Design, synthesis and cytotoxicity evaluation of pyrazolyl pyrazoline and pyrazolyl aminopyrimidine derivatives as potential anticancer agents

Article abstract of DOI:10.1007/s00044-017-2082-8

In an attempt to find bio-active heterocyclic analogues, a series of novel 1-(5-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazol-1-yl)ethanones 5a–i and 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-6-(pyridine-3-yl)pyrimidin-2-amines 6a–i were designed, synthesized, and evaluated for their in vitro cytotoxicity 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) cell lines. Most of these compounds exhibited moderate to good cytotoxicity against the tested cancer cell lines and weak toxicity against normal cell line. Analogs 5f, 5g, 5i, 6b–g showed significant cytotoxicity as compared to the standard drug etoposide. The compound 6g exhibited superior activity with IC₅₀ value of 5.47 ± 0.44 μM against Hela cancer cell line.

Full text of DOI:10.1007/s00044-017-2082-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2082-8
ORIGINAL RESEARCH
Design, synthesis and cytotoxicity evaluation of pyrazolyl
pyrazoline and pyrazolyl aminopyrimidine derivatives as potential
anticancer agents
1
Raquib Alam1,2 Aftab Alam3,4 Amulya K. Panda4 Rahisuddin
●
●
●
Received: 25 April 2017 / Accepted: 21 September 2017
© Springer Science+Business Media, LLC 2017
●
Abstract In an attempt to find bio-active heterocyclic
analogues, a series of novel 1-(5-(3-(aryl)-1-phenyl-1H-
pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazol-1-yl)
ethanones 5a–i and 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-
6-(pyridine-3-yl)pyrimidin-2-amines 6a–i were designed,
synthesized, and evaluated for their in vitro cytotoxicity
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) cell
lines. Most of these compounds exhibited moderate to good
cytotoxicity against the tested cancer cell lines and weak
toxicity against normal cell line. Analogs 5f, 5g, 5i, 6b–g
showed significant cytotoxicity as compared to the standard
drug etoposide. The compound 6g exhibited superior
activity with IC50 value of 5.47 0.44 µM against Hela
cancer cell line.
Keywords Pyrazolylpyrazolines Pyrazolyl
●
●
●
aminopyrimidines Michael addition Cytotoxicity
MTT assay
Introduction
Cancer has become a big threat to human beings as millions
of people are suffering by this disease in both the developed
and the developing countries. Although efforts have been
made in reducing cancer incidence in United States, the
numbers of cancer patients continue to increase (Siegel
et al. 2012). Chemotherapy is one of the most effective
approaches used for the treatment of cancer patients.
However, lack of selectivity and development of drug
resistance diminish the efficacy of cancer chemotherapy
(Wu et al. 2014). Therefore, development of effective and
safe anticancer agents with less toxicity and high potency
remains critically important.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2082-8) contains supplementary
material, which is available to authorized users.
The pyrazole tethered heterocycles have received con-
siderable attention owing to their diverse chemotherapeutic
potential (Raffa et al. 2015; Küçükgüzel and Senkardes
2015; Zhang et al. 2014). Drugs bearing 1,2-diaryl-sub-
stituted 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
apixaban, celecoxib, fipronil, remogliflazone etabonate,
lonazolac, tolpiprazole, deracoxib, etc. Few pyrazole-based
drugs are depicted in Fig. 1. Crizotinib and ruxolitinib are
important pyrazole-based anticancer drug (Bronson et al.
2012). Pyrazole derivatives showed anticancer activity due
to their inhibition of topoisomerase I/II, epidermal growth
factor receptor, telomerase, vascular endothelial growth
* Rahisuddin
rahisuddin@jmi.ac.in
1
Department of Chemistry, Jamia Millia Islamia (A Central
University), Jamia Nagar, New Delhi 110025, India
2
Applied Science & Humanities Section, University Polytechnic,
Jamia Millia Islamia (A Central University), Jamia Nagar, New
Delhi 110025, India
3
Department of Pharmacy, School of Medical & Allied Science,
Galgotias University, Greater Noida 201301 U.P., India
4
Product Development Cell, National Institute of Immunology,
New Delhi 110067, India

Med Chem Res
Fig. 1 Pyrazole-based drugs
factor, aurora-A kinase, Janus kinase 2 (JAK2), cMet,
anaplastic lymphoma kinase (ALK), mTOR, cyclin-
dependent kinases, B-Raf, p38 MAPK, glycogen synthase
kinase-3, PI3K, Src family kinase, etc. Crizotinib is a dual
inhibitor of cMet/ALK and is used for the treatment of non-
small cell lung carcinoma (Williams et al. 2000; Kulp et al.
2004). Ruxolitinib is a JAK2 inhibitor and is used for the
treatment of myeloproliferative neoplasm (Chen et al.
2015). The chalcones are α,β-unsaturated ketones that
belong to the flavonoid family and are widely distributed in
plants. Their importance may be explained in two branches,
the biological activity associated with them and their syn-
thetic utility for the preparation of pharmacologically
important heterocyclic derivatives. Chalcones of various
classes have been extensively studied owing to their wide
range of pharmacological activities, such as anti-inflam-
matory, anti-mitotic, anti-leishmanial, anti-invasive, anti-
malarial, anti-bacterial, anti-fungal, anti-protozoal, anti-
viral, anti-trypanosomal, anti-cancer activities, etc. Chal-
cones are versatile synthon for the preparation of five-
membered and six-membered ring heterocycles (Powers
et al. 1998). The synthetic utility of chalcones for the pre-
paration of pyrazolines and 2-aminopyrimidines is well
documented. The pyrazolines show various pharmacologi-
cal activities, like anti-hypertensive (Meyers et al. 2010;
Casimiro-Garcia et al. 2014), anti-inflammatory (Abdel-
Halim et al. 2014; Kharbanda et al. 2014), anti-depressent
(Acker et al. 2013), anti-neurodegenerative (van Loevezijn
et al. 2011; Badavath et al. 2016), anti-diabetic (Kharbanda
et al. 2015), anti-bacterial (Karad et al. 2014; Viveka et al.
2015; Singh et al. 2015), anti-fungal (Singh et al. 2015;
Fig. 2 Bioactive pyrazoline and aminopyrimidine derivatives
Yang et al. 2015; Altıntop et al. 2015), anti-cancer (Amin
et al. 2015; Insuasty et al. 2010; Alam et al. 2017) activities,
etc. 2-Aminoyrimidine derivatives displayed diverse biolo-
gical activities, such as anti-tubercular (Khunt et al. 2012;
Singh et al. 2011), anti-diabetic (Singh et al. 2011), anti-
microbial (Thanh and Mai 2009), anti-inflammotory, anti-
platelet (Giridhar et al. 2012), anti-leishmanial (Sharma
et al. 2013), and anti-cancer (Lee et al. 2011; Qin et al.
2015). Few bioactive pyrazolines and aminopyrimidines are
depicted in Fig. 2.
Herein, efforts are made on establishing some pyrazoline
and aminopyrimidine analogs integrated with pyrazole
scaffold to describe the relevance pharmacological activity.
Motivated by our previous findings of anticancer chalcones
(Alam et al. 2016a, b), and in continuation of our ongoing
research program to get potential anticancer agents, we have
synthesized some pyrazolylpyrazoline and pyrazolyl ami-
nopyrimidine derivatives and evaluated for their cytotoxi-
city against three different human cancer cell lines and a

Med Chem Res
1
1655, 1599, 1506; H NMR (300 MHz, CDCl3): δ 8.81 (s,
1H), 8.63 (d, J = 4.5 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.81
(s, 1H), 7.23–7.72 (m, 11H), 5.91 (dd, J = 11.7 Hz, 4.8 Hz,
1H), 3.63 (dd, J = 17.4 Hz, 11.7 Hz, 1H), 3.1 (dd, J = 17.4
Hz, 4.8 Hz, 1H), 2.45 (s, 3H); 13C NMR (125 MHz,
CDCl3): δ 169.07, 151.66, 151.06, 150.43, 147.86, 139.80,
133.46, 133.13, 129.33, 128.61, 128.47, 128.20, 127.36,
126.55, 125.99, 123.49, 122.17, 119.13, 52.43, 41.57,
22.08; anal. calcd. for C25H21N5O: C, 73.69; H, 5.19; N,
17.19. Found: C, 73.64; H, 5.26; N, 17.16; MS (ESI) [M
+H]+ calcd. for C29H23N5: 408.47. Found: 408.5 [M+H]+.
Fig. 3 Target compounds
normal cancer cell line. The structures of target compounds
are depicted in Fig. 3.
1-(5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-
(pyridin-3-yl)-4,5-dihydropyrazol-1-yl)ethanone
(5b)
White solid; yield: 76%; m.p.: 236–238 °C; IR (neat, νmax
cm−1): 3134, 3089, 1666, 1592, 1510; 1H NMR (300 MHz,
CDCl3): δ 8.82 (s, 1H), 8.63–8.65 (m, 1H), 7.99 (d, J = 8.1
Hz, 1H), 7.80 (s, 1H), 7.24–7.74 (m, 8H), 7.10 (t, J = 8.7
Hz, 2H), 5.85 (dd, J = 11.7 Hz, 4.5 Hz, 1H), 3.64 (dd, J =
17.4 Hz, 11.7 Hz, 1H), 3.10 (dd, 17.4 Hz, J = 4.5 Hz, 1H),
2.44 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 169.09,
163.79, 161.82, 151.54, 151.17, 149.59, 147.83, 139.71,
133.43, 130.36, 130.30, 129.37, 129.20, 127.29, 126.67,
125.94, 123.55, 122.24, 119.13, 115.69, 115.52, 52.28,
41.67, 22.06; anal. calcd. for C25H20FN5O: C, 70.58; H,
4.74; N, 16.46. Found: C, 70.55; H, 4.73; N, 16.48. MS
(ESI) [M+H]+ calcd. for C25H20FN5O: 426.46. Found:
426.8 [M+H]+.
Experimental
Chemistry
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 thin-layer chromatography (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 spec-
trometer. 1H NMR spectra of the compounds were recorded
on Bruker Avance-300 spectrometer and 13C NMR spectra
were recorded on Bruker Avance III-500 spectrometer using
CDCl3 or DMSO-d6 as a solvent and trimethylsilane as an
internal standard. Splitting patterns are designated as: s,
singlet; d, doublet; dd, doublet of doublet; t, triplet; m,
multiplet. Chemical shift values are represented by ppm.
Elemental analyses were performed on elementar vario
analyzer (Vario EL-III). Mass spectra were recorded on
ESI-MS (AB-Sciex 2000, Applied Biosystem).
1-(5-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-
(pyridin-3-yl)-4,5-dihydropyrazol-1-yl)ethanone
(5c)
White solid; yield: 71%; m.p.: 226–228 °C; IR (neat, νmax
cm−1): 3134, 3026, 1663, 1599, 1506; 1H NMR (300 MHz,
CDCl3): δ 8.84 (s, 1H), 8.64 (d, J = 4.5 Hz, 1H), 7.99 (d, J
= 8.1 Hz, 1H), 7.80 (s, 1H), 7.26–7.70 (m, 10H), 5.86 (dd,
J = 11.7 Hz, 4.8 Hz, 1H), 3.65 (dd, J = 17.4 Hz, 11.7 Hz,
1H), 3.09 (dd, 17.7 Hz, J = 4.8 Hz, 1H), 2.45 (s, 3H); 13C
NMR (125 MHz, CDCl3): δ 169.08, 151.57, 151.18,
149.28, 147.84, 139.68, 134.27, 133.45, 131.61, 129.79,
129.38, 128.84, 127.25, 126.74, 126.01, 123.56, 122.38,
119.16, 52.28, 41.70, 22.06; anal. calcd. for C25H20ClN5O:
C, 67.95; H, 4.56; N, 15.85. Found: C, 67.89; H, 4.61; N,
15.79; MS (ESI) [M+H]+ calcd. for C25H20ClN5O: 442.91.
Found: 442.7 [M+H]+.
General procedure for the synthesis of compounds 5a–i
To a solution of (E)-3-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-
1-(pyridin-3-yl)prop-2-en-1-ones 4a–i (5.0 mmol) in acetic
acid (10 ml) was added hydrazine hydrate (20 mmol) and
the reaction mixture was refluxed for 4 h. Upon cooling, the
reaction mixture was diluted with ethanol and allowed to
stand overnight. The precipitates were filtered, washed with
ethanol, and crystallized from ethanol, afforded compounds
5a–i.
1-(5-(3-(3-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-
(pyridin-3-yl)-4,5-dihydropyrazol -1-yl)ethanone (5d)
White solid; yield: 79%; m.p.: 232–234 °C; IR (neat, νmax
cm−1): 3130, 3070, 1663, 1599, 1514; 1H NMR (300 MHz,
CDCl3): 8.83 (s, 1H), 7.24–8.65 (m, 13H), 5.87 (dd, J =
11.7 Hz, 4.8 Hz, 1H), 3.66 (dd, J = 17.7 Hz, 12.0 Hz, 1H),
3.09 (dd, 17.7 Hz, J = 4.8 Hz, 1H), 2.45 (s, 3H); 13C NMR
(125 MHz, CDCl3): 169.13, 151.54, 151.16, 148.93,
1-(5-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-
dihydropyrazol-1-yl)ethanone (5a) White solid; yield:
86%; m.p.: 228–230 °C; IR (neat, νmax cm−1): 3074, 3033,

Med Chem Res
147.86, 139.64, 135.16, 133.52, 131.50, 131.19, 130.10,
129.39, 127.22, 127.03, 126.81, 126.31, 123.53, 122.74,
122.33, 119.20, 52.21, 41.57, 22.07; anal. calcd. for
C25H20BrN5O: C, 61.74; H, 4.14; N, 14.40. Found: C,
61.68; H, 4.16; N, 14.36; MS (ESI) [M+H]+ calcd. for
C25H20BrN5O: 486.36. Found: 488.4 ([M+H]++2).
4.46; N, 18.57. Found: C, 66.38; H, 4.49; N, 18.52; MS
(ESI) [M+H]+ calcd. for C25H20N6O3: 453.46. Found:
453.5 [M+H]+.
1-(5-(1-Phenyl-3-p-tolyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-
4,5-dihydropyrazol-1-yl) ethanone (5h) White solid; yield:
78%; m.p.: 220–222 °C; IR (neat, νmax cm−1): 3137, 3041,
1
1-(5-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-
(pyridin-3-yl)-4,5-dihydropyrazol -1-yl)ethanone (5e)
White solid; yield: 82%; m.p.: 238–240 °C; IR (neat, νmax
cm−1): 3134, 3063, 1659, 1599, 1506; 1H NMR (300 MHz,
CDCl3): δ 8.84 (s, 1H), 7–8.66.97 (m, 2H), 7.80 (s, 1H),
7.26–7.69 (m, 10H), 5.86 (dd, J = 12.0 Hz, 4.8 Hz, 1H),
3.65 (dd, J = 17.4 Hz, 11.7 Hz, 1H), 3.09 (dd, 17.7 Hz, J =
4.8 Hz, 1H), 2.45 (s, 3H); 13C NMR (125 MHz, CDCl3): δ
169.09, 151.58, 151.20, 149.30, 147.84, 139.67, 133.46,
132.07, 131.80, 130.08, 129.39, 127.24, 126.77, 126.04,
123.57, 122.51, 122.37, 119.17, 52.26, 41.70, 22.06; anal.
calcd. for C25H20BrN5O: C, 61.74; H, 4.14; N, 14.40.
Found: C, 61.77; H, 4.21; N, 14.37; MS (ESI) [M+H]+
calcd. for C25H20BrN5O: 486.36. Found: 488.3 ([M
+H]++2).
1670, 1599, 1510; H NMR (300 MHz, CDCl3): δ 8.79 (s,
1H), 8.62 (d, J = 4.8 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.80
(s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.58 (d, J = 7.8 Hz, 2H),
7.18–7.43 (m, 6H), 5.91 (dd, J = 11.7 Hz, 4.5 Hz, 1H), 3.61
(dd, J = 17.7 Hz, 12.0 Hz, 1H), 3.09 (dd, 17.7 Hz, J = 4.5
Hz, 1H), 2.45 (s, 3H), 2.34 (s, 3H); 13C NMR (125 MHz,
CDCl3): δ 169.03, 151.74, 150.99, 150.47, 147.86, 139.84,
138.01, 133.46, 130.23, 129.30, 128.34, 127.40, 126.45,
125.96, 123.44, 122.03, 119.08, 52.45, 41.43, 22.07, 21.25;
anal. calcd. for C26H23N5O: C, 74.09; H, 5.50; N, 16.62.
Found: C, 74.16; H, 5.54; N, 16.59; MS (ESI) [M+H]+
calcd. for C26H23N5O: 422.49. Found: 422.4 [M+H]+.
1-(5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-
(pyridin-3-yl)-4,5-dihydro pyrazol-1-yl)ethanone (5i)
White solid; yield: 76%; m.p. 224–226 °C; IR (neat, νmax
cm−1): 3141, 3056, 1652, 1603, 1506; 1H NMR (300 MHz,
CDCl3): δ 8.82 (s, 1H), 8.63 (d, J = 4.2 Hz, 1H), 7.14–8.15
(m, 10H), 8.92 (d, J = 7.8 Hz, 2H), 5.89 (dd, J = 11.7 Hz,
4.8 Hz, 1H), 3.73 (s, 3H), 3.61 (dd, J = 17.7 Hz, 12.0 Hz,
1H), 3.10 (dd, 17.1 Hz, J = 5.1 Hz, 1H), 2.38 (s, 3H); 13C
NMR (125 MHz, CDCl3): δ 169.06, 159.64, 151.72,
151.02, 150.28, 147.86, 139.83, 133.46, 129.73, 129.48,
129.30, 127.40, 126.42, 125.95, 125.61, 123.48, 121.89,
119.05, 114.06, 55.28, 52.45, 41.42, 22.08; anal. calcd. for
C26H23N5O2: C, 71.38; H, 5.30; N, 16.01. Found C, 71.35;
H, 5.36; N, 15.97; MS (ESI) [M+H]+ Calcd. for
C26H23N5O2: 438.49, Found: 438.3 [M+H]+.
1-(5-(3-(3-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyri-
din-3-yl)-4,5-dihydropyrazol-1-yl)ethanone
(5f) White
solid; yield: 82%; m.p. 264–266 °C; IR (neat, νmax cm−1):
3134, 3059, 1655, 1599, 1510; 1H NMR (300 MHz,
CDCl3): δ 8.91 (s, 1H), 8.61–8.64 (m, 2H), 8.54 (s, 1H),
8.25 (m, 2H), 8.12 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 8.4 Hz,
2H), 7.78 (t, J = 8.1 Hz, 1H) 7.31–7.53 (m, 4H), 5.89 (dd, J
= 12.0 Hz, 4.5 Hz, 1H), 3.76 (dd, J = 17.4 Hz, 11.7 Hz,
1H), 3.15 (dd, 17.7 Hz, J = 4.5 Hz, 1H), 2.45 (s, 3H); 13C
NMR (125 MHz, CDCl3): δ 169.19, 151.34, 151.24,
148.39, 148.00, 147.76, 139.51, 134.87, 134.39, 133.48,
129.66, 129.47, 127.13, 127.10, 126.43, 123.63, 123.28,
122.88, 122.75, 119.26, 52.12, 41.80, 22.04; anal. calcd. for
C25H20N6O3: C, 66.36; H, 4.46; N, 18.57. Found: C, 66.38;
H, 4.52; N, 18.53; MS (ESI) [M+H]+ calcd. for
C25H20N6O3: 453.46. Found: 453.5 [M+H]+.
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 (5 mmol) and guani-
dine hydrochloride (5 mmol) in ethanolic KOH (3%, 20 ml)
was refluxed for 10 h. On completion, the reaction mixture
was allowed to stand overnight. The precipitates were fil-
tered off, washed with water and dried, affording com-
pounds 6a–i. The compounds 6a–i were crystallized from
ethanol.
1-(5-(3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyri-
din-3-yl)-4,5-dihydropyrazol-1-yl)ethanone (5g) Yellow
solid; yield: 89%; m.p. 290–292 °C; IR (neat, νmax cm−1):
3137, 3085, 1666, 1599, 1506; 1H NMR (300 MHz,
CDCl3): δ 8.93 (s, 1H), 8.64 (d, J = 4.5 Hz, 1H), 8.48 (s,
1H), 8.33 (d, J = 8.7 Hz, 2H), 8.09–8.15 (m, 3H), 8.90 (d, J
= 7.8 Hz, 2H), 7.24–7.52 (m, 4H), 5.90 (dd, J = 11.7 Hz,
4.5 Hz, 1H), 3.74 (dd, J = 17.7 Hz, 11.7 Hz, 1H), 3.13 (dd,
17.7 Hz, J = 4.5 Hz, 1H), 2.46 (s, 3H); 13C NMR (125
MHz, CDCl3): δ 169.26, 151.37, 151.02, 148.04, 147.57,
147.49, 139.62, 139.47, 133.72, 129.49, 129.10, 127.26,
127.21, 126.30, 123.92, 123.75, 123.17, 119.33, 52.21,
41.99, 22.02; anal. calcd. for C25H20N6O3: C, 66.36; H,
4-(1,3-diphenyl-1H-pyrazol-4-yl)-6-(pyridine-3-yl)pyrimi-
dine-2-amine (6a) Light yellow solid; yield: 74%; m.p.
238–240 °C; IR (neat, νmax cm−1): 3312, 3149, 1655, 1573,
1
1544, 1503; H NMR (300 MHz, CDCl3): δ 8.87 (s, 1H),
8.60–8.64 (m, 1H), 8.59 (s, 1H), 7.28–8.08 (m, 12H), 6.92

Med Chem Res
(s, 1H), 5.18 (s, 2H); 13C NMR (125 MHz, DMSO-d6): δ
164.36, 162.30, 161.54, 151.57, 151.34, 148.33, 139.59,
134.45, 133.36, 133.23, 130.54, 130.13, 129.36, 128.82,
128.67, 127.35, 124.21, 121.53, 119.08, 104.93; anal.
calcd. for C24H18N6: C, 73.83; H, 4.65; N, 21.52. Found: C,
73.75; H, 4.59; N, 21.48; MS (ESI) [M+H]+ calcd. for
C24H18N6: 391.44. Found: 391.4 [M+H]+.
4-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-6-(pyri-
dine-3-yl)pyrimidin-2-amine (6e) Light yellow solid;
yield: 79%; m.p. 242–244 °C; IR (neat, νmax cm−1): 3324,
3164, 1652, 1581, 1547, 1503; 1H NMR (300 MHz,
CDCl3): δ 8.00 (s, 1H), 8.66–8.68 (m, 1H), 8.53 (s, 1H),
8.08–8.11 (m, 1H), 7.81 (d, J = 7.5, 2H), 7.33–7.59 (m,
8H), 6.96 (s, 1H), 5.17 (s, 2H); 13C NMR (125 MHz,
DMSO-d6): δ 164.26, 162.49, 161.40, 151.62, 150.07,
148.47, 139.50, 134.54, 133.22, 132.56, 131.50, 131.47,
131.16, 130.16, 127.49, 124.22, 122.12, 121.47, 119.11,
104.90; anal. calcd. for C24H17BrN6: C, 61.42; H, 3.65; N,
17.91. Found: C, 61.39; H, 3.64; N, 17.87; MS (ESI) [M
+H]+ calcd. for C24H17BrN6: 471.34. Found: 471.3 ([M
+H]+ +1).
4-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-6(pyri-
dine-3-yl)pyrimidin-2-amine (6b) Yellow solid; yield:
76%; m.p. 258–260 °C; IR (neat, νmax cm−1): 3320, 3160,
1
1655, 1577, 1547, 1510; H NMR (300 MHz, CDCl3): δ
8.95 (s, 1H), 8.65–8.66 (m, 1H), 8.56 (s, 1H), 7.13–8.11 (m,
11H), 6.95 (s, 1H), 5.13 (s, 2H); 13C NMR (125 MHz,
DMSO-d6): δ 164.26, 163.63, 162.42, 161.68, 161.45,
151.60, 150.37, 148.42, 139.54, 134.51, 133.23, 131.62,
131.55, 130.87, 130.14, 129.80, 129.78, 127.41, 124.23,
121.34, 119.07, 115.53, 115.36, 104.81; anal. calcd for
C24H17FN6: C, 70.58, H, 4.20; N, 20.58. Found: C, 70.61;
H, 4.16; N, 20.54; MS (ESI) [M+H]+ calcd for C24H18FN6:
409.43. Found: 409.6 [M+H]+.
4-(3-(3-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-6-(pyri-
dine-3-yl)pyrimidin-2-amine (6f) Pale yellow solid; yield:
78%; m.p. 266–268 °C; IR (neat, νmax cm−1): 3484, 3327,
1640, 1596, 1577, 1532, 1506; 1H NMR (300 MHz,
DMSO-d6): δ 9.34 (s, 1H), 9.29 (s, 1H), 8.78 (s, 1H), 8.71
(d, J = 4.5 Hz, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.33 (d, J =
7.8 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 8.01 (d, J = 7.8 Hz,
2H), 7.43–7.77 (m, 6H), 6.66 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): δ 164.10, 162.72, 161.01, 151.65, 149.05,
148.54, 148.00, 139.38, 135.71, 134.88, 134.59, 133.19,
131.72, 130.21, 129.94, 127.74, 124.23, 123.36, 121.41,
119.23, 104.50; anal. calcd. for C24H17N7O2: C, 66.20; H,
3.94; N, 22.52. Found: C, 66.16; H, 3.95; N, 22.51; MS
(ESI) [M+H]+ calcd. for C24H17N7O2: 436.44. Found:
436.4 [M+H]+.
4-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-6-(pyri-
dine-3-yl)pyrimidin-2-amine (6c) Light yellow solid;
yield: 69%; m.p. 254–256 °C; IR (neat, νmax cm−1): 3324,
3164, 1652, 1585, 1547, 1506; 1H NMR (300 MHz,
CDCl3): δ 8.99 (s, 1H), 8.7 (d, J = 3.3 Hz, 1H), 8.54 (s,
1H), 8.10 (d, J = 7.5 Hz, 1H), 7.82 (d, J = 7.8 Hz, 2H), 7.66
(d, J = 8.1 Hz, 2H), 7.35–7.52 (m, 6H), 6.96 (s, 1H), 5.13
(s, 2H); 13C NMR (125 MHz, DMSO-d6): δ 164.25, 162.49,
161.40, 151.61, 150.04, 148.46, 139.50, 134.54, 133.43,
133.22, 132.19, 131.19, 131.11, 130.15, 128.58, 127.48,
124.23, 121.47, 119.11, 104.89; anal. calcd. for
C24H17ClN6: C, 67.84; H, 4.03; N, 19.78. Found: C, 67.86;
H, 4.04; N, 19.81; MS (ESI) [M+H]+ calcd. for
C24H18ClN6: 425.88. Found: 425.5 [M+H]+.
4-(3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-6-(pyri-
dine-3-yl)pyrimidin-2-amine (6g) Yellow solid; yield:
81%; m.p. 274–276 °C; IR (neat, νmax cm−1): 3406, 3312,
1
1663, 1592, 1562, 1506; H NMR (300 MHz, DMSO-d6):
8.69–9.30 (m, 3H), 8.43 (d, J = 7.8 Hz, 1H), 8.32 (d, J =
9.0 Hz, 2H), 8.18 (d, J = 9.0 Hz, 2H), 8.01 (d, J = 7.8 Hz,
2H), 7.43–7.63 (m, 5H), 6.73 (s, 2H); 13C NMR (125 MHz,
DMSO-d6): δ 164.14, 162.63, 161.02, 151.52, 149.18,
148.44, 147.43, 140.02, 139.37, 134.71, 133.22, 131.71,
130.62, 130.18, 127.77, 124.26, 123.66, 121.96, 119.24,
104.77; anal. calcd. for C24H17N7O2: C, 66.20; H, 3.94; N,
22.52. Found: C, 66.18; H, 3.92; N, 22.56; MS (ESI) [M
+H]+ calcd. for C24H17N7O2: 436.44. Found: 436.5 [M
+H]+.
4-(3-(3-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-6-(pyri-
dine-3-yl)pyrimidin-2-amine (6d) White solid; yield:
83%; m.p. 240–242 °C; IR (neat, νmax cm−1): 3335, 3204,
1648, 1585, 1566, 15361H NMR (300 MHz, CDCl3): δ 8.97
(s, 1H), 8.66 (d, J = 4.8 Hz, 1H), 8.57 (s, 1H), 8.13 (d, J =
7.8 Hz, 1H), 7.95 (s, 1H), 7.82 (d, J = 7.5 Hz, 2H),
7.27–7.64 (m, 7H), 6.98 (s, 1H), 5.13 (s, 2H); 13C NMR
(125 MHz, DMSO-d6): δ 164.26, 162.54, 161.28, 151.63,
149.54, 148.44, 139.46, 135.61, 134.54, 133.19, 131.58,
131.46, 131.10, 130.75, 130.16, 128.47, 127.56, 124.23,
121.78, 121.55, 119.18, 104.96; anal. calcd. for
C24H17BrN6: C, 61.42, H, 3.65; N, 17.91. Found: C, 61.39;
H, 3.64; N, 17.89; MS (ESI) [M]+ calcd. for C24H17BrN6:
469.34. Found: 469.5 [M]+.
4-(1-phenyl-3-p-tolyl)-1H-pyrazol-4-yl)-6-(pyridine-3-yl)
pyrimidin-2-amine (6h) Light yellow solid; yield: 77%; m.
p. 260–262 °C; IR (neat, νmax, cm−1): 3312, 3152, 1655,
1
1581, 1547, 1503; H NMR (300 MHz, CDCl3): δ 8.88 (s,
1H), 8.63–8.64 (m, 1H), 8.58 (s, 1H), 8.08–8.11 (m, 1H),
7.82 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 7.8 Hz, 2H),
7.29–7.59 (m, 6H), 6.96 (s, 1H), 5.14 (s, 2H), 2.44 (s, 3H);

Med Chem Res
13C NMR (125 MHz, DMSO-d6): δ 164.34, 162.27, 161.68,
151.57, 151.29, 148.36, 139.62, 138.15, 134.47, 133.25,
130.48, 130.13, 129.21, 127.28, 124.23, 121.44, 119.05,
104.96; anal. calcd. for C25H20N6: C, 74.24; H, 4.98; N,
20.78. Found: C, 74.18; H, 4.92; N, 20.69; MS (ESI) [M
+H]+ calcd. for C25H20N6: 405.47. Found: 405.5 [M+H]+.
formazan to give a homogeneous blue solution. The
absorbance was measured at a test wavelength of 570 nm
using an ELISA plate reader, LMR-340 M (Labexim
International, Austria) with a microplate reader. The per-
centage inhibition was calculated by the formula:
Mean OD of treated cells
% Inhibition ¼ 100À
 100
Mean OD of the vehicle control cells ðnegative controlÞ
4-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-6-(pyr-
idine-3-yl)pyrimidin-2-amine (6i) White solid; yield: 79
%; m.p. 234–236 °C; IR (neat, νmax cm−1): 3454, 3175,
Each assay was repeated for three times. IC50 values
were calculated from the dose effect curves (Figs. 4 and 5)
and expressed as concentration (µM) of drug.
1
1644, 1581, 1526, 1506; H NMR (300 MHz, CDCl3): δ
8.94 (s, 1H), 8.64 (d, J = 4.5 Hz, 1H), 8.56 (s, 1H), 8.10 (d,
J = 7.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 8.7
Hz, 2H), 6.99–7.51 (m, 6H), 6.96 (s, 1H), 5.15 (s, 2H), 3.88
(s, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.33, 162.27,
161.72, 159.89, 151.56, 151.13, 148.37, 139.62, 134.48,
133.27, 130.69, 130.48, 130.11, 127.21, 125.68, 124.22,
121.24, 118.98, 114.09, 104.88, 55.66; anal. calcd. for
C25H20N6O: C, 71.41; H, 4.79; N, 19.99. Found: C, 71.38;
H, 4.82; N, 19.94; MS (ESI) [M+H]+ calcd for
C25H20N6O: 421.47. Found: 421.5 [M+H]+.
Results 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 which, in turn, were initially synthesized by
Vilsmeier–Haack formulation of phenylhydrazones 2a-i
(Yadlapalli et al. 2012; Desai et al. 2012; Elkady et al.
2012; Prakash et al. 2006, Alam et al. 2016a, b). Subse-
quently, the Claisen–Schmidt condensation of 3-aryl-1-
In vitro cytotoxicity evaluation
In vitro cytotoxicity of all the newly synthesized com-
phenylpyrazol-4-carboxaldehydes
3a–i
with
3-
pounds 5a–i and 6a–i were measured by MTT [3-(4,5-
acetylpyridine in methanolic NaOH afforded the corre-
sponding (E)-3-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-1-(pyr-
idin-3-yl)prop-2-en-1-ones 4a–i (Alam et al. 2016a, b).
Hydrazine hydrate reacted with chalcones 4a–i in the pre-
sence of acetic acid, afforded compounds 5a–i. Further,
treatment of chalcones 4a-–i with guanidine hydrochloride
in the presence of ethanolic KOH, afforded pyrazolyl ami-
nopyrimidine derivatives 6a–i.
dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide)]
assay against Hela, NCI-H460, PC-3 and NIH-3T3 cell
lines (Mosmann 1983). The MTT assay is based on con-
version 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, 5% CO2 in water
jacketed incubator shell) using DMEM 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 dis-
solved in DMSO. These concentrations were added in tri-
plicate to the single 96-well tissue culture plate. After 24 h
of treatment, the MTT assay was performed to check cell
viability. For the MTT assay, the media was removed from
all the wells, 10 µl of MTT reagent (Chemicon Interna-
tional, USA) per well from a working stock (5 mg/ml) was
added and the plates were incubated (37 °C and 5% CO2)
for 2–3 h, after that the reagent was removed and the
crystals were solubilised using dimethyl sulfoxide (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. The DMSO dissolves the
All the synthesized compounds were characterized by
1
detailed H and 13C NMR (Figs. S1–18), IR, mass spectral
data, and elemental analysis. IR spectra of compound 5a
shows absorption bands mainly at 1659 and 1603 cm−1
,
which were due to C=O and C=N functionalities, respec-
1
tively. In the H NMR spectra of compound 5a, methine
proton (5ʹ-H) resonated as a doublet of doublet at 5.91 ppm
with coupling constants of 11.7 and 4.8 Hz and two
methylene protons (4ʹ-H) displayed two signals; a doublet
of doublet at 3.63 ppm with coupling constants of 17.4 and
11.7 Hz and a doublet of doublet at 3.10 ppm with coupling
constants of 17.4 and 4.8 Hz. The main signal in the 13C
NMR for 5a corresponds to C-4′ at 41.57 ppm, C-5′ at
52.43 ppm. The elemental analysis and mass spectra sup-
ported the structure of pyrazolylpyrazoline derivative 5a. IR
spectra of the compound 6a exhibited characteristic strong
absorbance in the range of 3312–3149 cm−1 for the –NH2
1
group. In the H NMR spectra of aminopyrimidine 6a, a
singlet of NH2 group was observed at 5.18 ppm. 13C NMR

Med Chem Res
Fig. 4 Dose effect curves of compounds 5a–i and standard drug etoposide
spectra of the compound 6a was also in accordance with
their proposed structures. Furthermore, the mass spectra and
elemental analysis supported the structure of compound 6a.
Other spectroscopic data also supported the structure of rest
of the synthesized compounds.
NO2 group in the meta-substituted benzene ring showed
IC50 value of 15.42 0.80 µM against NCI-H460 and
23.66 1.10 µM against PC-3 cancer cell lines. Analog 5g
with NO2 group in the para-substituted benzene ring dis-
played IC50 value of 11.46 0.32 µM against Hela, 13.41
0.63 µM against NCI-H460, 22.47 0.80 µM against PC-
3 cancer cell lines. Compound 5i with OMe group in the
para-substituted benzene ring showed IC50 value of 16.56
0.66 µM against NCI-H460 and 22.04 0.48 µM against
PC-3 cancer cell lines. Analog 6b with F group present in
para position of the benzene ring showed IC50 value of
6.80 0.48 µM against Hela, 7.12 0.28 µM against NCI-
H460, and 9.89 0.44 µM against PC-3 cancer cell lines.
Compound 6c with Cl group present in para position of the
benzene ring displayed IC50 value of 7.84 0.65 µM
against Hela, 8.00 0.65 µM against NCI-H460, and 11.68
0.48 µM against PC-3 cancer cell lines. Analog 6d having
Br-substituted phenyl ring in meta position showed IC50
value of 10.77 0.44 µM against Hela and 16.69 0.61 µM
against PC-3 cancer cell lines. Compound 6e with Br in
para position of the benzene ring displayed IC50 value of
Biology
In vitro cytotoxicity of all the newly synthesized com-
pounds 5a–i and 6a–i were measured by MTT assay against
a panel of three different human cancer cell lines (HeLa,
NCI-H460, and PC-3). The IC50 values were calculated and
expressed as concentration (µM) of drug. Etoposide was
taken as reference compound and the results are summar-
ized 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-3T3 nor-
mal cell line. Among all the synthesized compounds, ana-
logs 5f, 5g, 5i, 6b–g showed significant cytotoxicity as
compared to standard drug etoposide. Compound 5f with

Med Chem Res
Fig. 5 Dose effect curves of compounds 6a–i and standard drug etoposide
9.31 0.69 µM against Hela, 11.53 0.67 µM against NCI-
H460, and 14.48 0.65 µM against PC-3 cancer cell lines.
Analog 6f having NO2-substituted phenyl ring in meta
position displayed IC50 value of 10.19 0.69 µM against
Hela, 11.55 0.69 µM against NCI-H460, and 15.36
1.13 µM against PC-3 cancer cell lines. Compound 6g with
NO2 in para position of the benzene ring displayed IC50
value of 5.47 0.44 µM against Hela, 6.19 0.69 µM
against NCI-H460, and 8.88 0.65 µM against PC-3 cancer
cell lines. Among all the synthesized compounds, analog 6g
displayed superior cytotoxicity against Hela cancer cell line
with an IC50 value of 5.47 0.44 µM. In our previous
study, various heterocyclic derivatives were synthesized
from pyrazolic chalcones 4a–i and evaluated their cyto-
toxicity in order to get potential anticancer agents (Alam
et al. 2016a, b). Ethyl 4-(3-(3-bromophenyl)-1-phenyl-1H-
pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarbox-
ylate was synthesized from pyrazolic chalcone 4d. This
compound displayed cytotoxicity of 14.31 0.90 µM
against MCF-7 cell lines. Similarly, 5-(3-(3-Bromophenyl)-
1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-
dihydropyrazole-1-carbothioamide, synthesized from pyr-
azolic chalcone 4d, displayed cytotoxicity of 13.51 1.01
µM against MCF-7 cell lines. On the other hand, pyrazolic
chalcone 4d showed cytotoxicity of 46.67 2.01 µM
against MCF-7 cell lines. These findings clearly demon-
strate that derivatization of chalcone significantly increased
the cytotoxicity.
Furthermore, structure–activity relationship (SAR) stu-
dies were performed to determine the effect of substituents
on cytotoxic activities of the synthesized compounds. SAR
in these derivatives 5a–i displayed that compounds with
para-substituted NO2 group in the benzene ring showed
superior cytotoxic activity. Substituents F, Cl, Br, CH3, and
OCH3 present 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 strength order is NO2 > F > Cl > Br for the
compounds with a para-substituted electron-withdrawing
group present in the benzene ring. Among the compounds
with meta-substituted electron-withdrawing group, the
cytotoxic activity order is NO2 > Br for the cancer cell lines.

Med Chem Res
Scheme 1 General procedure
for the synthesis of
pyrazolylpyrazoline pyrazolyl
aminopyrimidine derivatives: (i)
EtOH, H2SO4, reflux; (ii) a
POCl3/DMF, 80 °C; b NaHCO3/
H2O; (iii) 3-acetylpyridine,
MeOH, NaOH, r.t. (iv)
NH2NH2.H2O/CH3COOH,
reflux; and (v) guanidine
hydrochloride, KOH, EtOH,
reflux
Table 1 In vitro cytotoxicity of
compounds 5a–i, 6a–i, and
etoposide 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-3T3
5a
H
32.23 1.10
20.94 0.29
27.14 1.57
23.16 0.63
33.45 1.10
16.94 1.10
11.46 0.32
18.52 1.22
14.42 0.44
13.60 0.65
6.80 0.48
7.84 0.65
10.77 0.44
9.31 0.69
10.19 0.69
5.47 0.44
15.81 0.69
12.88 0.65
11.25 0.33
40.17 1.86
24.78 3.01
26.33 0.66
24.16 1.73
41.36 1.71
15.42 0.80
13.41 0.63
19.71 0.33
16.56 0.66
15.49 1.12
7.12 0.28
8.00 0.65
18.19 0.69
11.53 0.67
11.55 0.69
6.19 0.69
18.48 0.65
18.18 1.68
16.63 1.07
58.39 2.17
32.45 1.55
33.36 1.36
24.81 0.75
62.56 2.89
23.66 1.10
22.47 0.80
27.74 1.55
22.04 0.48
26.91 2.69
9.89 0.44
11.68 0.48
16.69 0.61
14.48 0.65
15.36 1.13
8.88 0.65
24.24 0.65
23.68 0.88
23.80 0.38
94.11 2.36
92.67 0.91
90.45 2.97
97.84 0.65
93.25 3.21
90.13 5.20
82.45 3.16
95.92 1.95
92.08 3.44
88.83 3.34
78.64 4.50
91.20 2.00
82.32 6.67
94.75 2.97
91.63 2.45
74.05 2.69
94.88 2.72
83.04 2.91
90.53 4.60
5b
5c
4-F
4-Cl
5d
5e
3-Br
4-Br
3-NO2
4-NO2
4-CH3
4-OCH3
H
5f
5g
5h
5i
6a
6b
6c
4-F
4-Cl
6d
6e
3-Br
4-Br
3-NO2
4-NO2
4-CH3
4-OCH3
–
6f
6g
6h
6i
Etoposide

Med Chem Res
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In continuation to get anticancer heterocyclic compounds,
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derivatives 6a–i were performed. All the synthesized
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tested cancer cell line and weak toxicity towards normal cell
line. The cytotoxicity evaluation data revealed that among
all the synthesized compounds studied, analogs 5f, 5g, 5i,
6b–g showed significant cytotoxicity as compared to stan-
dard drug etoposide. The compound 6g exhibited superior
activity with an IC50 value of 5.47 0.44 µM against Hela
cancer cell line.
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Acknowledgements The authors thank to the Head, Department of
Chemistry, Jamia Millia Islamia, New Delhi for providing research
facilities. AIRF, JNU is acknowledged for 13C NMR spectra. Raquib
Alam is also thankful to UGC, New Delhi for BSR fellowship.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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