Design, synthesis and biological evaluation of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides as CDK1/Cdc2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.06.011

A series of new (N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives (8–35) were designed, synthesized and evaluated as CDK1/Cdc2 inhibitors. Biological evaluation assays indicated that compounds 16 and 27 showed the most potent growth inhibitory activity against human cancer cell lines (MIAPaCa-2, MCF-7 and HeLa) with GI₅₀values ranging from 0.13 to 0.7?μM, compared with the positive control nocodazole (0.81–0.95?μM). Flow cytometric analysis revealed that these compounds induce cell cycle arrest in the G2/M phase and Western blot analysis suggested that compound treatment resulted in reduction of CDK1 expression levels in MCF-7?cell line. Moreover, the apoptosis inducing effect of the compounds was studied using Hoechst staining, Rhodamine 123 staining (MMP), carboxy-DCFDA staining (ROS), Annexin V-FITC assay. Based on these studies, two compounds 16 and 27 have been identified as promising new molecules that have the potential to be developed as leads.

Full text of DOI:10.1016/j.ejmech.2016.06.011

Accepted Manuscript
Design, synthesis and biological evaluation of N-((1-benzyl-1H-1,2,3-triazol-4-
yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides as CDK1/Cdc2 inhibitors
V. Ganga Reddy, T. Srinivasa Reddy, V. Lakshma Nayak, Budaganaboyina Prasad,
Adiyala Praveen Reddy, A. Ravikumar, Shaik Taj, Ahmed Kamal
PII:
S0223-5234(16)30488-3
10.1016/j.ejmech.2016.06.011
EJMECH 8672
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 March 2016
Revised Date: 5 June 2016
Accepted Date: 8 June 2016
Please cite this article as: V. Ganga Reddy, T. Srinivasa Reddy, V. Lakshma Nayak, B. Prasad,
A.P. Reddy, A. Ravikumar, S. Taj, A. Kamal, Design, synthesis and biological evaluation of N-((1-
benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides as CDK1/Cdc2 inhibitors,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.011.
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Graphical Abstract
Design, synthesis and biological evaluation of N-((1-benzyl-1H-1,2,3-triazol-4-
yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides as CDK1/Cdc2 inhibitors
V. Ganga Reddy, T. Srinivasa Reddy, V. Lakshma Nayak, Budaganaboyina Prasad,
Adiyala Praveen Reddy, A. Ravikumar, Shaik Taj, Ahmed Kamal
R1
N
N
N
O
Cell cycle arrest (G2/M)
CDK1 inihibition
R
NH
N
N
loss of MMP
Compound 16; R = F, R1 = Cl;
GI50 (MCFꢀ7) = 0.13 ꢁM
Compound 27; R = Cl, R1 = 3,5ꢀOMe;
GI50 (MCFꢀ7) = 0.15 ꢁM
ROS

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Design, synthesis and biological evaluation of N-((1-benzyl-1H-1,2,3-
triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides as
CDK1/Cdc2 inhibitors
V. Ganga Reddy,a T. Srinivasa Reddy,a,b V. Lakshma Nayak,a Budaganaboyina Prasad,a Adiyala
Praveen Reddy,a A. Ravikumar,a,c Shaik Taj,a Ahmed Kamal,*a,b,c,d
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India.
bIICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad 500
007, India.
cDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India.
dCatalytic Chemistry Research Chair, Chemistry Department, College of Science, King Saud
University, Riyadh 11451, Saudi Arabia.
Abstract
A series of new (N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-
carboxamide derivatives (8-35) were designed, synthesized and evaluated as CDK1/Cdc2
inhibitors. Biological evaluation assays indicated that compounds 16 and 27 showed the most
potent growth inhibitory activity against human cancer cell lines (MIAPaCa-2, MCF-7 and
HeLa) with GI50 values ranging from 0.13 to 0.7 µM, compared with the positive control
nocodazole (0.81-0.95 µM). Flow cytometric analysis revealed that these compounds induce cell
cycle arrest in the G2/M phase and Western blot analysis suggested that compound treatment
resulted in reduction of CDK1 expression levels in MCF-7 cell line. Moreover, the apoptosis
inducing effect of the compounds was studied using Hoechst staining, Rhodamine 123 staining
(MMP), carboxy-DCFDA staining (ROS), Annexin V-FITC assay. Based on these studies, two
compounds 16 and 27 have been identified as promising new molecules that have the potential to
be developed as leads.
Keywords:
Pyrazole; CDK1 inhibition; anti-cancer activity; cell cycle; apoptosis

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1. Introduction
Cyclin-dependent kinases (CDKs) belong to a family of serine/threonine protein kinases which
play a pivotal role in the regulation of the eukaryotic cell division, control of gene transcription
and other processes of the cell cycle. The loss of cell cycle control provides an abnormal cell
growth which is a major key characteristic point of cancer [1] and the inhibition of CDKs may
provide an effective method for controlling tumor growth in cancer chemotherapy [2, 3], hence
CDKs are known to play a key role in different types of human cancers [4]. In this progression,
CDKs do not participate alone, it is associated with cyclin subunits and its inhibition is
controlled by phosphorylation of the CDK subunits in different phases of cell cycle, such as G1,
S and G2/M [5, 6]. Each cell cycle phase transitions are regulated by different cdk/cyclin
complexes. From the known human CDKs, CDK1 (CDC2) is the most essential for the suitable
execution of cell cycle [7] and cell division [8, 9]. The predominant binding partner for CDK1 is
cyclin B, which activates CDK1 at the G2/M transition. Moreover, several small molecule
inhibitors of anti-CDK drugs are in clinical development as a new generation anticancer
chemotherapeutics [10, 11] and some of the selected CDK1 inhibitors are illustrated in Fig.1.
Heterocyclic moieties play an important role in synthetic organic chemistry due to their
extensive applications in pharmaceutical, veterinary and agrochemical fields [12]. Among them
pyrazole derivatives have gained considerable attention in the history of nitrogen heterocyclic
chemistry due to their wide variety of biological activities such as anticancer, antimicrobial,
antibacterial, antitubercular, antidepressant, antipyretic, antioxidant, analgesic and anti-
inflammatory etc [13-18]. They have taken a leading position in the medicinal chemistry and are
also being used as important pharmacophores in the field of drug design. In additon, triazole is a
unique template that is associated with a number of biological activities and this moiety is an
attractive option to use it as a structural component in the design of new molecules as it is stable
in acidic/basic conditions [19], also has a high dipole moment and affords the possibility of
forming hydrogen bonds with bimolecular targets [20].
Recently, our research group reported many pyrazole derivatives as potent anticancer and
apoptosis inducing agents against human cancer cell lines [21-23]. In continuation to extend our
research, herein we report the synthesis of a series of N-((1-benzyl-1H-1,2,3-triazol-4-
yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides and their biological evaluation. These

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studies indicated that some compounds like 16 and 27 showed potent antiproliferative activity
against some human cancer cell lines. The detailed biological and structure–activity relationship
studies have also been carried out.
< Fig. 1 >
2. Results and Discussion
2.1 Chemistry
The synthetic route for N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-
pyrazole-4-carboxamide analogs (8–35) is outlined in Scheme 1. The required precursors, 1,3-
diphenyl pyrazolocarboxaldehydes (4a-d) were prepared by following the previously reported
procedure [24]. These were oxidized to the corresponding 1,3-diphenyl-1H-pyrazole-4-
carboxylic acids (5a–d) employing sodium chlorite-sulphamic acid. The carboxylic acids (5a-d)
obtained were coupled with the propargylamine in the presence of N-(3-dimethylaminopropyl)-
N¢-ethylcarbodiimide hydrochloride (EDCI), 4-(dimethylamino)pyridine (DMAP) and 1-
hydroxybenzotriazole (HOBT) in dry DMF under nitrogen atmosphere to provide the
corresponding terminal alkynes (6a-d). By using click chemistry protocol, these terminal alkynes
were allowed to react with the benzyl azides (7a-g) in the presence of CuSO4.5H2O and sodium
ascorbate in t-butyl alcohol and water (1:1) at room temperature to afford the triazole derivatives
(8-35) in excellent yields (75-90%). To the best of our knowledge, these compounds are prepared
1
for the first time and all compounds were characterized by spectroscopic studies (IR, H NMR
and MS).
< Scheme 1 >
2.2 Antiproliferative activity
A sulforhodamine B assay was performed to evaluate the cytotoxic effects of these N-((1-
benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides (8–35) against
three tumor cell lines: MIAPaCa-2 (pancreatic), MCF-7 (breast carcinoma) and HeLa (cervical)
using nocodazole as the positive control and the growth inhibition values are listed in Table 1.
These carboxamides (8–35) showed notable antiproliferative effects and most of the compounds
from this series displayed potent broad-spectrum growth inhibitory activities against all the

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tested cell lines. Most of the compounds showed significant growth inhibition (GI50) values
compared to the standard, wherein some of the compounds like 13, 16, 17, 18, 20, 22, 23, 25, 26,
27, 28, 29, 32, 33 and 34 showed promising GI50 values against multiple cancer cell lines and
were comparable to the positive standard. From the Table 1, it is observed that they exhibit
significant antiproliferative activity with GI50 values ranging from 0.13 to ˃100 µM and some of
them showed enhanced activity compared to the positive control (ranging from 0.81 to 0.95 µM).
Among them, 16, 17, 20 and 27 possess remarkable GI50 values, i.e., 16 showed promising
cytotoxic activity with 0.28, 0.13 and 0.21 µM against MIAPaCa-2 (pancreatic), MCF-7 (breast
carcinoma) and HeLa (cervical) cancer cell lines. Similarly 17 exhibited 0.76, 0.81 and 0.25 µM,
whereas 20 showed 0.19, 2.5 and 0.36 µM and 27 showed 0.34, 0.15 and 0.73 µM values against
these human cancer cell lines (MIAPaCa-2, MCF-7 and HeLa), respectively.
< Table 1 >
Based on the antiproliferative activity data, the structure activity relationship (SAR) was
examined for these carboxamides (8–35). In this study, the pyarazole moiety has two aryl ring
systems i.e ring A and B, all the compounds having electron donating methoxy as well as
electron withdrawing groups like chloro, fluoro and trifluoro methyl groups on the ring systems
(A and B) have been investigated. It is demonstrated that compounds 16, 17, 18, 20, 22, 23, 25,
26, 27 and 28, with mono-substituted halogens (fluorine and chlorine) on the A ring, exhibited
more potent activities than compounds with methoxy substitutions, respectively. In case of
constant A ring substituents, and with a change of substituents on B ring the activities were
examined. Among these, electron withdrawing chloro and trifluoromethyl substituents on the B-
ring resulted in moderate to high cytotoxic activity (16 and 17) and whereas 3,5-disubstituted
compound 27 having an electron-donating group showed notable values. The interesting
biological activity exhibited by 16 and 27 prompted us to investigate their effects at the cellular
level and to understand the mechanism responsible for cell death.
2.3 Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by arresting the
cell or by induction of apoptosis or a combined effect of both cycle arrest and apoptosis [25, 26].
Moreover, regulation of the cell cycle and apoptosis are considered to be effective approaches in

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the development of cancer therapeutics [27]. The screening results revealed that compounds 16
and 27 showed significant cytotoxic activity against human breast cancer cell line, MCF-7.
Therefore, it was considered of interest to understand whether this inhibition of cell growth was
on account of cell cycle arrest. In this study MCF-7 cells were treated with these compounds at
50 and 100 nM concentrations for 48 h. nocodazole was used as a reference compound in this
study. The results shown in figure 2 indicated significant increase of DNA content in G2/M
phase and subsequent reduction in G0/G1 phase in comparison to the control cells. Moreover,
increasing the dose resulted in the increase in the amount of cells in G2/M phase, this indicates
that they induce G2/M phase cell cycle arrest in a dose dependent manner.
< Fig. 2 >
2.4 Effect of 16 and 27 on CDK1/Cdc2
The cyclin-dependent kinase (CDK) proteins play a key role in the cell-cycle regulation
in eukaryotic cells [28]. Orderly cell-cycle progression requires CDK activation, which is mainly
controlled by the expression of their activator subunit, cyclin. The complexation of CDK1
(Cdc2) with cyclin B is an important aspect in G2/M phase progression [29, 30]. In this context,
MCF-7 cells were treated with 16 and 27 at different concentrations (50 and 100 nM) for 48 h
and Western blot analysis was performed. Results indicated that there was a significant reduction
of CDK1 (Cdc2) expression level particularly by 27 as shown in Fig.3.
< Fig. 3 >
2.5 Molecular docking study
The promising cytotoxic activity shown by these pyrazolo-4-carboxamides prompted us
to perform molecular docking studies with a view to understand the binding poses on cyclin-
dependent kinase 1 (CDK-1) enzyme. The studies were performed at the LZ9 binding site of the
CDK-1 (PDB ID: 4Y72) [31]. Docking studies revealed that these molecules fit well in the LZ9
binding site of the CDK-1 (Figure 4a).
< Fig. 4 >

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These compounds (16 and 27) exhibit hydrogen bonding with Leu83 residue of the target
protein (red dashed line in Figs 4b, 4c and 4d). The amide proton from these compounds show
hydrogen bonding at the carbonyl group of Leu83. Moreover, the nitrogen present at the third
position of the triazole ring shows additional hydrogen bonding with amide proton of the same
amino acid residue in both the cases.
Besides this, these compounds (16 and 27) show hydrophobic interactions with the target
protein. The 4-chloro and 3,5-dimethoxy benzyl ring of 16 and 27 respectively, show interactions
with Thy15, Val18, Lys33, Val64, Phe80, Ala145 and Asp146. In addition, 27 also interacts with
Asn133 and leu134 due to the presence of methoxy groups. The phenyl ring of 16 and 27 shows
interactions with Gly11, Glu12 and Gln132, apart from the interaction with Asn133 by
compound 16. The 4-F and 4-Cl phenyl ring of 16 and 27 respectively interacts with Ile10,
Phe82, Ser84, Met85 and Lys89 whereas 16 interacts with Lys20. Pyrazole moiety interactions
with Asp86 and Leu135 and whereas the triazole ring interacts with Ala31, Glu81 and Leu83 in
both the cases.
2.6 Apoptotic studies
Apoptosis is an orderly cellular process that takes place in physiological and pathological
conditions, and plays an important role in tissue homeostasis. Disruption or inappropriate
regulation of different apoptotic pathways result in the overgrowth of malignant cells [32, 33].
Thus, it is necessary to consider inducing cellular apoptosis as an effective approach in cancer
treatment. Moreover, previous reports suggested that G2/M cell cycle arrest leads to apoptosis,
thus the apoptotic inducing effect of these compounds on MCF-7 cell line has been studied.
2.6.1 Hoechst 33242 staining
To investigate the morphological changes induced by these compounds in MCF-7 cells, Hoechst
staining has been carried out. Hoechst 33242 is a cell membrane permeable dye, which stains the
live cells nuclei as uniformly light blue and apoptotic cells nuclei as bright blue nuclei on
account of karyopyknosis and chromatin condensation [34]. MCF-7 cells were treated with
various concentrations (50 and 100 nM) of these compounds (16 and 27) for 48 h and stained
with Hoechst 33242. The results from Fig. 5 shows that control cells had no obvious
morphological changes with most cell nuclei appeared as uniformly light blue nuclei. On the

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contrary, the cells treated with 50 nM of 16 and 27 exhibited bright blue fluorescence because of
chromatin condensation which is a typical characteristic of apoptosis. These apoptotic nuclei
significantly increase with increasing the concentration of the compounds to 100 nM and this
observation demonstrates that they induce apoptosis.
< Fig. 5 >
2.6.2 Measurement of mitochondrial membrane potential (MMP)
Previous reports suggest that mitochondria plays an important role in the propagation of
apoptosis and its dysfunction within the apoptotic process, and is often associated with loss of
the mitochondrial inner transmembrane potential [35]. To investigate this possibility, MCF-7
cells were exposed to various concentrations (50 and 100 nM) of 16 and 27 for 48 h and were
analyzed for rhodamine-123 uptake using fluorescence microscope. Uptake of rhodamine-123
into the mitochondria is driven by mitochondrial transmembrane potential. Mitochondria with
normal cells retain the dye and gives strong green fluorescence, whereas apoptotic cells with
depolarized mitochondria cannot retain the dye, which results in reduced green fluorescence. The
results from the figure indicated that, more than 90% of the untreated control cells were
functionally active with high rhodamine-123 green fluorescence (Fig.6). Cells treated with 16
and 27 resulted in reduction of green fluorescence intensity which is significant with further
increase in the concentration of the compounds to 100 nM. Thus, decrease in DΨm by the 16 and
27 indicated that the mitochondrial apoptotic death-signal pathway plays a critical role in
induction of apoptosis in MCF-7 cells.
< Fig. 6 >
2.6.3 Measurement of reactive oxygen species (ROS)
Apoptosis can be induced by oxidative stress which may be mediated by the generation of
reactive oxygen species (ROS). As these compounds are cytotoxic and induce apoptosis in MCF-
7 cells. We then examined the effect of these compounds (16 and 27) on ROS generation using
non-fluorescent carboxy-2,7-dichlorofluorescein diacetate (carboxy-DCFDA) dye which gets
oxidized to the fluorescent dichlorofluorescein (DCF) by a variety of peroxides [36]. In this
assay, MCF-7 cells were treated with different concentrations of both 16 and 27 for 48 h and

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analyzed for DCF fluorescence. As seen from the Figure, there as not much DCF fluorescence
(ROS positive cells) observed in the untreated MCF-7 cells, and this status remained unchanged
when cells were incubated with the 50 nM concentrations of 16 and 27. Whereas, there was a
significant increase of DCF green fluorescence with 100 nM of by these compounds as compared
to the control. Therefore these results demonstrate that compounds are potent inducers of ROS.
< Fig. 7 >
2.6.4 Annexin V-FITC for apoptosis
The apoptotic effect of these compounds (16 and 27) was further evaluated by Annexin V
FITC/PI (AV/PI) dual staining assay [37] to examine the occurrence of phosphatidylserine
externalization and facilitates the detection of live cells (Q1-LL; AV-/PI-), early apoptotic cells (Q1-LR;
AVþ/PI-), late apoptotic cells (Q1-UR-AVþ/ PIþ) and necrotic cells (Q1-UL; AV-/PIþ). In this study,
MCF-7 cells were treated with these compounds for 48 h at 50 and 100 nΜ concentrations to
examine the apoptotic effect. As shown in the figure 8, the percentage of total apoptotic cells
significantly increased after treatment by these compounds. These results confirm a dose dependent
apoptotic inducing effect by these compounds.
< Fig. 8 >
3. Conclusion
In summary, we have designed and synthesized a series of N-((1-benzyl-1H-1,2,3-triazol-
4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides (8-35) and examined their cytotoxicity
on three human cancer cell lines (MIAPaCa-2, MCF-7 and HeLa). The antiproliferative activity
screening results indicated that most of these compounds exhibit potent growth inhibitory
activity (GI50 values ranging from 0.13 to >100 µM). Interestingly, compounds 16 and 27
showed potent cytotoxic efficacy against the MCF-7 human cancer cell line. Treatment of MCF-
7 cells with these compounds resulted in G2/M cell cycle arrest and down regulation of CDK1
expression in MCF-7 cells. Moreover, they induce apoptosis through depolarization of
mitochondrial membrane potential and increased ROS production. Therefore, compounds they
can be considered as interesting lead molecules for further development of more potent
anticancer agents against breast cancer cells.

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4. Experimental Section
All the starting materials and other reagents of the best grade were commercially
available and were used without further purification. TLC was performed on 0.25 mm silica gel
60-F254 plates. Spots were visualized by UV light. All melting points were taken and are
1
uncorrected. H and 13C NMR spectra were recorded on 75, 300 and 500 MHz spectrometers
using tetramethylsilane as the internal standard. Chemical shifts are reported in parts per million
(ppm) downfield from tetramethylsilane. Spin multiplicities are described as s (singlet), brs
(broad singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), or m (multiplet).
Coupling constants are reported in hertz (Hz). HRMS analyses were acquired on single
quadruple and carried out using the ESI techniques at 70 eV. Wherever required column
chromatography was performed using silica gel of 60–120 mm with hexane and ethyl acetate as
eluents.
4.1 General Procedure for the synthesis of 1,3-diphenyl-1H-pyrazole-4-carboxylic acids
(5a-d)
The 1,3-diphenyl-1H-pyrazole-4-carboxylic acids (4a-d) were synthesized based on a literature
method as following: para-substituted acetophenones (1a-d, 20 mmol) interact with phenyl
hydrazine (2, 25 mmol) in anhydrous ethanol to form 1-phenyl-2-(1-phenylethylidene)
hydrazine, which was added to a cold solution of DMF (25 mL) and POCl3 (5 mL), stirred at 50–
60 °C for 5 h. The resulting mixture was poured into ice-cold water, a saturated solution of
sodium bi carbonate was added to neutralize the mixture, the obtained solid (1, 3-diphenyl-1H-
pyrazole-4-carbaldehyde) precipitate was filtered, then after reaction with NaClO2 and sulphamic
acid in acetone to gave carboxylic acid derivatives (5a-d).
4.2 General Procedure for the synthesis of Alkynes (6a-d)
To a solution of 1, 3-diphenyl-1H-pyrazole-4-carboxylic acid (2 mmol) in 10 mL of dry DMF,
EDCI (2.4 mmol) and HOBT (2.4 mmol) were added at 0 °C and the reaction mixture was stirred
for 20 min. To the reaction mixture propargylamine (2 mmol) was added and stirred overnight at
room temperature for 12 h. The contents of the reaction mixture were poured into ice-cold water
(50 mL), extracted with ethyl acetate. The organic layer was washed with saturated NaHCO3 and

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1N HCl, dried over Na2SO4 and concentrated in vacuo. The crude product was purified by
column chromatography by using EtOAc/hexane as eluant.
4.2.1 1,3-diphenyl-N-(prop-2-ynyl)-1H-pyrazole-4-carboxamide (6a).
White solid, yield 74%, Mp: 131-133 °C; 1H NMR (500 MHz, CDCl3):
1H), 4.10 (dd, J = 2.4, 5.3 Hz, 2H), 5.89 (s, 1H), 7.35 (t, J = 7.3 Hz, 1H), 7.46-7.53 (m, 5H), 7.71
(dd, J = 1.5, 8.0 Hz, 2H), 7.75 (d, J = 7.4 Hz, 2H), 8.51 (s, 1H). 13C NMR (75 MHz, CDCl3):
δ 2.18 (t, J = 2.5 Hz,
δ
29.0, 71.5, 79.0, 117.3, 119.3, 127.3, 128.9, 129.1, 129.3, 129.5, 131.1, 131.9, 139.2, 150.9,
162.2. MS (ESI): m/z 302 [M+H]+.
4.2.2 3-(4-fluorophenyl)-1-phenyl-N-(prop-2-ynyl)-1H-pyrazole-4-carboxamide (6b).
White solid, yield 78%, Mp: 165-167 °C; 1H NMR (500 MHz, CDCl3):
δ 2.21 (t, J = 2.5 Hz,
1H), 4.13 (dd, J = 2.4, 5.3 Hz, 2H), 5.81 (s, 1H), 7.20 (t, J = 8.6 Hz, 2H), 7.36 (t, J = 7.4 Hz,
1H), 7.49 (t, J = 7.6 Hz, 2H), 7.76-7.71 (m, 4H), 8.47 (s, 1H). 13C NMR (75 MHz,
CDCl3+DMSO):
δ 28.1, 71.1, 80.0, 114.2 (d, JCF = 21.4 Hz), 116.0, 118.3, 126.5, 128.2, 129.0,
129.5, 130.3 (d, JCF = 7.6 Hz), 138.6, 150.6, 162.1 (d, JCF = 246.9 Hz), 162.1. MS (ESI): m/z 320
[M+H]+. HRMS (ESI) calcd for C19H15ON3F [M+H]+ 320.11937; found: 320.11903.
4.2.3 3-(4-chlorophenyl)-1-phenyl-N-(prop-2-ynyl)-1H-pyrazole-4-carboxamide (6c).
White solid, yield 80%, Mp: 151-153 °C; 1H NMR (500 MHz, CDCl3):
1H), 4.14 (dd, J = 2.4, 5.3 Hz, 2H), 5.82 (s, 1H), 7.36 (t, J = 7.3 Hz, 1H), 7.69 (m, 1H), 7.71 (m,
1H), 7.72-7.75 (m, 2H), 8.46 (s, 1H). 13C NMR (75 MHz, CDCl3+DMSO):
28.0, 71.7, 80.2,
δ 2.23 (t, J = 2.4 Hz,
δ
116.3, 118.3, 126.6, 127.4, 129.1, 129.8, 129.9, 130.7, 133.0, 138.6, 150.1, 161.9. MS (ESI): m/z
336 [M+H]+. HRMS (ESI) calcd for C19H15ON3Cl [M+H]+ 336.08982; found: 336.08940.
4.2.4 3-(4-methoxyphenyl)-1-phenyl-N-(prop-2-ynyl)-1H-pyrazole-4-carboxamide (6d).
White solid, yield 75%, Mp: 176-178 °C; 1H NMR (300 MHz, CDCl3+DMSO):
δ 2.53 (s, 1H),
2.98 (t, J = 2.2 Hz, 1H), 4.05 (dd, J = 2.2, 5.2 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 7.35 (t, J = 7.3
Hz, 1H), 7.53 (t, J = 7.9 Hz, 2H), 7.79-7.89 (m, 4H), 8.55 (t, J = 5.2 Hz, 1H), 8.86 (s, 1H). 13C
NMR (75 MHz, CDCl3+DMSO):
δ 28.0, 54.8, 72.4, 80.6, 113.0, 116.3, 118.3, 124.5, 126.5,

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129.3, 129.6, 129.7, 138.9, 150.8, 159.2, 162.3. MS (ESI): m/z 332 [M+H]+. HRMS (ESI) calcd
for C20H18O2N3 [M+H]+ 332.13935; found: 332.13852.
4.3 General Procedure for the synthesis of Click (8-35)
To a solution of alkynes (6a-d, 2 mmol) and corresponding aryl azides (7a-g, 2 mmol) in a
mixture of t-BuOH/H2O 1 : 1 (10 mL) was added CuSO4·5H2O (2 mmol) and sodium ascorbate
(10 mmol). The reaction mixture was stirred at room temperature overnight. After completion of
the reaction, the mixture was diluted with 50 ml of water and extracted with ethylacetate (2 X 25
ml), the combined organic phase was washed with brine, dried over anhydrous sodium sulfate,
filtered and evaporated in vacuo. The crude product was purified by column chromatography by
using EtOAc/hexane as eluant.
4.3.1
N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-
carboxamide (8).
Light brown solid, yield 79%, Mp: 134-136 °C; 1H NMR (500 MHz, CDCl3):
δ 4.54 (d, J = 5.9
Hz, 2H), 5.45 (s, 2H), 6.33 (t, J = 5.4 Hz, 1H), 7.04-7.08 (m, 2H), 7.23-7.26 (m, 1H), 7.32-7.36
(m, 1H), 7.40 (d, J = 1.6 Hz, 1H), 7.41 (d, J = 1.9 Hz, 2H), 7.45-7.49 (m, 3H), 7.61-7.64 (m,
2H), 7.72-7.75 (m, 2H), 8.47 (s, 1H).13C NMR (75 MHz, CDCl3):
δ 34.8, 53.3, 116.0 (d, JCF =
21.4 Hz), 117.4, 119.2, 122.2, 127.2, 128.7, 129.0, 129.1, 129.4, 129.9 (d, JCF = 8.2 Hz), 130.3
(d, JCF = 1.6 Hz), 130.7, 131.8, 139.1, 151.1, 162.7 (d, JCF = 248.0 Hz), 162.8. MS (ESI): m/z
453 [M+H]+. HRMS (ESI) calcd for C26H21ON6F [M+H]+ 453.18336; found: 453.18322.
4.3.2
N-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-
carboxamide (9).
Light brown solid, yield 76%, Mp: 158-160 °C; 1H NMR (500 MHz, CDCl3):
δ 4.53 (d, J = 5.7
Hz, 2H), 5.42 (s, 2H), 6.54 (t, J = 5.3 Hz, 1H), 7.17 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 2.1 Hz, 2H),
7.33 (s, 1H), 7.38 (d, J = 1.3 Hz, 1H), 7.39 (d, J = 2.1 Hz, 2H), 7.45 (t, J = 8.3 Hz, 2H), 7.49 (s,
1H), 7.62-7.66 (q, J = 2.1, 4.2 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H), 8.46 (s, 1H). 13C NMR (75
MHz, CDCl3):
δ 34.8, 53.3, 111.8, 117.4, 119.2, 122.4, 127.2, 128.7, 129.0, 129.1, 129.2, 129.3,

ACCEPTED MANUSCRIPT
129.4, 130.7, 131.8, 132.9, 134.7, 139.1, 151.0, 162.8. MS (ESI): m/z 469 [M+H]+. HRMS (ESI)
calcd for C26H21ON6Cl [M+H]+ 469.1544; found: 469.15341.
4.3.3
1,3-diphenyl-N-((1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-
pyrazole-4-carboxamide (10).
Light brown solid , yield 81%, Mp: 152-154 °C; 1H NMR (500 MHz, CDCl3):
δ 4.54 (d, J = 5.9
Hz, 2H), 5.52 (s, 2H), 6.53 (t, J = 5.4, 5.6 Hz, 1H), 7.30-7.35 (m, 3H), 7.39 (t, J = 3.3, 3.0 Hz,
3H), 7.45 (t, J = 8.2, 7.6 Hz, 2H), 7.52 (s, 1H), 7.61 (s, 1H), 7.62-7.65 (m, 3H), 7.72 (d, J = 7.9
13
Hz, 2H), 8.47 (s, 1H). C NMR (75 MHz, CDCl3):
δ 34.9, 53.4, 117.4, 119.2, 122.5, 126.0 (q,
JCF3 = 3.8 Hz), 127.3, 128.1, 128.8, 129.0, 129.1, 129.5, 130.7, 131.8, 138.4, 139.1, 145.3, 151.0,
162.8. MS (ESI): m/z 503 [M+H]+. HRMS (ESI) calcd for C27 H21ON6F3 [M+H]+ 503.18017;
found: 503.17995.
4.3.4 N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-
carboxamide (11).
White solid, yield 78%, Mp: 166-168 °C; 1H NMR (300 MHz, CDCl3):
δ 3.80 (s, 3H), 4.53 (d, J
= 5.8 Hz, 2H), 5.41 ( s, 2H), 6.32 (t, J = 4.9, 4.7 Hz, 1H), 6.89 (d, J = 8.6 Hz, 2H), 7.22 (d, J =
8.4 Hz, 2H), 7.32 (d, J = 7.5 Hz, 1H), 7.35-7.42 (m, 4H), 7.47 (t, J = 8.1, 7.5 Hz, 2H), 7.62 (dd, J
13
= 3.9, 1.8 Hz, 2H), 7.73 (d, J = 7.7 Hz, 2H), 8.47 (s, 1H). C NMR (75 MHz, CDCl3):
δ 34.9,
53.6, 55.2, 114.4, 117.5, 119.3, 121.9, 126.4, 127.2, 128.7, 129.0, 129.1, 129.4, 129.6, 130.7,
131.8, 139.2, 144.8, 151.0, 159.8, 162.7. MS (ESI): m/z 465 [M+H]+. HRMS (ESI) calcd for
C27H24O2N6 [M+H]+ 465.20335; found: 465.20293.
4.3.5 N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-
4-carboxamide (12).
White solid, yield 84%, Mp: 140-142 °C; 1H NMR (500 MHz, CDCl3):
δ 3.82 (s, 3H), 3.86 (s,
3H), 4.53 (d, J = 5.7 Hz, 2H), 5.40 (s, 2H), 6.46 (s, 1H), 6.78 (s, 1H), 6.84 (s, 2H), 7.32 (t, J =
7.3 Hz, 1H), 7.38 (s, 3H), 7.42-7.48 (m, 3H), 7.62 (dd, J = 3.8 Hz, 3H), 7.72 (d, J = 7.9 Hz, 2H),
13
8.46 (s, 1H). C NMR (75 MHz, CDCl3):
δ 34.9, 54.4, 55.9, 111.1, 111.2, 117.4, 119.2, 120.8,
122.0, 126.7, 127.2, 128.7, 128.9, 129.1, 129.4, 130.6, 131.8, 139.1, 144.8, 149.3, 149.4, 151.0,

ACCEPTED MANUSCRIPT
162.7. MS (ESI): m/z495 [M+H]+. HRMS (ESI) calcd for C28H26O3N6 [M+H]+ 495.21392;
found: 495.21333.
4.3.6 N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-
4-carboxamide (13).
Light brown solid, yield 76%, Mp: 171-173 °C; 1H NMR (500 MHz, CDCl3):
δ 3.76 (s, 6H),
4.55 (d, J = 5.8 Hz, 2H), 5.40 (s, 2H), 6.30 (t, J = 5.4 Hz, 1H), 6.39 (bs, 2H), 6.43 (t, J = 2.2, 1.8
Hz, 1H),7.34 (t, J = 7.3 Hz, 1H), 7.38-7.42 (m, 3H), 7.43-7.52 (m, 3H), 7.74 (d, J = 7.5 Hz, 2H),
13
8.48 (s, 1H). C NMR (75 MHz, CDCl3):
δ 34.9, 54.1, 55.3, 100.2, 106.1, 117.5, 119.2, 122.2,
127.2, 128.7, 129.0, 129.1, 129.4, 130.7, 131.8, 136.5, 139.2, 144.9, 151.1, 161.2, 162.7. MS
(ESI): m/z 495 [M+H]+. HRMS (ESI) calcd for C28H26O3N6 [M+H]+ 495.21392; found:
495.21236.
4.3.7
1,3-diphenyl-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-
pyrazole-4-carboxamide (14).
Light brown solid, yield 86%, Mp: 153-155 °C; 1H NMR (500 MHz, CDCl3):
δ 3.80 (s, 6H),
3.82 (s, 3H), 4.55 (d, J = 5.7 Hz, 2H), 5.39 (s, 2H), 6.48 (s, 2H), 7.33 (t, J = 7.4 Hz, 1H), 7.37-
7.41 (m, 3H), 7.45 (t, J = 8.2, 7.6 Hz, 2H), 7.52 (s, 1H), 7.63 (d, J = 4.2 Hz, 1H), 7.65 (d, J = 2.1
13
Hz, 1H), 7.73 (d, J = 7.6 Hz, 2H), 8.47 (s, 1H). C NMR (75 MHz, CDCl3):
δ 34.9, 54.4, 56.1,
60.7, 105.2, 117.4, 119.2, 122.2, 127.2, 128.7, 128.9, 129.1, 129.4, 129.8, 130.7, 131.8, 139.1,
145.0, 151.0, 153.6, 162.7. MS (ESI): m/z524 [M+H]+. HRMS (ESI) calcd for C29H28O4N6
[M+H]+ 525.22448; found: 525.22404.
4.3.8 N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-fluorophenyl)-1-phenyl-1H-
pyrazole-4-carboxamide (15).
White solid, yield 80%, Mp: 164–166 °C; 1H NMR (500 MHz, CDCl3):
δ 4.56 (s, 2H), 5.46 (s,
2H), 6.46 (s, 1H), 7.02-7.12 (m, 4H), 7.22-7.28 (m, 3H), 7.34 (t, J =7.3 Hz, 1H), 7.46 ( t, J = 7.6,
7.9 Hz, 3H), 7.66 (t, J = 8.2 Hz, 3H), 7.72 (d, J = 7.9 Hz, 2H), 8.44 (s, 1H). 13C NMR (75 MHz,
CDCl3):
δ 34.9, 53.6, 115.6 (d, JCF = 21.274 Hz), 116.1 (d, JCF = 22.0 Hz), 117.2, 119.2, 127.3,
128.0 (d, JCF = 2.9 Hz), 129.5, 129.9 (d, JCF = 8.0 Hz), 130.1 (d, JCF = 2.9 Hz), 130.4, 130.9 (d,

ACCEPTED MANUSCRIPT
JCF = 8.0 Hz), 139.1, 150.4, 162.8, 162.8 (d, JCF = 248.6 Hz), 163.2 (d, JCF = 248.6 Hz). MS
(ESI): m/z 471 [M+H]+. HRMS (ESI) calcd for C26H20ON6F2 [M+H]+ 471.17394; found:
471.17356.
4.3.9 N-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-fluorophenyl)-1-phenyl-1H-
pyrazole-4-carboxamide (16).
White solid, yield 82%, Mp: 174-176 °C; 1H NMR (500 MHz, CDCl3):
δ 4.56 (s, 2H), 5.46 (s,
2H), 6.56 (s, 1H), 7.10 (t, J = 8.5 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H),
7.46 (t, J = 7.9, 7.6 Hz, 2H), 7.65-7.70 (m, 3H), 7.72 ( d, J = 7.7 Hz, 2H), 8.45 (s, 1H). 13C NMR
(75 MHz, CDCl3):
δ 34.9, 54.1, 115.6, 115.7 (d, JCF = 20.8 Hz), 117.2, 117.3, 119.3, 128.0 (d,
JCF = 2.7 Hz), 128.8, 129.3, 129.4, 129.5, 130.8, 130.9 (d, JCF = 8.1 Hz), 132.6, 134.9, 139.1,
150.4, 162.8, 163.2 (d, JCF = 248.8 Hz). MS (ESI): m/z 487 [M+H]+. HRMS (ESI) calcd for
C26H20ON6ClF [M+H]+ 487.14439; found: 487.14295.
4.3.10
3-(4-fluorophenyl)-1-phenyl-N-((1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-
yl)methyl)-1H-pyrazole-4-carboxamide (17).
Off white solid, yield 77%, Mp: 195-197 °C; 1H NMR (300 MHz, CDCl3):
δ 3.36 (s, 2H), 4.54
(d, J = 5.28, 2H), 5.68 (s, 2H), 7.10 (t, J = 8.6 Hz, 2H), 7.34 (t, J = 7.3, 7.1 Hz, 1H), 7.46-7.54
(m, 4H), 7.65 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 7.9 Hz, 2H), 7.88-7.94 (m, 2H), 8.04 (s,1H), 8.62
13
(t, J = 5.0 Hz, 1H), 8.88 (s, 1H). C NMR (75 MHz, CDCl3):
δ 34.8, 53.3, 55.2, 114.2, 117.1,
119.1, 122.6, 124.0, 126.0 (q, JCF3 = 3.2 Hz), 127.1, 128.1, 129.4, 130.3, 130.6, 134.0, 138.3,
139.1, 145.3, 150.9, 160.2, 162.9. MS (ESI): m/z 521 [M+H]+. HRMS (ESI) calcd for
C27H20ON6F4 [M+H]+ 521.17075; found: 521.17106.
4.3.11 3-(4-fluorophenyl)-N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1-phenyl-
1H-pyrazole-4-carboxamide (18).
Off white solid, yield 84%, Mp: 184-186 °C; 1H NMR (300 MHz, CDCl3):
δ 3.80 (s, 3H), 4.60
(s, 2H), 5.40 (s, 2H), 6.84 (d, J = 8.1 Hz, 2H), 7.06 (t, J = 8.1, 8.3 Hz, 2H), 7.20 (d, J = 8.3 Hz,
13
2H),7.33 (t, J = 7.3 Hz, 1H), 7.46 (t, J = 7.7, 7.3 Hz, 2H), 7.64-7.74 (m, 4H), 8.46 (s, 1H). C
NMR (75 MHz, CDCl3):
δ 34.79, 54.2, 55.2, 114.4, 115.5 (d, JCF = 21.9 Hz), 117.2, 119.24,

ACCEPTED MANUSCRIPT
126.1, 127.2, 128.0, 129.4, 129.6, 130.4, 130.5, 130.9 (d, JCF = 8.2 Hz), 139.1, 150.5, 159.9,
162.7, 163.1 (d, JCF = 248.6 Hz). MS (ESI): m/z 483 [M+H]+. HRMS (ESI) calcd for
C27H23O2N6F [M+H]+ 483.19393; found: 483.19386.
4.3.12
N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-fluorophenyl)-1-
phenyl-1H-pyrazole-4-carboxamide (19).
Off white solid, yield 76%, Mp: 175-177 °C; 1H NMR (500 MHz, CDCl3):
δ 3.82 (s, 3H), 3.86
(s, 3H), 4.55 (d, J = 5.7 Hz, 2H), 5.40 (s, 2H), 6.78 (s, 2H), 6.82-6.85 (m, 2H), 7.06 (t, J = 8.6
Hz, 2H), 7.32 (t, J = 7.3, 7.4 Hz, 1H), 7.44 (t, J = 8.3, 7.4 Hz, 2H), 7.51 (s, 1H), 7.65-7.72 (m
,4H), 8.45 (s, 1H). 13C NMR (75 MHz, CDCl3):
(d, JCF = 21.9 Hz), 117.2, 119.2, 120.8, 122.1, 126.6, 127.2, 127.9, 129.4, 130.3, 130.9 (d, JCF
δ 34.8, 54.0, 55.8, 111.1 (d, JCF = 6.5 Hz), 115.6
=
8.2 Hz), 139.1, 144.8, 149.3, 149.4, 150.4, 162.7, 163.1 (d, JCF = 249.1 Hz); MS (ESI): m/z 513
[M+H]+. HRMS (ESI) calcd for C28H25O3N6F [M+H]+ 513.20449; found: 513.20435.
4.3.13
N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-fluorophenyl)-1-
phenyl-1H-pyrazole-4-carboxamide (20).
Off white solid, yield 88%, Mp: 181-183 °C; 1H NMR (300 MHz, CDCl3):
δ 3.74 (s, 6H), 4.56
(bs, 2H), 5.38 (s, 2H), 6.37 (s, 2H), 6.42 (s, 1H), 6.90 (s, 1H), 7.06 (t, J = 8.6, 8.4 Hz, 2H), 7.32
(d, J = 7.3 Hz, 1H), 7.43 (t, J = 7.9, 7.5 Hz, 2H), 7.64-7.74 (m ,4H), 8.47 (s, 1H). 13C NMR (75
MHz, CDCl3): δ 34.8, 54.2, 55.3, 100.2, 106.1, 115.5 (d, JCF = 21.9 Hz), 117.1, 119.2, 127.2,
128.0 (d, JCF = 2.7 Hz), 129.4, 130.2, 130.8 (d, JCF = 8.2 Hz), 136.3, 139.1, 150.5, 161.2, 162.8,
163.1 (d, JCF = 248.6 Hz); MS (ESI): m/z 513 [M+H]+. HRMS (ESI) calcd for C28H2O3N6F
[M+H]+ 513.20449; found: 513.20364.
4.3.14
3-(4-fluorophenyl)-1-phenyl-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)-1H-pyrazole-4-carboxamide (21).
Off white solid, yield 76%, Mp: 189-191 °C; 1H NMR (500 MHz, CDCl3):
δ 3.80(s, 6H), 3.82 (s,
3H), 4.57 (d, J = 5.7 Hz, 2H), 5.39 (s, 2H), 6.49 (s, 2H), 6.72 (t, J = 5.6 Hz, 1H), 7.08 (t, J = 8.6,
8.8 Hz, 2H), 7.33 (t, J = 7.4, 7.3 Hz, 1H), 7.45 (t, J = 8.3, 7.6 Hz, 2H), 7.55 (s, 1H), 7.65-7.77
13
(m, 1H), 7.72 (dd, J = 7.6, 1.2 Hz, 2H), 8.45 (s, 1H). C NMR (75 MHz, CDCl3):
δ 34.8, 54.4,

ACCEPTED MANUSCRIPT
56.1, 60.7, 105.2, 115.6 (d, JCF = 21.4 Hz), 117.1, 119.2, 122.3, 127.3, 127.9 (d, JCF = 2.7 Hz),
129.4, 129.7, 130.2, 130.9 (d, JCF = 8.7 Hz), 138.2, 139.1, 144.9, 150.4, 153.6, 162.8, 163.1 (d,
JCF = 248.6 Hz); MS (ESI): m/z 543 [M+H]+. HRMS (ESI) calcd for C29H27O4N6F [M+H]+
543.21506; found: 543.21488.
4.3.15
3-(4-chlorophenyl)-N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1-phenyl-
1H-pyrazole-4-carboxamide (22).
Off white solid, yield 82%, Mp: 183-185 °C; 1H NMR (500 MHz, CDCl3):
δ 4.58 (s, 2H), 5.42
(s, 2H), 6.70 (s, 1H), 7.06 (t, J = 8.3 Hz, 2H), 7.20 (d, J = 4.7 Hz, 1H), 7.36 (t, J = 9.1, 8.2 Hz,
3H), 7.42 (t, J = 7.6 Hz, 2H), 7.65 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 7.7 Hz, 2H), 8.44 (s, 1H). 13C
NMR (75 MHz, CDCl3):
δ 35.0, 53.8, 116.1 (d, JCF = 21.9 Hz), 116.5, 117.2, 119.2, 127.3,
128.6, 129.4, 129.7, 130.0 (d, JCF = 8.2 Hz), 130.2, 130.4, 131.5, 134.8, 139.0, 150.3, 162.8,
162.9 (d, JCF = 248.6 Hz); MS (ESI): m/z 487 [M+H]+. HRMS (ESI) calcd for C26H20ON6ClF
[M+H]+ 487.14439; found: 487.14549.
4.3.16
N-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-chlorophenyl)-1-phenyl-
1H-pyrazole-4-carboxamide (23).
Off white solid, yield 80%, Mp: 168-170 °C; 1H NMR (500 MHz, CDCl3):
δ 4.57 ( d, J = 5.7
Hz, 2H), 5.46 (s, 2H), 6.38-6.58 (m, 1H) 7.20(d, J = 8.3 Hz, 2H), 7.33-7.40 (m, 5H), 7.45-7.50
13
(m, 3H), 7.64 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H), 8.42 (s, 1H). C NMR (75 MHz,
CDCl3):
δ 34.8, 53.6, 115.7, 117.1, 119.2, 120.7, 127.3, 127.9, 128.0, 128.6, 129.2, 129.4, 130.4,
130.9, 132.7, 134.8, 139.0, 150.5, 163.8; MS (ESI): m/z 503 [M+H]+. HRMS (ESI) calcd for
C26H20ON6Cl2 [M+H]+ 503.11484; found: 503.11666.
4.3.17
3-(4-chlorophenyl)-1-phenyl-N-((1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-
yl)methyl)-1H-pyrazole-4-carboxamide (24).
Off white solid, yield 82%, Mp: 173-175 °C; 1H NMR (500 MHz, CDCl3):
δ 4.58 ( d, J = 5.7 Hz,
2H), 5.55 (s, 2H), 6.52 (t, J = 5.6, 5.4 Hz, 1H), 7.26 (t, J = 8.5, 7.4 Hz, 2H), 7.32-7.36 (m, 2H),
7.37 (s, 2H), 7.39 (s, 1H), 7.54 (s, 1H), 7.64 (t, J = 8.3, 8.5 Hz, 4H), 7.72 (d, J = 7.4 Hz, 2H),
8.42 (s, 1H). 13C NMR (75 MHz, CDCl3):
δ 34.8, 53.4, 117.1, 119.1, 122.7, 126.0 (q, JCF3 = 3.2

ACCEPTED MANUSCRIPT
Hz), 127.3, 128.1, 128.6, 129.4, 130.2, 130.4, 130.7, 131.2, 134.8, 138.2, 139.0, 145.3, 150.3,
162.9; MS (ESI): m/z 537 [M+H]+. HRMS (ESI) calcd for C27H20ON6ClF3 [M+H]+ 537.14120;
found: 537.14268.
4.3.18 3-(4-chlorophenyl)-N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1-phenyl-
1H-pyrazole-4-carboxamide (25).
Off white solid, yield 84%, Mp: 151-153 °C; 1H NMR (500 MHz, CDCl3):
δ 3.80 (s, 3H), 4.55
(d, J = 5.7 Hz, 2H), 5.42 (s, 2H), 6.55 (m, 1H), 6.88 (d, J = 8.5 Hz, 2H), 7.33 (m, 3H), 7.44 (m,
3H), 7.62 (d, J = 8.5 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 8.46 (s, 1H). 13C NMR (75 MHz, CDCl3):
δ
34.8, 53.7, 55.2, 114.4, 117.2, 119.2, 122.2, 126.2, 127.2, 128.6, 129.4, 129.6, 130.2, 130.4,
134.8, 139.1, 144.8, 150.2, 159.8, 162.8; MS (ESI): m/z 499 [M+H]+. HRMS (ESI) calcd for
C27H23O2N6Cl [M+H]+ 499.16438; found: 499.16414.
4.3.19
3-(4-chlorophenyl)-N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1-
phenyl-1H-pyrazole-4-carboxamide (26).
Off white solid, yield 77%, Mp: 155-157 °C; 1H NMR (300 MHz, CDCl3):
δ 3.80 (s, 3H), 3.82
(s, 3H), 4.58 (s, 2H), 5.40 (s, 2H), 6.78 (s, 1H), 6.84 (s, 2H), 7.00 (s, 1H), 7.28- 7.38 (m, 3H),
7.44 (t, J = 7.3, 7.5 Hz, 2H), 7.68 (q, J = 7.7, 8.1 Hz, 5H), 8.46 (s, 1H). 13C NMR (75 MHz,
CDCl3):
δ 34.7, 54.1, 55.8, 111.0, 111.1, 117.1, 119.1, 120.8, 126.5, 127.2, 128.5, 129.4, 130.2,
130.4, 134.7, 139.0, 149.2, 149.3, 150.4, 162.8; MS (ESI): m/z 529 [M+H]+. HRMS (ESI) calcd
for C28H25O3N6Cl [M+H]+ 529.17494; found: 529.17610.
4.3.20
3-(4-chlorophenyl)-N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1-
phenyl-1H-pyrazole-4-carboxamide (27).
Off white solid, yield 84%, Mp: 186-188 °C; 1H NMR (500 MHz, CDCl3):
δ 3.74 (s, 6H), 4.58
(s, 2H), 5.40 (s, 2H), 6.38 (s, 2H), 6.40 (s, 1H), 6.60 (s, 1H), 7.30-7.40 (m, 3H), 7.45 (t, J = 3.2,
7.6 Hz, 2H), 7.65 (d, J = 7.7 Hz, 2H), 7.72 (d, J = 7.6 Hz, 2H), 8.44 (s, 1H).13C NMR (75 MHz,
CDCl3+DMSO):
δ 33.1, 51.6, 53.5, 98.0, 104.4, 115.4, 117.0, 125.3, 126.1, 127.8, 128.5, 128.6,
129.6, 131.8, 136.0, 137.4, 148.8, 159.2, 160.9. MS (ESI): m/z 529 [M+H]+. HRMS (ESI) calcd
for C28H25O3N6Cl [M+H]+ 529.17494; found: 529.17604.

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4.3.21
3-(4-chlorophenyl)-1-phenyl-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)-1H-pyrazole-4-carboxamide (28).
Off white solid, yield 89%, Mp: 149-151 °C; 1H NMR (500 MHz, CDCl3):
δ 3.80 (s, 9H), 4.58
(d, J = 5.6 Hz, 2H), 5.40 (s, 2H), 6.50 (s, 2H), 6.56 (m, 1H), 7.32-7.40 (m, 3H), 7.46 (t, J = 7.1,
8.3 Hz, 2H), 7.54 (s, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.72 (d, J= 8.3 Hz, 2H), 8.43 (s, 1H). 13C
NMR (75 MHz, CDCl3):
δ 30.8, 34.8, 54.4, 56.0, 60.7, 105.2, 117.1, 119.2, 122.4, 127.3, 128.6,
129.4, 129.7, 130.1, 130.2, 130.3, 134.8, 139.0, 144.9, 150.3, 153.5, 162.8; MS (ESI): m/z 559
[M+H]+. HRMS (ESI) calcd for C29H27O4N6Cl [M+H]+ 559.18551; found: 559.18421.
4.3.22 N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)-1-phenyl-
1H-pyrazole-4-carboxamide (29).
White solid, yield 79%, Mp: 168-170 °C; 1H NMR (500 MHz, CDCl3):
δ 3.85 (s, 3H), 4.53 (d, J
= 5.7 Hz, 2H), 5.44 (s, 2H), 6.51 (t, J = 5.3 Hz, 1H), 6.93 (d, J = 7.3 Hz, 2H), 7.05 (t, J = 7.3, 8.3
Hz, 2H), 7.22-7.27 (m, 2H), 7.32 (t, J = 7.0, 7.7 Hz, 1H), 7.45 (t, J = 7.1 Hz, 2H), 7.50 (s, 1H),
7.56 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.2 Hz, 2H), 8.45 (s, 1H).13C NMR (75 MHz, CDCl3):
δ
34.8, 53.3, 55.3, 114.2, 116.0 (d, JCF = 21.7 Hz), 117.2, 119.2, 122.2, 124.0, 127.1, 129.4, 129.9
(d, JCF = 8.1 Hz), 130.3 (d, JCF = 2.7 Hz), 130.6, 139.2, 145.1, 150.9, 160.2, 162.7 (d, JCF = 247.9
Hz), 162.9 ; MS (ESI): m/z 483 [M+H]+. HRMS (ESI) calcd for C27H23O2N6F [M+H]+
483.19393; found: 483.19457.
4.3.23 N-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)-1-phenyl-
1H-pyrazole-4-carboxamide (30).
Off white solid, yield 82%, Mp: 160-162 °C; 1H NMR (300 MHz, CDCl3):
δ 3.82 (s, 3H), 4.52
(d, J = 5.8 Hz, 2H), 5.42 (s, 2H), 6.62 (t, J = 5.6, 5.4 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 7.17 (d, J
= 8.3 Hz, 2H), 7.32 (d, J = 8.1 Hz, 3H) 7.44 (t, J = 8.1, 7.5 Hz, 2H) 7.50 (s, 1H), 7.57 (d, J = 8.8
Hz, 2H), 7.70 (d, J = 7.9 Hz, 2H), 8.45 (s, 1H).13C NMR (75 MHz, CDCl3):
δ 34.8, 53.3, 55.3,
114.2, 117.2, 119.2, 122.3, 124.0, 127.1, 129.2, 129.3, 129.4, 130.3, 130.5, 132.9, 134.75, 139.2,
145.2, 150.9, 160.2, 162.9; MS (ESI): m/z 499 [M+H]+.HRMS (ESI) calcd for C27H23O2N6Cl
[M+H]+499.16438; found: 499.16555.

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4.3.24 3-(4-methoxyphenyl)-1-phenyl-N-((1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-
yl)methyl)-1H-pyrazole-4-carboxamide (31).
Off white solid, yield 80%, Mp: 145-147 °C; 1H NMR (500 MHz, CDCl3):
δ 3.84 (s, 3H), 4.55
(d, J = 5.7 Hz, 2H), 5.53 (s, 2H), 6.55 (s, 1H), 6.94 (d, J = 8.6 Hz, 2H), 7.30-7.36 (m, 3H), 7.45
(t, J = 7.6, 8.2 Hz, 2H), 7.54 (s, 1H), 7.56 (s, 1H), 7.58 (s, 1H), 7.62 (d, J = 7.9 Hz, 2H), 7.72 (d,
J = 8.3 Hz, 2H), 8.45 (s, 1H). 13C NMR (75 MHz, CDCl3):
δ 34.8, 53.3, 55.2, 114.2, 117.1,
119.1, 122.6, 124.0, 126.0 (q, JCF3 = 3.2 Hz), 127.1, 128.1, 129.4, 130.3, 130.6, 138.3, 139.1,
145.3, 150.9, 160.2, 162.9; MS (ESI): m/z 533 [M+H]+. HRMS (ESI) calcd for C28H23O2N6F3
[M+H]+ 533.19074; found: 533.19158.
4.3.25
N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazole-4-carboxamide (32).
Off white solid, yield 78%, Mp: 170-172 °C; 1H NMR (300 MHz, CDCl3):
δ 3.73 (s, 6H), 3.83
(s, 3H), 4.55 (bs, 2H), 5.38 (s, 2H), 6.35- 6.42 (m, 3H), 6.63 (s, 1H), 6.93 (d, J = 8.4 Hz, 2H),
7.30 (t, J = 7.3 Hz, 1H), 7.44 (t, J = 7.5, 7.9 Hz, 2H), 7.57 (d, J = 8.4 Hz, 3H), 7.70 (d, J = 7.7
Hz, 2H), 8.46 (s, 1H). 13C NMR (75 MHz, CDCl3):
δ 34.8, 53.7, 55.2, 55.3, 114.2, 114.4, 117.3,
119.2, 122.0, 124.0, 126.4, 127.1, 129.4, 129.6, 130.3, 130.6, 139.2, 150.9, 159.8, 160.2, 162.8.
MS (ESI): m/z 495 [M+H]+. HRMS (ESI) calcd for C28H26O3N6 [M+H]+ 495.21392; found:
495.21245.
4.3.26 N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazole-4-carboxamide (33).
White solid, yield 75%, Mp: 143-145 °C; 1H NMR (500 MHz, CDCl3):
δ 3.81 (s, 3H), 3.84 (s,
3H), 3.86 (s, 3H), 4.53 (d, J = 5.7 Hz, 2H), 5.40 (s, 2H), 6.55 (t, J = 5.3 Hz, 1H), 6.78 (s, 1H),
6.84 (s, 2H), 6.93 (d, J = 8.6 Hz, 2H), 7.32 (t, J = 7.4, 7.3 Hz, 1H), 7.45 (t, J = 8.0, 7.6 Hz, 2H),
7.49 (s, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 7.6 Hz, 2H), 8.45 (s, 1H). 13C NMR (75 MHz,
CDCl3):
δ 34.8, 54.0, 55.2, 55.8, 111.0, 111.1, 114.1, 117.1, 119.1, 120.7, 122.1, 124.0, 126.7,
127.1, 129.4, 130.3, 130.5, 139.1, 144.9, 149.3, 150.9, 160.2, 162.9; MS (ESI): m/z 525 [M+H]+.
HRMS (ESI) calcd for C29H28O4N6 [M+H]+ 525.22448; found: 525.22504.

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4.3.27 N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazole-4-carboxamide (34).
White solid, yield 81%, Mp: 165-167 °C; 1H NMR (500 MHz, CDCl3):
δ 3.75 (s, 6H), 3.86 (s,
3H), 4.55 (d, J = 5.0 Hz, 2H), 5.40 (S, 2H), 6.33-6.43 (m, 4H), 6.95 (d, J = 8.5 Hz, 2H), 7.33 (t, J
= 7.3 Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.49 (s, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 7.7 Hz,
2H), 8.45 (s, 1H). 13C NMR (75 MHz, CDCl3):
δ 34.9, 54.2, 55.3, 100.3, 106.1, 114.3, 117.4,
119.3, 124.0, 127.2, 129.5, 130.4, 130.7, 136.5, 139.2, 150.9, 160.3, 161.2. MS (ESI): m/z 525
[M+H]+. HRMS (ESI) calcd for C29H28O4N6 [M+H]+ 525.22448; found: 525.22506.
4.3.28
3-(4-methoxyphenyl)-1-phenyl-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)-1H-pyrazole-4-carboxamide (35).
White solid, yield 76%, Mp: 153-155 °C; 1H NMR (300 MHz, CDCl3):
δ 3.80 (s, 6H), 3.82 (s,
3H), 3.84 (s, 3H), 4.54 (d, J = 5.8 Hz, 2H), 5.38 (s, 2H), 6.48 (s, 2H), 6.60-6.66 (m, 1H), 6.93 (d,
J = 7.5 Hz, 2H), 7.32 (d, J = 7.3 Hz, 1H), 7.44 (t, J = 6.9 Hz, 2H), 7.55 (d, J = 4.3 Hz, 2H), 7.58
13
(s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 8.45 (s, 1H). C NMR (75 MHz, CDCl3):
δ 34.8, 54.3, 55.2,
56.1, 60.7, 105.2, 114.2, 117.1, 119.1, 122.2, 124.0, 129.4, 129.8, 130.3, 130.5, 138.2, 139.1,
145.0, 150.9, 153.5, 160.2, 162.9; MS (ESI): m/z 555 [M+H]+. HRMS (ESI) calcd for
C30H30O5N6 [M+H]+ 555.23504; found: 555.23647.
4.4 Biology
4.4.1 Cell culture
The cell lines, MIA PaCa-2 (pancreatic), MCF-7 (breast), HeLa (cervical) which were used in
this study were procured from American Type Culture Collection (ATCC), United States and
were maintained in the Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine
serum (FBS; HyClone, Logan, UT) supplemented with 100 units/mL of penicillin and
streptomycin (Sigma-Aldrich, St. Louis, MO). All cells were cultured at 37 ºC in a humidified
atmosphere with 5% CO2. Test compounds were dissolved in DMSO (Sigma-Aldrich) to prepare
10 mM stock solution for the following experiments. The stock was diluted with culture medium
to desired concentrations for drug treatment.

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4.4.2 Evaluation of anti-proliferative activity
The anti-proliferative activity of the synthesized pyrazole derivatives was evaluated using SRB
assay. A protocol of 48 h continuous drug exposure was used to estimate cell viability or growth.
MiaPaca-2, MCF-7 and HeLa cells were seeded in 96-well plates in 100 ꢀL aliquots at plating
densities depending on the doubling time of individual cell lines. The microtiter plates were
incubated at 37 °C, 5% CO2, 95% air, and 100% relative humidity for 24 h and then cells were
treated with different doses (0.01, 0.1, 1, 10, 100 µM) of synthesized compounds. After 48 hours
incubation at 37 °C, cells were fixed with 10% (wt/vol) cold trichloroacetic acid and incubated at
4 °C for 1h and were then stained with 0.057% SRB dissolved in 1% acetic acid for 30 min at
room temperature. Unbound SRB was washed with 1% acetic acid. The protein–bound dye was
dissolved in 10mM Tris base solution for OD determination at 510 nm using a micro plate reader
(Enspire, Perkin Elmer, USA). Using the seven absorbance measurements [time zero, (Tz),
control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)],
the percentage growth was calculated at each of the drug concentrations levels. Percentage
growth inhibition was calculated as: [(Ti-Tz)/(C-Tz)] x 100 for concentrations for which
Ti>/=Tz , [(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz. Growth inhibition of 50 %
(GI50) was calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration resulting
in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells
during the drug incubation.
4.4.3 Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells through
the cell-cycle phases. MCF-7 cells, breast cancer cells were incubated for 48 h with compounds
16 and 27 at concentrations of 50 and 100 nM. Untreated and treated cells were harvested,
washed with phosphate-buffered saline (PBS), fixed in ice-cold 70% ethanol, and stained with
propidium iodide (Sigma–Aldrich). Cell-cycle analysis was performed by flow cytometry
(Becton Dickinson FACS Caliber instrument) [38].

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4.4.4 Western blot analysis
After treatment with test compounds 16 and 27 at 50, 100 nM concentrations for 48 h. Protein
was isolated with RIPA (radioimmunoprecipitation assay) buffer. Protein (50 µg per lane)was
applied in 10 % SDS-PAGE (Sodium dodecyl sulfate Polyacrylamide gel electrophoresis). After
electrophoresis, the protein was transferred to a polyvinylidine difluoride (PVDF) membrane
(Thermo Scientific Inc.) and blocked with BSA (bovine serum albumin).The membrane was
washed with TBST for 5 min, then primary antibody was added. After 24 h, the membrane was
incubated with the corresponding horseradish peroxidase labeled secondary antibody and
incubated for another 1 h. Membranes were washed with TBST three times for 15 min and the
protein blots were visualized with chemiluminescence reagent (Thermo ScientificInc.). Images
were captured by using the chemiluminescence (vilberlourmat) [39].
4.4.5 Molecular Docking
The co-ordinates of the crystal structure were obtained from RCSB-Protein Data Bank. Required
corrections to the protein were carried out by using Protein Preparation Wizard from Schrödinger
package. Molecules were built using ChemBio3D Ultra 12.0 and geometries were optimized
using molecular mechanics. All docking studies were performed by using Auto Dock 4.2
docking software [40] and the results were visualized through PyMOL [41].
4.4.6 Hoechst staining
The MCF-7 cells were seeded in 24-well plates at the density of 1X105 cells per ml. After 24h,
cells were incubated with test compounds 16 and 27 for 24 h. Cells treated with vehicle (DMSO)
were included as controls for all experiment. After washing with PBS, cells were fixed with 4%
para formaldehyde (PFA) in PBS for 10 min at 4 ºC and stained with Hoechst 33242 (life
technologies) (2 µg/mL) for 10 min at room temp. Cells were washed twice with PBS to remove
the excess dye. Cells from each well were captured from randomly selected fields under
fluorescence microscope (Nikon).

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4.4.7 Measurement mitochondrial membrane potential
The MCF-7 cells were plated at 1X105 cells/mL in 24-well culture plates and allowed to adhere
overnight. Cells were treated with GI50 concentrations of the compounds 16 and 27 for 24 h.
After washing twice with PBS, cells were incubated with Rhodamine 123 (sigma Aldrich) (5
µg/ml) for 30 min in the dark at 37 ºC and cells were washed twice with PBS to remove the
excess dye. The decrease in intensity of fluorescence because of mitochondrial membrane
potential loss was analyzed using fluorescence microscope (Nikon).
4.4.8 Measurement of reactive oxygen species level
The MCF-7 cells were plated at 1X105 cells /mL in 24-well culture plates and allowed to adhere
for overnight. Cells were treated with compounds 16 and 27 for 24 h, washed with PBS and
fixed with 4 % para formaldehyde. Cells were exposed with Carboxy-DCFDA dye (life
technologies) (10 µM/mL) for 30 min in the dark at room temp and washed with PBS to remove
the excess dye. The increase in intensity of fluorescence because of generation of reactive
oxygen species was analyzed using fluorescence microscope (Nikon).
4.4.9 Annexin staining assay for apoptosis
MCF-7 (1×106) cells were seeded in six-well plates and allowed to grow overnight. The medium
was then replaced with complete medium containing compounds 16 and 27 at concentrations of
50 and 100 nM concentrations. After 48 h of drug treatment, cells from the supernatant and
adherent monolayer cells were harvested by trypsinization, washed with PBS at 5000 rpm. Then
the cells were stained with Annexin V/FITC and propidium iodide using the Annexin-V-FITC
apoptosis detection kit (Sigma aldrich).Flow cytometry was performed for this study as
described earlier [42].
Acknowledgement
The authors V.G.R, B.P. and A.P.R. acknowledge the Council of Scientific and Industrial
Research (CSIR), New Delhi (India) for the financial support under the 12th Five Year Plan
project “Affordable Cancer Therapeutics (ACT)” (CSC0301). This project was also supported by
King Saud University, Deanship of Scientific Research, Research Chair.

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References
[1] M. Hall, G. Peters, Genetic alterations of cyclins, cyclin dependent kinases and Cdk
inhibitors in human cancer, AdV. Cancer Res. 68 (1996) 67–108.
[2] S. van den Heuvel, Ed Harlow, Distinct roles for cyclin-dependent kinases in cell cycle
control, Science. 262 (1993) 2050–2054.
[3] M. Malumbres, M. Barbacid, Milestones in cell division: To cycle or not to cycle: a
critical decision in cancer, Nat. ReV. Cancer. 1 (2001) 222–231.
[4] J. Cicenas, M. Valius, The CDK inhibitors in cancer research and therapy, J. Cancer Res.
Clin. Oncol. 137 (2011) 1409–1418.
[5] D.O. Morgan, Cyclin-dependent kinases: engines, clocks, and microprocessors,
Annu.ReV. Cell DeV. Biol. 13 (1997) 261-291.
[6] Sherr CJ, Cancer cell cycles, Science. 274 (1996) 1672-1677.
[7] M. Malumbres, M. Barbacid, Cell cycle, CDKs and cancer: a changing paradigm, Nat.
Rev. 9 (2009) 153–166.
[8] Th’ng JP. et al. The FT210 cell line is a mouse G2 phase mutant with a temperature
sensitive CDC2 gene product, Cell. 63 (1990) 313–324.
[9] J.E. Itzhaki, C.S. Gilbert, A.C. Porter, Construction by gene targeting in human cells of a
‘conditional’ CDC2 mutant that rereplicates its DNA, Nat. Genet. 15 (1997) 258–265.
[10] P. M. Fischer, A. Gianella-Borradori, Recent progress in the discovery and development
of CDK inhibitors, Expert Opin.InVest. Drugs. 14 (2005) 457-477.
[11] C. Benson, S. Kaye, P. Workman, M. Garrett, M. Walton, J. de Bono, Clinical anti
cancer drug development: targeting the cyclin dependent kinases, Br. J. Cancer. 92
(2005)7-12.
[12] A. Czarnik, GuestꢁEditorial, Acc. Chem. Res. 29 (1996) 112-113.

ACCEPTED MANUSCRIPT
[13] G. Traquandi, M. Ciomei, D. Ballinari, E. Casale, N. Colombo, V. Croci, F. Fiorentini,
A. Isacchi, A. Longo, C. Mercurio, A. Panzeri, W. Pastori, P. Pevarello, D. Volpi, P.
Roussel, A. Vulpetti and M.G. Brasca, Identification of potent pyrazolo[4,3-
h]quinazoline-3-carboxamides as multi-cyclin-dependent kinase inhibitors, J. Med.
Chem. 53 (2010) 2171–2187.
[14] A.M. Vijesha, A.M. Isloor, S. Telkar, S.K. Peethambar, S. Rai, N. Isloor, Synthesis,
characterization and antimicrobial studies of some new pyrazole incorporated imidazole
derivatives, Eur. J. Med. Chem. 46 (2011) 3531-3536.
[15] Chang-Ji Zheng, Li-Li Xu, Liang-Peng Sun, Jing Miao, Hu-Ri Piao, Synthesis and
antibacterial activity of novel 1,3-diphenyl-1H-pyrazoles functionalized with
phenylalanine-derived rhodanines, Eur. J. Med. Chem. 58 (2012) 112-116.
[16] R.B. Pathaka, P.T. Chovatia, H.H. Parekh, Synthesis, antitubercular and antimicrobial
evaluation of 3-(4-chlorophenyl)-4-substituted pyrazole derivatives, Bioorganic &
Medicinal Chemistry Letters. 22 (2012) 5129–5133.
[17] D. Dias, B.S. Pacheco, W. Cunico, L. Pizzuti, C.M. Pereira, Recent advances on the
green synthesis and antioxidant activities of pyrazoles, Mini Rev Med Chem. 14 (2015)
1078-1092.
[18] P.D. Sauzem, P. Machado, M. A. Rubin, G.S. Sant’Anna, H.B. Faber, A.H. de Souza,
C.F. Mello, P. Beck, R.A. Burrow, H.G. Bonacorso, N. Zanatta, M.A.P. Martins,
Design and microwave-assisted synthesis of 5-trifluoromethyl-4,5-dihydro-1H-
pyrazoles: novel agents with analgesic and anti-inflammatory properties, Eur. J. Med.
Chem. 43 (2008) 1237-1247.
[19] A.H. Kategaonkar, P.V. Shinde, A.H. Kategaonkar, S.K. Pasale, B.B. Shingate and
M.S. Shingare, Synthesis and biological evaluation of new 2-chloro-3-((4-phenyl-1H-
1,2,3-triazol-1-yl)methyl)quinoline derivatives via click chemistry approach, Eur. J.
Med. Chem. 45 (2010) 3142–3146.

ACCEPTED MANUSCRIPT
[20] Y.-C. Duan, Y.-C. Ma, E. Zhang, X.-J. Shi, M.-M. Wang, X.-W.Ye and H.-M. Liu,
Design and synthesis of novel 1,2,3-triazole-dithiocarbamate hybrids as potential
anticancer agents, Eur. J. Med. Chem. 62 (2013) 11–19.
[21] T.S. Reddy, H. Kulhari, V.G. Reddy, V. Bansal, A. Kamal, R. Shukla, Design,
synthesis and biological evaluation of 1,3-diphenyl-1H-pyrazole derivatives containing
benzimidazole skeleton as potential anticancer and apoptosis inducing agents, Eur. J.
Med. Chem. 101 (2015) 790-805.
[22] T.S. Reddy, H. Kulhari, V.G. Reddy, A.V. Subba Rao, V. Bansal, A. Kamal and R.
Shukla, Synthesis and biological evaluation of pyrazolo–triazole hybrids as cytotoxic
and apoptosis inducing agents, Org. Biomol. Chem. 13 (2015) 10136-10149.
[23] T.S. Reddy, V.G. Reddy, H. Kulhari, R. Shukla, A. Kamal, V. Bansal, Synthesis of (Z)-
1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one
derivatives
as
potential anticancer and apoptosis inducing agents, Eur. J. Med. Chem. 117 (2016) 157-
166.
[24] O. Prakash, K. Pannu and A. Kumar, Synthesis of Some New 2-(3-Aryl-1-phenyl-4-
pyrazolyl)-benzoxazoles Using Hypervalent Iodine Mediated Oxidative Cyclization of
Schiff’s Bases, Molecules. 11 (2006), 43–48.
[25] K.T. Chan, F.Y. Meng, Q. Li, C.Y. Ho, T.S. Lam, Y. To, W.H. Lee, M. Li, K.H. Chu
and M. Toh, Cucurbitacin B induces apoptosis and S phase cell cycle arrest in BEL-
7402 human hepatocellular carcinoma cells and is effective via oral administration,
Cancer Lett. 294 (2010) 118-124.
[26] J.K. Shen, H.P. Du, M. Yang, Y.G. Wang and J. Jin, Casticin induces leukemic cell
deaththrough apoptosis and mitotic catastrophe, Ann. Hematol. 88 (2009) 743-752.
[27] M. Blank, Y. Shiloh, Programs for Cell Death: Apoptosis is Only One Way to Go, Cell
Cycle. 6 (2007) 686-695.
[28] (a) C.J. Sherr, Science. 274 (1996) 1672-1677; (b) C.J. Sherr, Cancer Res. 60 (2000)
3689-3695; (c) J. Pines, Adv. Cancer Res. 66 (1995) 181-212; (d) C.J. Sherr, J.M.

ACCEPTED MANUSCRIPT
Roberts, Genes Dev. 13 (1999) 1501-1512; (e) N. Dyson, Genes Dev. 12 (1998) 2245-
2262.
[29] I. Neganova, K. Tilgner, A. Buskin, I. Paraskevopoulou, SP Atkinson, D. Peberdy, JF
Passos and M. Lako, CDK1 plays an important role in the maintenance of pluripotency
and genomic stability in human pluripotent stem cells, Cell Death Dis. 5 (2014) e1508.
http://dx.doi.org/10.1038/cddis.2014.464.
[30] J.M Enserink and R.D Kolodner, Review An overview of Cdk1-controlled targets and
processes, Cell dev. 5 (2010) 11.
[31] N.R. Brown, S. Korolchuk, M.P. Martin, W.A. Stanley, R. Moukhametzianov, M.E.M.
Noble, J.A. Endicott, Nature Communications, doi:10.1038/ncomms7769. 6 (2015).
[32] A. Kamal, B. Shaik, V. L. Nayak, B. Nagaraju, J. S. Kapure, M. S. Malik, T. B. Shaik
and B. Prasad, Synthesis and biological evaluation of 1,2,3-triazole linked
aminocombretastatin conjugates as mitochondrial mediated apoptosis inducers, Bioorg.
Med. Chem. 22 (2014) 5155–5167.
[33] C. D. Fan, H. Su, J. Zhao, B. X. Zhao, S. L. Zhang and J. Y. Miao, A novel copper
complex of salicylaldehyde pyrazole hydrazone induces apoptosis through up-
regulating integrin β4 in H322 lung carcinoma cells, Eur. J. Med. Chem. 45 (2010)
1438–1446.
[34] A. H. Wyllie, lucocorticoid-induced thymocyte apoptosis is associated with endogenous
endonuclease activation, Nature. 284 (1980) 555-556.
[35] R. D. Miao, Y. Han, L. Z. An, J. B. Yang and Q. Wang, Seleno-podophyllotoxin
derivatives induce hepatoma SMMC-7721 cell apoptosis through Bax pathway, Cell
Biol. Int. 32 (2008) 217–223.
[36] D. Trachootham, J. Alexandre and P. Huang, Targeting cancer cells by ROS-mediated
mechanisms: a radical therapeutic approach?, Nat. Rev. Drug Discovery. 8 (2009) 579–
591.

ACCEPTED MANUSCRIPT
[37] N. M. Weir, K. Selvendiran, V. K. Kutala, L. Tong, S. Vishwanath, M. Rajaram, S.
Tridandapani, S. Anant and P. Kuppusamy, Curcumin induces G2/M arrest and
apoptosis in cisplatin-resistant human ovarian cancer cells by modulating akt and p38
mAPK, Cancer Biol. Ther. 6 (2007) 178–184.
[38] M. Szumilak, M. A. Szulawska, K. Koprowska, M. Stasiak, W. Lewgowd, A. Stanczak,
M. Czyz, Synthesis and in vitro biological evaluation of new polyamine conjugates as
potential anticancer drugs, Eur. J. Med. Chem. 45 (2010) 5744-5751.
[39] G. Morris, R. Huey, W. Lindstrom, M. Sanner, R. Belew, D. Goodsell, A. Olson,
Autodock4 And Autodocktools4: Automated Docking With Selective Receptor
Flexibility. J. Comput. Chem. 30 (2009) 2785-2791.
[40] G. Shafi, A. Munshi, T. N. Hasan, A. A. Alshatwi, A. Jyothy, D. K.Y. Lei. Induction of
apoptosis in HeLa cells by chloroform fraction of seed extracts of Nigella sativa. Cancer
Cell Int. 9 (2009) 1–8.
[41] W.L. Delano, The PyMOL Molecular Graphics System; DeLano Scientific: San Carlos,
California, 2002.
[42] L.J. Browne, C. Gude, H. Rodriguez, R.E. Steele, A.J. Bhatnager, Fadrozole
hydrochloride: a potent, selective, nonsteroidal inhibitor of aromatase for the treatment
of estrogen-dependent disease, J. Med. Chem. 34 (1991) 725-736.
Figure/Scheme Captions
Scheme 1. General synthesis of compounds 8-35. Reagents and conditions: (i) EtOH, 50–60 °C,
3 h; (ii) DMF, POCl3, 50–60 °C, 5 h; (iii) NaClO2/NH2SO3, 40–50 °C, 5 h; (iv) DMF, EDCI,
HOBt, propargyl amine, 0 °C- rt, 12 h; (v) substituted benzylazides, CuSO4.5H2O, sodium
ascorbate, t-BuOH/H2O (1:1), rt, 12 h.
Table 1. Antiproliferative activity (aGI50 µM) of compounds 8-35.
Fig. 1. Examples of chemical structures of CDK1 inhibitors.