Design, synthesis, and biological evaluation of 1,3,5-trisubstituted pyrazoles as tyrosine kinase inhibitors

Article abstract of DOI:10.1007/s00044-018-2282-x

We report herein, silica supported molybdic acid mediated oxidative C–N bond formation for the regioselective synthesis of new 1,3,5-trisubstituted pyrazole derivatives. This transformation furnishes a novel synthetic approach with solvent-free neat heat conditions, which was found to be flexible with wide substrate scope and better efficiency towards rapid synthesis of new 1,3,5-trisubstituted pyrazoles. Selected series of the synthesized derivatives were screened for their liability against carcinogenesis. A molecular docking study of the synthesized derivatives was performed in the active site of the tyrosine kinase enzymes. Based on the molecular docking study specific compounds were screened in vitro for their anticancer activity, which showed potent micro molar activity against human MDA-MB-231 breast cancer line and human leukemia cell line K-562 using 3-(4,5-dimethylthiazol-2-yl)–2, 5-diphenyltetrazolium bromide (MTT) assay. Compound 3l possesses higher inhibitory activity with IC₅₀ 0.58 ± 0.02 μM against the MDA-MB-231 cell line. Whereas compound 3k showed higher inhibitory activity with IC₅₀ value 0.78 ± 0.03 μM against the K-562 cell line. Fluorescence microscopic studies revealed that the compounds showed late apoptotic mode of cell death. These results can lead to further exploitation of tested pyrazole compounds to the highly active drug molecule. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-018-2282-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2282-x
ORIGINAL RESEARCH
Design, synthesis, and biological evaluation of 1,3,5-trisubstituted
pyrazoles as tyrosine kinase inhibitors
Sinthiya J. Gawandi1,2 Vidya G. Desai1,2 Sunil G. Shingade3
●
●
Received: 15 October 2018 / Accepted: 19 December 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
We report herein, silica supported molybdic acid mediated oxidative C–N bond formation for the regioselective synthesis of
new 1,3,5-trisubstituted pyrazole derivatives. This transformation furnishes a novel synthetic approach with solvent-free neat
heat conditions, which was found to be flexible with wide substrate scope and better efficiency towards rapid synthesis of
new 1,3,5-trisubstituted pyrazoles. Selected series of the synthesized derivatives were screened for their liability against
carcinogenesis. A molecular docking study of the synthesized derivatives was performed in the active site of the tyrosine
kinase enzymes. Based on the molecular docking study specific compounds were screened in vitro for their anticancer
activity, which showed potent micro molar activity against human MDA-MB-231 breast cancer line and human leukemia
cell line K-562 using 3-(4,5-dimethylthiazol-2-yl)–2, 5-diphenyltetrazolium bromide (MTT) assay. Compound 3l possesses
higher inhibitory activity with IC50 0.58 0.02 μM against the MDA-MB-231 cell line. Whereas compound 3k showed
higher inhibitory activity with IC50 value 0.78 0.03 μM against the K-562 cell line. Fluorescence microscopic studies
revealed that the compounds showed late apoptotic mode of cell death. These results can lead to further exploitation of tested
pyrazole compounds to the highly active drug molecule.
Graphical Abstract
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Keywords Hydrazones Pyrazoles Solvent-free Tyrosine kinase Apoptosis Anti-proliferative activity
Introduction
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-018-2282-x) contains supplementary
material, which is available to authorized users.
Cancer is one of the major chronic degenerative diseases
and a prominent cause of deaths world over. Cancers with
high prevalence includes lung, prostate, breast, uterine, and
stomach (Ayati et al. 2018). In normal cells, protein kinases
play a significant role in regulating the cellular functions of
cell metabolism, proliferation, motility, and apoptosis.
Inversely, in cancer cells, the expression of different kinases
is enhanced thereby, deregulating the functions leading to
cell growth and proliferation (Trejo-soto et al. 2018). For
instance, breast cancer is a complex and heterogeneous
* Vidya G. Desai
desai_vidya@gmail.com
1
Department of Chemistry, Dnyanprassarak Mandal’s College &
Research Centre, Assagao, Bardez 403507, India
2
Goa University, Taleigao, Panaji 403206, India
3
Department of Pharmaceutical Chemistry, PES’s Rajaram and
Tarabai Bandekar College of Pharmacy, Farmagudi, Ponda, Goa
403401, India

Medicinal Chemistry Research
NO2
Fig. 1 Structures of anticancer
drugs containing nitro group
N
N
O
CH3
HN
O
S
H
N
N
CF3
CF3
NO2
H
N
H3C
N
O
N
NO2
N
Flutamide
Nilutamide
Azathioprine
Cl
Fig. 2 Representative structures
of clinically important
compounds with pyrazole core
structures
O
O
CF3
S
O
N
N
N
Cl
HN
N
N
O
S
O
Cl
N
N
N
H2N
N
O
N
N
H3C
H
O
Celecoxib
Rimonabant
Sildenafil
disease, prevalent in women with around 1.7 million cases
diagnosed worldwide every year and the leading cause of
death by cancer in women. Existing therapies show detri-
mental side effects due to their lack of tumor targeting
specificity and the emergence of tumor multi-drug resis-
tance (Soudy et al. 2017; Holliday and Speirs 2011).
Enzyme inhibition represents an important phenomenon
that defines the drug potency and its efficacy. Potency of a
drug is measured in the way it can inhibit the activity of
enzymes that trigger the cause of disease. Tyrosine kinase,
proteasome (Piwowar et al. 2006), DNA topoisomerases
(Topcu 2001), histone deacetylases (Mottamal et al. 2015)
and so on enzymes have been widely used as a potential
target for anticancer agents. Boom of knowledge in mole-
cular sciences, bioinformatics, and proteomics have been an
influential factor in the designing of novel enzyme inhibi-
tors. In this aspect, variation in disubstitution pattern plays
an important role in the field of drug discovery. Besides the
presence of heterocyclic moiety, aryl substitution on a
molecule has an influence on the biological activity. Inter-
estingly, nitro group containing aromatic compounds have
proved to possess potent activity against different types of
cancer diseases. (Fig. 1) (Olender et al. 2018). Thus, with
the growing threat of cancer disease, there is an immense
need to synthesize a new drug like molecule that can be
developed as a better anticancer agent. Tracking on to
achieve this goal, we have synthesized a series of new N-
aryl hydrazones and converted them into its pyrazole deri-
vatives and evaluated its anticancer activity.
medicinal chemistry as it also constitutes the core structure
of clinically interesting compounds, such as Celecoxib
(Penning et al. 1997; Ahlstrom et al. 2007), Rimonabant,
Sildenafil (Mojzych et al. 2015) etc. (Fig. 2). They are also
known to possess enzyme inhibiting properties along with
diverse biological activities such as, COX inhibitors (Alam
et al. 2016), anticancer (Kumar et al. 2013), anti-microbial
(Hafez and El-Gazzar 2016), anti-inflammatory (Keche
et al. 2012), anti-oxidant (Renuka and Kumar 2013), anti-
convulsant, anti-depressant (Abdel-Aziz et al. 2009), anti-
pyretic (Malvar et al. 2014), urease inhibitors (Bole et al.
2011), and anti-viral (Tantawy et al. 2012) activity. They
also act as cytotoxic agents (Xia et al. 2007), inhibitors of
receptors for advanced glycation end products (Han et al.
2014), as dual inhibitors of acetylcholinesterase as well as
monoamine oxidase (Kumar et al. 2013).
We previously reported on the synthesis, screening, and
preliminary structure activity relationship studies of quinox-
alinyl chalcone hybrid scaffolds as tyrosine kinase inhibitors
(Desai et al. 2017). In our present work, Virtual screening of
the pyrazole compounds was carried out targeting tyrosine
kinase enzyme for anticancer activity. Very few pyrazole
derivatives containing N-2, 4-DNP group have been reported
(Deng and Mani 2008; Desai and Naik 2013). Our focus was
to achieve synthesis of differently substituted 1-(2, 4-dini-
trophenyl)-3,5-diaryl-1H-pyrazoles via oxidative cyclization
of stable N-(2,4-dinitrophenyl) hydrazones of chalcones.
The presence of 2,4-dinitrophenyl groups in hydrazones
retards the formation of pyrazole. As a result, there are no
generalized method for the synthesis of such compounds so,
a new ecofriendly protocol for the regioselective synthesis of
1,3,5-trisubstituted pyrazole has been devised (Scheme 1).
Pyrazoles represent a key structure in heterocyclic
chemistry (Abd El-Karim et al. 2015; Kheder et al. 2014;
Kumar et al. 2013) and occupies an important place in

Medicinal Chemistry Research
Scheme 1 Design for target
compounds
O
Ar''
O2N
NO2
2,4-DNP hydrazine
H
N
NO2
N
OConc. H SO , MeOH
2
4
50-60oC
Ar''
Ar'
SiO2 supported reagent
N
+
N
Solvent Free
Ar'
Ar''
O2N
Ar''
CHO
Ar'
Ar'
Chalcones
Cyclization
Highly Bioactive
Derivatives
Material and methods
Experimental procedure
Molecular modeling
Brunswick, Galaxy 170R, Germany) maintained at 37 °C,
5% CO2 with 95% humidity until the completion of
experiments. The absorption spectrum was measured on a
JASCO V670 spectrophotometer.
Cytotoxicity assay
Protein structure preparation The molecular docking
study was performed using Molegro Virtual Docker (MVD-
2013, 6.0). The crystal structure of the epidermal growth
factor receptor tyrosine kinase domain with 4-
anilinoquinazoline inhibitor erlotinib was downloaded
from Protein Data Bank PDB ID: 1M17. Molecular docking
studies of the synthesized compounds/ligands were per-
formed in order to understand various interactions between
the ligand and enzyme active site in detail.
The breast cancer cell line MDA-MB-231, leukemia cell
line K-562, and human non-cancerous cell line HEK-293
were cultured in DMEM medium, which was supplemented
with 10% heat inactivated fetal calf serum (FBS) and 1%
Antibiotic–Antimycotic 100X solution. The cells were
seeded at a density of approximately 5 × 103cells/well in a
96-well flat-bottom micro plate and maintained at 37 °C in
95% humidity and 5% CO2 for overnight. Different con-
centration (600, 300, 150, 75, 37.5, 18.75 µg/mL) of test
compounds were treated. The cells were incubated for
another 72 h. The cells in well were washed twice with
phosphate buffer solution, and 20 µL of the MTT staining
solution (5 mg/mL in phosphate buffer solution) was added
to each well and plate was incubated at 37 °C. After 4 h,
100 µL of dimethyl sulfoxide (DMSO) was added to each
well to dissolve the formazan crystals, and absorbance was
recorded with a 570 nm using micro plate reader. The IC50
values were calculated using graph Pad Prism Version5.1.
Molecular docking The synthesized compounds were built
using Chemdraw 11.0. The two-dimensional structures
were then converted into energy minimized three-
dimensional structures and were saved as MDL Molfile (.
mol2). The coordinate files and crystal structures of tyrosine
kinase (PDB ID: 1M17) were obtained from the RCSB
PDB website. The protein files were prepared by the
removal of water molecules, addition of polar hydrogens
and removal of other bound ligands. In the present study,
the binding sites were selected based on the amino acid
residues, which are involved in binding with tyrosine kinase
inhibitors as obtained from protein data bank, which would
be considered as the probable best accurate regions as they
are solved by experimental crystallographic data. The
docking protocol was carried out for the synthesized com-
pounds using MVD-2013 (6.0) software using the standard
operating procedures (Palkar et al. 2014).
Double staining (acridine orange-ethidium bromide)
The cells were seeded at a density of approximately 1 × 104
cells/well in a 24-well flat-bottom micro plate containing
cover slips and maintained at 37 °C in CO2 incubator for
overnight. IC50 concentration of synthesized compounds
was treated at 72 h. After the incubation, cells were washed
with PBS and fixed with 4% paraformaldehyde for 30 min.
Twenty microliters of dye mixture was incubated for half an
hour, examined under fluorescent microscope.
Cell culture
Breast cancer cell line MCF-7, leukemia cell line K-562,
and human embryonic kidney cell line HEK-293 were
purchased from the National Center for Cell sciences
(NCCS) Pune. The cell lines were cultured in DMEM
medium, i.e., Dulbecco’s modified Eagle medium, which
were supplemented with 10% heat inactivated fetal calf
serum (FBS) and 1% Antibiotic–Antimycotic 100X solu-
tion and incubated in CO2 incubator (Eppendorf, New
DAPI (4’,6’-diamine-2’-phenylindole dihydrochloride)
The cells were seeded at a density of approximately 1 × 105
cells/well in a 12-well flat-bottom micro plate containing
cover slips and maintained at 37 °C in CO2 incubator for
overnight. More than the IC50 of synthesized compounds
was treated at 72 h. After the incubation, cells were washed

Medicinal Chemistry Research
with PBS and fixed with 4% paraformaldehyde for 30 min.
Twenty microliters of DAPI was incubated for 20 min,
examined under fluorescent microscope.
product. The synthesized compounds were characterized on
the basis of IR, NMR and Mass spectroscopic data.
1-(2,4-dinitrophenyl)-3,5-diphenyl-1H-pyrazole (3a) Yel-
low solid, mp: 142–144 °C; IR (KBr) (νmax, cm–1): 3072,
1605, 1538, 1498, 1348 (Desai et al. 2012).
Chemistry
All commercially available reagents were purchased from
SD Fine, LobaChemie, and Avra synthesis and used with-
out further purification. Melting points were determined by
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.72 (d, 1H, J =
2.44, Ar–H), 8.33–8.35 (dd, 1H, J = 2.52, J = 8.4, Ar–H),
7.84 (d, 1H, J = 8.4, Ar–H), 7.25–7.48 (m, 10H, Ar–H),
6.90 (s, 1H, CH); 13C NMR (75 MHz, CDCl3) (δ, ppm):
154.6, 146, 145.4, 140.9, 139.6, 136.5, 130.3, 129, 128.5,
127, 120.6, 119.3, 105.3; HRMS (Electrospray ionisation
time-of-flight, [M]+): calcd for C21H14N4O4, 386.361;
found 386.354.
1
open capillary tube method and are uncorrected. H (13C)
nuclear magnetic resonance (NMR) spectra were recorded
at Bruker model 400 MHz instrument using CDCl3. The
chemical shifts (δ) are expressed in parts per million relative
to the residual deuterated solvent signal and coupling con-
stants (J) are given in hertz. Proton coupling patterns were
illustrated as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br). IR spectra were recorded on a
Schimadzu Fourier transform infrared spectrometer using
KBr pellets. High resolution mass spectroscopy data were
obtained in electron impact (EI) mode. Intensities are
reported as percentages relative to the base peak (I =
100%). Synthesis of new 2, 4-dinitrophenyl hydrazones has
been reported by our group (Desai and Gawandi 2016).
5-(4-chlorophenyl)-1-(2,4-dinitrophenyl)-3-(4-methoxyphe-
nyl)-1H-pyrazole (3b) Yellow solid, mp: 166-170 °C; IR
(KBr) (νmax,cm–1): 3093, 1608, 1537, 1498, 1348, 1215
(Desai et al. 2012).
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.73 (d, 1H, J =
2.4, Ar–H), 8.33–8.35 (dd, 1H, J = 2.44, J = 8.8, Ar–H),
7.75 (d, 2H, J = 8.4, Ar–H), 7.44 (d, 1H, J = 8.8, Ar–H)
6.97–7.36 (m, 6H, Ar–H), 6.82 (s, 1H, CH), 3.85 (s, 3H,
OCH3); 13C NMR (75 MHz, CDCl3) (δ, ppm): 162, 153.9,
146.2, 144.4, 140.3, 139.6, 134.6, 133.8, 130.3, 129.4,
128.8, 128.4, 128, 120.6, 119.3, 114.6, 106.3, 55.6; HRMS
(ESI-TOF, [M]+): calcd for C22H15N4O5Cl, 450.832; found
450.823.
Synthesis of silica chloride
A mixture of silica–gel (5 g) and thionyl chloride (20 mL)
was refluxed for 48 h in a round bottomed flask (100 mL)
fitted with a condenser and a guard tube containing CaCl2.
The resulting white-greyish powder was filtered and dried.
3-(4-bromophenyl)-1-(2,4-dinitrophenyl)-5-(4-methoxyphe-
nyl)-1H-pyrazole (3c) Yellow solid, mp: 174–176 °C; IR
(KBr) (νmax, cm–1): 3087, 1605, 1538, 1498, 1343, 1215,
594.
Synthesis of silica molybdic acid (SMA)
A mixture of silica chloride and sodium molybdate was
stirred under refluxing conditions for 3.5 h in n-hexane (5
mL). The reaction mixture was then filtered and washed
with distilled water followed by drying at 120 °C in an oven
for 6 h. The resulting mixture was then further stirred in 0.1
N HCl solution (40 mL) for 1 h, filtered, washed with dis-
tilled water, and dried in an oven at 120 °C for 6 h to obtain
the catalyst. Catalyst formation was characterized by XRD
analysis. (Ahmed and Siddiqui 2015)
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.72 (d, 1H, J =
2.4, Ar–H), 8.36–8.38 (dd, 1H, J = 0.4, J = 8.8, Ar–H),
7.77 (d, 2H, J = 8.4, Ar–H), 7.53 (d, 1H, J = 8.8, Ar–H)
6.97–7.48 (m, 6H, Ar–H), 6.85 (s, 1H, CH), 3.83 (s, 3H);
13C NMR (75 MHz, CDCl3) (δ, ppm): 160.8, 154.9, 146.2,
145.3, 144.3, 138.3, 133.6, 133, 130.1, 129.6, 129, 127.4,
124.2, 121.3, 114.2, 111.5, 106.5, 55.6; HRMS (ESI-TOF,
[M]+): calcd for C22H15N4O5Br, 494.022; found 494.023.
1-(2,4-dinitrophenyl)-3,5-bis(4-fluorophenyl)-1H-pyrazole
(3d) Yellow solid, mp. 172–174 °C; IR (KBr) (νmax, cm–1):
3090, 1603, 1535, 1496, 1347, 982.
General procedure for the synthesis of pyrazole derivatives
(3a–3o)
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.75 (d, 1H, J =
2.4, Ar–H), 8.31–8.33 (dd, 1H, J = 2.8, J = 8.8, Ar–H),
7.87 (d, 2H, J = 8.0, Ar–H), 7.56 (d, 1H, J = 8.8, Ar–H),
7.02–7.42 (m, 6H, Ar–H), 6.82 (s, 1H, CH); 13C NMR (75
MHz, CDCl3) (δ, ppm): 162.1, 158.6, 146.4, 144.6, 139.6,
132.1, 130.1, 128.4, 128., 120.2, 119.3, 116, 106.2; HRMS
(ESI-TOF, [M]+): calcd for C21H12F2N4O4, 422.082; found
422.084
Substituted hydrazones (1 mmole) and silica supported
molybdic acid (1 mmole) were placed in a conical flask and
heated at 120 °C. The progress of the reaction was monitored
by TLC. After the completion of the reaction, the product was
dissolved in ethyl acetate and filtered to recover the catalyst.
The solvent was then evaporated under vacuum and the crude
mixture was recrystallized from n-hexane to afford the pure

Medicinal Chemistry Research
3,5-bis(4-chlorophenyl)-1-(2,4-dinitrophenyl)-1H-pyrazole
(3e): Yellow solid, mp. 200-–206 °C; IR (KBr) (νmax, cm–1):
3075, 1602, 1532, 1499, 1344, 620
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.75 (d, 1H, J =
2.4, Ar–H), 8.35-8.38 (dd, 1H, J = 2.4, J = 8.8, Ar–H), 7.81
(d, 2H, J = 7.2, Ar–H), 7.48 (d, 1H, J = 8.8, Ar–H), 6.88–
7.26 (m, 6H, Ar–H), 6.79 (s, 1H, CH), 3.83 (s, 3H, OCH3);
13C NMR (75 MHz, CDCl3) (δ, ppm): 164.2, 160.2, 154.3,
145.9, 145.2, 144.6, 138.1, 130.1, 129.5, 127.8, 127.1,
120.9, 120.6, 115.9, 115.6, 114.6, 105.9, 55.8; HRMS (ESI-
TOF, [M]+): calcd for C22H15N4O5F, 434.103; found
434.101.
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.78 (d, 1H, J =
2.4, Ar–H), 8.39–8.41 (dd, 1H, J = 2.8, J = 8.8, Ar–H),
7.69 (d, 2H, J = 8.4, Ar–H), 7.55 (d, 1H, J = 8.8, Ar–H),
7.10–7.47 (m, 6H, Ar–H), 6.89 (s, 1H, CH); 13C NMR (75
MHz, CDCl3) (δ, ppm): 153.6, 146.4, 145.3, 144.6, 137.4,
135.2, 132.4, 129.6., 128.2, 123.1, 121.1, 106.5; HRMS
(ESI-TOF, [M]+): calcd for C21H12Cl2N4O4, 454.023; found
454.021
1-(2,4-dinitrophenyl)-3,5-bis(4-methoxyphenyl)-1H-pyra-
zole (3j) Yellow solid, mp. 168–170 °C; IR (KBr) (νmax
,
5-(4-chlorophenyl)-1-(2,4-dinitrophenyl)-3-(4-fluorophenyl)-
1H-pyrazole (3f) Yellow solid, mp. 178–182 °C; IR (KBr)
(νmax, cm–1): 3085, 1608, 1536, 1497, 1348, 989, 614.
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.79 (d, 1H, J =
2.4, Ar–H), 8.36–8.38 (dd, 1H, J = 2.42, J = 8.8, Ar–H),
7.77 (d, 2H, J = 8.72, Ar–H), 7.57 (d, 1H, J = 8.8, Ar–H),
6.98–7.42 (m, 6H, Ar–H), 6.83 (s, 1H, CH); 13C NMR (75
MHz, CDCl3) (δ, ppm): 162.4, 153.6, 146.6, 145.3, 144.5,
139.6, 135.3, 133.1, 132, 130.4, 129.4, 128.6, 128.4, 126.7,
120.6, 114.3, 106.5; HRMS (ESI-TOF, [M]+) : calcd for
C21H12N4O4ClF, 438.053; found 438.054.
cm–1): 3105, 1605, 1538, 1505, 1215 (Desai et al. 2012).
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.73 (d, 1H, J =
2.4, Ar–H), 8.32–8.34 (dd, 1H, J = 2.44, J = 8.8, Ar–H),
7.78 (d, 2H, J = 8.6, Ar–H), 7.50 (d, 1H, J = 8.8, Ar–H),
6.91–7.28 (m, 6H, Ar–H), 6.79 (s, 1H, CH), 3.87 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3)
(δ, ppm): 160.2, 154.5, 146, 145.2, 144.6, 137.8, 132.4,
130.1, 129.5, 127.4, 120.4, 114.2, 56.4; HRMS (ESI-TOF,
[M]+): calcd for C23H18N4O6, 446.122; found 446.126.
3,5-bis(4-bromophenyl)-1-(2,4-dinitrophenyl)-1H-pyrazole
(3k) Yellow solid, mp. 192–196 °C; IR (KBr) (νmax, cm–1):
3098, 1602, 1532, 1496, 1348, 572; 1HNMR(400 MHz,
CDCl3) (δ, ppm): 8.75 (d, 1H, J = 2.4, Ar–H), 8.31-8.33
(dd, 1H, J = 2.8, J = 8.8, Ar–H), 7.87 (d, 2H, J = 8.4, Ar–
H), 7.53 (d, 1H, J = 8.8, Ar–H), 7.03–7.45 (m, 6H, Ar–H),
6.85 (s, 1H, CH); 13C NMR (75 MHz, CDCl3) (δ, ppm):
154.6, 146.4, 145.3, 137.6, 132.4, 130.3, 128.6, 128.2,
123.1, 120.2, 119.3, 116.1, 106.2; HRMS (ESI-TOF, [M]
+): calcd for C21H12Br2N4O4, 541.922; found 541.920.
1-(2,4-dinitrophenyl)-5-(4-methylphenyl)-3-phenyl-1H-pyra-
zole (3g) Yellow solid, mp. 154–158 °C; IR (KBr) (νmax
,
cm–1): 3087, 1603, 1536, 1496, 1350.
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.73 (d, 1H, J =
2.4, Ar–H), 8.35-8.37 (dd, 1H, J = 2.4, J = 8.8, Ar–H), 7.84
(d, 2H, J = 8.4, Ar–H), 7.49 (d, 1H, J = 8.8, Ar–H), 7.05–
7.43 (m, 7H, Ar–H), 6.83 (s, 1H, CH), 2.38 (s, 3H, CH3);
13C NMR (75 MHz, CDCl3) (δ, ppm): 154.6, 146, 145.8,
145.2, 139.6, 138.2, 131.8, 129.8, 129.6, 128.9, 128.8,
128.5, 127, 126, 125.7, 120.9, 106.3, 21.37; HRMS (ESI-
TOF, [M]+): calcd for C22H16N4O4, 400.117; found
400.119.
3-(4-bromophenyl)-1-(2,4-dinitrophenyl)-5-(4-methylphe-
nyl)-1H-pyrazole (3l) Yellow solid, mp. 182–185 °C; IR
(KBr) (νmax, cm–1): 3087, 1605, 1538, 1498, 1343, 594.
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.72 (d, 1H, J =
2.4, Ar–H), 8.34-8.37 (dd, 1H, J = 2.4, J = 8.4, Ar–H), 7.81
(d, 2H, J = 8.0, Ar–H), 7.55 (d,1H, J = 8.4, Ar–H) 7.17–
7.50 (m, 6H, Ar–H), 6.85 (s, 1H, CH), 2.38 (s, 3H); 13C
NMR (75 MHz, CDCl3) (δ, ppm): 154.6, 146.2, 145.8,
145.18, 139.7, 138.3, 131.8, 129.8, 129.6, 128.9, 128.4,
127., 125.7, 123.5, 120.9, 106.4, 21.4; HRMS (ESI-TOF,
[M]+): calcd for C22H15N4O5Br, 494.022; found 494.023.
3-(4-bromophenyl)-5-(4-chlorophenyl)-1-(2,4-dinitrophe-
nyl)-1H-pyrazole (3h) Yellow solid, mp. 138–142 °C; IR
(KBr) (νmax, cm–1): 3085, 1608, 1539, 1495, 1348, 742, 552
(Desai et al. 2012).
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.79 (d, 1H, J =
2.4, Ar–H), 8.36–8.38 (dd, 1H, J = 2.44, J = 8.8, Ar–H),
7.69 (d, 2H, J = 8.4, Ar–H), 7.45 (d, 1H, J = 8.8, Ar–H),
7.10–7.58 (m, 6H, Ar–H), 6.89 (s, 1H, CH); 13C NMR (75
MHz, CDCl3) (δ, ppm): 153.6, 146.2, 145.3, 144.9, 137.7,
134.9, 132.5, 130.1, 129.6, 129.4, 129, 128.6, 127.3, 127.2,
124.1, 121.1, 106.6; HRMS (ESI-TOF, [M]+): calcd for
C21H12N4O4ClBr, 497.972; found 497.98.
1-(2,4-dinitrophenyl)-3-(4-methoxyphenyl)-5-(4-methylphe-
nyl)-1H-pyrazole (3m) Yellow solid, mp. 142–148 °C; IR
(KBr) (νmax, cm–1): 3097, 1603, 1542, 1445, 1349,
1217.1HNMR (400 MHz, CDCl3) (δ, ppm): 8.72 (d, 1H, J
= 2.4, Ar–H), 8.33-8.36 (dd, 1H, J = 2.8, J = 8.8, Ar–H),
7.77 (d, 2H, J = 8.4, Ar–H), 7.46 (d, 1H, J = 8.8, Ar–H)
6.94–7.26. (m, 6H, Ar–H), 6.80 (s, 1H, CH), 3.85 (s, 3H,
OCH3), 2.38 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) (δ,
1-(2,4-dinitrophenyl)-3-(4-fluorophenyl)-5-(4-methoxyphe-
nyl)-1H-pyrazole (3i) Yellow solid, mp. 142–144 °C; IR
(KBr) (νmax, cm–1): 3093, 1608, 1537, 1498, 1348, 1215.

Medicinal Chemistry Research
NO2
NH
NO2
O2N
NO2
R1
R2 2,4-DNP hydrazine
N
N
SiO2- MoO3
N
R1
50-60oC, MeOH
Neat Heat, 120oC
O
R1
R2
R2
1
2
3
Scheme 2 Synthesis of 1,3,5-trisubstituted pyrazoles
Table 1 List of 1,3,5-trisubstituted pyrazole derivatives
ppm): 160.8, 154.9, 146.2, 145.3, 144.3, 138.3, 133.6, 133,
130.1, 129.6, 129, 127.4, 124.2, 121.3, 114.2, 111.5, 106.5,
55.6, 21.3; HRMS (ESI-TOF, [M]+): calcd for C23H18N4O5,
430.128; found 430.129.
Compounds
R1
R2
Time in hours
% Yield
3a
3b
3c
3d
3e
3f
H
H
4
66
77
73
78
88
84
81
66
60
57
79
60
86
79
76
OMe
Br
Cl
3
OMe
F
4
1-(2,4-dinitrophenyl)-5-(4-fluorophenyl)-3-(4-hydroxyphe-
nyl)-1H-pyrazole (3n) Yellow solid, mp. 206–208 °C; IR
(KBr) (νmax, cm–1): 3596, 3076, 1601, 1532, 1489, 1342,
980.
F
2.5
3
Cl
Cl
F
Cl
3.5
4
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.73 (d, 1H, J =
2.4, Ar–H), 8.36-8.39 (dd, 1H, J = 2.4, J = 8.8, Ar–H), 7.75
(d, 2H, J = 6.4, Ar–H), 7.46 (d, 1H, J = 8.8, Ar–H) 6.88–
7.26 (m, 6H, Ar–H), 6.80 (s, 1H, CH); 13C NMR (75 MHz,
CDCl3) (δ, ppm): 160.8, 154.9, 152.4, 146.2, 145.3, 144.3,
138.3, 133.6, 132.2, 130.1, 129.6, 128.4, 127.2, 120.9,
116.2, 116.5, 106.5; HRMS (ESI-TOF, [M]+): calcd for
C21H13N4O5F, 420.087; found 420.080.
3g
3h
3i
H
CH3
Cl
Br
1.5
1.5
3.5
2.5
1.5
2
F
OMe
OMe
Br
3j
OMe
Br
3k
3l
Br
CH3
CH3
F
3m
3n
3o
OMe
OH
NO2
2.5
3
H
1-(2,4-dinitrophenyl)-5-(4-nitrophenyl)-3-phenyl-1H-pyra-
zole (3o) Yellow solid, mp. 224–226 °C. IR (KBr) (νmax
,
cm–1): 3089, 1603, 1540, 1484, 1350.
on the model reaction. It was observed that, catalyst not in
mol%, but in equimolar amount gave good yield of the
cyclized product at 120 °C.
1HNMR (400 MHz, CDCl3) (δ, ppm): 8.79 (d, 1H, J =
2.4, Ar–H), 8.41-8.44 (dd, 1H, J = 2.4, J = 8.6, Ar–H), 8.25
(d, 2H, J = 7.2, Ar–H), 7.84 (d, 1H, J = 8.6, Ar–H), 7.26–
7.51 (m, 7H, Ar–H), 7.03 (s, 1H, CH); 13C NMR (75 MHz,
CDCl3) (δ, ppm): 154.6, 148.3, 146.4, 145.8, 144.4, 139.6,
138.4, 131.8, 129.8, 129.4, 128.9, 128.8, 128.5, 127.5,
124.3, 120.9, 106.3; HRMS (ESI-TOF, [M]+): calcd for
C21H13N5O6, 431.086; found 431.102.
Once the optimized conditions had been developed, we
examined the substrate scope and generality of the desired
oxidative cyclization reaction. A wide range of newly
synthesized substituted 2,4-dinitrophenyl hydrazone deri-
vatives were used for the synthesis of 1,3,5–trisubstiuted
pyrazoles (3a–3o) (Scheme 2) (Desai and Gawandi 2016).
As shown in Table 1, respective hydrazones and equivalent
amount of catalyst were heated at 120 °C. After completion,
the reaction mixture was washed with ethyl acetate and
catalyst was filtered off. Solvent was removed on vacuum
and the crude product was recrystallized using n-hexane. It
is also worth noting that the purification procedure for the
synthesized compounds required only recrystallization
instead of tedious and prolonged column chromatographic
technique. In order to check the effectiveness of silica
molybdic acid, its recycling was performed and was infer-
red that it can be used over five cycles of reaction without
much loss in activity.
Results and discussion
Our preliminary investigations were focused on the devel-
opment of an ecofriendly reaction conditions for effecting
oxidative cyclization. To achieve this, screening of various
catalysts and solvents has been carried out. The results
revealed that the most suitable reagent for this transforma-
tion was SiO2-molybdic acid, which was then followed by
further optimization of reaction conditions. We investigated
the effect of the catalyst loading and effect of temperature

Medicinal Chemistry Research
Table 2 Molegro docking score
for pyrazole derivatives (3a–3o)
for anticancer activity
Code MolDock score Rerank score
E-inter
(protein-
ligand)
HBond
Heavy
atoms
count
Docking score
(Kcal/mol)
(Kcal/mol)
(Kcal/mol)
No. Kcal/mol
3a
3b
3c
3d
3e
3f
–136.051
–144.917
–149.881
–148.618
–144.812
–145.328
–137.869
–138.83
–90.2738
–98.7802
–101.725
–96.6649
–82.0598
–83.7235
–31.2169
–78.3858
–90.566
–132.572
–142.843
–143.373
–141.006
–137.168
–137.104
–135.422
–140.427
–144.484
–141.134
–152.841
–136.202
–124.899
–134.386
–153.638
3
3
4
5
3
3
0
7
3
1
1
3
6
6
7
–3.46377 29
–4.27694 32
–139.951
–148.045
–151.798
–150.723
–144.842
–145.484
–135.477
–146.55
–6.81212 32
–6.88374 31
–4.70957 31
–4.71109 31
3g
3h
3i
0
30
–10.4638 31
–4.57163 32
–2.05601 33
–1.90323 31
–4.71239 31
–9.61717 32
–9.80866 31
–9.10727 32
–152.306
–148.609
–161.223
–144.452
–128.389
–141.867
–158.518
–152.181
–148.955
–159.651
–144.564
–141.594
–152.178
–160.755
3j
–98.5868
–108.857
–83.159
3k
3l
3m
3n
3o
–33.1833
–47.023
–105.681
Under these experimental conditions, cyclization took
place better in the presence of electron withdrawing groups
on aromatic ring. Compound 3e with electron withdrawing
chloro group at 4 positions of 3 and 5 substituted aromatic
ring gave excellent yield in 88% in comparison to com-
pound 3a with no substituents in aromatic ring, obtained in
66% yield. On the other side, compound 3j with electron
donating methoxy substituent was obtained in 57% yield.
Thus, the nature of substituent alters the reactivity of the
substrate towards cyclization.
formation. Chemical shifts at 154.3, 146.8, 144.4, and 106.2
ppm corresponds to C5=N, C4-NO2, C3-N, and C4-H
carbon atoms. The exact molecular weight was also char-
acterized and confirmed by HRMS studies. The ultraviolet
spectroscopic studies showed absorbance values at 246 nm.
Anticancer activity
Virtual screening
All the synthesized compounds were characterized by
The synthesized pyrazole compounds were virtually
screened for its anticancer activity using tyrosine kinase as
the target enzyme (Table 2). The molecular docking study
revealed that the compounds 3i, 3k, and 3l act as a very
good inhibitors of tyrosine kinase enzyme. As depicted in
Fig. 3 compound 3i makes two hydrogen bonding interac-
tions at the active site of the enzyme, among them one
interaction comes from the oxygen atom of methoxy group
present at fourth position of aromatic ring with Gly772
(1.55 Å) and the other interaction from the oxygen atom of
nitro group present on the fourth position of another aro-
matic ring with Cys773 (2 Å).
The nitro group present on the second and fourth position
of aromatic rings in compound 3k makes five hydrogen
bonding interactions out of which two are observed between
nitrogen and oxygen of nitro group and Thr766 (1.52 and
1.55 Å). The oxygen atoms of nitro group show two inter-
actions with Asp831 (1.55 Å) and Thr830 (1.52 Å) and the
nitrogen of nitro group shows one interaction with Thr830
(1.52 Å).
1
IR, H NMR, 13C NMR spectroscopy, and mass spectro-
scopy. The IR spectra of the title products 3a–3o exhibited
characteristic absorption bands at 3095 to 3070 cm–1 indi-
cating the C–H bond of the pyrazole ring. Also dis-
appearance of absorption bands in the region of frequency
at 3300 cm−1 pertaining to N–H bond confirms the con-
version of hydrazone derivatives to pyrazole. Absorption
bands at 1600 to 1610 cm−1 could be attributed to the
presence of C=N bond. Characteristic absorption peak at
1540 to 1550 cm−1 corresponds to the (–NO2) nitro group
attached to the carbon atom. 1H NMR analysis further
confirms the pyrazole structure. The main distinctive peak
in the spectra of all the target compounds is the appearance
of the singlet δH at 6.5 to 7.5 ppm signifying the proton
attached to C4 of the five membered pyrazole ring. The
most deshielded proton was observed in case of the aro-
matic hydrogen present between the two nitro groups as a
doublet at δH 8.79–8.72 ppm. All the aromatic protons
showed multiplets at δH 7.50 to 7.10 ppm, thereby marking
the formation of pyrazole derivatives. Along with this, 13C
NMR spectroscopic study also signifies the pyrazole
Compound 3l makes two hydrogen bond interactions, the
nitrogen and oxygen atoms of nitro group present on fourth

Medicinal Chemistry Research
Fig. 3 Molecular docking data:
The compounds docked in best
of its conformation into the
binding site of 1M17. a Binding
mode of compound 3i forming
one H bond with Cys773, one H
bond with Gly772; b binding
mode of compound 3k forming
one H bond with Asp831, two H
bonds with Thr830, two H
bonds with Thr766; c binding
mode of compound 3l forming
two H bonds with Met76
Table 3 IC50 values (μM S.E.)
of compounds 3i, 3k, and 3l
against two different cancer cell
lines and normal human cell
lines
Compounds
MDA-MB-231a
K-562b
HEK-293c
SId-MDA-MB-231
SI K-562
3i
0.93 0.05
0.68 0.04
0.58 0.02
0.08 0.01
0.3 0.02
1.68 0.08
0.78 0.03
1.22 0.06
4.49 1.39
>100
4.41 0.09
4.70
8.86
66.96
–
2.62
7.72
32.16
–
3k
6.02 0.1
3l
39.24 0.12
Doxorubicin
Paclitaxel
–
–
–
–
aMDA-MB-231-triple negative breast cancer cell line
bK-562- Myelogenous leukemia cell line
cHEK-293-human embryonic kidney cells (human non-tumorous cells)
dSelective index was calculated as ratio of IC50 value of non-tumorous cell line to the IC50 value of cancer
cell line. IC50 values are obtained as the mean SD (μM) from three different experiments
position of aromatic ring shows interactions with Met769
(1.55 Å) (Fig 3).
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay method.
All the three selected pyrazole compounds inhibited cell
proliferation with IC50 values in the range of 0.58 to 1.68
μM. As shown in Table 3, the samples showed moderate to
good activity comparable to that of standard drugs,
especially compound 3l showed higher anti-proliferation
activity with IC50 = 0.58 0.02 μM against TNBC(triple
negative breast cancer) MDA-MB-231 cell lines. Also,
compound 3l exhibited moderate activity with IC50 value
1.22 0.06 μM against K-562-leukemia cell line.
Biological screening
Cancer cell inhibitory activities of the selected
compounds On the basis of the enzymatic inhibitory
potency against IM17, compounds 3i, 3k, and 3l were
selected and evaluated for their anti-proliferative activity
against breast cancer cell line MDA-MB-231 (ER-negative)
and myelogenous leukemia cell line K-562 using MTT(3-

Medicinal Chemistry Research
Compounds 3i and 3k showed moderate activity with IC50
values 0.93 0.05 and 0.68 0.04 μM against MDA-MB-
231 cell lines. In case of K-562 cell line, compound 3k
displayed higher anti-proliferative activity with IC50 value
0.78 0.03 μM as compared to the other two screened
samples, which was compatible with its docking study.
Accompanying this, the in vitro cytotoxic activity of all
the tested compounds was assessed against normal HEK-
293 cell lines by MTT colorimetric assay. The cytotoxic
results revealed that none of the three evaluated compounds
exhibited any significant toxicity effect on normal HEK-293
cells. SI, i.e., selectivity index exposes the differential
activity of a pure compound. Compound with a higher SI
value is said to be more selective and hence less cytotoxic.
On the other side, a compound with SI value < 2 indicates
cytotoxicity of the pure compound. As seen in Table 3 all
H
NO2
NO2
H
H
H
N
H
N
H
H
Br
H
H
CH3
H
H
Fig. 4 Structural features of compound 3l responsible for anticancer
activity
A
B
3i
3k
3l
3i
3k
3l
C
3i
3k
3l
Fig. 5 Graphical representation of cell viability exhibited by 3i, 3k, and 3l for three different cell lines. a For MDA-MB-231 cell line, b for K-562
cell line, and c HEK-293 (non-tumorous) cell line

Medicinal Chemistry Research
Fig. 6 Fluorescence microscopy
images of cells stained with
acridine orange/ethidium
a
b
bromide. a Viable untreated
cells, b cells treated with
compound 3i, c cells treated
with compound 3k, d cells
treated with compound 3l
3i
c
d
3l
3k
the samples were proved to be non toxic on the normal
tumor cells (Fig. 4).
showed an orange fluorescence with nuclear membrane
bebling Fig. 6b–d thereby signifying the apoptotic mode of
cell death.
Further, an apoptotic mode of cell death was confirmed
by fluorescence microscopy studies using DAPI staining
method wherein, the treated and untreated cells were stained
As seen in Fig. 5; the effect of pyrazole derivatives on the
viability of MDA-MB-231, K-562 human cancer cell lines
and HEK-293 was investigated and represented graphically
as variation in percentage of viability with respect to time
and concentration.
with
4’,6-diamidine-2’-phenylindole
dihydrochloride
(DAPI). As shown in Fig. 7 the cell shrinkage, nuclear
fragmentation characterizes the apoptotic mode of cell death
by cells treated with compound 3i, 3k, and 3l stained with
DAPI.
Cell morphology studies by fluorescence microscopy
Fluorescence microscopy study was used to study how cell
death was induced by selected pyrazole compounds 3i, 3k,
and 3l in comparison to the standard cis-platin by virtue of
acridine orange/ ethidium bromide staining. The fluores-
cence properties of dyes can help to characterize the mode
of cell death, whether early or late apoptosis. Acridine
orange is taken up by both viable and apoptotic (dead) cells
by emitting green fluorescence. Ethidium bromide will stain
only cells with disrupted membrane integrity, i.e., late
apoptotic cells and necrotic cells. Staining of acridine
orange on live cells showed green fluorescent nuclei, which
indicate early apoptosis, while late apoptotic cells have
orange to red nuclei with condensed or fragmented chro-
matin. Necrotic cells have uniform orange to red nuclei with
the organized structure. Thus, the results (Fig. 6) demon-
strated that the viable cells without treatment of pyrazole
compound stained with acridine orange appeared with green
fluorescent nucleus Fig. 6a, while late apoptotic cells
Conclusion
In conclusion, a novel series of 2,4-dinitrophenyl containing
pyrazole derivatives (3a–3o) were designed, synthesized,
and evaluated as anti-proliferative agents. Keeping in view
the significance of presence of electron withdrawing nitro
groups, our efforts of achieving oxidative cyclization of N-
aryl hydrazones has led to a novel class of bioactive pyr-
azoles of different substitution patterns in good yields.
Recycling of the reagent up to five cycles without any
higher loss in catalytic activity and time consumption
delivers the efficiency of the green method.
Considering the biological screening results, the pyrazole
derivatives showed potent anticancer activity against the
MDA-MB-231(ER-negative) human breast cancer cell line

Medicinal Chemistry Research
Fig. 7 Fluorescence microscopy
images of cells stained with
DAPI, showing a viable
a
b
untreated cells, b cells treated
with compound 3i, c cells
treated with compound 3k, d
cells treated with compound 3l
NC
3i
c
d
3k
3l
Maratha Mandal’s NGH Institute of Dental Sciences and Research
Center, Belgaum, Karnataka for biological screening facilities.
and K-562 leukemia cell line. It was noticed that the
selective compounds 3i, 3k, and 3l demonstrated significant
minimum inhibitory activity with IC50 values in the range
0.5–1.68 µM against cancer cell lines MDA-MB-231 and
K-562. Cytotoxicity against the human embryonic kidney
cell line HEK-293 was also evaluated and the selectivity
was thus assessed. The evaluated compounds displayed
moderate SI values ranging from 2.62 to 32.16. The dock-
ing studies revealed that compounds 3i, 3k, and 3l had good
tyrosine kinase binding interactions. Further, the mode of
cell death was characterized by fluorescence microscopy
study, to be late apoptosis, which was also supported by
DAPI results.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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