Unveiling novel diphenyl-1H-pyrazole based acrylates tethered to 1,2,3-triazole as promising apoptosis inducing cytotoxic and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bioorg.2019.03.071

Meagre and suboptimal therapeutic response along with the side effect profile associated with the existing anticancer therapy have necessitated the development of new therapeutic modalities to curb this disease. Bearing in mind the current scenario, a series of 1,2,3-triazole linked 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylates was synthesized following a multi-step reaction scheme. Initial screening for anticancer potential was done by in vitro sulforhodamine B assay against four human cancer cell lines- MCF-7 (breast), A549 (Lung) and HCT-116 and HT-29 (Colon). On evaluation, several compounds showed promising growth inhibition against all the cell lines, particularly compounds 6e, 6f and 6n. Among them, compound 6f displayed IC₅₀ values of 1.962, 3.597, 1.764 and 4.496 μM against A549, HCT-116, MCF-7 and HT-29 cell lines respectively. Furthermore, the apoptosis inducing potential of the compounds was determined by Hoechst staining and DNA fragmentation assay. Colony formation inhibition assay was also carried out to determine the long term cytotoxic potential of the molecules. Moreover, compounds 6e, 6f and 6n were also evaluated for anti-inflammatory activity by protein albumin denaturation assay and red blood cell membrane stabilizing assay.

Full text of DOI:10.1016/j.bioorg.2019.03.071

Accepted Manuscript
Unveiling Novel Diphenyl-1H-pyrazole Based Acrylates Tethered to 1,2,3-Tri-
azole as Promising Apoptosis Inducing Cytotoxic and Anti-inflammatory
Agents
Mohemmed Faraz Khan, Tarique Anwer, Afroz Bakht, Garima Verma, Wasim
Akhtar, M Mumtaz Alam, Moshahid Alam Rizvi, Mymoona Akhter,
Mohammad Shaquiquzzaman
PII:
S0045-2068(18)31310-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.071
YBIOO 2897
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
13 November 2018
27 March 2019
27 March 2019
Please cite this article as: M. Faraz Khan, T. Anwer, A. Bakht, G. Verma, W. Akhtar, M. Mumtaz Alam, M. Alam
Rizvi, M. Akhter, M. Shaquiquzzaman, Unveiling Novel Diphenyl-1H-pyrazole Based Acrylates Tethered to 1,2,3-
Triazole as Promising Apoptosis Inducing Cytotoxic and Anti-inflammatory Agents, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.03.071
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Unveiling Novel Diphenyl-1H-pyrazole Based Acrylates Tethered to 1,2,3-Triazole as
Promising Apoptosis Inducing Cytotoxic and Anti-inflammatory Agents
Mohemmed Faraz Khan1, Tarique Anwer2, Afroz Bakht3, Garima Verma1, Wasim Akhtar1,
M Mumtaz Alam1, Moshahid Alam Rizvi4, Mymoona Akhter1, Mohammad
Shaquiquzzaman1*
1Drug Design and Medicinal Chemistry Lab, Department of Pharmaceutical Chemistry, School of
Pharmaceutical Education and Research, Jamia Hamdard, New Delhi 110062, India.
2Department of Pharmacology, College of Pharmacy, Jazan University, PO Box 114, Gizan, KSA.
3 Department of Chemistry, College of Science and Humanities, Prince Sattam bin Abdulaziz
University, P.O. Box- 173, Al-Kharj, Saudi Arabia.
4The Genome Biology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi 110025,
India.
*Corresponding Author
Dr. Mohammad Shaquiquzzaman
Assistant Professor
Department of Pharmaceutical Chemistry
School of Pharmaceutical Education and Research
Jamia Hamdard, New Delhi-110062
Email: shaqiq@gmail.com
+91-9990663405

Abstract
Meagre and suboptimal therapeutic response along with the side effect profile associated with
the existing anticancer therapy have necessitated the development of new therapeutic
modalities to curb this disease. Bearing in mind the current scenario, a series of 1,2,3-triazole
linked 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylates was synthesized following a multi-step
reaction scheme. Initial screening for anticancer potential was done by in vitro
sulforhodamine B assay against four human cancer cell lines- MCF-7 (breast), A549 (Lung)
and HCT-116 and HT-29 (Colon). On evaluation, several compounds showed promising
growth inhibition against all the cell lines, particularly compounds 6e, 6f and 6n. Among
them, compound 6f displayed IC50 values of 1.962, 3.597, 1.764 and 4.496 µM against A549,
HCT-116, MCF-7 and HT-29 cell lines respectively. Furthermore, the apoptosis inducing
potential of the compounds was determined by Hoechst staining and DNA fragmentation
assay. Colony formation inhibition assay was also carried out to determine the long term
cytotoxic potential of the molecules. Moreover, compounds 6e, 6f and 6n were also evaluated
for anti-inflammatory activity by protein albumin denaturation assay and red blood cell
membrane stabilizing assay.
Key Words
1,2,3-Triazole; Diphenyl Pyrazole; Acrylates; Anticancer; Apoptosis; Anti-inflammatory.
List of Abbreviations
Copper (II) sulphate pentahydrate
Doublet
CuSO4.5H2O
d
Dichloromethane
DCM
N-N-Dimethyl formamide
Dimethyl sulfoxide
DMF
DMSO
Equipped with Electrospray
ESI
Hz
Hertz
Half Maximal Inhibitory Concentration
Infra-red
IC50
IR

Coupling Constant
J
Multiplet
m
National Cancer Institute
Nuclear Magnetic Resonance
NCI
NMR
Non-Steroidal Anti-Inflammatory Drugs
Protein Data Bank ID
NSAIDs
PDB ID
Phosphorus Oxychloride
Parts Per Million
POCl3
ppm
p-TsOH
RCSB
s
p-Toluenesulfonic acid
Research Collaboratory for Structural Bioinformatics
Singlet
Standard Precision
SP
Sulforhodamine B
SRB
t
Triplet
Thin Layer Chromatography
Tetramethylsilane
TLC
TMS

1. Introduction
Cancer, a broad term used to characterize uncontrolled proliferation of cells arising from
disruption or dysfunction of regulatory signalling pathways, which are normally under tight
control, is one of the foremost diseases responsible for worldwide mortality [1]. At present, it
is the major public concerned hotspot across the globe [2]. Deaths due to cancer are projected
to continue rising with an approximate 3 million deaths in 2030 [3]. In developing and
underdeveloped countries, lung, colon, breast and melanoma cancers are the most commonly
reported cases [4]. However, emergence and spread of resistance to the currently available
chemotherapeutic agents vindicates an urgent need for development of novel, more potent
and selective anticancer agents [5].
Inflammation is a well-known phenomenon that an organism utilizes for resolving infections,
tissue injury or restoring tissue functions via repair mechanism [6]. This response acts as a
double-edged response [7]. Acute inflammation is an immediate response produced in
response to tissue damage, which is generally useful for maintenance of homeostasis. This
plays a beneficial role in protection against injury. However, chronic inflammation i.e.
uncontrolled inflammatory reactions serve as a common etiologic factor for different ailments
including cancer [8-10].
Link between chronic inflammation and cancer may be associated with different mechanisms
like genomic instability induction, enhanced proliferation, inappropriate gene expression,
resistance to apoptosis, metastasis, invasion through tumor-associated basement membrane,
aggressive tumor neovascularization, etc. Elevated levels of reactive oxygen and nitrogen
species can serve as major contributors in malignant cell transformation. Abnormal activation
or overexpression of certain pro-inflammatory mediators like chemokines, cytokines,
prostaglandins, cyclooxygenase-2, nitric oxide and inducible nitric oxide synthase promote
tumor growth and progression. Certain pro-inflammatory mediators, particularly cytokines,
chemokines and prostaglandins are known to endorse angiogenesis, thereby leading to
metastasis and invasion [11].
Chronic inflammation plays a multifaceted role in carcinogenesis. Evidences from preclinical
and clinical studies suggest that persistent inflammation functions as a driving force in the
journey of cancer [12]. The inflammatory component contributes to tumor proliferation,
angiogenesis, metastasis and resistance to hormonal and chemotherapy [13]. Chronic
inflammation is also involved in the pathogenesis of insulin resistance, atherosclerosis,

neurodegeneration and tumor growth [14]. Work reported by Aleksandrova et al. provides an
evidence that elevated CRP concentrations (markers of inflammation) are related to a higher
risk of colon cancer [15]. Except for few drugs, long term use of anti-inflammatory drugs
including non-steroidal anti-inflammatory drugs (NSAIDs) results in adverse effects such as
bleeding, gastrointestinal ulceration and kidney damage [16].
Pyrazole moiety can be traced in number of anticancer agents viz. Crizotinib, Tartrazine,
Pyrazomycin, etc. [17]. This moiety is found in a number of anti-inflammatory drugs also. To
mention, these are Celecoxib, Ramifenazone, Deracoxib, Lonazolac, etc. [18]. Similarly,
triazole bearing compounds are also known to exhibit potent anticancer (Anastrazole,
Letrozole, Vorozole) and anti-inflammatory (Carboxyamidotriazole) activities [19]. Several
pyrazole [20,21] and triazole [22,23] bearing agents reported in literature possess dual
activity i.e., anticancer and anti-inflammatory.
The diphenyl pyrazole moiety is receiving significant attention now a days, as the structural
scaffold is found to make up the core structure of numerous anticancer (1) [24] and anti-
inflammatory (2) [25] agents. Recent literature is enriched with progressive findings on
anticancer (3) [26] and anti-inflammatory (4) [27] effects of 1,2,3-Triazole based compounds.
Hybrids of diphenyl-pyrazole and 1,2,3-triazole were also reported as both anticancer (5 and
6) [28, 29] and anti-inflammatory (7) [30] agents recently. Several studies also revealed the
anticancer (8, 9) [31, 32] and anti-inflammatory (10) [33] potential of acrylic acid derivatives,
particularly the acrylates (Figure 1).
(a) Diphenyl Pyrazole Containing Compounds

(b) 1,2,3-Triazole Containing Compounds
(c) Diphenyl Pyrazole and 1,2,3-Triazole Containing Compounds
(d) Acrylate Linkage Containing Compounds
Figure 1: Anticancer and anti-inflammatory compounds bearing (a) diphenyl pyrazole, (b)
1,2,3-triazole, (c) diphenyl pyrazole and 1,2,3-triazole and (d) acrylate linkage.
Despite significant potency of acrylic linkage, literature search revealed that diphenyl
pyrazol-4-yl acrylic acid’s analogs are found to be rarely synthesized [34] and never found to
be clubbed with 1,2,3-triazoles. Henceforth, the present work reports synthesis of novel 3-
(1,3-diphenyl-1H-pyrazol-4-yl)acrylates linked with 1,2,3-Triazole (Figure 2). The

synthesized molecules were evaluated for their anticancer and anti-inflammatory activities.
On evaluation, several compounds were found to be potent against both cancer and
inflammation.
Figure 2: Structure of title compounds (Scheme 1).
2. Results and Discussion
2.1 Chemistry
Requisite compounds for Scheme I were obtained by employing multi-step reactions. Several
substituted acetophenones were subjected to addition reaction with phenylhydrazine in the
presence of glacial acetic acid. Subsequently, utilizing Vilsmeier-Haack reaction, formation
of the pyrazole ring took place in the presence of N-N-dimethyl formamide (DMF) and
phosphorus oxychloride (POCl3). DMF served as acylating agent while POCl3 acted as
activating agent. Vilsmeier reagent, also known as Vilsmeier complex was formed during this
reaction [35]. An aldol condensation’s modified form, Knoevenagel condensation reaction
was used to convert pyrazole carbaldehydes into pyrazole carboxylic acids. In this, aldehyde
derivatives were reacted with malonic acid in the presence of pyridine and piperidine. In this
reaction, pyridine was used as the solvent whereas piperidine served as the catalyst. This
reaction is based on nucleophilic addition between an active hydrogen compound (malonic
acid in this case) and an aldehyde, resulting in the formation of -unsaturated dicarboxylic
acid [36]. Furthermore, monocarboxylic acid derivatives were obtained by decarboxylation of
dicarboxylic acids via Doebner modification [37]. The acids were further reacted with
propargyl bromide in the presence of potassium carbonate to yield terminal alkyne bearing
pyrazole acrylate derivatives [38].

Aromatic azides were formed in high yields by aromatic amines via diazotization with
sodium nitrite in water in the presence of p-toluenesulfonic acid (p-TsOH) and subsequent
addition of sodium azide [39].
These synthesized terminal alkyne bearing pyrazole acrylates and aromatic azides were
coupled in the final step in the presence of catalytic copper (II) sulphate pentahydrate
(CuSO4.5H2O) and sodium ascorbate in a H2O/Dichloromethane (DCM) mixture at room
temperature to yield the final compounds, triazoles [40].
2.2 Spectral Characterization
Spectral data for all the synthesized derivatives was found to be in concordance with the
reported data and is discussed in detail in the Supporting Information section.
2.3 Biological Activity
Anticancer potential was assessed of all the prepared compounds (4a-e, 6a-o). For assessing
anticancer potential, the synthesized compounds were screened against a panel of four human
cancer cell lines using the sulforhodamine B assay. Three of the most active compounds (6e,
6f and 6n) were further subjected to mechanistic studies including colony formation
inhibition assay, Hoechst staining and DNA fragmentation assay.
Based on the reported facts that there exists a crucial link between cancer and inflammation.
We also moved on the same aisle and got our most potent anticancer compounds (6e, 6f and
6n) evaluated for anti-inflammatory potential as well. Anti-inflammatory activity was
performed using protein albumin denaturation assay and red blood cell membrane stabilizing
assay.
2.3.1 Anticancer
2.3.1.1 Sulforhodamine B Assay
The newly synthesized compounds (4a-e and 6a-o) were assessed for their in vitro cytotoxic
potential against a panel of four human cancer cell lines i.e., MCF-7 (breast), A549 (Lung)
and HCT-116 and HT-29 (Colon).
Initial anticancer screening was performed at concentration of 1 and 10 µM (Table 1,
Supplementary Information). The results of the preliminary anticancer screening were
found to be in concordance with results of in silico studies. Half maximal inhibitory

concentration (IC50) value was determined for the compounds showing more than 50%
inhibition at a concentration of 10 µM. Amongst the compounds of this series, compounds
6e, 6f and 6n were tested at concentrations of 1, 3, 5, 7 and 10 µM in order to determine IC50
values against the aforementioned panel of cell lines. On evaluation, compound 6f emerged
as the most active compound with IC50 values of 1.962, 3.597, 1.764 and 4.496 µM against
A549, HCT-116, MCF-7 and HT-29 respectively. Another compound, 6n also displayed
promising activity profile against HCT-116, MCF-7 and HT-29 with IC50 values of 4.940,
1.851 and 4.362 µM respectively (Table 1). Depending on the results, colony formation
inhibition assay, DNA fragmentation assay and fluorescence microscopy studies were also
performed for compounds 6e, 6f and 6n.
Table 1: IC50 values of compounds 6e, 6f and 6n against a panel of Human Cancer Cell Lines
Cell Line Type
Tissue
A549
Lung
HCT-116
Colon
MCF-7
Breast
HT-29
Colon
S.No. Compd.
IC50 µM
10.607 ± 0.08 15.159 ± 0.34 10.184 ± 0.24 8.629 ± 0.20
1.
2.
3.
4.
5.
6e
1.962 ± 0.16
9.463 ± 0.24
0.030 ± 0.17
0.180 ± 0.22
3.597 ± 0.26
4.940 ± 0.45
0.050 ± 0.14
0.006 ± 0.06
1.764 ± 0.18 4.496 ± 0.26
1.851 ± 0.42 4.362 ± 0.13
0.200 ± 0.24 0.800 ± 0.27
6f
6n
Camptothecin
Combretastatin A4
0.033 ± 0.26
>10
2.3.1.2 Colony formation inhibition assay
Clonogenic cell survival assay is performed to assess the ability of a single cell to grow into a
colony. It helps us in investigating the long term cytotoxic potential. Since the method
reflects all modes of cell death or arrest, it is considered to be a standard for determining long
term cell viability [41]. The colony formation of MCF-7 cells was found to be significantly
inhibited by the exposure of MCF-7 cells to the compounds (Figure 3). The clonogenic cell
survival is found to be reduced approximately to its half, and was found to be in agreement
with IC50 values of the compounds. The result indicates that the molecules can efficiently
inhibit proliferation and growth of MCF-7 cells.

Figure 3: Colony formation inhibition assay of compounds 6e, 6f and 6n in MCF-7 cancer
cell line.
2.3.1.3 Hoechst staining
In order to determine apoptosis inducing potential of compounds, MCF-7 cells were
incubated with IC50 concentration of compounds 6e, 6f and 6n for a period of 24 h.
Furthermore, the morphological changes were studied after Hoechst 33242 staining. Control
cells treated with DMSO demonstrated uniformly dispersed chromatin with almost negligible
apoptotic characteristics (Figure 4). On the contrary, 30-40% cells treated with compounds
6e, 6f and 6n exhibited typical apoptotic characteristics, like condensation of chromatin
(brightly stained cells) and advent of fragmented apoptotic nucleus (indicated by
arrowheads). Amongst the tested compounds, compound 6f was found to be most potent
apoptosis inducer (45% apoptosis cells) compared to the other two compounds, i.e., 6n (28%
apoptotic cells) and 6e (35% apoptotic cells). The results demonstrated the apoptosis
inducing potential of the compounds against MCF-7 cancer cell line.

Figure 4: Hoechst staining of MCF-7 cells treated with compounds 6e, 6f and 6n.
2.3.1.4 DNA fragmentation assay
The higher order chromatin structure of DNA is degraded by the activated nucleases into
mono and oligo nucleosomal DNA-fragments during the apoptotic process. Therefore, a
characteristic DNA ladder on agarose gel electrophoresis is formed as a result of loss of DNA
content due to fragmentation of DNA [42]. Thus, in order to further confirm the apoptosis
inducing effect of these compounds, DNA fragmentation assay was performed. MCF-7 cells
were treated with the IC50 concentration of compounds 6e, 6f and 6n for 24 h and
chromosomal DNA was extracted. Agarose based gel electrophoresis was used to examine
the apoptotic degradation of DNA. DNA from the cells treated by compounds exhibited a
typical DNA smeared ladder pattern, an indicative of DNA fragmentation. In case of
treatment with the synthesized compound 6f, a typical DNA smeared ladder pattern was
observed that clearly indicated fragmentation (Figure 5). DNA breaks at several positions
across the chromosomal DNA resulted in smear of this type. Significantly intense smear was
seen in DNA of cells treated with compounds 6f and 6n. However, moderate smear formation
was seen in case of compound 6e. DNA from control cells showed little or no signs of DNA

degradation. Results obtained using MCF-7 cells clearly indicate that compounds 6f and 6n
are apoptosis inducing agents.
Figure 5: DNA ladder assay of compounds 6e, 6f and 6n in MCF-7 cell line, C is Control
(Untreated) and M is Molecular Weight Marker.
2.3.2 Anti-inflammatory
2.3.2.1 Protein Albumin Denaturation Assay
Protein (egg albumin) denaturation assay was used to evaluate the in vitro anti-inflammatory
activity of three most active compounds 6e, 6f and 6n. Diclofenac sodium was used as the
standard drug for performing assay. All three of the compounds demonstrated significant
anti-inflammatory activity with IC50 values of 60.56, 57.24 and 69.15 µg/ml for compounds
6e, 6f and 6n respectively (Table 2). However, the IC50 value for the standard, Diclofenac
Sodium was found to be 54.65 µg/mL.
Table 2: Anti-inflammatory activity of compounds 6e, 6f and 6n on the basis of protein
albumin denaturation assay.

% Inhibition
Comp.
IC50 (µg/mL)
25 (µg/mL) 50 (µg/mL) 100 (µg/mL) 200 (µg/mL)
04.99 ± 1.08 68.21 ± 0.67 84.60 ± 0.64 100 ± 0.00
0.820 ± 0.69 74.43 ± 0.53 88.68 ± 0.29 100 ± 0.00
19.51 ± 0.89 48.28 ± 0.59 74.01 ± 0.49 100 ± 0.00
7.47 ± 0.66 67.31 ± 0.43 92.62 ± 0.58 100 ± 0.00
60.56 ± 0.46
57.24 ± 0.95
69.15 ± 0.45
54.65 ± 0.71
6e
6f
6n
Diclofenac
Sodium
2.3.2.2 Red Blood Cell Membrane Stabilizing Assay
In vitro anti-inflammatory activity was also assessed by another method called to be as red
blood cell membrane stabilizing assay. Consequently, the results thus obtained revealed that
the molecules inhibited RBC haemolysis in a concentration dependent manner (Table 3).
Aspirin was used as standard. All of the compounds displayed noteworthy anti-inflammatory
potential with compound 6f as the most promising one.
Table 3: Anti-inflammatory activity of compounds 6e, 6f and 6n on the basis of red blood
cell membrane stabilizing assay.
Compounds
6e
Concentration (µg/mL)
% Protection
40.25 ± 3.72
70.24 ± 1.02
94.67 ± 1.84
51.06 ± 1.09
74.25 ± 2.65
97.25 ± 3.27
46.24 ± 2.65
68.41 ± 3.01
90.48 ± 2.08
60.14 ± 1.65
84.23 ± 2.95
100
200
500
100
200
500
100
200
500
100
200
6f
6n
Aspirin

2.4. Molecular Docking
In order to assess the mechanism of action of the synthesized novel anticancer derivatives,
molecular docking studies were performed on different anticancer targets particularly,
tubulin.
Table 4: Dock Score of Synthesized compounds 4a-4e and 6a-6o against tubulin protein
(PDB ID: 3E22)
S. No.
Compd.
S. No.
Compd.
Compd.
Dock
Score
Dock
S.
Dock
Score
Score No.
-7.722 15.
-7.703 16.
1.
2.
3.
4.
5.
6.
7.
4a
4b
4c
4d
4e
6a
6b
-6.611 8.
-7.368 9.
-6.290 10.
-7.463 11.
-6.116 12.
-7.241 13.
-7.377 14.
6c
6d
6e
6f
6j
-7.192
-7.708
-5.442
-7.847
-7.737
-7.478
-8.376
6k
6l
17.
18.
-7.812
-8.047
6m
6n
6o
6g
6h
6i
-7.633 19.
-7.474 20.
-7.166 21.
Combretas
tatin A4
As a result of the molecular docking study, almost all of the compounds have shown
noteworthy and remarkable interactions with the tubulin protein. Among all the compounds
used in the study, triazoles (6a-6o) were found to have better interactions than the terminal
alkyne containing propargyl derivatives (4a-4e). The dock scores ranged from -8.047 to -
5.442 kCal/mol, with 6f having the best score (-8.047) compared to the dock score of
colchicine (-7.876). Table 4 represents the Dock Score of different synthesized compounds
against tubulin protein.
Cartoon representation of the overall structure of the complex formed between 6f and tubulin
is shown in Figure 6a. -tubulin is shown in grey () and green (, whereas -tubulin is
represented by red ( and blue ( color. RB3 Stathmin-like domain is shown in yellow
color. GTP (green) and GDP (orange) molecules bound to the -tubulin and -tubulin are

displayed as CPK model. Also the most active compound (6f) can be seen bound to the -
tubulin in purple color CPK model form.
In order to have a deeper look into the molecular interactions of the most active compound 6f
and tubulin, the expanded image of the active site of -tubulin bearing compound 6f is
displayed in Figure 6b. Figure 6c represent the Ligand Interaction Diagram (LID) depicting
the molecular interactions of 6f with tubulin protein. To understand the molecular
interactions displayed in LID more evidently, legends for the same are also represented below
the diagram.
6a

6b
6c
Figure 6: Docking Analysis for most active anticancer compound 6f with tubulin (PDB ID:
3E22). (a) Cartoon representation of the overall structure of the complex formed between 6f
and tubulin. (b) Dock pose of 6f bound in the colchicine binding site of tubulin. (c) 2-D
Ligand Interaction Diagram of 6f bound in the colchicine binding site of tubulin.
From the results, it is quite evident that compound 6f demonstrated interactions with a
number of amino acid residues (Figure 6c). The bridging Oxygen of the ester group was
found to form a hydrogen bond with the amino acid residue Asn 258. Compound 6f
interacted hydrophobically with numerous amino acid residues like Cys 241, Leu 242, Leu
248, Ala 250, Leu 252, Ala 316, Met 259, Pro 261, Trp 346, Ile 347, Val 355, etc. 6f also
exhibited significant polar interactions with amino acid residues like Thr 239, Gln 247, Asn
249, Asn 258, Thr 314, Asn 349, etc. The compound was found to have interaction with
positively charged residue Lys 352 as well. Sufficient amount of solvent exposure is also
found to be contributing towards good affinity of the molecule with the protein.

2.5 Structure-Activity Relationship
On comparing the in vitro anticancer results of the synthesized molecules (4a-e, 6a-o),
triazole containing compounds (6a-o) clearly demonstrated better results than the predecessor
terminal alkyne containing acrylate derivatives (4a-e). The superior effect of di-phenyl
pyrazole and triazole conjugates (6a-o) over di-phenyl pyrazole containing terminal alkynes
(4a-e) may be attributed to the synergistic action of two pharmacophores when used in
combination. Substitution of electron donating groups at –R position (over one of the phenyl
ring of diphenyl pyrazole moiety) favored anticancer potential of the compounds as seen in
compounds 6f, 6e and 6m having methyl, methoxy and methoxy groups respectively. On the
contrary, compounds bearing electron withdrawing groups like Chloro and Fluoro (4c, 4e, 6j
and 6l) at position –R were found to have diminished anticancer activity. Unsubstituted
compounds (6a, 6b, 6d, 6g and 6k) were found to possess mediocre anticancer potential.
In case of substitutions over –X position (over phenyl ring directly attached to the triazole
ring), the situation was found to be just opposite. Here, substitution of electron withdrawing
groups resulted in boosting the anticancer potential. This is quite evident from compounds 6f
and 6n (containing Nitro group), 6e (containing Chloro group) and compounds 6k and 6m
(containing 3-Chloro and 4-Fluoro substitutions). Whereas, compounds bearing electron
donating groups like Methyl group (6i and 6l) were found to possess poor anticancer
potential.
3. Experimental
3.1 Chemistry
All the required chemicals and reagents used in the reactions were purchased from
commercial vendors. These chemicals were not purified further. Aluminum backed silica
plates (Merck, silica gel 60 F254) were used to perform Thin layer chromatography (TLC).
Melting points were reported by using open capillaries and are uncorrected. Bruker alpha-T
spectrophotometer was used to record Infra-red (IR) spectra. Bruker Avance-400 was used
for recording Nuclear Magnetic Resonance (NMR) spectra. Frequencies of 400 and 100 MHz
1
were used for recording H and 13C spectra respectively. CDCl3 served as the solvent to
dissolve triazole derivatives whereas Tetramethylsilane (TMS) was taken as an internal
standard. Chemical shift values (δ) are reported in parts per million (ppm) whereas coupling
constants (J) are reported in Hertz (Hz). Xevo G2-XS QTOF, equipped with electrospray
(ESI) ionization spray source was used to record high resolution mass spectra (HRMS). This

was operated in full scan positive mode. For a few compounds, mass spectra were recorded
on Synapt mass spectrometer (UPLCMS/MS). Purity of certain compounds was determined
by UPLC (WATERS INC.). Perkin-Elmer-240 was used for elemental analysis. Column
chromatography was done wherever required using silica gel of 60-120 mm taking hexane
and ethyl acetate in the ratio of 7 : 3 v/v respectively, as the solvent system.
3.1.1 Synthesis of 1,3-Diphenyl-1H-pyrazole-4-carbaldehyde (2a-e)
1,3-Diphenyl-1H-pyrazole-4-carbaldehydes (2a-e) were synthesized as per the method
reported in literature [43, 44]. The aldehydes thus obtained were found to have spectral and
other analytical data in agreement with the data reported in the literature.
3.1.2 Synthesis of 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylic acid derivatives (3a-e)
Synthesis of 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylic acid derivatives was carried out
according to our previously reported method. Details for derivatives 3a-c have already been
published by our group [34]. Spectral details for newly synthesized derivatives 3d and 3e
have been incorporated in the Supplementary Information section.
3.1.3 Synthesis of Prop-2-ynyl-3-(1,3-diphenylpyrazol-4-yl)prop-2-enoate (4a-e)
Pyrazole acids (1 mM) (3a-e) were dissolved in dried acetone, and fused potassium carbonate
(4 mM) was added to it. The resulting mixture was then heated with stirring for 30 min. After
that, propargyl bromide (1 mM) was added and stirring with heating was continued till the
completion of reaction. Potassium carbonate was then filtered from the hot reaction mixture,
and the filtrate was poured in ice cold water. The precipitate thus obtained was filtered,
washed with water and dried. Spectral details for compounds 4a-e are depicted in the
Supplementary Information section.
3.1.4 Synthesis of aryl azides (5a-h)
Derivatives 5a-h were synthesized in accordance with the reported methods [39].
3.1.5 Synthesis of (1-phenyl-1H-1,2,3-triazol-4-yl)methyl 3-(1,3-diphenyl-1H-pyrazol-4-
yl)acrylate (6a-6o)
Scheme 1 represents the method adopted for the preparation of triazoles reported in this
paper.
Different derivatives synthesized by this method are enlisted in Table 5. To a solution of
terminal alkyne containing pyrazole acrylate derivatives (1 mM) (4a-e) and aryl azides (1.1
mM) (5a-h) in 1:1 mixture of water and DCM, sodium ascorbate (0.1 mM) and copper (II)
sulphate (0.05 mM) were added sequentially. Reaction was stirred overnight at room
temperature. Solvents were concentrated under vacuum and mixture was extracted with

DCM, dried and concentrated under vacuum. Further, all the compounds were purified by
column chromatography using silica gel and solvent system hexane and ethyl acetate in the
ratio 7 : 3 v/v , respectively.
(1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl
3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylate
(6a)
Compound 6a was synthesized by reaction of 4a with 5a as per the procedure given above.
1
Appearance: White powder; Yield (%): 72; m.p. 176-178ºC; H NMR (400 MHz, CDCl3):
5.43 (s, 2H, CH2), 6.29 (d, 1H, H-, J=16.0 Hz), 7.33 (t, 1H, H-4, J=7.2 Hz), 7.43-7.51 (m,
5H, H-3,5, 3’,4’,5’), 7.61-7.81 (m, 8H, H-2,6,2’,6’,4”,5”,6”,), 7.97 (s, 1H, H-Triazole), 8.09
(d, 2H, H-3”, J=7.6 Hz), 8.25 (s, 1H, H-Pyrazole); IR (cm-1): 1716 (C=O), 1523,1330 (NO2),
1239 (C-O); Mass (m/z): 493.2 [M+H]+; Elemental analysis (%) of C27H20N6O4,
calculated/found: C (65.85/65.86), H (4.09/4.12), N (17.06/17.08).
(1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl
3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylate
(6b)
Compound 6b was synthesized by reaction of 4a with 5b as per the procedure given above.
1
Appearance: White powder; Yield (%): 70; m.p. 196-198ºC; H NMR (400 MHz, CDCl3):
5.44 (s, 2H, CH2), 6.29 (d, 1H, H-, J=16.0 Hz), 7.33 (t, 2H, H-4, J=7.6 Hz), 7.42-7.51 (m,
5H, H-3,5,3’,4’,5’), 7.66 (d, 2H, H-2,6, J=8.4 Hz), 7.74-7.82 (m, 4H, H-2’,6’,5”,), 8.17 (dd,
1H, H-6”, J=1.6 Hz), 8.20 (s, 1H, H-Triazole), 8.25 (s, 1H, H-Pyrazole), 8.31 (dd, 1H, H-4”,
J= 8.8 & 1.6 Hz), 8.61 (t, 1H, H-2”, J=2 Hz); IR (cm-1): 1719 (C=O), 1534, 1328 (NO2), 1219
(C-O); Mass (m/z): 493.5 [M+H]+; Elemental analysis (%) of C27H20N6O4, calculated/found:
C (65.85/65.84), H (4.09/4.08), N (17.06/17.08).
(1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl 3-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-
4-yl)acrylate (6c)
Compound 6c was synthesized by reaction of 4e with 5a as per the procedure given above.
1
Appearance: white powder; Yield (%): 66; m.p. 182-184ºC; H NMR (400 MHz, CDCl3):
5.44 (s, 2H, CH2), 6.30 (d, 1H, H-, J= 16.0 Hz), 7.34 (t, 1H, H-4, J= 7.6 Hz), 7.45-7.52 (m,
4H, H-3,5,3’,5’), 7.60-7.64 (m, 3H, H-2,6,4”), 7.72-7.76 (m, 4H, H-2’,6’,6”,β), 7.79 (t, 1H,
H-5”, J= 8.8 Hz), 7.97 (s, 1H, H-Triazole), 8.10 (d, 1H, H-3”, J= 7.6 Hz), 8.24 (s, 1H, H-
Pyrazole); IR (cm-1): 1721 (C=O), 1535, 1336 (NO2), 1267 (C-O); Mass (m/z): 527.7

[M+H]+; Elemental analysis (%) of C27H19ClN6O4, calculated/found: C (61.54/61.52), H
(3.63/3.64), N (15.95/15.96).
(1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylate
(6d)
Compound 6d was synthesized by reaction of 4a with 5c as per the procedure given above.
1
Appearance: White powder; Yield (%): 69; m.p. 102-104ºC; H NMR (400 MHz, CDCl3):
5.44 (s, 2H, CH2), 6.30 (d, 1H, H-, J=16.0 Hz), 7.33 (t, 1H, H-4, J=7.6 Hz), 7.41-7.51 (m,
7H, H-3,5,3’,4’,5’,2”,4”), 7.58-7.64 (m, 2H, H-5”,6”), 7.66 (d, 2H, H-2,6, J= 6.8 Hz), 7.75 (d,
2H, H-2’,6’, J= 7.2 Hz), 7.77 (d, 1H, H-, J= 15.6 Hz), 8.09 (s, 1H, H-Triazole), 8.24 (s, 1H,
H-Pyrazole); 13C NMR (100 MHz, CDCl3): (57.48, 116.61, 117.50, 119.39, 125.98, 126.55,
127.29, 127.81, 127.96, 128.60, 128.72, 128.81, 129.59, 130.81, 130.89, 132.13, 134.75,
136.21, 139.39, 142.90, 153.46, 166.84); IR (cm-1): 1714 (C=O), 1265 (C-O); Mass (m/z):
482.3 [M+H]+; Elemental analysis (%) of C27H20ClN5O2, calculated/found: C (67.29/67.30),
H(4.18/4.15), N(14.53/14.55).
(1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl
3-(3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)acrylate (6e)
Compound 6e was synthesized by reaction of 4d with 5c as per the procedure given above.
Appearance: White powder; Yield (%): 74; m.p. 88-90ºC; 1H NMR (400 MHz, CDCl3): 3.87
(s, 3H, OCH3), 5.44 (s, 2H, CH2), 6.29 (d, 1H, H-, J=16.0 Hz), 7.00 (d, 2H, H-3’,5’, J= 8.4
Hz), 7.32 (t, 1H, H-4, J=7.2 Hz), 7.45-7.52 (m, 4H, H-3,5,2”,4”), 7.59-7.64 (m, 4H, H-
2,6,5”,6”), 7.74 (d, 2H, H-2’,6’, J= 8.0 Hz), 7.76 (d, 1H, H-, J= 16.0 Hz), 8.10 (s, 1H, H-
Triazole), 8.22 (s, 1H, H-Pyrazole); IR (cm-1): 1722 (C=O), 1263 (C-O); Mass (m/z): 512.6
[M+H]+; Elemental analysis (%) of C28H22ClN5O3, calculated/foun2d: C (65.69/65.66), H
(4.33/4.35), N (13.68/13.69).
(1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl
3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-
yl)acrylate (6f)
Compound 6f was synthesized by reaction of 4b with 5d as per the procedure given above.
1
Appearance: White powder; Yield (%): 75; m.p. 194-196ºC; H NMR (400 MHz, CDCl3):
2.42 (s, 3H, CH3), 5.43 (s, 2H, CH2), 6.28 (d, 1H, H-, J=16.0 Hz), 7.34 (t, 1H, H-4, J= 7.2
Hz), 7.44-7.52 (m, 4H, H-3,5,3’,5’), 7.65 (d, 2H, H-2,6, J= 6.8 Hz), 7.75 (d, 2H, H-2’,6’, J=

8.0 Hz), 7.80 (d, 1H, H-, J= 16.0 Hz), 7.97 (d, 2H, H-2”,6”, J= 9.2 Hz), 8.20 (s, 1H, H-
Triazole), 8.24 (s, 1H, H-Pyrazole), 8.41 (d, 2H, H-3”,5”, J= 9.2 Hz); IR (cm-1): 1727 (C=O),
1540, 1342 (NO2), 1252 (C-O); Mass (m/z): 507.2 [M+H]+; Elemental analysis (%) of
C28H22N6O4, calculated/found: C (66.40/66.43), H (4.38/4.36), N (16.59/16.60).
(1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl
3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylate
(6g)
Compound 6g was synthesized by reaction of 4a with 5e as per the procedure given above.
1
Appearance: White powder; Yield (%): 67; m.p. 150-152ºC; H NMR (400 MHz, CDCl3):
5.41 (s, 2H, CH2), 6.28 (d, 1H, H-, J=16.0 Hz), 7.20 (t, 2H, H-3”,5”, J= 8.8 Hz), 7.33 (t, 1H,
4, J=7.2 Hz), 7.42-7.52 (m, 5H, H-3,5,3’,4’,5’), 7.66 (d, 2H, H-2,6, J= 6.8 Hz), 7.70-7.73 (m,
2H, H-2”,6”), 7.75 (d, 2H, H-2’,6’, J= 6.4 Hz), 7.77 (d, 1H, H-, J= 15.6 Hz), 8.04 (s, 1H, H-
13
Triazole), 8.24 (s, 1H, H-Pyrazole); C NMR (100 MHz, CDCl3): (57.47, 116.49, 116.67,
116.90, 117.45, 119.40, 122.43, 122.59, 122.68, 126.55, 127.33, 128.73, 128.82, 129.60,
132.13, 136.32, 139.38, 143.95, 153.48, 166.93); IR (cm-1): 1727 (C=O), 1259 (C-O); HRMS
m/z measured for C27H20FN5O2 [M+H]+ 466.1699, m/z calculated [M+H]+ 466.1679; UPLC:
99.92% purity; Elemental analysis (%) of C27H20FN5O2, calculated/found: C (69.67/69.65),
H(4.33/4.34), N(15.05/15.07).
(1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl
3-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)acrylate (6h)
Compound 6h was synthesized by reaction of 4d with 5f as per the procedure given above.
1
Appearance: White powder; Yield (%): 68; m.p. 126-128ºC; H NMR (400 MHz, CDCl3):
2.43 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 5.41 (s, 2H, CH2), 6.27 (d, 1H, H-, J=16.0 Hz), 7.01
(d, 2H, H-3’,5’, J= 8.8 Hz), 7.31-7.35 (m, 3H, H-4,3”,5”), 7.46 (t, 2H, H-3,5, J= 8.0 Hz),
7.59-7.62 (m, 4H, H-2,6,2”,6”), 7.74 (d, 2H, H-2’,6’, J= 8.0 Hz), 7.75 (d, 1H, H-, J= 15.6
Hz), 8.04 (s, 1H, H-Triazole), 8.21 (s, 1H, H-Pyrazole); 13C NMR (100 MHz, CDCl3): (21.13,
55.39, 57.55, 114.09, 114.28, 116.33, 117.30, 119.34, 119.58, 120.54, 120.59, 122.21,
126.49, 127.19, 129.44, 129.57, 129.97, 130.28, 130.59, 134.64, 136.40, 139.07, 139.42,
143.66, 153.29, 160.07, 166.98); IR (cm-1): 1728 (C=O), 1258 (C-O); HRMS m/z measured
for C29H25FN5O3 [M+H]+ 492.2047, m/z calculated [M+H]+ 492.2036; UPLC: 99. 83%
purity; Elemental analysis (%) of C29H25N5O3, calculated/found: C (70.86/70.85),
H(5.13/5.11), N(14.25/14.27).

(1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylate (6i)
Compound 6i was synthesized by reaction of 4a with 5f as per the procedure given above.
1
Appearance: White powder; Yield (%): 66; m.p. 118-120ºC; H NMR (400 MHz, CDCl3):
2.42 (s, 3H, CH3), 5.41 (s, 2H, CH2), 6.28 (d, 1H, H-, J=16.0 Hz), 7.31-7.36 (m, 3H, H-
4,3”,5”), 7.43-7.51 (m, 5H, H-3,5,3’,4’,5’), 7.59 (d, 2H, H-2,6, J= 8.4 Hz), 7.65 (d, 2H, H-
2”,6”, J= 8.4 Hz), 7.75 (d, 2H, H-2’,6’, J= 7.6 Hz), 7.76 (d, 1H, H-, J= 16.0 Hz), 8.04 (s,
13
1H, H-Triazole), 8.24 (s, 1H, H-Pyrazole); C NMR (100 MHz, CDCl3): (21.23, 57.57,
116.61, 117.49, 119.40, 120.54, 122.21, 126.55, 127.30, 128.81, 129.59, 130.27, 132.13,
134.63, 136.21, 139.39, 143.64, 153.47, 166.92); IR (cm-1): 1719 (C=O), 1270 (C-O); Mass
(m/z): 462.3 [M+H]+; Elemental analysis (%) of C28H23N5O2, calculated/found: C
(72.87/72.89), H(5.02/5.00), N(15.17/15.16).
(1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl 3-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-
4-yl)acrylate (6j)
Compound 6j was synthesized by reaction of 4c with 5b as per the procedure given above.
1
Appearance: White powder; Yield (%): 68; m.p. 168-170ºC; H NMR (400 MHz, CDCl3):
5.44 (s, 2H, CH2), 6.28 (d, 1H, H-, J=16.0 Hz), 7.16 (t, 2H, H-3’,5’, J=8.8 Hz), 7.34 (t, 1H,
H-4, J=7.6 Hz), 7.47 (t, 2H, H-3,5), 7.63-7.66 (m, 2H, H-2,6), 7.73-7.78 (m, 4H, H-
2’,6’,5”,β), 8.18 (dd, 1H, H-6”, J= 6.8 Hz & 1.6 Hz), 8.20 (s, 1H, H-Triazole), 8.24 (s, 1H,
H-Pyrazole), 8.31 (dd, 1H, H-4”, J= 7.6 Hz & J= 1.6 Hz), 8.61 (t, 1H, H-2”, J= 2.0 Hz); IR
(cm-1): 1714 (C=O), 1526, 1329 (NO2), 1219 (C-O); Mass (m/z): 511.6 [M+H]+; Elemental
analysis (%) of C27H19FN6O4, calculated/found: C (63.53/63.55), H(3.75/3.77),
N(16.46/16.43).
(1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl
3-(1,3-diphenyl-1H-pyrazol-4-
yl)acrylate (6k)
Compound 6k was synthesized by reaction of 4a with 5g as per the procedure given above.
1
Appearance: Brick red powder; Yield (%): 74; m.p. 160- 162ºC; H NMR (400 MHz,
CDCl3): 5.41 (s, 2H, CH2), 6.28 (d, 1H, H-, J=16.0 Hz), 7.29-7.37 (m, 2H, H-4,5”), 7.44-
7.51 (m, 5H, H-3,5,3’,4’,5’), 7.61-7.67 (m, 3H, H-2,6,2”), 7.75 (d, 2H, H-2’,6’, J= 6.8 Hz),
7.77 (d, 1H, H-, J= 16.0 Hz), 7.84 (dd, 1H, H-6”, J= 6.4 Hz & 2.4 Hz), 8.05 (s, 1H, H-
Triazole), 8.24 (s, 1H, H-Pyrazole); IR (cm-1): 1720 (C=O), 1230 (C-O); Mass (m/z): 500.1

[M+H]+; Elemental analysis (%) of C27H19ClFN5O2, calculated/found: C (64.87/64.86), H
(3.83/3.85), N(14.01/14.04).
(1-(o-tolyl)-1H-1,2,3-triazol-4-yl)methyl
3-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-
yl)acrylate (6l)
Compound 6l was synthesized by reaction of 4e with 5h as per the procedure given above.
1
Appearance: White powder; Yield (%): 76; m.p. 172-174ºC; H NMR (400 MHz, CDCl3):
2.18 (s, 3H, CH3), 5.30 (s, 2H, CH2), 6.55 (d, 1H, H-, J= 16.0 Hz), 6.90 (d, 1H, H-3”, J= 8.4
Hz), 7.36 (t, 1H, H-4, J=7.2 Hz), 7.46 (dd, 1H, H-5”, J=8 Hz & 2.4 Hz), 7.53-7.66 (m, 9H, H-
2,3,5, 6,3’,5’,4”,6”,β), 7.88 (d, 2H, H-2’,6’, J=8.8 Hz), 8.66 (s, 1H, H-Triazole), 9.25 (s, 1H,
H-Pyrazole); IR (cm-1): 1725 (C=O), 1238 (C-O); Mass (m/z): 496.4 [M+H]+; Elemental
analysis (%) of C28H22ClN5O2, calculated/found: C (67.81/67.82), H (4.47/4.45), N
(14.12/14.13).
(1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl 3-(3-(4-methoxyphenyl)-1-phenyl-
1H-pyrazol-4-yl)acrylate (6m)
Compound 6m was synthesized by reaction of 4d with 5g as per the procedure given above.
1
Appearance: White powder; Yield (%): 72; m.p. 170-172ºC; H NMR (400 MHz, CDCl3):
3.88 (s, 3H, OCH3), 5.40 (s, 2H, CH2), 6.26 (s, 1H, H-, J= 16.0 Hz), 7.00 (d, 2H, H-3’,5’, J=
8.8 Hz), 7.15 (d, 1H, H-5”, J=8.8 Hz), 7.32 (t, 1H, H-4, J=6.8 Hz), 7.46-7.60 (m, 6H, H-
2,3,5,6,2’,6’), 7.74-7.79 (m, 3H, H-2’,6’,β), 8.00 (s, 1H, H-Triazole), 8.21 (s, 1H, H-
Pyrazole); IR (cm-1): 1721 (C=O), 1248 (C-O); Mass (m/z): 530.1 [M+H]+; Elemental
analysis (%) of C28H21ClFN5O3, calculated/found: C (63.46/63.44), H (3.99/4.01), N
(13.22/13.24).
(1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl
3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylate
(6n)
Compound 6n was synthesized by reaction of 4a with 5d as per the procedure given above.
1
Appearance: White powder; Yield(%): 80; m.p. 208-210ºC; H NMR (400 MHz, CDCl3):
5.43 (s, 2H, CH2), 6.28 (d, 1H, H-, J=16.0 Hz), 7.33 (t, 1H, H-4, J=7.6 Hz), 7.42-7.51 (m,
5H, H-3,5,3’,4’,5’), 7.65 (d, 2H, H-2,6, J=7.2 Hz), 7.75 (d, 2H, H-2’,6’, J=8.4 Hz), 7.77 (d,
1H, H-β, J=15.6 Hz), 7.97 (d, 2H, H-2”,6”, J=8.8 Hz), 8.20 (s, 1H, H-Triazole), 8.24 (s, 1H,
H-Pyrazole), 8.41 (d, 2H, H-3”,5”, J=9.2 Hz); IR (cm-1): 1726 (C=O), 1545, 1330 (NO2),
1236 (C-O); HRMS m/z measured for C27H20FN6O4 [M+H]+ 493.1602, m/z calculated

[M+H]+ 493.1624; Elemental analysis (%) of C27H20N6O4, calculated/found: C (65.85/65.83),
H(4.09/4.11), N(17.06/17.07).
(1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl
3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)acrylate
(6o)
Compound 6o was synthesized by reaction of 4b with 5f as per the procedure given above.
Appearance: Brick red powder; Yield(%): 68; m.p. 128-130ºC; 1H NMR (400 MHz, CDCl3):
2.42 (s, 6H, 2 × CH3), 5.41 (s, 2H, CH2), 6.27 (d, 1H, H-, J= 16.0 Hz), 7.28-7.36 (m, 5H, H-
4,3’,5’,3”,5”), 7.46 (t, 2H, H-3,5, J= 7.6 Hz), 7.54 (d, 2H, H-2”,6”, J= 8.4 Hz), 7.60 (d, 2H,
H-2,6, J= 8.0 Hz), 7.74 (d, 2H, H-2’,6’, J= 7.6 Hz), 7.76 (d, 1H, H-, J= 14.4 Hz), 8.04 (s,
1H, H-Triazole), 8.22 (s, 1H, H-Pyrazole); 13C NMR (100 MHz, CDCl3): (21.13, 21.37,
57.54, 116.40, 117.43, 119.37, 119.61, 120.55, 122.21, 126.50, 127.21, 128.59, 129.24,
129.32, 129.51, 129.56, 130.27, 134.64, 136.38, 138.62, 139.07, 139.43, 143.67, 153.54,
166.95); IR (cm-1): 1718 (C=O), 1251 (C-O); Mass (m/z): 476.3 [M+H]+; Elemental analysis
(%) of C29H25N5O2, calculated/found: C (73.25/73.27), H (5.30/5.27), N (14.73/14.75).

Table 5: List of Derivatives Synthesized Under Scheme 1
Compound
6a
R
X
Compound
6f
R
X
Compound R
X
2-NO2
-CH3
4-NO2
-H
-H
3-Cl,4-F
6k
6l
3-NO2
2-NO2
3-Cl
2-CH3
3-Cl,4-F
4-NO2
4-CH3
-H
-H
4-F
-Cl
6b
6c
6d
6e
6g
6h
6i
-OCH3 4-CH3
-OCH3
-H
-Cl
6m
6n
6o
4-CH3
3-NO2
-H
-H
-F
-OCH3
-CH3
3-Cl
6j
3.2 Biological Activity
3.2.1 Anticancer
3.2.1.1. Sulforhodamine B Assay
For evaluating anticancer potential of the synthesized compounds, certain human cancer cell
lines- MCF-7 (breast), A549 (Lung) and HCT-116 and HT-29 (Colon) were obtained from
National Cancer Institute (NCI). Growth of all these cell lines was achieved in tissue culture
flasks in complete medium (RPMI-1640) supplemented with100 μg/mL streptomycin, 10%
fetal bovine serum and 100 units/mL penicillin in carbon dioxide incubator (New Brunswick,
Galaxy 170R, Eppendorf) at 5% CO2, 37°C and 98% RH.
96-well cell culture plates were employed for performing sulforhodamine B (SRB) assay. To
each well, 100 L of the cell suspension of desired density was added and incubated (5%
CO2, 37ºC, 90 % RH) for 24 h. Following incubation for the stipulated time period, 100 μL of
the test samples were added into the wells supplemented with cells. These culture plates were
again incubated for a period of 48 h. Depending on the requirements, controls, blanks and
positive controls were also included in every experiment. Following incubation for the
stipulated period, cells were fixed using 50 μL of ice-cold TCA (50%) for 1 h at a
temperature of 4ºC. Further, plates were washed using distilled water and then air dried.
Following complete drying, addition of SRB dye (100 μL) was made to every well and was
then allowed to stain for 30 min at room temperature. In order to remove excessive dye,
plates were washed with 1 % acetic acid for five times. These plates were then air dried.
Solubilisation of adsorbed dye was done by addition of 100 μL of 10 mM Tris Buffer (pH
10.5) to each well. Following this, these plates were shaken on shaker platform for a duration

of 5 min. 96-well ELISA plate reader (Molecular Devices, Sunnydale, USA) was used for
reading these plates. Every sample was run in triplicate fashion. Mean value of these readings
was taken for getting the results. Concentration of DMSO in cultures was kept < 1% [45].
% Cell Viability= 100 × (T – T0) / (C – T0)
% Growth inhibition = 100 - % Cell viability.
T: Absorbance of Test sample
T0: Absorbance of Blank
C: Absorbance of Control
3.2.1.2 Colony formation inhibition assay
Plating of MCF-7 cells at a density of 500 cells/well was done into 6-well culture plates. An
adherence period of 24 h was given before treatment. Incubation of the cells was done with
culture medium containing compounds 6e, 6f and 6n at IC50 concentration. Following a
period of 24h, medium was replaced with fresh medium and further, the cells were incubated
for a period of 14 days. Washing of cells was then done using 150 mM PBS (pH 7.4)
followed by fixing using 4% paraformaldehyde, staining with 0.5% methylene blue in 10%
ethanol for 30 min and finally rinsing with distilled water for removing excess dye. Plates
were then photographed using a digital camera.
3.2.1.3 Hoechst staining
For Hoechst staining, seeding of MCF-7 cells was done at a density of 5 × 104 cells/well
using 12 well tissue culture plates. Incubation period was set to 24 h. After this, media
containing IC50 concentrations of the compounds 6e, 6f and 6n was used to replace the
culture medium. It was then incubated for 24 h. Following this, 4% para formaldehyde was
used to fix the cells. Further, Hoechst 33242 (5 µg/mL) was used for 30 min at room temp to
stain the cells. Washing of cells was then done using 150 mM PBS (pH 7.4) to remove excess
dye. Fluorescence microscopy (filters, excitation 350 nm and emissions 460 nm) was used to
capture the images of stained nuclei from each well from randomly selected fields to find
apoptotic cells.
3.2.1.4 DNA fragmentation assay

In six-well plates, MCF-7 cells were seeded (1 × 106 cells/well) and then incubated for 24 h.
Following this, cells were subjected to the treatment with IC50 concentration of compounds
6e, 6f and 6n for 24 h. Trypsinisation was used to harvest the cells and centrifugation at 2500
rpm for 5 min at 4ºC was done. Then the pellet was collected and washing was done using
phosphate buffered saline (150 mM PBS; pH 7.4). 400 µg/mL DNase free RNase A
containing 250 µL of lysis buffer (100 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0,
0.25% SDS) was added and incubated for 90 min at 37ºC. This was followed by incubation
with proteinase K (200 µg/mL) for 1 h at 50ºC. The samples were then centrifuged for 5 min
at 3000 rpm at 4ºC and the supernatant was then collected. 65 µL of ammonium acetate
(10M) and ice cold ethanol (500 µL) was added and mixed well. The samples were then
incubated for 1 h at -80ºC and were further subjected to centrifugation at 12000 rpm at 4ºC
for 20 min. Washing of the pellet with 80% ethanol was done followed by air-drying for 10
min at room temperature. After drying, the pellet was dissolved in 50 µL of TBE buffer.
Finally, 1.5% agarose gel electrophoresis in TBE Buffer was used to visualize DNA
laddering after staining with ethidium bromide, which was followed by photography using
digital camera.
3.2.2 Anti-inflammatory
3.2.2.1 Protein Albumin Denaturation Assay
In vitro protein denaturation method was adopted for determining anti-inflammatory potential
of the most active anticancer compounds 6e, 6f and 6n. Method described by Mizushima and
Kobayashi was used for in vitro assessment. Bovine serum albumin and egg albumin proteins
were dissolved in 50 mM sodium phosphate buffer (pH 6.4) at a concentration of 1%. The
reaction mixture comprised test sample (0.1 mL, 1mg/mL) and 0.2 mL albumin protein. Final
volume of the reaction mixture was made to 5 mL using buffer. Incubation of the reaction
mixture was done at 37°C for a period of 20 min. This mixture was then heated to 95°C for
20 min. Following this, the reaction mixture was brought to room temperature and turbidity
was measured using UV-visible spectrophotometer at 660 nm. The entire experiment was
done in triplicate and average values were calculated. Finally, percentage inhibition of protein
denaturation was calculated using the following formula.
Percent Inhibition = [(Abs. Control - Abs. Sample)/Abs. Control] X 100
Here:

Abs. Control: Absorbance of Control
Abs. Sample: Absorbance of Test sample
3.2.2.2 Red Blood Cell Membrane Stabilizing Assay
In this method, 5 mL of blood was collected from healthy adult rat and mixed with 5.0 mL of
Alsever solution. Alsever solution comprised of 2.0% dextrose, 0.8% sodium citrate, 0.5%
citric acid and 0.42% NaCl in water. The complete mixture was then centrifuged at 300 rpm.
Packed cells were washed with isosaline (0.85%, pH 7.2) thrice for removal of cell debris.
Assay mixture comprised of different concentrations of test sample (100, 200 and 500
µg/mL), 1.0 mL phosphate buffer (0.15 M, pH 7.4), 2.0 mL hyposaline (36%) and 0.5 mL red
blood cell suspension. The complete mixture was incubated at 37°C for 30 min. followed by
centrifugation at 3000 rpm for 20 min. Spectrophotometer was used for determining
haemoglobin content of the supernatant at 560 nm. Aspirin (100 and 200 µg/mL) served as
the standard. Percentage of RBC membrane stabilization was determined using formula.
% Protection: [(OD1-OD2)/OD1)] X 100
Here:
OD1: Optical density of test sample
OD2: Optical density of control
3.3 Molecular Docking
Catalytic domain of tubulin with PDB ID 3E22 was downloaded from RCSB protein data
bank. [46]. Protein preparation was done using protein preparation wizard [47], a module of
Schrödinger 2016-1. Water molecules without 3H bonds were removed. Following this,
addition of hydrogen bonds corresponding to pH 7 was made considering proper ionization
states for both basic and acidic amino acid residues. The energy of the crystal structure was
minimized by using force field, OPLS_2005. Further, colchicine bound to tubulin protein was
selected as its center for generating the grid box at active site’s centroid. Around the centroid,
a radius of 16Å was selected in order to define the active site.

Finally, docking of all low energy conformations into the catalytic domain of protein was
done using Grid based Ligand Docking with Energetics (Glide v7.0, Schrödinger 2016-1) in
standard precision (SP) mode in the absence of any constraints.
4. Conclusion
In the present study, synthesis and anticancer evaluation of series of 1,2,3-triazole linked 3-
(1,3-diphenyl-1H-pyrazol-4-yl)acrylates have been carried out. Amongst all the synthesized
compounds, compound 6f showed most promising anticancer effects with IC50 value of 1.962,
3.597, 1.764 and 4.496 µM against A549, HCT-116, MCF-7 and HT-29 cell lines
respectively. Further, studies like Hoechst staining and DNA fragmentation assay
strengthened the preliminary results and ascertained the role of apoptosis induction as the
mechanism of action of the synthesized compounds. Anti-inflammatory assessment for the
three most potent anticancer compounds was performed by protein albumin denaturation
assay and red blood cell membrane stabilizing assay. On the whole, it was observed that all
the synthesized compounds demonstrated significant anti-inflammatory potential in addition
to anticancer effects.
Acknowledgements
Mr. Mohemmed Faraz Khan would like to acknowledge University Grant Commision
(UGC), Government of India as he is the recipient of Research Fellowship of the UGC-
MANF scheme of UGC, Government of India. The authors would also like to thank Jamia
Hamdard, New Delhi and DST-PURSE, Government of India.
Conflict of Interest
Authors declare no conflict of interest.