Design, synthesis and pharmacological evaluation of 2-phenyl quinazolin-4-one derivatives as anticolorectal cancer and anti-inflammatory agent

Article abstract of DOI:10.14233/ajchem.2018.21547

Quinazoline derivatives are heterocyclic compounds that acts as important structural lead for the discovery of effective therapeutic agents. The anti-inflammatory activity along with cytotoxicity helps to reduce the inflammation and pain associated with carcinoma. Derivatives of quinazoline-4-one were preliminary screened and in silico molecular modelling studies using Autodock were performed. In the docking study, ligands were docked against anticancer and anti-inflammatory targets. In silico analysis revealed that the compounds with aromatic substitution at 3rd and halogen substitution at 6th or 7th positions possess lesser side effects and have more potent antitumor activity. Based upon these results 12 compounds were selected, synthesized, characterized and screened for their in vitro, anti-inflammatory, antioxidant and anticancer activities. From the in vitro studies, compounds QA4, QA7 and QB1 showed good anticancer, anti-inflammatory and antioxidant activity when compared to the standard.

Full text of DOI:10.14233/ajchem.2018.21547

Asian Journal of Chemistry; Vol. 30, No. 12 (2018), 2677-2685
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2018.21547
Design, Synthesis and Pharmacological Evaluation of 2-Phenyl Quinazolin-4-one
Derivatives as Anticolorectal Cancer and Anti-Inflammatory Agent
*
DEENA BOSCO, ASHITHA BALAKRISHNAN, ROHAN MISHRA and T.P. ANEESH
Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Amrita University, AIMS Health Sciences Campus, Kochi-682 041, India
*Corresponding author: E-mail : aneeshtp@aims.amrita.edu
Received: 28 June 2018;
Accepted: 1 September 2018;
Published online: 31 October 2018;
AJC-19131
Quinazoline derivatives are heterocyclic compounds that acts as important structural lead for the discovery of effective therapeutic agents.
The anti-inflammatory activity along with cytotoxicity helps to reduce the inflammation and pain associated with carcinoma. Derivatives
of quinazoline-4-one were preliminary screened and in silico molecular modelling studies usingAutodock were performed. In the docking
study, ligands were docked against anticancer and anti-inflammatory targets. In silico analysis revealed that the compounds with aromatic
substitution at 3rd and halogen substitution at 6th or 7th positions possess lesser side effects and have more potent antitumor activity. Based
upon these results 12 compounds were selected, synthesized, characterized and screened for their in vitro, anti-inflammatory, antioxidant
and anticancer activities. From the in vitro studies, compounds QA4, QA7 and QB1 showed good anticancer, anti-inflammatory and anti-
oxidant activity when compared to the standard.
Keywords: Quinazolin-4-one, Colorectal cancer, Drug designing, Molecular docking, Anticancer activity.
[2,3], antibacterial [4], analgesia [2], antivirus [5], anti-
cytotoxin [6], antioxidation [7] and antidiabetes [8] effects.
Quinazolinones are also the fundamental part of about 150
natural alkaloids occurring in some families of plants, animals
and microorganisms [9]. In addition, the nucleus constructs
an intrinsic part in many drugs currently used in numerous
clinical medicine [10]. The relevance of quinazoline derivatives
in biology has been thoroughly examined they earned
increasing attentions since they were proven to be promising
anticancer agents.
INTRODUCTION
The practice of cancer medicine has faced drastic
transformations over the past decade with the development of
a large number of curative and palliative medicines. However,
none of the available therapy has proven to alleviate cancer
and prevent its remission. Also with the advancement of the
modern lifestyle, newer forms of cancer and related mutations
are cropping up. This provides a need for development of a
novel therapeutic agent for cancer therapy.
Quinazoline is one of the widest spread scaffolds among
natural and synthetic bioactive compounds. With the chemical
formula C8H6N2, this heterocyclic compound is made up of
two fused rings a benzene ring and a pyrimidine ring which
are both six-membered aromatic ring. Quinazolines are
insoluble in most of the organic solvents and water but soluble
in aqueous alkali and are always high melting crystalline solids.
Keto quinazolines otherwise simply termed as quinazolinones
is one of the most important therapeutically active derivative
of quinazoline system. Quinazolin-4-(3H)-one has been consi-
dered as an important research compound as it carries several
therapeutic uses such as anticancer [1], anti-inflammatory
Colorectal cancer starts in the colon or rectum, which can
be either inherited or due to alterations in the genomes. First
stage in colorectal cancer is the formation of polyps on the
inner lining of colon or rectum and develops into cancer slowly.
The development of cancer may take several years. The major
cause of morbidity and mortality all over the world is colorectal
cancer. Of all cancer cases it accounts for over 9 %. Structure
activity relationship studies showed that substitutions at 2 and
3 positions of quinazolin-4-one ring are related to cytotoxic
activity of these derivatives. In the present investigation we
reported some 2-phenyl 3-(substituted) quinazolin-4-(3H)-one
derivatives and screened them for anticancer properties and
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2678 Bosco et al.
Asian J. Chem.
anti-inflammatory properties.Antioxidant activity of the active
derivatives was also examined.
Anthranilic acid was completely precipitated by the gradual
addition of 25 mL of glacial acetic acid. The crude product
was recrystallized from hot water with the addition of little
amount of charcoal. The recrystallized product was collected
and dried at 100 °C [11]. m.f.: C7H7N2O, m.w.: 137, colour
and appearance: light yellow colour crystals, m.p.: 144 °C,
yield: 80 %, Rf value: 0.6 (hexane:ethyl acetate (8:2)), UV
EXPERIMENTAL
Design of the derivatives: Software’s used for ligand
designing are ChemSketch, Corina, Mol inspiration, Molsoft,
Swiss PDBViewer, Cygwin andAutoDock. From the literature
review, quinazoline-4-one was selected for the further study.
By giving different substitutions at 3rd, 6th and 7th positions of
2-phenyl quinazolin-4-one ligands were designed. From the
online software CORINA 3D structure were generated and the
molecular structure were drawn by using ChemSketch software.
Lipinski’s rule of five by using Molinspiration online soft-
ware was calculated by giving the structures of the molecules as
the input. The druglikeness of the molecule in terms of hydrogen
bond acceptors, donors, molecular weight and physiological
log P would be provided as the output. AutoDock was used
for the docking studies, which is a free molecular docking
package. Docking score of each drug molecule against all the
targets were analyzed.
λ
max (methanol) 310 nm, IR (KBr, νmax, cm-1): 3608 (NH2) 3030
(OH), 2916 (C-H, Ar-H), 1764 (C=O).
Synthesis of 2-phenyl-4(3H)-3,1-benzoxazinone: 0.05
mol of benzoyl chloride was added to a solution of equimolar
anthranilic acid in 25 mL of pyridine drop-wise with the
temperature maintained between 0-5 °C for 1 h. The stirring
was continued for another 2 h at room temperature to get a
solid product. Upon neutralization of the above reaction
mixture with sodium bicarbonate solution yields pale yellow
solid crude product (compound 1). This separated product can
be filtered, washed with water and then recrystallized from
ethanol [12]. m.f.: C21H16N2O, m.w.: 223, Colour and Appea-
rance: cream colour crystals, m.p.: 120 °C, yield: 78 %, Rf
value: 0.70 [toluene:chloroform (8:2)], UV λmax (methanol)
313 nm, IR (KBr, νmax, cm-1): 3030 (C-H, Ar-H), 1764 (C=O),
1614 (C=N), 1254 (C-O).
Synthesis of QA1-QA9: An equimolar mixture of 2-phenyl-
4(3H)-3,1-benzoxazinone and substituted anilines were taken
in a round bottom flask containing glacial acetic acid and
refluxed for 6 h. TLC was performed in a timely manner to
determine the progress and completion of the reaction. Upon
completion of the reaction, the reaction mixture was poured
onto crushed ice to for solid mass. This crude product obtained
was filtered, washed with water and recrystallized from warm
ethanol [11].
Synthesis and characterization
General information for synthesis and characterization:
All the chemicals used were commercially procured from
Spectrum Reagents and Chemicals Pvt. Ltd. Cochin, Nice
Chemicals Pvt. Ltd. Cochin, Fisher Scientific Mumbai, Sigma-
Aldrich Bangalore, Loba Chemie Pvt. Ltd. Mumbai andAlfa-
Aesar USA and were of analytical grade. Using thermo electric
melting point apparatus, melting point apparatus, roy capillary,
melting points were determined and were uncorrected. For
TLC Silica gelG plates (3 cm × 8 cm) were used and using
UV and iodine chamber spots were located. The derivatives
were purified by performing column chromatography in silica
gel (100-200 mesh). The IR spectra were determined on FT-
IR Shimadzu spectrometer and the values were expressed in
cm-1. 1H NMR and 13C NMR were recorded in DMSO solvents
using TMS as internal reference with Bruker Fourier-NMR
spectrometer in 400 MHz. On ABSCIEX triple quadrupole
MS mass spectra were recorded on with Waters ACQUITY
ultra performance LC.
Synthesis of anthranilic acid: Weighed accurately 30 g
of sodium hydroxide and transferred into a 350 mL conical
flask containing 120 mL distilled water. This was allowed
to cool to 0 °C or below in a bath of ice and salt. 8.4 mL of
bromine was added in portions to the above mixture with
continuous shaking till all the bromine is reacted to provide
with the sodium hypobromite solution. Upon the addition of
bromine, the temperature of the reaction will rise and again
cool the solution to 0 °C or below. Meanwhile a solution was
prepared by dissolving 22 g of sodium hydroxide in 80 mL
water. To the cold solution of sodium hypobromite, added 24
g of powdered pthalimide in one portion to form a smooth
paste with continuous stirring. The flask was removed from
the ice bath and shaken vigorously for approximately 5 min
to get a clear yellow solution. The NaOH solution prepared
earlier was added in one portion and the solution was heated
to 80 °C for 2 min. The solution was cooled in ice and neutra-
lized by slow addition of concentrated hydrochloric acid.
3-(2-Methylphenyl)-2-phenylquinazolin-4(3H)-one
(QA1): m.f.: C21H16N2O; m.w.: 312.46; colour and appearance:
cream colour crystals; m.p.: 110 °C; yield: 80 %, Rf value:
0.82 (acetonitrile:ethyl acetate (1:1)); IR (KBr, νmax, cm-1): 3030
(Ar-H str), 1764 (C=O), 1614 (C=N), 1180 (C-N), 1254 (C-
1
O), 2850 (C-H str), C-C (1269), 2732 (C-CH3); H NMR
(DMSO, 400 MHz) δ 8.28 (s, 2H), 7.39 (d, J = 6 Hz, 1H),
7.10 (d, J = 6.2 Hz, 1H), 6.86 (s, 2H), 6.62 (d, J = 7.4 Hz, 1H);
13C NMR (DMSO, 100 MHz) δ 160.33, 157.28, 149.09,
148.79, 130.30, 129.94, 129.87, 129.57, 126.56, 121.80,
120.43, 112.94, 112.51, 102.24, 55.99, 45.60; MS m/z (%):
312 (100), 313 (M+1, 20), 314 (M+2, 4).
3-(4-Chlorophenyl)-2-phenylquinazoline-4(3H)-one
(QA2): m.f.: C20H13N2OCl; m.w.: 332; colour and appearance:
off white crystals; m.p.: 190 °C; yield: 68.56 %; Rf value: 0.75
(n-hexane:ethyl acetate [1:1]); IR (KBr, νmax, cm-1): 3026 (Ar-
H str), 822 (C-C), 1179 (C-N), 1607 (C=N), 1665 (C=O), 686
(C-Cl); 1H NMR (DMSO, 400 MHz) δ 9.62 (s, 2H), 8.15 (d, J
= 7.6 Hz, 1H), 7.18 (d, J = 6.2 Hz, 1H), 6.80 (s, 2H); 13C NMR
(DMSO, 100 MHz) δ 160.33, 157.27, 148.83, 148.80, 133.10,
130.57, 129.86, 127.08, 126.67, 126.16, 120.43, 112.94,
112.51, 102.23, 55.99, 45.58; MS m/z (%): 332 (100), 334
(M+4,34).
3-(2-Ethyl-6-methylphenyl)-2-phenylquinazolin-4(3H)-
one (QA3): m.f.: C23H20N2O; m.w.: 340; colour and appearance:
pinkish white crystals; m.p.: 130 °C; yield: 74.83 %; Rf value:

Vol. 30, No. 12 (2018)
Design, Synthesis and Pharmacological Evaluation of 2-Phenyl Quinazolin-4-one Derivatives 2679
0.98 (acetonitrile:ethyl acetate (1:1)); IR (KBr, νmax, cm-1): 3016
Rf value: 0.71 (ethyl acetate: chloroform (1:1)); IR (KBr, νmax,
(Ar-H str), 819 (C-C), 1180 (C-N), 1603 (C=N), 1720 (C=O),
cm-1): 3020 (Ar-H str), 822 (C-C), 1179 (C-N), 1607 (C=N),
1665 (C=O), 606 (C-Cl); 1H NMR (DMSO, 400 MHz) δ 9.64
(s, 2H), 8.18 (d, J = 7.6 Hz, 1H), 7.16 (d, J = 6.2 Hz, 1H),6.70
(s, 2H); 13C NMR (DMSO, 100 MHz) δ 160.33, 157.28,
149.09, 148.79, 130.30, 129.94, 129.87, 129.57, 126.56,
121.80, 120.43, 112.94, 112.51, 102.24, 55.99, 45.60; MS m/z
(%) 366 (100), 368 (M+2, 64).
1
2732 (C-CH3); H NMR (DMSO, 400 MHz) δ 7.42 (s, 3H),
7.36 (s, 2H), 7.29 (t, J = 6.24 Hz, 1H), 7.10 (d, J = 8.6 Hz,
1H), 6.81 (s, 2H); 13C NMR (DMSO, 100 MHz) δ 160.28,
159.04, 149.09, 148.89, 133.07, 130.94, 129.96, 129.66,
126.56, 121.84, 120.33, 114.61, 111.43, 100.98, 55.57, 55.37,
45.53; MS m/z (%) 340 (100), 341 (M+1, 28), 342 (M+2, 4).
7-Chloro-3-(4-methylphenyl)-2-phenylquinazolin-
4(3H)-one (QA4): m.f.: C21H15N2OCl; m.w.: 346.8; colour and
appearance: light brown crystals; m.p.: 150 °C; yield: 65.35
6-Chloro-2-(2-ethyl-6-methylphenyl)-3-phenylquina-
zolin-4(3H)-one (QA9): m.f.: C23H19N2OCl; m.w.: 374.86;
colour and appearance: light violet crystals; m.p.: 228 °C; yield:
28.07 %; Rf value: 0.64 (n-hexane:ethanol (1:1)); IR (KBr,
%; Rf value: 0.71 (n-hexane:ethyl acetate (1:1)); IR (KBr, νmax
,
cm-1): 3024 (Ar-H str), 819 (C-C), 1180 (C-N), 1603 (C=N),
1662 (C=O), 646 (C-Cl); 1H NMR (DMSO, 400 MHz) δ 8.18
(s, 2H), 7.29 (d, J = 6 Hz, 1H), 7.08 (d, J = 6.2 Hz, 1H), 6.92
(s,2H), 6.72 (d, J = 7.4 Hz, 1H); 13C NMR (DMSO, 100 MHz)
δ 160.29, 159.04, 148.88, 148.63, 133.15, 130.62, 129.93,
127.10, 126.76, 126,16, 120.32, 114.61, 111.42, 100.98, 55.57,
55.37, 45.50; MS m/z (%) 346 (100), 347 (M+1, 22), 348 (M+2,
34).
ν
max, cm-1): 3010 (Ar-H str), 819 (C-C), 1180 (C-N), 1603
1
(C=N), 1662 (C=O), 2732 (C-CH3); H NMR (DMSO, 400
MHz) δ 7.42 (s, 3H), 7.36 (s, 2H), 7.29 (t, J = 6.24 Hz, 1H),
7.10 (d, J = 8.6 Hz, 1H), 6.81 (s, 2H); 13C NMR (DMSO, 100
MHz) δ 160.28, 159.04, 149.09, 148.89, 133.07, 130.94,
129.96, 129.66, 126.56, 121.84, 120.33, 114.61, 111.43,
100.98, 55.57, 55.37, 45.53; MS m/z (%) 374 (8) 369 (2), 354
(15), 340 (8), 325 (2), 262 (16), 160 (100), 120 (20).
7-Chloro-3-(4-chlorophenyl)-2-phenylquinazolin-4(3H)-
one (QA5): m.f.: C20H12N2OCl2; m.w.: 366.22; colour and
appearance: off white crystals; m.p.: 210 °C; yield: 45.26 %;
Synthesis of 3-amino-2-phenyl-4(3H)-quinazolinone:
To a solution of 0.05 mol of compound 1 in 20 mL pyridine,
added 0.15 mol of 80 % hydrazine hydrate solution. The
mixture was stirred well and refluxed at 117 °C for about 2 h.
after refluxed is stopped, the reaction mixture was cooled and
filtered to get the crude product. The pure compound (comp-
ound 2) can be obtained by recrystallization from warm ethanol
[12]. m.f.: C14H11N3O, m.w.: 237.25, colour and appearance:
lemon yellow crystals, m.p.: 172 °C, yield: 83 %, Rf value:
0.72 (toluene:chloroform (8:2)), UV λmax (methanol) 270 nm,
IR (KBr, νmax, cm-1): 3316 (NH2), 1659 (C=O), 1602 (C=N).
Synthesis of QB1-QB3: 0.47 g of compound 2, 0.002
mol of substituted aryl aldehydes and 2-3 drops of glacial acetic
acid was taken in a round bottom flask and refluxed for 3-4 h.
After the completion of the reaction, which is monitored by
performing TLC, the solvent was removed and the final product
was recrystallized from ethanol [13].
3-{(Z)-[2-(3-Chlorophenyl)ethylidene]amino}-2-
phenylquinazolin-4(3H)-one (QB1): m.f.: C21H14N3OCl;
m.w.: 359.8; colour and appearance: white crystals; m.p.: 214 °C;
yield: 13.89 %; Rf value: 0.72 (dimethyl formamide:ethyl
acetate (1:1)); IR (KBr, νmax, cm-1): 3028(Ar-H str), 1654 (C=O),
1604 (C=N), 1180 (C-N), 697 (C-Cl); 1H NMR (DMSO, 400
MHz) δ 8.96 (s, 2H), 7.83 (d, J = 7.4 Hz, 1H), 6.50 (d, J = 2.4,
2H), 6.21 (d, J = 2.4 Hz, 2H); 13C NMR (DMSO, 100 MHz) δ
163.43, 161.01, 160.34, 157.23, 149.27, 149.20, 148.81,
130.61, 130.52, 129.83, 123.44, 123.42, 120.45, 114.09,
113.88, 113.55, 113.34, 113.05, 112.45, 102.02, 55.99, 45.66;
MS m/z, 359 (M+, 100), 360 (M+1, 22), 361 (M+2, 34).
3-[(E)-(3-Bromobenzylidene)amino]-7-chloro-2-
phenylquinazolin-4(3H)-one (QB2): m.f.: C21H13N3OBrCl;
m.w.: 438.7; colour and appearance: cream crystals; m.p.: 210
°C; yield: 24.28 %; Rf value: 0.71 (acetonitrile:ethyl acetate
(1:1)); IR (KBr, νmax, cm-1): 3030 (Ar-H str), 1654 (C=O), 1604
(C=N), 1174 (C-N), 697 (C-Cl); 1H NMR (DMSO, 400 MHz)
δ 8.86 (s, 2H), 7.73 (d, J = 9.4 Hz, 1H), 6.60 (d, J =4.4, 2H),
6.51 (d, J = 6.4 Hz, 2H); 13C NMR (DMSO, 100 MHz) δ
160.33, 157.27, 148.83, 148.80, 133.10, 130.57, 129.86,
Rf value: 0.74 (ethyl acetate:chloroform (1:1)); IR (KBr, νmax
,
cm-1): 3020 (Ar-H str), 822 (C-C), 1179 (C-N), 1607 (C=N),
1665 (C=O), 606 (C-Cl); 1H NMR (DMSO, 400 MHz) δ 9.62
(s, 2H), 8.15 (d, J = 7.6 Hz, 1H), 7.18 (d, J = 6.2 Hz, 1H), 6.80
(s, 2H); 13C NMR (DMSO, 100 MHz) δ 163.43, 161.01,
160.34, 157.23, 149.27, 149.20, 148.81, 130.61, 130.52,
129.83, 123.44, 123.42, 120.45, 114.09, 113.88, 113.55,
113.34, 113.05, 112.45, 102.02, 55.99, 45.66; MS m/z (%)
366 (100), 368 (M+2, 64).
7-Chloro-3-(2-ethyl-6-methylphenyl)-2-phenylquina-
zolin-4(3H)-one (QA6): m.f.: C23H19N2OCl; m.w.: 374.86;
colour and appearance: pinkish white crystals; m.p.: 185 °C,
yield: 23.28 %; Rf value: 0.72 (n-hexane:ethanol(1:1)); IR
(KBr, νmax, cm-1): 3010 (Ar-H str), 819 (C-C), 1180 (C-N),
1603 (C=N), 1662 (C=O), 648(C- Cl); 1H NMR (DMSO, 400
MHz) δ 7.44 (s, 3H), 7.38 (s, 2H), 7.24 (t, J = 6.24 Hz, 1H),
7.10 (d, J = 8.6 Hz, 1H), 6.81 (s, 2H); 13C NMR (DMSO, 100
MHz) δ 163.44, 161.01, 160.29, 159.02, 149.06, 149.00,
148.88, 130.68, 130.59, 129.91, 123.46, 120.35, 114.70,
114.12, 113.91, 113.65, 113.43, 111.36, 100.96, 55.60, 55.37,
45.67; MS m/z (%) 374 (100) 376 (M+2, 36).
6-Chloro-3-(4-methylphenyl)-2-phenylquinazolin-
4(3H)-one (QA7): m.f.: C21H15N2OCl; m.w.: 346.8; colour and
appearance: off white crystals; m.p.: 155 °C; yield: 20.49 %;
Rf value: 0.64 (ethyl acetate:chloroform (1:1)); IR (KBr, νmax
,
cm-1): 3012 (Ar-H str), 819 (C-C), 1180 (C-N), 1603 (C=N),
1662 (C=O), 2732 (C-CH3); 1H NMR (DMSO, 400 MHz) δ
8.18 (s, 2H), 7.60 (d, J = 6 Hz, 1H), 7.12 (d, J = 6.4 Hz, 1H),
6.94 (s, 2H), 6.66 (d, J = 7.2 Hz, 1H); 13C NMR (DMSO, 100
MHz) δ 160.49, 150.31, 149.12, 148.92, 133.07, 130.51,
129.37, 127.04, 126.56, 126.13, 120.59, 109.61, 109. 45,
98.48, 55.72, 45.94, 45.43; MS m/z (%) 346 (100), 348 (M+2, 34).
6-Chloro-2-(4-chlorophenyl)-3-phenylquinazolin-4(3H)-
one (QA8): m.f.: C20H12N2OCl2; m.w.: 366.22; colour and
appearance: off white crystals; m.p.: 190 °C; yield: 53.05 %;

2680 Bosco et al.
Asian J. Chem.
127.08, 126.67, 126.16, 120.43, 112.94, 112.51, 102.23, 55.99,
45.58; MS m/z, 439 (M+, 100), 441 (M+2, 26), 437 (74).
3-[(E)-(3-Bromobenzylidene)amino]-6-chloro-2-phenyl-
quinazolin-4(3H)-one (QB3): m.f.: C21H13N3OBrCl; m.w.:
438.7; colour and appearance: cream crystals; m.p.: 210 °C;
yield: 24.28 %; Rf value: 0.71 (acetonitrile:ethyl acetate (1:1));
IR (KBr, νmax, cm-1): 3024 (Ar-H str), 1684 (C=O), 1624 (C=N),
1180 (C-N), 697 (C-Cl); 1H NMR (DMSO, 400 MHz) δ 8.76
(s, 2H), 7.43 (d, J = 7.2 Hz, 1H), 6.40 (d, J = 2.8, 2H), 6.11 (d,
J = 2.6 Hz, 2H); 13C NMR (DMSO, 100 MHz) δ 160.33,
157.28, 149.09, 148.79, 130.30, 129.94, 129.87, 129.57,
126.56, 121.80, 120.43, 112.94, 112.51, 102.24, 55.99, 45.60;
MS m/z: 439 (M+, 100), 441 (M+2, 26), 437 (74).
different test concentrations of the corresponding test drugs
were added on the partial monolayer in microtitre plates. Then
it was incubated for 3 days at 37 °C in 5 % CO2 atmosphere
and microscopic examinations were carried out every 24 h.
The drug solutions in the wells were discarded after 72 h and
50 µL of MTT in PBS was added to each well. Then the plates
were shaken gently and incubated at 37 °C for 3 h in 5 % CO2
atmosphere. The supernatant was removed and then propanol
of volume 100 µL was added and the plates were gently shaken
to solubilize the formed formazan. The absorbance was mea-
sured at a wavelength of 540 nm using a micro plate reader
[15,16]. The percentage growth inhibition was calculated and
from the dose-response curves concentration of test drug req-
uired to inhibit cell growth by 50 % (CTC50) values for each
cell line are generated [17].
Antioxidant activity
Nitric acid free radical scavenging method: Nitric oxide
was generated from sodium nitroprusside and measured by
the Griess reagent. 0.5 mL of the sample was mixed with 0.5 mL
of phosphate buffer (pH 7.4) and 2 mL sodium nitroprusside
solution. The mixture was incubated at 25 °C for 2 h. 0.5 mL
of the reaction mixture was pipetted out and mixed with 1 mL
pf sulphanilic acid and allowed to stand for 5 min for complete
diazotization. Then 1 mL of 0.1 % N-naphthylethylenediamine
dihydrochloride was added, mixed and allowed to stand for
30 min to form pink coloured chromophore. Absorbance was
then measured at 530 nm against the corresponding blank
solution.
Hydrogen peroxide free radical scavenging method:
1 mL of standard ascorbic acid and test solution was added to
0.6 mL hydrogen peroxide solution. After 10 min the absor-
bance of the solution was measured at 230 nm using UV/visible
spectrophotometer against a blank solution containing phos-
phate buffer without hydrogen peroxide. The percentage scaven-
ging of hydrogen peroxide of both and test compound were
determined.
RESULTS AND DISCUSSION
Design of novel quinazoline-4-ones: 2-Phenyl-quina-
zolin-4-one derivatives were docked against the three known
targets, thymidylate synthase (1JU6) and focal adhesion kinase
receptor (1MP8) were anticancer targets and cyclooxygenase
2 receptor (1CX2) was the anti-inflammatory protein target.
The ligands were validated by docking operation usingAutoDock.
Reference standards were used to compare docking score of
the ligands. The bound analogues were examined for their
hydrogen bonding and binding energies, which was shown in
the Table-1.
TABLE-1
AUTODOCK DOCKING SCORES
Docking score (kcal/mol)
Compd.
code
S. No.
1JU6
1MP8
-6.41
-7.23
-7.44
-6.45
-7.06
-6.26
-5.89
-6.34
-6.99
-7.21
-7.24
-6.48
-4.31
–
1CX2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
-7.41
-7.10
-6.23
-6.16
-5.99
-5.98
-7.45
-7.05
-6.35
-6.10
-6.92
-6.99
-4.83
–
-5.86
-6.41
-6.82
-6.61
-5.49
-6.37
-5.16
-6.83
-6.81
-6.84
-6.42
-5.86
-3.21
-4.12
QA1
QA2
QA3
QA4
QA5
QA6
QA7
QA8
QA9
In vitro anti-inflammatory activity: 2 mL of each sample
was mixed with 2 mL of 2 mM of BSA (1.329 g in 10 mL of
phosphate buffer) and incubated at 27 1 °C for 15 min. Denatu-
ration was induced by keeping the reaction mixture at 60
1
°C in electronic water bath for 10 min. After cooling, the
turbidity was measured at 660 nm. The percentage inhibitions
of all test samples were determined.
QB1
QB2
QB3
In vitro cytotoxicity-MTT assay: Cell lines used were
HT-29 cell line was obtained from National Centre for Cell
Sciences, Pune, India. Stock cells was cultured in culture medium
composed of DMEM supplemented with 10 % inactivated
foetal bovine serum, streptomycin (100 µg/mL), penicillin (100
IU/mL) and amphotericin B (5 µg/mL) in a moist atmosphere
of 5 % CO2 at 37 °C until consistent. The cells were disasso-
ciated with TPVG solution (0.2 % trypsin, 0.02 % EDTA, 0.05 %
glucose in PBS). The stock cultures were grown in 25 cm2
culture flasks and 96 microtitre plates were used to carry out
the experiment [14-18].
Procedure: The cell culture was trypsinized and by using
DMEM containing 10 % FBS the cell count was adjusted to
1.0 × 105 cells/mL. 0.1 mL of the diluted cell suspension were
added to each well of 96 well microtitre plate.A partial mono-
layer was developed after 24 h, the supernatant flicked off,
washed the monolayer once with medium and 100 µL each of
Raltitrexed
Ibuprofen
AutoDock results showed that most of the derivatives have
hydrogen bonding between the ligand-target interactions (Fig.
1). These hydrogen bonding interactions helped to increase
the binding energy of ligand-protein interactions.
Lipinski rule of 5 and their extension parameters like
number of rotatable bonds and TPSA were used to evaluate
the drug likeness of the derived analogues. The drug-likeness
assessments of the compounds were shown in Table-2.
These results revealed that the value of all the derivatives
were within the optimal range of molecular weight less 500
Daltons and possessed number of hydrogen bond donors (HBD)
and hydrogen bond acceptors (HBA) of all the analogues below
5 and 10, respectively. All the number of rotatable bonds and

Vol. 30, No. 12 (2018)
Design, Synthesis and Pharmacological Evaluation of 2-Phenyl Quinazolin-4-one Derivatives 2681
QA3-IJU6
QA7-IJU6
QB1-IJU6
QA1-IMP8
QA3-IMP8
QA6-IMP8
QA4-ICX2
QB3-ICX2
Fig. 1. Ligand-target complexes with H2 bonding interactions by AutoDock 4.0 program
TABLE-2
DRUG LIKENESS ASSESSMENTS OF THE 2-PHENYL-QUINAZOLIN-4-ONE DERIVATIVES
Number of
rotatable
bonds
Compd.
code
Number of
HBA
Number of
HBD
R1
H
R2
H
R3
C log P
4.37
TPSA (Å2)
69.277
CH3
2
2
0
0
2
2
QA1
QA2
H
H
H
H
4.80
3.15
74.277
62.283
Cl
CH3
2
0
2
QA3
H3C
Cl
Cl
H
H
2
2
0
0
4.20
3.52
2
2
69.283
76.277
QA4
QA5
CH3
Cl
CH3
Cl
H
2
0
3.86
3
62.287
QA6
H3C
H
H
Cl
Cl
2
2
0
0
4.20
3.52
2
2
69.283
76.277
QA7
QA8
CH3
Cl

2682 Bosco et al.
Asian J. Chem.
CH3
H
Cl
2
0
3.86
3
62.287
QA9
H3C
N
Cl
Br
Br
H
Cl
H
H
H
3
3
3
0
0
0
4.91
4.76
4.76
3
3
3
68.682
70.471
70.471
QB1
QB2
QB3
N
N
Cl
values of partition coefficient were under the limit of 10 and
5, respectively. The polarity of the molecules is explained by
the physico-chemical property called topological polar surface
area, none of the analogues exhibited TPSA greater than 140
Å2. This parameter has been found to associate with the gastro-
intestinal absorption, Caco-2 permeability and blood-brain
barrier penetration.All these data revealed that having no more
violations makes it likely to be an orally active drug.
Substituted or unsubstituted anthranilic acid reacts with
benzoyl chloride followed by dehydration to form the benzo-
xazinone intermediate. The subsequent addition of an amine
or an aldehyde to the above mixture provides the fused quinazo-
linones. Reaction for the synthesis was shown in Fig. 2.
IR spectrum showed characteristic bands at 3150-3050
and 1600-1400 cm-1 indicating the presence of aromatic –CH
stretching and C=C stretching respectively. The characteristic
OH
R
2
R
R
N
1
2
O
O
N
+
H
O
stirring 2 hrs
R
N
H
Cl
1
O
Benzoyl chloride
Substituted Anthranilic acid
substituted 2-phenyl benzoxazin-4-one
(R
3
)
6 hrs reflux
COOH
4hrs reflux
2
Ar-NH
NH2NH2.H2O
glacial CH
3
O
H
R
R
N
O
1
2
R
R
N
O
1
2
+
N
NH
NH
2
Ar
X
(R4)
(QA1 - QA9)
4 -7 hrs
reflux
C2H5OH
R
N
1
2
Ar
X
C6H5CH3, C6H5CH2CH3,C6H5Cl
Cl, Br
N
R
N
O
X
(QB1-QB3)
Fig. 2. Preparation of 2-phenyl-3-substituted quinazoline-4-one

Vol. 30, No. 12 (2018)
Design, Synthesis and Pharmacological Evaluation of 2-Phenyl Quinazolin-4-one Derivatives 2683
TABLE-3
bands for alkyl –CH were seen at 3000-2800 cm-1. The charac-
teristic bands for halogen groups like chlorine and bromine
were found at 800-680 cm-1 and 616-620 cm-1, respectively.
For C=O of amide, the characteristic peaks were observed at
1670-1630 cm-1. The characteristic peak for C-N and C=N
were found at 1280-1180 and 1640-1600 cm-1, respectively.
This clearly suggests the formation of expected compounds.
1H and 13C were carried out using DMSO as solvent. The
characteristic signals for the protons nearer to electron
withdrawing groups were found at higher chemical shift range
and signlas for the proton nearer to electron donating groups
INHIBITION (%) SHOWED BY SYNTHESIZED
COMPOUNDS (ALBUMIN DENATURATION METHOD)
Percentage inhibition SEM
Compound
code
IC50 µg/mL
10 µg/mL
20 µg/mL
30 µg/mL
QA1
QA2
QA3
QA4
QA5
QA6
QA7
QA8
QA9
QB1
QB2
QB3
Ibuprofen
33.20 0.28 48.40 0.22 59.73 0.26
36.19 0.69 52.00 0.52 66.94 0.39
32.67 0.13 56.66 0.38 67.79 0.33
29.41 0.08 54.13 0.04 67.77 0.39
31.64 0.38 54.31 0.61 63.81 0.48
31.66 0.39 52.30 0.67 58.24 0.62
35.41 0.48 69.99 0.77 83.19 0.69
37.62 0.17 47.76 0.20 64.66 0.32
41.44 0.22 56.24 0.66 64.58 0.08
41.18 0.68 65.12 0.34 70.34 0.16
31.56 0.21 45.76 0.25 68.46 0.32
40.70 0.01 48.53 0.01 56.12 0.03
80.35 0.07 80.80 0.04 84.22 0.06
20.8
18.4
16.8
14.2
18.0
16.6
13.8
20.4
14.4
16.2
22.6
20.2
11.8
1
were found at lower chemical shift range. H NMR showed
peaks for the protons of alkyl group at 2-4 δppm. For aryl protons
peaks were found at 6-10 δ ppm. First and second hydrogens
singlet or doublet peaks depended upon the substitutions on
the adjacent carbon atom. For 4th, 5th and 6th hydrogens a single
triplet peak and for 3rd and 5th hydrogens a single doublet peaks
were seen. The δ value were found at 6.5-8.1 ppm for above
hydrogens. 13C NMR signals assigned to confirm the number
of carbon atoms present in the prepared compound. Aromatic
carbon aatoms on the ring were seen in the range of 120-150
δ ppm. Aliphatic carbon atoms were found at 2-4 δ ppm. This
1H and 13C NMR spectral data led to confirm the structure of
the targeted compounds.
Formation of the derivatives were also confirmed by mass
spectrometric analysis. Mass spectroscopy of these compounds
were recorded in the positive ion mode. Base peak and mole-
cular ion peak were same. Halogens were present in majority
of the compound and gave characteristic M+2 peaks.
In vitro analysis: The in vitro anti-inflammatory activity
of 2-phenyl quinazolin-one derivatives was evaluated by
albumin denaturation method and is compared with that of the
standard ibuprofen. Results were summarized in the Table-3.
All the compounds showed significant level of anti-infla-
mmatory activity, but the level of percentage inhibition was
less than the reference standard. The results states that the mole-
cules QA7, QA4 and QA9 having a very low IC50 value when
compared to the other molecules.
hydroxytoluene (BHT) were used as reference standards. Per-
centage inhibitions of all the derivatives found out by hydrogen
peroxide method and nitric oxide radical scavenging method
were shown in the Tables 4 and 5, respectively.
Nitric oxide scavenging activity displayed by QA4, QA3
and QA1 were higher when compared to the other compounds.
From the results of hydrogen peroxide scavenging activity, it
was observed that maximum scavenging activity was exhibited
by QA4, QA7 and QA1 when compared to standard ascorbic
acid in hydrogen peroxide scavenging method.
In vitro anticancer screening was carried out by MTT assay
in colorectal cancer (HT-29) cell lines. Twelve of the synthe-
sized compounds were submitted to cytotoxicity study. Medium
of cancer cell lines without samples were served as control.
From the dose-response curves concentration of test drug
needed to inhibit cell growth by 50 % (CTC50) was identified.
Dose dependent curve was plotted between % Cell inhibition
and concentration and CTC50 was determined from the curve
by using Graph Pad Prism software. The results were shown
in Table-6.
In vitro activity screening was done for all the compounds
at different concentrations of 100, 50, 25, 12.5 and 6.25 µg/mL.
All the compounds possessed moderate to good anticancer
In vitro antioxidant activity of 2-phenyl quinazoln-4-one
derivatives was assessed by hydrogen peroxide and nitric oxide
free radical scavenging activity. Ascorbic acid and butylated
TABLE-4
PERCENTAGE INHIBITION SHOWED BY SYNTHESIZED DERIVATIVES (N2O SCAVENGING METHOD)
Percentage inhibition SEM
Compound code
IC50 µg/mL
10 µg/mL
20 µg/mL
30 µg/mL
40 µg/mL
50 µg/mL
QA1
QA2
QA3
QA4
QA5
QA6
QA7
QA8
QA9
QB1
QB2
QB3
BHT
39.45 0.78
25.45 0.07
39.57 0.95
26.82 0.10
30.30 0.20
39.29 0.59
32.91 0.85
36.16 0.11
28.51 0.14
29.42 0.82
33.24 0.58
29.60 0.18
31.75 0.14
34.77 0.05
45.60 0.18
39.38 0.10
47.72 0.58
44.13 0.09
46.18 0.14
44.36 0.67
40.25 0.97
43.63 0.47
45.41 0.14
38.76 0.20
40.23 0.69
42.06 0.04
38.55 0.08
50.53 0.05
53.77 0.86
48.61 0.11
53.94 0.20
55.50 0.11
59.41 0.13
52.18 0.47
49.08 0.06
51.00 0.43
54.28 0.18
50.37 0.48
53.32 0.44
53.47 0.12
49.65 0.04
68.47 0.06
58.52 0.83
61.57 0.11
62.95 0.81
65.42 0.26
67.42 0.06
62.17 0.32
54.32 0.41
55.95 0.68
68.63 0.14
55.76 0.95
56.35 0.25
66.61 0.11
58.40 0.48
77.82 0.04
63.18 0.79
70.65 o.11
72.92 0.20
72.69 0.09
76.67 0.09
74.09 0.27
60.39 0.38
63.61 0.09
74.68 0.10
65.05 0.36
66.85 0.06
75.37 0.18
66.28 0.06
89.55 0.17
25.4
30.4
24.8
23.8
26.2
29.4
30.2
29.2
25.6
29.2
29.2
27.3
30.4
20.6
Ascorbic acid

2684 Bosco et al.
Asian J. Chem.
TABLE-5
PERCENTAGE INHIBITION SHOWED BY SYNTHESIZED DERIVATIVES (H2O2 SCAVENGING METHOD)
Percentage inhibition SEM
Compound code
IC50 (µg/mL)
10 µg/mL
20 µg/mL
30 µg/mL
40 µg/mL
50 µg/mL
QA1
QA2
QA3
QA4
QA5
QA6
QA7
QA8
QA9
QB1
QB2
QB3
BHT
25.46 0.06
35.72 0.94
23.64 0.06
27.26 0.05
32.04 0.43
31.70 0.19
29.88 0.79
35.44 0.77
29.35 0.19
34.55 0.14
26.44 0.07
28.21 0.04
29.85 0.22
26.89 0.88
42.75 0.06
41.20 0.64
37.26 0.07
46.46 0.08
42.19 0.81
38.21 0.46
42.65 0.60
40.42 0.52
44.55 0.04
38.77 0.12
41.75 0.22
43.65 0.04
37.86 0.05
41.25 0.06
56.35 0.05
49.77 0.45
49.14 0.05
55.45 0.08
47.50 0.64
48.41 0.77
52.93 0.47
52.29 0.55
56.37 0.17
53.04 0.48
53.61 0.21
54.57 0.08
48.44 0.07
56.76 0.23
65.35 0.09
56.08 0.43
60.83 0.05
63.69 0.06
59.16 0.99
53.95 0.11
63.48 0.66
60.02 0.91
68.35 0.14
58.45 0.61
66.45 0.05
67.33 0.05
59.35 0.08
68.83 0.03
71.83 0.08
64.92 0.86
70.84 0.04
74.83 0.08
69.03 0.46
62.31 0.68
68.41 0.39
70.29 0.49
76.46 0.18
66.71 0.34
74.55 0.04
75.47 0.08
68.14 0.08
86.72 0.06
25.4
31.6
30.4
24.2
30.8
30.2
24.6
26.4
24.6
26.0
29.4
28.0
31.4
24.8
Ascorbic acid
TABLE-6
PERCENTAGE CYTOTOXICITY OF PREPARED COMPOUNDS AGAINST COLORECTAL CANCER (HT-29) CELL LINES
Compound
code
Test concentration
(µg/mL)
Cytotoxicity
(%)
Compound
code
Test concentration
(µg/mL)
Cytotoxicity
(%)
CTC50 (µg/mL)
25.8
CTC50 (µg/mL)
100
50
25
12.5
6.25
100
50
70.73
62.87
48.97
33.64
21.70
76.50
61.89
41.68
28.66
11.71
74.81
64.31
45.73
20.38
10.38
81.14
71.37
66.85
51.87
35.71
68.50
56.19
37.68
28.27
10.31
71.85
67.24
44.96
20.31
16.18
100
50
25
12.5
6.25
100
50
89.37
70.94
67.33
44.50
33.16
71.40
53.78
32.86
27.97
15.21
77.51
62.90
42.69
29.67
12.72
80.59
70.21
59.22
41.96
34.65
83.80
76.14
58.53
32.77
28.64
84.97
66.83
53.17
43.64
21.76
14.8
QA1
QA2
QA3
QA4
QA5
QA6
QA7
QA8
QA9
QB1
QB2
QB3
25
33.6
28.2
11.2
41.6
29.4
25
46.4
33.4
17.6
19.8
19.8
12.5
6.25
100
50
12.5
6.25
100
50
25
25
12.5
6.25
100
50
12.5
6.25
100
50
25
25
12.5
6.25
100
50
12.5
6.25
100
50
25
25
12.5
6.25
100
50
25
12.5
6.25
12.5
6.25
100
50
25
12.5
6.25
activity. Compounds QA4, QA7 and QB1 were the most active
compounds against HT-29 cell lines with IC50 of 11.2, 14.8
and 17.6 /mL, respectively. So these compounds were found
to be more potent than the remaining compounds.
Conclusion
Few quinazoline derivatives were successfully synthesized
in this work. Analytical and spectral analysis were performed
for the same. The structural elucidation was done with the
help of spectral data. These synthesized molecules were
screened for anti-inflammatory, antioxidant and anticancer
activity. It was concluded that all the compounds showed potent
anticancer activity against HT-29 cell line with IC50 less than
50 µg/mL. These compounds possessed significant level of
anti-infla-mmatory and antioxidant activity. Compounds QA4,
QA7 and QB1 showed good anticancer and anti-inflammatory
activity compared to other compounds. Cytotoxic effects of
these compounds in cancer cells indicated that they were good
candidates for further pharmacological studies to discover
effective chemotherapeutic agents for the treatment of human
colorectal carcinoma.

Vol. 30, No. 12 (2018)
Design, Synthesis and Pharmacological Evaluation of 2-Phenyl Quinazolin-4-one Derivatives 2685
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