–7.33 (1H, m),
7.29–7.25 (2H, m), 7.19–7.16 (1H, m), 7.06 (2H, d,
J = 8 Hz), 4.78–3.59 (2H, m), 2.73–2.43 (4H, m),
1.75–1.66 (3H, m), 1.41–0.71 (2H, m). 13C-NMR (CDCl3)
d: 173.4, 150.2, 140.5, 139.9, 133.5, 129.5, 129.3, 128.9,
128.4, 126.3, 121.5, 120.2, 114.0, 45.3, 41.8, 37.4, 29.5.
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Med Chem Res (2013) 22:4893–4908
4905
HRMS C27H27N4O [M?H]? Calc. 423.2185, Found
m/z 423.2175.
3.03–2.99 (2H, m). 13C-NMR (CDCl3) d: 169.3, 152.4,
150.6, 150.2, 140.5, 139.9, 133.5, 129.5, 129.3, 126.3,
121.5, 120.2, 114.0, 107.2, 53.6, 49.5. HRMS C24H23N6O
[M?H]? Calc. 411.1933, Found m/z 411.1915.
(4-Methyl-piperazin-1-yl)-[1-phenyl-3-(pyridin-4-yl)-
1H-pyrazol-4-yl]methanone (5d)
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic
White solid. Yield 66 %, mp 136–138 °C; IR (FTIR/
FTNIR-ATR): 1616 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.66 (2H, d, J = 6 Hz), 8.10 (1H, s), 7.74 (2H, d,
J = 8.4 Hz), 7.68 (2H, d, J = 6 Hz), 7.52–7.47 (2H, m),
7.39–7.34 (1H, m), 3.83–3.80 (2H, m), 3.29–3.25 (2H, m),
2.45–2.41 (2H, m), 2.22 (3H, s), 2.06–2.02 (2H, m). 13C-
NMR (CDCl3) d: 169.3, 150.2, 140.5, 139.9, 133.5, 129.5,
129.3, 126.3, 121.5, 120.2, 114.0, 51.7, 49.5, 46.8. HRMS
C20H22N5O [M?H]? Calc. 348.1824, Found m/z 348.1817.
acid 4-methoxy-phenyl ester (5h)
White solid. Yield 67 %, mp 160–162 °C; IR (FTIR/
FTNIR-ATR): 1728 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.71 (1H, s), 8.67 (2H, d, J = 5.6 Hz), 7.92 (2H, d,
J = 6 Hz), 7.82 (2H, d, J = 7.2 Hz), 7.55–7.51 (2H, m),
7.43–7.37 (1H, m), 7.09 (2H, d, J = 7.2 Hz), 6.92 (2H, d,
J = 7.2 Hz), 3.80 (3H, s). 13C-NMR (CDCl3) d: 165.5,
157.7, 150.2, 142.0, 140.5, 139.9, 133.5, 129.5, 129.3,
126.3, 122.9, 121.5, 120.2, 114.9, 115.5, 114.9, 55.9.
HRMS C22H18N3O3 [M?H]? Calc. 372.1348, Found
m/z 372.1330.
(4-Phenyl-piperazin-1-yl)-[1-phenyl-3-(pyridin-4-yl)-
1H-pyrazol-4-yl]methanone (5e)
White solid. Yield 54 %, mp 185–187 °C; OC IR (FTIR/
FTNIR-ATR): 1636 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.66 (2H, d, J = 6 Hz), 8.15 (1H, s), 7.76 (2H, d,
J = 7.2 Hz), 7.71 (2H, d, J = 6 Hz), 7.53–7.49 (2H, m),
7.39–7.35 (1H, m), 7.27–7.23 (2H, m), 6.91–6.83 (3H, m),
3.99–3.94 (2H, m), 3.43–3.38 (2H, m), 3.25–3.21 (2H, m),
2.87–2.82 (2H, m). 13C-NMR (CDCl3) d: 169.3, 150.2,
149.9, 140.5, 139.9, 133.5, 129.9, 129.5, 129.3, 126.3,
122.3, 121.5, 120.2, 114.6, 114.0, 53.6, 49.5. HRMS
C25H24N5O [M?H]? Calc. 410.1981, Found m/z 410.1986.
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic
acid 4-ethyl-phenyl ester (5i)
White solid. Yield 60 %, mp 101–103 °C; IR (FTIR/
FTNIR-ATR): 1728 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.74 (1H, s), 8.67 (2H, d, J = 6 Hz), 7.98 (2H, d,
J = 5.6 Hz), 7.84 (2H, d, J = 7.6 Hz), 7.56–7.41 (3H, m),
7.25 (2H, d, J = 8.8 Hz), 7.09 (2H, d, J = 8.8 Hz), 2.68
(2H, q, J = 7.6 Hz), 1.24 (3H, t, J = 7.6 Hz). 13C-NMR
(CDCl3) d: 165.5, 150.2, 146.9, 141.4, 140.5, 139.9, 133.5,
129.5, 129.3, 126.3, 121.8, 121.5, 120.2, 115.5, 29.3, 14.8.
HRMS C23H20N3O2 [M?H]? Calc. 370.1556, Found
m/z 370.1543.
[1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl]-[4-(4-
trifluoromethyl-phenyl)-piperazin-1-yl]methanone (5f)
White solid. Yield 56 %, mp 172–174 °C; IR (FTIR/FTNIR-
ATR): 1615 cm-1 (C=O) 1H-NMR (CDCl3) d: 8.67 (2H, d,
J = 6 Hz), 8.16 (1H, s), 7.76 (2H, d, J = 7.6 Hz), 7.71 (2H,
d, J = 6 Hz), 7.53–7.49 (2H, m), 7.48 (2H, d, J = 8.8 Hz),
7.41–7.38 (1H, m), 6.85 (2H, d, J = 8.4 Hz), 3.97–3.93 (2H,
m), 3.41–3.31 (4H, m), 2.93–2.91 (2H, m). 13C-NMR
(CDCl3) d: 169.3, 153.2, 150.2, 140.5, 139.9, 133.5, 129.5,
129.3, 126.3, 124.7, 121.5, 120.2, 114.0, 113.6, 53.6, 49.5.
HRMS C26H23N5OF3 [M?H]? Calc. 478.1855, Found m/
z 478.1855.
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic
acid 3-isopropyl-phenyl ester (5j)
White solid. Yield 48 %, mp 128–130 °C; IR (FTIR/FTNIR-
ATR): 1725 cm-1 (C=O) 1H-NMR (CDCl3) d: 9.03 (1H, s),
8.99 (2H, d, J = 6.4 Hz), 8.24 (2H, d, J = 6.4 Hz), 8.13
(2H, d, J = 7.2 Hz), 7.87–7.83 (2H, m), 7.75–7.71 (1H, m),
7.63 (1H, t, J = 7.6 Hz), 7.43 (1H, d, J = 8 Hz), 7.34–7.28
(2H, m), 3.25–3.21 (1H, m), 1.57 (6H, s). 13C-NMR (CDCl3)
d: 165.5, 153.5, 150.7, 150.2, 140.5, 139.9, 133.5, 129.5,
129.3, 129.1, 126.3, 123.2, 121.5, 120.2, 119.9, 119.0, 115.5,
33.5, 23.5. HRMS C24H22N3O2 [M?H]? Calc. 384.1712,
Found m/z 384.1703.
[1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl]-(4-
pyridin-4-yl-piperazin-1-yl)methanone (5g)
White solid. Yield 56 %, mp 207–209 °C; IR (FTIR/
FTNIR-ATR): 1632 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.66 (2H, d, J = 6.4 Hz), 8.27 (2H, d, J = 6.8 Hz), 8.16
(1H, s), 7.73 (2H, d, J = 8 Hz), 7.69 (2H, d, J = 6 Hz),
7.52–7.48 (2H, m), 7.40–7.37 (1H, m), 6.58 (2H, d,
J = 6.8 Hz), 3.93–3.87 (2H, m), 3.41–3.37 (4H, m),
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic
acid 4-tert-butyl-phenyl ester (5k)
White solid. Yield 62 %, mp 160–162 °C; IR (FTIR/FTNIR-
ATR): 1746 cm-1 (C=O) 1H-NMR (CDCl3) d: 8.71 (1H, s),
8.67 (2H, d, J = 6.4 Hz), 7.92 (2H, d, J = 6 Hz), 7.80 (2H,
123

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d, J = 7.6 Hz), 7.55–7.50 (2H, m), 7.43–7.37 (3H, m), 7.09
(2H, d, J = 8.8 Hz), 1.31 (9H, s). 13C-NMR (CDCl3) d:
165.5, 150.2, 148.5, 146.7, 140.5, 139.9, 133.5, 129.5, 129.3,
126.3, 125.9, 121.5, 120.2, 115.5, 34.5, 31.5. HRMS
C25H24N3O2 [M?H]? Calc. 398.1869, Found m/z 398.1861.
receptor negative breast carcinoma (MDA-MB-231), Bur-
kitt’s lymphoma (Raji), and human promyelocytic leuke-
mia (HL60), were obtained from ATCC.
Cytotoxicity assay
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic
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-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium
bromide) assay (Cell Proliferation Kit I, Roche, Germany).
Briefly, cells were seeded in a 96-well plate at 10,000 cells
per well, cultured overnight in growth medium containing
1 % FBS. Then the cells were treated with 50 lM of test
compounds for 48 h. As for solvent control, cells were also
treated with dimethyl sulfoxide (DMSO) at a final con-
centration 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 was 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 the crystals. The absorbance values of
samples were 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
test compounds to the cell viability were calculated by
comparing mean values obtained from compound treated
culture wells with those of the solvent controls. This
experiment was performed in quadruplicate. The IC50
values were calculated from the concentration–response
curves by means of the PRISM 5, GraphPad Software
(GraphPad Prism Version 5.04 for Windows 2010).
acid 2-isopropyl-5-methyl-phenyl ester (5l)
White solid. Yield 43 %, mp 142–144 °C; IR (FTIR/
FTNIR-ATR): 1729 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.74 (1H, s), 8.68 (2H, d, J = 6 Hz), 7.97 (2H, d,
J = 6 Hz), 7.84 (2H, d, J = 7.6 Hz), 7.57–7.42 (3H, m),
7.25 (H, d, J = 8 Hz), 7.06 (1H, d, J = 7.6 Hz), 6.92 (1H,
s), 3.05–3.01 (1H, m), 2.34 (3H, s), 1.20 (6H, s). 13C-NMR
(CDCl3) d: 165.5, 150.2, 147.7, 140.5, 139.9, 137.6, 136.5,
133.5, 129.5, 129.3, 126.3, 126.0, 125.6, 121.5, 120.2,
115.5, 27.5, 23.5, 21.4. HRMS C25H24N3O2 [M?H]? Calc.
398.1869, Found m/z 398.1869.
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic
acid biphenyl-4-yl ester (5m)
White solid. Yield 50 %, mp 195–197 °C; IR (FTIR/FTNIR-
ATR): 1733 cm-1 (C=O) 1H-NMR (CDCl3) d: 8.77 (1H, s),
8.71 (2H, d, J = 6.4 Hz), 7.98 (2H, d, J = 6 Hz), 7.85 (2H,
d, J = 8.8 Hz), 7.65–7.62 (2H, d, J = 8.8 Hz), 7.59–7.53
(4H, m), 7.47–7.42 (3H, m), 7.39–7.35 (1H, m), 7.26 (2H, d,
J = 8.8 Hz). 13C-NMR (CDCl3) d: 165.5, 150.2, 148.5,
141.3, 140.5, 139.9, 137.9, 133.5, 129.8, 129.5, 129.3, 128.3,
127.9, 126.3, 122.5, 121.5, 120.2, 115.5. HRMS C27H20
N3O2 [M?H]? Calc. 418.1556, Found m/z 418.1551.
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carboxylic acid
naphthalen-2-yl ester (5n)
White solid. Yield 53 %, mp 182–184 °C; IR (FTIR/
FTNIR-ATR): 1734 cm-1 (C=O) 1H-NMR (CDCl3) d:
8.79 (1H, s), 8.68 (2H, d, J = 6.4 Hz), 7.95 (2H, d,
J = 6.4 Hz), 7.90–7.81 (5H, m), 7.66 (1H, d, J = 2.4 Hz),
7.57–7.41 (5H, m), 7.35–7.30 (1H, m). 13C-NMR (CDCl3)
d: 165.5, 153.4, 150.2, 148.5, 141.3, 140.5, 139.9, 137.9,
134.9, 133.5, 130.3, 129.8, 129.5, 129.3, 128.3, 128.0,
127.9, 126.9, 126.7, 126.3, 124.3, 122.5, 121.5, 120.2,
117.9, 115.5, 109.7. HRMS C25H18N3O2 [M?H]? Calc.
392.1399, Found m/z 392.1384.
Statistical significances of the effects of compounds to the cell
viabilities were analyzed by one-way ANOVA andTukey’s
post hoc test using SigmaStat v3.5 software. p\0.05 were
considered statistically significant (* p B 0.05, ** p B 0.01,
*** p B 0.001 compared with control).
Lipinski’s rule of five and drug-likeness profile
Anticancer activity
In order to explore the bioavailability of synthesized deriva-
tives, theoretical calculations were carried out to predict
some physicochemical properties of synthesized compounds.
The bioavailabilities of the compounds were assessed using
ADME(absorption, distribution, metabolism, and elimination)
Cell lines and cell culture
The human cancer cell lines, cervical carcinoma (HeLa),
estrogen receptor positive breast carcinoma (MCF7), estrogen
123

Med Chem Res (2013) 22:4893–4908
4907
giving improved sensitivity and reliability. J Immunol Methods
89:271–277
prediction methods. In particular, we calculated the compli-
ance ofcompounds to the Lipinski’s rule of five (Lipinski et al.,
2001). Poorabsorptionand permeation are more likely to occur
when there are more than 5 hydrogen-bond donors (HBD),
more than 10 hydrogen-bond acceptors (HDA), when the
molecular mass (MM) is greater than 500, or the log P value
(clog P) is greater than 5. The TPSA should be smaller than
Ding X-L, Meng N, Xia Y, Wang L-J, Zhang X-F, Zhao B-X, Miao J-Y
(2007) Synthesis of 3-phenyl-1-[(aryl)methyl]-1H-pyrazole-5-car-
boxylic acid ethyl ester derivatives and determination of their
activityaspromoters ofcellapoptosis.YoujiHuaxue27:1542–1546
Ding XL, Zhang HY, Qi L, Zhao B-X, Lian S, Lv HS, Miao J-Y
(2009) Synthesis of novel pyrazole carboxamide derivatives and
discovery of modulators for apoptosis or autophagy in A549 lung
cancer cells. Bioorg Med Chem Lett 19:5325–5328
˚
90 A (Brueggemeier et al., 2005). The method for calculation
El-Shafei A, Fadda AA, Khalil AM, Ameen TAE, Badria FA (2009)
Synthesis, antitumor evaluation, molecular modeling and quan-
titative structure-activity relationship (QSAR) of some novel
arylazopyrazolodiazine and triazine analogs. Bioorg Med Chem
17:5096–5105
Ertl P, Rohde B, Selzer P (2000) Fast calculation of molecular polar
surface area as a sum of fragment-based contributions and its
application to the prediction of drug transport properties. J Med
Chem 43:3714–3717
Gaston MA, Dias LRS, Freitas ACC, Miranda ALP, Barreiro EJ
(1996) Synthesis and analgesic properties of new 4-arylhydraz-
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of molecule volume developed by Molinspiration is based on
group contributions. These have been obtained by fitting sum
of fragmentcontributionsto ‘‘real’’3D volumefor atrainingset
of about twelve thousand, mostly drug-like molecules. Mole-
cules violating more than one of these rules may have problems
in terms of bioavailability.
Acknowledgments This investigation was partially supported by
Scientific Research Grant 02/2011-08, awarded by Gazi University
BAP.
GraphPad Prism Version 5.04 for Windows (2010), G. S., San Diego,
CA, USA. www.graphpad.com
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