Design, synthesis and biological evaluation of some new 2-Pyrazoline derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2020.104063

A new series of N-(4-(1-Phenyl-5-aryl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-substitutedbenzamide derivatives were designed and synthesized from new chalcone derivatives. All newly synthesized compounds were determined by using IR, ¹H-NMR,

Full text of DOI:10.1016/j.bioorg.2020.104063

Journal Pre-proofs
Design, Synthesis And Biological Evaluation Of Some New 2-Pyrazoline De-
rivatives As Potential Anticancer Agents
Fatih Tok, Burçin İrem Abas, Özge Çevik, Bedia Koçyiğit-Kaymakç ıoğlu
PII:
S0045-2068(20)31360-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.104063
YBIOO 104063
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
14 April 2020
28 May 2020
28 June 2020
Please cite this article as: F. Tok, B. İrem Abas, O. Çevik, B. Koçyiğit-Kaymakç ıoğlu, Design, Synthesis And
Biological Evaluation Of Some New 2-Pyrazoline Derivatives As Potential Anticancer Agents, Bioorganic
Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104063
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Design, Synthesis And Biological Evaluation Of Some New 2-Pyrazoline Derivatives As
Potential Anticancer Agents
Fatih Toka, Burçin İrem Abasb, Özge Çevikb, Bedia Koçyiğit-Kaymakçıoğlua,*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University,
Istanbul, Turkey
b Department of Biochemistry, School of Medicine, Aydin Adnan Menderes University,
Aydın, Turkey
*Corresponding author:
Bedia Koçyiğit-Kaymakçıoğlu, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Marmara University, Istanbul 34668, Turkey. Tel: +90-216-4142963/1023 E-mail:
bkaymakcioglu@marmara.edu.tr

Highlights
New chalcone and pyrazoline derivatives were designed and synthesized.
Anticancer activity of all synthesized compounds was investigated.
Their pharmacokinetic and drug-like properties were tested using swissadme online server.

GA

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

Abstract
A
new
series
of
N-(4-(1-Phenyl-5-aryl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-
substitutedbenzamide derivatives were designed and synthesized from new chalcone
1
13
derivatives. All newly synthesized compounds were determined by using IR, H-NMR, C-
NMR spectroscopic methods, elemental analysis and evaluated for their in vitro
antiproliferative activities on HeLa, MCF-7, MKN-45 cancer cell lines and NIH-3T3 cell line
using MTT assay. Expression of Bax and Bcl-2 proteins was detected by Western-blot analysis
and caspase-3 enzyme activity was measured. Notably, compounds 1f and 2f showed a
significant cytotoxic effect in all three cancer cells and did not display cytotoxicity on NIH-3T3
normal cells. (IC50 = 26.66±2.73 μM on HeLa, IC50 = 9.41±2.19 μM on MCF-7, IC50
=
5.17±3.54 μM on MKN-45 for 1f. IC50 = 17.96±3.34 μM on HeLa, IC50 = 0.69±0.13 μM on
MCF-7, IC50 = 0.88±0.16 μM on MKN-45 for 2f.) Moreover, 1f and 2f upregulated protein
expression level of Bax and downregulated protein expression level of Bcl-2 in cells. Similarly,
caspase-3 activity was increased in cells via 1f and 2f. It can be concluded that 1f and 2f
activated apoptosis by inducing mitochondrial apoptotic proteins in HeLa, MCF-7, MKN-45.
This could be potentially new anti-cancer derivatives and used to contribute to new therapeutic
development.
Keywords: Pyrazoline; anticancer; caspase activity; apoptosis; chalcone; MTT assay; HeLa.
1 .Introduction
It is predicted that cancer incidence will reach from 18.1 million to 29.5 million, cancer
mortality from 9.6 million to 16.3 million burdens worldwide from the current estimates in
2018 up until 2040 by World Health Organisation [1]. Therefore chemotherapeutic agents play
an important role in the cancer therapy. Many anticancer drugs have potential disadvantages
such as reduced bioavailability, toxicity, drug-resistance, few drug targets, higher cost.
Medicinal chemists aim to develop new anticancer drugs with better properties [2].
Pyrazolines are very important examples of biologically active five-membered ring compounds
in the area of drug design [3]. N-N bond linkage of the pyrazoline ring is regarded as the key
factor in their biological activities. Living organisms establish these bonds more difficulty,
therefore N-N bonds are less available in natural compounds [4]. Pyrazolines have a variety of

biological activities such as antibacterial, antifungal, antiviral, analgesic, antidepressant, anti-
inflammatory and antitumor activities [5-11]. Many drug molecules carrying pyrazole and
pyrazoline ring with different activities are currently available in the market. For example,
antipyrine, ramifenazone, morazone, celecoxib, fezolamine and tepoxalin is adducible [12-14]
(Figure 1).
Figure 1
In this study, we aimed the synthesis of new pyrazoline derivatives from new chalcones and
these synthesized compounds were screened potential anticancer effects on HeLa (human
cervical adenocarcinoma), MCF-7 (human breast adenocarcinoma), MKN-45 (human gastric
carcinoma) cancer cell lines. The tumor selectivity of the synthesized compounds was also
determined on NIH-3T3 mouse embryonic fibroblast cell line using MTT assay.
2. Results and discussion
2.1. Chemistry
New pyrazoline derivatives were synthesized in three steps and synthetic route for the
preparation of target compounds as described in Figure 2. In the first step, amide derivatives
were synthesized with 4-aminoacetophenone and different benzoyl chloride in chloroform.
Then, new chalcone derivatives were prepared according to Claisen-Schmidt condensation by
N-(4-acetylphenyl)-4-substitutedbenzamide with different aromatic aldehydes in aqueous
ethanolic KOH. Finally, new pyrazoline derivatives were obtained with chalcones and
phenylhydrazine hydrochloride in acetic acid (Figure 2). The structures of chalcone and
pyrazoline (1a-s, 2a-s) were established on the basis IR, 1H-NMR, 13C-NMR spectral data and
elemental analysis.
Figure 2
IR spectra of chalcone derivatives afforded C=C stretching (1550-1599 cm-1), amide group N-
1
H stretching (3281-3418 cm-1) and C=O stretching bands (1646-1680 cm-1). In the H-NMR
spectrum of chalcone derivatives, olefinic protons were observed with aromatic protons as
multiplet in the range of 6.17-8.40 ppm.
IR spectra of pyrazoline derivatives afforded C=N stretching (1589-1624 cm-1), amide group
1
N-H stretching (3290-3392 cm-1) and C=C stretching bands (1579-1597 cm-1). The H-NMR
spectrum of pyrazoline ring showed three different signals as doublet of doublet attributed Ha,
Hb and Hx protons due to the ABX spin system in the structures. The Ha, Hb and Hx protons

resonated at 3.07-3.38 ppm (Jab: 17.24-17.88 Hz), 3.77-3.98 ppm (Jax: 5.98-6.41 Hz) and 5.41-
5.68 ppm (Jbx: 11.90-12.36 Hz), respectively. NH protons of amide groups appeared as a singlet
peak at 10.29-10.71 ppm. All other aliphatic and aromatic protons were observed at expected
regions. In 13C-NMR spectra, the C3, C4 and C5 carbons of the pyrazoline ring were found at
147.5-148.7 ppm, 41.5-43.5 ppm and 57.1-63.5 ppm, respectively. Amide C=O and chalcone
C=O peaks appeared at 161.7-166.3 ppm and 186.7-188.7 ppm, respectively. The values of
elemental analysis were within ± 0.4% of the theoretical values.
2.2. Biology
2.2.1. Cytotoxicity of the compound
The cytotoxicity of the synthesized new chalcone and pyrazoline derivatives was assessed in
three cancer cells lines: cervix (HeLa), breast (MCF-7) and gastric (MKN-45). Cancer cells
were incubated with these newly synthesized compounds (0-1000 µM) at different
concentrations for 24 hours. Cell viability was measured as a colorimetric assay based on
mitochondrial dehydrogenase activities changes and into a colored formazan production.
According to our results, the compounds exhibit the highest concentrations of toxicity. (Table
1). However, the compounds were found to be different IC50 values in cells when compared
with having an effect below 50 µM concentration between cells. Therefore, compounds with
the appropriate IC50 value must exhibit less than 50 µM, a clinically significant concentration,
to become a drug candidate. Potential anticancer drug candidates should also be cytotoxic to
cancer cells but harmless to normal cells. Toxicity profiles of the newly synthesized chalcone
and pyrazoline derivatives were tested against NIH-3T3 normal mouse fibroblast cells and were
found not to harm healthy cells. Cell survival was performed with compound IC50 values of 1f,
2f, 1h, 2h, 1m and 2m on HELA cells and 1f, 2f, 1i, 2i, 1r and 2r on MKN-45 cells and 1d,
2d, 1f and 2f on MCF-7 cells for 6h, 24 h and 48h. Compound 1f and 2f appeared to have the
same anti-survival effect in all three cells (Figure 3a-c). Compound 1f and 2f selected from
synthesized chalcone and pyrazoline derivatives were treated with cells and changes in the cells
were observed under a light microscope. The selected compound 1f and 2f had an effect on the
cells and cellular contractions occurred in HELA, MKN-45 and MCF7 cells. At the same time,
the interconnection of the cells with each other decreased and exhibited a more rounded
morphology rather than spreading adhesive cells (Figure 3d). Looking at the data, the
compounds 1f and 2f were found to show strong anticancer activity and were potentially
potential drug candidates.

Figure 3
Heterocyclic compounds are extremely important for the development of active molecules.
Biological molecules such as DNA, RNA bear some heterocyclic ring in their major skeleton.
These rings interact enzyme or receptor easily due to the ability to form hydrogen bond
acceptors and donors. Therefore the probability of showing strong activity increases.
Compounds 2f, 2l and 2s contain pyrazoline ring as well as furan ring demonstrated strong
cytotoxic effect on cancer cells. Moreover these compounds 2f, 2l and 2s showed higher activity
than chalcone analogs such as 1f, 1l and 1s.
Table 1
2.2.2. Cell apoptosis of the compounds
Apoptosis occurs in the cell in two ways, one is called extrinsic and the other is intrinsic ways,
apoptosis is determined depending on the initiator signal. The extrinsic pathway is initiated in
the cell membrane through death receptors. On the other hand, the intrinsic pathway is
controlled through the Bcl-2 protein family and affects the permeability of the mitochondrial
outer membrane. Both ways It is controlled by cysteine proteases specific to aspartic acid
residues and its common enzyme caspase-3 in two ways. To investigate the effects of
compounds 1f and 2f on HeLa, MCF7, MKN-45 cell apoptosis, these cells were treated with
IC50 μM values for 24h and then analysis of caspase-3 activity and mitochondrial apoptotic
proteins were performed. The caspase-3 activity showed a significant increase (compare to
control) in the 1f and 2f compounds treated HeLa, MCF7, MKN-45 cells (Figure 4a). Western
blotting was performed to evaluate the mitochondrial apoptosis expression levels of the Bax,
Bcl-2 and GAPDH (control) proteins. As expected, the proapoptotic Bax levels were increased
in the 1f and 2f compounds and the antiapoptotic Bcl-2 levels were decreased in the 1f and 2f
compounds treated HeLa, MCF7, MKN-45 cells (Figure 4b-e). Importantly, alterations in the
relative levels of Bax and Bcl-2 are important in determining whether cells will undergo
apoptosis. The ratio of Bax/Bcl-2 protein expression levels in cells treated with compounds 1f
and 2f was significantly increased in HeLa, MCF7, MKN-45 cells (Figure 4f). The present
findings suggest that the cell apoptosis induced by treatment with 1f and 2f is dependent on
alterations in the expression of Bcl-2 family proteins and caspase-3 activity, and is associated
with the mitochondrial pathway in HeLa, MCF7, MKN-45 cells.
Figure 4

2.3. Physicochemical properties and pharmacokinetic profile predictions
Prediction of physicochemical and pharmacokinetic properties of the compounds is very
important for the development of new drug candidates. If these properties of molecules are not
good enough, it can not succeed in clinical candidate drugs for the treatment of illness.
Bioavailability of synthesized compounds can be tested with determining druglike properties
by using the Lipinski rule of five and the Veber rule. Compounds should have at least three of
the following properties according to Lipinski rule: molecular weight should be lower than 500,
less than 5 logP, number of hydrogen bond donors below 5 and number of hydrogen bond
acceptors below 10. Furthermore, the number of rotatable bonds should be lower than the
maximum value of 10 and the Polar Surface Area should be less than the maximum value of
140 Å2 according to Veber rule [15-17]. The screening results are presented in Table 2. These
values demonstrate that all the synthesized compounds are not violating the Lipinski and Veber
rules. Especially compound 1f and 2f, the most promising agent, exhibited hopeful
bioavailability score which was defined as good permeability. Compound 1f and 2f were
moderately soluble and showed without any violations of other drug-like properties Egan and
Ghose rules (Table 3).
Table 2
Table 3
Swissadme program provides important information about pharmacokinetic properties (such as
gastrointestinal absorption, brain access and P-glycoprotein) of molecules. BOILED-Egg server
predicts these pharmacokinetics by using wLogP and TPSA. The yellow region (BBB) shows
molecules which are for a high probability of passively permeated through the blood brain
barrier. The white region (HIA) shows molecules that are for a high probability of passively
absorbed by the gastrointestinal tract. However, molecules on the gray region have low
absorption and limited brain access. Blue dots (PGP+) are for molecules predicted to be flowed
out from the central nervous system by the P-glycoprotein. Red dots (PGP-) are for molecules
predicted not to be effluated from the central nervous system by the P-glycoprotein [15]. It was
predicted that compound 1j from chalcones and 2j from pyrazolines had low gastrointestinal
absorption and brain penetration because both molecules carried steric hindrance from bulky
substituents such as nitro groups on aromatic rings. All compounds except 1j and 2j exhibited
good bioavailability. Compound 1f and 2f, the most promising agents, displayed high blood-
brain permeability and gastrointestinal absorption. Compound 1f was not subject to active

efflux (red dot), however compound 2f was pumped-out from the brain (blue dot) easily because
of being a P-glycoprotein substrate. Compound 1f and 2f can be potential candidates for drug
discovery because of their good drug-likeness and pharmacokinetic properties The boiled egg
predictive models of all compounds were given in Figure 5.
Figure 5
3. Conclusion
In this article, we synthesized new chalcone and pyrazoline derivatives which have exhibited
cytotoxic activities against HeLa, MCF7, MKN-45 cancer cell lines, and NIH-3T3 normal cell.
Among them, compounds 2l and 2s containing pyrazole and furan ring demonstrated the highest
cytotoxic activity on MCF7 and MKN-45 cancer cells. (IC50 = 0.06±0.04 μM on MCF-7, IC50
= 0.02±0.01 μM on MKN-45 for 2l. IC50 = 0.16±0.04 μM on MCF-7, IC50 = 0.02±0.01 μM on
MKN-45 for 2s.) On the other hand, 1f and 2f having furan moiety showed strong anticancer
activity against all cancer cells with good selectivity. (IC50 = 26.66±2.73 μM on HeLa, IC50 =
9.41±2.19 μM on MCF-7, IC50 = 5.17±3.54 μM on MKN-45 for 1f. IC50 = 17.96±3.34 μM on
HeLa, IC50 = 0.69±0.13 μM on MCF-7, IC50 = 0.88±0.16 μM on MKN-45 for 2f.) Cytotoxic
effect of 1f and 2f on NIH-3T3 normal cell did not observe. We observed that compound 1f and
2f were a potent inducer of apoptosis in HeLa, MCF7, MKN-45 cells and caused an alteration
in the induction of mitochondrial apoptotic events. At the same time, the promising compound
1f and 2f decreased the level of Bcl-2 and increased the level of Bax, which was consistent
with the result of caspase-3 enzyme activity analysis in inducing apoptosis. Therefore,
compounds 1f and 2f can be potential anticancer agents with further investigation.
4. Experimental
4.1. Chemistry
Chemicals and solvents were purchased from Sigma Aldrich (St. Louis, MO, USA),
Merck(Darmstadt, Germany). The progress of reactions was monitored via thin layer
chromatography (TLC), performed on commercially available silica gel (Kieselgel 60, F254)
coated aluminium sheets (Merck) by using petroleum ether:ethyl acetate (10:90) as solvent
system. The visualization on TLC was done under ultra-violet (UV) light (λ= 254 nm). Melting
points were determined by the Schmelzpunktbestimmer SMP II apparatus. Infrared spectra
were recorded on a Shimadzu FTIR 8400 S Spectrometer. Proton nuclear magnetic resonance
(NMR) (400 MHz) and carbon NMR (150 MHz) spectra were obtained (DMSO-d6) were run

on a Bruker ACP 200 spectrometer (Bruker Corp., Billerica, MA, USA). It was used
deuterodimethylsulfoxide (DMSO-d6) as the solvent and tetramethylsilane (TMS) as an internal
standard. Elemental analysis was determined by CHNS-932 (LECO).
4.1.1. General procedure of amide synthesis
Firstly, 1mmol 4-aminoacetophenone was dissolved in 20 mL chloroform. 1mmol
Substitutedbenzoyl chloride was added to the reaction liquid and it was stirred and refluxed in
a water bath over a period of 8 h. After evaporation of the solvent, amide derivatives were
washed with distilled water and filtered [18].
4.1.2. General procedure of chalcone synthesis
1mmol N-(4-acetylphenyl)-4-substitutedbenzamide and different aromatic aldehydes were
dissolved in ethanol. %40 KOH (1mL) was added to the reaction liquid. It was stirred on room
temperature for 10 h and poured into ice-cold water. The precipitated product was washed with
water, filtered and recrystallized from methanol [19].
4.1.2.1. N-(4-Cinnamoylphenyl)-4-fluorobenzamide (1a)
Yield 75%; white solid; m.p. 201.2-202.0ºC; IR (max, cm-1): 3362 (N-H), 3061 (=C-H), 1654
(C=O), 1585, 1573 (C=C); 1H-NMR (400 MHz, DMSO-d6):  7.36-8.21 (m, 15H, CH=CH and
Ar-H), 10.59 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  188.0, 165.5, 144.1, 143.9, 135.2,
133.1, 131.5, 131.1, 131.0, 129.4, 129.2, 122.5, 120.1, 119.9, 116.1, 115.9, 115.7; Anal. calcd.
for C22H16FNO2: C, 76.51; H, 4.67; N, 4.06; Found: C, 76.47; H, 4.65; N, 4.09%.
4.1.2.2. 4-Fluoro-N-(4-(3-(4-fluorophenyl)acryloyl)phenyl)benzamide (1b)
Yield 77%; white solid; m.p. 222.3-222.9ºC; IR (max, cm-1): 3369 (N-H), 3086 (=C-H), 1656
(C=O), 1593, 1579 (C=C); 1H-NMR (400 MHz, DMSO-d6):  7.27-8.20 (m, 14H, CH=CH and
Ar-H), 10.61 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6):  188.0, 165.5, 164.6, 163.9,
144.2, 142.6, 133.1, 132.5, 131.9, 131.6, 131.5, 131.1, 130.2, 129.7, 122.3, 120.1, 119.9, 116.4,
115.9; Anal. calcd. for C22H15F2NO2: C, 72.72; H, 4.16; N, 3.85; Found: C, 72.62; H, 4.14; N,
3.82%.
4.1.2.3. N-(4-(3-(4-Chlorophenyl)acryloyl)phenyl)-4-fluorobenzamide (1c)
Yield 72%; yellow solid; m.p. 164.5-165.4ºC; IR (max, cm-1): 3281 (N-H), 3030 (=C-H), 1647
(C=O), 1597, 1587 (C=C); 1H-NMR (400 MHz, DMSO-d6):  7.36-8.21 (m, 14H, CH=CH and

Ar-H), 10.59 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  186.8, 164.4, 162.8, 143.2, 141.3,
134.4, 133.1, 131.9, 130.4, 130.0, 129.9, 128.7, 128.3, 122.1, 119.0, 118.9, 114.9, 114.7; Anal.
calcd. for C22H15ClFNO2: C, 69.57; H, 3.98; N, 3.69; Found: C, 69.65; H, 3.95; N, 3.72%.
4.1.2.4. 4-Fluoro-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)benzamide (1d)
Yield 70%; yellow solid; m.p. 236.7-237.5ºC; IR (max, cm-1): 3363 (N-H), 3080 (=C-H), 1666
(C=O), 1589 (C=C); 1H-NMR (400 MHz, DMSO-d6):  7.37-8.30 (m, 14H, CH=CH and Ar-
13
H), 10.63 (s, 1H, NH); C-NMR (150 MHz,DMSO-d6):  188.7, 164.5, 162.8, 147.4, 143.6,
140.7, 139.9, 131.6, 130.4, 130.1, 129.3, 125.4, 123.3 119.0, 114.9, 114.7; Anal. calcd. for
C22H15FN2O4: C, 67.69; H, 3.87; N, 7.18; Found: C, 67.77; H, 3.85; N, 7.23%.
4.1.2.5. 4-Fluoro-N-(4-(3-p-tolylacryloyl)phenyl)benzamide (1e)
Yield 85%; yellow solid; m.p. 203.9-204.5ºC; IR (max, cm-1): 3404 (N-H), 3032 (=C-H), 1666
(C=O), 1591, 1566 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.35 (s, 3H, CH3), 7.26-8.12 (m,
14H, CH=CH and Ar-H), 10.49 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  186.9, 164.4,
162.8, 143.1, 142.9, 139.9, 132.1, 131.5, 130.4, 130.1, 129.1, 128.9, 120.3, 119.0, 118.9, 114.9,
114.8, 20.5; Anal. calcd. for C23H18FNO2: C, 76.86; H, 5.05; N, 3.90; Found: C, 76.74; H, 5.06;
N, 3.89%.
4.1.2.6. 4-Fluoro-N-(4-(3-(furan-2-yl)acryloyl)phenyl)benzamide (1f)
Yield 83%; yellow solid; m.p. 180.3-181.1ºC; IR (max, cm-1): 3366 (N-H), 3095 (=C-H), 1651
(C=O), 1599, 1581 (C=C); 1H-NMR (400 MHz, DMSO-d6):  7.40-8.19 (m, 13H, CH=CH and
Ar-H), 10.61 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  187.5, 165.5, 163.9, 151.7, 146.6,
144.0, 133.1, 131.5, 131.1, 130.5, 129.9, 120.1, 119.1, 117.3, 115.9, 113.6; Anal. calcd. for
C20H14FNO3: C, 71.64; H, 4.21; N, 4.18; Found: C, 71.77; H, 4.19; N, 4.20%.
4.1.2.7. N-(4-Cinnamoylphenyl)-4-nitrobenzamide (1g)
Yield 75%; yellow solid; m.p. 235.6-236.3ºC; IR (max, cm-1): 3335 (N-H), 3076 (=C-H), 1672,
1
1649 (C=O), 1591, 1573 (C=C); H-NMR (400 MHz, DMSO-d6):  7.45-8.37 (m, 15H,
CH=CH and Ar-H), 10.81 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  187.5, 165.5, 163.9,
151.7, 146.6, 144.0, 133.1, 131.5, 131.1, 130.5, 129.9, 120.1, 119.1, 117.3, 115.9, 113.6; Anal.
calcd. for C22H16N2O4: C, 70.96; H, 4.33; N, 7.52; Found: C, 70.87; H, 4.30; N, 7.57%.
4.1.2.8. N-(4-(3-(4-Fluorophenyl)acryloyl)phenyl)-4-nitrobenzamide (1h)

Yield 78%; yellow solid; m.p. 204.5-205.3ºC; IR (max, cm-1): 3418 (N-H), 3093 (=C-H), 1680,
1660 (C=O), 1587 (C=C); 1H-NMR (400 MHz, DMSO-d6):  7.27-8.39 (m, 14H, CH=CH and
Ar-H), 10.85 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  187.5, 165.5, 163.9, 151.7, 146.6,
144.0, 133.1, 131.5, 131.1, 130.5, 129.9, 120.1, 119.1, 117.3, 115.9, 113.6; Anal. calcd. for
C22H15FN2O4: C, 67.69; H, 3.87; N, 7.18; Found: C, 67.75; H, 3.84; N, 7.23%.
4.1.2.9. N-(4-(3-(4-Chlorophenyl)acryloyl)phenyl)-4-nitrobenzamide (1i)
Yield 79%; yellow solid; m.p. 186.6-187.3ºC; IR (max, cm-1): 3381 (N-H), 3080 (=C-H), 1670,
1
1646 (C=O), 1586, 1566 (C=C); H-NMR (400 MHz, DMSO-d6):  6.17-8.38 (m, 14H,
CH=CH and Ar-H), 10.91 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  187.5, 165.5, 163.9,
151.7, 146.6, 144.0, 133.1, 131.5, 131.1, 130.5, 129.9, 120.1, 119.1, 117.3, 115.9, 113.6; Anal.
calcd. for C22H15ClN2O4: C, 64.95; H, 3.72; N, 6.89; Found: C, 64.87; H, 3.69; N, 6.94%.
4.1.2.10. 4-Nitro-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)benzamide (1j)
Yield 73%; yellow solid; m.p. 226.7-227.4ºC; IR (max, cm-1): 3381 (N-H), 3093 (=C-H), 1672,
1655 (C=O), 1575 (C=C); 1H-NMR (400 MHz, DMSO-d6):  7.80-8.40 (m, 14H, CH=CH and
Ar-H), 10.92 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  187.5, 165.5, 163.9, 151.7, 146.6,
144.0, 133.1, 131.5, 131.1, 130.5, 129.9, 120.1, 119.1, 117.3, 115.9, 113.6; Anal. calcd. for
C22H15N3O6: C, 63.31; H, 3.62; N, 10.07; Found: C, 63.19; H, 3.59; N, 10.02%.
4.1.2.11. 4-Nitro-N-(4-(3-p-tolylacryloyl)phenyl)benzamide (1k)
Yield 74%; yellow solid; m.p. 201.2-202.1ºC; IR (max, cm-1): 3379 (N-H), 3093 (=C-H), 2983,
2887 (C-H), 1672, 1651 (C=O), 1573 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.35 (s, 3H,
CH3), 7.27-8.39 (m, 14H, CH=CH and Ar-H), 10.88 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-
d6):  187.5, 165.5, 163.9, 151.7, 146.6, 144.0, 133.1, 131.5, 131.1, 130.5, 129.9, 120.1, 119.1,
117.3, 115.9, 113.6; Anal. calcd. for C23H18N2O4: C, 71.49; H, 4.70; N, 7.25; Found: C, 71.32;
H, 4.67; N, 7.26%.
4.1.2.12. N-(4-(3-(furan-2-yl)acryloyl)phenyl)-4-nitrobenzamide (1l)
Yield 81%; yellow solid; m.p. 212.3-212.9ºC; IR (max, cm-1): 3377 (N-H), 3093 (=C-H), 1674,
1656 (C=O), 1573 (C=C); 1H-NMR (400 MHz, DMSO-d6):  6.69-8.40 (m, 13H, CH=CH and
Ar-H), 10.89 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  187.5, 165.5, 163.9, 151.7, 146.6,
144.0, 133.1, 131.5, 131.1, 130.5, 129.9, 120.1, 119.1, 117.3, 115.9, 113.6; Anal. calcd. for
C20H14N2O5: C, 66.30; H, 3.89; N, 7.73; Found: C, 66.41; H, 3.88; N, 7.76%.

4.1.2.13. N-(4-Cinnamoylphenyl)-4-methylbenzamide (1m)
Yield 82%; white solid; m.p. 210.8-211.2ºC; IR (max, cm-1): 3371 (N-H), 3056 (=C-H), 2968,
2904 (C-H), 1666, 1651 (C=O), 1593, 1573 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.40 (s,
3H, CH3), 7.35-8.21 (m, 15H, CH=CH and Ar-H), 10.51 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  188.1, 166.3, 144.4, 143.8, 142.4, 135.2, 132.9, 130.9, 129.7, 129.4, 129.2,
128.4, 122.4, 120.0, 119.9, 21.51; Anal. calcd. for C23H19NO2: C, 80.92; H, 5.61; N, 4.10;
Found: C, 81.01; H, 5.59; N, 4.13%.
4.1.2.14. N-(4-(3-(4-Fluorophenyl)acryloyl)phenyl)-4-methylbenzamide (1n)
Yield 80%; white solid; m.p. 181.5-182.3ºC; IR (max, cm-1): 3373 (N-H), 3082 (=C-H), 2970,
2912 (C-H), 1672, 1651 (C=O), 1593, 1573 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.39 (s,
3H, CH3), 7.01-8.21 (m, 14H, CH=CH and Ar-H), 10.49 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  187.9, 161.7, 144.3, 144.1, 143.8, 142.6, 133.2, 132.9, 131.9, 131.6, 130.2,
130.1, 129.7, 129.3, 128.4, 127.9, 122.3, 119.9, 116.5, 114.8, 21.51; Anal. calcd. for
C23H18FNO2: C, 76.86; H, 5.05; N, 3.90; Found: C, 76.72; H, 5.08; N, 3.88%.
4.1.2.15. N-(4-(3-(4-Chlorophenyl)acryloyl)phenyl)-4-methylbenzamide (1o)
Yield 77%; white solid; m.p. 208.3-208.9ºC; IR (max, cm-1): 3355 (N-H), 3086 (=C-H), 2978,
2904 (C-H), 1651 (C=O), 1597, 1572 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.38 (s, 3H,
CH3), 7.33-8.20 (m, 14H, CH=CH and Ar-H), 10.47 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-
d6):  186.8, 165.2, 143.3, 141.4, 134.3, 133.2, 131.7, 129.9, 128.7, 128.3, 127.3, 122.1, 118.9,
20.4; Anal. calcd. for C23H18ClNO2: C, 73.50; H, 4.83; N, 3.73; Found: C, 73.60; H, 4.85; N,
3.75%.
4.1.2.16. 4-Methyl-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)benzamide (1p)
Yield 85%; white solid; m.p. 200.9-201.4ºC; IR (max, cm-1): 3348 (N-H), 3086 (=C-H), 2978,
2906 (C-H), 1658 (C=O), 1587, 1573 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.39 (s, 3H,
CH3), 7.34-8.30 (m, 14H, CH=CH and Ar-H), 10.51 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-
d6):  186.7, 165.2, 147.4, 143.7, 141.4, 140.7, 139.9, 131.4, 129.3, 128.6, 128.3, 127.3, 125.4,
123.3, 118.9, 20.45; Anal. calcd. for C23H18N2O4: C, 71.49; H, 4.70; N, 7.25; Found: C, 71.62;
H, 4.71; N, 7.23%.
4.1.2.17. 4-Methyl-N-(4-(3-p-tolylacryloyl)phenyl)benzamide (1r)

Yield 82%; white solid; m.p. 155.3-155.9ºC; IR (max, cm-1): 3352 (N-H), 3032 (=C-H), 2982
(C-H), 1688, 1649 (C=O), 1591, 1579 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.35 (s, 3H,
CH3), 2.39 (s, 3H, CH3), 7.69-8.20 (m, 14H, CH=CH and Ar-H), 10.49 (s, 1H, NH); 13C-NMR
(150 MHz,DMSO-d6):  188.0, 166.2, 144.2, 143.9, 142.4, 141.0, 133.0, 132.5, 132.1, 130.1,
129.7, 128.3, 121.3, 119.9, 26.9, 21.5; Anal. calcd. for C24H21NO2: C, 81.10; H, 5.96; N, 3.94;
Found: C, 80.97; H, 5.97; N, 3.93%.
4.1.2.18. N-(4-(3-(furan-2-yl)acryloyl)phenyl)-4-methylbenzamide (1s)
Yield 82%; white solid; m.p. 187.6-188.2ºC; IR (max, cm-1): 3331 (N-H), 3095 (=C-H), 2980
(C-H), 1680, 1651 (C=O), 1597, 1550 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.39 (s, 3H,
CH3), 6.69-8.12 (m, 13H, CH=CH and Ar-H), 10.50 (s, 1H, NH); 13C-NMR (150 MHz,DMSO-
d6):  187.5, 166.2, 151.7, 146.5, 144.2, 142.5, 132.9, 130.4, 129.9, 129.4, 128.3, 120.0, 119.8,
117.2, 113.57, 21.51; Anal. calcd. for C21H17NO3: C, 76.12; H, 5.17; N, 4.23; Found: C, 76.21;
H, 5.15; N, 4.25%.
4.1.3. General procedure of pyrazoline synthesis
10 mmol chalcone derivatives and 10 mmol phenylhydrazine hydrochloride were stirred in
glacial acetic acid (10 mL) and heated under reflux for 12 h. The precipitated product was
washed with water, filtered and recrystallized from ethanol [20].
4.1.3.1. N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-fluorobenzamide (2a)
Yield 75%; yellow solid; m.p. 250.8-251.2ºC; IR (max, cm-1): 3336 (N-H), 3063 (=C-H), 1651
1
(C=O), 1589 (C=N), 1579 (C=C); H-NMR (400 MHz, DMSO-d6):  3.08 (dd, Jax 6.36 Hz,
Jab 17.40 Hz, 1H, Ha), 3.92 (dd, Jbx 12.16 Hz, Jab 17.39 Hz, 1H, Hb), 5.46 (dd, Jax 6.34 Hz,
Jbx 12.12 Hz, 1H, Hx), 6.68-8.07 (m, 18H, Ar-H), 10.40 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  165.4, 163.7, 147.5, 144.8, 143.1, 140.0, 131.7, 130.9, 129.4, 129.3, 128.0,
127.8, 126.6, 126.3, 120.6, 118.9, 115.9, 115.7, 113.3, 63.5, 43.5; Anal. calcd. for C28H22FN3O:
C, 77.22; H, 5.09; N, 9.65; Found: C, 77.25; H, 5.10; N, 9.67%.
4.1.3.2.
4-Fluoro-N-(4-(5-(4-fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-
yl)phenyl)benzamide (2b)
Yield 78%; yellow solid; m.p. 270.8-271.5ºC; IR (max, cm-1): 3336 (N-H), 3049 (=C-H), 1651
1
(C=O), 1593 (C=N), 1579 (C=C); H-NMR (400 MHz, DMSO-d6):  3.10 (dd, Jax 6.26 Hz,
Jab 17.42 Hz, 1H, Ha), 3.90 (dd, Jbx 12.15 Hz, Jab 17.41 Hz, 1H, Hb), 5.49 (dd, Jax 6.21 Hz,

Jbx 12.10 Hz, 1H, Hx), 6.72-8.06 (m, 17H, Ar-H), 10.39 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  165.4, 164.9, 163.7, 162.6, 160.9, 147.5, 144.7, 140.1, 139.2, 131.6, 130.9,
129.3, 128.3, 128.0, 126.7, 120.6, 119.0, 116.3, 115.9, 113.3, 62.8, 43.4; Anal. calcd. for
C28H21F2N3O: C, 74.16; H, 4.67; N, 9.27; Found: C, 74.33; H, 4.64; N, 9.25%.
4.1.3.3.
N-(4-(5-(4-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-
fluorobenzamide (2c)
Yield 81%; yellow solid; m.p. 303.1-303.9ºC; IR (max, cm-1): 3331 (N-H), 3049 (=C-H), 1651
1
(C=O), 1595 (C=N), 1579 (C=C); H-NMR (400 MHz, DMSO-d6):  3.12 (dd, Jax 6.23 Hz,
Jab 17.44 Hz, 1H, Ha), 3.91 (dd, Jbx 12.16 Hz, Jab 17.41 Hz, 1H, Hb), 5.50 (dd, Jax 6.20 Hz,
Jbx 12.14 Hz, 1H, Hx), 6.70-8.07 (m, 17H, Ar-H), 10.40 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  165.4, 164.9, 163.7, 147.6, 144.6, 142.0, 140.1, 132.3, 131.6, 130.9, 129.4,
128.3, 127.9, 126.7, 120.6, 119.0, 115.9, 113.3, 62.8, 43.3; Anal. calcd. for C28H21ClFN3O: C,
71.56; H, 4.50; N, 8.94; Found: C, 71.71; H, 4.48; N, 8.98%.
4.1.3.4.
4-Fluoro-N-(4-(5-(4-nitrophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-
yl)phenyl)benzamide (2d)
Yield 83%; red solid; m.p. 277.2-278.1ºC; IR (max, cm-1): 3336 (N-H), 3082 (=C-H), 1649
1
(C=O), 1593 (C=N), 1579 (C=C); H-NMR (400 MHz, DMSO-d6):  3.14 (dd, Jax 6.28 Hz,
Jab 17.49 Hz, 1H, Ha), 3.98 (dd, Jbx 12.28 Hz, Jab 17.51 Hz, 1H, Hb), 5.68 (dd, Jax 6.26 Hz,
Jbx 12.28 Hz, 1H, Hx), 6.71-8.30 (m, 17H, Ar-H), 10.41 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  165.5, 164.9, 163.7, 150.6, 148.7, 144.4, 141.7, 140.9, 132.7, 131.2, 131.0,
130.5, 129.5, 127.9, 126.7, 128.8, 124.2, 120.1, 119.3, 115.7, 113.3, 62.8, 42.5; Anal. calcd. for
C28H21FN4O3: C, 69.99; H, 4.41; N, 11.66; Found: C, 70.11; H, 4.40; N, 11.68%.
4.1.3.5.
4-Fluoro-N-(4-(1-phenyl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)benzamide
(2e)
Yield 84%; yellow solid; m.p. 265.5-266.3ºC; IR (max, cm-1): 3321 (N-H), 3084 (=C-H), 2989,
2895 (C-H), 1649 (C=O), 1591 (C=N), 1579 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.24
(s, 3H, CH3), 3.11 (dd, Jax 6.20 Hz, Jab 17.85 Hz, 1H, Ha), 3.89 (dd, Jbx 11.90 Hz, Jab 17.81
Hz, 1H, Hb), 5.42 (dd, Jax 6.25 Hz, Jbx 11.92 Hz, 1H, Hx), 6.69-8.08 (m, 17H, Ar-H), 10.41
(s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  165.4, 163.7, 147.5, 144.8, 140.1, 137.0, 131.7,
130.9, 130.0, 129.2, 128.1, 126.2, 120.6, 118.8, 115.9, 115.7, 113.3, 63.3, 43.5, 21.1; Anal.
calcd. for C29H24FN3O: C, 77.49; H, 5.38; N, 9.35; Found: C, 77.57; H, 5.39; N, 9.40%.

4.1.3.6.
4-Fluoro-N-(4-(5-(furan-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-
yl)phenyl)benzamide (2f)
Yield 77%; yellow solid; m.p. 272.7-273.6ºC; IR (max, cm-1): 3338 (N-H), 3057 (=C-H), 1654
1
(C=O), 1589 (C=N), 1579 (C=C); H-NMR (400 MHz, DMSO-d6):  3.37 (dd, Jax 5.98 Hz,
Jab 17.28 Hz, 1H, Ha), 3.78 (dd, Jbx 12.16 Hz, Jab 17.28 Hz, 1H, Hb), 5.58 (dd, Jax 5.98 Hz,
Jbx 12.17 Hz, 1H, Hx), 6.37-8.08 (m, 16H, Ar-H), 10.41 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  165.4, 163.7, 153.7, 147.9, 144.9, 143.3, 140.1, 131.6, 130.8, 129.3, 127.9,
126.8, 120.7, 119.3, 116.0, 115.8, 115.6, 113.6, 111.0, 108.3, 57.1, 42.5; Anal. calcd. for
C26H20FN3O2: C, 73.40; H, 4.74; N, 9.88; Found: C, 73.29; H, 4.78; N, 9.91%.
4.1.3.7. N-(4-(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-nitrobenzamide (2g)
Yield 84%; red solid; m.p. 220.1-220.8ºC; IR (max, cm-1): 3331 (N-H), 3059 (=C-H), 1691
1
(C=O), 1622 (C=N), 1589 (C=C); H-NMR (400 MHz, DMSO-d6):  3.11 (dd, Jax 6.41 Hz,
Jab 17.39 Hz, 1H, Ha), 3.91 (dd, Jbx 12.19 Hz, Jab 17.39 Hz, 1H, Hb), 5.46 (dd, Jax 6.37 Hz,
Jbx 12.16 Hz, 1H, Hx), 6.68-8.38 (m, 18H, Ar-H), 10.68 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  164.3, 149.6, 147.4, 144.7, 143.0, 140.8, 139.6, 129.7, 129.3, 128.5, 127.8,
126.7, 124.0, 120.7, 118.9, 113.3, 63.5, 43.5; Anal. calcd. for C28H22N4O3: C, 72.71; H, 4.79;
N, 12.11; Found: C, 72.66; H, 4.80; N, 12.14%
4.1.3.8.
N-(4-(5-(4-Fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-
nitrobenzamide (2h)
Yield 80%; red solid; m.p. 226.3-226.9ºC; IR (max, cm-1): 3319 (N-H), 3057 (=C-H), 1691
1
(C=O), 1620 (C=N), 1597 (C=C); H-NMR (400 MHz, DMSO-d6):  3.11 (dd, Jax 6.28 Hz,
Jab 17.86 Hz, 1H, Ha), 3.90 (dd, Jbx 12.36 Hz, Jab 17.88 Hz, 1H, Hb), 5.50 (dd, Jax 6.28 Hz,
Jbx 12.36 Hz, 1H, Hx), 6.69-8.38 (m, 17H, Ar-H), 10.69 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  164.3, 162.6, 161.0, 149.6, 147.4, 144.6, 140.8 139.6, 139.1, 129.7, 128.4,
128.3, 126.7, 124.0, 120.7, 119.0, 116.1, 113.4, 62.8, 43.4; Anal. calcd. for C28H21FN4O3: C,
69.99; H, 4.41; N, 11.66; Found: C, 69.89; H, 4.43; N, 11.69%.
4.1.3.9.
N-(4-(5-(4-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-
nitrobenzamide (2i)
Yield 78%; red solid; m.p. 232.8-233.5ºC; IR (max, cm-1): 3344 (N-H), 3085 (=C-H), 1697
1
(C=O), 1620 (C=N), 1589 (C=C); H-NMR (400 MHz, DMSO-d6):  3.09 (dd, Jax 6.20 Hz,

Jab 17.40 Hz, 1H, Ha), 3.92 (dd, Jbx 12.17 Hz, Jab 17.41 Hz, 1H, Hb), 5.51 (dd, Jax 6.19 Hz,
Jbx 12.15 Hz, 1H, Hx), 6.70-8.38 (m, 17H, Ar-H), 10.70 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  164.3, 149.6, 147.5, 144.5, 141.9, 140.8, 139.6, 132.3, 129.7, 129.4, 128.4,
126.7, 124.0, 120.7, 119.1, 113.3, 62.8, 43.3; Anal. calcd. for C28H21ClN4O3: C, 67.67; H, 4.26;
N, 11.27; Found: C, 67.78; H, 4.22; N, 11.25%.
4.1.3.10.
4-Nitro-N-(4-(5-(4-nitrophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-
yl)phenyl)benzamide (2j)
Yield 80%; red solid; m.p. 207.4-208.2ºC; IR (max, cm-1): 3309 (N-H), 3095 (=C-H), 1687
1
(C=O), 1622 (C=N), 1593 (C=C); H-NMR (400 MHz, DMSO-d6):  3.14 (dd, Jax 6.33 Hz,
Jab 17.44 Hz, 1H, Ha), 3.97 (dd, Jbx 12.24 Hz, Jab 17.44 Hz, 1H, Hb), 5.66 (dd, Jax 6.32 Hz,
Jbx 12.24 Hz, 1H, Hx), 6.71-8.37 (m, 17H, Ar-H), 10.71 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  164.4, 150.5, 149.6, 147.6, 144.4, 140.8, 139.8, 129.8, 129.7, 128.1, 127.9,
126.9, 124.8, 124.3, 124.0, 120.8, 119.4, 113.3, 62.9, 43.3; Anal. calcd. for C28H21N5O5: C,
66.27; H, 4.17; N, 13.80; Found: C, 66.37; H, 4.20; N, 13.75%.
4.1.3.11.
4-Nitro-N-(4-(1-phenyl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)benzamide
(2k)
Yield 82%; red solid; m.p. 240.1-240.8ºC; IR (max, cm-1): 3348 (N-H), 3095 (=C-H), 2987,
2906 (C-H), 1697 (C=O), 1620 (C=N), 1595 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.24
(s, 3H, CH3), 3.07 (dd, Jax 6.37 Hz, Jab 17.38 Hz, 1H, Ha), 3.88 (dd, Jbx 12.15 Hz, Jab 17.38
Hz, 1H, Hb), 5.41 (dd, Jax 6.34 Hz, Jbx 12.12 Hz, 1H, Hx), 6.67-8.38 (m, 17H, Ar-H), 10.68
(s, 1H, NH); 13C-NMR (150 MHz,DMSO-d6):  164.3, 149.6, 147.3, 144.7, 140.8, 139.5, 137.0,
130.0, 129.7, 128.5, 126.8, 126.2, 124.0, 120.8, 118.9, 113.3, 63.4, 42.5, 21.1; Anal. calcd. for
C29H24N4O3: C, 73.09; H, 5.08; N, 11.76; Found: C, 73.15; H, 5.08; N, 11.73%.
4.1.3.12.
N-(4-(5-(furan-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-
nitrobenzamide (2l)
Yield 82%; red solid; m.p. 209.4-210.1ºC; IR (max, cm-1): 3290 (N-H), 3084 (=C-H), 1687
1
(C=O), 1622 (C=N), 1591 (C=C); H-NMR (400 MHz, DMSO-d6):  3.38 (dd, Jax 5.98 Hz,
Jab 17.28 Hz, 1H, Ha), 3.78 (dd, Jbx 12.19 Hz, Jab 17.28 Hz, 1H, Hb), 5.60 (dd, Jax 5.98 Hz,
Jbx 12.17 Hz, 1H, Hx), 6.38-8.39 (m, 16H, Ar-H), 10.71 (s, 1H, NH); 13C-NMR (150
MHz,DMSO-d6):  164.4, 153.7, 149.6, 147.8, 144.8, 143.2, 140.9, 139.6, 129.7, 128.4, 126.7,

124.0, 120.8, 119.2, 113.6, 110.9, 108.2, 57.1, 41.5; Anal. calcd. for C26H20N4O4: C, 69.02; H,
4.46; N, 12.38; Found: C, 69.15; H, 4.49; N, 12.24%.
4.1.3.13. N-(4-(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-methylbenzamide (2m)
Yield 83%; yellow solid; m.p. 217.7-218.5ºC; IR (max, cm-1): 3338 (N-H), 3072 (=C-H), 2987,
2906 (C-H), 1656 (C=O), 1624 (C=N), 1593 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.39
(s, 3H, CH3), 3.11 (dd, Jax 6.37 Hz, Jab 17.39 Hz, 1H, Ha), 3.91 (dd, Jbx 12.17 Hz, Jab 17.39
Hz, 1H, Hb), 5.46 (dd, Jax 6.39 Hz, Jbx 12.15 Hz, 1H, Hx), 6.68-7.90 (m, 18H, Ar-H), 10.29
(s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6):  165.8, 147.5, 144.8, 143.1, 142.2, 140.2,
132.3, 129.4, 129.3, 128.2, 127.8, 126.6, 126.3, 120.5, 118.8, 113.3, 63.5, 43.5, 21.5; Anal.
calcd. for C29H25N3O: C, 80.72; H, 5.84; N, 9.74; Found: C, 80.75; H, 5.85; N, 9.77%.
4.1.3.14.
N-(4-(5-(4-Fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-
methylbenzamide (2n)
Yield 75%; yellow solid; m.p. 212.8-213.6ºC; IR (max, cm-1): 3296 (N-H), 3063 (=C-H), 2960,
2895 (C-H), 1691 (C=O), 1622 (C=N), 1593 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.39
(s, 3H, CH3), 3.09 (dd, Jax 6.30 Hz, Jab 17.40 Hz, 1H, Ha), 3.90 (dd, Jbx 12.08 Hz, Jab 17.39
Hz, 1H, Hb), 5.46 (dd, Jax 6.26 Hz, Jbx 12.09 Hz, 1H, Hx), 6.69-7.96 (m, 17H, Ar-H), 10.29
(s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6):  165.8, 147.6, 144.8, 144.7, 142.2, 140.2,
132.8, 129.4, 129.4, 128.4, 127.5, 126.6, 126.3, 120.6, 118.8, 113.3, 63.0, 43.5, 21.5; Anal.
calcd. for C29H24FN3O: C, 77.49; H, 5.38; N, 9.35; Found: C, 77.61; H, 5.41; N, 9.32%.
4.1.3.15.
N-(4-(5-(4-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-
methylbenzamide (2o)
Yield 73%; yellow solid; m.p. 216.7-217.7ºC; IR (max, cm-1): 3387 (N-H), 3095 (=C-H), 2974,
2906 (C-H), 1687 (C=O), 1620 (C=N), 1595 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.39
(s, 3H, CH3), 3.12 (dd, Jax 6.21 Hz, Jab 17.42 Hz, 1H, Ha), 3.91 (dd, Jbx 12.15 Hz, Jab 17.41
Hz, 1H, Hb), 5.56 (dd, Jax 6.21 Hz, Jbx 12.11 Hz, 1H, Hx), 6.70-7.90 (m, 17H, Ar-H), 10.29
(s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6):  165.8, 147.6, 144.6, 142.2, 142.0, 140.3,
132.3, 130.6, 129.6, 129.4, 128.3, 127.7, 126.7, 120.6, 119.0, 113.3, 62.8, 43.3, 21.5; Anal.
calcd. for C29H24ClN3O: C, 74.75; H, 5.19; N, 9.02; Found: C, 75.69; H, 5.17; N, 9.05%.
4.1.3.16.
4-Methyl-N-(4-(5-(4-nitrophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-
yl)phenyl)benzamide (2p)

Yield 77%; red solid; m.p. 169.9-170.2ºC; IR (max, cm-1): 3308 (N-H), 3072 (=C-H), 2960,
2906 (C-H), 1691 (C=O), 1622 (C=N), 1595 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.24
(s, 3H, CH3), 3.17 (dd, Jax 6.31 Hz, Jab 17.47 Hz, 1H, Ha), 3.98 (dd, Jbx 12.24 Hz, Jab 17.50
Hz, 1H, Hb), 5.67 (dd, Jax 6.34 Hz, Jbx 12.22 Hz, 1H, Hx), 6.71-8.23 (m, 17H, Ar-H), 10.30
(s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6):  165.8, 150.6, 147.8, 147.3, 144.5, 142.2,
140.4, 132.3, 129.7, 129.4, 128.3, 127.8, 126.8, 124.7, 120.5, 119.8, 119.3, 113.3, 62.8, 43.1,
21.5; Anal. calcd. for C29H24N4O3: C, 73.09; H, 5.08; N, 11.76; Found: C, 72.99; H, 5.05; N,
11.78%.
4.1.3.17. 4-Methyl-N-(4-(1-phenyl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)benzamide
(2r)
Yield 76%; yellow solid; m.p. 208.3-209.3ºC; IR (max, cm-1): 3392 (N-H), 3088 (=C-H), 2974,
2916 (C-H), 1691 (C=O), 1621 (C=N), 1597 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.25
(s, 3H, CH3), 2.39 (s, 3H, CH3), 3.08 (dd, Jax 6.32 Hz, Jab 17.36 Hz, 1H, Ha), 3.89 (dd, Jbx
12.14 Hz, Jab 17.37 Hz, 1H, Hb), 5.41 (dd, Jax 6.32 Hz, Jbx 12.14 Hz, 1H, Hx), 6.67-7.90 (m,
17H, Ar-H), 10.29 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6):  165.8, 147.5, 144.8, 142.2,
140.2, 140.1, 137.0, 132.3, 130.0, 129.7, 129.4, 128.3, 127.9, 126.6, 120.6, 119.8, 118.8, 113.3,
63.3, 43.5, 21.4, 21.1; Anal. calcd. for C30H27N3O: C, 80.87; H, 6.11; N, 9.43; Found: C, 80.91;
H, 6.15; N, 9.41%.
4.1.3.18.
N-(4-(5-(furan-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-4-
methylbenzamide (2s)
Yield 80%; red solid; m.p. 246.2-246.9ºC; IR (max, cm-1): 3362 (N-H), 3095 (=C-H), 2982,
2916 (C-H), 1655 (C=O), 1622 (C=N), 1595 (C=C); 1H-NMR (400 MHz, DMSO-d6):  2.39
(s, 3H, CH3), 3.11 (dd, Jax 5.98 Hz, Jab 17.24 Hz, 1H, Ha), 3.77 (dd, Jbx 12.12 Hz, Jab 17.24
Hz, 1H, Hb), 5.58 (dd, Jax 5.98 Hz, Jbx 12.16 Hz, 1H, Hx), 6.37-7.91 (m, 16H, Ar-H), 10.30
(s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6):  165.8, 153.7, 147.9, 144.9, 143.2, 142.2,
140.3, 132.3, 129.4, 129.2, 128.2, 127.8, 126.6, 120.6, 119.2, 113.6, 110.9, 108.2, 57.1, 43.5,
21.5; Anal. calcd. for C27H23N3O2: C, 76.94; H, 5.50; N, 9.97; Found: C, 77.05; H, 5.49; N,
10.00%.
4.2. Anticancer activity
The changes in the anti-cancer activities of the synthesized new chalcone and pyrazoline
derivatives were tested on three human cancer cell lines as human breast adenocarcinoma cell

line (MCF-7), gastric cancer cell line (MKN-45) and cervical cancer cell line (HeLa) using
MTT assay [21]. Mouse normal fibroblasts (NIH-3T3) cell line was used as control. All cell
lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA,
USA). Cells were maintained in DMEM medium containing 10% FBS, glutamine and
pen/strep. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 and
experiments were performed at 70-80% confluence at 75 cm cell culture flask. To determine
the anticancer activity, cells were trypsinized with Trypsin-EDTA (0.25%) solution (Gibco,
USA) and seeded into 96-well plates (1 × 104 cells/well). The cells were incubated new
chalcone and pyrazoline derivatives at different concentrations for 24 h. After 24h, cells washed
with PBS for elimination of derivatives and added to fresh 100 μL DMEM. Freshly prepared
10 μL MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Vybrant,
Invitrogen) used for viability testing. After adding MTT solution, plate was incubated for 4 h
in a humidified atmosphere at 37°C incubator with 5% CO2 in the air. At the end of the
incubation, 100 μL of solubilization solution was added into each well. The color changes that
occur after dissolving the formazan precipitate were measured using a microplate reader at 570
nm (Epoch, Biotek,USA). Cell viability was carried out in triplicate of each assay.
4.2.1. Caspase-3 activity assay
In order to determine the levels of the cell caspase-3 activity, cells were lysed with cell lysis
buffer (50mM HEPES, pH 7.4, 0.1% CHAPS, 5mM dithiothreitol, 0.1mM
ethylenediaminetetraacetic acid) and centrifuged for 10 min at 9000 rpm at 4°C after treatment
of compounds for 24 h [21]. Using a commercial kit (Calbiochem 235419, San Diego, CA,
USA) and following the manufacturer's instructions, the supernatant was used for measuring
caspase-3 activity with acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) substrate.
Release of the pNA moiety was detected at 405 nm (εmM=10.5) by ELISA reader (Epoch,
Biotek, USA) and concentration was calculated with pNA standards.
4.2.2. Western blot analysis for protein expression
Cells were treated with the final concentration of IC50 of synthesized compound 1f and 2f in 6
well plates and incubated for 24 h [22]. Treated cells were collected and lysed with RIPA lysis
buffer (Thermo Scientific 89900, USA) included protease inhibitor cocktail (Sigma P2714,
USA). Total cellular protein concentrations were measured by BCA protein assay (Thermo
Scientific 23227, USA). Cellular proteins were denaturated with SDS-Laemmli buffer pH 6.8
(0.125 M Tris-HCl, 20% glycerol, 10% β-mercaptoethanol, 4% SDS, and 0.004% bromphenol

blue) and 25 μg protein was loaded to each well. SDS-PAGE (4–12%) gels were prepared and
celular proteins resolved. Gels were transferred onto polyvinylidene fluoride membrane
(PVDF) using transfer sytsem (Trans-Blot Turbo Transfer System, Biorad, USA) and blocked
with 3% BSA (Capricorn Scientific, BSA-1T). After the blocking, the PVDF membranes were
incubated with overnight 1:250 dilution specific primary antibodies (anti-Bax sc-7480, anti-
Bcl2 sc-492, anti-GAPDH sc-25778, Santa Cruz Biotechnology, Heidelberg, Germany) at 4°C.
Then, the PVDF membranes were washed with TBST (containing 0.1% Tween 20) and
incubated with the 1:1000 dilution HRP-conjugated secondary antibodies antibody (anti-
mouse; sc-2060 or anti-rabbit; sc-2004, Santa Cruz Biotechnology) for 2 h. After that, the
PVDF membranes were incubated with ECL substrate (sc2048, Santa Cruz Biotechnology,
Texas, USA) for the detection of the bands using imaging system (Syngene, Cambridge, UK).
For the normalization of each band, GAPDH was used as a housekeeping protein as control.
Finally, the expression levels of proteins were analyzed and quantified using Image J software
(NIH, USA).
4.3. Physicochemical properties and pharmacokinetic profile predictions
All synthesized compounds were tested for their drug-like properties such as Lipinski rule of
five and Veber rule. Their pharmacokinetic properties such as gastrointestinal absorption, brain
permeation and P-glycoprotein substrate were predicted using Swissadme online server
(http://www.swiss adme.ch/) for calculations.
4.4. Statistical Analysis
Student’s t-test or analysis of variance (ANOVA) were used to analyze the differences among
group means as appropriate. A p-value of 0.05 or less was considered as significant. All data is
represented as standard deviation from the mean, unless otherwise indicated. All experiments
were repeated in triplicate. Data from representative experiments are shown.
Funding sources
This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
Conflict of interest
The authors declare that there are no conflicts of interest.
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Table 1. Half maximal inhibitory concentrations (IC50) for the drugs under study at 24 h in
HeLa, MCF-7, MKN-45 and NIH-3T3 cells.
IC-50 (µM) values
Compound #
1a
HELA
45.61±2.62
>50
MCF-7
>50
MKN-45
>50
NIH-3T3
>50
1b
1c
1d
1e
1f
>50
>50
>50
>50
>50
24.34±3.17
>50
>50
>50
10.72±1.68
>50
>50
>50
>50
>50
26.66±2.73
>50
9.41±2.19
>50
5.17±3.54
>50
>50
1g
1h
1i
>50
15.21±2.15
>50
>50
>50
>50
24.06±4.58
>50
0.04±0.02
49.63±6.62
0.07±0.03
27.90±3.46
>50
22.01±3.71
>50
1j
>50
1k
1l
>50
0.66±0.17
17.34±1.41
>50
0.79±0.21
3.13±1.11
>50
>50
1m
1n
1o
1p
1r
1s
15.64±3.10
>50
>50
1.19±1.01
0.13±0.10
0.03±0.01
1.21±0.64
46.01±3.14
>50
1.10±1.03
0.02±0.01
0.24±0.18
>50
>50
0.81±0.25
34.19±6.13
>50
>50
>50
>50
10.76±4.22
8.38±3.41
2.30±1.18
>50
0.60±0.22
1.26±1.09
0.04±0.02
>50
2a
2b
2c
2d
2e
2f
>50
>50
>50
>50
>50
>50
7.17±1.19
>50
>50
>50
>50
>50
>50
17.96±3.34
>50
0.69±0.13
4.01±2.43
>50
0.88±0.16
0.53±0.04
>50
>50
2g
2h
2i
1.51±0.14
>50
5.14±1.66
>50
>50
0.50±0.22
>50
>50
2j
>50
>50
>50
2k
2l
>50
>50
>50
>50
>50
0.06±0.04
>50
0.02±0.01
>50
0.02±0.01
>50
2m
2n
3.33±1.02
>50
>50
>50
>50

2o
2p
2r
2s
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.06±0.02
0.02±0.01
>50
0.16±0.04
0.05±0.02

Table 2. Lipinski and Veber Parameters of the synthesized compounds.
Lipinski rule of five
n-ON n-OHNH
Veber rule
n-ROTB TPSA
Lipinski rule of five
Veber rule
n-ROTB TPSA
Code
Code
2a
2b
2c
MW
Log P
4.29
4.66
4.77
3.21
4.50
2.64
2.83
3.21
3.32
1.89
3.05
1.24
4.13
4.50
4.61
3.05
4.34
2.49
MW
Log P
5.21
5.58
5.68
4.19
5.41
4.02
3.82
4.19
4.29
2.91
4.02
2.67
5.04
5.41
5.50
4.02
5.24
3.85
n-ON
n-OHNH
1a
1b
1c
1d
1e
1f
345.37
363.36
379.81
390.36
359.39
335.33
372.37
390.36
406.82
417.37
386.40
362.34
341.40
359.39
375.85
386.40
355.43
331.36
3
4
3
5
3
4
4
5
4
6
4
5
2
3
2
4
2
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
6
6
7
6
6
7
7
7
8
7
7
6
6
6
7
6
6
46.17
46.17
46.17
91.99
46.17
59.31
91.99
91.99
91.99
137.81
91.99
105.13
46.17
46.17
46.17
91.99
46.17
59.31
435.49
453.48
469.94
480.49
449.52
425.45
462.50
480.49
496.94
507.50
476.53
452.46
431.53
449.52
465.97
476.53
445.55
421.49
3
4
3
5
3
4
4
5
4
6
4
5
2
3
2
4
2
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
6
6
7
6
6
7
7
7
8
7
7
6
6
6
7
6
6
44.70
44.70
44.70
90.52
44.70
57.84
90.52
90.52
90.52
136.34
90.52
103.66
44.70
44.70
44.70
90.52
44.70
57.84
2d
2e
2f
1g
1h
1i
2g
2h
2i
1j
2j
1k
1l
2k
2l
1m
1n
1o
1p
1r
1s
2m
2n
2o
2p
2r
2s

Table 3. Drug-like properties of compound 1f and 2f.
Drug-likeness parameters
Compound 1f
Compound 2f
Log S
-4.38
-5.80
(Moderately soluble) (Moderately soluble)
Egan
Yes
Yes
logP<5.88
TPSA<131.6
Ghose
Yes
Yes
160<MW<480
-0.4<logP<5.6
40<MR<130
20<atoms<30
Bioavailability score
MR: Molar refractivity
0.55
0.55