Synthesis and characterization of 2-phenylpyrazoline derivatives and evaluation of their activities against antimicrobial and breast cancer cell line in vitro and in silico studies

Article abstract of DOI:10.14233/ajchem.2019.21915

The new series of 2-phenylpyrazoline derivatives (2a-j) were synthesized and evaluated for their antimicrobial, in silico and in vitro anticancer activity was performed by MTT assay using MDA-MB-231 (human breast adenocarcinoma) cell line. The 2-phenylpyr

Full text of DOI:10.14233/ajchem.2019.21915

Asian Journal of Chemistry; Vol. 31, No. 6 (2019), 1311-1320
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.21915
Synthesis and Characterization of 2-Phenylpyrazoline Derivatives and Evaluation of their
Activities against Antimicrobial and Breast Cancer Cell Line in vitro and in silico Studies
*,
RAJA CHINNAMANAYAKAR and M.R. EZHILARASI
Department of Chemistry, Karpagam Academy of Higher Education, Coimbatore-641021, India
*Corresponding author: E-mail: mrezhilarasi@gmail.com
Received: 4 January 2019;
Accepted: 18 February 2019;
Published online: 29 April 2019;
AJC-19374
The new series of 2-phenylpyrazoline derivatives (2a-j) were synthesized and evaluated for their antimicrobial, in silico and in vitro
anticancer activity was performed by MTT assay using MDA-MB-231 (human breast adenocarcinoma) cell line. The 2-phenylpyrazoline
derivatives (2a-j) were obtained by the cyclization of chalcones with phenylhydrazine hydrochloride. Synthesized compounds were
confirmed using FT-IR, 1H NMR and 13C NMR spectral data. Molecular docking studies were carried out using Auto Dock Tool version
1.5.6 and Auto dock version 4.2.5.1 docking program. in silico Docking study, compound 2d showed good binding score and good
binding interaction with selected bacterial proteins and breast cancer protein. Based on this result, compound 2d was performed the
anticancer activity by MTT assay method. From this result, compound 2d shown the LC50 value is 185.30 1. 469 µg/mL. From the
antibacterial activity compound 2i (2,3-dichloro substituted 2-pyrazoline derivative) showed a good zone of inhibition at high concentration
(100 mg/mL) as compared to other derivatives (2a-j) and compound 2c (fluoro substituted 2-phenylpyrazoline derivative) showed a good
zone of inhibition at low concentration (25 mg/mL) compared to other derivative (2a-j).
Keywords: Biphenyl Chalcone, Phenylhydrazine hydrochloride, Antimicrobial activity, Anticancer activity.
the presence of a large number of anticancer drugs, no currently
available agents can eradicate cancer cells without harming
normal tissues. Thus, the development of newer chemothera-
peutic scaffolds which with selectively act on the target without
side effects has become a primary objective of medicinal
chemists [4].
The chalcones are the convenient intermediate for the syn-
thesis of pyrazoline derivatives. It has been reported various
biological activities, such as muscle relaxant, tranquilizing,
psychoanalytic, anticonvulsant, antidepressant and antihyper-
tensive activities [5,6]. Pyrazolines are very useful synthone
in organic synthesis. Among various pyrazoline derivatives,
2-pyrazolines derivative of compounds is mostly studied [7].
Pyrazoline compounds have adjacent two nitrogen atom occur
from saturated and partially saturated pyrazoles in biologically
active compounds and natural products [8]. Pharmacologically
interesting heterocyclic systems like pyrazoline derivatives
have been widely studied to their pharmacological activities
such as antitumor, anti-inflammatory, antiparasitary, anti-
convulsant, antimicrobial, antinociceptives, antimalarial,
INTRODUCTION
In 20th century the most prevailing disease is cancer. The
spreading of the disease is rising rapidly day by day. Surgery,
radiation and chemotherapy are the most common treatment
for this disease. Despite these treatments, cancer is uncontrollable
and medical field needs a new approach to treat this dreadful
disease. One of the most commonly used treatment methods
is chemotherapy. In this method, it spreads throughout the body
because the medicine and radiation used in this method travel
to an entire part of the body and have some affect on the human
being [1]. Therefore, we have to develop and pay more atten-
tion to update, modify and recreate the drugs and treatment
methods for cancer. For medicinal chemistry and drug dis-
covery, it is one of the urgent requirements to introduce new
drugs and treatment methods for the disease [2]. The breast
cancer as the second leading cause of death throughout the
world is still the most frequently identified cancer in woman
[3]. The literature clearly indicated that more than 90 % of
cancer patients die due to chronic tumor metastases. Despite
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1312 Chinnamanayakar et al.
Asian J. Chem.
Alzheimer, Huntington and inflammatory arthritis, anticancer,
antiviral, antioxidant, antiamoebic, antifungal, antidiabetic,
cytotoxic and pesticidal properties [9]. In our previous studies,
we synthesized several 1-thiocarbamoyl derivatives and tested
them for antimicrobial activity and in silico activity.We reported
that in vitro antimicrobial activity and in silico activity of
1-thiocarbamoyl substituted pyrazole derivatives [10].
A new series of 2-phenylpyrazoline derivatives are formed
by cyclization of chalcones with phenylhydrazine hydrochloride.
The chemical structures of 2-phenylpyrazoline derivatives was
confirmed using FT-IR, 1H NMR and 13C NMR spectral data.
in silico Studies were carried out for synthesized 2-phenyl -
pyrazoline compounds (2a-j) using bacterial proteins (1UAG,
3UDI and 2X5O) and breast cancer protein (1OQA). Based
on the high binding score, the best compound was performed
to in vitro anticancer activity by MTT assay. Furthermore,
antimicrobial activity was carried out using different strains
(Staphylococcus aureus, Klebsiella pneumonia, Escherichia
coli and Candida albicans) by agar disc diffusion method.
in 10 mL acetic acid in a 100 mL beaker. Then, the solution
was added to the round bottom flask. After that the reaction
mixtures was refluxed for 14 -16 h. Completion of the reaction
was monitored by TLC using CHCl3 used as a solvent. This
reaction mixture was poured into crushed ice and kept in over-
night at room temperature. Then these precipitate was filtered,
dried and recrystallized using ethanol. The purity of the compound
was checked by TLC using CHCl3 as a solvent (Scheme-I).
Spectral data
4,5-Dihydro-1-phenyl-3-diphenyl-5-phenyl-1H-pyrazole
(2a): Yield 81%; m.p: 181 ºC; solid yellow colour; IR (KBr,
ν
max, cm-1): 1598.71 (C=N), 1513.85 (C=C), 1426.01 (C-N)
1
3067.13 (Ar-CH); H NMR (CDCl3); 400 MHz, δ, ppm (J,
Hz): 3.06 (1H, dd, H4a, J4a,4b 17.4 Hz, J4a,5a 6.2 Hz); 3.91 (1H, dd,
H4b, J4b,4a 16.4 Hz, J4b,5a 12.8 Hz); 5.67 (1H, dd, H5a, J5a,4a, 12 Hz,
J5a,4b 6 Hz); 6.92-7.87 (Ar-H). 13C NMR δ: 149.72 (C3 of pyrazole
ring), 43.14 (C4 of pyrazole ring), 62.51 (C5 of pyrazole ring);
145.71, 144.52, 143.87, 143.21 (IPSO carbon); 113.21, 118.78,
122.90, 124.35, 124.90, 126.35, 126.86, 126.97, 127.12, 127.83,
1278.49, 128.55, 129.30, 129.73, 129.91, 131.16 (Ar-C). Elem-
ental analysis of C27H22N2 calcd. (found) %: C, 86.59 (86.60);
H, 5.87 (5.92); N, 7.47 (7.48).
EXPERIMENTAL
General procedure for the synthesis of E-3-(phenyl)-
1-(diphenyl-2-yl)-3-arylprop-2-en-1-one derivatives (1a-j):
Various substituted aldehydes (1a-j, 0.1 mol) and 4-acetyl
biphenyl (0.1 mol) were taken in a 250 mL Erlenmeyer flask
and to this approximately added nearly 30-35 mL of ethanol
containing 20 % of NaOH solution. This mixture was stirred
well for 3 h, then this mixture was transferred to 500 mL beaker
containing pieces of ice cubes and kept overnight at room tempe-
rature. The solid obtained is a chalcone, which was filtered,
dried and recrystallized using ethanol. Finally, the purity of
the compound was checked by TLC using CHCl3 as a solvent
[10].
General procedure for the synthesis of 4,5-dihydro-1-
phenyl-3-diphenyl-5-phenyl-1H-pyrazole derivatives (2a-j):
Substituted chalcone (1 mol) and phenylhydrazine hydrochloride
(1 mol) was taken in a 250 mL round bottom flask containing
40 mL ethanol. After, 1 mol of sodium acetate was dissolved
4,5-Dihydro-1-phenyl-3-diphenyl-5-(4-nitrophenyl)-1H-
pyrazole (2b):Yield 84 %; m.p: 186 ºC; solid yellow colour;
IR (KBr, νmax, cm-1): 1595.81 (C=N), 1521 (C=C), 1410 (C-N),
1
3053.73 (Ar-CH); H NMR (CDCl3); 400 MHz, δ, ppm (J,
Hz): 3.14 (1H, dd,H4a, J4a,4b 17 Hz, J4a,5a 7 Hz); 3.93 (1H, dd,
H4b, J4b,4a 16.8 Hz, J4b,5a 12.8 Hz); 5.38 (1H, dd, H5a, J5a,4a 12.2
Hz, J5a,4b 7 Hz); 6.81-8.21 (Ar-H). 13 C NMR δ: 149.81 (C3 of
pyrazole ring), 43.25 (C4 of pyrazole ring), 63.81 (C5 of pyrazole
ring); 147.46, 146.58, 144.35, 141.58 (IPSO carbon); 113.48,
119.85, 123.88, 124.25, 124.61, 125.50, 126.35, 126.66, 126.97,
127.00, 127.31, 127.49, 127.75, 129.00, 129.13, 129.24, 129.43,
131.18, 140.31 (Ar-C). Elemental analysis of C27H21N3O2 calcd.
(found) %: C, 77.30 (77.31); H, 5.00 (5.05); N, 10.01 (10.02);
O, 7.62 (7.63).
4,5-Dihydro-1-phenyl-3-diphenyl-5-(4-fluorophenyl)-
1H-pyrazole (2c): Yield 74 %;; m.p: 169 ºC; solid yellow
O
O
C
Ar C CH
O
NaOH in EtOH
+
C
H3C
H
3 h refluxed,
0 oC
(1a-j)
X
Chalcone
Substituted aldehyde
4-Acetyl biphenyl
Ar = 1a = C6H5
Sodium acetate,
Acetic acid
Ar = 1b = C6H4NO2
Ar = 1c = C6H4F
Ar = 1d = C6H4Br
Ar = 1e = C7H7
Ar = 1f = C7H7O
Ar = 1g = C6H4Cl
Ar = 1h = C6H4Cl
Ar = 1i = C6H3Cl2
Ar = 1j = C6H4Br
Ethanol,
Phenyl hydrazine hydrochloride
N
N
Ar
(2a-j)
4,5-dihydro-1-phenyl-3-diphenyl-5-substituted phenyl-1H-pyrazole
Scheme-I: Synthetic pathway for 2-phenyl pyrazoline derivatives (2a-j)

Vol. 31, No. 6 (2019)
Synthesis and Characterization of 2-Phenylpyrazoline Derivatives 1313
colour; IR (KBr, νmax, cm-1): 1599.66 (C=N), 1513.85 (C=C),
(J, Hz): 3.15 (1H, dd, H4a, J4a,4b 17.1 Hz, J4a,5a 7.2 Hz); 3.81
(1H, dd, H4b, J4b,4a 16.4 Hz, J4b,5a 12.4 Hz); 5.22 (1H, H5a, J5a,4a
11.8 Hz, J5a,4b 7.4Hz); 6.68-7.79 (Ar-H). 13C NMR δ: 150.00
(C3 of pyrazole ring), 43.65 (C4 of pyrazole ring), 64.23 (C5
of pyrazole ring); 146.44, 145.04, 141.01, 140.61 (IPSO carbon);
113.04, 113.52, 118.96, 126.18, 126.81, 127.21, 127.52, 128.91,
128.93, 130.31, 132.06 (Ar-C). Elemental analysis of C27H21N2Cl
calcd. (found) %: C, 79.29 (79.30); H, 5.13 (5.18); N, 6.84
(6.85); Cl, 8.66 (8.67).
4,5-Dihydro-1-phenyl-3-diphenyl-5-(4-chlorophenyl)-
1H-pyrazole (2h):Yield 81 %; m.p.: 181 ºC; solid yellow colour;
IR (KBr, νmax, cm-1): 1599.66 (C=N), 1542.77 (C=C), 1407.78
(C-N), 3032.51 (Ar-CH); 1H NMR (CDCl3): 400 MHz, δ, ppm
(J, Hz): 3.12 (1H, dd, H4a, J4a,4b 16.8 Hz, J4a,5a 6.8 Hz); 3.86 (1H,
dd, H4b, J4b,4a 16.4 Hz, J4b,5a 12.8 Hz); 5.27 (1H, H5a, J5a,4a 11.8
Hz, J5a,4b 7 Hz); 3.65 (S, 3H, OCH3); 6.80-8.11 (Ar-H). 13C NMR
δ: 149.89 (C3 of pyrazole ring), 43.78 (C4 of pyrazole ring),
64.36 (C5 of pyrazole ring); 145.72, 144.58, 143.34, 143.27
(IPSO carbon); 113.45, 119.43, 122.40, 125.42, 126.99, 127.03,
127.25, 127.34, 127.37, 127.42, 128.32, 128.86, 128.91, 129.04,
129.15, 129.18, 129.39, 130.01, 131.52, 133.37, 136.47, 139.89,
140.46, 141.07 (Ar-C). Elemental analysis of C27H21N2Cl calcd.
(found) %: C, 79.29 (79.30); H, 5.13 (5.18); N, 6.86 (6.85);
Cl, 8.66 (8.67).
4,5-Dihydro-1-phenyl-3-diphenyl-5-(2,3-dichloro-
phenyl)-1H-pyrazole (2i): Yield 61 %; m.p; 183 ºC; solid
yellow colour; IR (KBr, νmax, cm-1): 1597.74 (C=N), 1515.67
(C=C), 1427.04 (C-N), 3031.86 (Ar-CH); 1H NMR (CDCl3):
400 MHz, δ, ppm (J, Hz): 3.19 (1H, dd, H4a, J4a,4b 16.6 Hz,
J4a,5a 6.4 Hz); 3.81 (1H, dd, H4b, J4b,4a 16.9 Hz, J4b,5a 13.4 Hz);
5.38 (1H, H5a, J5a,4a 13.8 Hz, J5a,4b 6.8 Hz); 6.73- 8.21 (Ar-H).
13C NMR δ: 146.63 (C3 of pyrazole ring), 43.47 (C4 of pyrazole
ring), 63.88 (C5 of pyrazole ring);56.17 (s, 3H, OCH3); 145.23,
144.61, 143.14, 143.27 (IPSO carbon); 113.75, 115.43, 119.40,
125.42, 126.00, 127.61, 127.85, 128.01, 128.86, 128.97, 129.00,
129.34, 129.77, 129.89, 130.01, 131.32, 132.37, 133.51, 134.89,
138.46, 139.31,139.79, 140.56, 141.07, 159.03 (Ar-C). Elemental
analysis of C27H20N2Cl2 calcd. (found) %: C, 73.13 (73.14); H,
4.51 (4.55); N, 6.32 (6.31); Cl, 15.99 (15.99).
4,5-Dihydro-1-phenyl-3-diphenyl-5-(3-bromophenyl)-
1H-pyrazole (2j):Yield 86 %; m.p.: 172 ºC; solid yellow colour;
IR (KBr, νmax, cm-1); 1H NMR (CDCl3); 400 MHz, δ, ppm (J,
Hz): 3.12 (1H, dd, H4a, J4a,4b 16.8 Hz, J4a,5a 6.8 Hz); 3.83 (1H, dd,
H4b, J4b,4a 14.4 Hz, J4b,5a 12.8 Hz); 5.34 (1H, H5a, J5a,4a 12.8 Hz,
J5a,4b 7.2 Hz); 6.78-8.14 (Ar-H). 13CNMR δ: 146.47 (C3 of pyrazole
ring), 43.76 (C4 of pyrazole ring), 63.45 (C5 of pyrazole ring);
143.17, 143.38, 142.78, 141.03 (IPSO carbon); 113.17, 119.65,
122.14, 123.19, 124.39, 125.37, 126.98, 127.00, 127.25, 127.47,
127.98, 128.00, 128.66, 128.81, 128.87, 129.13, 129.54, 130.01,
131.43, 138.81, 139.67 (Ar-C). Elemental analysis of C27H21N2Br
calcd. (found) %: C, 71.52 (71.53); H, 4.63 (4.67); N, 6.17
(6.18); Br, 17.62 (17.62).
1
1401.03 (C-N), 3034.44 (Ar-CH); H NMR (CDCl3); 400
MHz, δ, ppm (J, Hz): 3.19 (1H, dd, H4a, J4a,4b 17.3 Hz, J4a,5a
6.9 Hz); 3.91 (1H, dd, H4b, J4b,4a16.9 Hz, J4b,5a 12.2 Hz); 5.28
(1H, H5a, J5a,4a 12.6 Hz, J5a,4b 6.7 Hz); 6.79-8.18 (Ar-H). 13C
NMR δ: 149.34 (C3 of pyrazole ring), 45.87 (C4 of pyrazole
ring), 61.53 (C5 of pyrazole ring);145.24, 145.09, 144.23,
141.58 (IPSO carbon); 115.28, 117.32, 122.88, 124.25, 124.31,
125.67, 125.95, 126.13, 127.97, 128.00, 128.18, 128.77, 129.20,
129.63, 129.74, 130.43, 141.18, 141.73 (Ar-C). Elemental
analysis of C27H21N2F calcd. (found) %: C, 82.62 (82.63); H,
5.35 (5.39); N, 7.13 (7.14); F, 4.83 (4.84).
4,5-Dihydro-1-phenyl-3-diphenyl-5-(4-bromophenyl)-
1H-pyrazole (2d):Yield 86 %; m.p.: 168 ºC; solid yellow colour;
IR (KBr, νmax, cm-1): 1593 (C=N), 1544 (C=C), 1405 (C-N),
1
3057.58 (Ar-CH); H NMR (CDCl3); 400 MHz, δ, ppm (J,
Hz): 3.13(1H, dd, H4a, J4a,4b 17 Hz, J4a,5a 7 Hz); 3.87 (1H, dd,
H4b, J4b,4a 14.8 Hz, J4b,5a 12.6 Hz); 5.38 (1H, H5a, J5a,4a 12.4 Hz,
J5a,4b 7.2 Hz); 6.79-8.11 (Ar-H). 13C NMR δ: 146.39 (C3 of
pyrazole ring), 43.42 (C4 of pyrazole ring), 63.93 (C5 of
pyrazole ring);144.53, 143.38, 141.58, 141.39 (IPSO carbon);
113.41, 119.42, 122.75, 125.42, 126.19, 126.27, 126.98, 127.02,
127.25, 127.40, 127.57, 127.69, 127.86, 128.81, 128.87, 129.14,
129.32, 130.24, 131.79, 140.46, 140.73 (Ar-C). Elemental analysis
of C27H21N2Br calcd. (found) %: C, 71.52 (71.53); H, 4.63
(4.67); N, 6.17 (6.18); Br, 17.62 (17.62).
4,5-Dihydro-1-phenyl-3-diphenyl-5-(p-tolyl)-1H-pyrazole
(2e): Yield 84 %; m.p:. 174 ºC; solid yellow colour; IR (KBr,
ν
max, cm-1): 1589.32 (C=N), 1523.46 (C=C), 1414.61 (C-N),
1
3073.98 (Ar-CH); H NMR (CDCl3); 400 MHz, δ, ppm (J,
Hz) : 3.14 (1H, dd, H4a, J4a,4b 16.8 Hz, J4a,5a 7.2 Hz); 3.84 (1H,
dd, H4b, J4b,4a 16.8 Hz, J4b,5a 12.4 Hz); 5.26 (1H, H5a, J5a,4a 12 Hz,
J5a,4b 7.2 Hz); 2.36 (S, 3H, CH3); 6.76-7.79 (Ar-H). 13C NMR
δ: 146.41 (C3 of pyrazole ring), 43.64 (C4 of pyrazole ring),
64.34 (C5 of pyrazole ring); 21.35 (S, 3H , CH3); 144.86,
141.16, 140.56, 139.67 (IPSO carbon); 113.45, 119.12, 125.85,
126.19, 127.00, 127.05, 127.21, 127.43, 127.54, 128.68, 128.85,
128.89, 128.95, 129.93, 129.86, 131.84, 137.28, (Ar-C). Elemental
analysis of C28H24N2 calcd. (found) %: C, 86.55 (86.56); H, 6.17
(6.18); N, 7.20 (7.21).
4,5-Dihydro-1-phenyl-3-diphenyl-5-(4-methoxyphenyl)
-1H-pyrazole (2f): Yield 71 %; m.p.:167 ºC; solid yellow
colour; IR (KBr, νmax, cm-1): 1596.77 (C=N), 1544.77 (C=C),
1407.78 (C-N), 3061.44 (Ar-CH); 1H NMR (CDCl3); 400 MHz,
δ, ppm (J, Hz): 3.14 (1H, dd, H4a, J4a,4b 17.14 Hz, J4a,5a 7 Hz); 3.84
(1H, dd, H4b, J4b,4a 13.4 Hz, J4b,5a 9 Hz); 5.26 (1H, H5a, J5a,4a 12
Hz, J5a,4b 7.2 Hz); 3.77 (s, 3H, OCH3); 6.76-7.77 (Ar-H); 13CNMR
δ: 146.43 (C3 of pyrazole ring), 43.63 (C4 of pyrazole ring),
64.04 (C5 of pyrazole ring); 55.31 (s, 3H, OCH3); 144.84, 141.15,
140.54, 134.67 (IPSO carbon); 113.49, 114.02, 114.54, 119.14,
125.47, 126.20, 126.35, 127.00, 127.05, 127.11, 127.22, 127.40,
127.56, 128.86, 128.97, 129.02, 130.12, 131.85, 159.02 (Ar-C).
Elemental analysis of C28H24N2O calcd. (found) %: C, 83.13
(83.14); H, 5.93 (5.98); N, 6.92 (6.93); O, 3.95 (3.96).
4,5-Dihydro-1-phenyl-3-diphenyl-5-(2-chlorophenyl)-
1H-pyrazole (2g):Yield 77 %; m.p.: 177 ºC; solid yellow colour;
IR (KBr, νmax, cm-1): 1596.77 (C=N), 1521.56 (C=C), 1447.31
(C-N), 3055.66 (Ar-CH); 1H NMR (CDCl3): 400 MHz, δ, ppm
in silico Studies: in silico Study has been carried out for
synthesized new 2-phenylpyrazoline derivatives (2a-j) using
bacterial proteins (1UAG, 3UDI, and 2X5O) and breast cancer
protein (1OQA). Different software is used in molecular docking
studies, they are Chem draw, Pyrx, Chimera and Discovery
studies.

1314 Chinnamanayakar et al.
Asian J. Chem.
Molecular docking studies: Molecular docking studies
have been carried out using ADT (Auto Dock Tool) version
1.5.6. and Auto Dock version 4.2.5.1 docking program.
Preparation of protein: The bacterial proteins and breast
cancer protein were downloaded from Protein Data Bank (PDB
id: 1UAG, 3UDI, 2X50 and 10QA).
Ligand preparation: 2D structure of 2-phenylpyrazoline
derivatives (2a-j) is drawn using ChemDraw Ultra 8.0 (Chem-
office 2002).After that Chem 3D Ultra 8.0 was used to convert
the 2D structure into the 3D structure and the energy is mini-
mized using semi-empirical AM1 method. All the structures
are saved as PDB file format for input to ADT. Finally, all the
ligand structures are saved as PDB file format to carry out
molecular docking in Auto dock Vina.
Grid formation: A grid box with a dimension of 40 × 40
× 40 Å3 in 0.375 Å spacing and centered on 30.473, 47.997,
9.563 has created around the binding site of protein usingADT.
The center of the box was set at ligand center and grid energy
calculations have been carried out.
Docking protocol: The auto dock calculation is such as
default parameters have been used and 10 docked confirma-
tions are generated for each compound. The energy calculation
is done using genetic algorithms. The outputs are exported to
Chimera 1.10 and discovery studio 4.5 for visual inspection
of the binding modes and interaction of the compounds with
amino acid residues in the active site [11].
lved and sterilized by filter sterilization.After 24 h of incubation
period, the sample content in wells was removed and 30 µL of
reconstituted MTT solution was added to all test and cell control
wells, the plate was gently shaken well and incubated at 37 ºC
in a humidified 5 % CO2 incubator for 4 h.After the incubation
period, the supernatant was removed and 100 µL of MTT solubi-
lization solution (DMSO, SigmaAldrich, USA) was added and
the wells were mixed gently by pipetting up and down in order
to solubilize the formazan crystals. The absorbance values were
measured by using microplate reader at a wavelength of 540
nm [12]. The percentage of growth inhibition was calculated
using the following formula:
Mean OD samples
Viability (%) =
×100
Mean OD of control group
Antimicrobial activity: The synthesis of 2-phenyl-
pyrazoline derivatives was subjected to antimicrobial activity
by agar disk diffusion method. In these studies, four different
strains were used are Staphylococcus aureus, Streptococcus,
Escherichia coli and Klebsiella.
Antifungal activity: The synthesized new 2-phenyl-
pyrazoline derivatives were screened for antifungal activity
using Candida albicans by agar disc diffusion method.
RESULTS AND DISCUSSION
In this work, 2-phenylpyrazoline derivatives (2a-j) were
prepared by the cyclization of phenylhydrazine hydrochlo-
ride with chalcones (1a-j) in turn generated as intermediates
by Aldol condensation reaction between the corresponding
4-acetyl biphenyl and substituted aldehydes in ethanolic NaOH
solution.
Molecular docking studies for 4,5-dihydro-1-phenyl-
3-diphenyl-5-substituted phenyl-1H-pyrazole derivatives
(2a-j): In the present study, in silico studies were carried out
byAuto Dock Tool version 1.5.6 andAuto dock version 4.2.5.1
docking program using bacterial proteins (1UAG, 3UDI and
2X50) and breast cancer protein (1OQA) (Figs. 1 and 2). The
bacterial proteins (1UAG, 3UDI and 2X5O) which is involved
in cell wall synthesis mechanism.
Anticancer activity
MTT assay: MDA-MB-231 (human breast adenocarci-
noma) cell was initially procured from National Centre for
Cell Sciences (NCCS), Pune, India and maintained Dulbecco′s
modified Eagles medium, DMEM (Sigma Aldrich, USA).
The cell line was cultured in a 25 cm2 tissue culture flask
with DMEM supplemented with 10 % FBS, L-glutamine, sodium
bicarbonate (Merck, Germany) and the antibiotic solution
containing penicillin (100 U/mL), streptomycin (100 µg/mL),
and amphotericin B (2.5 µg/mL). Cultured cell lines were kept
at 37 ºC in a humidified 5 % CO2 incubator (NBS Eppendorf,
Germany). The viability of cells was evaluated by direct obser-
vation of cells by inverted phase contrast microscope and followed
by MTT assay method.
The docking studies which are reported in terms of binding
affinity value, which mean the lower, the score, the better and
the interactions. In ligand 2-phenylpyrazoline derivatives (2a-j)
were individually docked with the bacterial proteins and breast
cancer protein. The molecular docking results are shown in
Table-1, which showed that 2-phenylpyrazoline derivatives (2a-j)
exhibited good binding affinity score as compared to the standard
drug. Based on binding affinity score, hydrophobic and hydro-
philic interactions and 2-phenylpyrazoline derivatives showed
better binding affinity as compared to standard drug.
The molecular docking studies show that these 2-phenyl-
pyrazoline derivatives bind well in the active site pocket of
bacterial proteins and interact with the active site of amino acid
residues. The best compound of these derivatives (2a-j) have
been explained as follows:
Cells seeding in 96 well plate: A two-day old confluent
monolayer of cells trypsinized and the cells were suspended
in 10 % growth medium, 100 µL cell suspension (5 × 104 cells/
well) was seeded in 96 well tissue culture plate and incubated
at 37 ºC in a humidified 5 % CO2 incubator.
Preparation of compound stock: 1 mg of sample was
weighed and dissolved in 1 mL of DMEM using a cyclomixer.
The sample solution was filtered through 0.22 µm Millipore
syringe filter to ensure the sterility.
Anticancer evaluation: After 24 h the growth medium
was removed, freshly prepared each compounds in 5 % DMEM
was five times serially diluted by two-fold dilution (100, 50,
25, 12.5 and 6.25 µg) in 500 µL of 5 % DMEM and each concen-
tration of 100 µL were added in triplicates to the respective
wells and incubated at 37 ºC in a humidified 5 % CO2 incubator.
Non-treated control cells were also maintained.
Binding affinity score:The synthesized 2-phenylpyrazoline
derivatives (2a-j) were docked with bacterial protein 1UAG,
2X5O and 3UDI. From this result, 2-phenylpyrazoline derivative
Anticancer assay by MTT method: 15 mg of MTT (Sigma,
M-5655) was reconstituted in 3 mL PBS until completely disso-

Vol. 31, No. 6 (2019)
Synthesis and Characterization of 2-Phenylpyrazoline Derivatives 1315
Interactions
van der Waals
Conventional hydrogen bond
Amide-Pi-stacked
Alkyl
Pi-Cation
Pi-Sigma
Pi-Alkyl
(a)
Interactions
Alkyl
van der Waals
Conventional hydrogen bond
Pi-Pi stacked
Pi-Alkyl
(b)
Interactions
Pi-Sigma
van der Waals
Pi-Pi stacked
Amide-Pi stacked
Pi-Alkyl
Halogen (Fluorine)
Pi-Cation
Pi-Donor hydrogen bond
(c)
Fig. 1. (a) 2D and 3D image of compound 2d docked with 1UAG protein; (b) 2D and 3D image of compound 2d docked with 2X5O protein,
(c) compound 2d and 3D image of compound 2d docked with 3UDI protein

1316 Chinnamanayakar et al.
Asian J. Chem.
Interactions
van der Waals
Pi-Anion
Pi-Pi T-shaped
Pi-Alkyl
Pi-Sigma
Fig. 2. 2D and 3D image of compound 4,5-dihydro-1-phenyl-3-diphenyl-5-(4-bromophenyl)-1H-pyrazole (2d) docked with breast cancer
protein (1OQA)
TABLE-1
MOLECULAR DOCKING RESULTS FOR 2- PHENYL PYRAZOLINE
DERIVATIVES (2a-j) DOCKED WITH DIFFERENT BACTERIAL PROTEINS
Binding
Protein affinity value
(kcal/mol)
Conventional
H-Bond
interaction
H-Bond
length
Hydro-phobic
interaction
Compound
Other interactions
1UAG -8.7
Nil
Nil
ALA A: 414,
LYS A: 348,
LEU A: 416
LYS A: 262
ALA A: 66,
LYS A: 137,
ILE A: 140,
PRO A: 184
LYS A: 319,
LYS A: 115
N
N
2X5O
3UDI
-8.6
-10.7
PHE A: 303
Nil
2.40
Nil
PHE A: 303
PHE A: 72, ILE A:
140, TYR A: 144,
LYS A: 147
1UAG -9.3
LYS A: 115,
ARG A: 302,
LYS A: 319
ASN A: 271
2.75,
2.42,
2.53
LYS A: 348,
ALA A: 414,
LEU A: 416
PHE A: 422, ASP
A: 185,
LEU A: 416
N
N
2X50
3UDI
-8.6
-9.2
2.62
ALA A: 328,
LEU A: 216,
ALA A: 215
ASP A: 214, GLU
A: 327
LYS A: 137,
ARG A: 202
2.93,
1.90
PRO A: 184
PHE A: 72, LYS
A: 147, ILE A:
148TYR A: 144
O2N
1UAG -8.8
Nil
Nil
LEU A: 416,
ALA A: 414
LEU A: 416,
LYS A: 319, PHE
A: 422, ASP A:
185
N
2X5O
3UDI
-8.7
-9.0
Nil
Nil
Nil
Nil
LYS A: 262
PHE A: 303, GLU
A: 304, HIS A: 267
N
ALA A: 66,
PRO A: 184
PHE A: 72, LYS
A: 137, ILE A:
140, TYR A: 144,
LYS A: 147, ILE
A: 148
F
1UAG -9.6
ASN A: 138
2.40
LEU A: 15,
PRO A: 41,
PRO A: 142
LYS A: 115,
GLYA: 137,
LEU A: 15
N
2X5O
3UDI
-8.7
-11.1
PHE A:303,
Nil
2.45
Nil
LYS A: 262
PHE A: 303
N
ALA A: 66,
PRO A: 184,
LYS A: 137,
ILE A: 140
PHE A: 72, TYR
A: 144, LYS A:
147, LYS A: 137,
ILE A: 140, ILE A:
148
Br

Vol. 31, No. 6 (2019)
Synthesis and Characterization of 2-Phenylpyrazoline Derivatives 1317
1UAG -8.9
Nil
Nil
LYS A: 319,
AL A: 414,
LEU A: 416
LYS A: 262
LEU A: 416,
ASP A: 185,
PHE A: 422
HIS A: 267, PHE
A: 303
PHE A: 72, LYS
A: 137, TYR A:
144, LYS A: 147,
ILE A: 148
N
2X5O
3UDI
-8.6
Nil
Nil
Nil
Nil
N
-10.5
LYS A: 137,
ILE A: 140,
PRO A: 184
H3C
1UAG -9.1
LYS A: 319
Nil
2.86
Nil
LEU A: 416
LYS A: 262
ASP A: 185, LEU
A: 416, ALA A:
414, PHE A: 422
HIS A: 267, PHE
A: 303, GLU A:
304
N
N
2X5O
3UDI
-8.4
-8.1
GLU A: 252,
TYR A: 567
1.98,
2.13
LEU A: 426,
ILE A: 534
HIS A: 256, PHE
A: 554
H3CO
1UAG -8.8
Nil
Nil
LYS A: 348,
ALA A: 414,
LEU A: 416
LYS A: 262
ALA A: 66,
PHE A: 72,
TYR A: 144,
ILE A: 140,
LYS A:
LYS A: 115,
LYS A: 319
2X50
3UDI
-8.5
-11.0
PHE A: 303
Nil
2.42
Nil
PHE A: 303
N
PHE A: 72, TYR
A: 144, LYS A:
147, ILE A:
N
148,LYS A: 137
Cl
137,PRO A:
184
1UAG -8.9
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
LYS A: 348,
LEU A: 416
LEU A: 416, ALA
A: 414, PHE A:
422, ASP A: 185
LYS A: 147,PHE
A: 72, TYR A: 144,
ILE A: 148
TYR A: 144, ILE
A: 148, LYS A:
147
N
2X5O
3UDI
-8.4
-8.9
ILE A: 140,
PRO A: 184
N
ILE A: 140,
PRO A: 184
Cl
1UAG -8.7
LEU A: 15,
LYS A: 420,
ALA A: 414
LYS A: 262
LYS A: 420
N
2X5O
3UDI
-8.7
HIS A: 267, PHE
A: 303, GLU A:
304
LYS A: 147, ILE
A: 148, PHE A: 72
N
-10.4
ALA A: 66,
PRO A: 184,
PHEA: 63, ILE
A: 148
Cl
Cl
1UAG -9.1
Nil
Nil
LEU A: 15,
PRO A: 142
LEU A: 15, LYS
A: 115, GLY A:
137
N
N
2X5O
3UDI
-8.6
PHE A: 303
Nil
2.41
Nil
LYS A: 262,
LEU A: 333
ILE A: 140,
PHE A: 72,
ALA A: 66,
PRO A: 184
PHE A: 303
-10.8
LYS A: 147, ILE
A: 148, PHE A: 72,
TYR A: 144
Br
1UAG -7.8
LEU A: 416,
SER A: 415,
HIS A: 183,
LYS A: 115,
LYS A: 319
ASN A: 315,
TYR A: 317,
ALA A: 314,
ASN A: 416,
GLU A: 281
VAL A: 305,
PHE A: 303,
SER A: 264,
ALA A: 328,
ALA A: 329
2.78,
2.68,
2.29,
2.42,
2.10
2.84,
1.78,
2.33,
2.16,
2.74
2.66,
2.44,
2.26,
2.81,
1.88
LYS A:
319,PHE A:
422
ALA A: 414
H
H
N
H
N
H
S
3UDI
2X5O
-7.6
-8.3
Nil
Nil
GLU A: 281
H
O
O
O
O
O
HIS A: 267, PHE
A: 303
H

1318 Chinnamanayakar et al.
Asian J. Chem.
compound (2d) showed good binding affinity score (-9.6 kcal/
mol, -8.7 kcal/mol and -11.1 kcal/mol) compared to other
synthesized 2-phenylpyrazoline derivatives (2a-j except 2d)
and a standard drug (amoxicillin). Other derivatives binding
affinity scores are given in Table-1.
Conventional hydrogen bond interaction: Based on high
binding affinity score, compound 2d has one conventional
hydrogen bond interaction (ASNA: 138) formed with the pyrazole
moiety (1UAG). The hydrogen bond distance is 2.40 Å. Compound
2d has one hydrogen bond interaction (PHE A: 303) with the
pyrazole moiety (2X5O). The hydrogen bond distance is 2.45
Å. Compound 2d has no hydrogen bond interaction with the
3UDI protein.
Hydrophobic interaction: Based on high binding affinity
score, compound 2d have three hydrophobic interaction (LEU
A: 15, PRO A: 41, PRO A: 142) formed with the benzene ring
and bromo moiety (1UAG). Compound 2d have one hydrophobic
interaction (LYSA: 262) formed with the benzene ring and bromo
moiety. Compound 2d have four hydrophobic interaction
(ALAA: 66, LYSA: 137, ILEA: 140, PROA: 184) formed with
2-phenylpyrazoline benzene ring and substituted bromo-
benzene ring.
Molecular docking study with breast cancer protein
(1OQA): The synthesized new 2-phenylpyrazoline derivatives
(2a-j) was docked with breast cancer protein (1OQA). The results
are shown in Table-2. Thus, compound 2d have high binding
TABLE -2
MOLECULAR DATA FOR 2- PHENYL PYRAZOLINE DERIVATIVES (2a-j) DOCKED WITH BREAST CANCER PROTEIN
Binding
Protein affinity score
(kcal/mol)
Conventional H-Bond
Hydrophobic
interaction
Compound
hydrogen
bond
length
(Å)
Other interactions
PRO A: 59, ILE
A: 102, PRO A:
103
N
N
ASN A: 66, GLY
A: 67, PHE A: 68
1OQA
-7.8
Nil
Nil
N
N
PRO A: 103,
PRO A: 59
THR A: 81, PHE
A: 68
1OQA
1OQA
-8.1
-7.9
Nil
Nil
Nil
Nil
O2N
N
N
PRO A: 59,
PRO A: 103
PHE A: 68, THR
A: 81
F
N
PRO A: 103,
PRO A: 59,
TYR A: 100
GLU A: 83, PHE
A: 68, THR A: 81,
PRO A: 59
N
1OQA
1OQA
1OQA
-8.8
-8.2
-7.7
Nil
Nil
Nil
Nil
Nil
Nil
Br
PRO A: 59,
PHE A: 68,
PRO A: 103,
ILE A: 102
N
N
Nil
H3C
N
PRO A: 59, ILE
A: 102, PRO A:
103
N
GLU A: 83, THR
A: 81
H3CO
PRO A: 59, ILE
A: 102, PRO A:
103
N
N
ASN A: 66, GLY
A: 67
1OQA
1OQA
-7.8
-7.8
Nil
Nil
Nil
Nil
Cl
N
N
PRO A: 103,
PRO A: 59
PHE A: 68, HIS A:
69, THR A:81
Cl

Vol. 31, No. 6 (2019)
Synthesis and Characterization of 2-Phenylpyrazoline Derivatives 1319
N
N
ILE A: 102,
PRO A: 59,
PRO A: 103
GLY A: 67, PHE
1OQA
1OQA
-8.0
-7.9
Nil
Nil
Nil
Nil
A: 68
Cl
Cl
N
N
PRO A: 103,
PRO A: 59, ILE
A: 102
PHE A: 68, GLY
A: 67, ILE A: 102
Br
affinity score (-8.8 kcal/mol) compared to other compounds
(2a-j) of this series. Other compounds binding affinity scores
are shown in Table-2. Based on the high binding affinity score,
compound 2d have three hydrophobic interactions (PRO A:
59, PRO A: 103, TYR A:100) formed with pyrazole moiety.
Other compounds hydrophobic interactions are shown in Table-2.
Based on this result, compound 2d wasperformed in vitro anti-
cancer activity against MDA-MB-231 cell line.
Among the compounds (2a-j) subjected to in silico analysis
against the bacterial cell wall proteins 1UAG, 2X5O and 3UDI.
Compound 2d showed good binding affinity score -9.6, -8.7
and -11.1 kcal/mol when docked with these three proteins,
indicating a very good affinity. In a similar way, these synthe-
sized compounds (2a-j) were docked with the human breast
cancer protein (1OQA). Compound 2d showed a binding score
of -8.8 kcal/mol and thereby indicated it is a good candidate
for further studies. So in vitro studies (MTT assay of antimicro-
bial studies) were carried out to confirm its activities.
MTT assay: Based on high binding affinity score, compound
2d (4,5-dihydro-1-phenyl-3-diphenyl-5-(4-bromophenyl)-1H-
pyrazole) were screened for in vitro anticancer studies (human
breast cancer). The in vitro anticancer activity was performed
by MTT assay method. It was done by various concentration
(100, 50, 25, 12.5, 6.25 µg/mL) of compound 2d against MDA-
MB-231 cell line. From this result, compound 2d showed
moderate activity in all concentration except 6.25 µg/mL. The
LC50 value for this compound is -185.309 1.469 µg/mL.
Antimicrobial activity:Antibacterial activity were screened
for 2-phenylpyrazoline derivatives (2a-j) using different strains
(S. aureus, Klebsiella, E. coli and Streptococcus) at different
concentrations (100, 50 and 25 mg/mL). The results are shown
in Table-3. It is seen that all 2-phenylpyrazoline compounds
showed a good zone of inhibition against these four strains.
At high concentration (100 mg/mL), compound 2i showed
a good zone of inhibition (34 mm) against Streptococcus;
Compounds 2b, 2d and 2i showed a good zone of inhibition
(13 mm) against E. coli; Compound 2j showed good zone of
inhibition against Klebsiella strain (28 mm); Compounds 2b,
2d and 2i have shown a good zone of inhibition (13 mm) against
S. aureus.
At low concentration (25 mg/mL), compounds 2b, 2c, 2d
and 2i shown good zone of inhibition (10 mm) against S. aureus;
Compound 2a showed a good zone of inhibition (15 mm)
against Klebsiella; Compounds 2b, 2c and 2d (10 mm) showed
a good zone of inhibition against E. coli, while compound 2c
have shown a good zone of inhibition against Streptococcus.
Finally from these results, compound 2i (2,3-dichloro)
substitution shown a good zone of inhibition at high concen-
tration compared to other derivatives of this series (2a-j). At
low concentration, compound 2c (fluoro substitution) shown
a good zone of inhibition compared to other derivatives of
this series (2a-j).
Antifungal activity: 2-Phenylpyrazoline derivatives were
screened for antifungal activity at different concentrations (100,
TABLE-3
ANTIBACTERIAL ACTIVITY TEST FOR 2-PHENYL PYRAZOLINE DERIVATIVES
WITH DIFFERENT STRAINS BY AGAR DISK DIFFUSION METHOD
S. aureus
Klebsiella
E. coli
Streptococcus
Comd.
100
50
25
100
50
25
100
50
25
100
50
25
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
mg/mL
12
13
12
13
–
10
–
11
13
10
10
11
11
12
–
–
–
8
12
5
5
10
10
10
–
–
–
5
10
–
18
17
13
16
12
24
24
20
23
28
15
>10
>10
11
11
11
14
15
16
16
12
–
>10
>10
>10
10
14
13
13
14
12
13
12
13
–
10
–
11
13
10
10
11
11
12
–
–
–
8
12
5
5
10
10
10
–
–
–
5
0
21
23
24
20
20
25
30
32
34
22
16
13
22
13
18
13
20
27
19
13
14
–
2a
2b
2c
2d
2e
2f
2g
2h
2i
20
11
17
12
18
16
–
–
12
2j
– which means there is no zone of inhibition.

1320 Chinnamanayakar et al.
Asian J. Chem.
50 and 25 mg/mL), the results are shown in Table-4. It is found
that 2-phenylpyrazoline derivatives did not showed any zone
of inhibition against Candida albicans.
and 2,3-dichloro substitution showed a good zone of inhibition
at high concentration (100 mg/mL) against Streptococcus.
CONFLICT OF INTEREST
TABLE-4
The authors declare that there is no conflict of interests
regarding the publication of this article.
ANTIFUNGAL ACTIVITY TEST FOR 2-PHENYL
PYRAZOLINE DERIVATIVES (2a-j)
Antimicrobial susceptibility test against Candida albicans
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Conclusion
A new series of 2-phenylpyrazoline derivatives were synthe-
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data. The synthesized compounds were screened for molecular
docking studies using bacterial proteins and breast cancer
protein. Among the synthesized compounds, compound 2d
(4-bromo substitution) showed a good binding score and good
binding interaction as compared to other derivative compounds.
Thus, compound 2d was selected for the anticancer activity
using MTT assay method, which showed a moderate activity
of LC50 value (185.30 1.469 µg/mL). The antimicrobial activity
was screened for synthesized 2-phenylpyrazoline derivatives
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