Design, Synthesis, and Cytotoxicity Evaluation of 3-(5-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)pyridine and 5-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbaldehyde Derivatives as Potential Anticancer Agents

Article abstract of DOI:10.1002/jhet.2768

In an attempt to find bio-active small molecules, a series of novel 3-(5-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)pyridine 5a–i and 5-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbaldehyde 6a–i were designed, synthesized, and evaluated for their in vitro cytotoxic activity against a panel of human cancer cell lines namely; HeLa (human cervix), NCI-H460 (human lung), PC-3 (human prostate), and NIH-3T3 (mouse embryo fibroblasts) normal cell line. Most of these compounds exhibited moderate to good cytotoxic activity against the tested cancer cell lines and weak toxicity against normal cell line. Analogues 5b, 5f, 5g, 6b, and 6g showed significant cytotoxicity as compared to standard drug etoposide. Among all the synthesized compounds, compound 6g displayed superior cytotoxicity with an IC₅₀ value of 7.98 ± 1.08 μM for Hela cancer cell line.

Full text of DOI:10.1002/jhet.2768

Month 2016 Design, Synthesis, and Cytotoxicity Evaluation of 3-(5-(3-(aryl)-1-phenyl-
H-pyrazol-4-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)pyridine and
-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-
dihydropyrazole-1-carbaldehyde Derivatives as Potential Anticancer Agents
1
5
a
b,c
c
a
Raquib Alam, Md. Aftab Alam, Amulya K. Panda, and Rahisuddin *
a
Department of Chemistry, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi 110025, India
b
Department of Pharmacy, School of Medical and Allied Science, Galgotias University, Greater Noida 201301, UP, India
c
Product Development Cell, National Institute of Immunology, New Delhi 110067, India
*
E-mail: rahisuddin@jmi.ac.in
Additional Supporting Information may be found in the online version of this article.
Received May 1, 2016
DOI 10.1002/jhet.2768
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
In an attempt to find bio-active small molecules, a series of novel 3-(5-(3-(aryl)-1-phenyl-1H-
pyrazol-4-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)pyridine 5a–i and 5-(3-(aryl)-1-phenyl-1H-pyrazol-4-
yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbaldehyde 6a–i were designed, synthesized, and evaluated
for their in vitro cytotoxic activity against a panel of human cancer cell lines namely; HeLa (human cervix),
NCI-H460 (human lung), PC-3 (human prostate), and NIH-3T3 (mouse embryo fibroblasts) normal cell line.
Most of these compounds exhibited moderate to good cytotoxic activity against the tested cancer cell lines
and weak toxicity against normal cell line. Analogues 5b, 5f, 5g, 6b, and 6g showed significant cytotoxicity
as compared to standard drug etoposide. Among all the synthesized compounds, compound 6g displayed
superior cytotoxicity with an IC50 value of 7.98 ± 1.08 μM for Hela cancer cell line.
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
based on heterocyclic compounds bearing 1, 2-diaryl
substituted pyrazole ring have often been occupying a
position in the list of best selling pharmaceutical
products. The important pyrazole based drugs available
in the market are Celecoxib, Apixaban, Fipronil,
Tolpiprazole, Remogliflazone etabonate, Lonazolac,
Deracoxib, and many more. The important pyrazole-
based antitumor drugs are Ruxolitinib and Crizotinib [8].
Few pyrazole-based drugs are depicted in Figure 1.
Celecoxib is a typical model of pyrazole entethered
diaryl heterocyclic small molecule [9]. The antitumor
activity of celecoxib is reported against prostate tumors
in experimental models [10–12]. Pyrazoles show
anticancer activity because of their inhibition of various
targets, including topoisomerase II [13–15], EGFR
[16,17], VEGF [18,19], HDAC [20,21], IGF-1R [22],
Aurora-A kinase [23], cMet [24], Tubulin [25], mTOR
[26], B-raf [27,28], ROS 1 [29], CDKs [30], PI3K [31],
JAK2 [32], and ALK [33]. Crizotinib is a dual inhibitor
of cMet/ALK and is used for the treatment of non-small
cell lung carcinoma. Ruxolitinib is a Janus kinase 2
(JAK2) inhibitor and is used for the treatment of
myeloproliferative neoplasm.
Cancer is a major public health problem after the
cardiovascular disorder across the world which affected
millions of lives. Although major advances have been
made in reducing cancer incidence in United States, the
numbers of cancer patients continue to increase [1].
Chemotherapy is one of the most effective approaches used
for treating cancer patients. However, lack of selectivity
and development of drug resistance reduce the efficacy of
cancer chemotherapy [2]. Despite enormous efforts in
developing newer chemotherapeutic strategies for the de-
velopment of potential anticancer drug, it remains critically
important. The search for effective and safe anticancer
agents attracted the attention of scientists globally.
In last few decades, research has been focussed on
development of chemically synthesized compounds as
anticancer agents. Recently, interest in pyrazole
chemistry has significantly increased mainly because of
the discovery of large number of interesting pyrazole
derivatives [3,4]. The heterocyclic compounds containing
pyrazole ring have received considerable attention owing
to their diverse chemotherapeutic potential [5–7]. Drugs
©
2016 Wiley Periodicals, Inc.

R. Alam, Md. A. Alam, A. K. Panda, and Rahisuddin
Vol 000
Figure 1. Pyrazole containing drugs.
Chalcones of various classes have been extensively
studied owing to their wide range of pharmacological
activities such as anti-inflammatory, antimitotic,
antileishmanial, anti-invasive, antimalarial, antibacterial,
antifungal, antiprotozoal, antiviral, antitrypanosomal,
and anticancer activities. The synthetic utility of
chalcones for the preparation of pyrazolines has been
recognized for decades. The pyrazolines have shown
various pharmacological activities like anti-hypertensive
cytotoxic activity against three different human cancer
cell lines and a normal cancer cell line. The structures of
target compounds are shown in Figure 3.
RESULT AND DISCUSSION
Chemistry.
Synthesis of intermediates and target
compounds has been outlined in Scheme 1. Pyrazolic
chalcones were prepared by corresponding 3-aryl-1-
phenylpyrazol-4-carboxaldehydes 3a–i [50,51] which, in
turn, were synthesized by Vilsmeier–Haack formylation
of phenylhydrazones 2a–i. Subsequently, the Claisen–
Schmidt condensation of 3-aryl-1-phenylpyrazol-4-
carboxaldehydes 3a–i with 3-acetylpyridine in methanolic
NaOH afforded the corresponding (E)-3-(3-(aryl)-1-
phenyl-1H-pyrazol-4-yl)-1-(pyridin-3-yl)prop-2-en-1-ones
4a–i [50]. With the objective of synthesizing pyrazolyl-
pyrazoline derivatives, the pyrazolic chalcones 4a–i were
treated with phenylhydrazine and hydrazine hydrate by
nucleophilic cycloaddition reaction by conventional
heating method. The formation of pyrazoline ring
[
[
34,35], anti-inflammatory [36,37], antidepressant
38], antineurodegenerative [39,40], antimalarial [41],
antidiabetic [42], antibacterial [43–45], antifungal [45–47],
and anticancer [42,48,49], among others. Some bioactive
pyrazolines are given in Figure 2.
Herein, efforts are made on establishing some
heterocyclic analogues integrated with pyrazole and
pyrazoline scaffold to describe the relevance
pharmacological activity. Motivated by our previous
findings of anticancer chalcones [50], and in continuation
of our ongoing research program to get potential
anticancer agents, we have synthesized some
pyrazolylpyrazoline derivatives and evaluated for their
Figure 2. Bioactive pyrazolines.
Figure 3. Target compounds.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Design, Synthesis, and Cytotoxicity Evaluation of Pyrazolylpyrazoline
Derivatives as Potential Anticancer Agents
Scheme 1. General procedure for the synthesis of pyrazolylpyrazoline derivatives (i) EtOH, H
2
SO
NH
4
, reflux; (ii) a. POCl
3 3 2
/DMF, 80°C; b. NaHCO /H O;
(iii) 3-Acetylpyridine, MeOH, NaOH, r.t.; (iv) Phenylhydrazine, glacial AcOH, reflux; (v) NH
2
2
.H O/HCOOH, reflux.
2
involves a Michael addition of hydrazine on chalcone,
followed by cyclization and dehydration. In the present
study, we were intended to investigate two series of
compounds. Compounds 5a–i were synthesized by
refluxing phenyl hydrazine with chalcones in the
presence of glacial acetic acid. Further, hydrazine hydrate
reacted with chalcones in the presence of formic acid,
afforded compounds 6a–i. The progress of the reaction
(5 -H) resonated as a doublet of doublet at 5.58ppm with
coupling constants of 12 and 6.8 Hz and the two
methylene protons (4 -H) displayed two signals; a doublet
of doublet at 3.86ppm with coupling constants of 17.2
and 12.4 Hz and a doublet of doublet at 3.26ppm with
coupling constants of 16.8 and 6.8 Hz. The main signals
13
in the C NMR for 5a correspond to C-4 at 42.45, C-5
at 56.65. The IR spectra of compound 6a show
-
1
was monitored by thin-layer chromatography (TLC). All
absorption bands mainly at 1666, 1599, and 1503 cm
1
the synthesized compounds were characterized by
H
which were because of C¼O, C¼N, and C¼C
13
1
NMR and C NMR (Figs. S1–S18), IR spectroscopy and
mass spectrometry. The elemental analysis of the
synthesized compounds was ascertained by CHNS
analyzer.
functionalities, respectively. In the H NMR spectra of
compound 6a, methine proton (5 -H) resonated as a
doublet of doublet at 5.84ppm with coupling constants of
12 and 4.4 Hz and two methylene protons (4 -H)
displayed two signals; a doublet of doublet at 3.65ppm
with coupling constants of 17.6 and 12.0Hz and a
doublet of doublet at 3.12ppm with coupling constants of
The formation of pyrazoline ring in the target
compounds 5a–i and 6a–i was confirmed by the
1
appearance of interesting AMX pattern in their H NMR
13
spectrum. The IR spectra of compound 5a show
17.6 and 5.2 Hz. The main signals in the C NMR for 6a
correspond to C-4 at 42.81, C-5 at 51.42. The elemental
analysis and mass spectra supported the structure of
pyrazolyl-pyrazoline derivatives 5a and 6a.
-
1
absorption bands mainly at 1596 and 1501 cm which
were because of C¼N and C¼C functionalities,
1
respectively. In the H NMR spectra of compound 5a,
the stereogenic 5 -H proton of the pyrazolinic moiety and
the two methylenic 4 -H protons displayed a typical
AMX spin system of doublet of doublet. Methine proton
Cytotoxic activity. In vitro cytotoxicity of all the newly
synthesized compounds 5a–i and 6a–i were measured by
MTT
[(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

R. Alam, Md. A. Alam, A. K. Panda, and Rahisuddin
Vol 000
tetrazolium bromide)] assay against a panel of three
different human cancer cell lines namely; HeLa (human
cervix), NCI-H460 (human lung), PC-3 (human prostate),
and NIH-3T3 (mouse embryo fibroblasts) normal cell
line [52]. The MTT assay is a colorimetric assay for
measuring the cellular growth that reduces the
tetrazolium yellow dye MTT to its insoluble purple color
formazan by mitochondrial dehydrogenases of living
cells. MTT assay is used to determine the cytotoxicity of
toxic compounds and potential drugs. The insoluble
purple formazan is dissolved into a colored solution by
the addition of a suitable solvent. At a particular
wavelength, the absorbance of this colored solution can
be measured, and the IC50 values were calculated from
the dose effect curve (Figs. S19 and S20) and expressed
as concentration (μM) of drug. Etoposide was taken as
reference compound, and the results are summarized in
terms of IC50 values (Table 1). From the IC50 values, it
is clear that most of these compounds showed moderate
to good cytotoxicity against intestinal, pancreatic, and
breast cancer cell lines and weak toxicity towards NIH-
ring represented IC50 value of 21.26 ±2.40 μM against
PC-3 cancer cell lines. The compound, 3-(5-
(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-
4,5-dihydro-1H-pyrazol-3-yl)pyridine (5g) having NO2
in the para-substituted benzene ring showed IC50
value of 8.62 ±0.66 μM against Hela, 9.44 ± 0.64 μM
against NCI-H460, 16.66 ±0.82 against PC-3 cancer cell
lines. 5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-
(pyridin-3-yl)-4,5-dihydropyrazole-1-carbaldehyde (6b) with
F group at para-substituted phenyl in parent analogue
exhibited IC50 value of 9.72 ±0.98μM against Hela and
22.97± 0.91 μM against PC-3 cancer cell lines. The
analogue, 5-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-
(pyridin-3-yl)-4,5-dihydropyrazole-1-carbaldehyde (6g) with
NO group in the para-substituted benzene ring displayed
2
IC50 value of 7.98 ±1.08μM against Hela, 8.41±0.61μM
against NCI-H460, 9.53± 0.62μM against PC-3 cancer cell
lines. In this study, analogue 6g demonstrated superior
cytotoxic activity with the IC50 value of 7.98± 1.08 μM for
Hela cancer cell line. Subsequently, structure activity
relationship (SAR) studies were performed to determine the
effect of substituents on cytotoxic activities of the
synthesized compounds. SAR in these derivatives displayed
3
T3 normal cell line. Among all the synthesized
compounds, analogues 5b, 5f, 5g, 6b, and 6g showed
significant cytotoxicity as compared to standard drug
etoposide. The compound, 3-(5-(3-(4-fluorophenyl)-1-
phenyl-1H-pyrazol-4-yl)-1-phenyl-4,5-dihydro-1H-
that compounds with para substituted NO group in the
2
benezene ring showed superior cytotoxicity against cancer
cell lines. Substituents F, Cl, Br, CH , and OCH present
3
3
pyrazol-3-yl)pyridine (5b) with F group in the para-
substituted benzene ring showed IC50 value of 9.75
in the benzene ring of these compounds showed moderate
effect, which is clearly seen in Table 1. The cytotoxic
activity against the tested cancer cell lines shows that the
±
0.79μM against Hela cancer cell lines. The analogue,
3
-(5-(3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-
strength order is NO > F> Cl > Br for the compounds
2
phenyl-4,5-dihydro-1H-pyrazol-3-yl)pyridine (5f) with
NO2 present in meta position of the benzene
with a para substituted electron-withdrawing group present
in the benzene ring. Among the compounds with meta
Table 1
In vitro cytotoxicity evaluation of the synthesized compounds against a panel of human cancer cell lines and a normal cell line in terms of IC50
value in μM.
Compounds
R
Hela
NCI-H460
PC-3
NIH-3 T3
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
g
h
i
a
b
c
d
e
f
g
h
i
H
4-F
27.66 ± 1.22
9.75 ± 0.79
32.43 ± 2.19
44.15 ± 1.98
36.50 ± 2.02
13.70 ± 0.96
8.62 ± 0.66
42.53 ± 3.55
27.85 ± 1.86
37.52 ± 1.94
9.72 ± 0.98
16.75 ± 0.79
24.09 ± 0.75
25.71 ± 2.44
17.27 ± 0.32
7.98 ± 1.08
25.09 ± 1.27
19.30 ± 1.49
11.25 ± 0.33
40.36 ± 1.36
19.80 ± 0.47
41.60 ± 1.55
32.23 ± 2.36
45.05 ± 2.17
18.68 ± 0.48
9.44 ± 0.64
39.36 ± 2.26
31.62 ± 1.07
43.94 ± 1.97
17.80 ± 1.73
23.59 ± 0.63
34.67 ± 3.40
32.24 ± 3.40
21.54 ± 0.54
8.41 ± 0.61
37.62 ± 0.76
28.14 ± 2.29
29.68 ± 2.33
51.96 ± 4.59
49.41 ± 4.22
21.26 ± 2.40
16.66 ± 0.82
53.18 ± 2.47
28.66 ± 1.68
62.44 ± 2.49
22.97 ± 0.91
31.33 ± 2.52
43.62 ± 2.07
37.52 ± 1.55
24.68 ± 1.07
9.53 ± 0.62
>100
94.73 ± 0.76
>100
96.85 ± 1.55
>100
95.44 ± 1.33
97.35 ± 1.68
>100
97.78 ± 0.91
>100
93.73 ± 2.29
91.45 ± 1.60
89.25 ± 1.07
>100
90.05 ± 0.63
87.30 ± 1.08
>100
95.63 ± 0.37
90.53 ± 4.60
4-Cl
3-Br
4-Br
3-NO
4-NO
4-CH
4-OCH
H
2
2
3
3
4-F
4-Cl
3-Br
4-Br
3-NO
4-NO
4-CH
4-OCH
—
2
2
3
36.40 ± 2.33
22.47 ± 1.82
16.63 ± 1.07
46.17 ± 1.27
34.67 ± 1.61
23.80 ± 0.38
3
Etoposide
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Design, Synthesis, and Cytotoxicity Evaluation of Pyrazolylpyrazoline
Derivatives as Potential Anticancer Agents
+
substituted electron-withdrawing group, the cytotoxic
(ESI) [M + H] Calcd. for C H N : 442.2, Found:
2
9 23 5
+
activity order is NO > Br in cancer cells. Finally,
442.8 [M + H] .
2
cytotoxic activity against cancer cells shows that the
3-(5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-
strength order is OCH >CH for the compounds with
electron-donating substituent present in para position of the
benzene ring.
4,5-dihydro-1H-pyrazol-3-yl)pyridine (5b).
Yellow solid;
cm ): 3069,
max
3
3
-
1
Yield: 73%; mp 178–180°C; IR (neat, ν
1
1598, 1501, 1391, 1223; H NMR (400MHz, CDCl ):
3
8
.87 (d, J= 2.0 Hz, 1H), 8.55–8.57 (m, 1H), 8.06–8.08
m, 1H), 7.75–7.78 (m, 3H), 7.63 (d, J= 7.6, 2H), 7.19–
7.41 (m, 8H), 7.11 (d, J = 8 Hz, 2H), 6.86 (t, J = 8 Hz,
H), 5.53 (dd, J= 6.8 Hz, 12Hz, 1H), 3.85 (dd,
J = 16.8Hz, 12Hz, 1H), 3.24 (dd, 16.8 Hz, J =6.8Hz,
(
EXPERIMENTAL
1
All the starting materials and the solvents were
purchased from commercial sources and are used without
further purification. Melting points were determined in
open capillaries using an electrothermal melting point
apparatus and are uncorrected. The progress of the
reactions was monitored by TLC. Precoated aluminum
sheets (Silica gel 60 F254, Merck Germany) were used for
TLC, and spots were visualized under UV light. IR spectra
were recorded on Agilent Cary 630 FT-IR spectrometer.
13
1H); C NMR (100 MHz, CDCl ): 164.12, 161.61,
3
160.21, 149.51, 147.11, 144.25, 144.23, 139.62, 132.60,
129.84, 129.76, 129.41, 129.18, 128.65, 126.67, 126.44,
123.51, 122.45, 120.06, 118.91, 116.06, 115.84, 113.77,
56.65, 42.45; Anal. Calcd. for C H FN : C, 75.80; H,
29
22
5
4.83; N, 15.24. Found: C, 75.76; H, 4.86; N, 15.25. MS
+
(ESI) [M + H] Calcd. for C H FN : 460.2, Found:
29
22
5
+
460.4 [M + H] .
1
13
H and C NMR spectra were recorded on Bruker Avance
00-MHz and Bruker Avance 100-MHz spectrometer, re-
spectively, using CDCl or DMSO-d as a solvent and
3-(5-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-
4
4,5-dihydro-1H-pyrazol-3-yl)pyridine (5c).
Yellow solid;
-
1
Yield: 61%; mp 182–184°C; IR (neat, ν
cm ):
3
6
max
1
trimethylsilane (TMS) as an internal standard. Splitting
patterns are designated as: s, singlet; d, doublet;dd, doublet
of doublet; t, triplet; m, multiplet. Chemical shift values (δ)
are represented in ppm. Elemental analyses were
performed on Elementar Vario analyzer (Vario EL-III).
Mass spectra were recorded on ESI-MS (AB-Sciex 2000,
Applied Biosystem).
3041, 1600, 1503, 1393, 1220; H NMR (400MHz,
CDCl ): 8.88 (s, 1H), 8.56 (d, J = 4.4 Hz, 1H), 8.06–
3
8.08 (m, 1H), 7.77 (s, 1H), 7.74 (d, J = 8.4 Hz, 2H),
7.63 (d, J= 8 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.21–
7.41 (m, 6H), 7.11 (d, J= 8 Hz, 2H), 6.86 (t, J =7.6Hz,
1H), 5.55 (dd, J= 12Hz, 6.8 Hz, 1H), 3.86 (dd,
J = 16.8Hz, 12Hz, 1H), 3.23 (dd, J = 16.8Hz, 6.8 Hz,
13
General procedure for the synthesis of compounds 5a–i.
1H); C NMR (100 MHz, CDCl ): 149.55, 148.80,
3
A mixture of (E)-3-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-1-
147.13, 144.28, 144.21, 139.58, 134.40, 132.60,
131.54, 129.42, 129.23, 129.19, 129.14, 128.61,
126.75, 126.59, 123.50, 122.60, 120.09, 118.95,
113.76, 56.67, 42.44; Anal. Calcd. for C H ClN : C,
(pyridin-3-yl)prop-2-en-1-ones 4a–i (5.0 mmol) and
phenylhydrazine (7.5 mmol) was refluxed in glacial acetic
acid for 4–5 h. Upon completion, ethanol was added to
the cool reaction mixture and allowed to stand overnight.
The precipitates obtained were filtered off, washed with
ethanol, and dried, affording compounds 5a–i. The
compounds 5a–e and 5h–i were precipitated out in
ethanol, and compounds 5f–g were precipitated out in
acetic acid.
29
22
5
73.18; H, 4.66; N, 14.71. Found: C, 73.13; H, 4.69; N,
+
14.72; MS (ESI) [M + H] Calcd. for C H ClN :
29
22
5
+
476.2, Found: 476.4 [M + H] .
3-(5-(3-(3-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-
4,5-dihydro-1H-pyrazol-3-yl)pyridine (5d).
Yellow solid;
-
1
Yield: 82%; mp 168–170°C; IR (neat, ν
cm ):
max
1
3
-(5(1,3-diphenyl-1H-pyrazol-4-yl)-1-phenyl-4,5-dihydro-1H-
3030, 1593, 1501, 1391, 1218; H NMR (400MHz,
pyrazol-3-yl)pyridine (5a). Yellow solid; Yield: 76%; mp
CDCl ): 8.89 (s, 1H), 8.56 (d, J =4.4 Hz, 1H), 8.05 (d,
3
-1
1
1
8
88–190°C; IR (neat, ν
398, 1228; H NMR (400MHz, CDCl ): 8.88 (s, 1H),
.56 (d, J = 4.4 Hz, 1H), 7.78–8.08 (m, 4H), 7.65 (d,
cm ): 3129, 1596, 1501,
J = 8Hz, 1H), 7.99 (s, 1H), 7.78 (s, 1H), 7.70 (d,
J = 7.6 Hz, 1H), 7.64 (d, J= 7.6 Hz, 2H), 7.57 (d,
J = 7.2 Hz, 1H), 7.21–7.41 (m, 7H), 7.11 (d, J =8.4 Hz,
2H), 6.86 (t, J= 7.2 Hz, 1H), 5.54 (dd, J = 12Hz,
6.8 Hz, 1H), 3.85 (dd, 16.8Hz, J= 12Hz, 1H), 3.22
max
1
3
J =8 Hz, 2H), 7.20–7.55 (m, 9H), 7.12 (d, J= 8 Hz, 2H),
6
3
.84 (t, J= 7.2Hz, 1H), 5.58 (dd, J= 12Hz, 6.8 Hz, 1H),
.86 (dd, J= 17.2 Hz, 12.4Hz, 1H), 3.26 (dd,
13
(dd, 16.8 Hz, J = 6.8 Hz, 1H):
C NMR (100MHz,
1
3
J =16.8 Hz, 6.8Hz, 1H); C NMR (100MHz, CDCl3):
CDCl3): 149.50, 148.40, 147.10, 144.22, 144.15,
139.53, 135.05, 132.67, 131.36, 130.99, 130.38,
129.44, 129.19, 128.61, 126.85, 126.65, 126.39,
123.52, 123.11, 122.74, 120.10, 119.01, 113.75, 56.60,
42.47; Anal. Calcd for C H BrN : C, 66.93; H, 4.26;
1
1
1
5
5
50.00, 149.46, 147.14, 144.29, 144.23, 139.70, 133.03,
32.62, 129.40, 129.18, 128.98, 128.72, 128.45, 128.02,
26.59, 126.34, 123.52, 122.62, 119.97, 118.90, 113.75,
6.69, 42.55; Anal. Calcd. for C H N : C, 78.89; H,
2
9
23
5
29 22
5
.25; N, 15.86. Found: C, 78.44; H, 5.58; N, 15.98; MS
N, 13.46. Found: C, 66.88; H, 4.29; N, 13.48; MS
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

R. Alam, Md. A. Alam, A. K. Panda, and Rahisuddin
ESI) [M + H] Calcd for C H BrN : 520.1, Found: Yield: 83%; mp 186–188°C; IR (neat, ν cm ): 3043,
max
Vol 000
+
-1
(
2
9
22
5
+
1
5
22.7 ([M+ H] +2).
1592, 1503, 1397; H NMR (400MHz, CDCl ): 8.88 (s,
3
3
-(5-(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-
1H), 8.55 (d, J= 4 Hz, 1H), 8.06 (d, J =8 Hz, 1H), 7.75
(s, 1H), 7.69 (d, J= 8 Hz, 2H), 7.64 (d, J = 7.6Hz, 2H),
7.20–7.40 (m, 8H), 7.12 (d, J= 8 Hz, 2H), 6.84 (t,
J = 7.6 Hz, 1H), 5.57 (dd, J= 12Hz, 6.8 Hz, 1H), 3.85
(dd, J = 16.8Hz, 12Hz, 1H), 3.25 (dd, J= 16.8 Hz, 6.8 Hz,
4
,5-dihydro-1H-pyrazol-3-yl)pyridine (5e).
Yield: 83%; mp 196–198°C; IR (neat, ν
594, 1492, 1395, 1302; H NMR (400MHz, CDCl ):
.87 (s, 1H), 8.56 (d, J = 4 Hz, 1H), 8.06 (d, J = 8Hz,
H), 7.77 (s, 1H), 7.21–7.69 (m, 12H), 7.11 (d, J = 8Hz,
H), 6.86 (t, J= 8.8Hz, 1H), 5.55 (dd, J= 12Hz, 6.8 Hz,
H), 3.86 (dd, J= 16.8Hz, 12Hz, 1H), 3.23 (dd,
Yellow solid;
-
1
cm ): 3041,
max
1
1
8
1
2
1
3
13
1H), 2.44 (s, 3H); C NMR (100 MHz, CDCl ): 150.08,
3
149.34, 147.06, 144.24, 139.73, 138.29, 132.67, 130.11,
129.64, 129.35, 129.14, 128.79, 127.88, 126.49, 123.51,
122.44, 119.95, 118.90, 113.75, 56.76, 42.51, 21.39;
Anal. Calcd for C H N : C 79.10; H, 5.53; N, 15.37.
1
3
J =16.8 Hz, 6.8Hz, 1H); C NMR (100MHz, CDCl3):
1
1
1
4
1
49.41, 148.82, 146.99, 144.19, 139.56, 132.72, 132.09,
31.95, 129.49, 129.41, 129.18, 128.66, 126.79, 126.63,
23.55, 122.62, 122.57, 120.12, 118.99, 113.75, 56.70,
2.44; Anal. Calcd for C H BrN : C 66.93; H, 4.26; N,
3.46. Found: C, 66.99; H, 4.22; N, 13.44; MS (ESI) [M
H] Calcd for C H BrN : 520.1, Found: 522.7 ([M
3
0 25 5
+
Found: C, 79.06; H, 5.55; N, 15.39; MS (ESI) [M + H]
+
Calcd for C H N : 456.2, Found: 456.5 [M +H] .
3
0 25 5
3-(5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phe-
2
9
22
5
nyl-4,5-dihydro-1H-pyrazol-3-yl)pyridine (5i). Yellow solid;
+
-1
+
+
Yield: 76%; mp 184–186°C; IR (neat, ν
cm ): 3043,
2
9
22
5
max
+
1
H] +2).
1600, 1505, 1397, 1251; H NMR (400MHz, CDCl ):
3
3
-(5-(3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-
8.88 (d, J= 1.2 Hz, 1H), 8.54 (d, J = 4.8 Hz, 1H), 7.73–
8.05 (m, 4H), 7.63 (d, J = 8.0 Hz, 2H), 7.37 (t, J =8.4 Hz,
2H) 7.19–7.31 (m, 4H), 7.13 (d, J = 8 Hz, 2H), 7.06 (d,
J = 8.8 Hz, 2H), 6.85 (t, J= 7.2 Hz, 1H), 5.53 (dd,
J = 12Hz, 6.8 Hz, 1H), 3.87 (s, 3H), 3.80 (dd, J= 16.8Hz,
4
,5-dihydro-1H-pyrazol-3-yl)pyridine (5f).
Yield: 79%; mp 176–178°C; IR (neat, νmax cm ): 3114,
598, 1536, 1499, 1352, 1326, 1225;
400 MHz, CDCl ): 8.88 (s, 1H), 8.66 (s, 1H), 8.56 (d,
Orange solid;
-
1
1
1
(
H NMR
3
13
J =4 Hz, 1H), 8.26–8.29 (m, 1H), 8.17 (d, J= 8 Hz, 1H),
12.4 Hz, 1H), 3.22 (dd, J = 16.8Hz, 6.8 Hz, 1H);
C
8
7
.06 (d, J= 8 Hz, 1H), 7.85 (s, 1H), 7.21–7.71 (m, 9H),
.11 (d, J =8 Hz, 2H), 6.87 (t, J= 6.8, 1H), 5.60 (dd,
NMR (100MHz, CDCl ): 159.82, 149.89, 149.41,
3
149.12, 144.29, 139.75, 132.60, 129.35, 129.28, 129.15,
128.76, 126.42, 126.21, 125.56, 123.48, 122.25, 119.95,
118.82, 114.38, 113.78, 56.77, 55.41, 42.45ppm; Anal.
Calcd for C H N O: C 76.41; H, 5.34; N, 14.85. Found
J =12.4 Hz, 6.8Hz, 1H), 3.95 (dd, J = 16.8Hz, 12Hz,
1
3
1
H), 3.28 (dd, J = 16.8Hz, 6.8Hz, 1H);
C NMR
(100 MHz, CDCl ): 149.54, 148.41, 147.16, 144.25,
3
30 25 5
+
1
1
1
44.19, 139.55, 135.08, 132.62, 131.35, 131.00, 130.36,
29.43, 129.18, 128.60, 126.83, 126.64, 126.40, 123.48,
23.10, 122.75, 120.10, 119.01, 113.78, 56.62, 42.47;
C, 76.35; H, 5.38; N, 14.87; MS (ESI) [M + H] Calcd
+
for C H N O: 472.2, Found: 472.4 [M + H] .
3
0 25 5
General procedure for the synthesis of compounds 6a–i.
A mixture of (E)-3-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-1-
(pyridin-3-yl)prop-2-en-1-ones 4a–i (1 mmol) and
hydrazine hydrate (0.5 mL) in formic acid (5 mL) was
refluxed for 8–10h. On completion, the reaction mixture
was allowed to attain room temperature, poured into
crushed ice and neutralized by dilute NaOH. The
precipitate obtained was filtered off, washed with water,
and dried, affording compounds 6a–i. The compounds
6a–i were precipitated out in DMF:Ethanol.
Anal. Calcd for C H N O : C, 71.59; H, 4.56; N,
1
+
+
2
9 22 6 2
7.27. Found: C, 71.64; H, 4.53; N, 17.25; MS (ESI) [M
+
H] Calcd for C H N O : 487.2, Found: 487.2 [M
29 22 6 2
+
H] .
3
-(5-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenyl-
4
,5-dihydro-1H-pyrazol-3-yl)pyridine (5g).
Yellow solid;
-
1
Yield: 82%; mp 198–200°C; IR (neat, ν
1
CDCl ): 8.88 (s, 1H), 8.57 (s, 1H), 8.37 (d, J= 8.4 Hz,
cm ): 3153,
max
1
600, 1562, 1503, 1397, 1343, 1229; H NMR (400MHz,
3
2
H), 8.13 (d, J = 8Hz, 1H), 8.00 (d, J= 8.4Hz, 2H), 7.82
5-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihyd-
(
s, 1H), 7.66 (d, J =8 Hz, 2H), 7.22–7.44 (m, 6H), 7.11 (d,
ropyrazole-1-carbaldehyde (6a). White solid; Yield: 83%;
-
1
J =8 Hz, 2H), 6.88 (t, J= 7.2Hz, 1H), 5.63 (dd,
J =12.4 Hz, 6.8Hz, 1H), 3.92 (dd, J =16.8 Hz, 12 Hz, 1H),
3
DMSO-d ): 149.86, 147.90, 147.38, 145.90, 144.63,
1
1
Anal. Calcd for C H N O : C, 71.59; H, 4.56; N, 17.27.
Found: C, 71.67; H, 4.51; N, 17.24; MS (ESI) [M+ H]
Calcd for C H N O : 487.2, Found: 487.5 [M +H] .
mp 218–220°C; IR (neat, ν
1562, 1506, 1421; H NMR (400MHz, CDCl ): 9.00 (s,
cm ): 3059, 1666, 1599,
max
1
3
1
3
.25 (dd, J = 16.8Hz, 6.8Hz, 1H); C NMR (100MHz,
1H), 8.80 (s, 1H), 8.64 (d, J= 3.6 Hz, 1H), 7.98 (d,
J = 8Hz, 1H), 7.86 (s, 1H), 7.69 (d, J =7.6 Hz, 4H), 7.26–
7.43 (m, 7H), 5.85 (dd, J =12Hz, 4.4 Hz, 1H), 3.68 (dd,
J = 17.6Hz, 12Hz, 1H), 3.14 (dd, J = 17.6Hz, 5.2Hz,
6
39.59, 139.41, 133.19, 130.05, 129.38, 128.48, 127.35,
24.37, 124.16, 119.92, 118.93, 114.02, 56.38, 42.41;
13
1H); C NMR (100MHz, DMSO-d ): 160.42, 154.39,
2
9
22
6
2
6
+
151.46, 150.11, 148.17, 139.78, 134.33, 133.19, 129.94,
129.09, 128.58, 127.57, 127.46, 126.80, 124.31, 122.63,
118.65, 51.42, 42.81; Anal. Calcd. for C H N O: C,
+
29 22 6 2
3
-(1-phenyl-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-1-phenyl-
24 19 5
4
,5-dihydro-1H-pyrazol-3-yl)pyridine (5h).
Yellow solid;
73.27; H, 4.87; N, 17.80. Found: C, 73.25; H, 4.86; N,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Design, Synthesis, and Cytotoxicity Evaluation of Pyrazolylpyrazoline
Derivatives as Potential Anticancer Agents
+
1
7.78; MS (ESI) [M +H] Calcd for C H N O: 394.2,
1H), 8.03 (d, J= 8 Hz, 1H), 7.85 (s, 1H), 7.68 (d,
J = 8Hz, 2H), 7.26–7.69 (m, 8H), 5.82 (dd, J = 12Hz,
4.8 Hz, 1H), 3.72 (dd, J =17.6 Hz, 11.6Hz, 1H), 3.15 (dd,
2
4 19 5
+
Found: 394.7 [M+ H] .
5
-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-
-yl)-4,5-dihydropyrazole-1-carbaldehyde (6b). White solid;
Yield: 86%; mp 226–228°C; IR (neat, ν
666, 1599, 1532, 1503, 1421; H NMR (400MHz,
CDCl ): 9.00 (s, 1H), 8.83 (s, 1H), 8.67 (s, 1H), 8.03 (d,
J =8 Hz, 1H), 7.86 (s, 1H), 7.26–7.72 (m, 8H), 7.11 (t,
J =8.4 Hz, 2H), 5.81 (dd, J = 12Hz, 4.4Hz, 1H), 3.71
dd, J = 17.6Hz, 12Hz, 1H), 3.16 (dd, J = 17.6Hz, 4.8 Hz,
H); C NMR (100MHz, CDCl ): 164.10, 161.63,
13
3
J = 17.6Hz, 4.8 Hz, 1H; C NMR (100 MHz, CDCl3):
160.33, 153.33, 153.36, 150.98, 149.30, 147.46, 139.58,
134.02, 131.90, 131.82, 130.02, 129.44, 127.06, 126.93,
126.30, 123.86, 122.67, 121.06, 119.21, 51.44, 41.95;
Anal. Calcd for C H BrN O: C, 61.03; H, 3.84; N,
-
1
cm ): 3093,
max
1
1
3
24
18
5
14.83. Found: C, 60.79; H, 4.74; N, 14.78; MS (ESI) [M
+
(
+ H] Calcd for C H BrN O: 472.1, Found: 472.2 [M
24
18
5
13
+
1
1
1
1
4
+ H] .
3
60.28, 153.45, 151.36, 149.59, 147.79, 139.61, 133.69,
30.33, 130.25, 129.43, 129.00, 128.97, 126.92, 126.83,
26.22, 123.70, 120.98, 119.16, 115.84, 115.62, 51.41,
1.93; Anal. Calcd for C H FN O: C, 70.06; H, 4.41;
5-(3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-
yl)-4,5-dihydropyrazole-1-carbaldehyde (6f).
White solid;
cm ): 3100,
-
1
Yield: 91%; mp 235–237°C; IR (neat, ν
max
1
1666, 1599, 1540, 1510, 1424; H NMR (400MHz,
2
4
18
5
N, 17.02. Found: C, 69.98; H, 4.36; N, 17.16; MS (ESI)
CDCl ): 9.00 (s, 1H), 8.85 (s, 1H), 8.67 (d, J = 4.4Hz,
3
+
[
[
M + H] Calcd for C H FN O: 412.1, Found: 412.3
M + H] .
1H), 8.59 (s, 1H), 8.18–8.24 (m, 2H), 8.06 (d, J = 8 Hz,
1H), 7.90 (s, 1H), 7.70 (d, J = 8Hz, 2H), 7.32–7.65 (m,
5H), 5.85 (dd, J =11.6 Hz, 4.8 Hz, 1H), 3.83 (dd,
2
4
18
5
+
5
-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-
3
-yl)-4,5-dihydropyrazole-1-carbaldehyde (6c). White solid;
J = 17.6Hz, 12Hz, 1H), 3.22 (dd, J = 17.6Hz, 4.8Hz,
-
1
13
Yield: 89%; mp 214–216°C; IR (neat, ν
1
CDCl ): 9.00 (s, 1H), 8.83 (s, 1H), 8.66 (d, J= 4 Hz, 1H),
8
cm ): 3070,
1H); C NMR (100MHz, DMSO-d ): 160.34, 154.26,
max
6
1
655, 1603, 1547, 1510, 1417; H NMR (400MHz,
151.26, 148.52, 148.05, 139.58, 134.84, 134.29, 130.53,
129.81, 128.19, 127.36, 127.05, 124.14, 123.07, 122.74,
118.86, 51.22, 42.62; Anal. Calcd for C H N O : C,
3
.01 (d, J = 7.6Hz, 1H), 7.85 (s, 1H), 7.26–7.69 (m,
0H), 5.82 (dd, J =11.6 Hz, 4.8 Hz, 1H), 3.71 (dd,
24 18 6 3
1
65.75; H, 4.14; N, 19.17. Found: C, 65.64; H, 4.35; N,
+
J =17.6 Hz, 11.6Hz, 1H), 3.15 (dd, J = 17.6Hz, 4.8 Hz,
19.38; MS (ESI) [M +H] Calcd for C H N O : 439.1,
Found: 439.3 [M +H] .
2
4 18 6 3
13
+
1
1
1
1
6
1
4
H); C NMR (100MHz, CDCl ): 160.25, 153.45,
3
51.43, 149.27, 147.85, 139.59, 134.43, 133.65, 131.39,
29.73, 129.43, 128.94, 126.89, 126.25, 123.68, 121.14,
19.17, 51.40, 41.98; Anal. Calcd for C H ClN O: C,
5-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-
yl)-4,5-dihydropyrazole-1-carbaldehyde (6g). Yellow solid;
-
1
Yield: 89%; mp 238–240°C; IR (neat, νmax cm ): 3093,
2
4
18
5
1
7.37; H, 4.24; N, 16.37. Found: C, 67.42; H, 4.34; N,
1670, 1599, 1562, 1506, 1469, 1343, 1320; H NMR
+
6.28; MS (ESI) [M +H] Calcd for C H ClN O:
(400 MHz, CDCl ): 9.01 (s, 1H), 8.86 (s, 1H), 8.67 (d,
2
4
18
5
3
+
28.1, Found: 428.4 [M +H] .
J = 4.0 Hz, 1H), 8.31 (d, J= 8.8 Hz, 2H), 8.06 (d,
J = 8.0 Hz, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.88 (s, 1H),
7.70 (d, J = 8 Hz, 2H), 7.30–7.47 (m, 4H), 5.86 (dd,
5
-(3-(3-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-
3
-yl)-4,5-dihydropyrazole-1-carbaldehyde (6d). White solid;
-
1
Yield: 82%; mp 208–210°C; IR (neat, ν
1
cm ): 3067,
J = 11.6Hz, 4.8 Hz, 1H), 3.81 (dd, J= 17.6Hz, 12Hz,
max
1
13
678, 1599, 1547, 1506, 1424; H NMR (400MHz,
1H), 3.19 (dd, J= 17.6 Hz, 4.8 Hz, 1H);
C NMR
CDCl ): 9.01 (s, 1H), 8.83 (s, 1H), 8.67 (d, J= 4.4 Hz,
(100 MHz, DMSO-d ): 160.16, 153.75, 151.09, 147.86,
3
6
1
7
3
H), 8.03 (d, J =8 Hz, 1H), 8.87 (d, J = 6Hz, 2H),
.26–7.70 (m, 9H), 5.83 (dd, J =11.6 Hz, 4.8 Hz, 1H),
.73 (dd, J= 16.8 Hz, 11.6Hz, 1H), 3.15 (dd,
147.30, 139.64, 139.45, 134.11, 129.54, 129.17, 127.37,
127.13, 127.07, 123.91, 123.86, 122.65, 118.96, 51.30,
42.52; Anal. Calcd for C H N O : C, 65.75; H, 4.14;
24 18 6 3
1
3
J =17.6 Hz, 4.8Hz, 1H); C NMR (100MHz, CDCl3):
N, 19.17. Found: C, 65.97; H, 4.21; N, 19.46; MS (ESI)
+
1
1
1
60.29, 153.45, 151.40, 148.90, 147.86, 139.56, 134.96,
33.74, 131.40, 130.22, 129.45, 126.96, 126.84, 126.52,
23.66, 122.86, 121.09, 119.22, 51.34, 41.83; Anal.
[M + H] Calcd for C H N O : 439.1, Found: 439.6 [M
+ H] .
2
4 18 6 3
+
5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-
Calcd for C H BrN O: C, 61.03; H, 3.84; N, 14.83.
dihydropyrazole-1-carbaldehyde (6h). White solid; Yield:
24
18
5
+
-1
Found: C, 60.89; H, 4.64; N, 14.68; MS (ESI) [M+ H]
Calcd for C H BrN O: 472.1, Found: 474.6 ([M
78%; mp 180–182°C; IR (neat, ν , cm ): 3137, 1659,
max
1
1603, 1547, 1510, 1462; H NMR (400MHz, CDCl ):
24
18
5
3
+
+
H] +2).
9.01 (s, 1H), 8.80 (s, 1H), 8.65 (d, J = 4.4 Hz, 1H), 8.00
(d, J = 8.0 Hz, 1H), 7.85 (s, 1H), 7.69 (d, J =8.0 Hz, 2H),
7.57 (d, J = 8.0 Hz, 2H), 7.35–7.58 (m, 6H), 5.87 (dd,
5
-(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-
3
-yl)-4,5-dihydropyrazole-1-carbaldehyde (6e). White solid;
-
1
Yield: 87%; mp 216–218°C; IR (neat, ν
1
cm ): 3059,
J = 11.6Hz, 4.8 Hz, 1H), 3.67 (dd, J = 17.6Hz, 11.6 Hz,
max
1
13
674, 1603, 1551, 1506, 1417; H NMR (400MHz,
1H), 3.15 (dd, J= 17.6 Hz, 4.4 Hz, 1H);
C NMR
CDCl ): 9.00 (s, 1H), 8.84 (s, 1H), 8.67 (d, J= 4.0 Hz,
(100 MHz, CDCl ): 160.25, 153.61, 151.20, 150.46,
3
3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

R. Alam, Md. A. Alam, A. K. Panda, and Rahisuddin
Vol 000
1
1
5
7
1
47.83, 139.76, 138.20, 133.70, 130.01, 129.40, 129.36,
28.29, 127.03, 126.60, 126.16, 123.59, 120.81, 119.12,
1.60, 41.74, 21.27; Anal. Calcd for C H N O: C
solubilised using DMSO. The DMSO converts the phenol
red in the tissue culture medium to a yellow color that
does not interfere with the MTT formazan measurement.
2
4 21 5
3.69; H, 5.19; N, 17.19. Found: C, 74.02; H, 5.08; N,
The DMSO dissolves the formazan to give
a
+
7.34; MS (ESI) [M+ H] Calcd for C H N O : 408.2,
homogeneous blue solution. The absorbance was
measured at a test wavelength of 570 nm using an ELISA
plate reader, LMR-340M (Labexim International, Austria)
with a microplate reader. The percentage inhibition was cal-
culated by the formulae:
2
4 18 6 3
+
Found: 408.4 [M+ H] .
5
-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyrid-
in-3-yl)-4,5-dihydropyrazole-1-carbaldehyde (6i).
solid; Yield: 78%; mp 210–212°C; IR (neat, ν
White
-
1
cm ):
H NMR
max
1
3
041, 1666, 1599, 1536, 1506, 1421;
Mean OD of treated cells
Mean OD of the vehicle control cells ðnegative controlÞ
%Inhibition ¼ 100-
ꢀ100:
(
400 MHz, CDCl ): 9.01 (s, 1H), 8.81 (s, 1H), 8.65 (d,
Each assay was repeated for three times. IC50 values
were calculated from the dose effect curve and expressed
as concentration (μM) of drug.
3
J =4.0 Hz, 1H), 8.00 (d, J = 8.0Hz, 1H), 7.85 (s, 1H),
7
7
.69 (d, J = 8.0Hz, 2H), 7.62 (d, J = 8.4Hz, 2H), 7.24–
.44 (m, 4H), 6.93 (d, J = 8.4Hz, 2H), 5.84 (dd,
J =11.6 Hz, 4.8Hz, 1H), 3.80 (s, 3H), 3.67 (dd,
J =17.6 Hz, 12.0Hz, 1H), 3.15 (dd, J = 17.6Hz, 4.8 Hz,
CONCLUSION
13
1
1
1
1
H); C NMR (100MHz, CDCl ): 160.27, 159.74,
3
53.54, 151.11, 150.29, 147.72, 139.75, 133.79, 129.70,
29.37, 127.09, 126.59, 126.16, 125.37, 123.66, 120.66,
19.09, 114.16, 55.31, 51.61, 41.71; Anal. Calcd for
In this study, a series of 5a–i and 6a–i were
synthesized and evaluated in vitro cytotoxicity against a
panel of three human cancer cell lines namely; HeLa
C H N O : C, 70.91; H, 5.00; N, 16.54, Found: C,
2
5 21 5 2
(
human cervix), NCI-H460 (human lung), PC-3 (human
+
7
0.62; H, 5.04; N, 16.86; MS (ESI) [M +H] Calcd for
prostate), and NIH-3T3 (mouse embryo fibroblasts)
normal cell line. These compounds exhibited moderate
to good cytotoxic activity against the above mentioned
cancer cell lines and weak toxicity against normal cell
line. Among all the synthesized compounds, analogue
+
C H N O : 424.2, Found: 424.4 [M + H] .
24 18 6 3
Cytotoxic activity. The MTT [3-(4,5-dimethylthiazolyl-
2
)-2,5-diphenyltetrazolium bromide)] assay is based on
conversion of yellow water soluble tetrazolium dye to a
water-insoluble purple formazan by living cells. The
amount of formazan crystals generated is directly
proportional to the number of viable cells. The Hela,
NCI-H460, PC-3, and NIH-3T3 cells were grown (37°C,
6
g demonstrated superior cytotoxic activity with the
IC50 value of 7.98± 1.08 μM for Hela cancer cell line.
So, the present study offers preliminary pointers which
broaden the research scope for further design, modify,
and investigate the structure of these molecules to
expand the utility of such novel compounds as potential
anticancer agents.
5
% CO in water jacketed incubator shell) using DMEM
2
media with 10% fetal bovine serum (FBS) and seeded on
a single 96 well plate (Falcon Plate, Corning costar,
USA) and allowed to adhere for MTT assays. The plate
was treated with increasing concentration made in medium
of 1, 12.5, 25, 50, and 100 μM of synthesized compounds
and etoposide dissolved in dimethyl sulfoxide (DMSO).
These concentrations were added in triplicate to the single
Acknowledgments. The authors thank to the Head, Department
of Chemistry, JMI, New Delhi for providing research facilities.
We thank to Dr. Mohammad Abid for helpful discussions.
Raquib Alam is thankful to UGC, New Delhi for research
fellowship.
9
6 well tissue culture plate. After 24h of treatment, the
MTT assay was performed to check cell viability. For the
MTT assay, the media was removed from all the wells,
1
0 μL of MTT reagent (Chemicon International, USA) per
REFERENCES AND NOTES
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plates were incubated (37°C and 5% CO ) for 2–3 h; after
that, the reagent was removed, and the crystals were
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