Synthesis and in vitro cytotoxicity study of novel 4-substituted quinazolines encompassed with thiazolidinone and azetidinone

Article abstract of DOI:10.14233/ajchem.2020.22837

A series of quinazolines encompassed with thiazolidinone and azetidinone have been synthesized and evaluated for their antioxidant, anticancer and DNA binding studies. All the synthesized compounds were characterized by IR, 1H & 13C NMR and mass spectra. Antioxidant activity was carried out using % free radical scavenging by DPPH assay. Compounds 4b, 5b and 5d have shown better antioxidant activity (60, 67 and 66%, respectively) among the tested compounds. Compounds having % free radical scavenging activity more than 55% were evaluated for anticancer activity by MTT assay towards cell lines A-549 (lung carcinoma) and MDA-231 (human breast cancer). Results revealed that the tested compounds exhibited moderate to low anticancer activity. Further, DNA binding activity was studied by absorption titration method for all the synthesized compounds, and compound 5b showed a good binding constant of 70.05 and % hyperchromicity of 82.93%.

Full text of DOI:10.14233/ajchem.2020.22837

Asian Journal of Chemistry; Vol. 32, No. 10 (2020), 2617-2623
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2020.22837
Synthesis and in vitro Cytotoxicity Study of Novel 4-Substituted
Quinazolines Encompassed with Thiazolidinone and Azetidinone
*
AKASH JORI, SHESHAGIRI R. DIXIT and GURUBASAVRAJ V. PUJAR
Department of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru-570015, India
*Corresponding author: Fax: +91 821 2548359; E-mail: gvpujar@jssuni.edu.in
Received: 26 May 2020;
Accepted: 18 July 2020;
Published online: 25 September 2020;
AJC-20079
A series of quinazolines encompassed with thiazolidinone and azetidinone have been synthesized and evaluated for their antioxidant,
anticancer and DNA binding studies.All the synthesized compounds were characterized by IR, 1H & 13C NMR and mass spectra.Antioxidant
activity was carried out using % free radical scavenging by DPPH assay. Compounds 4b, 5b and 5d have shown better antioxidant activity
(60, 67 and 66%, respectively) among the tested compounds. Compounds having % free radical scavenging activity more than 55% were
evaluated for anticancer activity by MTT assay towards cell lines A-549 (lung carcinoma) and MDA-231 (human breast cancer). Results
revealed that the tested compounds exhibited moderate to low anticancer activity. Further, DNA binding activity was studied by absorption
titration method for all the synthesized compounds, and compound 5b showed a good binding constant of 70.05 and % hyperchromicity of
82.93%.
Keywords: Quinzolines, Thiazolidinones, Azetidinones, in vitro anticancer activity, MTT assay.
[5], fungicidal [6], antituberculosis [7], antimalarial [8] and anti-
viral [9]. Several clinically useful drugs such as doxazosinmes-
ylate, quinoxalinephthalazine, cinnoline, methotrexate, dipro-
qualone, gefitinib possess quinazoline moiety. The anticancer
activity of quinazoline derivatives is one of the most important
properties because of their behaviour as a multi target molecules
[10], some of the derivatives causes the polymerization of tubulin
after interaction [11]. Numerous quinazolines impress apoptosis
inducers or influence acute phase in the cell cycle [12], apart
from these they are inhibitors of dihydrofolate reductase [13],
topoisomerase I [14], checkpoint kinase [15], and protein
kinase [16] such as gefitinib. In view of these findings on quina-
zolines, herein the synthesis, characterization of novel quinazo-
line moiety linked with thiazolidin-4-ones and azetidin-2-one
and evaluated for in vitro antioxidant, anticancer activities and
DNA binding studies is proposed.
INTRODUCTION
Today, cancer is a growing problem in developed and
undeveloped countries and one of the principal causes of
fatality around the globe [1]. Many researchers have focused to
work on the development of newer anticancer agents by various
approaches and among them one of most commonly employed
strategies of new drug design is molecular exploitation invol-
ving the efforts to coalesce group having comparable activity
or by eliminating or substituting new moiety to a parent lead
compound. Structural variation brings about new physical,
biological and chemotherapeutic properties in the lead mole-
cule [2]. The improvement of multifunctional remedial tools,
i.e. a drug candidate to interact with manifold distorted patho-
genetic pathways for the multifactorial mechanistic nature of
cancer cells, is of current interest.
A nitrogen-rich heterocyclic compound characterizes a
distinctive category of chemicals with wide variety of biolo-
gical activities and has been modified to the design novel pharm-
aceutically active compounds [3,4]. Among nitrogen contain-
ing heterocyclic compounds, quinazolines are known for their
wide spectrum of biological activities like, anti-inflammatory
EXPERIMENTAL
The reagents used for synthesis were of laboratory grade
and solvents of analytical grade. The melting point of the
synthesized compounds was determined in open capillary method
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2618 Jori et al.
Asian J. Chem.
O
N
NHNH2
and are uncorrected. A periodic monitoring of the reaction was
made through TLC with the solvent system toluene:ethyl acetate
(3:7). IR spectra were recorded on Shimadzu FT-IR 8400-S
spectrophotometer by KBr pellet technique and are expressed
in cm-1. UV absorbance was recorded using Shimadzu-1800
NH2
NH2NH2 H2O
NH
HCONH2
N
FUSION
140 °C
ZnCl2/Dioxane
COOH
N
(1)
(2)
1
UV Spectrophotometer. H NMR spectra were recorded on
R-CHO
C2H5OH
Bruker 400 MHz FT-NMR spectrophotometer using DMSO
(d6) and CDCl3 as the solvent and TMS as internal standard (δ
ppm). Mass spectra were recorded on GC-MS with electron
impact ionization technique.
CH3COOH
R
N
N
HN
R
H
N
General procedure for the synthesis of quinazoline-
4(3H)-one (1):An equimolar quantity of anthranilic acid (0.01
mol) and formamide (0.01 mol) were fused together at 140 ºC
for 2 h. Further, the resulting reaction mixture was cooled to
room temperature and extracted with ethyl acetate. The organic
layer was treated with sodium bicarbonate to remove the excess
of anthranilic acid and the organic layer was evaporated to
dryness under vacuum to obtain solid product, dried and
recrystallized from ethanol to get quinazolin-4(3H)-one (1)
[17].
HSCH2COOH
N
S
ZnCl2
Dioxane
N
N
N
O
(3a-e)
(4a-e)
ClCH2COCl
Dioxane
R
Phenyl
a
b
c
O
2-Hydroxy phenyl
2-Chloro phenyl
H
N
N
d
e
4-Pyridyl
N
N
Thiophen-2-yl
General procedure for the synthesis of 1-(quinazolin-
4-yl) hydrazine (2): 0.01 mol of quinazoline-4(3H)-one (1),
hydrazine hydrate (0.02mol) and zinc chloride (1.0 g) were
fused at 150ºC for 8 h. Then the reaction mixture was poured
in dichloromethane with stirring and solid product formed was
filtered, washed with cold water to remove the excess of zinc
chloride, dried and recrystallized from ethanol to gave 1-
(quinazolin-4-yl) hydrazine (2).Yield: 72%, m.p.: 132-136 ºC,
m.w.: 160. FT-IR (KBr, νmax, cm-1): 3450 (NH str.); 2924 (Ar-H);
Cl
R
(5a-e)
Scheme-I
2H, -OH and NH), 8.54 (s, 2H, N=Ch and quinazoline C2-H),
7.90-8.25 (m, 3H, quinazoline C5, C7, C8-H), 6.95-7.68 (m,
5H, quinazoline C6-H and 2-OHph C3, C4, C5, C6-H). 13C NMR
(100 MHz, DMSO-d6): (δ, ppm) 170, 156, 150, 144, 133, 129,
127, 115. Mass (GC-MS) m/z = 265 (M+1).
1
4-(2-(2-Chlorobenzylidene)hydrazinyl)quinazoline
(3c): Yield: 65%, m.p.: 155-158 ºC, m.w.: 282. FT-IR (KBr,
1620 (Ar-C=C); 1543 (C=N). H NMR (400 MHz, DMSO-
d6) (δ, ppm): 9.01 (s, 1H, C2-H), 7.38 (m, 2H, C6, C7-H), 6.98
(m, 2H, C5, C8-H), 5.42 (m, 3H, C4-H and 2-NH), 4.20 (s, 2H,
-NH2). Mass (GC-MS) m/z = 161 (M+1)
ν
max, cm-1): 3448 (NH), 3039 (Ar-H), 1620 (N=CH), 1556 (Ar
C=C), 1306(C-N), 748 (Cl). 1H NMR (400 MHz, DMSO-d6):
(δ, ppm) 10.66 (s, 1H, NH), 8.50 (s, 1H, quinazoline C2-H),
8.10-8.28 (m, 2H, quinazoline C8-H and N=CH), 7.24-7.86
(m, 7H, quinazoline C5, C6, C7-H and 2-Clph C3, C4, C5, C6-H).
Mass (GC-MS) m/z = 283 (M+1).
General procedure for the synthesis of (E)-4-(2-benzyli-
denehydrazinyl)quinazoline (3a): To an equimolar concen-
tration solution of 1-(quinazolin-4-yl)hydrazine (2), substituted
benzaldehyde in ethanol and catalytic amount of glacial acetic
acid was added. Then the mixture was refluxed for 2-4 h on
water bath. The completion of reaction was monitored by TLC.
The excess of solvent was distilled off and then remaining
residue was poured in to ice cold water. The separated solid
was filtered, washed and recrystalized from ethanol to give 2-
benzylidene-1-(quinazolin-4-yl)hydrazine (3a) (Scheme-I).A
similar procedure was followed to synthesize other quinazoline
Schiff bases (3b-e) using appropriate aldehydes. Yield: 60%,
m.p.: 140-143 ºC, m.w.: 248. FT-IR (KBr, νmax, cm-1): 3039
(Ar-H); 1689 (N=CH); 1620 (Ar C=C), 1311 (C-N). 1H NMR
(400 MHz, DMSO-d6): (δ, ppm) 10.48 (s, 1H, NH), 8.40 (s,
2H, quinazoline C2-H and N=CH), 7.80-8.20 (m, 5H, quina-
zoline C5, C7, C8-H and ph C2, C6-H), 7.50-7.65 (m, 4H, quina-
zoline C6-H and ph C3, C4, C5-H). 13C NMR (100 MHz, DMSO-
d6): (δ, ppm) 170, 157, 150, 145, 134, 130, 129, 128, 126, 117.
Mass (GC-MS) m/z = 249 (M+1).
4-(2-(Pyridin-4-ylmethylene)hydrazinyl)quinazoline
(3d): Yield: 63%, m.p.: 200-202 ºC, m.w.: 249. FT-IR (KBr,
ν
max, cm-1): 3425 (NH ); 3047 (Ar-H); 1612(N=CH); 1530 (Ar
C=C); 1311(C-N). 1H NMR (400 MHz, DMSO-d6): (δ, ppm)
10.50 (s, 1H, NH), 8.66 (s, 2H, pyridine C2, C6-H), 8.40 (s, 2H,
N=CH and quinazoline C2-H), 7.60-8.08 (m, 6H, pyridine C2,
C6-H and quinazoline C5, C6, C7, C8-H).
4-(2-(Thiophen-2-ylmethylene)hydrazinyl)quinazoline
(3e): Yield: 62%, m.p.: 170-173 ºC, m.w.: 254. FT-IR (KBr,
ν
max, cm-1): 3386 (NH), 3070 (Ar-H), 1612 (N=CH), 1535 (Ar
C=C), 1311 (C-N), 1265 (C=S). 1H NMR (400 MHz, DMSO-
d6): (δ, ppm) 10.52 (s, 1H, NH), 8.66 (s, 1H, quinazoline C2-H),
8.25 (s, 2H, quinazoline C8-H and N=CH), 7.80-7.88 (m, 3H,
thiophen C5-H and quinazoline C5, C7-H), 7.60 (s, 2H, thipohen
C3-H and quinazoline C6-H), 7.24 (s, 1H, thiophen C4-H).
General procedure for the synthesis of 2-phenyl-3-
(quinazolin-4-yl amino)thiazolidin-4-one (4a):A mixture of
0.01 mol of 2-benzylidene-1-(quinazolin-4-yl) hydrazine (3a)
and thioglycolic acid (0.02 mol) in presence of zinc chloride
and 1,4-dioxane were refluxed for 12 h. The completion of
4-(2-(2-Hydroxybenzylidene)hydrazinyl)quinazoline
(3b): Yield: 58%, m.p.: 220-222 ºC, m.w.: 264. FT-IR (KBr,
ν
max, cm-1): 3600 (OH); 3424 (NH); 3008 (Ar-H); 1612
(N=CH). 1H NMR (400 MHz, DMSO-d6): (δ, ppm) 11.40 (s,

Vol. 32, No. 10 (2020)
Synthesis and in vitro Cytotoxicity Study of Novel 4-Substituted Quinazolines 2619
reaction was monitored by TLC. After completion, reaction
mass was poured onto ice cold water. The formed crude product
was filtered, washed with water and recrystallized from ethanol
to give 2-phenyl-3-(quinazolin-4-ylamino)thiazolidin-4-ones
(4a). A similar procedure was followed to synthesize other 2-
substituted-3-(quinazolin-4-ylamino) thiazolidin-4-ones (4b-
e) using appropriate Schiff base. m.p.: 118-120 ºC.Yield: 62%,
m.w.: 322. FT-IR (KBr, νmax, cm-1): 3630 (NH str), 2996 (Ar-
H), 1637 (C=O), 1565 (Ar C=C), 1232 (C-S), 830 (C-N). 1H
NMR (400 MHz, DMSO-d6): (δ, ppm) 8.40 (s, 2H, quinazoline
C2-H and -NH), 7.83 (s, 2H, quinazoline C5, C7-H), 7.48 (s,
1H, quinazoline C6-H), 7.38 (m, 5H, Ar-H), 6.02 (s, 1H,
thiazolidinone C2-H), 3.75 (m, 2H, thiazolidinone C4-H). 13C
NMR (400 MHz, DMSO-d6): (δ, ppm) 168, 155, 148, 140,
131, 129, 128, 126, 115, 66, 36. Mass (GC-MS) m/z = 323
(M+1).
2-(2-Hydroxyphenyl)-3-(quinazolin-4-ylamino)thiazo-
lidin-4-one (4b): Yield: 60%, m.p.: 205-208 ºC, m.w.: 338.
FT-IR (KBr, νmax, cm-1): 3630 (OH), 3416 (NH), 3045 (Ar-H),
1620 (C=O), 1582 (N=CH), 1572 (Ar C=C), 1240 (C-S), 856
(C-N). 1H NMR (400 MHz, DMSO-d6): (δ, ppm) 9.50 (s, 1H,
OH), 8.45 (s, 2H, quinazoline C2-H and -NH), 8.10 (s, 1H, quina-
zoline C8-H), 7.75 (s, 2H, quinazoline C5, C7-H), 7.40 (s, 1H,
quinazoline C6-H), 7.11 (s, 2H, 2-OHph C4, C6-H), 6.80 (s,
2H, 2-OHph C3, C5-H), 5.80 (s, 1H, thiazolidinone C2-H), 3.75
(s, 2H, thiazolidinone C4-H). Mass (GC-MS) m/z = 339 (M+1).
2-(2-Chlorophenyl)-3-(quinazolin-4-ylamino)thia-
zolidin-4-one (4c):Yield: 60%, m.p.: 136-138 ºC, m.w.: 356.
FT-IR (KBr, νmax, cm-1): 3676 (NH), 3049 (Ar-H), 1682 (C=O),
1487 (Ar C=C), 1271 (C-S), 854 (C-N), 748 (C-Cl). 1H NMR
(400 MHz, DMSO-d6): (δ, ppm) 8.50 (s, 1H, quinazoline C2-H),
8.25 (s, 1H, NH and quinazoline C8-H), 7.90-7.94 (s, 2H, quina-
zoline C5, C7-H), 7.65-7.62 (m, 2H, 2-OHph C3-H and quina-
zoline C6-H); 7.11-7.18 (m, 3H, quinazoline C4, C5, C6-H), 5.76
(s, 1H, thiazolidinone-C2-H); 3.55-3.61 (m, 2H, thiazolidinone-
CH2). 13C NMR (400 MHz, DMSO-d6): (δ, ppm) 170, 157, 150,
133, 131, 128, 127, 125, 117, 105, 60, 36. Mass (GC-MS) m/z
= 355 (M-1).
2-(Pyridin-4-yl)-3-(quinazolin-4-ylamino)thiazolidin-
4-one (4d): Yield: 70%, m.p.: 180-182 ºC, m.w.: 323. FT-IR
(KBr, νmax, cm-1): 3446 (NH), 3068 (Ar-H), 1680 (C=O), 1541
(Ar C=C), 1269 (C-S), 840 (C-N). 1H NMR (400 MHz, DMSO-
d6): (δ, ppm) 8.65-8.50 (m, 3H, pyridine C2, C6-H and quinazo-
line C2-H), 8.25 (s, 2H, quinazoline C8-H and NH), 7.80-7.84
(m, 2H, quinazoline C5, C7-H), 7.30-7.60 (m, 3H, pyridine C3,
C5-H and quinazoline C6-H), 6.02 (s, 1H, thiazolidinone C2-H),
3.75-3.80 (m, 2H, thiazolidinone C4-H). Mass (GC-MS) m/z
= 323 (M+).
3-(Quinazolin-4-ylamino)-2-(thiophen-2-yl)thiazolidin-
4-one (4e): Yield: 70%, m.p.: 154-157 ºC, m.w.: 328. FT-IR
(KBr, νmax, cm-1): 3402(NH), 3043 (Ar-H), 1610 (C=O), 1552
(Ar C=C), 1234 (C-S), 830 (C-N). 1H NMR (400 MHz, DMSO-
d6): (δ, ppm) 8.50 (s, 1H, quinazoline C2-H), 8.25-8.30 (m, 2H,
quinazoline C8-H and NH), 7.35-7.80 (m, 4H, quinazoline C5,
C6, C7-H and thiophene C5-H), 6.80 (s, 2H, thiophene C3, C4-H),
5.80 (s, 1H, thiazolidinone C2-H), 3.75-3.80 (m, 2H, thiazoli-
dinone C4-H).
General procedure for the synthesis of 3-chloro-4-phenyl-
1-(quinazolin-4-ylamino)azetidin-2-one (5a): An equimolar
quantity of 2-benzylidene-1-(quinazolin-4-yl) hydrazine (3a)
and chloroacetyl chloride were dissolved in minimum quantity
of 1,4-dioxane was stirred for 48 h at room temperature. The
completion of reaction was monitored byTLC.The solid obtained
was filtered, dried and recrystallized from absolute ethanol to
give 3-chloro-4-phenyl-1-(quinazolin-4-ylamino)azetidin-2-
one (5a). A similar procedure was followed to synthesize other
3-chloro-4-substituted-1-(quinazolin-4-ylamino)-azetidin-2-
one (5b-e) using appropriate Schiff bases. Yield: 70%, m.p.:
120-122 ºC, m.w.: 324. FT-IR (KBr, νmax, cm-1): 3345 (NH
str), 3030 (Ar-H), 1676(C=O), 1538 (Ar C=C), 1350 (C-N),
698 (Cl). 1H NMR (400 MHz, DMSO-d6): (δ, ppm) 8.50 (s,
1H, quinazoline C2-H), 8.25-8.30 (m, 2H, quinazoline C8-H
and NH), 7.67-7.52 (m, 3H, quinazoline C5, C6, C7-H), 7.20-
7.38 (m, 5H, Ar-H), 5.30 (s, 1H, azetidinone C3-H), 4.99 (s,
1H, azetidinone C4-H). Mass (GC-MS) m/z = 323 (M-1).
3-Chloro-4-(2-hydroxyphenyl)-1-(quinazolin-4-yl-
amino)azetidin-2-one (5b): Yield: 60%, m.p.: 210-212 ºC,
m.w.: 340. FT-IR (KBr, νmax, cm-1): 3400 (NH), 3043 (Ar-H),
1650 (C=O), 1550 (Ar C=C), 790 (C-N), 704 (Cl). 1H NMR
(400 MHz, DMSO-d6): (δ, ppm) 10.03 (s, 1H, OH), 8.40 (s, 1H,
quinazoline C2-H), 7.80-7.99 (m, 5H, quinazoline C5, C6, C7,
C8-H and NH), 6.80-7.15 (m, 4H, 2-OHph C3, C4, C5, C6-H),
5.30 (s, 1H, azetidinone C3-H), 4.99 (s, 1H, azetidinone C4-H).
Mass (GC-MS) m/z = 340 (M+).
3-Chloro-4-(2-chlorophenyl)-1-(quinazolin-4-yl-
amino)azetidin-2-one (5c): Yield: 60%, m.p.: 130-132 ºC,
m.w.: 358. FT-IR (KBr, νmax, cm-1): 3446 (NH), 3070 (Ar-H),
1664 (C=O), 1585 (Ar C=C), 1271 (C-N), 700 (Cl). 1H NMR
(400 MHz, DMSO-d6): (δ, ppm) 8.50 (s, 1H, quinazoline C2-H),
8.23 (s, 2H, NH and quinazoline C8-H), 7.91-7.58 (m, 4H,
quinazoline C5, C6, C7-H and 2-Clph C3-H), 7.18-7.22 (m, 3H,
2-Clph C4, C5, C6-H), 5.30 (s, 1H, azetidinone C3-H), 4.99 (s,
1H, azetidinone C4-H). Mass (GC-MS) m/z = 358 (M+).
3-Chloro-4-(pyridin-4-yl)-1-(quinazolin-4-ylamino)-
azetidin-2-one (5d):Yield: 60%, m.p.: 173-175 ºC, m.w.: 325.
FT-IR (KBr, νmax, cm-1): 3445 (NH), 3070 (Ar-H), 1635 (C=O),
1
1535 (Ar C=C), 1284 (C-N), 794 (Cl). H NMR (400 MHz,
DMSO-d6): (δ, ppm) 8.55-8.50 (m, 3H, quinazoline C2-H and
pyridine C2, C6-H), 8.23 (s, 2H, NH and quinazoline C8-H), 7.91
(s, 2H, quinazoline C5, C7-H), 7.30-7.52 (m, 3H, pyridine C3,
C5-H), 5.30 (s, 1H, azetidinone C3-H), 4.99 (s, 1H, azetidinone
C4-H). 13C NMR (100 MHz, DMSO-d6): (δ, ppm) 170, 157, 148,
132, 128, 126, 123, 115, 68, 65. Mass (GC-MS) m/z = 325 (M+).
3-Chloro-1-(quinazolin-4-ylamino)-4-(thiophen-2-yl)-
azetidin-2-one (5e):Yield: 70%, m.p.: 165-168 ºC, m.w.: 330.
FT-IR (KBr, νmax, cm-1): 3331 (NH), 2922 (Ar-H), 1633 (C=O),
1568 (Ar C=C), 779 (C-N), 704 (Cl). 1H NMR (400 MHz, DMSO-
d6): (δ, ppm) 8.55-8.50 (m, 1H, quinazoline C2-H), 8.23 (s, 2H,
NH and quinazoline C8-H), 7.88 (s, 2H, quinazoline C5, C7-H),
7.30-7.52 (m, 2H, quinazoline C6-H and thiophen C5-H), 7.10
(s, 2H, thiophen C3, C4-H), 5.30 (s, 1H, azetidinone C3-H), 4.99
(s, 1H, azetidinone C4-H). 13C NMR (100 MHz, DMSO-d6):
(δ, ppm) 170, 165, 157, 148, 132, 129, 128, 127, 115, 68, 65.
Mass (GC-MS) m/z = 331 (M+1).

2620 Jori et al.
Asian J. Chem.
Biological activity
given complex with DNA was obtained from a plot of D/∆εapp
versus D according to the following equation:
in vitro DPPH radical scavenging assay:All the synthe-
sized molecules were analyzed for antioxidant activity using
DPPH assay method. To 1 mL of synthesized compounds (40
µg/mL) mixed with 1 mL DPPH solution (40 µg/mL) in methanol
and incubated at 37 ºC for 30 min and the control was prepared
as above without sample was used for the baseline correction.
The absorption of each solution was measured at 517 nm [18].
Experiment was performed in triplicate.
D
D
1
=
+
∆εapp ∆ε ∆ε×K
where D = concentration of DNA in base molarities.
∆εapp = εa − εf and ∆ε = εb − εf
where εa and εf are respective extinction coefficient of the complex
in the presence and absence of DNA. The apparent extinction
coefficient εa is obtained by calculating Aobs/[Drug]. The data
were fitted to the equation with a slope equal to 1/∆ε and
Y-intercept equal to 1/(∆ε × K). The intrinsic binding constant
(Ki) is determined from the slope ofY-intercept. The percentage
hypochromism was calculated as follows [21]:
Acontrol − Asample
Radical scavenging activity (%) =
×100
Acontrol
where control absorbance is the measurement of DPPH radical
solution without compound and sample absorbance is the
measurement of DPPH radical solution with the compound.
Ascorbic acid (40 µg/mL) solution was used as a standard for
the comparison of antioxidant activity.
εfree − εbound
Hypochromism (%) =
×100
εfree
MTT based cytotoxicity activity: Cellular conversion
of MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium
bromide] into a formazan product [19] was used to evaluate
cytotoxic activity (IC50) of the compounds againstA549 (lung
adenocarcinoma) and MDA 231 (breast cancer) cell-line up
to concentrations ranging from 31.25-500 µg/mL using Promega
Cell Titer 96 non-radioactive cell proliferation assay [20] with
cisplatin as the positive control. The IC50 values are the averages
SEM of three independent experiments.
A-549 (Lung carcinoma) and MDA-231 (breast cancer)
cell lines were plated 5000-10,000 cells/well in 100 µL was
seeded into the wells of 96 well plate. The plates were incubated
overnight in a humidifier air atmosphere at 37 ºC with 5%
CO2. After overnight incubation, 100 µL of the compounds in
DMSO were added to each well containing media and missed
thoroughly at 150 rpm for 5 min. The plates were incubated
further 48 h to allow the drug to produce effect. After comple-
tion of the treatment, 20 µL MTT solution was added to each
well and mixed well at 150 rpm for 5 min followed by 3 h
incubation.After incubation, the media was then replaced with
200 µL DMSO and the absorbance of each well was measured
at 570 nm. For each compound, the concentration causing 50%
cell growth inhibition (IC50) compared with the control was
calculated from concentration response curves by regression
analysis.
DNA binding activity by absorption titration:The spectro-
metric titration was conducted by UV spectrophotometer at
room temperature. The CT-DNA was dissolved in double distilled
de-ionized water with 50 mM NaCl and dialyzed against buffer
solution for 2 days. Its concentration was determined by absor-
ption spectrometry at 340 nm using a molar extinction coeffi-
cient 6600 M-1 cm-1. The ratio A325/A355 > 1.80 was used as an
indication of a protein-free DNA. Absorption titration was
performed at a fixed concentration of drugs (10 µM) in a sodium
phosphate buffer (20 mM sodium phosphate, 150 mM NaCl,
pH 74). Small aliquots of concentrated CT-DNA (5 mM) were
added into the solution at final concentrations from 0 to 100
mM and stirred for 5 min before measurement. The parameters,
hypochromicity and binding constant were found from the
absorption spectra. The intrinsic binding constant (Ki) for a
RESULTS AND DISCUSSION
A series of quinazoline encompassed with thiazolidinones
(4a-e) and azetidinones (5a-e) were synthesized using synthetic
procedure as per Scheme-I. The precursor, quinazoline-4(3H)-
one (1)was synthesized by fusing anthranilic acid with formamide
which follows Niementowski reaction, further the formation
of hydrazide (2) was straight forward by reacting it with hydra-
zine hydrate in presence of ZnCl2 and 1,4-dioxane. Hydrazide
derivative (2) was then made to react with different aldehydes
in presence of ethanol and AcOH to yield quinazoline Schiff
bases (3a-e). Schiff bases (3a-e) were then made to react with
thioglycolic acid and chloroacetyl chloride to respectively yield
thiazolidinones (4a-e) and azetidinones (5a-e) and compounds
were characterized.
FTIR spectrum of compound 3a, as a representative of
Schiff bases, showed absorption band at 1689 cm-1 indicates
the presence of -N=CH- of Schiff base, further the structure
of compound 3a was confirmed by 1H NMR, which showed a
singlet peak at 10.48 ppm representing the proton of -NH group
and a singlet peak appeared at 8.40 ppm indicating the presence
of quinazoline C2-H and imine group (-N=CH-) and mass spectra
confirms the formation of compound 3a, a molecular ion peak
at 249 (M+1).
FTIR spectrum of compound 4c, showed absorption band
at 3676 cm-1 attributed to the presence of -NH, further presence
of carbonyl and C-S group were confirmed by the peaks at
1682 and 1271 cm-1, respectively. Further the structure of
compound 4c was confirmed by 1H NMR which showed the
presence of 2 protons of thiazolidinone-CH2 as a multiplet
between δ 3.55-3.61 ppm and a singlet for a proton of thiazoli-
dinone-CH was observed at 5.76 ppm. Further, mass spectra
showed a molecular ion peak at 355 (M-1).
FTIR spectrum of compound 5c showed absorption band
at 3446 cm-1, which attributed to the -NH stretching and a
peak at 1664 cm-1 for carbonyl group. Further, structural 5c
was confirmed by 1H NMR which showed a singlet peaks at 5.30
ppm and 4.99 ppm indicating C3 & C4 protons, respectively of
azetidinone. Further, mass spectra showed a molecular ion peak
at 355 (M+).

Vol. 32, No. 10 (2020)
Synthesis and in vitro Cytotoxicity Study of Novel 4-Substituted Quinazolines 2621
TABLE-2
Biological activity
% CYTOTOXICITY ACTIVITY OF THE TESTED COMPOUNDS
in vitro DPPH radical scavenging assay: in vitro DPPH
radical scavenging assay was performed spectrophotometri-
cally with ascorbic acid as positive control. The activity of
quinazoline derivatives was compared with standard ascorbic
acid (Table-1). The synthesized compounds showed significant
free radical scavenging activity ranging from 51-67% while
standard, ascorbic acid has 91% (Fig. 1). Compounds 4b, 5b,
5c and 5d have shown good antioxidant activity than the rest
of the compounds. The compounds exhibited more than 55%
of radical scavenging activity were selected for in vitro anti-
cancer activity by MTT assay method. This test was performed
to understand the radical scavenging activity of the synthesized
compounds as this activity is associated with anti-proliferative
property of molecules.
A-549
(Lung carcinoma)
MDA-231
(Breast cancer)
Compound
Control
Cisplatin
4b
–
9.9
–
8.0
153.20
144.08
142.91
134.77
127.25
137.77
129.25
134.73
149.06
138.39
146.19
142.91
137.40
145.14
139.41
145.37
4c
4d
4e
5b
5c
5d
5e
method using CT-DNA. This study was performed to study the
intercalation of molecule with DNA as they are analogues of
quinaolines. To examine this aspect, the intercalation of synthe-
sized compounds with duplex DNA was examined by monitoring
the changes in the UV-visible spectra of quinazoline analogues
upon addition of CT-DNA. The DNA binding properties of
the compounds were studied by monitoring the change in the
absorption spectra of the quinazoline analogues upon addition
of CT-DNA.
As shown in Figs. 2-4, the absorption spectra of compounds
4b, 5b and 5d, respectively in the absence and presence of
varying amount of CT-DNA. The percentage hypochromism
was found to be 52.46, 73.54 and 82.93. Similarly, the absor-
ption spectra of all the compounds were also obtained. The
binding of the compounds to duplex DNA led to strong decrease
in the absorption intensities in the absorption maxima. The
progressive addition of DNA leads to strong hypochromism
in the absorption intensity. The half reciprocal plots for binding
of compounds 4b, 5b and 5d with CT-DNA are shown in
Figs. 5-7. The binding constants (Ki) of synthesized compounds
were in the following order 4a > 4d > 4e > 4b > 4c > 5a > 5b
> 5e > 5c > 5d. The tested compounds showed good to moderate
DNA binding activity ranging from 49.31 to 80. The % hyper-
chromicity and DNA binding constant value are given in Table-
3.
TABLE-1
ANTIOXIDANT ACTIVITY DATA OF
THE SYNTHESIZED COMPOUNDS
% Free radical scavenging
Compounds
activity at 40 µg/mL
51
64
57
59
56
52
67
60
66
58
91
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
Ascorbic acid (std)
100
90
80
70
60
50
40
30
20
10
0
2.000
1.500
1.000
0.500
0
4a 4b 4c 4d 4e 5a 5b 5c 5d 5e Std.
Concentration at 40 µg/mL
Fig. 1. in vitro free radical scavenging activity of the synthesized compounds
MTT based cytotoxicity activity: The selected synthe-
sized compounds (4b, 4c, 4d, 4e, 5b, 5c, 5d and 5e) were
evaluated for in vitro anticancer activity against two human
cancer cell lines, respectively A-549 (lung carcinoma) and
MDA-231 (human breast cancer). The percentage cytotoxicity
for the tested compounds was calculated as per the MTT standard
protocol using cisplatin as standard drug. The IC50 values
reported in Table-2 indicates that thiazolidinone derivatives
are comparatively shows less significant activity whereas the
azitidinone derivatives in particular compound 5b exhibited
better cytotoxic activity against all the tested cell lines.
DNA binding activity: The binding properties of all the
synthesized compounds were evaluated by UV absorptiometry
-0.500
305
320
340
360
380
400
Wavelength (nm)
Fig. 2. Absorption titration of 4b at 10 µM in 20 mM sodium phosphate
buffer with 150 mM NaCl at increasing CT-DNA concentration

2622 Jori et al.
Asian J. Chem.
1.000
0.030
0.025
0.020
0.015
0.010
0.005
0
0.800
0.600
0.400
0.200
y = 0.270x – 0.000
R2 = 0.991
0
0.02
0.04
0.06
0.08
0.10
0.12
-0.005
D × 10
Fig. 6. Half reciprocal plot for binding of 5b with CT-DNA
0.030
0.025
0.020
0.015
0.010
0.005
0
0
310
320
330
340
350
Wavelength (nm)
Fig. 3. Absorption titration of 5b at 10 µM in 20 mM sodium phosphate
buffer with 150 mM NaCl at increasing CT-DNA concentration
y = 0.270x – 0.000
R2 = 0.991
1.000
0.800
0.600
0.400
0.200
0
0
0.02
0.04
0.06
D × 10
0.08
0.10
0.12
-0.005
Fig. 7. Half reciprocal plot for binding of 5d with CT-DNA
TABLE-3
DNA BINDING RESULTS OF SYNTHESIZED COMPOUNDS
Compound
% Hyperchromicity
51.32
Ki
49.31
56.28
55.32
52.34
54.23
62.56
70.05
63.54
68.75
64.55
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
53.12
52.46
48.54
51.62
68.32
82.93
71.98
73.54
305
320
340
360
380
400
Wavelength (nm)
Fig. 4. Absorption titration of 5d at 10 µM in 20 mM sodium phosphate
67.89
buffer with 150 mM NaCl at increasing CT-DNA concentration
0.18
0.16
0.14
0.12
0.10
Conclusion
The present study was focused on the synthesis and charac-
terization of new quinazolines encompassed with thiazoli-
diones and azetidinones with the hope of developing new chemical
entities serving as potential lead for anticancer agents. The
antioxidant, anticancer and DNA binding studies were also
evaluated. The results of antioxidant activity of the compounds
4b, 5b and 5d (64, 67 and 66%, respectively @ 40 µg/mL)
showed a better % free radical scavenging activity then other
synthesized compounds as compared to standard, ascorbic acid
has 91% @ 40 µg/mL. The in vitro cytotoxic potential of the
compounds were tested against two human cancer cell lines
namely,A-549 lung carcinoma and MDA-231 breast carcinoma
0.08
0.06
0.04
0.02
0
y = 1.437x + 0.026
R2 = 0.776
0
0.02
0.04
0.06
0.08
0.10
0.12
D × 10
Fig. 5. Half reciprocal plot for binding of 4b with CT-DNA

Vol. 32, No. 10 (2020)
Synthesis and in vitro Cytotoxicity Study of Novel 4-Substituted Quinazolines 2623
9. Z. Wang, M. Wang, X.Yao,Y. Li, J. Tan, L. Wang, W. Qiao,Y. Geng, Y.
by using MTT based assay method. Compound 5b was found
to be more active than other compounds in comparison with
standard, cisplatin. Further, DNA binding assay was studied
to understand the possible role of cytotoxic nature of the
compounds by DNA affinity and studies indicated that comp-
ound 5b have showed better binding constant among all the
synthesized compounds. From the results, it was observed that
in azetidinone, 4th position is substituted with hydroxy phenyl
group shows a better activity as compared to the substitution
with unsubstituted phenyl ring or heterocyclic compounds,
which showed comparatively less active. In case of thiazolidi-
nones, 2nd position of ring is substituted with unsubstituted
aromatic ring or heterocyclic ring decreases the activity as
compared to substitution with hydroxyphenyl ring.
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CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
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