Design, synthesis, cytotoxicity, HuTopoIIα inhibitory activity and molecular docking studies of pyrazole derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2016.10.001

In an attempt to find potential anticancer agents, a series of novel ethyl 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylates 5a-i and 5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamides 6a-i were designed, synthesized and evaluated for their topoisomerase IIα inhibitory activity and in vitro cytotoxicity against a panel of cancerous cell lines (MCF-7, NCI-H460, HeLa) and a normal cell line (HEK-293T). Molecular docking studies of all the synthesized compounds into the binding site of topoisomerase IIα protein (PDB ID: 1ZXM) were performed to gain a comprehensive understanding into plausible binding modes. These compounds were also screened for in silico drug-likeliness properties on the basis of the absorption, distribution, metabolism and excretion (ADME) prediction. Among all the synthesized compounds, analogue 5d showed superior cytotoxicity with an IC₅₀ value of 7.01?±?0.60?μM for HeLa, 8.55?±?0.35?μM for NCI-H460 and 14.31?±?0.90 for MCF-7 cancer cell lines. Further, compound 5d showed 70.82% inhibition of topoisomerase IIα at a concentration of 100?μM with maximum docking score of ?8.24. Results of ADME prediction revealed that most of these compounds showed in silico drug-likeliness properties within the ideal range.

Full text of DOI:10.1016/j.bioorg.2016.10.001

Accepted Manuscript
Design, Synthesis, Cytotoxicity, HuTopoIIα Inhibitory Activity and Molecular
Docking Studies of Pyrazole Derivatives as Potential Anticancer Agents
Raquib Alam, Divya Wahi, Raja Singh, Devapriya Sinha, Vibha Tandon,
Abhinav Grover, Rahisuddin
PII:
S0045-2068(16)30129-8
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.10.001
YBIOO 1956
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
14 July 2016
3 October 2016
4 October 2016
Please cite this article as: R. Alam, D. Wahi, R. Singh, D. Sinha, V. Tandon, A. Grover, Rahisuddin, Design,
Synthesis, Cytotoxicity, HuTopoIIα Inhibitory Activity and Molecular Docking Studies of Pyrazole Derivatives as
Potential Anticancer Agents, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.10.001
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Design, Synthesis, Cytotoxicity, HuTopoIIα Inhibitory Activity and Molecular Docking Studies of Pyrazole Derivatives as
Potential Anticancer Agents
a
b
c
d
c,d
b,
a,
Raquib Alam , Divya Wahi , Raja Singh , Devapriya Sinha , Vibha Tandon , Abhinav Grover *, Rahisuddin *
a
Department of Chemistry, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi 110025, India
b
School of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India
c
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi-110 067, India
d
Department of Chemistry, University of Delhi, Delhi-110007, India
*
Authors for correspondence

Abstract
In an attempt to find potential anticancer small molecules, a series of novel ethyl 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-
yl)cyclohex-3-enecarboxylates
5a-i
and
5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-
carbothioamides 6a-i were designed, synthesized and evaluated for their topoisomerase IIα inhibitory activity and in vitro cytotoxicity against a
panel of cancerous cell lines (MCF-7, NCI-H460, HeLa) and a normal cell line (HEK-293T). M
α
PDB ID
hese compounds were also screened for in silico drug-likeliness properties on the basis of the absorption, distribution,
metabolism and excretion (ADME) prediction. Among all the synthesized compounds, analogue 5d showed superior cytotoxicity with an IC50
value of 7.01±0.60 μM for HeLa, 8.55±0.35 μM for NCI-H460 and 14.31±0.90 for MCF-7 cancer cell lines. Further, compound 5d showed
7
0.82% inhibition of topoisomerase IIα at a concentration of 100 μM with maximum docking score of -8.24. Results of ADME prediction
revealed that most of these compounds showed in silico drug-likeliness properties within the ideal range.

1
. Introduction
Cancer, being the second most dreaded disease causing mortality in the world after cardiovascular disorder, is a major problem of health in both
developed and developing countries. Although there has been considerable progress to reduce cancer incidence in the United States, the number
of cancer patients continues to increase [1]. Chemotherapy is one of the most effective approaches used to treat cancer patients. However, lack of
selectivity and development of drug-resistance reduce the efficacy of cancer chemotherapy [2]. Therefore, development of effective and safe
anticancer agents with high potency and less toxicity is a major focus for researchers across the world.
In last few decades, anticancer drugs have been developed from chemically synthesized compounds. Recently, large number of interesting
pyrazole derivatives have been synthesized [3]. The pyrazole tethered heterocyclic compounds have received considerable attention owing to
their diverse chemotherapeutic potential [4-6]. Drugs 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
Apixaban, Celecoxib, Fipronil, Remogliflazone etabonate, Lonazolac, Tolpiprazole, Deracoxib and many more. Crizotinib and Ruxolitinib are
important pyrazole tethered anticancer drug [7]. Few pyrazole based drugs are depicted in Figure 1. Celecoxib a typical model of pyrazole
tethered diaryl heterocyclic small molecule [8]. The antitumor activity of celecoxib is reported against prostate tumors in experimental models
[
9-11]. Pyrazole derivatives show anticancer activity due to their inhibition of various targets such of topoisomerase II [12-14], EGFR [15, 16],
VEGF [17, 18], HDAC [19, 20], IGF-1R [21], Aurora-A kinase [22], cMet [23], Tubulin [24], mTOR [25], B-raf [26, 27], ROS 1 [28], CDKs
19], PI3K [30], JAK2 [31], ALK [32], among others. Hence, incorporation of the pyrazole moiety is an important synthetic strategy in rational
[

drug development process. The chalcones are α,β-unsaturated ketones that acts as a central core for a variety of pharmacologically important
compounds. Their true importance may be explained in two branches, the biological activity associated with them and their synthetic 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-inflammatory [33], antimitotic [34], antileishmanial [35, 36], anti-invasive [37],
antimalarial [38, 39], antibacterial [40], antifungal [41], antiprotozoal [42], antiviral [43], antitrypanosomal [44] and anticancer activity [45-50].
Chalcones are versatile synthon for the preparation of five and six-membered ring systems [51]. Di-aryl cyclohexenone derivatives are a class of
cyclic compounds prepared from chalcone, shows interesting biological activities, among which its antimicrobial activity is well known [52, 53].
Recently, anticancer activity of chalcone thiosemicarbazone derivatives have been reported [54].
HuTopoIIα is an important target in anticancer drug development process. There are wide range of topoisomerase IIα inhibitors reported in the
literature such as Doxorubicin, Mitoxanthrone, Etoposide, Amonafide, Daunomycin, Ellipticine, Aclarubicin, Merbarone and others [55].
Majority of the cancer treatment regimens rely on the use of at least one drug that inhibits topoisomerases [56]. Topoisomerases are essential
nuclear enzymes that maintain the topological changes of DNA and play key role in replication, transcription and chromosome segregation [55,
5
7-59]. Inhibitors of HuTopoIIα are generally classified into two groups namely poisons and catalytic inhibitors. Poisons act by stabilizing a
transient covalent DNA-enzyme complex, thereby causing damage to the DNA molecule. The catalytic inhibitors interfere with a single step of
the topo II catalytic cycle. Many topoisomerase inhibitors have been effectively used in clinical trial for the curing of various neoplasm [60, 61].

The development of topoisomerase IIα inhibitors as an anticancer drug is emerging [62]. The pyrazoles have been reported to show anticancer
activity by inhibiting human topoisomerase IIα [12-14]. Few pyrazole based topoisomerase II inhibitors are depicted in figure 2.
In this way, efforts are made on establishing some heterocyclic analogues integrated with pyrazole scaffold to describe the relevance
pharmacological activity. Motivated by our previous findings [63], and in continuation of our ongoing research program to get potential
anticancer agents, we have synthesized ethyl 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylates 5a-i and
5
-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamides 6a-i. The structures of target
compounds are depicted in Figure 3. These compounds were evaluated for their topoisomerase IIα inhibitory activity and in vitro cytotoxicity
against a panel of cancerous cell lines and a normal cell line. To
in silico drug-likeliness properties of these compounds on the basis of the absorption,
distribution, metabolism and excretion (ADME) prediction.
m
2
. Results and Discussion
2
.1.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 [63, 64] which, in turn, were initially synthesized by Vilsmeier-Haack formylation of phenylhydrazones
2
a-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 [63]. 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. The chalcones 4a-i on treatment with ethyl
acetoacetate in the presence of NaOH in ethanol afforded pyrazolylcyclohex-3-enecarboxylate derivatives 5a-i. Further, treatment of chalcones
with thiosemicarbazide in presence of concentrate H
2
SO in acetic acid afforded pyrazolylcarbothioamide derivatives 6a-i. The formation of
4
pyrazoline ring involves a Michael addition of thiosemicarbazide on chalcone followed by cyclization and dehydration. The progress of the
1
13
reaction was monitored by TLC. All the synthesized compounds were characterized by detailed H NMR and C NMR (Figure S1-S18), IR,
mass spectral data and elemental analysis.
-1
The IR spectra of the compound 5a revealed a sharp strong absorption band at 1737 cm that is due presence of the ester group in
-1
cyclohexenones. Another sharp strong absorption band appeared at 1674 cm that was assigned to the carbonyl group in conjugation with a
1
carbon-carbon double bond. The H NMR spectra 5a showed characteristic triplet at 1.13 ppm for methyl proton at C-3ʹ that confirmed the
presence of the ester group. The signal for vinylic proton at C-2 of the cyclohexenone ring appeared as singlet at 6.55 ppm. The two protons at
C-4 being non-equivalent appeared as two different signals. The axial proton appeared as multiplet at around 2.79-2.86 ppm and the equatorial
proton appeared as double of doublet at 3.07 ppm. The signal for the proton at C-6 appeared as doublet at 3.78 ppm. The signals for the protons
1
3
at C-2ʹ and C-5 appeared as multiplets at 4.07-4.16 ppm. C NMR spectra of 5a showed two signals at 193.21 and 168.95 due to presence
carbonyl carbons. The elemental analysis and mass spectra supported the structure of compound 5a.

-
1
IR spectra of the compound 6a showed strong absorption bands at 3424 and 3242 cm due to presence of primary amine group. The
1
characteristic signal in H NMR spectra of 6a was due to presence of three pyrazoline protons which displayed an interesting AMX pattern of
doublet of doublet. Methine proton of pyrazolines displayed signal at 6.06 ppm as a doublet of doublet with coupling constants of 11.6 and 4.4
Hz. The methylene proton displayed doublet of doublet at 3.96 ppm with coupling constants of nearly 18.0 Hz and 11.6 Hz. The peak for another
1
3
methylene proton has not been observed clearly because of merging of this particular proton with DMSO-d solvent peak at 3.29-3.34 ppm. C
6
NMR spectra of compound 6a displayed a signal at 176.01 ppm assigned to thiocarbamoyl carbon (C=S). Carbon of methine showed a chemical
shift at 55.68 ppm. Carbon of methylene displayed a chemical shift at 42.04 ppm. Furthermore, the mass spectra and elemental analysis
supported the structure of compound 6a.
2
.2. Evaluation of in vitro cytotoxicity
In vitro cytotoxicity of all the newly synthesized compounds 5a-i and 6a-i was determined by the MTT [(3-(4, 5-dimethyl-2-thiazolyl) 2,5-
diphenyl-2H-tetrazolium bromide)] assay against a panel of three different human cancer cell lines namely; MCF-7 (human breast), NCI-H460
(
human lung), HeLa (human cervix) and HEK-293T (Human embryonic kidney cells) normal cell line [65]. The MTT assay is a colorimetric
assay for measuring the cellular growth that reduces the yellow tetrazolium dye (MTT) to its insoluble purple colour formazan by mitochondrial
dehydrogenases of living cells, was employed for the analysis of the cytotoxic potential of the designed compounds and to assess their potential
as drugs. The insoluble purple formazan that is formed by living cells is dissolved into a coloured solution by the addition of DMSO as a suitable
solvent. At a particular wavelength, the absorbance of this coloured solution can be measured and the IC50 values were calculated from the dose

effect curve (S 19-20) and expressed as concentration (µM) of drug activity. 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 lung, cervix and breast cancer cell lines and weak toxicity towards normal human embryonic kidney cells. Analogues 5d, 5e,
5
f, 5g, 6d, 6e and 6g showed significant cytotoxicity as compared to standard drug etoposide. Subsequently, structure activity relationship
SAR) studies were performed to determine the effect of substituents on cytotoxic activities of the synthesized compounds. Compounds 5d and
d with meta substituted Br group in the benzene ring showed most significant cytotoxic activity. Substituents Br and NO present in the
benzene ring of compounds 5e, 5g, 6e and 6g showed good effect, which is clearly seen in Table 1. Compounds with meta substituted electron-
withdrawing group, the cytotoxic activity order for the cancerous cell lines is Br > NO . The cytotoxic strength is NO > Br > F > Cl for the
compounds with para substituted electron-withdrawing in cancerous cell lines. Finally, compounds with electron-donating substituent present in
para position of the benzene ring, cytotoxic strength order for the cancerous cell line is OCH > CH . Furthermore, the majority of compounds in
this series showed moderate selectivity index values. Compound 5d, 5e, 5f, 5g, 6d, 6g and 6h displayed good selectivity index.
(
6
2
2
2
3
3
2
.3. HuTopoIIα inhibitory Activity
All the synthesized compounds were evaluated for their effect on HuTopIIα in terms of percentage inhibition at varying concentration of 100-
000 μM and IC50 values. The percentage inhibitions of relaxation of super coiled DNA at a concentration of 100 μM and IC50 value (µM) for
1
the inhibitions of relaxation of supercoiled DNA of these compounds are summarized in Table 2. Out of all the synthesized compounds, four
compounds (5c, 5d, 5e and 5f) have shown significant inhibition of HuTopIIα (Figure S21). Gel densitometry analysis was performed to

calculate the % inhibition of relaxation activity of HuTopIIα by the synthesized compounds. Compounds 5b-f displayed IC50 value of < 100 μM
against HuTopIIα inhibitory activity. As per our result, at 100 μM concentration, compound 5c showed 89% inhibition of relaxation of
supercoiled DNA by HuTopII α. Out of all the synthesized molecules, compound 5c was observed to be the most potent against HuTopIIα.
Compound 5d and 5e was also observed to inhibit significantly with 70.82 % and 70.45 % inhibition of relaxation activity at a concentration of
1
00 μM respectively. Furthermore, 60.83% inhibition of relaxation of supercoiled DNA by HuTopIIα at a concentration of 100 μM was
observed by 5f. Our observation suggests Cl and Br at both meta and para position could inhibit HuTopIIα significantly as observed in
compound 5c, 5d and 5e whereas compound 5f with NO at meta position was also found to be potent. Our observation suggests Cl and Br at
2
both meta and para position could inhibit HuTopIIα significantly as observed in compound 5c, 5d and 5e whereas compound 5f with NO
2
at
meta position was also found to be potent.
2
.4. Molecular docking Studies
Molecular docking of all the synthesized compounds into the binding site of topoisomerase IIα was performed to gain a comprehensive
understanding into plausible binding modes. All the synthesized compounds exhibited good glide energy and docking scores towards the
receptor active pocket and bind to the active of the amino acid residue with H-bonding and hydrophobic interaction (Table 2, Figure S22-S24) as
compared to the standard drug Etoposode (Figure S25). Among them, the conformations with lowest binding energies and those ligands
exhibiting well established H-bonds with available oxygen and nitrogen in the closest range of ꢀ-3.8 ꢁ with one or more amino acids in the
receptor active pocket (Figure 4), were chosen as best docked ligand orientations. Among all the synthesized compounds, compound 5d was

energetically favoured as this ligand bind to active site of topoisomerase IIα and are exhibiting interactions with amino acids of active pocket of
the receptor and considered as the best docking poses as compared to the others. The ligand 5d with Br group at m-substituted phenyl in parent
analogue exhibited H-bonding with Asn150, Ser148, Lys168, Thr147 and Gly164 amino acid residues as well as hydrophobic interaction with
Gly166, Tyr165, Gly161, Ser149, Phe142, Ile141, Val137, Ile125, Arg98, Asn95 and Asn91 amino acid residues and represent the most
-1
effective docking score of -8.24 and glide energy of -59.08 Kcal mol . All other ligands show comperative glide energy as well as moderate
docking scores. The topoisomerase IIα used in the docking studies was derived from the X-ray crystal structure of the ATPase domain of human
topoisomerase IIα (1ZXM) and the potential inhibitors obtained can primarily be classified as catalytic inhibitors, since they have been shown to
disrupt the binding at the ATP Binding catalytic site of 1ZXM.
2
.5. In silico physicochemical properties for “druglikeliness”
All the synthesized compounds 5a-i and 6a-i were screened for “drug-likeliness” profile on the basis of the absorption, distribution, metabolism
and excretion (ADME) properties, calculated in silico using Qikprop module of Schrodinger 9.0 [66, 67].The ADME prediction studies data of
the synthesized compounds are shown in Table 3. The range of various important parameters, like molecular weight lied in range between
4
24.52-508.52, the value of total solvent accessible surface area (SASA) ranged 704.82-836.80, estimated number of hydrogen bonds donated
(
(
Donor HB) by the solute to water molecules in an aqueous solutions was found between 0.0-2.0, estimated number of hydrogen bonds accepted
accept HB) by the solute to water molecules in an aqueous solutions ranged 6.00-7.50, predicted octanol/gas partition coefficient (QPlogPoct)
ranged 22.35-27.44, predicted water/gas partition coefficient (QPlogPw) ranged 11.26-15.86 predicted octanol/water partition coefficient

(
QPlogPo/w) ranged 4.04-6.10, predicted aqueous solubility (QPlogS), ranged -8.46 to -6.00, predicted number of rotable bond (#rotor) ranged
0
0
-3, apparent Caco-2 cell permeability (QPPCaco) ranged 100.07- 1503.50, predicted brain/blood partition coefficient (QPlogBB)ranged -1.58 to
.00, apparent MDCK cell permeability (QPPMDCK) ranged 41.10 - 4672.21, predicted skin permeability (QPlogKp) ranged -3.71 to -1.37,
predicted human serum albumin binding (QPlogKhsa) ranged 0.66-1.25, percentage human oral absorption (%HOA) ranged 73.47-100, total
polar surface area (PSA) ranged 70.59-129.71 and Lipinski violations were 0-2. Results of ADME studies revealed that most of these
compounds showed physiochemical properties within the ideal range [66].
3
. Conclusion
In this study, a series of novel ethyl 4-(3-(aryl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylates 5a-i and 5-(3-(4-
fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamides 6a-i have been synthesized. Structures of all
1
13
the newly synthesized compounds were fully characterized by detailed IR, H NMR, C NMR and mass spectroscopy and elemental analysis.
These compounds exhibited potent anti-proliferative activities against MCF-7 (human breast), NCI-H460 (human lung) HeLa (human cervix)
and cancer cell lines. Among the synthesized compounds, analogues 5c, 5d, 5f, 5g, 6b, 6f and 6g showed significant cytotoxicity as compared to
standard drug doxorubicin. The compound 5d represented the most potent cytotoxic activity with the IC50 value of 7.01±0.60 μM for HeLa
cancer cell lines and 70% inhibition of relaxation of negatively supercoiled DNA by human topoisomerase IIα with highest docking score of -
8
.24. ADME prediction revealed that most of the synthesized compounds showed physiochemical properties within the ideal range. So, the

present study offers preliminary pointers which broaden the research scopes for further design, modify and investigate the structure of these
molecules to expand the utility of such novel compounds as potential anticancer agents.
4
. Experimental Protocol
4
.1. General
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 Aluminium sheets (Silica gel 60 F254, Merck Germany) were used for thin-layer chromatography (TLC) and spots
1
were visualized under UV light. IR spectra were recorded on Agilent Cary 630 FT-IR spectrometer. H NMR spectra were recorded on Bruker
1
3
Avance 400 MHz spectrometer, using CDCl
3
or DMSO-d
6
as a solvent and tetramethyl silane (TMS) as an internal standard. C NMR spectra
were recorded on Jeol 100 MHz spectrometer, using CDCl
3
or DMSO-d as a solvent. Splitting patterns are designated as: s, singlet; d, doublet;
6
dd, doublet of doublet; t, triplet; m, multiplet. Chemical shift values (δ) 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).
4
.2. General Procedure for the synthesis of compounds 5a-i
To an ethanolic NaOH suspension of 4a-i (2 mmol) was added ethylacetoacetate (2.2 mmol) drop by drop and the reaction mixture was stirred at
room temperature for one hour and heated at 70 °C for two hours. Upon completion, reaction mixture was allowed to stand overnight. The

precipitates obtained were filtered off, washed with ethanol and water, affording compounds 5a-i. The compounds 5a-i were recrystallized in
AcOH:EtOH.
4
.2.1. Ethyl 4-(1,3-diphenyl-1H-Pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5a).White solid; Yield: 68%; m.p. 170-172
-
1
1
°
C; IR (neat, νmax cm ): 1737, 1674; H NMR (400 MHz, CDCl
s, 1H), 4.07-4.16 (m, 3H), 3.78 (d, J = 12 Hz, 1H), 3.07 (dd, J = 18 Hz, 4.4 Hz, 1H), 2.79-2.86 (m, 1H), 1.13 (t, J = 7.2 Hz, 3H); C NMR
100 MHz, CDCl ): 193.21, 168.95, 155.35, 151.70, 151.26, 147.25, 139.70, 133.33, 133.23, 132.99, 129.42, 129.24, 128.74, 128.60, 128.36,
26.61, 125.20, 123.56, 121.56, 118.91, 118.67, 61.43, 59.58, 35.81, 33.67, 13.97; Anal. Calcd. for C29 : C, 75.14; H, 5.44; N, 9.00.
3
): 8.71 (s, 1H), 8.63 (d, J = 4.8 Hz, 1H), 7.96 (s, 1H), 7.28-7.73 (m, 12H), 6.55
1
3
(
(
3
1
H
25
N
3
O
3
+
+
Found: C, 75.19; H, 5.38; N, 8.98; MS (ESI) [M + H] Calcd. for C29
H
25
N
3
O
3
: 463.2, Found: 464.2 [M + H] .
4
.2.2. Ethyl 4-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5b). White solid; Yield: 63%;
-1
1
m.p. 160-162°C; IR (neat, νmax cm ): 1734, 1674; H NMR (400 MHz, CDCl
3
): 8.73 (d, J = 2 Hz, 1H), 8.64-8.66 (m, 1H), 7.96 (s, 1H), 7.67-
7
.78 (m, 5H), 7.47 (t, J = 7.6 Hz, 2H); 7.32-7.38 (m, 2H), 7.16 (t, J = 8.4 Hz, 2H), 6.56 (d, J = 1.6 Hz, 1H); 4.08-4.15 (m, 3H), 3.77 (d, J = 12
1
3
Hz, 1H), 3.05 (dd, J = 18.4 Hz, 4.8 Hz, 1H), 2.80-2.88 (m, 1H), 1.14 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl
3
): 193.09, 175.73, 168.96,
1
1
55.00, 150.85, 146.85, 139.63, 133.70, 133.40, 130.25, 130.16, 130.03, 129.48, 129.31, 126.76, 125.38, 125.32, 123.79, 121.46, 118.98,
18.75, 115.89, 115.67, 61.52, 59.57, 35.94, 33.63, 14.00; Anal. Calcd. for C29
H24FN
3
3
O : C, 72.34; H, 5.02; N, 8.73. Found: C, 75.26; H, 5.16;
+
+
N, 8.65. MS (ESI) [M + H] Calcd. for C29
H24FN
3
O
3
: 481.2. Found: 482.3 [M + H] .

4
.2.3. Ethyl 4-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5c).White solid; Yield: 68%;
-1
1
m.p.: 158-160 °C; IR (neat, νmax cm ):1737, 1670; H NMR (400 MHz, CDCl
3
): 8.73 (d, J = 2 Hz, 1H), 8.65 (d, J = 3.6 Hz, 1H), 7.97 (s, 1H),
7
1
1
.30-7.78 (m, 11H), 6.57 (d, J = 2 Hz, 1H), 4.09-4.19 (m, 3H), 3.77 (d, J = 12 Hz, 1H), 3.05 (dd, J = 18.4 Hz, 4.8 Hz, 1H), ), 2.80-2.88 (m,
1
3
H), 1.14 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ): 193.04, 175.67, 168.92, 154.96, 150.84, 146.83, 133.71, 133.39, 131.40, 129.67,
3
29.49, 129.32, 128.98, 128.85, 126.83, 125.42, 125.38, 123.79, 121.56, 118.99, 118.77, 61.52, 59.56, 35.96, 33.61, 13.99; Anal. Calcd. for
+
C
29
H24ClN
3
O
3
: C, 69.95; H, 4.86; N, 8.44. Found: C, 70.13; H, 4.79; N, 8.42; MS (ESI) [M + H] Calcd. for C29
H24ClN
3
3
O : 497.2. Found: 498.2
+
[
M + H] .
4
.2.4. Ethyl 4-(3-(3-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5d). White solid; Yield: 72%;
-1
1
m.p. 184-186 °C; IR (neat, νmax cm ): 1737, 1663; H NMR (400 MHz, CDCl
3
): 8.73 (s, 1H), 8.64 (d, J = 4.4 Hz, 1H), 7.97 (s, 1H), 7.88 (s, 1H),
7
.30-7.76 (m, 10H), 6.56 (s, 1H), 4.08-4.19 (m, 3H), 3.77 (d, J = 12 Hz, 1H), 3.07 (dd, J = 18 Hz, 4.4 Hz, 1H), ), 2.80-2.87 (m, 1H), 1.14 (t, J
13
=
7.2 Hz, 3H); C NMR (100 MHz, CDCl ): 193.00, 168.86, 155.21, 151.36, 150.13, 147.28, 139.56, 135.01, 133.36, 133.19, 131.38, 130.25,
3
1
4
4
29.51, 126.85, 125.42, 125.29, 123.60, 122.88, 121.71, 119.00, 61.54, 59.60, 35.99, 33.58, 14.01; Anal. Calcd. for C29
H
24BrN
3
O : C, 64.21; H,
3
+
+
.46; N, 7.75. Found C, 64.18; H, 4.49; N, 7.82; MS (ESI) [M + H] Calcd. for C29
H24BrN
3
O
3
: 541.1. Found: 542.1 [M + H] .
.2.5. Ethyl 4-(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5e). White solid; Yield: 75%;
-1
1
m.p.: 162-164 °C; IR (neat, νmax cm ): 1737, 1674; H NMR (400 MHz, CDCl
3
): 8.73 (s, 1H), 8.65 (d, J = 4.0 Hz, 1H), 7.96 (s, 1H), 7.30-7.78
(
m, 11H), 6.55 (s, 1H), 4.08-4.20 (m, 3H), 3.77 (d, J = 12 Hz, 1H), 3.05 (dd, J = 18 Hz, 4.4 Hz, 1H), 2.81-2.88 (m, 1H), 1.13 (t, J = 7.2 Hz,

1
3
3
1
H); C NMR (100 MHz, CDCl ): 193.04, 175.83, 168.92, 154.94, 150.72, 150.51, 146.72, 146.37, 139.56, 133.80, 131.91, 131.79, 129.94,
3
29.77, 129.48, 129.31, 126.84, 125.45, 125.37, 123.83, 121.52, 118.98, 118.77, 61.53, 59.56, 33.99, 33.58, 14.04, 13.99; Anal. Calcd. for
+
C
29
H24BrN
3
O
3
: C, 64.21; H, 4.46; N, 7.75. Found: C, 64.16; H, 4.51; N, 7.68; MS (ESI) [M + H] Calcd. for C29
H24BrN
3
3
O : 541.1. Found: 542.1
+
[
M + H] .
4
5
.2.6. Ethyl 4-(3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5f). Pale yellow solid; Yield:
-
1
1
9%; m.p. 168-170 °C; IR (neat, νmax cm ): 1745, 1655; H NMR (400 MHz, CDCl ): 8.74 (s, 1H), 8.65 (d, J = 4.4 Hz, 1H), 8.60 (s, 1H), 8.26
3
(
(
d, J = 7.6 Hz, 1H), 8.12 (d, J = 7.2 Hz, 1H), 8.02 (s, 1H), 7.33-7.80 (m, 8H), 6.58 (s, 1H), 4.09-4.21 (m, 3H), 3.79 (d, J = 12 Hz, 1H), 3.11
1
3
dd, J = 18.4 Hz, 4.4 Hz, 1H), 2.86-2.93 (m, 1H), 1.14 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl
3
): 192.77, 168.81, 155.06, 151.40,
1
1
49.17, 148.48, 147.21, 139.39, 134.65, 134.17, 133.35, 133.07, 129.80, 129.57, 129.39, 127.16, 125.74, 125.30, 123.63, 123.25, 123.00,
21.89, 119.04, 118.87, 61.58, 59.66, 36.22, 33.57, 14.00; Anal. Calcd. for C29
H
24
N
4
5
O : C, 68.49; H, 4.76; N, 11.02. Found: C, 68.54; H, 4.63;
+
+
N, 11.15; MS (ESI) [M + H] Calcd. for C29
H
24
N O
4 5
: 508.2. Found: 509.2 [M + H] .
4
7
2
=
.2.7. Ethyl 4-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5g). Pale Yellow solid; Yield:
-1
1
2%; m.p. 182-184 °C; IR (neat, νmax cm ): 1741, 1659; H NMR (400 MHz, CDCl
3
): 8.74 (s, 1H), 8.65 (d, J = 3.6 Hz, 1H), 8.32 (d, J = 8.8 Hz,
H), 8.03 (s, 1H), 7.96 (d, J = 8.8 Hz, 2H), 7.34-7.78 (m, 7H), 6.59 (d, J = 1.6 Hz, 1H), 4.11-4.18 (m, 3H), 3.79 (d, J = 12 Hz, 1H), 3.08 (dd, J
1
3
18 Hz, 4.4 Hz, 1H), 2.85-2.93 (m, 1H), 1.14 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO-d
6
): 193.70, 169.86, 156.74, 151.19, 148.68,
1
47.48, 146.85, 139.65, 139.25, 134.16, 132.93, 129.80, 129.11, 128.53, 126.94, 123.95, 123.88, 123.75, 122.96, 118.28, 60.29, 60.19, 35.38,

+
3
3.33, 14.32; Anal. Calcd. for C29
: 508.2. Found: 509.1 [M + H] .
.2.8. Ethyl 2-oxo-4-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5h). White solid; Yield: 63%; m.p. 156-
H
24
N
4
O
5
: C, 68.49; H, 4.76; N, 11.02. Found: C, 68.53; H, 4.69; N, 11.06; MS (ESI) [M + H] Calcd. for
+
C
29
H
24
N
4
O
5
4
1
6
-1
1
58 °C; IR (neat, νmax cm ): 1737, 1674; H NMR (400 MHz, CDCl
3
): 8.71 (s, 1H), 8.64 (d, J = 4.00 Hz, 1H), 7.94 (s, 1H), 7.28-7.76 (m, 11H),
.55 (s, 1H), 4.10-4.19 (m, 3H), 3.78 (d, J = 12 Hz, 1H), 3.06 (dd, J = 18.00 Hz, 4.4 Hz, 1H), 2.79-2.86 (m, 1H), 2.39 (s, 3H), 1.14 (t, J = 6.8
1
3
Hz, 3H); C NMR (100 MHz, CDCl
3
): δ 193.31, 175.77, 168.99, 155.18, 151.76, 150.69, 146.79, 139.72, 138.17, 133.78, 133.51, 130.00,
1
29.43, 129.41, 129.24, 128.25, 128.10, 126.54, 125.33, 125.19, 123.78, 121.40, 118.92, 118.67, 61.47, 59.56, 33.82, 33.69, 21.28, 13.97; Anal.
+
Calcd. for C30
H
27
N
3
O
3
: C, 75.45; H, 5.70; N, 8.80. Found: C, 75.39; H, 5.65; N, 8.74; MS (ESI) [M + H] Calcd. for C30
H
27
N
3
3
O : 477.2. Found:
+
4
4
5
1
78.2 [M + H] .
.2.9. Ethyl 4-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-2-oxo-6-(pyridin-3-yl)cyclohex-3-enecarboxylate (5i). White solid; Yield:
-1
1
6%; m.p.: 150-152 °C; IR (neat, νmax cm ): 1734, 1670; H NMR (400 MHz, CDCl
3
): 8.72 (d, J = 2.0 Hz, 1H), 8.64 (d, J = 3.6 Hz, 1H), 7.93 (s,
H), 7.28-7.76 (m, 9H), 6.99 (d, J = 8.8 Hz, 2H), 6.55 (d, J = 1.6 Hz, 1H), 4.12-4.18 (m, 3H), 3.84 (s, 3H), 3.80 (d, J = 12 Hz, 1H), 3.06 (dd,
1
3
J = 18.4 Hz, 4.8 Hz, 1H), 2.78-2.86 (m, 1H), 1.14 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl
3
): 193.29, 168.99, 159.64, 155.20, 151.50,
1
1
7
50.81, 146.87, 139.77, 139.69, 133.68, 133.42, 129.60,.129.39, 129.22, 126.48, 126.01, 125.34, 125.27, 125.13, 123.73, 121.29, 118.84,
18.60, 114.14, 114.03, 61.44, 60.70, 59.52, 55.24, 35.81, 33.69, 13.98; Anal. Calcd. for C30
H
27
N
3
4
O : C, 73.01; H, 5.51; N, 8.51. Found C,
+
+
3.05; H, 5.58; N, 8.48; MS (ESI) [M + H] Calcd for C30
H
27
N
3
O
4
: 493.2. Found: 494.2 [M + H] .

4
.3. 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 (2 mmol) and thiosemicarbazide (3 mmol) in
acetic acid (10 ml) was added 0.5 ml of Conc. HCl and the reaction mixture was refluxed for 12 hrs. 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 ethanol, and dried, affording compounds 6a-i. The compouds 6a-i were crystallized in DMF:MeOH.
4
.3.1. 5-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide (6a). White solid; Yield: 86%; m.p. 258-260
-1
1
°
C; IR (neat, νmax cm ): 3424, 3242, 3089, 1599, 1510, 1339; H NMR (400 MHz, DMSO-d
6
): δ 9.04 (d, J = 1.6 Hz, 1H), 8.60-8.62 (m, 1H),
8
.15-8.23 (m, 3H), 7.96 (s, 1H), 7.85 (d, J = 7.6 Hz, 2H), 7.78 (d, J = 7.2 Hz, 2H), 7.38-7.49 (m, 6H ), 7.27 (t, J = 7.2 Hz, 1H), 6.06 (dd, J = 11.6
1
3
Hz, 4.4 Hz, 1H), 3.96 (dd, J = 18.0 Hz, 11.6 Hz, 1H), C NMR (100 MHz, DMSO-d
6
): 176.24, 152.79, 150.95, 149.37, 148.36, 139.32, 134.35,
S: C, 67.90; H, 4.75; N, 19.80.
1
29.49, 128.59, 127.96, 127.21, 126.20, 125.99, 124.21, 123.76, 118.17, 55.73, 42.04; Anal. Calcd. for C24
H
20
N
6
+
+
Found: C, 67.86; H, 4.68; N, 19.78; MS (ESI) [M + H] Calcd for C24
H
20
N
6
S: 424.2. Found: 425.5 [M + H] .
4
.3.2. 5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide (6b). White solid; Yield: 89%;
-
1
1
m.p. 270-172 °C; IR (neat, νmax cm ): 3263, 3138, 1601, 1508, 1337; H NMR (400 MHz, DMSO-d
H), 8.17-8.22 (m, 3H), 7.95 (s, 1H), 7.25-7.86 (m, 10H), 6.02 (dd, J = 11.6 Hz, 4.0 Hz, 1H), 3.94 (dd, J = 17.6 Hz, 11.6 Hz, 1H); C NMR
100 MHz, DMSO-d ): 176.11, 152.62, 151.03, 148.55, 148.24, 139.33, 134.19, 130.07, 129.51, 127.17, 126.26, 126.16, 124.16, 123.77, 118.10,
6
): 9.03 (d, J = 1.6 Hz, 1H), 8.61-8.62 (m,
1
3
1
(
6

+
1
15.38, 55.96, 42.00; Anal. Calcd. for C24
H19FN
6
S: C, 65.14; H, 4.33; N, 18.99. Found: C, 65.98.05; H, 4.36; N, 18.86; MS (ESI) [M + H]
+
Calcd. for C24
H19FN
6
S: 442.1. Found: 443.1 [M + H] .
4
.3.3. 5-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide (6c). White solid; Yield: 85 %;
-1
1
m.p. 280-282 °C; IR (neat, νmax cm ): 3265, 3129, 1601, 1505, 1330; H NMR (400 MHz, DMSO-d
6
): 9.04 (s, 1H), 8.62 (d, J = 3.6 Hz, 1H),
7
.93-8.23 (m, 3H), 7.85 (d, J = 8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.43-7.48 (m, 3H ), 7.27 (t, J = 7.2 Hz, 1H), 6.03
1
3
(
dd, J = 11.2 Hz, 3.6 Hz, 1H), 3.95 (dd, J = 18.0 Hz, 12.0 Hz, 1H); C NMR (100 MHz, DMSO-d
6
): 175.74, 152.94, 150.77, 148.26, 139.30,
1
34.15, 132.70, 132.11, 129.83, 129.56, 128.64, 127.09, 126.30, 124.19, 123.59, 118.05, 55.48, 41.92; Anal. Calcd. for C24
H
19ClN S: C, 62.81;
6
+
+
H, 4.17; N, 18.31. Found: C, 62.79; H, 4.24; N, 18.28; MS (ESI) [M + H] Calcd. for C24
H19ClN
6
S: 458.1. Found: 459.0 [M + H] .
4
.3.4. 5-(3-(3-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide (6d). White solid; Yield: 88 %;
-1
1
m.p.: 242-244 °C; IR (neat, νmax cm ): 3423, 3235, 3084, 1598, 1508, 1341; H NMR (400 MHz, DMSO-d
6
): 9.04 (d, J = 1.6 Hz, 1H), 8.61-8.62
(
m, 1H), 7.95-8.26 (m, 5H), 7.86 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.42-7.49 (m, 4H), 7.28 (t, J = 7.2 Hz,
1
3
1
1
H), 6.03 (dd, J = 11.6 Hz, 4.0 Hz, 1H), 3.96 (dd, J = 17.6 Hz, 11.6 Hz, 1H); C NMR (100 MHz, DMSO-d ): 176.22, 152.77, 151.00, 148.34,
6
47.81, 139.14, 135.46, 135.00, 130.77, 130.40, 129.52, 127.20, 127.05, 126.45, 124.55, 123.61, 121.96, 118.24, 55.42, 42.05; Anal. Calcd. for
+
C
24
H19BrN
6
S: C, 57.26; H, 3.80; N, 16.69. Found: C, 57.34; H, 3.74; N, 16.75; MS (ESI) [M + H] Calcd. for C24
H
19BrN S: 502.1. Found: 505.1
6
+
(
[M + H] +2).

4
.3.5. 5-(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide (6e). White solid; Yield: 84 %;
-1
1
m.p. 282-284 °C; IR (neat, νmax cm ): 3267, 3131, 1601, 1505, 1330; H NMR (400 MHz, DMSO-d
6
): 9.04 (s, 1H), 8.61-8.62 (d, J = 4.4 Hz,
1
7
1
H), 8.15-8.23 (m, 3H), 7.97 (s, 1H), 7.85 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 7.43-7.49 (m, 3H ), 7.27 (t, J =
1
3
.6 Hz, 1H), 6.03 (dd, J = 10.8 Hz, 3.2 Hz, 1H), 3.95 (dd, J = 18.0 Hz, 11.6 Hz, 1H); C NMR (100 MHz, DMSO-d ): 176.45, 152.66, 150.91,
6
48.23, 139.22, 134.22, 132.32, 131.48, 129.96, 129.48, 127.16, 126.26, 124.23, 123.63, 121.35, 118.10, 55.49, 41.96; Anal. Calcd. for
+
C
24
H19BrN
6
S: C, 57.26; H, 3.80; N, 16.69. Found: C, 57.18; H, 3.74; N, 16.73; MS (ESI) [M + H] Calcd. for C24
H
19BrN S: 502.1. Found: 505.2
6
+
(
[M + H] +2).
4
.3.6. 5-(3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide (6f). Pale yellow solid; Yield: 76
-1
1
%
; m.p.: 272-274 °C; IR (neat, νmax cm ): 3261, 3131, 1600, 1505, 1344; H NMR (400 MHz, DMSO-d ): 9.02 (s, 1H), 8.61-8.62 (m, 2H), 8.36
6
(
s, 1H), 8.17-8.26 (m, 4H), 7.96 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.77 (t, J = 7.6 Hz, 1H), 7.45-7.49 (m, 3H), 7.30 (t, J = 7.2 Hz, 1H), 6.07 (dd, J
1
3
=
11.6 Hz, 4.0 Hz, 1H), 3.96 (dd, J = 17.6 Hz, 11.2 Hz, 1H); C NMR (100 MHz, DMSO-d ): 176.05, 152.61, 150.87, 148.16, 147.96, 147.34,
6
1
39.13, 134.63, 134.37, 134.19, 130.18, 129.54, 127.07, 126.81, 126.56, 124.61, 123.66, 122.72, 122.38, 118.31, 55.33, 41.91; Anal. Calcd. for
+
C
24
H
19
N
7
O
2
S: C, 61.39; H, 4.08; N, 20.88. Found: C, 61.34; H, 4.15; N, 20.91; MS (ESI) [M + H] Calcd. for C24
H
19
N
7
2
O S: 469.1. Found:
+
4
70.1 [M + H] .
4
.3.7. 5-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide (6g). Greenish yellow solid;
-
1
1
Yield: 79 %; m.p.: 292-294 °C; IR (neat, νmax cm ): 3263, 3138, 1600, 1510, 1335; H NMR (400 MHz, DMSO-d ): 9.04 (d, J = 1.6 Hz, 1H),
6

8
.61-8.63 (m, 1H), 8.32 (d, J = 8.0 Hz, 3H), 8.19-8.24 (m, 2H), 8.08 (d, J = 8.8 Hz, 2H), 7.99 (s, 1H), 7.89 (d, J = 8.0 Hz, 2H), 7.45-7.49 (m, 3H
1
3
)
, 7.31 (t, J = 7.2 Hz, 1H), 6.11 (dd, J = 11.2 Hz, 4.0 Hz, 1H), 4.00 (dd, J = 18.4 Hz, 12.0 Hz, 1H); C NMR (100 MHz, DMSO-d
6
): 176.01,
1
52.61, 150.89, 148.22, 146.78, 139.61, 139.11, 134.23, 129.48, 128.91, 127.14, 126.90, 126.68, 125.11, 123.87, 123.65, 118.31, 55.50, 41.88;
+
Anal. Calcd. for C24
H
19
N
7
O
2
S: C, 61.39; H, 4.08; N, 20.88. Found: C, 61.47; H, 4.11; N, 20.76; MS (ESI) [M + H] Calcd. for C24
H
19
N
7
2
O S:
+
4
4
2
8
4
1
69.1. Found: 470.4 [M + H] .
.3.8. 5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1- carbothioamide (6h). White solid; Yield: 86 %; m.p.
-1
1
66-ꢀ68 °C; IR (neat, νmax, cm ): 3263, 3119, 1600, 1506, 1330; H NMR (400 MHz, DMSO-d
6
): 9.03 (d, J = 1.2 Hz, 1H), 8.60-8.62 (m, 1H),
.21 (d, J = 8.0 Hz, 1H), 8.16 (s, 2H), 7.96 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.24-7.48 (m, 6H), 6.05 (dd, J = 11.6 Hz,
1
3
.0 Hz, 1H), 3.94 (dd, J = 18.0 Hz, 11.6 Hz, 1H); 2.37 (s, 3H); C NMR (100 MHz, CDCl ): 176.33, 152.66, 150.85, 149.23, 148.15, 139.31,
3
37.37, 134.27, 130.14, 129.48, 129.15, 127.86, 127.22, 126.13, 125.91, 124.04, 123.62, 118.03, 55.58, 41.98, 20.89; Anal. Calcd for
+
C
25
H
22
N
6
S: C, 68.47; H, 5.06; N, 19.16. Found: C, 68.42; H, 5.08; N, 19.24; MS (ESI) [M + H] Calcd. for C25
H
22
6
N S: 438.2. Found: 439.2 [M
+
+
H] .
4
.3.9. 5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide (6i). White solid; Yield: 81
-
1
1
%
; m.p. 264-ꢀ66 °C; IR (neat, νmax cm ): 3425, 3239, 3045, 1596, 1506, 1335; H NMR (400 MHz, DMSO-d ): 9.03 (d, J = 1.6 Hz, 1H), 8.60-
6
8
.61 (m, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.15 (s, 2H), 7.96 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.41-7.48 (m, 3H ), 7.25 (t, J
1
3
=
7.6 Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H), 6.03 (dd, J = 11.6 Hz, 4.4 Hz, 1H), 3.93 (dd, J = 18.0 Hz, 12.0 Hz, 1H), 3.78 (s, 3H); C NMR (100

MHz, DMSO-d
6
): 176.11, 159.08, 152.63, 150.82, 148.19, 139.35, 134.24, 129.46, 129.24, 127.18, 126.02, 125.86, 123.81, 123.63, 117.97,
+
1
13.98, 55.69, 55.14, 41.98; Anal. Calcd. for C25
Calcd. for C25 OS: 454.2. Found: 455.2 [M + H] .
.4. In vitro cytotoxicity
H
22
N OS: C, 66.06; H, 4.88; N, 18.49. Found: C, 66.13; H, 4.94; N, 18.46; MS (ESI) [M + H]
6
+
H
22
N
6
4
The MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)] assay is based on the conversion of the yellow water-soluble
tetrazolium dye to purple water-insoluble formazan crystals by living cells. The amount of formazan crystals generated is directly proportional to
the number of viable cells. The NCI-H460 (human lung), HeLa (human cervix) and MCF-7 (human breast) and a normal HEK-293T (Human
embryonic kidney) cells were grown as per the standard protocol (37°C, 5% CO
2
in water jacketed incubator shell) using DMEM media with
1
0% FBS (Foetal bovine serum) and seeded onto a single 96 well plate (Falcon Plate, Corning costar, USA) and allowed to adhere for 18 hours
for the assays. The plate was treated with increasing concentration of the compounds to be tested at 1, 10, 25, 50, 100, 150 and 200 µM of the
synthesized compounds dissolved in DMSO. These concentrations were added in triplicates to the 96 well tissue culture plate. After 24 hours of
treatment, the MTT assay was performed to check cell viability. For the MTT assay, the media was removed from all the wells and 10 µl of
MTT reagent (Sigma Aldrich, USA) per well from a working stock of 5 mg/ml was added and the plates were incubated (37°C and 5% CO
-3 h. Then the solution from the plates was removed and the crystals were solubilised using equal volume (100µl) of dimethyl sulfoxide
DMSO) in each well. The DMSO converts the phenol red in the tissue culture medium to a yellow colour that does not interfere with the MTT
2
) for
2
(

formazan measurement. The DMSO dissolves the formazan crystals to give a homogeneous blue solution. The absorbance was measured at a
test wavelength of 570 nm using an ELISA plate reader (Tecan, Switzerland). The percentage inhibition was calculated by the formulae:
Mean OD of treated cells
100
Inhibition =
x 100
%
Mean OD of the vehicle control cells
negative control)
(
Each assay was repeated for three times. IC50 values were calculated from the dose effect curve and expressed as concentration (µM) of drug.
4
.5. Relaxation assay of human topoisomerase IIα
Relaxation of negatively supercoiled plasmid DNA by human topoisomerase IIα was assayed in ꢀ0 μL of reaction buffer (10 mM Tris-HCl, pH
.9, 150 mM NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol and ATP) containing 250 ng of supercoiled pHOT1 plasmid DNA and 1 unit of
7
enzyme. After incubation at 37 °C for 30 min, the reactions were terminated and analysed by agarose gel electrophoresis. The ethidium bromide-
stained gel was photographed over UV light for densitometry analysis. The percentage relaxation was determined by dividing the distance
between the negatively supercoiled band (SC) and the weighted centre of the partially relaxed band (PR) by the distance between the supercoiled
band (SC) and the fully relaxed band (FR) to obtain percent relaxation = (SC−PR)/(SC−FR) [68]. Percent inhibition by different compounds was
then calculated by subtracting percent relaxation in the presence of compounds from 100% relaxation obtained with enzyme only.
4
.6. Molecular Docking Study

The three dimensional (3D) X-ray crystal structures of the ATPase domain of human topoisomerase IIα (PDB ID: 1ZXM; resolution: 1.87 A)
was obtained from Protein Data Bank (PDB) [68]. The topoisomerase IIα (1ZXM) is a homodimer consisting of two identical subunits, each
2
+
contains AMPPNP (50-adenylyl-b,c-imidodiphosphate) and Mg ion at the ATP binding pocket. Before docking, ligand present in the structure,
obtained from PDB, was deleted and the crystal structure was pre-processed by removing water molecules and all non-bonded heteroatoms using
Accelyrs Viewerlite 5.0 and made clean. The energy of the protein molecule was minimized by Steepest Descent and Conjugate Gradient
method using the Accelrys Discovery Studio, one of the most comprehensive suites for molecular modelling and simulation solutions. The
minimization process was carried out using CHARMM force field and the protein was then prepared for docking using Schrödinger’s protein
preparation wizard LigPrep [70]. The protein preparation included the assigning of correct bond orders, addition of hydrogens, creation of
disulphide bonds, conversion of seleno-methionine to methionine and capping of terminal residues. The Glide docking module of Schrödinger
was used to investigate the interactions between the synthesized compounds and human topoisomerase IIa [71, 72]. A grid was generated at the
active site of the prepared protein structure using the Glide docking module of Schrödinger and the size of the grid was 20 cubic Å. The three
dimensional grid was generated with centre around the critical residues of the human topoisomerase IIα, which were composed of residues
namely: Glu-87, Asn-91, Asn-120, Ser-148, Ser-149, Asn-150, Arg-162, Lys-168 and Lys-378. The prepared library of the synthesized
compounds were subjected to the high throughput virtual screening (HTVS) protocol followed by Glide's extra precision (XP) docking protocol
for further docking refinement [73]. The compounds were investigated to study their molecular interactions with the protein molecule and the
hydrophobic interactions and H-bonds were calculated using the Ligplot program. H-bonds were taken into consideration when the distance

between acceptor-donor atoms was less than 3.3 Å and acceptor-H-donor angle greater than 90°. The docking calculations were performed using
the XP (extra precision) mode of Glide. It performs systematic search of conformational, orientation and positional space of docked ligand,
discarding unwanted conformations using scoring followed by energy optimization. The conformations are further refined via Monte Carlo
sampling of pose conformations. All the docking runs were performed using Intel(R) Core™ ꢀ Duo CPU, T5870@ ꢀ.00GHz of hp origin, 1.99
GB of RAM.
4
.7. Physicochemical properties for “druglikeliness”
All the synthesized compounds 5a-i and 6a-i were screened for their drug-likeliness on the basis of the absorption, distribution, metabolism and
excretion (ADME) properties, calculated in silico using Qikprop module of Schrodinger 9.0 [74].
Acknowledgement
The authors thank to the Head, Department of Chemistry, JMI, New Delhi for providing research facilities. We also thank to Dr. Raghibul
1
3
Hussan for helpful discussions. USIC, University of Delhi is acknowledged for C NMR spectral analysis. Raquib Alam is thankful to UGC,
New Delhi for research fellowship and Divya Wahi is thankful to DST, New Delhi for the DST-INSPIRE fellowship.
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